大鼠骨髓间充质干细胞向子宫内膜细胞分化的体外实验

发布时间：2018-07-04 19:23

  本文选题：骨髓间充质干细胞 + 子宫内膜间质细胞　； 参考：《中南大学》2012年博士论文

【摘要】：目的：体外诱导骨髓间充质干细胞(BMSCs)向子宫内膜细胞分化,为干细胞移植治疗薄型子宫内膜提供实验基础。 方法：分离、培养、鉴定大鼠BMSCs和人子宫内膜间质细胞(hECs)。将BMSCs和hECs在Transwell培养板中共培养,添加雌激素(E2,1×10-7mol/L)和生长因子(GF,10ng/ml TGF-β、10ng/ml EGF、 lOng/ml PDGF-BB),在培养的第7天免疫荧光法检测BMSCs的波形蛋白、角蛋白表达情况。在普通培养板中培养,将BMSCs和hECs按细胞组成分为3组：co-culture组、BMSCs组和hECs组(其中co-culture组为BMSCs和hECs直接共培养,另外两组仅培养一种细胞)。每组中采用3种不同的诱导方式,分别加入:E2+GF(1×10-7mol/L E2,10ng/ml TGF-β、10ng/ml EGF、10ng/ml PDGF-BB); cAMP (1mmol/ml8-Br-cAMP); E2+cAMP (1×10-7mol/L E2、1mmol/ml8-Br-cAMP)。在培养的第14天和第20天通过细胞免疫荧光法检测各组细胞波形蛋白和角蛋白表达情况,第3天和第19天酶联免疫吸附法(ELISA)检测蜕膜化标记泌乳素(PRL)和胰岛素样结合蛋白1(IGFBP1)浓度。 结果：通过细胞培养获得了稳定增殖的大鼠BMSCs和人子宫内膜间质细胞。在Transwell培养板中共培养的BMSCs第7天波形蛋白表达阳性,角蛋白表达阴性。普通培养板中各组细胞在培养的第14天和第20天细胞波形蛋白表达均为阳性,角蛋白表达均为阴性。第3天和第19天两种蛋白在组间及组内的表达均没有显著统计学差异(P0.05)；PRL和IGFBP1的表达第19天明显高于第3天,结果有统计学差异(P0.001)。 结论：①骨髓间充质干细胞可以向子宫内膜间质细胞分化；②雌激素、生长因子、cAMP类似物在骨髓间充质干细胞向子宫内膜间质细胞分化中起到诱导作用,且诱导能力相当；③本实验条件下骨髓间充质干细胞未能向子宫内膜上皮细胞分化。
[Abstract]:Aim: to induce the differentiation of bone marrow mesenchymal stem cells (BMSCs) into endometrial cells in vitro. Methods: rat BMSCs and human endometrial stromal cells (hECs) were isolated, cultured and identified. BMSCs and hECs were co-cultured on Transwell culture plate. The expression of vimentin and keratin of BMSCs were detected by immunofluorescence on the 7th day after adding estrogen (E2N 1 脳 10-7mol / L) and growth factor (GFF10ngr / ml TGF- 尾 -10ng / ml EGF-, lOng / ml PDGF-BB). BMSCs and hECs were divided into three groups according to cell composition: BMSCs and hECs (co-culture group was directly co-cultured with hECs, and only one cell was cultured in the other two groups). In each group, three different induction methods were used, respectively, with 1 脳 10 ~ (-7) mol / L E _ (2) E _ (2) O _ (10) ng / ml TGF- 尾 (10 ~ (-10) ng / ml EGFN _ (10) ng / ml PDGF-BB); camp (1 mmol / ml 8-Br-cAMP); E _ (2) camp (1 脳 10 ~ (-7) mol / L E _ 2 路mol / ml 路ml ~ (-1) Br-cAMP). The expression of vimentin and keratin was detected by cell immunofluorescence on the 14th and 20th day of culture. The concentrations of decidualized prolactin (PRL) and insulin-like binding protein 1 (IGFBP1) were detected by enzyme-linked immunosorbent assay (Elisa) on day 3 and day 19. Results: rat BMSCs and human endometrial stromal cells were obtained by cell culture. BMSCs co-cultured on Transwell culture plate showed positive expression of vimentin and negative expression of keratin on day 7. The expression of vimentin was positive on the 14th and 20th day, and keratin expression was negative in the normal culture plate. There was no significant difference in the expression of PRL and IGFBP1 between groups on day 3 and day 19 (P0.05). The expression of PRL and IGFBP1 on day 19 was significantly higher than that on day 3 (P0.001). Conclusion Bone marrow mesenchymal stem cells (BMSCs) can differentiate into endometrial stromal cells. Growth factor camp analogues play an important role in inducing the differentiation of BMSCs into endometrial stromal cells. 3 in this experiment, bone marrow mesenchymal stem cells could not differentiate into endometrial epithelial cells.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R329

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