体内外定向诱导人脐带间充质干细胞分化为男性生殖细胞的实验研究

【作者】 马廉； 林丽敏； 唐秋灵； 吴小英； 林广裕； 谢庆东； 黄天华；

【机构】 汕头大学医学院第二附属医院儿科； 汕头大学医学院生殖医学研究中心；

【摘要】 目的探讨人脐带间充质干细胞(HUMSC)向男性生殖细胞分化可能性,为细胞移植治疗男性不育提供细胞来源。方法 HUMSC 纯化培养后,体外采取人绝经期促性腺激素进行诱导培养,用光学显微镜、电子显微镜和扫描电镜观察诱导后分化细胞形态变化, 用免疫组织化学、Western-blot、PT-PCR 检测诱导后的细胞男性生殖细胞特异性标记物的表达。体内经显微注射方法移植到不育小鼠睾丸曲细精管,对体内移植后的细胞行免疫组化、免疫荧光观察其存活、迁移及男性生殖细胞的特异性标记物表达等情况。结果 HUMSC 经人绝经期促性腺激素诱导后可向精原细胞分化,分化的细胞免疫组化证实表达精原细胞的标记α6、c-Kit 和 VASA:Western-blot、PT-PCR 检测证实表达α 6,而诱导前的细胞均不表达上述的标记;HUMSC 体内移植后免疫组化显示其在睾丸微环境中可以存活至少120天并发生从管腔→基底膜→管腔迁移;免疫荧光证明 HUMSCs 移植后在睾丸微环境中可以进一步分化,表达生殖细胞特异性标记物 VASA。结论 HUMSCs 能定向诱导分化为早期男性生殖细胞,表达生殖细胞的相关标记;体内移植 HUMSC 到小鼠曲细输精管后能在异种睾丸中存活、迁移并进一步分化为早期男性生殖细胞,提示 HUMSCs 将可能为治疗男性不育提供一种新的细胞来源。更多还原

【Abstract】 Objective: To investigate the possibility of human umbilical cord mesenchymal stem cells(HUMSCs) differentiated into human male germ cells, and find a new source of cells for the transplantation to the male infertility. Methods: HUMSCs were induced by human menopausal gonadotropin(HMG) in vitro. The morphologic changes of the differentiated cells were detected by phase contrast microscopy, electron microscopy and transmission electron microscope; the male germ cell markers were detected by immunohistochemistry、Western-blot and PT—-PCR. HUMSCs were transplanted into the seminiferous tubules of the infertility mice testis by micminjection.The survival、migration and germ cell markers of the HUMSCs in the infertility mice testis were detected by immunohistochemistry、immunofluorescence. Results: HUMSCs can differentiate into spermatogonium-like cells induced by HMG in vitro. The expression of the germ cell markers:α 6、c-kit and VASA were detected by immunohistochcmistry;while α 6 was also detected by Western-blot and PT— PCR .Immunohistochemistry revealed that HUMSCs can survive in the mice testis at least 120 days, and they can migrate from the lumens to the basement membrane then to lumens again. Immunofluorescence showed that HUMSCs can go further differentiation in the mice favorable testicular environment, and express the germ cell marker VASA. Conclusions: HUMSCs can differentiate into early male germ cells after induction by HMG intro. HUMSCs derived germ cells expressed the known molecular markers of germ cells. When HUMSCs were transplanted into the infertility mice testis, they can survive、migrate and differentiated into early male germ cells. This clinically relevant finding raises the possibility for treatment of male infertility, and this reveals novel aspects of germ cell development and opens the possibilities for use of these cells in reproductive medicine.更多还原