脂肪间充质干细胞向肝细胞横向分化的研究

A study on the transdifferentiation of adipose mesenchymal stem cells into hepatocytes

目的探讨脂肪源性间充质干细胞（AMSC）向肝细胞横向分化的可能性。方法胶原酶消化脂肪组织,贴壁培养,体外扩增后以流式细胞仪鉴定其表面标志。取扩增3代的AMSC分为2组,诱导分化组在含有2％FBS的DMEM-F12培养基中加入肝细胞生长因子20 ng/ml和成纤维细胞生长因子4 10 ng/ml、1×ITS和地塞米松0．1μmol/L,培养14 d；空白对照组则不加任何细胞因子。RT-PCR检测诱导分化过程中肝细胞核因子1、GATA4等基因转录水平的变化。2周后,采用流式细胞术检测AFP和Alb阳性细胞在两组细胞中的比例,检测肝细胞特异性细胞角蛋白（CK） 18、CK19的表达。结果分离、培养的AMSC呈成纤维细胞样生长,可以稳定传代。流式细胞术检测结果显示第3代脂肪间充质干细胞高表达表面CD29、CD44；不表达CD34、CD45。RT-PCR检测诱导5、8、11、14 d的细胞,显示有肝细胞特异性转录因子GATA4和肝细胞核因子1A基因的表达,并随时间延长而逐渐增多。流式细胞术检测诱导14 d的细胞,发现30．0％的细胞表达Alb,17．8％细胞表达AFP,双阳性的细胞为6．9％；免疫荧光检测发现诱导细胞表达CK18、CK19。空白对照组脂肪间充质干细胞则未见上述各项变化。结论在低血清培养体系中,采用细胞因子联合诱导,显示脂肪间充质细胞在体外能定向分化为肝细胞样细胞。

Objective To investigate the possibility of transdifferentiation of adipose mesenchymal stem cells （AMSCs） into hepatocytes. Methods Human omentum adipose tissue was dispersed with collegenase i. Cells collected were cultured in a DMEM-F12 medium containing 2% FBS supplemented with 20 ng/ml HGF, 10 ng/ml FGF4, 1×ITS and 0.1 μmol/L dexasmison. The cells of the control group were also cultured in the same kind of medium but without any cytokines serving as a control. The expression of hepatic transcriptional factors such as GATA4 and HNF1 were checked by RT-PCR. At the end of the induction, hepatocyte markers were analysed by flow cytometry, and cytokeratin expressions were examined using cytoimmunofluorescence methods. Results AMSCs grew like fibroblasts and were passaged easily. Most of the third passaged AMSCs were positive against anti-CD29, anti-CD44 antibodies, but negative for the anti- CD34 and anti-CD45 ones. The hepatic transcriptional factor was expressed gradually to higher levels during the induction time. AFP and Alb positive cells were 30.0% and 17.8% of the total cultured cells, and the rate of cells positive to the two markers was 6.9%. The inducted cells were positive for CK18 and CK19 antibodies at the end of the induction. Thecells in the control group were negative when checked by these methods. Condusiom AMSCs could be directed to differentiate into hepatocytes in vitro by a cytokine cocktail with a low concentration FBS culture system.