C-kit^+细胞体外增殖、分化的研究

Proliferation and Differentiation of C-kit^+ Cell In Vitro

分离纯化卵圆细胞并对其体外增殖、分化进行研究。SD大鼠喂饲2-乙酰氨基芴(2-acetainofluorene,2-AAF)10mg/kg,以促进肝脏干细胞增殖,通过免疫磁珠法(Magnetic activated cell sorting,MACS)标记C-kit阳性细胞以纯化卵圆细胞,进行体外培养,采用免疫细胞化学、逆转录聚合酶链反应(RT-PCR)等方法观察其体外增殖、分化特性。以MACS纯化的C-kit阳性卵圆细胞,90%以上为卵圆细胞抗原(OV6)阳性,大部分甲胎蛋白(AFP)阳性。C-kit阳性细胞在体外增殖能力较强,能形成集落样生长,并向胆管细胞及肝细胞分化,表达角蛋白19(CK19)和/或白蛋白(ALB)标记。细胞集落RT-PCR结果显示AFP、CK19及ALB基因均有表达。以上结果表明利用C-kit标记通过MACS纯化的C-kit+细胞为具有双向分化潜能的肝脏干细胞。

This study sought to isolate and purify C-kit^＋ cells from rat 2-AAF/PH model and to investigate the proliferation and differentiation of C-kit^＋ cells in vitro. C-kit positive oval cells were enriched by using magnetic activated cell sorting （MACS） . The sorted oval cells were cultured in a low density, and then colony formation was observed. The capacity of proliferation and differentiation of C-kit positive cells were examined in vitro by immunocytochemistry and RT-PCR. By using C-kit antibody in conjunction with MACS, we developed a rapid oval cell isolation protocol. The sorted cells formed colony when cultured in vitro. Cells in the colony expressed albumin or cytokeratin 19（CK19） or coexpressed both, and BrdU incorporation test was positive. RTPCR on colony showed expression of albumin and CK19 gene. The results demonstrate that by means of MACS we have established a method to isolate oval ceils. The sorted hepatic oval ceils can form colony in vitro which expresses different combinations of phenotypic markers and genes from both hepatocytes and cholangiocyte lineage.