CAJ | 학술논문

目的研究肝细胞生长因子激活物激活肝细胞生长因子后对肝星状细胞(HSC)凋亡的影响及对Rho表达的调控.方法用免疫组织化学方法检测HSC中α-平滑肌肌动蛋白的表达量；四甲基偶氮唑盐法检测Y-27632的最佳干预浓度；流式细胞仪检测HSC凋亡情况；免疫荧光法检测肝细胞生长因子-α链的表达水平；PCR法检测HSC中RhoA mRNA的表达；Western blot检测HSC中RhoA蛋白的表达,计量资料采用方差分析,组间比较采用t检验.结果 (1) Y-27632以10 μmol/L作用于HSC引起细胞受抑制最明显(0.96±0.51),与其他各浓度比较,差异均有统计学意义(P值均＜ 0.05)；(2)肝细胞生长因子-α链表达量均随培养时间的延长而增加,24、48、72 h空白对照组(545.25±41.43、585.57±56.87、618.65±55.82)与50 ng/ml外源性HGF干预的实验对照组a(557.34±52.84、589.46±67.21、625.59±54.68)比较,差异无统计学意义(P＞ 0.05),其他各组在各时间段比较,差异均有统计学意义(P值均＜0.01)；(3) HSC凋亡率随培养时间延长而增加,各组各时间段比较,以实验组最高(38.68±3.44、46.93±4.30、59.03±4.25),差异均有统计学意义(P值均＜0.05);(4)实验组RhoA mRNA和蛋白表达量均随时间延长而减少,以实验组表达量最低(0.88±0.01、0.64±0.01、0.51±0.01；0.83±0.04、65±0.06、0.51±0.07)与其他各组在相同时间段比较,差异有统计学意义(P值均＜0.01).结论通过下调Rho通路,激活肝细胞生长因子能促进HSC凋亡.
목적 연구간세포생장인자격활물격활간세포생장인자후대간성상세포(HSC)조망적영향급대Rho표체적조공.방법 용면역조직화학방법검측HSC중α-평활기기동단백적표체량；사갑기우담서염법검측Y-27632적최가간예농도；류식세포의검측HSC조망정황；면역형광법검측간세포생장인자-α련적표체수평；PCR법검측HSC중RhoA mRNA적표체；Western blot검측HSC중RhoA단백적표체,계량자료채용방차분석,조간비교채용t검험.결과 (1) Y-27632이10 μmol/L작용우HSC인기세포수억제최명현(0.96±0.51),여기타각농도비교,차이균유통계학의의(P치균＜ 0.05)；(2)간세포생장인자-α련표체량균수배양시간적연장이증가,24、48、72 h공백대조조(545.25±41.43、585.57±56.87、618.65±55.82)여50 ng/ml외원성HGF간예적실험대조조a(557.34±52.84、589.46±67.21、625.59±54.68)비교,차이무통계학의의(P＞ 0.05),기타각조재각시간단비교,차이균유통계학의의(P치균＜0.01)；(3) HSC조망솔수배양시간연장이증가,각조각시간단비교,이실험조최고(38.68±3.44、46.93±4.30、59.03±4.25),차이균유통계학의의(P치균＜0.05);(4)실험조RhoA mRNA화단백표체량균수시간연장이감소,이실험조표체량최저(0.88±0.01、0.64±0.01、0.51±0.01；0.83±0.04、65±0.06、0.51±0.07)여기타각조재상동시간단비교,차이유통계학의의(P치균＜0.01).결론 통과하조Rho통로,격활간세포생장인자능촉진HSC조망.
Objective To investigate the role of activated hepatocyte growth factor (HGF) in apoptosis of hepatic stellate cells (HSCs) and in modulating the Rho signaling pathway.Methods HSCs were divided into the following groups:blank control,consisting of HSCs without treatment; two treatment controls,consisting of HSCs exposed to exogenous HGF at 50 ng/ml and HSCs exposed to exogenous HGF activator (HGFA) at 70 ng/ml; three experimental groups,consisting of HSCs exposed to both exogenous HGF and HGFA,HSCs pre-incubated with the HGF inhibitor c-met at 500 ng/ml for 6 hours and then exposed to exogenous HGF and HGFA,and HSCs preincubated with the Rho pathway inhibitor Y-27632 at 10 ng/ml and then exposed to exogenous HGF and HGFA.Activation status of the cultured HSCs was determined by change in expression of alpha-smooth muscle actin (SMA).The optimal intervention concentration ofY-27632 was determined by MTT assay.The apoptotic status of HSCs was determined by flow cytometry.Expression of the HGF-alpha chain was detected by immunofluorescence.The expression of RhoA was evaluated by PCR (for mRNA) and by immunohistochemical staining and Western blot analysis (for protein).Results Exposure to 10 μmol/L Y-27632 led to obvious growth inhibition ofHGF + HGFA-induced HSCs,compared with the other concentrations tested (P ＜ 0.05).HGF + HGFA induced the expression of the HGF-alpha chain in a time-dependent manner (P ＜ 0.01); however,the increases in expression of HGF-alpha chain induced by HGF alone and HGFA alone were not significantly different from the level in the blank controls (P＞ 0.05).Exposure to HGF alone and HGFA alone led to a time-dependent increase in apoptosis (24 h,48 h,72 h) but exposure to HGF + HGFA led to the highest levels of apoptosis (P ＜ 0.05).Exposure to HGF + HGFA led to a time-dependent decrease in RhoA mRNA and protein expression (P ＜ 0.01).Conclusion Activation of hepatocyte growth factor promotes apoptosis of hepatic stellate cells by suppressing RhoA expression and down-regulating the Rho signaling pathway.