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

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

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f Luc,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.AFPLuc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2 d迁移并聚集于肝脏,注射后5 d肝脏内发光信号进一步增强;MSC.AFPLuc肝内注射后24 h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。

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.