猪骨髓间充质干细胞对共培养肝细胞的作用及其机制

Study on the effects and mechanisms of bone marrow mesenchymal stem cells on porcine primary hepatocyte culture in vitro

目的探索细胞外基质（ECM）在猪肝细胞与骨髓间充质干细胞（MSCs）体外共培养中的表达与分布规律。方法自中华实验猪髂前上棘抽取骨髓，采用密度梯度离心法分离单个核细胞，贴壁传代培养至第3代；原位两步胶原酶法分离猪肝细胞后与MSCs随机混合培养，观察肝细胞形态和功能的变化情况。用免疫细胞化学染色法观察ECM的表达和分布情况。RNA干扰MSCs后继续观察共培养肝细胞的功能变化情况。多组间比较采用单因素方差分析，两两比较采用LSD法。结果第3代MSCs纯度〉90％；肝细胞活率〉95％，纯度〉99％。共培养组肝细胞迅速黏附于MSCs表面，呈肝细胞岛样分布。共培养组肝细胞白蛋白分泌水平和尿素合成能力自共培养第1天起均明显高于单纯肝细胞组（P值均〈0．01），并在第2天达到高峰，分别为（457．71±22．62）ng和（69．05±2．12）μg。免疫细胞化学检测结果显示共培养组MSCs表达多种ECM；RNA干扰实验进一步证实ECM的存在与共培养中肝细胞的白蛋白分泌和尿素合成功能有关。结论骨髓MSCs通过分泌多种ECM改善共培养肝细胞的形态与功能。

Objective To investigate the expression and distribution of extracelluar matrix （ECM） in the co-culture of porcine primary hepatocytes and bone marrow mesenchymal stem cells （MSCs） in vitro. Methods Mononuclear cells were isolated from bone marrow of swines by density gradient centrifugation, MSCs of passage 3 and primary hepatocytes harvested by a two-step in situ collagenase perfusion technique were co-cultured, and the morphological and functional changes of heterotypic interactions were characterized, Immunocytochemical analysis was performed to monitor the expression and distribution of ECM. Results The purity of the third passage MSCs and primary hepatocytes was more than 90% and 99%, respectively. More than 95% of the hepatocytes were viabile. Compared to hepatocytes culture, co-culture with MSCs significantly enhanced hepatic function： including albumin secretion and urea synthesis （P 〈 0.01）. The best hepatic function level was achieved on day 2 and gradually decreased in the following co-culture days. Immunocytochemical staining suggested that higher amounts of naturally occurring ECM proteins including fibronectin, laminin, and several kinds of collagens were produced in co-culture group compared to hepatocyte homo-culture （P 〈 0.01）. RNAi experiments verified that there was a correlation between ECM and hepatic functions. Conclusion ECM may indeed play a key role in the up-regulation of hepatocyte functions in MSC/hepacytes co-culture.