人脐带来源间充质干细胞向HepG2肝癌细胞原位移植瘤的归巢及分化

目的:观察人脐带来源的间充质干细胞(human umbilical cord derived mesenchymal stem cell,HUMSC)向HepG2肝癌细胞原位移植瘤的归巢及分化.方法:二步法体外诱导HUMSC向肝细胞分化,通过Real-time PCR和Western blotting检测不同时间点HUMSC中肝细胞特异性基因甲胎蛋白(alpha-fetoprotein,AFP)及白蛋白(albumin,ALB)mRNA和蛋白水平的变化;Transwell实验观察HUMSC在HepG2肝癌细胞条件培养基作用下的趋化现象;采用皮下-原位移植的方法建立BALB/c裸鼠的HepG2肝癌细胞原位移植瘤模型,利用慢病毒感染的方法将HUMSC标记CMV或AFP启动子驱动的fLuc,制备成MSC.CMVLuc或MSC.AFPLuc,并将其注射入荷瘤肝组织,IVIS成像系统观察MSC.CMVLuc向肿瘤部位的迁移归巢及MSC.AF-PLuc在肝癌微环境中向肝细胞分化.结果:HUMSC在体外诱导培养过程中出现细胞形态学变化证明其向肝细胞分化,同时诱导培养后AFP、ALB mRNA和蛋白的表达明显升高(均P＜0.05);HUMSC向HepG2肝癌细胞的趋化呈瘤细胞密度依赖性(P ＜0.01);MSC.CMVLuc注射后2d迁移并聚集于肝脏,注射后5d肝脏内发光信号进一步增强;MSC.AFPLuc肝内注射后24h已向肝细胞分化,注射第7天AFP启动子活性最强,注射第9天活性消失.结论:在小鼠体内外证明了HUMSC具有向肝脏归巢及分化的能力,为今后MSC应用于肝癌基因靶向治疗提供了一种新的策略.

Tropism and differentiation of human umbilical cord derived mesenchymal stem cells to tumor transplanted by hepatocarcinama HepG2 cells in situ

Objective:To study the tropism and differentiation of human umbilical cord derived mesenchymal stem cell (HUMSC) to HepG2 orthotopic hepatocarcinoma.Methods:The in vitro hepatic differentiation of HUMSCs was induced by a two-step protocol;the changes in mRNA and protein expression of hepatic alpha-fetoprotein (AFP) and albumin (ALB) at different time points were examined by Real-time PCR and western blotting;the tropism of HUMSCs stimulated by conditional culture medium with HepG2 cells was identified by transwell assay.Subcutaeous-orthotopic transplantation was used to build the HepG2 orthotopic hepatocarcinoma model in athymic BALB/c nude mice.HUMSCs were labeled with MSC.CMV-Luc or MSC.AFP-Luc through lentivirus transduction before orthotopically injected into hepatic parenchyma;the tropism of MSC.CMV-Luc to tumor location as well as the hepatic differentiation of MSC.AFP-Luc in tumor-bearing liver was monitored by using IVIS living imaging system.Results:The in vitro hepatic differentiation of HUMSCs was successfully induced in vitrowith morphological changes,and the mRNA and protein levels of AFP and ALB significantly elevated after the culture (P ＜ 0.05);the tropism of HUMSCs to HepG2 cells was in a dose-dependent manner (P ＜0.01).MSC.CMV-Luc,which injected intravenously,migrated to liver after 48 h;and the luciferase signal in the liver of rats was enhanced on day 5.MSC.AFP-Luc developed hepatic differentiation after 24 h of in situ transplantation;at day 7 of post-transplantation,the activity of AFP promoter was at the highest,while,its activity was undetectable at day 9.Conclusion:It was confirmed the tropism and differentiation of HUMSCs to liver by in vitro and in vivo experiments.This study may provide a new strategy of target gene therapy for hepatocarcinoma by using MSCs.