大鼠肝卵圆细胞增殖模型建立及其纯化鉴定

Establishment of Hepatic Oval Cell Proliferation Model and Purification of Hepatic Oval Cell

为建立大鼠卵圆细胞增殖模型,分离纯化卵圆细胞并对其特性进行研究,SD大鼠喂饲2-乙酰氨基芴(2-acetyailamifluorene,2-AAF),剂量分别为5、10、15、20mg/kg,连续给药6d,于第7天行三分之二部分肝切除,术后继续给药1周,每隔3d取肝组织行常规组织学观察,作细胞增殖试验及免疫组化染色。通过免疫磁珠标记C-kit阳性细胞以纯化卵圆细胞(Magneticactivatedcellsorting,MACS),用免疫细胞化学及RT-PCR对纯化细胞进行鉴定。结果:10、15、20mg/kg三种剂量2-AAF均可成功建立卵圆细胞增殖模型,卵圆细胞增殖于第8天较明显,至第11天达高峰,第14天有所减少。卵圆细胞胞核Brdu染色阳性,其除表达卵圆细胞特异性抗原OV6外,尚表达肝细胞标记白蛋白及胆管细胞标记CK19。胚胎期肝细胞抗原AFP、连接蛋白43在卵圆细胞中亦有高表达,同时还表达造血干细胞标记C-kit。以MACS纯化的C-kit阳性卵圆细胞,90%以上为OV6阳性,并有AFP、白蛋白、CK19mRNA转录。结论:2-AAF剂量为10～20mg/kg时可获得满意的大鼠卵圆细胞增殖模型,增生的卵圆细胞为具有双向分化潜能的肝干细胞/前体细胞,利用C-kit标记通过MACS可纯化这种肝干细胞/前体细胞。

To establish a rat model for hepatic oval cells proliferation and to analyze the characterization of oval cell, SD rats (about 150 g) were given 2-acetylaminofluorene (2-AAF)6 days before partial hepatectomy and 7 days after it. Liver tissue specimens were obtained every 2~3 days after partial hepatectomy and were investigated by HE staining and immunohistochemical staining for Brdu,OV6, cytokeratin-19, albumin, AFP, connexin-43 and C-kit. Oval cells were enriched by magnetic activated cell sorting(MACS) using C-kit antibody. RT-PCR was performed on sorted cells for the albumin,AFP and CK19 messenger RNA expression. Results: Obvious oval cell proliferation was seen in the liver in 10 mg/kg,15 mg/kg,20 mg/kg groups and oval cell Brdu incorporation test is positive. Oval cells expressed the specific marker OV6, hepatic cell marker albumin, biliary cell marker CK19 ,fetal liver cell marker AFP and connexin-43 as well as hematopoietic stem cell receptor C-kit. More than 90% sorted oval cells were OV6 positive and all expressed the albumin, AFP and CK19 gene. Conclusions: Rat model for hepatic oval cell proliferation can be established with 2-AAF/partial hepatectomy at dosage of 10~20 mg/kg body weight. And such oval cells were hepatic stem/progenitor cells which expressed OV6/CK19/albumin/AFP/connexin43/C-kit. Oval cell population can be highly enriched with MACS using C-kit.