Gu Jin-yang, Shi Xiao-lei, Zhang Yue, Ding Yi-tao
Department of Hepatobiliary Surgery, Affiliated Drum Tower Hospital of Medical School, Nanjing University, Nanjing 210008, China.
Zhonghua Gan Zang Bing Za Zhi. 2009 Nov;17(11):867-71.
To investigate the expression and distribution of extracellular matrix (ECM) in the co-culture of porcine primary hepatocytes and bone marrow mesenchymal stem cells (MSCs) in vitro.
Mononuclear cells were isolated from bone marrow of swine 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.
The purity of the third passage MSCs and primary hepatocytes was more than 90% and 99%, respectively. More than 95% of the hepatocytes were viable. 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.
ECM may indeed play a key role in the up-regulation of hepatocyte functions in MSC/hepatocytes co-culture.
研究猪原代肝细胞与骨髓间充质干细胞（MSCs）体外共培养时细胞外基质（ECM）的表达及分布情况。
采用密度梯度离心法从猪骨髓中分离单个核细胞。将第3代MSCs与通过两步原位胶原酶灌注技术收获的原代肝细胞进行共培养，并对异型相互作用的形态和功能变化进行表征。进行免疫细胞化学分析以监测ECM的表达和分布。
第3代MSCs和原代肝细胞的纯度分别超过90%和99%。超过95%的肝细胞存活。与肝细胞培养相比，与MSCs共培养显著增强了肝功能：包括白蛋白分泌和尿素合成（P<0.01）。在第2天达到最佳肝功能水平，在随后的共培养天数中逐渐下降。免疫细胞化学染色表明，与肝细胞同型培养相比，共培养组产生了更多天然存在的ECM蛋白，包括纤连蛋白、层粘连蛋白和几种胶原蛋白（P<0.01）。RNA干扰实验证实ECM与肝功能之间存在相关性。
在MSCs/肝细胞共培养中，ECM确实可能在肝细胞功能上调中起关键作用。
