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Objective To investigate the operation status and service level of radiation occupational health inspection institutions in Zhejiang Province, China, and to provide a basis for administrative departments and quality management departments to develop policies. Methods The investigation data of radiation occupational health inspection institutions in Zhejiang Province were collected for descriptive analysis of the regional distribution, nature, and service qualification of the institutions. Results There were 27 radiation occupational health inspection institutions in Zhejiang Province. These institutions were located in 11 cities, of which 85.18% were public institutions and 14.72% were private institutions. For the physical examination workload of radiation workers in Zhejiang Province in 2021, general hospitals accounted for 75.90%, private institutions accounted for 4.51%, and occupational prevention and treatment hospitals accounted for 19.59%. In the radiation occupational health inspection institutions, the stand-alone and online software installation rates were 33.33% and 37.04%, respectively. A total of 26 986 individuals (82.97%) underwent chromosome aberration examination. The examination rates of thyroid color Doppler ultrasound examination and eye lens examination were 41.24% and 82.97%, respectively. Pre-job, on-job, and off-job physical examination accounted for 25.81%, 70.52%, and 3.67%, respectively. For radiation workers who underwent on-job physical examination, diagnostic radiology workers accounted for the highest proportion of 34.90%. The excellent, qualified, and unqualified rates of 27 radiation occupational health inspection institutions were 7.41%, 88.89%, and 3.70%, respectively. Conclusion The network of radiation occupational health inspection institutions in Zhejiang Province is well-established and located mainly in general hospitals, occupational prevention and control institutions, and private institutions. To enhance the quality and proficiency of occupational health examinations, it is imperative to prioritize self-improvement and management, reinforce law enforcement supervision, actively engage in blind sample assessments, and advance the application of information technology and standardized services.
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Objective:To investigate the levels of gross radioactivity in drinking water in Hangzhou city before and after Qiandao Lake as the water source to the main urban area of Hangzhou.Methods:Since 2012, water samples were collected from water source, factory water and peripheral water in rainy and dry seasons, respectively, to determine their gross α and β activity concentrations for comparison and analysis.Results:The gross radioactivity levels in drinking water in Hangzhou are lower than the limits specified in the national standard "Standards for drinking water quality" (GB 5749-2006), without statistically significant difference for these water sources between the rainy and dry season ( P>0.05). The gross α(0.008±0.000)and gross β(0.034±0.013)levels in Qiandao lake were both less than those in Qiantang river ( Z=-3.235, -4.058, P<0.05), with significant difference ( Z=-2.181, -4.577, P<0.05). There was no significant difference in gross α and gross β in factory water and peripheral water before and after the operation of Qiandao Lake water supply project ( P>0.05). Conclusions:The gross radioactivity in drinking water in downtown Hangzhou are low from 2012 to 2020. The gross radioactivity levels in Qiandao Lake are lower than in the lower reaches of Qiantang river and Dongtiao steam. No impact was generated on radioactivity levels in drinking water after Qiandao lake supplied water to Hangzhou.
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Objective:To investigate and analyze the radioactivity levels of 90Sr and 137Cs in drinking water and 137Cs in food after the installation of the first AP1000 nuclear power unit in China. Methods:From 2012 to 2019, four drinking water monitoring points around AP1000 nuclear power unit located at Sanmen nuclear power plant site were collected during the wet season and dry season, 90Sr and 137Cs and radioactivity concentrations were determined in drinking water. Local rice, cabbage, crucian and mullet were collected to determine the radioactivity concentration of 137Cs. Results:From 2012 to 2019, the radioactivity concentrations of 90Sr and 137Cs in drinking water were 1.2-9.8 mBq/L and 0.2-8.1 mBq/L, respectively. The radioactivity concentration of 137Cs in food were 1.1×10 -2-2.8×10 -1 Bq/kg, lower than the limits specified in the Limited concentrations of radioactive materials in foods (GB 14882-94). Conclusions:After the installation of the first AP1000 nuclear power unit in China, the radioactivity levels of 90Sr and 137Cs in drinking water and 137Cs in foods are stable, without environmental impact identified.
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Objective@#To explore the effects of transient receptor potential vanilloid 1 (TRPV1) on autophagy in early hypoxic mouse cardiomyocytes and the mechanism in vitro.@*Methods@#The hearts of 120 C57BL/6 mice aged 1-2 days, no matter male or female, were isolated, and then primary cardiomyocytes were cultured and used for the following experiments, the random number table was used for grouping. (1) The cells were divided into normoxia group and hypoxia 3, 6, and 9 h groups, with one well in each group. The cells in normoxia group were routinely cultured (the same below), the cells in hypoxia 3, 6, and 9 h groups were treated with fetal bovine serum-free and glucose-free Dulbecco′ s modified Eagle medium under low oxygen condition in a volume fraction of 1% oxygen, 5% carbon dioxide, and 94% nitrogen for 3, 6, and 9 h, respectively. The protein expressions of microtubule-associated protein 1 light chain 3 (LC3), Beclin-1, TRPV1 were determined with Western botting. (2) The cells were divided into normoxia group and hypoxia group, with two coverslips in each group. The cells in hypoxia group were treated with hypoxia for 6 h as above. The positive expression of TRPV1 was detected by immunofluorescence assay. (3) The cells were divided into 4 groups, with one well in each group. The cells in simple hypoxia group were treated with hypoxia for 6 h as above, and the cells in hypoxia+ 0.1 μmol/L capsaicin group, hypoxia+ 1.0 μmol/L capsaicin group, and hypoxia+ 10.0 μmol/L capsaicin group were respectively treated with 0.1, 1.0, 10.0 μmol/L capsaicin for 30 min before hypoxia for 6 h. The protein expressions of LC3, Beclin-1, and TRPV1 were detected by Western blotting. (4) The cells were divided into 5 groups, with 5 wells in each group. The cells in hypoxia group were treated with hypoxia for 6 h as above, the cells in hypoxia+ chloroquine group, hypoxia+ capsaicin group, and hypoxia+ capsaicin+ chloroquine group were treated with hypoxia for 6 h after being cultured with 50 μmol/L chloroquine, 10.0 μmol/L capsaicin, and 50 μmol/L chloroquine+ 10.0 μmol/L capsaicin for 30 min, respectively. Viability of cells was detected by cell counting kit 8 assay. (5) The cells were divided into simple hypoxia group and hypoxia+ 10.0 μmol/L capsaicin group, with one well in each group. The cells in hypoxia group were treated with hypoxia for 6 h as above, the cells in hypoxia+ 10.0 μmol/L capsaicin group were treated with 10.0 μmol/L capsaicin for 30 minutes and then with hypoxia for 6 h. The protein expressions of lysosomal associated membrane protein 1 (LAMP-1) and LAMP-2 were detected by Western blotting. Each experiment was repeated for 3 or 5 times. Data were processed with one-way analysis of variance, least significant difference t test, and Bonferroni correction.@*Results@#(1) Compared with those of normoxia group, the protein expressions of LC3, Beclin-1, and TRPV1 were significantly increased in cardiomyocytes of hypoxia 3, 6, and 9 h groups (t3 h=4.891, 5.890, 4.928; t6 h=9.790, 6.750, 10.590; t9 h=6.948, 6.764, 5.049, P<0.05 or P<0.01), which of hypoxia 6 h group were the highest (1.08±0.05, 1.12±0.10, 0.953±0.071, respectively). (2) The density of TRPV1 in cell membrane and inside the cardiomyocytes in hypoxia group was significantly increased with lump-like distribution, and the expression of TRPV1 was higher than that in normoxia group. (3) Compared with those of simple hypoxia group, the protein expression of Beclin-1 in cardiomyocytes of hypoxia+ 0.1 μmol/L capsaicin group was increased (t=10.488, P<0.01), while the protein expressions of LC3 and TRPV1 were increased without statistically significant differences (t=4.372, 3.026, P>0.05); the protein expressions of LC3, TRPV1, and Beclin-1 in cardiomyocytes of hypoxia+ 1.0 μmol/L capsaicin group and hypoxia+ 10.0 μmol/L capsaicin group were significantly increased (t=15.505, 5.773, 13.430; 20.915, 8.054, 16.384; P<0.05 or P<0.01), which of hypoxia+ 10.0 μmol/L capsaicin group were the highest (2.33±0.09, 1.34±0.07, 1.246±0.053, respectively). (4) Compared with 0.585±0.045 in normoxia group, the cardiomyocyte viability in hypoxia group was significantly decreased (0.471±0.037, t=4.365, P<0.05). Compared with that in hypoxia group, the cardiomyocyte viability in hypoxia+ chloroquine group was further decreased (0.350±0.023, t=6.216, P<0.01), while 0.564±0.047 in hypoxia+ capsaicin group was significantly increased (t=3.489, P<0.05). Compared with that in hypoxia+ chloroquine group, the cardiomyocyte viability in hypoxia+ capsaicin+ chloroquine group did not significantly change (0.364±0.050, t=0.545, P>0.05). (5) Compared with 0.99±0.04 and 0.54±0.04 in simple hypoxia group, the protein expressions of LAMP-1 and LAMP-2 in hypoxia+ 10.0 μmol/L capsaicin group were significantly increased (1.49±0.06, 0.81±0.05, t=12.550, 7.442, P<0.01).@*Conclusions@#TRPV1 can further promote the expression of autophagy-related proteins in hypoxic cardiomyocytes through autophagy-lysosomal pathway, enhance autophagy activity, and improve autophagic flow for alleviating early hypoxic cardiomyocyte injury.
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Objective@#To investigate the effect of human antigen R on lysosomal acidification during autophagy in mouse cardiomyocytes cultured in vitro.@*Methods@#The hearts of 20 C57BL/6 mice aged 1-2 days no matter male or female were isolated to culture primary cardiomyocytes which were used in the following experiments. (1) The cells were divided into 5 groups according to the random number table (the same grouping method below), i. e., normal control group and sugar-free serum-free 0.5, 1.0, 3.0, and 6.0 h groups. The cells in normal control group were routinely cultured for 54.0 h with Dulbecco′s modified Eagle medium/nutrient mixture F12 (DMEM/F12) medium (the same regular culture condition below), and the cells in sugar-free serum-free 0.5, 1.0, 3.0, and 6.0 h groups were firstly regularly cultured for 53.5, 53.0, 51.0, 48.0 h and then cultured with replaced sugar-free serum-free medium for 0.5, 1.0, 3.0, and 6.0 h, respectively. The protein expressions of microtubule-associated protein 1 light chain 3 Ⅱ (LC3Ⅱ), autophagy-related protein 5, and adenosine triphosphatase V1 region E1 subunit (ATP6V1E1) were detected by Western blotting. (2) The cells were divided into normal control group and sugar-free serum-free 3.0 h group. The cells in corresponding groups were treated the same as those in experiment (1), and the cell lysosomal acidification level was observed and detected under a laser scanning confocal microscope. (3) Two batches of cells were grouped and treated the same as those in experiment (1). The protein expression of human antigen R in the whole protein of cells of one batch and its protein expression in the cytoplasm and nucleus protein of cells of the other batch were detected by Western blotting. (4) The cells were divided into normal control group, simple control small interfering RNA (siRNA) group, simple human antigen R-siRNA1 (HuR-siRNA1) group, simple HuR-siRNA2 group, sugar-free serum-free 3.0 h group, sugar-free serum-free+ control siRNA group, sugar-free serum-free+ HuR-siRNA1 group, and sugar-free serum-free+ HuR-siRNA2 group. After 48 hours of regular culture, the cells in simple control siRNA group and sugar-free serum-free+ control siRNA group were transfected with negative control siRNA for 6 h, the cells in simple HuR-siRNA1 group and sugar-free serum-free+ HuR-siRNA1 group were transfected with HuR-siRNA1 for 6 h, and the cells in simple HuR-siRNA2 group and sugar-free serum-free+ HuR-siRNA2 group were transfected with HuR-siRNA2 for 6 h. Hereafter, the cells in these 8 groups were continuously cultured for 48 h with regular conditon, and then the cells in normal control group and each simple siRNA-treated group were replaced with DMEM/F12 medium, the cells in the other groups were replaced with sugar-free serum-free medium, and they were cultured for 3 h. The protein expression of human antigen R in the whole protein of cells was detected by Western blotting. (5) Two batches of cells were divided into sugar-free serum-free+ control siRNA group and sugar-free serum-free+ HuR-siRNA1 group, and the cells in corresponding groups were treated the same as those in experiment (4). The distribution and expression of human antigen R in the cells of one batch were observed and detected by immunofluorescence method, and the lysosomal acidification level in the cells of the other batch was observed and detected under a laser scanning confocal microscope. (6) Three batches of cells were divided into sugar-free serum-free 3.0 h group, sugar-free serum-free+ control siRNA group, sugar-free serum-free+ HuR-siRNA1 group, and sugar-free serum-free+ HuR-siRNA2 group, and the cells in corresponding groups were treated the same as those in experiment (4). The protein expressions of cathepsin D in the whole protein of cells of one batch, human antigen R in the cytoplasm protein of cells of one batch, and ATP6V1E1 in the whole protein of cells of the other batch were detected by Western blotting. (7) The cells were divided into normal control group, sugar-free serum-free 3.0 h group, sugar-free serum-free+ control siRNA group, and sugar-free serum-free+ HuR-siRNA1 group, and the cells in corresponding groups were treated the same as those in experiment (4). The mRNA expression of ATP6V1E1 in cells was detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction. The sample number of each experiment was 3. Data were processed with independent data t test, one-way analysis of variance, least significant difference t test, and Bonferroni correction.@*Results@#(1) Compared with those of normal control group, the protein expressions of LC3Ⅱ and ATP6V1E1 in the whole protein of cells of sugar-free serum-free 1.0, 3.0, and 6.0 h groups were significantly increased (t=12.16, 4.05, 4.82, 11.64, 3.29, 8.37, P<0.05 or P<0.01). Compared with that of normal control group, the protein expression of autophagy-related protein 5 in the whole protein of cells of sugar-free serum-free 0.5, 1.0, 3.0, and 6.0 h groups was significantly increased (t=6.88, 10.56, 5.76, 9.91, P<0.05 or P<0.01). (2) Compared with 1.03±0.08 of normal control group, the lysosomal acidification level in the cells of sugar-free serum-free 3.0 group (2.92±0.30) was significantly increased (t=6.01, P<0.01). (3) There was no statistically significant difference in the overall comparison of protein expression of human antigen R in the whole protein of cells among the 5 groups (F=1.09, P>0.05). Compared with that of normal control group, the protein expression of human antigen R in the cytoplasm protein of cells was significantly increased in sugar-free serum-free 1.0, 3.0, and 6.0 h groups (t=43.05, 11.07, 5.39, P<0.05 or P<0.01), while the protein expression of human antigen R in the nucleus protein of cells was significantly decreased in sugar-free serum-free 3.0 and 6.0 h groups (t=11.18, 12.71, P<0.01). (4) Compared with that of simple control siRNA group, the protein expression of human antigen R in the whole protein of cells of simple HuR-siRNA1 group and simple HuR-siRNA2 group was significantly decreased (t=4.82, 4.44, P<0.05). Compared with that of sugar-free serum-free+ control siRNA group, the protein expression of human antigen R in the whole protein of cells of sugar-free serum-free+ HuR-siRNA1 group and sugar-free serum-free+ HuR-siRNA2 group was significantly decreased (t=4.39, 6.27, P<0.05). (5) Compared with those of sugar-free serum-free+ control siRNA group, the distribution of human antigen R in the cytoplasm of cells and its expression level were significantly decreased in sugar-free serum-free+ HuR-siRNA1 group (t=10.13, P<0.01). Compared with 1.00±0.06 of sugar-free serum-free+ control siRNA group, the lysosomal acidification level (0.73±0.06) in the cells of sugar-free serum-free+ HuR-siRNA1 group was significantly decreased (t=3.28, P<0.01). (6) Compared with those of sugar-free serum-free+ control siRNA group, the protein expressions of cathepsin D in the whole protein of cells, human antigen R in the cytoplasm protein of cells, and ATP6V1E1 in the whole protein of cells were significantly decreased in sugar-free serum-free+ HuR-siRNA1 group and sugar-free serum-free+ HuR-siRNA2 group (t=4.16, 3.99, 4.81, 5.07, 11.68, 12.97, P<0.05 or P<0.01). (7) Compared with that of normal control group, the mRNA expression of ATP6V1E1 in the cells of sugar-free serum-free 3.0 h group was significantly increased (t=5.51, P<0.05). Compared with that of sugar-free serum-free+ control siRNA group, the mRNA expression of ATP6V1E1 in the cells of sugar-free serum-free+ HuR-siRNA1 group was significantly decreased (t=5.97, P<0.05).@*Conclusions@#After sugar-free serum-free treatment in vitro, the autophagy in mouse primary cardiomyocytes is activated, the lysosomal acidification is enhanced, and the expression of human antigen R in cytoplasm is increased. Human antigen R function is activated and involved in maintaining lysosomal acidification during autophagy in mouse cardiomyocytes.
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Objective@#To investigate the role of hexokinase Ⅱ in the changes of autophagic flow in cardiomyocytes of mice with ischemia-hypoxia in vitro.@*Methods@#The hearts of totally six male and female C57BL/6 mice aged from 1 to 2 days were isolated to culture primary cardiomyocytes which were used for the following experiments. (1) The cells were divided into 6 groups according to the random number table (the same grouping method below), i. e., normal control 3, 6, and 9 h groups and ischemia-hypoxia 3, 6, and 9 h groups, with 4 wells in each group. After being regularly cultured for 48 h with Dulbecco′s modified Eagle medium/nutrient mixture F12 (DMEM/F12) medium (the same regular culture condition below), the cells in normal control 3, 6, and 9 h groups were cultured with replaced fresh DMEM/F12 medium for 3, 6, and 9 h, respectively, and the cells in ischemia-hypoxia 3, 6, and 9 h groups were cultured with replaced sugar-free serum-free medium in the low-oxygen incubator with a volume fraction of 1% oxygen and a volume fraction of 5% carbon dioxide at 37 ℃ (the same hypoxic culture condition below) for 3, 6, and 9 h, respectively. Cell viability was measured by the cell counting kit 8 (CCK-8) method. (2) The cells were grouped and treated the same as those in experiment (1), with 1 well in each group. Western blotting was used to detect the protein expressions of microtubule-associated protein 1 light chain 3 Ⅰ (LC3Ⅰ), LC3Ⅱ, p62, and hexokinase Ⅱ. (3) The cells were divided into normal control group, simple ischemia-hypoxia 9 h group, and ischemia-hypoxia 9 h+ 2-deoxyglucose (2-DG) group, with 4 wells in each group. After a regular culture for 48 h, the cells in normal control group were cultured with replaced fresh DMEM/F12 medium for 9 h; the cells in simple ischemia-hypoxia 9 h group were replaced with sugar-free serum-free medium, and the cells in ischemia-hypoxia 9 h+ 2-DG group were replaced with sugar-free serum-free medium in which 2-DG was dissolved in a concentration of 10 mmol/L (20 μmol), and then they were cultured with hypoxia for 9 h. Cell viability was measured by CCK-8 method. (4) The cells were grouped and treated the same as those in experiment (3), with 1 well in each group. Western blotting was used to detect the protein expressions of LC3Ⅰ, LC3Ⅱ, and p62. (5) The cells were grouped and treated the same as those in experiment (3), with 2 wells in each group. Transmission electron microscope was used to observe autophagosomes/autolysosomes in cardiomyocytes. (6) The cells were divided into normal control group, simple ischemia-hypoxia 9 h group, ischemia-hypoxia 9 h+ hexosinase Ⅱ small interfering RNA1 (HK-ⅡsiRNA1) group, and ischemia-hypoxia 9 h+ HK-ⅡsiRNA2 group, with 4 wells in each group. The cells in normal control group and simple ischemia-hypoxia 9 h group were regularly cultured for 48 h, and the cells in ischemia-hypoxia 9 h+ HK-ⅡsiRNA1 group and ischemia-hypoxia 9 h+ HK-ⅡsiRNA2 group were respectively transfected with 200 nmol/L HK-ⅡsiRNA1 and HK-ⅡsiRNA2 and then also cultured for 48 h. The cells in normal control group were cultured with replaced fresh DMEM/F12 medium for 9 h, and the cells in simple ischemia-hypoxia 9 h group, ischemia-hypoxia 9 h+ HK-ⅡsiRNA1 group, and ischemia-hypoxia 9 h+ HK-ⅡsiRNA2 group were cultured with replaced sugar-free serum-free medium and hypoxia for 9 h. Cell viability was measured by CCK-8 method. (7) The cells were grouped and treated the same as those in experiment (6), with 1 well in each group. Western blotting was used to detect the protein expressions of LC3Ⅰ, LC3Ⅱ, p62, and hexokinase Ⅱ. Except for experiment (5), each experiment was repeated 3 times. Data were processed with one-way analysis of variance and lest significant difference t test, and Bonferroni correction.@*Results@#(1) The viabilities of cardiomyocytes in ischemia-hypoxia 3, 6, and 9 h groups were 0.450±0.022, 0.385±0.010, and 0.335±0.015, respectively, which were significantly lower than 0.662±0.026, 0.656±0.028, and 0.661±0.021 of the corresponding normal control 3, 6, and 9 h groups, respectively (t=6.21, 9.12, 12.48, P<0.01). (2) Compared with those of corresponding normal control 3, 6, and 9 h groups, the LC3Ⅱ/Ⅰ ratio and protein expressions of p62 and hexokinase Ⅱ in cardiomyocytes of ischemia-hypoxia 3, 6, and 9 h groups were significantly increased (t3 h=16.15, 10.99, 5.30, t6 h=6.79, 10.42, 9.42, t9 h=15.76, 16.51, 7.20, P<0.05 or P<0.01). (3) The viability of cardiomyocytes in simple ischemia-hypoxia 9 h group was 0.353±0.022, which was significantly lower than 0.673±0.027 of normal control group (t=9.29, P<0.01). The viability of cardiomyocytes in ischemia-hypoxia 9 h+ 2-DG group was 0.472±0.025, which was significantly higher than that of simple ischemia-hypoxia 9 h group (t=3.60, P<0.05). (4) Compared with those of normal control group, the LC3Ⅱ/Ⅰ ratio and protein expression of p62 in cardiomyocytes of simple ischemia-hypoxia 9 h group were significantly increased (t=9.45, 8.40, P<0.01). Compared with those of simple ischemia-hypoxia 9 h group, the LC3Ⅱ/Ⅰratio and protein expression of p62 in cardiomyocytes of ischemia-hypoxia 9 h+ 2-DG group were significantly decreased (t=4.39, 4.74, P<0.05). (5) In cardiomyocytes of normal control group, only single autophagosome/autolysosome with bilayer membrane structure was observed. Compared with that of normal control group, the number of autophagosome/autolysosome with bilayer membrane structure in cardiomyocytes of simple ischemia-hypoxia 9 h group was increased significantly. Compared with that of simple ischemia-hypoxia 9 h group, the number of autophagosome/autolysosome with bilayer membrane structure in cardiomyocytes of ischemia-hypoxia 9 h+ 2-DG group was significantly decreased. (6) The viability of cardiomyocytes in simple ischemia-hypoxia 9 h group was 0.358±0.023, which was significantly lower than 0.673±0.026 in normal control group (t=9.12, P<0.01). The viabilities of cardiomyocytes in ischemia-hypoxia 9 h+ HK-ⅡsiRNA1 group and ischemia-hypoxia 9 h+ HK-ⅡsiRNA2 group were 0.487±0.027 and 0.493±0.022, respectively, which were significantly higher than the viability in simple ischemia-hypoxia 9 h group (t=3.63, 4.28, P<0.05). (7) Compared with those of normal control group, the LC3Ⅱ/Ⅰratio and protein expressions of p62 and hexokinase Ⅱ in cardiomyocytes of simple ischemia-hypoxia 9 h group were significantly increased (t=6.08, 6.31, 4.83, P<0.05 or P<0.01). Compared with those of simple ischemia-hypoxia 9 h group, the LC3Ⅱ/Ⅰ ratio and protein expressions of p62 and hexokinase Ⅱ in cardiomyocytes of ischemia-hypoxia 9 h+ HK-ⅡsiRNA1 group and ischemia-hypoxia 9 h+ HK-ⅡsiRNA2 group were significantly decreased (t=5.10, 7.76, 15.33, 4.17, 8.42, 12.11, P<0.05 or P<0.01).@*Conclusions@#Ischemia-hypoxia upregulates the expression level of hexokinase Ⅱ protein in mouse cardiomyocytes cultured in vitro, which decreases the viability of cardiomyocytes by impairing autophagic flow. To inhibit the activity of hexokinase Ⅱ or its expression can alleviate the ischemia-hypoxia damage of cardiomyocytes.
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Objective To investigate the effects of edaravone on improving the prognosis of TBI rats.Methods A total of 150 SD male rats were divided into normal control group (10 rats),TBI group (70 rats) and edaravone group (70 rats).In the edaravone treatment group,the rats were injected intraperitoneally once a day continously for 2 weeks with the injection dose of 5.4 mg · kg-1 · d-1.At 6 hours,12 hours,24 hours,48 hours,72 hours,1 week and 2 weeks after injury,the neurobehavioral and motor function scores of rats were monitored respectively,with 10 rats monitored at each time point.Serum and cerebrospinal fluid samples were collected and the levels of β-endorphin and gonadotropin-releasing hormone (GnRH) were determined by radioimmunoassay (RIA).Results In the edaravone group,the neurobehavioral and motor function scores were higher than those of the TBI group at 6 hours,12 hours,24 hours,48 hours,72 hours,1 week and 2 weeks after injury.At 48 hours after injury,the neurobehavioral scores of the TBI group and the edaravone treatment group were (8.2 ±0.9) points and (10.3 ±0.7) points,respectively (P ＜ 0.05),and the motor function scores were (5.9 ± 1.0) points and (6.9 ± 1.2) points respectively (P ＜ 0.05).Meanwhile,the contents of β-endorphin in blood and cerebrospinal fluid of the normal control group were (50.2 ± 9.5) pg/ml and (16.2 ± 2.8) pg/ml,and the contents of GnRH were (75.2 ± 11.2) pg/ml and (36.2 ± 10.8)pg/ml,respectively.The levels of β-endorphin and GnRH in serum and cerebrospinal fluid were significantly increased at 6 hours,12 hours,24 hours,48 hours,72 hours,l week and 2 weeks after injury.The levels of β-endorphin and GnRH in the edaravone group were lower than those of TBI group.At 72 hour after injury,the levels of β-endorph in serum in TBI group and edaravone group were (165.2 ± 8.5) pg/ml and (109.5 ± 6.3) pg/ml respectively (P ＜ 0.05),and the levels of β-endorph in cerebrospinal fluid were (63.3 ± 3.1) pg/ml and (38.2 ± 2.3) pg/ml respectively (P ＜ 0.05).At 72 hour after injury,the levels of GnRH in serum in TBI group and edaravone group were (203.7 ± 17.1)pg/ml and (110.4 ± 19.2)pg/ml respectively (P ＜0.05),and the levels of GnRH in cerebrospinal fluid is (153.0 ± 13.4) pg/ml and (93.2 ± 10.5) pg/ml respectively (P ＜ 0.05).Conclusion During acute and recovery periods after TBI,continuous treatment with edaravone can obviously reduce the levels of β-endorphin and GnRH,which is beneficial to alleviate the secondary brain injury after TBI in rats,promote the recovery of nerve and function,and improve the prognosis.
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Objective@#To explore the effects of decline of pH value on cardiomyocyte viability of rats, and to analyze the possible mechanism.@*Methods@#Hearts of five newborn Sprague-Dawley rats were isolated, and then primary cardiomyocytes were cultured and used in the following experiments. (1) The primary cardiomyocytes were divided into pH 7.4+ 6 h, pH 7.0+ 6 h, pH 6.5+ 6 h, pH 6.0+ 6 h, pH 6.5+ 1 h, and pH 6.5+ 3 h groups according to the random number table, with 4 wells in each group. After being routinely cultured for 48 h (similarly hereinafter), cells in pH 7.4+ 6 h, pH 7.0+ 6 h, pH 6.5+ 6 h, and pH 6.0+ 6 h groups were cultured with pH 7.4, pH 7.0, pH 6.5, and pH 6.0 DMEM-F12 medium (similarly hereinafter), respectively, and then they were cultured for 6 h. Cells in pH 6.5+ 1 h and pH 6.5+ 3 h groups were cultured with pH 6.5 medium, and then they were cultured for 1 h and 3 h, respectively. Viability of cells was detected by methyl-thiazolyl-tetrazolium (MTT) method. (2) The primary cardiomyocytes were divided into pH 7.4, pH 6.5, and pH 6.5+ taxol groups according to the random number table, with 2 wells in each group. Cells in pH 7.4 group were cultured with pH 7.4 medium, while cells in pH 6.5 and pH 6.5+ taxol groups were cultured with pH 6.5 medium. Cells in pH 6.5+ taxol group were added with taxol of a final molarity of 0.2 μmol/L in addition, and then they were cultured for 6 h. Morphology and density of microtubule of cells was detected by immunofluorescence assay. (3) The primary cardiomyocytes were grouped and treated as in experiment (2), with 2 wells in each group. The expressions of polymerized microtubulin and free microtubulin were determined with Western blotting. (4) The primary cardiomyocytes were grouped and treated as in experiment (2), with 4 wells in each group. Viability of cells after treated with taxol was detected by MTT method. Data were processed with one-way analysis of variance and LSD-t test.@*Results@#(1) The viability of cells in pH 7.4+ 6 h, pH 7.0+ 6 h, pH 6.5+ 6 h, pH 6.0+ 6 h, pH 6.5+ 1 h, and pH 6.5+ 3 h groups were 1.00±0.08, 0.90±0.08, 0.85±0.06, 0.83±0.04, 0.91±0.10, and 0.89±0.10, respectively. Compared with that in pH 7.4+ 6 h group, viability of cells in pH 7.0+ 6 h, pH 6.5+ 6 h, pH 6.0+ 6 h, pH 6.5+ 1 h, and pH 6.5+ 3 h groups were all decreased in different degrees (t=2.476, 4.002, 4.996, 2.168, 2.400, P<0.05). (2) Microtubules of cells in pH 7.4 group were radially distributed around the nucleus with clear tubular structure. Compared with that in pH 7.4 group, the skeleton of microtubules of cells in pH 6.5 group was obviously damaged, with broken structure of microtubule and reduced density. Compared with that in pH 6.5 group, the damage degree of microtubules of cells in pH 6.5+ taxol group was obviously alleviated, and the structure of microtubules basically returned to normal. (3) Compared with that in pH 7.4 group, the expression of free microtubulin of cells in pH 6.5 group was significantly increased (t=3.030, P<0.05), while the expression of polymerized microtubulin of cells was significantly decreased (t=8.604, P<0.05). Compared with that in pH 6.5 group, the expression of free microtubulin of cells in pH 6.5+ taxol group was significantly decreased (t=4.559, P<0.05), while the expression of polymerized microtubulin of cells was significantly increased (t=5.472, P<0.05). (4) Viability of cells in pH 7.4, pH 6.5, and pH 6.5+ taxol groups were 1.00±0.10, 0.83±0.04, and 0.93±0.10, respectively. Compared with that in pH 7.4 group, the viability of cells in pH 6.5 group was obviously declined (t=4.412, P<0.05). Compared with that in pH 6.5 group, the viability of cells in pH 6.5+ taxol group was obviously increased (t=2.461, P<0.05).@*Conclusions@#The decline of pH value reduces the viability of cardiomyocytes of rats through destroying the skeleton of microtubule. Stabilizing microtubule skeleton can significantly reduce acidic treatment-induced damage and ameliorate cardiomyocyte viability.
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Objective@#To compare and analyze the epidemiological characteristics of hospitalized elderly, young and middle-aged patients with severe burn in recent years, so as to provide reference for the prevention and treatment of elderly patients with severe burn.@*Methods@#Relying on the entry system of epidemiological case data and biological sample of severe burn from multicenter in clinic, medical records of patients with severe burn, aged above 18, hospitalized in 8 burn wards from January 2012 to December 2015 were collected. Six hundred and fifteen patients who were more than 18 years old and less than or equal to 65 years old were included in young and middle-aged group (YM). Eighty-two patients aged more than 65 years old were included in elderly group (E). Data of age, gender, residence, education level, cause of injury, location of injury, season of injury, total burn area, occurrence and area of full-thickness burn injury, wound site, inhalation injury incidence and severity, post burn admission time, proportion of delayed resuscitation, proportion of escharectomy or tangential excision and skin grafting, preinjury systemic disease, system complication during hospitalization, length of hospital stay, outcome of treatment, and reason of abandoning treatment of patients were analyzed. Data were processed with chi-square test and Mann-Whitney U test. The odds ratios of preinjury systemic disease, system complication during hospitalization, and adverse outcome of patients in group YM were compared with those in group E.@*Results@#(1) The majority of patients in the two groups were male, but the proportion of male patients in group YM was higher. There was statistically significant difference in gender distribution of patients between the two groups (χ2=18.727, P<0.001). The majority of patients in the two groups were from rural areas, but the proportion of rural patients in group E was higher. There was statistically significant difference in residence distribution of patients between the two groups (χ2=9.306, P=0.002). Patients in group YM mainly had secondary education, while patients in group E mainly had primary education. There was statistically significant difference in distribution of education level of patients between the two groups (χ2=146.797, P<0.001). (2) The most common causes of injury of patients in the two groups were both flame, but the proportion of patients with flame burn injury in group E was higher. There was statistically significant difference in distribution of cause of injury of patients between the two groups (χ2=25.063, P<0.001). The main locations of injury of patients in groups YM and E were respectively public place and private residence. There was statistically significant difference in location distribution of injury of patients between the two groups (χ2=46.313, P<0.001). The main seasons of injury of patients in groups YM and E were respectively summer and winter. There was statistically significant difference in season distribution of patients between the two groups (χ2=23.143, P<0.001). There was statistically significant difference in distribution of total burn area of patients between the two groups (χ2=25.799, P=0.002). The occurrences of full-thickness burn injury of patients in the two groups were similar (χ2=2.685, P=0.101), while there was statistically significant difference in area of full-thickness burn injury of patients between the two groups (χ2=26.702, P=0.002). There was no statistically significant difference in distribution of wound site of patients between the two groups (χ2=3.954, P=0.785). There were no statistically significant differences in incidence and severity distribution of inhalation injury of patients between the two groups (with χ2 values respectively 0.425 and 0.672, P values above 0.05). (3) There was statistically significant difference in distribution of admission time of patients between the two groups (χ2=6.632, P=0.036), but there was no statistically significant difference in proportion of delayed resuscitation of patients between the two groups (χ2=1.261, P=0.261). The proportion of escharectomy or tangential excision and skin grafting of patients in group YM was 72.0% (443/615), which was significantly higher than 35.4% (29/82) of group E (χ2=44.498, P<0.001). The incidence of preinjury systemic disease of patients in group YM was 13.2% (81/615), which was significantly lower than 61.0% (50/82) of group E (χ2=108.337, P<0.001). The risk of preinjury systemic disease of patients in group E was 10.30 times of that of patients in group YM [with 95% confidence interval (CI) of 6.24-17.01, P<0.001]. During hospitalization, 59.8% (49/82) of patients in group E suffered from system complications, which was significantly higher than 36.6% (225/615) of group YM (χ2=16.282, P<0.001). The risk of system complication of patients in group E was 2.57 times of patients in group YM (with 95% CI of 1.61-4.12, P<0.001). The length of hospital stay of patients in group E was significantly shorter than that of group YM (U=36 735, P<0.001). There was statistically significant difference in treatment outcome of patients between the two groups (χ2=106.251, P<0.001). The risk of adverse outcome of patients in group E was 7.52 times of group YM (with 95% CI of 4.40-12.88, χ2=67.709, P<0.001). The proportion of abandoning treatment of patients in group E was significantly higher than that of group YM (χ2=150.670, P<0.001). The risk of abandoning treatment of patients in group E was 15.86 times of that of group YM (with 95% CI of 9.36-26.88, P<0.001). There was no statistically significant difference in distribution of reason of abandoning treatment of patients between the two groups (χ2=4.178, P=0.243).@*Conclusions@#There were significant differences in the epidemiological characteristics of patients in groups E and YM. In elderly burn patients, the proportion of rural population was higher and the education level was lower. Flame burn was common and burns mostly occurred in private residences and in winter. The total burn area was slightly lower but the area of full-thickness burn injury was larger. The length of hospital stay was shorter and the proportion of surgical treatment was lower. The incidences of preinjury systemic disease and system complication during hospitalization were higher, and therefore the risks of adverse outcome and abandoning treatment were higher.
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Objective@#To explore the effects of change of activity of vacuolar adenosine triphosphatase (V-ATPase) of myocardial lysosome on myocardial damage in rats after severe burn and its mechanism.@*Methods@#The myocardial lysosomes were extracted from the hearts of 12 SD rats with ultra-high speed gradient density centrifugation, then Western blotting and transmission electron microscope observation were conducted for identification. One hundred and twenty rats were divided into pure burn group, ATP group, normal control group, and bafilomycin group according to the random number table, with 30 rats in each group. Rats in pure burn group and ATP group were inflicted with 40% TBSA full-thickness scald on the back. Immediately after injury, rats in pure burn group were intraperitoneally injected with lactated Ringer′s solution in 4 mL·%TBSA-1·kg-1, and rats in ATP group were intraperitoneally injected with ATP in 0.4 mg/kg at 12 h before burn, immediately after burn, and 12 h after burn. Rats in normal control group did not receive any treatment, and rats in bafilomycin group were intraperitoneally injected with bafilomycin A1 in 0.3 mg/kg at the same time points as those of ATP group. At 24 h after burn, 30 rats from each group were collected for determining activity of V-ATPase of myocardial lysosome with coupled-enzyme assay and the expression of myocardium autophagy-related proteins microtubule-associated protein 1 light chain 3 (LC3) and P62 by Western blotting. Left ventricular arterial blood was collected to detect the content of 5 items of myocardial enzyme spectrum and cardiac troponin T (cTnT). Data were processed with one-way analysis of variance and t test.@*Results@#(1) After identification, both the expression level of lysosome-related membrane protein 1 and purity of lysosome in the sample were high, and the structure of lysosome was intact. (2) At 24 h after burn, the activity values of V-ATPase of myocardial lysosome in rats of pure burn group, ATP group, normal control group, and bafilomycin group were (2.03±0.67), (3.01±0.58), (4.29±0.26), and (1.83±0.52) μmol·mg-1·h-1, respectively. The activity value of V-ATPase of myocardial lysosome in rats of pure burn group was significantly lower than the values in ATP group and normal control group (with t values respectively 3.14 and 8.87, P values below 0.01). The activity values of V-ATPase of rats in normal control group were significantly higher than those in bafilomycin group (t=11.87, P<0.01). At 24 h after burn, the expressions of myocardial LC3 and P62 in pure burn group were significantly higher than those in ATP group and normal control group (with t values from 3.73 to 5.88, P values below 0.01). The expressions of myocardial LC3 and P62 in normal control group were significantly lower than those in bafilomycin group (with t values respectively 2.64 and 3.07, P<0.05 or P<0.01). At 24 h after burn, the content of 5 items of myocardial enzyme spectrum and cTnT in pure burn group was significantly higher than that in ATP group and normal control group (with t values from 3.24 to 16.72, P values below 0.01). The content of 5 items of myocardial enzyme spectrum and cTnT in normal control group was significantly lower than that in bafilomycin group (with t values from 2.39 to 10. 70, P values below 0.01).@*Conclusions@#The activity of V-ATPase of myocardial lysosome decreased in rats after severe burn, which can result in myocardial damage by inhibiting myocardial autophagy flux.
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<p><b>OBJECTIVE</b>To explore the effects of direct current electric fields on directional migration and arrangement of dermal fibroblasts in neonatal BALB/c mice and the related mechanisms.</p><p><b>METHODS</b>Twelve neonatal BALB/c mice were divided into 4 batches. The skin on the back of 3 neonatal mice in each batch was obtained to culture fibroblasts. Fibroblasts of the second passage were inoculated in 27 square cover slips with the concentration of 5 × 10(4) cells per mL. (1) Experiment 1. Six square cover slips inoculated with fibroblasts of the second passage were divided into electric field group (EF) and sham electric field group (SEF), with 3 cover slips in each group. The cover slips were put in live cell imaging workstation. The cells in group EF was treated with electric power with EF intensity of 200 mV/mm, while simulating process without actual power was given to SEF group (the same below) for 6 h. Cell proliferation rate was subsequently counted. (2) Experiment 2. Six cover slips were divided and underwent the same processes as in experiment 1. Cell movement locus within EF hour (EFH) 6, direction change of cell migration at EFH 0 (immediately), 1, 2, 3, 4, 5, and 6 which was denoted as cos(α), cell migration velocity within EFH 6, direction change of long axis of cell within EFH 6, and direction change of cell arrangement at EFH 0, 1, 2, 3, 4, 5, and 6 which was denoted as polarity value cos[2(θ-90)] were observed under live cell imaging workstation. After EFH 6, the morphological changes in microtubules and microfilaments were observed with immunofluorescent staining. (3) Experiment 3. Six cover slips were divided into cytochalasin D group (treated with 1 μmol/L cytochalasin D for 10 min) and colchicine group (treated with 5 μmol/L colchicine for 10 min), with 3 cover slips in each group. The morphological changes in microfilaments and microtubules were observed with the same method as in experiment 2. (4) Experiment 4. Nine cover slips were divided into control group (no reagent was added), cytochalasin D group and colchicine group (added with the same reagents as in experiment 3), with 3 cover slips in each group. Cells in the 3 groups were exposed to an EF of 200 mV/mm for 6 h. Cell movement locus within EFH 6, cell migration velocity within EFH 6, cell polarity values at EFH 0, 3, and 6, and morphological changes of cells at EFH 0 and 6 were observed. Data were processed with independent samples t-test, one-way analysis of variance, and LSD test.</p><p><b>RESULTS</b>(1) There was no statistically significant difference in cell proliferation rate in group EF and group SEF (t=-0.24, P﹥0.05). (2) Within EFH 6, cells in group EF migrated towards the anode of EF, while cells in group SEF moved randomly. At EFH 0, the values of cos(α) of cells in the 2 groups were both 0. The absolute value of cos(α) of cells in group EF (-0.57 ± 0.06) was significantly higher than that in group SEF (0.13 ± 0.09, t=6.68, P<0.01) at EFH 1, and it was still higher than that in group SEF from EFH 2 to 6 (with t values from 5.33 to 6.83, P values below 0.01). Within EFH 6, migration velocity of cells in group EF was (0.308 ± 0.019) μm/min, which was significantly higher than that in group SEF [(0.228 ± 0.021) μm/min, t=-2.76, P<0.01]. Within EFH 6, long axis of cells in group EF was perpendicular to the direction of EF, while arrangement of cells in group SEF was irregular. Cell polarity values in group EF were significantly higher than that in group SEF from EFH 2 to 6 (with t values from -7.52 to -0.90, P values below 0.01). At EFH 6, the morphology of microfilaments and microtubules of cells in EF group was similar to that in SEF group. (3) The fluorescent intensity of microfilaments of cells in cytochalasin D group became weakened, and the filamentary structure became fuzzy. The microtubules of cells in colchicine group became fuzzy with low fluorescent intensity. (4) Within EFH 6, cells in control group migrated towards the anode of EF, while cells in cytochalasin D group and colchicine group moved randomly. Within EFH 6, there was statistically significant difference in migration velocity of cells in the 3 groups (F=6.36, P<0.01). Migration velocity of cells in cytochalasin D group and colchicine group was significantly slower than that in control group (P<0.05 or P<0.01). At EFH 0, 3, and 6, cell polarity values in the 3 groups were close (with F values from 0.99 to 1.51, P values above 0.05). At EFH 0, cells in control group were spindle; cells in cytochalasin D group were polygonal or in irregular shapes; cells in colchicine group were serrated circle or oval. At EFH 6, no morphological change was observed in cells in control group; cells in cytochalasin D group were spindle with split ends on both ends; cells in colchicine group were serrated oval.</p><p><b>CONCLUSIONS</b>The physiologic strength of exogenous direct current EF can induce directional migration and alignment of dermal fibroblasts in neonatal BALB/c mice. Microfilaments and microtubules are necessary skeleton structure for cell directional migration induced by EF, while they are not necessary for cell directional arrangement induced by EF.</p>
Subject(s)
Animals , Mice , Cell Movement , Cells, Cultured , Electricity , Fibroblasts , Cell Biology , Mice, Inbred BALB C , Microtubules , Skin , Cell BiologyABSTRACT
<p><b>OBJECTIVE</b>To retrospectively analyze the risk factors and clinical manifestations of myocardial damage of patients with severe burn in order to provide evidence for its prevention and treatment.</p><p><b>METHODS</b>Two hundred and fifty-two patients with severe burn admitted to 5 burn centers from January 2010 to June 2015, conforming to the study criteria, were treated in accordance with the fluid resuscitation formula of the Third Military Medical University. According to the creatine kinase isoenzyme-MB (CK-MB) level before treatment on admission, patients were divided into non-myocardial damage group (n=118, CK-MB level less than 75 U/mL) and myocardial damage group (n=134, CK-MB level higher than or equal to 75 U/mL). Data of patients in two groups were collected and evaluated such as gender, age, body mass, number of patients with chemical burn, admission time after injury, total burn area, full-thickness burn area, number of patients with inhalation injury, levels of haemoglobin, hematocrit, and blood lactate on admission and at post injury hour (PIH) 24 and 48, volumes of urine output and fluid input at PIH 24 and 48, levels of creatinine, urea nitrogen, total bile acid, diamine oxidase on admission and at PIH 24 and 48, and mortality. Furthermore, patients were divided into three groups, i. e. less than 50% total body surface area (TBSA) group (n=110), larger than or equal to 50% TBSA and less than 80% TBSA group (n=83), and larger than or equal to 80% TBSA group (n=59) according to the total burn area, and the incidence rates of myocardial damage in patients of three groups were recorded. Data were processed with chi-square test, t test, Wilcoxon test, analysis of variance for repeated measurement, and the values of P were adjusted by Bonferroni. Basic data of 252 patients were processed with binary logistic regression analysis. Receiver operating characteristic curve of total burn area of 252 patients was drawn to predict myocardial damage.</p><p><b>RESULTS</b>(1) There were no statistically significant differences in age, body mass, number of patients with chemical burn, number of patients with inhalation injury, and full-thickness burn area between two groups (with t values respectively 0.20 and 0.31, χ(2) values respectively 0.49 and 4.10, Z=1.42, P values above 0.05). There were statistically significant differences in gender, admission time after injury, and total burn area of patients between two groups (χ(2)=5.00, with t values respectively 2.44 and 3.13, P<0.05 or P<0.01). (2) Gender, admission time after injury, and total burn area were independent risk factors related to myocardial damage in the patients (with odds ratios respectively 2.608, 3.620, and 1.030; 95% confidence intervals respectively 1.315-5.175, 1.916-6.839, and 1.011-1.049; P values below 0.01). (3) The incidence rates of myocardial damage of patients in less than 50% TBSA group, larger than or equal to 50% TBSA and less than 80% TBSA group, and larger than or equal to 80% TBSA group were 38.2% (42/110), 54.2% (45/83), and 61.0% (36/59) respectively, and there was statistically significant difference among them (χ(2)=9.46, P<0.05). (4) The total area under receiver operating characteristic curve of total burn area to predict myocardial damage of 252 patients was 0.706 (with 95% confidence interval 0.641-0.772, P<0.01), and 51.5% TBSA was chosen as the optimal threshold value, with sensitivity of 62.6% and specificity of 65.3%. (5) Compared with those in non-myocardial damage group, except the levels of haemoglobin and hematocrit at PIH 48 (with t values respectively -0.76 and -0.61, P values above 0.05), the levels of haemoglobin, hematocrit, and blood lactate of patients in myocardial damage group were significantly increased at each time point (with t values from -2.80 to -2.06, P<0.05 or P<0.01). Compared with those in non-myocardial damage group, the volume of urine output of patients was significantly declined (with t values respectively 2.05 and 3.68, P<0.05 or P<0.01), while the volume of fluid input of patients was not obviously changed in myocardial damage group at PIH 24 and 48 (with t values respectively 1.01 and 1.08, P values above 0.05). (6) Compared with those in non-myocardial damage group, the level of creatinine of patients was significantly increased on admission and at PIH 24 and 48 (with Z values from -2.91 to -1.99, P<0.05 or P<0.01), the level of urea nitrogen of patients was only significantly increased at PIH 24 and 48 (with t values respectively -4.75 and -5.24, P values below 0.01), the level of total bile acid of patients was not obviously changed on admission and at PIH 24 and 48 (with t values from -0.81 to -0.20, P values above 0.05), and the level of diamine oxidase of patients was only significantly increased on admission and PIH 24 in myocardial damage group (with t values respectively -3.97 and -2.02, P<0.05 or P<0.01). (7) Compared with that in myocardial damage group, the mortality of patients in non-myocardial damage group was significantly declined (χ(2)=5.81, P<0.05).</p><p><b>CONCLUSIONS</b>Patients with severe burn have high incidence of myocardial damage, which may be predicted by total burn area. Severely burned patients with myocardial damage are more likely to suffer from decline of effective circulating volume, tissue oxygenation disorders, and damage in other organs in shock stage.</p>
Subject(s)
Humans , Body Surface Area , Burn Units , Burns , Pathology , Fluid Therapy , Hematocrit , Hemoglobins , Lactic Acid , Blood , Myocardium , Pathology , Retrospective Studies , ShockABSTRACT
<p><b>OBJECTIVE</b>To study the effects of hypoxia of different duration on movement and proliferation of human epidermal cell line HaCaT.</p><p><b>METHODS</b>(1) HaCaT cells in logarithmic phase were cultured in RPMI 1640 medium containing 10% FBS (the same culture method below). Cells were divided into control group (routine culture) and hypoxia for 1, 3, 6 h groups according to the random number table (the same grouping method below), with 6 wells in each group. Cells in the 3 hypoxia groups were cultured in incubator containing 5% CO2, 2% O2, and 93% N2 (the same hypoxic condition below) for corresponding duration. Range of movement of cells in 3 hours was observed under live cell imaging workstation, and their curvilinear and rectilinear movement speeds were calculated at post observation hour (POH) 1, 2, 3. (2) HaCaT cells in logarithmic phase were divided into control group (routine culture) and hypoxia for 1, 3, 6, 9, 12, 24 h groups, with 20 wells in each group. Cells in the 6 hypoxia groups were cultured under hypoxic condition for corresponding duration. Proliferation of cells was examined with cell counting kit and microplate reader (denoted as absorbance value). (3) HaCaT cells in logarithmic phase were divided into control group (routine culture) and hypoxia for 1, 3, 6, 24 h groups, with 5 wells in each group. Cells in the 4 hypoxia groups were cultured under hypoxic condition for corresponding duration. Protein expression of proliferating cell nuclear antigen (PCNA) was determined with Western blotting. Data were processed with one-way analysis of variance and Dunnett- t test.</p><p><b>RESULTS</b>(1) Compared with that of control group, the movement area of cells was obviously expanded in hypoxia for 1, 3, 6 h groups. The longer the hypoxic treatment, the greater the increase was. At POH 1, 2, 3, the curvilinear movement speeds of cells in hypoxia for 1, 3, 6 h groups were respectively (43 ± 18), (44 ± 17), (43 ± 16) µm/h; (44 ± 16), (44 ± 14), (45 ± 14) µm/h; (55 ± 19), (54 ± 17), (56 ± 18) µm/h. They were significantly higher than those of control group [(33 ± 13), (33 ± 12), (33 ± 10) µm/h, with t values from 2.840 to 9.330, P < 0.05 or P < 0.01]. The curvilinear movement speed of cells was significantly higher in hypoxia for 6 h group than in hypoxia for 1 or 3 h group (with t values from 3.474 to 4.545, P < 0.05 or P < 0.01). There was no significant difference in the curvilinear movement speed among the observation time points within each group (with F values from 0.012 to 0.195, P values above 0.05). At POH 1, the rectilinear movement speed of cells in hypoxia for 1 h group was (22 ± 11) µm/h, which was obviously higher than that of control group [(15 ± 10) µm/h, t = 2.697, P < 0.01]. At POH 1, 2, 3, rectilinear movement speeds of cells in hypoxia for 3 and 6 h groups were respectively (19 ± 14), (12 ± 8), (10 ± 6) µm/h; (32 ± 19), (21 ± 13), (17 ± 12) µm/h. They were significantly higher than those of control group [(9 ± 7) and (6 ± 5) µm/h at POH 2 and 3, with t values from 1.990 to 8.231, P < 0.05 or P < 0.01]. The rectilinear movement speed of cells in hypoxia for 6 h group was obviously higher than that of hypoxia for 1 or 3 h group (with t values from 3.394 to 6.008, P < 0.05 or P < 0.01). The rectilinear movement speed of cells in each group decreased at POH 2 or 3 in comparison with POH 1 (with t values from -8.208 to -4.232, P values below 0.01). The rectilinear movement speed of cells in control group at POH 3 was significantly different from that at POH 2 (t = -1.967, P < 0.05). (2) The proliferation levels of cells in control group and hypoxia for 1, 3, 6, 9, 12, 24 h groups were respectively 1.11 ± 0.08, 1.36 ± 0.10, 1.39 ± 0.05, 1.38 ± 0.05, 1.10 ± 0.14, 1.06 ± 0.09, 0.99 ± 0.06 (F = 39.19, P < 0.01). Compared with that of control group, the rate of proliferation of cells was obviously increased in hypoxia for 1, 3, 6 h groups (with t values respectively 6.639, 7.403, 7.195, P values below 0.01), but obviously decreased in hypoxia for 24 h group (t = -3.136, P < 0.05). The proliferation of cells decreased in hypoxia for 9, 12, 24 h groups in comparison with hypoxia for 1, 3, 6 h groups (with t values from -10.538 to -6.775, P values below 0.01). (3) The protein expressions of PCNA of cells in control group and hypoxia for 1, 3, 6, 24 h groups were respectively 0.93 ± 0.12, 0.97 ± 0.14, 1.62 ± 0.18, 0.95 ± 0.09, 0.66 ± 0.21 (F = 20.11, P < 0.01). Compared with that of control group, the expression of PCNA was obviously increased in hypoxia for 1, 3, 6 h groups (with t values respectively 2.339, 5.783, 2.235, P < 0.05 or P < 0.01), but obviously decreased in hypoxia for 24 h group (t = -1.998, P < 0.05). The protein expression of PCNA was higher in hypoxia for 3 h group than in hypoxia for 1 or 6 h group (with t values respectively 4.312 and 3.947, P values below 0.01), and it was increased in the 3 groups in comparison with that of hypoxia for 24 h group (with t values respectively 2.011, 6.193, 3.287, P < 0.05 or P < 0.01).</p><p><b>CONCLUSIONS</b>Short-time hypoxia (1, 3, 6 h) treatment can promote the movement and proliferation of HaCaT cells. Hypoxia for 6 h is the best condition to promote their movement, while hypoxia for 3 or 6 h is better for their proliferation.</p>
Subject(s)
Humans , Carbon Dioxide , Pharmacology , Cell Cycle , Cell Line , Cell Movement , Physiology , Cell Proliferation , Physiology , Cells, Cultured , Epithelial Cells , Cell Biology , Hypoxia , Nitric Oxide , Pharmacology , Oxygen , Pharmacology , Phosphorylation , Proliferating Cell Nuclear Antigen , Signal TransductionABSTRACT
BACKGROUND:Compared with the conventional composite resin, the 3M Z350 nano-resin has good wear resistance, physical mechanical properties, and polishing, and exerts a lower irritation to the dental pulp. Besides fil ing materials, a reliable tooth-prosthesis bonding interface is necessary for resin bonded repairs. OBJECTIVE:To compare the clinical effects of self-etch bonding Adper Easy One and total-etch bonding Single Bond 2 on nano-resin bonding restoration of the anterior teeth. METHODS:120 anterior teeth with vital pulp, which had defects at the incisal ends and were to be restored with nano resins, were divided into two groups randomly. Two kinds of adhesives, self-etch adhesive and total-etch adhesive, combined with nano-resin were used to restore the teeth. The patients were re-examined immediately, 6 months, 1 year and 2 years after the treatment. The fil ings, teeth and pulps of patients were examined, including whether the prosthesis and tooth color were coordinated, whether the gap between the prosthesis and the teeth were sealed, whether the surface of the prosthesis was intact with no loose, whether the prosthesis and teeth had no staining and secondary caries, whether the condition of the tooth pulp had hot or cold stimulation-induced pain. RESULTS AND CONCLUSION:No significant difference in the fil ing effects was found between the two groups when the patients were re-examined immediately, 6 months and 1 year after the treatment (P>0.05). The pulp lesions of the self-etching group were fewer than those of the total-etch group 2 years after the treatment (Pincomplete restoration and secondary caries, respectively. No statistical y significant differences were found in these four aspects between the two groups (P>0.05). The 2-year fol ow up showed a low incidence of pulp lesions and satisfactory clinical performance after 3M Z350 nano-resin working with self-etching bonding system in the nano-resin fil ing of anterior teeth with vital pulp.
ABSTRACT
Objective To investigate whether microtubule disassembly plays an important role in the pathogenesis of the opening of mitochondria permeability transition pore (MPTP) in hypoxic cardiomyocytes and the decrease of its activity, resulting in its hypoxic injury. Methods Neonatal rat cardiomyocytes in primary culture were randomized as normoxia group (A), hypoxic group (B), normoxia treated with microtubule destabilizing agent (Colchicine) group (C), hypoxia treated with microtubule stabilizing agent (Taxol) group (D). At 0.5, 1, 3, 6, 12 h after treatment, polymeric tubulin was detected by immunofluorescence and Western blotting, mitochondria permeability transition pore (MPTP) open by coloading with calcein AM and cobalt chloride, and the activity of cells by measuring the mitochondrial-dependent reduction of MTT to formazan. Results Early microtubule disassembly, MPTP open and activity decrease of cardiomyocytes in both groups B and C were observed at 0.5 h after treatment. These phenomena all became more and more significant with the prolongation of treatment. However, microtubule disassembly, MPTP open and activity decrease of cardiomyocytes of group D were significantly lower than those of group B. Conclusion Microtubule disassembly happened at 0.5 h after hypoxic treatment. Microtubule stabling agent Taxol and destabilizing agent Colchicine can regulate microtubule integrity efficiently. The microtubule damage plays an important role in the hypoxic injury of cardiomyocytes.