组织工程化胆管中平滑肌及内皮细胞的来源及培养研究

The study of the source and the cultivation of the smooth muscle cells and the endothelial cells for the tissue engineering bile duct

目的解决组织工程化胆管的支架材料及细胞来源⑴体外培养人胚动脉平滑肌细胞(VSMCs),并观察人胚VSMCs在乳酸与羟基乙酸共聚物(PLGA)上的三维培养情况。⑵探讨体外诱导大鼠骨髓间充质干细胞(rBMSCs)定向分化为肝干细胞的可行性。方法⑴成功培养人胚VSMCs后,将其与PLGA(膜材和三维材料)进行三维培养,通过倒置显微镜、扫描电镜和MTT法观察细胞的生长情况。⑵采用全骨髓培养法体外培养rBMSCs(设实验组及对照组)。应用肝细胞生长因子(HGF)诱导分化,采用倒置相差显微镜观察细胞形态变化,ELISA等方法检测甲胎蛋白,白(清)蛋白及碱性磷酸酶。结果⑴体外培养的人胚VSMCs生长旺盛,与PLGA具有良好的相容性,在PLGA上生长良好。⑵rBMSCs经体外培养诱导后呈多角形、卵圆形改变,白(清)蛋白,AFP表达阳性,碱性磷酸酶出现明显改变。结论⑴体外培养的人胚VSMCs成功培养纯化并与PLGA有良好的相容性,⑵在HGF诱导下,rBMSCs体外具有向肝干细胞分化的能力,为进一步分化为胆管内皮细胞提供实验依据。

Objective In order to solve the scaffold material and the cell source of the tissue engineering bile duct. This experiment was divided into two parts： （1）To cultivate the vascular smooth muscle cell（VSMCs） in vitro and observe the cultivate status of the VSMCs in 3D environment of polylatic acid- polynlycolic acid （PLGA）. （2）To explore the feasibility of differentiation of rat bone marrow stromal cells towards liver stem cells in vitro. Method （1）To cultivate the VSMCs with PLGA（the film material with 3D material） after they were successfully cultivated, and to observe the cell growth status by inverted microscope, scanning electron microscope and MTT. （2）To culture rBMSCs in vitro by culturing the full bone marrow stem cells in a system containing hepatocte growth factor（HGF）. The morphology of the cells was observed by inverted microscope, and the expressions of albumin, AFP were detected with ELISA and immunohistochemistry technique. Result （1）The VSMCs growth is prosperous in vitro with good compatibility with PLGA. （2）Mter being cultured with HGF, the rBMSCs showed multilateral and ovary tansformation. The differentiated cells expressed albumin and AFP. Conclusion （1）The VSMCs were successfully cultivated and purified in vitro and have good compatibility with PLGA. （2） The rBMSCs have the ability to-differentiate into liver stem cells in vitro. The current studies provide the experimental basis for rBMSCs to differentiate into endothelial cells of the bile duct.