摘要
目的:研究SOX9的表达在诱导肝细胞向胰腺干祖样细胞转化过程中的意义。方法:分离培养小鼠原代肝细胞,包装、浓缩表达OCT4的慢病毒并感染肝细胞,使用RT-PCR监测慢病毒感染后肝细胞中SOX9的表达,并根据SOX9表达情况尝试向胰腺干祖样细胞诱导。使用PCR和免疫荧光鉴定生成的胰腺干祖样细胞。结果:肝细胞感染OCT4-慢病毒后,肝细胞内颗粒逐渐释出,细胞折光性增强。SOX9表达从第6 d持续至第8 d。从SOX9阴性的第4 d开始向胰腺诱导,无集落样的胰腺干祖样细胞生成,RT-PCR检测其标志物CK19为阴性;从SOX9阳性的第6 d开始向胰腺诱导,可见集落样生长的胰腺干祖样细胞,RT-PCR检测CK19为阳性,免疫荧光染色显示CK19表达在胞浆中。结论:肝细胞向胰腺干祖样细胞转化过程中SOX9的表达提示了"兼性肝细胞"的出现,此细胞具有向胰腺谱系诱导分化的潜能,这为进一步生成有功能的胰岛内分泌细胞和实现糖尿病的细胞替代治疗提供了理论和实验依据。
Objective： To investigate the significance of SOX9 expression during the induction of hepatocyte towards stem/progenitor-like cell.Methods： Mouse primary hepatocytes were isolated by two-step collagenase method and cultivated in vitro.Lentivirus expressing OCT4 were packaged and concentrated.Hepatocytes were transduced by OCT4-lentivirus.Expression of SOX9 was monitored by RT-PCR.Infected hepatocytes were introduced towards stem/progenitor-like cell according to the SOX9 expression.RT-PCR and immunofluoresence staining were used to identify stem/progenitor-like cell.Results： After hepatocytes were infected by OCT4-lentivirus,the intracellular particles were gradually released and the cell reflectivity was enhanced.SOX9 expression lasted from the 6th day to the 8th day.If induction towards stem/progenitor-like cell started on the 4th day when SOX9 wasn＇t expressed,no colony-like stem/progenitor-like cell grew and its marker CK19 was negative tested by RT-PCR.If induction started on the 6th day when SOX9 began to express,stem/progenitor-like cell presented colon-like growth and its marker CK19 was positive tested by RT-PCR.Immunofluorescent staining revealed that CK19 was expressed in the cytoplasm.Conclusion： During the conversion of hepatocyte into stem/progenitor-like cell,SOX9 expression indicated the emergence of ＂facultative hepatocyte＂,which had the potential to be induced and differentiated towards the pancreas lineage.It provided experimental basis for the further production of functional pancreatic endocrine cells and the realization of cell replacement therapy in diabetes.
出处
《现代生物医学进展》
CAS
2013年第15期2852-2856,共5页
Progress in Modern Biomedicine
基金
国家高技术研究发展计划(863计划)(2011AA020113)
关键词
SOX9
胰腺干细胞
胰腺祖细胞
SOX9
Pancreatic stem cell
Pancreatic progenitor cell
