骨髓间充质干细胞体外向子宫内膜上皮细胞的分化

Differentiation of bone marrow mesenchymal stem cells into endometrial epithelial cells in vitro

背景:以往研究表明,骨髓干细胞是子宫内膜细胞的子宫外来源,参与损伤后子宫内膜的再生功能重建。目的:探讨小鼠骨髓间充质干细胞在体外能否向子宫内膜上皮细胞方向分化。方法:将第2代骨髓间充质干细胞、子宫内膜间质细胞分别进行以下4组不同处理:1第1组为单纯小鼠骨髓间充质干细胞,用含体积分数为2%胎牛血清的DMEM/F12培养液培养;2第2组为单纯小鼠骨髓间充质干细胞,用含体积分数为2%胎牛血清、10-7 mol/Lβ-雌二醇、10μg/L表皮生长因子的DMEM/F12培养液培养;3第3组为用Transwell小室进行骨髓间充质干细胞与子宫内膜间质细胞共培养,用含体积分数为2%胎牛血清的DMEM/F12培养液培养;4第4组为用Transwell小室进行骨髓间充质干细胞与子宫内膜间质细胞共培养,用含体积分数为2%胎牛血清、10-7 mol/Lβ-雌二醇、10μg/L表皮生长因子的DMEM/F12培养液培养。上述各组处理第5天,收集培养板底部骨髓间充质干细胞,RT-PCR检测上皮细胞标记(CK7,CK18,CK19,EMA)的基因表达,免疫荧光化学方法检测角蛋白表达。第3组细胞共培养后的第1,3,5,7天,RT-PCR检测上皮细胞标记(CK7,CK18,CK19,EMA)的基因表达。结果与结论:1CK7 m RNA表达在各组中依次上升,并在第4组中达到最高,各组之间经比较差异有显著性意义。第2,3,4组CK18,CK19 m RNA的表达均较第1组显著升高,且第2,3,4组间比较差异无显著性意义。EMA m RNA在第2,4组的表达比第1,3组升高,但组间比较差异也无显著性意义。2第4组角蛋白呈强阳性表达,第3组呈弱阳性表达,第1,2组呈阴性表达。3随着时间的推移,CK7 m RNA表达量不断上升,且第5,7天的表达量显著高于第1,3天,不同时间经比较差异有显著性意义。CK18,CK19,EMA m RNA在不同时间的表达量差异无显著性意义。4实验结果表明,小鼠骨髓间充质干细胞在一定条件下可向子宫内膜上皮细胞方向分化。外源性因素和内源性间质细胞分泌因子联合作用下,其促进小鼠骨髓间充质干细胞向子宫内膜上皮细胞方向分化的作用最显著。

BACKGROUND： Previous studies have confirmed that as an exogenous origin of endometrial epithelial cells, bone marrow stem cells are involved in the functional reconstruction of the injured endometrium. OBJECTIVE： To discuss whether bone marrow mesenchymal stem cells can differentiate into endometrial epithelial cells in vitro. METHODS： Passage 2 bone marrow mesenchymal stem cells and endometrial stromal cells were collected and subjected to different treatments： bone marrow mesenchymal stem cells were cultured alone in the DMEM/F12 medium containing 2% fetal bovine serum or in the DMEM/F12 medium containing 2% fetal bovine serum, 10-7 mol/L β-estradiol and 10 μg/L epidermal growth factor as groups 1 and 2, respectively; bone marrow mesenchymal stem cells co-cultured with endometrial stromal cells by Transwell chamber were cultured in the DMEM/F12 medium containing 2% fetal bovine serum or in the DMEM/F12 medium containing 2% fetal bovine serum, 10^-7 mol/L β-estradiol and 10 μg/L epidermal growth factor as groups 3 and 4, respectively. After 5 days culture, bone marrow mesenchymal stem cells at the bottom of the culture plate were collected to detect expressions of epithelial cell markers, such as CK7, CK18, CK1 and EMA by RT-PCR technology, and to determine keratin expression using immunofluorescence assay. Besides, expressions of these epithelial cell markers in the group 3 were detected using RT-PCR at 1, 3, 5 and 7 days of culture, respectively. RESULTS AND CONCLUSION： mRNA expression of CK7 showed a significant successive rise in the groups 1, 2, 3, 4 and it was highest in the group 4. mRNA expressions of CK18 and CKl9 in the later three groups had no significant differences, but all significantly higher than those in the group 1. Compared with the groups 1 and 3, mRNA expression of EMA in the groups 2, 4 were significantly higher, but no significant differences existed between groups. Expression of keratin was strongly positive in the group 4, weakly positive in the group 3 and negative in the groups 1, 2, respectively. Furthermore, with the increase of culture time, mRNA expression of CK7 exhibited a constant increase, which was significantly higher at 5 and 7 days than at 1 and 3 days; but there were no significant differences in expressions of CKl9, CKl8 and EMA at different time points. These results show that bone marrow mesenchymal stem cells can differentiate into endometrial epithelial cells in vitro under certain conditions. Moreover, it can remarkably promote the endometrial differentiation under the combined effects of some exogenous and endogenous factors.