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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 observe the homing and differentiation of human umbilical cord derived mesenchymal stem cells (HUMSCs) into HepG2 hepatoma cells in situ. Methods: HUMSC cells were induced to differentiate into hepatocytes in vitro by two-step method. The expression of hepatocyte-specific genes alpha-fetoprotein (AFP) and albumin (ALB) in HUMSCs were detected by Real-time PCR and Western blotting. Transwell experiments were used to observe the chemotactic effect of HUMSC on HepG2 hepatoma cells conditioned medium. The in situ xenograft model of HepG2 hepatoma cells was established by subcutaneous-orthotopic transplantation in BALB / c nude mice. Methods of Lentiviral Infection. Fluorescently transfected with HUMSC-labeled CMV or AFP-driven f Luc was prepared into MSC.CMVLuc or MSC.AFPLuc and injected into tumor-bearing liver tissue. The IVIS imaging system was used to observe the migration of MSC.CMVLuc to the tumor site Nest and MSC.AFPLuc differentiate into hepatocytes in liver cancer microenvironment. Results: The morphological changes of HUMSC during in vitro culture showed that HUMSC differentiated into hepatocytes and the expression of AFP, ALB mRNA and protein were significantly increased (all P <0.05) after HUMSC culture. The tendency of HUMSC to HepG2 hepatoma cells (P <0.01). MSC.CMVLuc migrated and accumulated in the liver 2 days after injection, and the luminescence signal in the liver was further enhanced 5 days after the injection of MSC.CMVLuc. After hepatic injection of MSC.AFPLuc, Differentiation, AFP promoter activity was the strongest on the 7th day after injection, and disappeared on the 9th day after injection. CONCLUSION: The ability of HUMSC to homing and differentiating into liver has been demonstrated both in vitro and in vivo, providing a new strategy for targeted therapy of hepatocellular carcinoma by MSC in the future.