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Objective To investigate the photoregulation of transient receptor potential ankyrin 1 (TRPA1) in HaCaT cells,and to explore its mechanisms.Methods Cultured HaCaT cells were divided into 225-mJ/cm2 UVA radiation groups and 25-mJ/cm2 UVB radiation groups.HaCaT cells in the UVA radiation groups were further classified into 6 groups:blank control group 1 receiving no treatment,retinal group 1 treated with 12 μmol/L retinal alone,UVA group treated with 225 mJ/cm2 UVA radiation alone,retinal + UVA group (UVA-TRPA1 control group),retinal + UVA + 500 μmol/L cinnamaldehyde group (UVA-TRPA1 agonist group) and retinal + UVA + 1 mmol/L camphor group (UVA-TRPA1 antagonist group).Additionally,HaCaT cells in the UVB radiation groups were also further classified into 6 groups:blank control group 2 receiving no treatment,retinal group 2 treated with 12 μmol/L retinal alone,UVB group treated with 25-mJ/cm2 UVB radiation,retinal + UVB group (UVB-TRPA1 control group),retinal + UVB + 500 μmol/L cinnamaldehyde group (UVB-TRPA1 agonist group) and retinal + UVB + 1 mmol/L camphor group (UVB-TRPA1 antagonist group).Real-time fluorescence-based quantitative PCR (qPCR) and Western blot analysis were performed to determine the mRNA and protein expression of TRPA 1 respectively.Flow cytometry was conducted to investigate changes of calcium influx in HaCaT cells in the above groups.Results qPCR and Western blot analysis showed that TRPA1 mRNA and protein were expressed in HaCaT cells.The fluorescence intensity of calcium influx significantly differed among the blank control group 1,retinal group 1,UVA group and retinal + UVA group (155.06 ± 7.62,148.37 ± 18.77,166.92 ± 3.71 and 331.333 ± 40.563;F =44.509,P ＜ 0.01),as well as among the blank control group 2,retinal group 2,UVB group and retinal + UVB group (150.20 ± 1.73,171.66 ± 56.23,147.56 ± 6.60 and 250.44 ± 9.13;F =85.261,P ＜ 0.01).Additionally,retinal + UVA/UVB groups showed significantly higher fluorescence intensity of calcium influx compared with the blank control groups (q =18.442,6.052,P ＜ 0.01).The TRPA1 agonist cinnamaldehyde and its antagonist camphor could regulate the UVA-and UVB-induced calcium influx (P ＜ 0.001).Compared with the blank control group 1 and 2 respectively,the fluorescence intensity of retinal-dependent calcium influx was significantly higher in the UVA/UVB-TRPA1 agonist group (q =14.934,32.770,P ＜ 0.001),and significantly lower in the UVA/UVB-TRPA1 antagonist group (q =7.986,14.596,P ＜ 0.001).Conclusion TRPA1 is expressed in HaCaT cells,and UVA or UVB can regulate the calcium influx in HaCaT cells by adjusting the activity of TRPA 1.
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Objective To investigate the photoregulation of transient receptor potential ankyrin 1 (TRPA1) in HaCaT cells,and to explore its mechanisms.Methods Cultured HaCaT cells were divided into 225-mJ/cm2 UVA radiation groups and 25-mJ/cm2 UVB radiation groups.HaCaT cells in the UVA radiation groups were further classified into 6 groups:blank control group 1 receiving no treatment,retinal group 1 treated with 12 μmol/L retinal alone,UVA group treated with 225 mJ/cm2 UVA radiation alone,retinal + UVA group (UVA-TRPA1 control group),retinal + UVA + 500 μmol/L cinnamaldehyde group (UVA-TRPA1 agonist group) and retinal + UVA + 1 mmol/L camphor group (UVA-TRPA1 antagonist group).Additionally,HaCaT cells in the UVB radiation groups were also further classified into 6 groups:blank control group 2 receiving no treatment,retinal group 2 treated with 12 μmol/L retinal alone,UVB group treated with 25-mJ/cm2 UVB radiation,retinal + UVB group (UVB-TRPA1 control group),retinal + UVB + 500 μmol/L cinnamaldehyde group (UVB-TRPA1 agonist group) and retinal + UVB + 1 mmol/L camphor group (UVB-TRPA1 antagonist group).Real-time fluorescence-based quantitative PCR (qPCR) and Western blot analysis were performed to determine the mRNA and protein expression of TRPA 1 respectively.Flow cytometry was conducted to investigate changes of calcium influx in HaCaT cells in the above groups.Results qPCR and Western blot analysis showed that TRPA1 mRNA and protein were expressed in HaCaT cells.The fluorescence intensity of calcium influx significantly differed among the blank control group 1,retinal group 1,UVA group and retinal + UVA group (155.06 ± 7.62,148.37 ± 18.77,166.92 ± 3.71 and 331.333 ± 40.563;F =44.509,P ＜ 0.01),as well as among the blank control group 2,retinal group 2,UVB group and retinal + UVB group (150.20 ± 1.73,171.66 ± 56.23,147.56 ± 6.60 and 250.44 ± 9.13;F =85.261,P ＜ 0.01).Additionally,retinal + UVA/UVB groups showed significantly higher fluorescence intensity of calcium influx compared with the blank control groups (q =18.442,6.052,P ＜ 0.01).The TRPA1 agonist cinnamaldehyde and its antagonist camphor could regulate the UVA-and UVB-induced calcium influx (P ＜ 0.001).Compared with the blank control group 1 and 2 respectively,the fluorescence intensity of retinal-dependent calcium influx was significantly higher in the UVA/UVB-TRPA1 agonist group (q =14.934,32.770,P ＜ 0.001),and significantly lower in the UVA/UVB-TRPA1 antagonist group (q =7.986,14.596,P ＜ 0.001).Conclusion TRPA1 is expressed in HaCaT cells,and UVA or UVB can regulate the calcium influx in HaCaT cells by adjusting the activity of TRPA 1.
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Objective To analyze the laboratory index characteristics of pulmonary hypertention (PH ) patients with different function states and pressure stages .Methods 2 752 patients diagnosed with PH in the outpatient department ,emergency depart-ment and inpatient department of this hospital from January 2006 to December 2011 were retrospectively analyzed .The clinical as-sessment of the disease condition was done according to the function state and pressure stage of PH recommended by WHO .The characteristics of hematological indexes ,inflammatory indicators and blood gas analysis were observed as well .Results The most common hematological abnormal indexes were the serum brain natriuretic peptide (BNP) elevation(89 .12% ) ,abnormal liver func-tion(58 .32% ) and abnormal hemoglobin(58 .32% ) .The proportion of the PH patients with the elevation of erythrocyte sedimenta-tion rate(ESR) and high-sensitivity C-reactive protein (hs-CRP) were 78 .52% and 73 .59% respectively .The constituent ratio of the BNP increase ,liver function abnormality ,abnormal hemoglobin ,elevation of UA and ESR had statistical differences among the PH patients with different function states and pressure stages (P<0 .05) .The most commonly blood gas analysis abnormality was hypoxemia(83 .08% ) .Respiratory alkalosis had the highest incidence rate in the acid-base imbalance(24 .58% ) .Conclusion The most common hematological abnormal indexes among PH patients are the elevation of serum BNP ,abnormal liver function and he-moglobin abnormality .The laboratory abnormal indexes of above 3 items and the increase of UA and ESR are always related with the severity of disease ,which should to be followed-up .
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Objective To investigate the roles of glutamate signaling pathway in the activation of human peripheral blood lymphocytes(PBLs) and pathogenesis of vitiligo.Methods Peripheral blood lymphocytes (PBLs) isolated from 5 patients with generalized vitiligo and 5 healthy controls were cultured in vitro.Flow cytometry was performed to quantify the expression of CD25 and interferon-γ on PBLs derived from healthy controls and treated with MK801 (a non-competitive antagonist of N-methyl-D-aspartic acid receptor,NMDAR) at 100 μmol/L or phosphate buffered saline (PBS) for 48 hours,as well as the level of reactive oxygen species (ROS) in the controlderived PBLs treated with MK801 at 100 μmol/L,NMDA (an agonist of N-methyl-D-aspartic acid receptor) at 0.5 mmol/L or PBS for 48 hours.The protein and mRNA expressions of NMDAR1 and NMDAR2A were measured by flow cytometry and real-time PCR,respectively,in PBLs from the healthy controls and vitiligo patients.Immunohistochemistry was used to observe the expressions of NMDAR1 and NMDAR2A in tissue specimens from depigmented and postinflammatory hyperpigmentation lesions of the patients with vitiligo and from normal skin of the healthy controls.Results Compared with the PBS-treated PBLs from the healthy controls,the MK801-treated PBLs showed a downregulated expression of CD25 (7.28％ ± 0.18％ vs.16.02％ ± 0.42％,P ＜ 0.01),but an upregulated proportion of CD25+IFN-γ+ lymphocytes (1.79％ ± 0.09％ vs.0.78％ ± 0.06％,P ＜ 0.01),and the NMDA-treated PBLs displayed a higher ROS level (101.1 ± 3.50 vs.69.80 ± 2.08,P＜ 0.01 ).The protein expression of NMDAR1 in PBLs was significantly higher in vitiligo patients than in the healthy controls (3.85 ± 2.17 vs.0.97 ±0.55,P ＜ 0.05).Conclusion Glutamate signaling pathway may be involved in the immunopathogenesis of vitiligo via affecting the secretion of interferon-γ by,and ROS level in,activated lymphocytes.
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Objective To investigate the diagnosis value of immunophenotype and karyotypes in newly diagnosed chronic lymphocytic leukemia (CLL).Methods To retrospect the flow cytometry (FCM) immunophenotype and karyotypes characteristics in newly diagnosed 70 CLL cases.Results In all cases,the positive rates of CD19,CD20,CD5,CD23,CD22 were 100 ％,88.5 ％ (54/61),77.1％ (54/70),67.6 ％ (23/34)and 51.9 ％ (27/52),respectively.And 6 were misdiagnosed,2 was CD+5CD+19(+),but CD20,CD22 were strongly positive,final diagnosed as mantle cell lymphoma (MCL) by FISH t(11;14) examination and CyclinDl; CD+5CD+19(-) CLL were 16 cases (22.9 ％),but 4 were misdiagnosed,the misdiagnosis rate was 25 ％,significantly higher than that of CD+5CD+19(-) CLL (P =0.030).59 cases were examined by conventional cytogcnetic (CC),and 13 cases were with abnormal karyotypes,positive rate was 22.0 ％,with complex karyotypes in 5 cases (8.5 ％); 10 cases combined with FISH abnormalities karyotype examination rate was 60 ％.Conclusion Typical CLL immunophenotypic characteristics were CD5,CD19,CD23 co-expression,and CD－5 CLL with higher misdiagnosis rate,combined with CD20 (,) CD22 expression and karyotype analysis helps to CLL and other B lymphoid proliferative diseases (B-LPD) identification.Conventional cytogenetic detection combined with FISH scan can improve the recognition ability of abnormal chromosome.
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Objective To investigate the action mechanism of glutamate-mediated signaling pathway in malignant melanoma. Methods WM451LU melanoma cells in log phase were classified into 6 groups, negative control group treated with PBS (100 μl), MK801 group treated with the non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 (100 μmol/L), CPCCOEt group treated with non-competitive metabotropic glutamate receptor 1 (mGluR1) antagonist CPCCOEt, MAP2 group transfected with adenovirus vector containing microtubule associated protein 2a (Ad-MAP2a), MK801 + MAP2 group treated with MK801 of 100 μmol/L and transfected with Ad-MAP2a, CPCCOEt + MAP2 group treated with CPCCOEt of 10 μmol/L and transfected with Ad-MAP2a. Western blot was performed to detect the expression of an ionotropic glutamate receptor, i.e., N-methyl-D-aspartate receptor type 2A (NMDAR2A) in WM451LU cells transfected with Ad-MAP2a. Scratch motility assay and cell invasion assay were conducted in vitro to detect the changes in migration and invasion ability of WM451LU cells after treated with Ad-MAP2a, MK-801, CPCCOEt alone or in combination. In vivo study was carried out to compare the inhibitory effect of the above treatments on melanoma. Results Western blot revealed a decrease in the expression of NMDAR2A in WM451LU cells after transfected with Ad-MAP2a. The scratch motility assay showed that the number of migrating cells per high power field was 117.04 ± 2.76 in MAP2 group,107.64 ± 6.50 in MK801 group,97.36 ± 4.79 in CPCCOEt group, 43.28 ± 3.02 in MK801 + MAP2 group,30.76 ± 3.97 in CPCCOEt + MAP2 group,significantly different from that in the negative control group (152.3 ± 5.75,all P ＜ 0.01 ). Cell invasion assay demonstrated that the average number of invading cells per high power field in the negative control was significantly higher than that in MAP2 group, MK801 group, CPCCOEt group, MK801+MAP2 group and CPCCOEt + MAP2 group (170.43 ±8.72 vs. 98.26 ± 3.84, 97.22 ± 5.54, 112.23 ± 7.21, 42.89 ± 5.06, 58.25 ± 6.68, P ＜ 0.05, 0.05, 0.05, 0.01and 0.01, respectively).A significant decrease was observed in the average volume of experimental melanoma in mice of MAP2 group, MK801 group, MK801 + MAP2 group, CPCCOEt group and CPCCOEt + MAP2 group compared with the negative control group (224.02 ± 46.19 mm3, 160.33 ± 33.91 mm3, 91.49 ± 21.48 mm3,202.30 ± 52.37 mm3, 111.13 ± 69.81 mm3 vs. 342.70 ± 60.92 mm3, all P ＜ 0.01 ). Conclusions To block the glutamate signaling pathway in vitro can inhibit the invasion and migration of melanoma cells, and to block the pathway in vivo can inhibit the growth of malignant melanoma and alter the morphology of melanoma cells.
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Objective To study the foxml gene and its protective effect on the lung tissue of rats with acute lung injury (ALI) induced by lipopolysaccharide (LPS), and to observe the dexamethason' s (DEX) impacts on foxml gene and the prognosis of ALI. Method Seventy-two healthy mice were randomly(random number) divid-ed into three groups: control group (A group, n = 24), model group (B group, n = 24) and DEX treatment group (C group, n = 24). The observing intervals were respectively set in 24 h, 48 h and 72 hours. At each ob-serving interval, the foxml protein in lung tissue of mice was detected by using immunohistochemistry (IHC), and the expression of foxml gene in lung tissue was detected by using RT-PCR, as well as to observe the pathological changes in lung tissue. Results Comparisons were made between paired groups at 24 h,48 h and 72 h intervals in which the expression of foxml mRNA and the level of foxml protein in lung tissue of mice in C group were signifi-cantly higher than those in B group (P < 0.05), and those in B group were significantly higher than those in A group (P < 0.05). The expression of foxml mRNA and the level of foxml protein in lung tissue of mice in B group at 48 h interval were significantly higher than those both at intervals of 72 h and 24 h (P < 0.05), and the those at 72 interval were significantly higher than those at 24 h interval (P < 0.05). Compared with B group, the pathologi-cal changes in lung tissue of mice in C group were lessened. Conclusions In both model group and dexamethasone treatment group, the expression of foxml mRNA and the level of foxml protein in lung tissue of mice are increased significantly. Dexamethasone lessens the injury of both vascular endothelial cells and alveolar epithelial ceils of lung tissue, and it also significantly increases the expression of foxml mRNA and the level of foxml protein.
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Objective To study the correlation between the obstructive sleep apnea-hypopnea syndrome (OSAHS) and hypertension by monitoring nocturnal blood pressure fluctuation in patients.Methods 95 patients with OSAHS and hypertension (group A) and 95 patients with OSAHS only (group B) were selected and their body mass index, the ratio of neck circumference and height , the blood pressure at time of visit were recorded.At least 7 hours, polysomnography(PSG) was performed for every patient , meanwhile, the blood pressure before sleep, at night and right after wake-up were also collected.The two groups' data was compared and analyzed.Re-sults There was no statistical difference in the body mass index and neck circumference/height between the two groups.No significant difference was found in the highest blood pressure in patients with different severity of OS-AHS.There was no statistical correlation between AHI and systolic blood pressure(SBP) difference of sleep and right after wake, so was AHI and diastolic blood pressure (DBP) difference of before sleep and sleep and bedtime (P > 0.05).But there was correlation between AHI and the different value of SBP before sleep and sleep, so is AHI and DBP diferenee of right after wake and sleep (P > 0.05).Conclusions There is correlation between AHI and the different value of SBP before sleep and sleep, so was AHI and DBP diferenee of right after wake and sleep.Measure of different time-points blood pressure at night can avoid interrupting the sleep state of patients, will help better evaluate cardiovascular complications and prognosis of patients with OSAHS .
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Objective To explore the role of p170, MRP, bcl-2 in clinical drug- resistance and their correlation. Methods There are 44 acute leukemia(AL) patients, among them ANLL is 29, ALL is 15.The expression of p170, MRP, bcl-2 were detected. At last, the patients were divided into two groups: CR and NR according to efficacy after two periods standard chemotherapy. Results Overexpression of p170, MRP,bcl-2 were found in AL (P ＜0.05). The expression of p170 and MRP were lower in CR group than that in NR groups in AML and ALL; there were 22 of 24 patients expressed one or two proteins in NR groups and the expression of p170 in patients was concordant with that of MRP to certain extent (P ＜0.01). The overexpression of bcl-2 was related with the worse prognosis. The expression of bcl-2 was lower significantly in CR group than in CR and NR (P＜0.05). Conclusion p170, bcl-2, MRP are all associated with clinical MDR. High expression of these proteins is an unfavorable factor to prognosis. The expression of MRP and p170 showed coincidence, but showed no significance with expression of bcl-2 in untreated patients, it indicated that the drug-resistance mediated by p170 was different from bcl-2.