体外诱导大鼠骨髓间充质干细胞向肝细胞分化的实验研究  

Induced differentiation of rat bone marrow mesenchymal stem cells into hepatocytes in vitro

作　　者：鲁学恒[1] 马力[2] 刘沛[1] 

机构地区：[1]中国医科大学附属第一医院感染科,辽宁沈阳110001 [2]中国医科大学附属盛京医院感染科

出　　处：《中国医科大学学报》2007年第3期256-258,共3页Journal of China Medical University

基　　金：卫生部临床重点资助项目(97100252)

摘　　要：目的:探讨大鼠骨髓间充质干细胞(mMSCs)在体外定向诱导分化为肝细胞及其方法。方法:采用梯度离心法分离纯化雄性SD大鼠的mMSCs。A组原代培养;B组原代培养分别加刺激因子50ng/ml肝细胞生长因子(HGF)和50ng/ml成纤维细胞生长因子4(FGF-4),倒置显微镜连续观察细胞分化过程的形态学变化。于培养的7、14、21、28d,细胞免疫荧光法检测各组细胞甲胎蛋白(AFP)、白蛋白(ALB)、细胞角蛋白18(CK-18)、细胞角蛋白19(CK-19)的表达。采用过碘酸希夫(PAS)染色法检测各组细胞糖原染色情况。结果:分离纯化B组大鼠的mMSCs,应用HGF和FGF-4诱导10d后变为肝细胞样圆形。细胞免疫荧光测定B组显示,培养第7dAFP即出现阳性,第21d表达明显减弱;第7dCK-19染色阳性,以后增强;第14d白蛋白与CK-18染色开始阳性,以后表达逐渐加强。A组在培养过程中不表达AFP、ALB、CK-18和CK-19。糖原染色显示,B组第14d开始阳性,一直持续到第28d;A组未见阳性染色。结论:大鼠mMSCs在较高浓度的HGF及FGF-4诱导下可分化为肝细胞。Objective： To explore whether rat bone marrow mesenchymal stem cells （mMSCs） can differentiate into hepatocytes and the condition of differentiation in vitro. Methods： mMSCs were isolated and purified from male SD rats by using density gradient centrifngation, mMSCs were cultured primarily in group A and treated with 50 ng/ml of hepatocyte growth factor （HGF） and 50 ng/ml of fibroblast growth factor-4 （FGF-4） in group B, respectively. The morphological changes in mMSCs were observed under phase contrast microscope. On days 7, 14, 21, and 28 of culture, the expressions of album （ALB） , alpha-fetoprotein （AFP） , cytokeratin-18 （CK- 18）, and cytokeratin-19 （CK-19） in mMSCs were determined after immnnoflnorescence staining. The glycogen storage was detected with periodic acid-Schiff （PAS） reaction. Results： The spindle-shaped mMSCs became round-shaped and differentiated into hepatocyte-like cells after treated with HGF and FGF-4 for 10 days in group B. The immnnoflnorescence staining showed that in group B, the expression of AFP appeared on day 7 and decreased on day 21 ; the expression of CK-19 was found on day 7 and then continued to increase; the expression of ALB and CK-18 appeared on day 14 and increased since then. No expressions of ALB, AFP, CK-18, and CK-19 were found in group A. Glycogen storage was found from day 14 to day 28 in group B, and no glycogen storage was found in group A. Conclusion： Rat mMSCs cultured in the presence of high concentrations of HGF and FGF-4 can differentiate into hepatocytes in vitro.

关 键 词：细胞分化 肝细胞生长因子 骨髓间充质干细胞 肝细胞 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]