雌二醇诱导体外立体培养仿生羊膜及分化为子宫内膜的可行性

Feasibility of estradiol-induced three-dimensional culture of bionic amniotic membrane in vitro and differentiation into endometrium

目的探讨雌二醇诱导体外立体培养仿生羊膜及分化为子宫内膜的可行性。方法体外立体培养人羊膜间充质干细胞制成仿生羊膜,将细胞按加入雌二醇浓度进行分组,未加入雌二醇(以等剂量的磷酸盐缓冲液代替)为对照组,实验1组与实验2分别加入1×10^-8 mol/L和1×10^-7 mol/L的雌二醇。定期观察培养瓶中人羊膜间充质干细胞外观变化,细胞经处理24 h、36 h和48 h后,采用CCK-8法检测细胞增殖情况,采用荧光定量PCR检测技术检测CK19 mRNA水平表达情况,采用酶联免疫法检测细胞中上皮细胞间粘附分子(EPCAM)、基质金属蛋白酶-9(MMP-9)表达水平。结果分离后的人羊膜间充质干细胞传代后生长较快,人羊膜间充质干细胞表达干细胞表面分子CD29,鉴定为仿生羊膜。细胞经雌激素处理24 h、36 h和48 h后,实验1组与实验2组的人羊膜间充质干细胞增殖指数、CK19 mRNA相对表达水平、EPCAM与MMP-9表达水平高于对照组,实验2组也高于实验1组,差异有统计学意义(P<0.05),且作用呈时间依赖性。结论雌二醇诱导下可在体外立体培养仿生羊膜,促进人羊膜间充质干细胞的增殖,促进EPCAM与MMP-9的分泌,从而有利于诱导分化为子宫内膜,且作用呈时间剂量依赖性。

Objective To explore the feasibility of estradiol-induced bionic amniotic membrane culture in vitro and differentiation into endometrium.Methods Human amniotic mesenchymal stem cells were cultured in vitro to prepare biomimetic amnion.According to estrogen concentrations,the cells were divided into control group(estradiol were not added,but with equal dose of phosphate buffer,experiment group 1 and experiment group 2(added 1×10^-8 mol/L and 1×10^-7 mol/L of estradiol,respectively).The appearance of human amniotic mesenchymal stem cells in culture flasks were observed regularly.Cell proliferation were detected by CCK-8 method,the expression of CK19 mRNA were detected by real-time PCR,and the expression levels of molecules(Epithelial cell adhesion molecule,EPCAM)and matrix metalloprotein(MMP)-9 were detected by enzyme-linked immunosorbent assay.Results After isolated,human amniotic mesenchymal stem cells grew faster after passage,the human amniotic mesenchymal stem cells expressed stem cell surface molecule CD29 and were identified as bionic amniotic membrane.After treated with estrogen for 24 h,36 h and 48 h,the proliferation index,CK19 mRNA expression level and EPCAM and MMP-9 expression levels of human amniotic mesenchymal stem cells in experimental group 1 and experimental group 2 were higher than those in control group,and those in the experimental group 2 were also higher than those in the experimental group 1 with significant difference(P<0.05).Conclusion Estradiol can induce the biomimetic amnion in vitro,promote the proliferation of human amniotic mesenchymal stem cells,promote the secretion of EPCAM and MMP-9,and facilitate the differentiation into the endometrium.