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ObjectiveTo investigate the expression and role of response gene to complement 32 （RGC32） in liver regeneration after partial hepatectomy （PH）. MethodsA total of 42 male C57BL/6 mice， aged 10 weeks， were randomly divided into control group， postoperative day 1 group （1－d group）， postoperative day 2 group （2－d group）， postoperative day 4 group （4－d group）， postoperative day 6 group （6－d group）， postoperative day 8 group （8－d group）， and postoperative day 10 group （10－d group）， with 6 mice in each group. In the control group， the complete liver of the mice was resected for weighing and photography as the normal control group （sham group）； further， the left and middle lobes of the liver were resected for weighing and photography as the surgical control group （0－day group）； the sham group and the 0－day group shared the same group of mice. After successful modeling by PH， the mice were sacrificed on days 1， 2， 4， 6， 8， and 10 after surgery， and the liver was collected to measure the change in size. HE staining and oil red O staining were used to evaluate liver histomorphological changes； serum alanine aminotransferase （ALT） and aspartate aminotransferase （AST） were measured to evaluate the changes in liver function； immunohistochemical staining was used to measure the expression of proliferating cell nuclear antigen （PCNA） and Ki67 and analyze the change in cell proliferation during liver regeneration； quantitatie real－time PCR and immunohistochemical staining were uused to measure the expression and subcellular distribution of RGC32 during liver regeneration； EdU cell proliferation assay was used to analyze the effect of RGC32 overexpression or knocknout on hepatocyte proliferation in L02 cells. For continuous data， comparison between multiple groups was made by analysis of variance， and further pairwise comparisons were conducted using the LSD－t test. The independent samples t－test was used for comparison of continuous data between two groups. A Pearson correlation analysis was performed. ResultsThe liver gradually enlarged after PH， and the liver/body weight ratio rose to the peak from days 0 to 6， with significant differences between different time points （all P<0.05）， while there was no significant change in liver size from days 6 to 10. The number of liver lipid droplets significantly increased after PH surgery and gradually decreased with liver regeneration， with a significant difference between the portal vein region and the central vein region （all P<0.05）. Compared with the sham group， the 1d group had significant increases in the serum levels of ALT and AST （all P<0.05）， which gradually returned to the levels of the sham group on day 6 and day 2 after surgery， respectively （P>0.05）. Immunohistochemical staining showed that there were rapid increases in the numbers of PCNA－ and Ki67－positive liver parenchymal cells after PH surgery， with the highest numbers of 86±5 and 89±5， respectively， on day 2， which then gradually decreased； however， there were gradual increases in the numbers of PCNA－ and Ki67－positive nonparenchymal cells， with the peak numbers of 34±5 and 25±3， respectively， on day 6， which then gradually decreased. The total expression of RGC32 increased to the highest level on day 2 after PH surgery and then gradually decreased， and the changing trend of RGC32 expression in cytoplasm was consistent with that of total RGC32 expression； however， the expression of RGC32 in nucleus decreased to the lowest level on day 2 after PH surgery and then increased gradually. The correlation analysis showed that the expression of RGC32 in nucleus was negatively correlated with the proliferation of liver parenchymal cells （R2=0.308 3， P=0.016 7）， and the expression of RGC32 in cytoplasm was positively correlated with the proliferation of liver parenchymal cells （R2=0.808 6， P<0.000 1）. Cell experiments showed that compared with the control group， the EdU－positive rate was reduced by 15.6% after RGC32 overexpression （P<0.01） and was increased by 19.2% after RGC32 knockdown （P<0.01）. ConclusionLiver parenchymal cells and nonparenchymal cells show asynchronous proliferation and participate in liver regeneration together. During liver regeneration after hepatectomy， there are differences in the expression of RGC32 between nucleus and cytoplasm， and RGC32 in nucleus may inhibit hepatocyte proliferation.
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Objective:To investigate the implementation effect of micro-video assisted physiological theory teaching based on functional experiments.Methods:There were 140 clinical undergraduates (control group) from Class 1 and Class 2, and 123 clinical undergraduates (experimental group) from Class3 and Class 4 of Batch 2017 in our university who were involved in this study. The control group adopted traditional teaching method, the experimental group adopted micro-videos to assist traditional teaching in the teaching of selected chapters, and these micro-videos were collected from the recording and editing of functional experiments. After the course, questionnaire survey in terms of course design, implementation and effect, as well as final exanimation performance analysis were conducted to evaluate the teaching effect. SPSS 17.0 was used for t test. Results:The final examination scores of the experimental group were significantly higher than those of the control group [(81.02±9.64) vs. (73.41±11.39)], with significant differences ( t=-5.805, P<0.001). Among them, the scores of Chapter 2 of the two groups were [(8.07±0.94) vs. (6.14±1.05), t=-15.616, P<0.001)], the scores of Chapter 4 were [(16.16±1.79) vs. (10.90±2.23), t=-20.903, P<0.001)], and the scores of Chapter 6 were [(6.04±0.53) vs. (5.82±0.78), t=-2.638, P=0.009)], all with significant differences. 100% of questionnaires were recovered, and more than 90% of students were interested in this teaching method which could strengthen their understanding of the key and difficulties in physiology and was also helpful to cultivate their ability of induction and summarization. Conclusion:Micro-videos based on functional experiments assisted teaching can improve the teaching effect of physiology, and it's worth popularizing.
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Objective To investigate the expression of Situin 1 ( SIRT1) in 5 strains of human lung adenocarcinoma cell lines, inclu-ding HCC827, H1650, H1975, A549 and H1299, and its relation to the susceptibility of nedaplatin ( NDP ) . Methods The SIRT1 mRNA and protein levels in 5 strains of human lung adenocarcinoma cells were detected by real-time quantitative PCR and Western blot, respectively. The viability of cells treated with NDP was detected by the CCK-8 method and the half growth inhibition concentra-tion ( IC50 ) was calculated. After the expressions of SIRT1 in A549, H1299, H1650 and H1975 cells were down-regulated by the siR-NA interference, the effects of NDP on the viability and apoptosis of these cells were determined by the CCK-8 method and flow cytom-etry, respectively.Results The expression levels of SIRT1 mRNA (4.53 ± 0.74, 3.11 ± 0.64, 15.76 ± 2.28 and 18.09 ± 1.17) and protein (0.23 ± 0.03, 0.21 ± 0.02, 0.52 ± 0.11 and 0.56 ± 0.08) in H1650, H1975, A549 and H1299 cells were significantly higher than that in HCC827 cells (1.00 for SIRT1 mRNA and 0.11 ± 0.02 for SIRT1 protein, F=122.10 and 26.50, respectively, P<0.01). The susceptibility of A549 and H1299 cells to NDP [IC50=(7.38 ± 1.59) and (8.14 ± 1.43) μmol/L, respectively] was significantly higher than that of HCC827, H1650 and H1975 cells [IC50=(26.16±4.35),(22.29±3.26) and (24.41 ± 2.58), respectively, F=30.86, P<0.01].The survivals of A549 and H1299 cells transfected by siSIRT1 and treated with NDP were significantly higher than that in the NC group ( F=235.10 and 39.20, respectively,P<0.01) , and the apoptotic rates were the reverse ( t=7.29 and 6.68, re-spectively, P<0.05) . However, the survivals of H1650 and H1975 cells transfected by siSIRT1 and treated with NDP were significantly lower than that in the NC group ( F=185.40 and 60.09, respectively,P<0.01) , and the apoptotic rates were the reverse ( t=6.15 and 31.36, respectively,P<0.01).Conclusion The expression of SIRT1 in A549 and H1299 cells with high expression of SIRT1 increases their susceptibility to NDP , while that in H1650 and H1975 cells with moderate expression of SIRT1 decreases their susceptibility to NDP, indicating that SIRT1 may play dual roles in the resistance of human lung adenocarcinoma cells to platinum.
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Objective To investigate the effects of liver X receptor (LXR) agonist on the proliferation of mouse pancreatic β cell line MIN6 cells.Methods The viability,changes of cell cycle,mRNA levels of S phase kinase associated protein 2 (Skp2) and p27,and protein levels of Skp2 and p27 in MIN6 cells treated with LXR agonist T0901317 were determined by the CCK-8 method,flow cytometry,real-time RT-PCR and western blot,respectively.Results The viability of MIN6 cells treated with 1 μmol/L,5 μmol/L and 10 μnol/L of T0901317 were (98.54 ±0.94)％,(87.03 ±0.93)％ and (75.57 ± 1.85)％ of the controls,respectively,and there was significant difference among them (F =301.90,P ＜ 0.01).The percentages of G1 phase cells in the MIN6 cells treated with 0 μmol/L,1 μmol/L,5 μmol/L and 10 μmol/L of T0901317 were (35.93 ±2.25)％,(38.45 ±0.91)％,(45.46±1.34)％ and (53.28 ± 1.14) ％,respectively,and there was significant difference among them (F =80.83,P ＜ 0.01).Similarly,the percentages of S phase cells in the MIN6 cells treated with 0 μmol/L,1 μmol/L,5 μmol/L and 10 μmoi/L of T0901317 were (52.87 ± 1.19) ％,(48.65 ± 0.85) ％,(36.31 ± 1.37) ％ and (31.45 ± 1.22) ％,respectively,and there was also significant difference among them (F =221.30,P ＜ 0.01).The protein levels of p27 in the MIN6 cells treated with 10 μmol/L of T0901317 (2.84 ± 0.14) were significantly higher than that in the controls (2.28 ± 0.10) (t =4.54,P ＜ 0.05),while there was no significant difference in the mRNA levels of p27 between them (t =0.28,P ＞ 0.05).However,10 μmol/L of T0901317 significantly decreased mRNA (0.52 ± 0.02,t =29.22,P ＜ 0.01) and protein levels (0.98 ± 0.12 vs 1.89 ± 0.01,t =10.98,P ＜ 0.01) of Skp2 in MIN6 cells.Based on the control siRNA transfection group as a reference (100％),the cell survival rates of the p27 siRNA transfection group,10 μmol/L of T0901317 treatment group and the intervention group (p27 siRNA transfection + T0901317 treatment) were (100.97 ± 1.08) ％,(75.03 ± 1.83) ％ and (86.67 ± 2.45) ％,respectively.There was no significant difference between the control siRNA and p27 siR-NA transfection groups (t =1.542,P ＞ 0.05).Compared with the control siRNA transfection group,the cell survival rates of the T0901317 treatment group decreased (t =23.58,P ＜ 0.01).There was also significant difference in the cell survival rates between the T0901317 treatment group and the intervention group (t =7.77,P ＜ 0.01).Conclusion The activation of LXR may induce pancreatic β cell cycle arrest by up-regulating the expression of p27 and down-regulating the expression of Skp2.
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Objective To explore the protective effects of GYY4137, a new hydrogen sulfide donor, on intestinal mucosa in a neonatal rat model of necrotizing enterocolitis (NEC), and its potential mechanism.Methods Sixty SD rats were randomly assigned into 4 groups: group A (control group), group B (NEC group), group C (NEC with GYY4137 treatment, H2S donor group), and group D (NEC with GYY4137 and Znpptreatment, HO-1 inhibitor group). The SD rat models of NEC were established using simulated milk feeding-hypoxia-cold stress-Lipopolysaccharides. The injury degree of intestinal mucosa was evaluated using HE-staining, and its mechanisms were investigated using biochemical indicators and Western blotting. Results Compared with control group, the pathology score and the total superoxide dismutase (T-SOD) in the NEC group was significantly higher, the concentrations of methane dicarboxylic aldehyde (MDA) and necrosis factor α (TNF-α) were lower(P<0.05). Compared with those in NEC group, the pathology score and the concentration of MDA and TNF-α in the H2S donor group were signiflcantly lower, the T-SOD, and the HO-1 expression was higher. The pathology score and the level of MDA and TNF-α were signiflcantly increased after treated with HO-1 inhibitor Znpp, and T-SOD was signiflcantly decreased.. Conclusions The GYY4137, as a new H2S donor, could attenuate the injury of intestinal mucosa in a neonatal rat model of NEC by upregulating the expression of HO-1.
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In order to observe the therapeutic effect of azithromycin combined with IFN-γ on mouse toxoplasmosis and its impact on the cellular immune function of mouse, a total of 100 BALB/c mice were selected and divided into 5 groups, namely an infection control group (Group A) , azithromycin treatment group (Group B) , azithromycin combined with IFN-γ treatment group (Group C) , IFN-γ treatment group (Group D) and blank control group (Group E). The mice in Group A, B, C, D were infected by Toxoplasma tachyzoites through intraperitoneal injection and those in Group B, C, D were treated with relative drugs 24 h later for S days. The survival time of mice in each group and the levels of CD4 ~+ and CD8~+ T cells in blood were observed. The results showed that azithromycin combined with IFN-γ could improve the therapeutic effect of mouse toxoplasmosis and the cellular immune function of mice.
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Objective To investigate the in vivo transduction capability of fusion protein PEP-1-EGFP with mice.Methods Two prokaryotic expression plasmids pET15b-EGFP and pET15b-PEP-1-EGFP were constructed and transformed into E.coli BL21(DE3) to express EGFP and fusion protein PEP-1-EGFP,respectively.The expressed EGFP and PEP-1-EGFP were purified with Ni2+-resin affinity chromatography.Five hundred micrograms of EGFP and PEP-1-EGFP fusion protein were injected into mouse through caudal vein,respectively,the mice were euthanized and perfused with PBS 2 hours after administration.Then,the heart,brain,liver,spleen and kidney were removed and sectioned with a cryostat at 7 ?m for visualization with a inverted fluorescent microscope.ResultsThe brain,heart,liver,spleen and kidney injected with PEP-1-EGFP showed bright and homogenous green fluorescence whereas that with EGFP showed no green fluorescence at all.Conclusion The successful expression and purification of PEP-1-EGFP fusion protein and its efficient transduction into mice in vivo provide a basis for the research on transmembrane delivery of macromolecule drugs mediated by the cell-penetrating peptide,PEP-1.
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Objective To investigate the effects of pyrroloquinoline quinone(PQQ) on the hippocampal neurons of aged rats induced by D-galactose(D-gal).Methods D-gal was used to induce the model of aging rat,PQQ was administered into rat lateral intracerebroventricle.After 50 days the metamorphosis of hippocampal neurons was observed by H-E and Nissl's staining.The apoptosis rate of hippocampus was tested by flow cytometry.The contents of free radical and C-FOS protern were measured.Results Compared with the control group,the size of the neurosoma was slightly changed,the optical density of Nissl's was decreased,the content of free radical and the apoptosis rate increased markedly in D-gal group.After PQQ injection with D-gal,the size of neurosoma and the optical density of Nissl's were markedly increased,the content of free radical and the apoptosis rate of hippocampus did not change.PQQ improved the expression of C-FOS protern.Conclusion PQQ can slow down the aging progress of hippocamal neurons induced by D-gal.
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Objective To establish rSAG1-IgM-ELISA with purified rSAG1 fusion protein for immunodiagnosis of toxoplasmosis. Methods The rSAG1 fusion protein was purified by Ni 2+ column. The ELISA plate was coated with different concentrations of rSAG1, reacted with pooled positive and negtive human sera. Goat anti-human IgM conjugated to horseradish peroxidase was used as the second antibody. The appropriate detecting condition of the rSAG1-IgM-ELISA assay was determined by orthogonal experiment. The reproducibility, sensitivity and specificity of the assay were assessed. Thirty-five IgM-positive and 57 IgM-negative human sera detected by the imported IgM-ELISA kit were detected with the rSAG1-IgM-ELISA. Results The purity of rSAG1 was above 90%. The appropriate detecting condition was that the coated rSAG1 was 2 5 ?g/ml, the human serum was in 1∶100 dilution, and the second antibody was in 1∶4000 dilution. The coefficient of variation (CV) value of IgM-positive and IgM-negative pooled sera were 13 8% and 7 7% respectively. The inhibition rate of the assay was 62 0% The positive correspondence rate and negative correspondence rate were 82 9% (29/35) and 91 2% (52/57) respectively,the total correspondence rate was 88 0%, compared with the imported IgM-ELISA kit. Conclusions The rSAG1-IgM-ELISA has high sensitivity and specificity, and good correspondence rate with the imported IgM-ELISA kit. It indicates that rSAG1-IgM-ELISA has potential value for early diagnosis of toxoplasmosis.