骨髓间充质干细胞调控肝星状细胞增殖和胶原合成的实验研究

目的研究骨髓间充质干细胞（BMSCs）对肝星状细胞（HSCs）增殖、胶原合成的影响。方法 （1）BMSCs对HSCs增殖的影响：利用transwell共培养体系,按1：1细胞比例在6孔塑料培养板上室接种BMSCs（1×105cells/well）,下室接种HSCs;对照组成纤维细胞代替BMSCs接种于上室;空白组为HSCs单独培养。培养24、48、72h后MTT法检测HSCs增殖抑制率;（2）BMSCs对HSCs胶原合成的影响：实验分为3组,A：HSCs组,B：HSCs＋TGF-β1组,C：共培养＋TGF-β1组。按上述方法进行BMSCs、HSCs共培养,培养液中加入TGF-β1诱导活化。培养72h后,取出BMSCs并换液,24h后收集培养液,用ELISA方法测定Ⅰ型胶原浓度,收集HSCs,RT-PCR检测HSCsα-SMA以及Ⅰ型胶原的基因表达。结果培养24、48、72 h后,共培养组较对照组可明显提高HSCs增殖抑制率（P〈0.01）;比较B组,C组Ⅰ型胶原浓度显著下降（P〈0.01）,同时HSCsα-SMA及Ⅰ型胶原基因表达出现显著下调（P〈0.01）。结论 BMSCs在体外可明显抑制HSCs增殖以及抑制TGF-β1诱导的α-SMA表达和Ⅰ型胶原合成。

Experimental study on proliferation and collagen synthesis in hepatic stellate cells regulated by bone marrow mesenchymal stem cells

Objective To investigate the effects of bone marrow mesenchymal stem cells（BMSCs）on proliferation and collagen synthesis in hepatic stellate cells（HSCs）.Methods（1）The effects on proliferation of HSCs by BMSCs： At a 1：1 co-culture ratio,BMSCs and HSCs（1×105cells/well） were seeded on the upper and lower compartment of 6-well Transwell co-culture plates,respectively.HSCs co-cultured with fibroblast cell lines and HSCs alone were served as the control group and blank group,respectively.After 24,48 and 72h culture,MTT method was used to detect the inhibition rate of HSCs proliferation.（2） The effects on collagen synthesis of HSCs by BMSCs： The experiments were divided into 3 groups：A：HSCs group B：HSCs ＋TGF-β1group C：co-culture ＋TGF-β1group.HSCs co-cultured with BMSCs for 72h in the presence of TGF-β1,BMSCs was removed and the medium on the HSCs was replaced with fresh medium.24 hours later,the medium was collected and Type 1 collagen（COL1） was measured by ELISA.The gene expressions of α-SMA and COL1 in HSCs were detected by RT-PCR.Results Compared with control group,the inhibition rates of HSCs proliferation in co-culture group were significantly increased at different time points of 24,48 and 72h（P0.01）.Compared with group B,Levels of COL1 secretion by HSCs in group C were significantly reduced and the mRNA expressions of α-SMA and COL1 in HSCs were decreased significantly（P0.01）.Conclusion BMSCs could significantly inhibit HSCs proliferation and reduce TGF-β1-induced α-SMA expression and COL1 synthesis in vitro.