胆汁淤积性微环境中甘氨鹅脱氧胆酸钠促进肝脏干细胞凋亡

Effect of sodium glycosaminodeoxycholate on apoptosis of hepatic stem cells in cholestatic microenvironment

目的探讨在胆汁淤积性肝硬化病理微环境下对肝脏干细胞存活率的影响及体外甘氨鹅脱氧胆酸钠(GCDC)对肝脏干细胞凋亡的影响。方法以Balb/c小鼠胆总管结扎模型模拟胆汁淤积性肝硬化的病理环境并回输肝脏干细胞HP14-19,检测细胞定植存活情况;通过细胞计数试剂盒8(CCK-8)、结晶紫染色检测甘氨鹅脱氧胆酸钠对增殖的影响,流式细胞术检测细胞凋亡,通过Western blotting检测凋亡标志物Bax、Bcl-2、Caspase-3表达和磷酸化腺嘌呤核苷二磷酸依赖性蛋白激酶α(p-AMPKα)、腺嘌呤核苷二磷酸依赖性蛋白激酶α(AMPKα)等的表达。结果通过CCK-8检测和结晶紫染色发现,随着甘氨鹅脱氧胆酸钠作用浓度的增加,HP14-19细胞的增殖抑制;流式细胞术提示,GCDC处理组细胞凋亡率明显增加(P<0. 05);Western blotting检测发现,与对照组相比,实验组凋亡基因Bax、Caspase-3蛋白表达上调,抗凋亡基因Bcl-2蛋白表达下调,AMPKα活化增加(P<0. 05)。结论胆汁淤积性肝硬化所导致的胆汁淤积微环境诱导肝脏干细胞发生凋亡。

Objective To investigate the effects of hepatic stem cells survival in the pathological microenvironment of cholestatic cirrhosis,and the effects of sodium glycosaminodeoxycholate( GCDC) on apoptosis of hepatic stem cells in vitro.Methods Balb/c mice were subjected to bile duct ligation( BDL) to simulate the pathological environment of cholestatic cirrhosis;Liver stem cells HP14-19 were transplanted back into liver by the splenic vein and survival of cell colonization was detected;Effects of sodium glycosaminodeoxycholate on the viability of HP14-19 cells at different concentrations by cell counting kit-8( CCK-8) and crystal violet staining,flow cytometry was used to detect the apoptosis of HP14-19 cells treated with 600 μmol/L GCDC 24 hours later,and the expression of Bax,Bcl-2,Capase-3,phosphorylated adenosine 5 ’-monophosphate-activated protein kinase α( p-AMPKα) and adenosine 5 ’-monophosphate-activated protein kinase α( AMPKα) were detected by Western blotting. Results The results of CCK-8 and crystal violet staining showed that the proliferation of HP14-19 cells was inhibited with the increase of the concentration of sodium glycosaminodeoxycholate. Flow cytometry showed that the apoptosis rate of GCDC treated group was higher than that of control group. Compared with the control group,the expression of Bax,Capase-3 was up-regulated and the expression of Bcl-2 protein was down-regulated in the experimental group. The results showed that GCDC could induce apoptosis of HP14-19( P<0. 05) AMPK was activated.Conclusion Microenvironment of cholestatic cirrhosis induced apoptosis of liver stem cells.