骨髓间充质干细胞移植对模型大鼠生精功能障碍的作用

发布时间：2018-04-01 09:11

本文选题：骨髓间充质干细胞　切入点：60Coγ射线　出处：《郑州大学》2014年硕士论文

【摘要】：研究背景及目的：不育是泌尿男科门诊常见疾病，影响约15%的夫妇，并且男性因素作为主要因素的约占30%，而大约40%的夫妇双方都有问题。雄性不育的发病机制尚未完全明确，病因明确者采用对症治疗，而原因不明者，多采用经验治疗，效果多不满意。近些年来，辅助生育技术发展迅速，为广大不育患者提供了福音，但辅助生育技术违反了生物界自然选择的规律，而且辅助生育技术都是有创的，经济负担较大，并具有潜在的健康风险。而且对于那些一方不能产生有功能配子的夫妇，除了求助于异体供体没有其他的治疗方案。近些年来随着干细胞在雄性不育中的研究深入，发现干细胞可以分化为生殖细胞，为雄性不育的治疗和发病机制研究提供了新的途径。本文就骨髓间充质干细胞移植是否对大鼠生精障碍研究具有治疗作用，并初步探讨其潜在的机制，为临床上雄性不育的治疗提供更多的治疗方法。方法： 75只健康雄性Wistar大鼠随机均分为5组，每组15只。A组为正常对照组,B组为低剂量照射对照组，C组高剂量照射对照组，D组为低剂量照射治疗组，E组为高剂量照射治疗组。B组和D组两组大鼠均以60Co γ射线局部照射1次，C组和E组大鼠均局部照射2次（间隔一周照射）。造模成功后，D组和E组给予大鼠睾丸骨髓间充质干细胞移植，饲养8周。8周后，称取体重及睾丸重量，并计算睾丸指数。采用ELISA法检测血清中FSH、LH、T的水平，应用HE染色观察大鼠睾丸组织形态学变化，应用RT-PCR检测GFP、Inhibin B、P450scc和VASA表达情况,双重免疫荧光检测GFP和VASA的表达情况。结果：照射对照组体重及睾丸指数均明显下降，并具有统计学意义（P＜0.05），而治疗组体重则与正常对照组相比无统计学意义（P＞0.05），而睾丸指数仍明显下降，并具有统计学意义（P＜0.05）。性激素方面，模型组及治疗组血清FSH显著提高，T含量明显降低，LH升高，差异均有统计学意义（P＜0.05）；治疗后，FSH、LH下降，T升高，两组LH和T变化均具有统计学意义（P＜0.05）；而高剂量治疗组FSH下降无统计学意义（P＞0.05）。睾丸组织病理学方面，照射对照组曲细精管结构破坏明显，可见极少量的支持细胞和精原细胞，以高剂量对照组破坏更为明显；治疗后，低剂量干细胞组大鼠生精功能有所恢复，而高剂量干细胞组生精功能恢复欠佳。RT-PCR示:照射模型组睾丸组织中InhibinB、P450scc和VASA mRNA几乎不表达；给予治疗后，低剂量治疗组大鼠InhibinB、P450scc和VASA mRNA表达水平升高，而高剂量治疗组仅有P450scc表达水平升高，而VASA和Inhibin B表达水平改变不明显。双重免疫荧光检测VASA和GFP结果同RT-PCR。结论：骨髓间充质干细胞移植后可以明显改善生精功能轻度受损大鼠睾丸的生精功能，而对于损伤较重，支持细胞恢复欠佳者生精功能恢复不明显。
[Abstract]:Background and objectives:. Infertility is a common disease in urological andrology clinics, affecting about 15% of couples, and male factors account for about 30% of the main factors, and about 40% of couples have problems with each other. The pathogenesis of male sterility is not completely clear. Those with definite etiology are treated with symptomatic treatment, while those with unknown causes are treated with experience, and the results are not satisfactory. In recent years, assisted reproductive technology has developed rapidly, providing good news for the vast number of infertile patients. But assisted reproductive technology violates the laws of natural selection in the biological world, and it is invasive, financially burdensome, and has potential health risks. And for couples who are not able to produce functional gametes on one side, There is no treatment other than the use of allogeneic donors. In recent years, with the further study of stem cells in male sterility, it has been found that stem cells can differentiate into germ cells. This study provides a new approach for the treatment of male sterility and the study of pathogenesis of male sterility. In this paper, we discuss the therapeutic effect of bone marrow mesenchymal stem cell transplantation on rat spermatogenesis disorder, and explore its potential mechanism. To provide more clinical treatment for male sterility. Methods:. 75 healthy male Wistar rats were randomly divided into 5 groups. There are 15 rats in each group. Group A is normal control group, group B is low dose irradiation, control group, group C, group C, high dose irradiation control group, group D, low dose radiation treatment group, group E, high dose irradiation treatment group, group B and group D, all rats are treated with 60Co 纬 rays. Rats in group C and group E were given local irradiation twice (interval of one week). Group D and group E were transplanted with mesenchymal stem cells (MSCs) of testis after successful modeling. After 8 weeks of feeding, weight of testis and weight of testis were measured and testicular index was calculated. ELISA method was used to detect the level of FSHLHX T in serum, HE staining was used to observe the morphological changes of testis, and RT-PCR was used to detect the expression of P450 SCC and VASA in rat testis. Double immunofluorescence was used to detect the expression of GFP and VASA. Results:. The body weight and testicular index of the irradiated control group were significantly decreased (P < 0.05), while the weight of the treatment group was not significantly higher than that of the normal control group (P > 0.05), while the testicular index was still significantly decreased (P < 0.05). In model group and treatment group, serum FSH level was significantly increased and the level of LH was significantly decreased (P < 0.05), and the serum FSH level was significantly lower than that in control group (P < 0.05). The changes of LH and T in both groups were statistically significant (P < 0.05), but the decrease of FSH in high-dose treatment group was not statistically significant (P > 0.05). In testicular histopathology, the structure of seminiferous tubules was destroyed obviously in the irradiated control group, and a very small number of Sertoli cells and spermatogonocytes were observed. After treatment, the spermatogenic function of rats in the low-dose stem cell group was recovered, while the spermatogenic function in the high-dose stem cell group was poor. RT-PCR showed that there was almost no expression of VASA mRNA and Inhibin Bon P450scc in the testis of the model group. After treatment, the expression levels of Inhibin Bon P450 SCC and VASA mRNA increased in the low dose group, but only in the high dose group, but the expression level of VASA and Inhibin B did not change significantly. The results of double immunofluorescence detection of VASA and GFP were similar to those of RT-PCR. Conclusion:. After transplantation of bone marrow mesenchymal stem cells, the spermatogenic function of the testis of the rats with slightly impaired spermatogenic function was obviously improved, but the spermatogenic function of the rats with severe injury and poor recovery of Sertoli cells was not obvious.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R698.2

【参考文献】