Objective: To explore the effect of substrate mechanical microenvironment and cell-cell interaction on differentiation of bone marrow mesenchymal stem cells (BMSCs), intrahepatic cellular function and phenotype. Methods: Bone marrow mesenchymal stem cells (BMSCs)-hepatocytes (HCs) and BMSCs-hepatic stellate cells (HSCs) were co-cultured on polyvinyl alcohol (PVA) hydrogel substrates at different stiffness (4.50 ± 0.47 kPa, 19.00 ± 3.51 kPa and 37.00 ± 2.09 kPa) by non-contact co-culture method. Furthermore, the effect of substrate mechanical microenvironment on BMSCs, HCs and HSCs and the activation and proliferation of HCs under different co-cultured condition was studied. A Student's t-test was used to compare the two groups. Results: The expression ofα-smooth muscle actin (α-SMA) and collagenα1- I (Col1A1) in BMSCs and HSCs cultured on its own increased with increase of substrate stiffness. After 72 h, the expression of albumin (ALB) of HCs on three stiff substrates was significantly higher than that of 24 and 48 h. Moreover, the expression of ALB of HCs increased with the increase of substrate stiffness. During the co-culture of BMSCs and HSCs, BMSCs of all three stiffness substrates promoted the expression ofα-SMA, Col1A1 in HSCs, but reduced the expression of PPARγin HSCs cells, thererby promoted the activation of HSCs, with apparent stiffness at 37 kPa. HSCs promoted the expression of ABL in BMSCs at three stiff substrates, but inhibited the expression of alpha-SMA and Col1A1 in BMSCs at 37 kPa, suggesting that co-culture had inhibited the differentiation of BMSCs myofibroblasts, and promoted the differentiation of hepatocyte-like cells, especially at high stiff substrates. In the co-culture of BMSCs and hepatic parenchymal cells, BMSCs had promoted the proliferation of hepatic parenchymal cells at 4.5 kPa. Further, hepatic parenchymal cells had inhibited the expression ofα-SMA in BMSCs, and promoted the expression of Alb, with inhibition of BMSCs differentiation towards myofibroblasts. Conclusion: The differentiation of BMSCs affects the substrate mechanical microenvironment, co-culture of HCs and HSCs. Simultaneously, affecting the function of hepatocytes in relation to the mechanical state of the substrates.
目的： 探索基底力学微环境和细胞间相互作用对骨髓间充质干细胞（BMSCs）分化，以及肝内细胞功能和表型的影响。 方法： 制备不同硬度的聚乙烯醇水凝胶基底[（4.50±0.47）kPa、（19.00±3.51）kPa、（37.00±2.09）kPa],采用非接触共培养的方法，将BMSCs-肝实质细胞（HCs）、BMSCs-肝星状细胞（HSCs）共培养于不同硬度的聚乙烯醇水凝胶基底上。研究基底力学微环境对BMSCs、HCs和HSCs的功能影响；研究不同基底力学微环境中共培养下BMSCs对HSCs的活化和HCs增殖的影响；以及HSCs和HCs分别对BMSCs分化的影响。两组间比较采用t检验。 结果： 随着基底硬度的增加，单独培养的BMSCs和HSCs的α-平滑肌肌动蛋白、α1-I型胶原的表达增加；在72 h后，3个硬度上HCs的白蛋白表达量均明显高于24 h、48 h，且随硬度增加，HCs的白蛋白表达量增加。在BMSCs与HSCs共培养中，3个硬度上BMSCs都促进HSCs细胞α-平滑肌肌动蛋白、α1-I型胶原的表达，降低过氧化物酶体增殖物激活受体的表达，促进HSCs的活化,且37.00 kPa更加明显。HSCs在3个硬度上均促进BMSCs的白蛋白表达，在37.00 kPa则抑制BMSCs的α-平滑肌肌动蛋白、α1-I型胶原的表达，提示共培养具有抑制BMSCs肌成纤维细胞分化，而促进其肝样细胞分化的可能，高基底硬度下则更为明显。在BMSCs与肝实质细胞共培养中，BMSCs能够促进肝实质细胞增殖，且4.50 kPa更加明显。肝实质细胞则能够抑制BMSCs的α平滑肌肌动蛋白表达，并促进白蛋白的表达，有抑制BMSCs向肌成纤维细胞方向分化的可能。 结论： BMSCs的分化会受到基底力学微环境、HCs及HSCs共培养的影响，同时BMSCs也会对肝内细胞功能造成影响，且这些影响和基底力学状态有关。.
Keywords: Cell communication; Liver cirrhosis; Mesenchyma stem cells.