人胎盘胎儿来源间充质干细胞通过旁分泌肝细胞生长因子减轻脂多糖诱导的人肺微血管内皮细胞损伤

Hepatocyte growth factor paracrined from human placental mesenchymal stem cells of fetal origin alleviates the injury of human pulmonary microvascular endothelial cells induced by lipopolysaccharide

目的探讨无血清培养人胎儿来源胎盘间充质干细胞(hfPMSC)旁分泌肝细胞生长因子(HGF)对脂多糖(LPS)诱导内皮细胞损伤的保护作用。方法用无血清培养基培养hfPMSC,流式细胞术检测CD73、 CD90、 CD105、 CD14、 CD34、 CD45、 HLA-DR的表达以确定其干细胞属性;利用Transwell^(TM)共培养体系[人肺微血管内皮细胞(HPMEC)接种于Transwell^(TM)上室, hfPMSC接种于下室],检测hfPMSC旁分泌HGF对LPS处理的HPMEC通透性的影响。实验分为LPS处理组、 hfPMSC共培养组、 HGF中和抗体组、正常HPMEC对照组。在Transwell^(TM)培养上室加入100μg异硫氰酸荧光素标记的葡聚糖(FITC-dextran),荧光酶标仪检测染料渗透情况; Western blot法检测渗透相关蛋白血管内皮钙黏素(VE-cadherin)和陷窝蛋白1(caveolin-1)及凋亡相关裂解型蛋白胱天蛋白酶3(c-caspase-3)和裂解型多腺苷二磷酸核糖聚合酶1(c-PARP1)的蛋白水平。结果所培养细胞具备间充质干细胞形态,为CD73、 CD90、 CD105阳性及CD14、 CD34、 CD45、 HLA-DR阴性细胞;与LPS处理组相比, hfPMSC共培养可显著抑制LPS环境中HPMEC的通透性,显著上调VE-cadherin的表达水平,降低caveolin-1、 c-caspase-3和c-PARP1的蛋白水平。相反,中和共培养体系中的HGF,可逆转hfPMSC对HPMEC的上述作用。结论 hfPMSC通过分泌HGF抑制LPS诱导的HPMEC通透性增加。

Objective To investigate the protective function of paracrine hepatocyte growth factor( HGF) derived from human placental mesenchymal stem cells of fetal origin( hf PMSCs) cultured in serum-free medium against endothelial cell injury induced by lipopolysaccharide( LPS). Methods The hf PMSCs were cultured in serum-free medium and surface antigen CD73,CD90,CD105,CD14,CD34,CD45 and HLA-DR were analyzed by flow cytometry. Using TranswellTM co-culture system [human pulmonary microvascular endothelial cell( HPMECs) were added into the upper chambers of TranswellTMinserts,and hf PMSCs were added into the lower chambers of TranswellTMinserts],the influence of hf PMSCs paracrine HGF on the permeability of HPMECs in LPS condition was identified. Then four different co-culture conditions were used as follows: 100 ng/m L LPS treatment group;hf PMSCs co-culture group;HGF neutralization group;HPMECs normal control. After 100 μg FITC-dextran was added into the upper chambers of TranswellTMinserts,the effect of stain permeability was detected by fluorescence microplate reader. The expression of VE-cadherin,caveolin-1 and cleaved caspase-3,cleaved PARP-1 in HPMECs were measured by Western blot analysis. Results The hf PMSCs showed the classic morphology of mesenchymal stem cells and expressed the surface markers CD73,CD90 and CD105,but did not express CD14,CD34,CD45 and HLA-DR. Compared with LPS treatment group,the co-culture with hf PMSCs dramatically inhibited the permeability of HPMECs,significantly up-regulated the expression of VE-cadherin,and reduced the expression of caveolin-1,cleaved caspase-3,cleaved PARP1. In contrast, neutralizing HGF with anti-HGF antibody reversed the above effects of hf PMSCs-HPMECs co-culture. Conclusion Owing to paracrine HGF,hf PMSCs possess the capability to availably inhibit the permeability of HPMECs induced by LPS.