体外诱导人羊膜间充质细胞向子宫内膜上皮细胞分化

Inducing differentiation of human amniotic mesenchymal cells into endometrial epithelial cells in vitro

目的体外分离培养人羊膜间充质细胞（hAMCs），初步探讨其向子宫内膜上皮细胞分化的可行性。方法原代分离培养hAMCs和人子宫内膜细胞并鉴定，采用添加50%的子宫内膜细胞培养液，并补充生长因子（TGF-β10 ng/mL，EGF 10 ng/mL，PDGF-BB 10 ng/mL）和雌激素（17-β雌二醇1x10-7 mol/L）作为诱导剂培养第3代hAMCs，倒置显微镜下观察细胞形态的改变，应用细胞免疫组化检测分化的hAMCs是否表达上皮细胞特异标记角蛋白，并抽提诱导第8天细胞的mRNA，采用荧光定量PCR方法检测分化细胞的CK19基因水平。结果分离提纯得到的hAMCs可体外长期传代培养，表达间质细胞特异性波形蛋白及胚胎干细胞的表面标记蛋白Oct-3/4，流式检测CD29表达阳性；诱导后的部分细胞形态由成纤维样向上皮样细胞变化，细胞免疫组化染色表达角蛋白，荧光定量PCR检测实验组角蛋白CK19 mRNA表达量明显增加（P〈0.05）。结论建立了人羊膜间充质细胞的分离培养方法， hAMCs在ESCs分泌和外源性因素诱导下可以向子宫内膜上皮细胞方向分化。

Objective To isolate and culture human amniotic mesenchymal cells （hAMCs） in vitro,and to investigate their differentiation into endometrial epithelial cells. Methods The hAMCs and human endometrial stromal cells （ESCs） were isolated for primary culture,and identified. The third generation of hAMCs was cultured with medium,which included 50%of ESCs,and the growth factors（TGF-β 10 ng/mL,EGF 10 ng/mL,PDGF-BB 10 ng/mL） and estrogen （17-β estradiol 1 x10-7mol/L） as the inducer. The morphological changes of the cells were determined under inverted microscope. Immunohistochemistry was used to determine whether the differentiated hAMCs expressed the specific marker of epithelial cells,keratin. The mRNA of the induced cells at 8 d were extracted. The level of CK19 gene in the differentiated cells was determined by fluorescent quantitative PCR. Results The isolated and purified hAMCs could be long passaged in vitro,expressed specific vimentin of interstitial cells and surface marker protein Oct-3/4 of embryonic stem cells. The CD29 expressed negative by flow cytometry. The morphology of some cells was changed from fibroblast-like cells to fibroblastoid to epithelioid cells. The expression of keratin was determined by immunohistochemical staining. Fluorescent quantitative PCR verified that the expression of keratin CK19 mRNA in the experimental group was significantly increased（P〈0.05）. Conclusion The isolation and culture of hAMCs can be established,and hAMCs can be differentiated into endometrial epithelial cells inducing by ESCs secretion and exogenous factors.