内皮前体细胞促骨髓间充质干细胞向肝样细胞转化中的VEGF-Notch信号通路

Vascular endothelial growth factor-Notch signaling pathways in endothelial precursor cells in promoting the transformation of bone marrow mesenchymal stem cells into hepatocyte-like cells

背景:在前期研究中发现骨髓血管内皮前体细胞在体外培养过程中通过旁分泌的方式产生了能够促进骨髓间充质干细胞分化的因子,血管内皮生长因子是其中之一。目的:探讨骨髓血管内皮前体细胞促进骨髓间充质干细胞向肝样细胞转化中是否有VEGF-Notch信号通路参与。方法:①分离培养SD大鼠骨髓间充质干细胞和骨髓血管内皮前体细胞,将第3代骨髓间充质干细胞分为3组:实验组使用培养骨髓血管内皮前体细胞的不含血清及因子的上清液,按照体积1∶1的比例与肝细胞转化培养基混合;模型对照组使用无血清的α-MEM培养基,按照体积1∶1比例与肝细胞转化培养基混合;空白对照组使用正常α-MEM培养基。在培养3,5,7,9 d,应用免疫荧光检测白蛋白、甲胎蛋白的表达。②取第3代骨髓间充质干细胞向肝样细胞转化分组培养,在肝细胞转化培养基中按体积1∶1比例分别加入培养骨髓血管内皮前体细胞的无血清及因子的上清液、血管内皮生长因子、培养血管内皮生长因子干扰质粒转染骨髓血管内皮前体细胞的无血清及因子的上清液;对照组使用肝细胞转化培养基培养。培养48 h后,使用实时定量RT-PCR和Western blot方法检测骨髓间充质干细胞中Notch下游基因Dll4、Hes1及Hey1的表达变化。结果与结论:①空白对照组骨髓间充质干细胞中未见白蛋白、甲胎蛋白的表达,实验组和模型对照组骨髓间充质干细胞中甲胎蛋白、白蛋白表达水平均增高(P<0.05),其中实验组甲胎蛋白、白蛋白表达水平高于模型对照组(P<0.05);②肝细胞转化培养基中加入培养过骨髓血管内皮前体细胞的无血清及因子的上清液或血管内皮生长因子诱导骨髓间充质干细胞,Hey1、Dll4蛋白和mRNA表达均增高(P<0.05),Hes1蛋白和mRNA表达均降低(P<0.05);而在肝细胞转化培养基中加入血管内皮生长因子干扰质粒转染骨髓血管内皮前体细胞的无血清及因子的上清液诱导骨髓间充质干细胞,Dll4、Hey1蛋白和mRNA的表达均降低,Hes1蛋白和mRNA表达均增高(P<0.05);③结果表明,骨髓血管内皮前体细胞在骨髓间充质干细胞向肝样细胞转化中可能起一定促进作用,在此过程中可能有VEGF-Notch信号通路参与。

BACKGROUND:Previous studies have shown that endothelial precursor cells produced factors that could promote bone marrow mesenchymal stem cells differentiation by paracrine in vitro,and vascular endothelial growth fa ctor was one of them.OBJECTIVE:To investigate whether vascular endothelial growth factor-N otch signaling pathway participates in the way that endothelial precursor cells promote the transformation of bone marrow mesenchymal stem cells to hepatocyte-like cells.METHODS:(1) Bone marrow mesenchymal stem cells and endothelial precursor cells of Sprague-Dawley rats were isolated and cultured.The bone marrow mesenchymal stem cells at passage 3 were divided into three groups.In the experimental group,the serum-free and fa ctor endothelial precursor cellsconditioned medium was mixed with hepatocyte transfo rmation medium in a volume ratio of 1:1.In the model control group,serum-free alpha-minimal essential medium was mixed with the hepatocyte transformation medium in a volume ratio of 1:1.In the blank control group,normal alpha-minimal essential medium was used.The expression levels of alpha-fetoptotein and albumin were detected by immunofluorescence at 3,5,7,and 9 days of culture.(2) The third generation of bone marrow mesenchymal stem cells was taken to transform into hepatocyte-like cells and grouped.The serum-free and fa ctor endothelial precursor cells-conditioned medium,vascular endothelial growth factor,and serum-free and factor vascular endothelial growth fa ctor-siRNA-endothelial precursor cells-conditioned medium were added in hepatocyte transformation medium in a volume ratio of 1:1.In the control group,the samples were cultured with the hepatocyte transformation medium.At 48 hours after culture,the expression changes of DⅡ4,Hes1,and Hey1(downstream gene of Notch)in bone marrow mesenchymal stem cells were detected by RT-PCR and western-blot assay.RESULTS AND CONCLUSION:(1) Alpha-fetoprotein and albumin were not found in the blank control group.Alpha-fetoprotein and albumin levels were increased in the model control group and the experimental group(P<0.05),and alpha-fetoprotein and albumin levels were significantly increased in the experimental group compared with the model control group(P<0.05).(2) After adding serum-free and fa ctor endothelial precursor cells-conditioned mediumor vascular endothelial growth factor,the expression of DⅡ4 and Hey1 protein and mRNA increased(P <0.05),but the expression of Hes1 protein and mRNA decreased(P <0.05).After adding serum-free and fa ctor vascular endothelial growth facto r-siRNA-endothelial precursor cells-conditioned medium,the expression of DⅡ4 and Heyl protein and mRNA decreased,and the expression of Hes1 protein and mRNA increased(P <0.05).(3) The results show that the bone marrow endothelial precursor cells may play a certain role in the transformation of bone marrow mesenchymal stem cells into hepatocyte-like cells,and the vascular endothelial growth factor-N otch signaling pathway may be involved in this process.