肝脏干细胞生长及凋亡特性的研究

Study on growth and apoptotic characteristics of hepatic stem cells

目的了解大鼠肝脏干细胞(WB-F344)的基本特征,探讨各种理化因素对肝脏干细胞凋亡的影响。方法观察肝干细胞的形态特征并绘制细胞生长曲线;采用细胞DNA琼脂糖凝胶电泳图检测饥饿、紫外线及乙醇对肝干细胞凋亡的影响;RT-PCR法、Western-blottting法及免疫组化法检测肝干细胞ALB、AFP的表达;结果WB细胞倍增时间约为48h,与正常人肝细胞系LO2细胞的倍增时间相近,是人肝癌细胞系HepG2细胞的2倍;紫外线和缺乏营养可引起WB细胞凋亡,而乙醇则引起WB细胞坏死;WB细胞较HepG2细胞和LO2细胞对乙醇、紫外线及饥饿的耐受性强;免疫组化、RT-PCR法及Western-blot法的结果相似,提示WB细胞和LO2细胞均表达AFP、ALBmRNA和蛋白,HepG2细胞仅表达AFPmRNA和蛋白,而不表达ALBmRNA和蛋白。结论肝干细胞倍增时间与正常肝细胞相仿;紫外线、乙醇和饥饿等理化因素可引起肝干细胞凋亡或坏死,但肝干细胞对它们的耐受性明显高于正常肝细胞及肝肿瘤细胞。

[Objective]To explore the growth characteristic of hepatic stem cells and investigate the effects of ultraviolet rays, ethanol and cells culturing without serum on apoptosis and death of hepatic stem cells. The morphologic characteristic of hepatic stem cells were observed, and its growth curve was protracted; apoptosis of WB, LO2 and HepG2 cells were induced by ultraviolet rays, ethanol and culturing without serum. Apoptotic cells were detected by DNA ladder. Expression of ALB and AFP mRNA was detected by RT-PCR and protein by Western-blotting and immunohistochemistry. The double time of WB cells was about 48 hours, resembled that of LO2 cells, was longer than that of HepG2 cells. Ultraviolet rays and culturing without serum could make the three cells apoptosis, and ethanol induced the cells death. WB cells were more sensitive than LO2 cells and HepG2 cells to ultraviolet rays, ethanol and culturing without serum. The results of RT-PCR, Western-blotting and immunohistochemistry were similar and showed that WB cells and LO2 cells expressed ALB and AFP mRNA and protein, HepG2 cells expressed only AFP but not ALB. [Conclusion] The double time of hepatic stem cells was similar with that of hepatocytes and longer than that of HepG2 cells. Ultraviolet rays and culturing without serum can induced Hepatic stem cells apoptosis or death, and Hepatic stem cells were sensitive to them than LO2 cells and HepG2 cells.