人脐带间充质干细胞异种移植不育小鼠睾丸的研究

发布时间：2018-06-06 23:48

本文选题：脐带 + 间充质干细胞　；参考：《汕头大学》2009年硕士论文

【摘要】： 背景与目的:不孕症的发生率日益升高,约占育龄夫妇的15%,其中由男方原因引起者约占一半。男性不育大部分可以通过辅助生殖技术得到纠正,但由于生殖细胞缺乏所致的不育无法通过辅助生殖技术治疗。干细胞具有多向分化的潜能,可以分化为各种组织的细胞包括生殖细胞;已有研究证实胚胎干细胞、成体干细胞在一定的条件下可以分化为雄性生殖细胞,因此干细胞移植为治疗男性不育提供了新的解决途径。人脐带间充质干细胞(HUMSCs)也是一种成体干细胞,理论上也可以向男性生殖细胞分化。本课题主要研究HUMSCs体外在人绝经期促性腺激素及维甲酸诱导培养下向男性生殖细胞方向分化的情况;并将HUMSCs移植到不育小鼠睾丸曲细输精管内,观察其在不育小鼠体内存活及向男性生殖细胞方向分化的能力。方法:采取组织块贴壁培养方法获得HUMSCs,体外采取人绝经期促性腺激素及维甲酸进行诱导培养,诱导后在显微镜下观察细胞形态变化,诱导培养8-14天后行细胞免疫化学、PT-PCR检测诱导后的细胞生殖细胞标记物的表达情况。体内研究方面,脂质体介导增强绿色荧光蛋白质粒pIRES2-EGFP转染HUMSCs,转染后经显微注射方法移植到不育小鼠睾丸曲细输精管,细胞移植40-120天后行免疫组化、免疫荧光观察移植后的HUMSCs在受体小鼠睾丸内存活及向男性生殖细胞方向分化的情况。结果:HUMSCs在人绝经期促性腺激素及维甲酸进行诱导培养8-14天后,在光镜显微镜下观察可见部分细胞形态发生改变,部分长条型的细胞逐渐变圆,细胞器增多,行细胞免疫化学染色显示HUMSCs诱导后能表达生殖细胞的部分标记VASA,Oct4,而未诱导的细胞成阴性表达;RT-PCR显示诱导后的细胞表达α6基因,未诱导的细胞未见α6基因的表达。HUMSCs移植到不育小鼠睾丸曲细输精管40-120天后行免疫组化、免疫荧光观察。免疫组化观察可见细胞移植40-70天能表达生殖细胞的标记:VASA、α6、C-kit和Oct4成阳性表达,而未移植细胞的睾丸组织未见VASA、α6、C-kit和Oct4.表达。免疫荧光显示细胞移植40-60天在受体睾丸内可以观察到绿色荧光信号,少部分表达绿色荧光的细胞同时表达生殖细胞特异性标记VASA、α6、C-kit和Oct4。对照组未移植细胞的小鼠睾丸组织未见阳性表达。结论:HUMSCs在人绝经期促性腺激素及维甲酸进行诱导培养下能表达生殖细胞的部分特征;移植到不育小鼠睾丸曲细输精管后能在异种小鼠睾丸中存活,并表达生殖细胞部分特征。体内外定向诱导HUMSCs可能可以向男性生殖细胞方向分化,HUMSCs将可能为细胞移植治疗男性不育提供一种新的细胞来源,提供一条新的治疗途径。
[Abstract]:Background and objective: the incidence of infertility is increasing, accounting for about 15% of the couples of childbearing age, of which about half of them are caused by male causes. Most male infertility can be corrected by assisted reproductive technology, but infertility can not be treated by assisted reproductive technology because of the lack of germ cells. Stem cells have the potential for multiple differentiation. The cells that can be differentiated into various tissues include germ cells. Studies have shown that embryonic stem cells, adult stem cells can differentiate into male reproductive cells under certain conditions, so stem cell transplantation provides a new solution for the treatment of male infertility. Human umbilical cord mesenchymal stem cells (HUMSCs) are also a kind of adult stem cells. It can also differentiate into male germ cells. This topic mainly studies the differentiation of HUMSCs in human menopause gonadotropin and retinoic acid induced differentiation to male germ cells, and transplants HUMSCs into the testicular fine vas deferens in sterile mice to observe the survival of the male sterile mice and the direction of male germ cells in the male sterile mice. The ability to differentiate.
Methods: HUMSCs was obtained by tissue block wall culture. Human menopause gonadotropin and retinoic acid were used to induce culture in vitro. After induction, the morphological changes of cells were observed under the microscope. Cell immunocytochemistry was performed for 8-14 days after induction of culture. PT-PCR was used to detect the expression of markers of cell germ cells after induction. The liposome mediated green fluorescent protein particles pIRES2-EGFP transfected to HUMSCs, and transplanted into the testicular fine vas deferens in sterile mice after transfection. After 40-120 days of transplantation, immunofluorescence was performed. The immunofluorescence was used to observe the survival of the transplanted HUMSCs in the testis of the recipient mice and the direction of the male germ cells.
Results: after 8-14 days of induction and culture of gonadotropin and retinoic acid in human menopause, the morphologic changes of some cells were observed under light microscope, and some of the cells became round and organelles increased. Cell immunocytochemical staining showed that the partial markers of VASA and Oct4 could be expressed after HUMSCs induction. The uninduced cells expressed negative expression, RT-PCR showed that the induced cells expressed the alpha 6 gene, and the uninduced cells did not have the expression of alpha 6 gene expression.HUMSCs transplanted to the testicular fine vas deferens in sterile mice for 40-120 days after immunofluorescence, immunofluorescence observation. The immunofluorescence observation showed that the cell transplantation could express the marker of germ cells for 40-70 days: VASA, The positive expression of alpha 6, C-kit and Oct4 was positive, while no VASA, alpha 6, C-kit and Oct4. were found in the testis of untransplanted cells. Immunofluorescence showed that green fluorescent signals could be observed in the testicles of the recipient 40-60 days, and a few cells expressing green fluorescence were also expressed in the germ cell specific marker VASA, and the control group of alpha 6, C-kit and Oct4. was not. No positive expression of transplanted cells in mouse testis was observed.
Conclusion: HUMSCs can express some characteristics of reproductive cells in the induced culture of human menopause gonadotropin and retinoic acid, and can survive in the testis of xenogeneic mice after transplantation to the testicular fine vas deferens in sterile mice, and express the characteristics of germ cells. In vivo and in vivo, HUMSCs may be able to differentiate into male germ cells in vivo and in vivo. HUMSCs may provide a new cell source for cell transplantation in the treatment of male infertility, and provide a new way of treatment.
【学位授予单位】：汕头大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

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