力学刺激参与WJ-MSC在全肝脱细胞支架内肝样分化的基础研究

Liver transplantation is the only effective treatment for end stage liver disease（ESLD）. However, donor shortage has limited its application all around the world. Tissue engineered liver and cell therapy are increasingly focused as a bridge or replacement therapeutic methods for ESLD. Seeding cell is one of the three key factors in liver tissue-engineering. Wharton’s jelly mesenchymal stem cell (WJ-MSC) is one of the idealest potential choices due to its simple procurement, low immunogenicity and pluripotential differentiation. At present, the mechanism of WJ-MSC’s differentiation is not yet clear. The living conditions they need for differentiation and their application potential still need to be studied. Researchers have shown that mechanical stimulation promoted stem cell differentiation. Our previous work showed also that mechanical stimulation promoted WJ-MSC’s hepatocyte-like differentiation, up-regulated integrin related signaling pathway, and that its downstream nucleus protein, lamin A, was activated as well. We thus suppose that mechanical stimulation regulate WJ-MSC’s differentiation on hepatocyte-like cells by integrin-cytoskeleton-lamin mechanical force transfer axis. To verify this hypothesis, we will recellularize the decellularized rat liver scaffolds with WJ-MSC, which will be cultured and differentiated into hepatocyte-like cells in a self-designed bioreactor with different hydraulic pressure applied. Firstly, the influence of mechanical stimulation on WJ-MSC’s hepatocyte-like cell differentiation will be observed and the best mechanical stimulation for WJ-MSC’s hepatocyte-like differentiation will be found. Secondly, the tissue engineered liver, that means, WJ-MSC seeded in decellularized whole liver scaffold and cultured in vitro, will be transplanted in rats to verify their application potential on clinic. Thirdly, we will also make systematic researches on proteins related to mechanical stimulation and integrin-cytoskeleton-lamin force transfer axis in WJ-MSC in order to find the mechanism of WJ-MSC’s differentiation. This study will provide a theoretical and experimental foundation for liver tissue engineering. And a new point of observation will be applied in exploring the mechanism of WJ-MSC’s hepatocyte-like differentiation.

肝移植是终末期肝病的唯一治疗手段，供肝缺乏限制其推广，构建组织工程肝是潜在解决途径。脐带间充质干细胞（WJ-MSC）是种子细胞的理想选择之一，但其肝样分化条件、分化机制和应用前景尚需研究。前期研究发现，力学刺激促进WJ-MSC肝样分化，同时上调整合素信号通路及其下游核蛋白Lamin的表达，推测力学刺激可能通过整合素-细胞骨架-Lamin力学传导轴调控WJ-MSC肝样分化。因此拟在全肝脱细胞支架内种植WJ-MSC，用体外持续灌流培养生物反应器，维持肝内不同压力，诱导WJ-MSC肝样分化，并将体外培养的生物人工肝移植给大鼠。研究1)力学刺激诱导WJ-MSC肝样分化的最适条件；2）用大鼠肝移植在体内验证构建的组织工程肝；3)确立整合素-细胞骨架-Lamin力学传导通路在力学刺激诱导WJ-MSC分化中的可能作用机制。籍此,为探索干细胞分化的分子机制提供新思路，为构建组织工程肝提供理论和实验基础。

肝移植是终末期肝病的唯一治疗手段，供肝缺乏限制其推广，构建组织工程肝是潜在解决途径。脐带间充质干细胞（WJ-MSC）是构建组织工程肝潜在种子细胞。本研究在全肝脱细胞支架内移植WJ-MSC，采用不同的力学条件下（二维、静态三维、生理灌注压），利用条件培养液诱导WJ-MSC肝样分化，筛选最适力学分化条件。进一步研究WJ-MSC分化机制，抑制整合素亚基的表达，研究细胞骨架信号通路对WJ-MSC分化的影响，发现抑制整合素亚基的表达可促进WJ-MSC在二维和三维支架中的肝向分化。另外，采用脂质体转染方法，在WJ-MSC内过表达miRNA122 可促进WJ-MSC肝向分化，过表达miRNA185可抑制WJ-MSC肝向分化。因此，我们推测，整合素-细胞骨架-Lamin力学传导通路在力学刺激诱导WJ-MSC分化中可能起到作用，miRNA122具备提高WJ-MSCs肝向分化的调控的潜在应用价值。最后，将组织工程肝植入大鼠皮下，检测免疫排斥相关指标，对其生物相容性进行验证，未发现免疫排斥迹象。目前正在用大鼠肝移植在体内验证构建的组织工程肝的功能及生物相容性。该项目研究了影响干细胞肝向分化可能的信号通路，提出抑制整合素信号通路和Wnt/-catenin信号通路可以促进肝细胞肝向分化， miRNA122可以促进干细胞肝向分化，miRNA185会抑制干细胞肝向分化，为揭示细胞分化和发育提供部分理论基础，为研究干细胞分化规律、干扰干细胞分化效率提供实验基础，为肝组织工程构建提供种子细胞层面理论和实验基础。