【作者】 邢柳；

【导师】 范立青；

【作者基本信息】 中南大学 ， 生殖工程学， 2010， 硕士

【摘要】 目的：通过比较脂质体转染法与慢病毒介导法转导小鼠精原干细胞,确立适宜小鼠精原干细胞的体外转基因操作方法,获得GFP-阳性精原干细胞后通过睾丸输出小管显微注射法进行小鼠精原干细胞移植,以期观察GFP阳性小鼠精原干细胞在受体小鼠睾丸中的存活情况。方法：利用本所已建立的ICR品系新生小鼠精原干细胞系,比较了脂质体介导法和慢病毒介导法转染精原干细胞的效率。确立了适宜的方法,即通过脂质体介导,载体质粒与慢病毒包装质粒pCMV、pMD. G共转染293FT细胞,制备慢病毒(Lentivirus-GFP),检测病毒滴度。通过慢病毒将带有绿色荧光蛋白(GFP)标记的病毒表达载体转导入小鼠精原干细胞内,使其发出绿色荧光。收集GFP阳性精原干细胞后进行体外培养与扩增,于移植前消化成单细胞悬液,经睾丸输出小管显微注射技术,对白消安腹腔注射处理的不育小鼠行同种异体移植,观察GFP阳性精原干细胞在受体小鼠睾丸内的存活情况。结果：慢病毒可有效转导小鼠精原干细胞,较普通脂质体转染法获得更多GFP阳性细胞。移植术后30天荧光解剖显微镜下可见睾丸组织有散在光点,但未见曲细精管发出明显绿色荧光,睾丸组织冰冻切片内可见少量绿色荧光蛋白表达阳性的精原干细胞沿着曲细精管基底膜生长增殖。结论：1、证实了慢病毒介导法是一种适宜小鼠精原干细胞的转导方法。2、建立了白消安腹腔注射法不育受体小鼠模型。3、成功运用输出小管显微注射技术完成精原干细胞移植。4、初步探讨了ICR品系新生小鼠精原干细胞移植术后细胞存活情况。更多还原

【Abstract】 Objective:By comparing the lipofection method with lentivirus-mediated transduction of mouse spermatogonial stem cells,to establish appropriate method of mice spermatogonial stem cells genetically modified in vitro,to obtain GFP-positive spermatogonial stem cells. Establish the platform of mice spermatogonial stem cell transplantation technology through the method of testicular efferent duct microscopy injection, in order to observe the survival situation of GFP-positive spermatogonial stem cells in the recipient mice testes.Method:Through the lentivirus, virus vector marked with the green fluorescent protein (GFP)gene is transducted into the spermatogonial stem cells which established before from newborn male ICR mice, and make it radiate Green fluorescent. Cultured and proliferated in vitro after collecting the GFP-positive cells, and then digested into cell suspension. Take an allotransplantation via efferent ductules of testis microinjection on the sterile mice which has been intraperitoneal injected with busulfan. Finally, observe whether or not the GFP-positive spermatogonial stem cell survival in the mouse testis and the process of spermatogenesis.Results:lentivirus transduction could be a efficient method to obtain GFP-positive spermatogonial stem cells.30 days after transplantation,the testis with scattered points of light could be seen under fluorescent dissecting microscope, however, no significant green fluorescent issued from seminiferous tubule, and a few of spermatogonial stem cells expressed GFP growing and proliferating along with the basement membrane of contorted seminiferous tubules in the testis frozen sections.Conclusions:1. It confirmed that Lentivirus-mediated is a suitable method for mice spermatogonial stem cell transduction.2. Established the mouse infertility receptor model by intraperitoneal injection of busulfan.3.Successfully completed the spermatogonial stem cell transplantation by using the efferent ductules of testis microinjection.4. Preliminarily investigated in the newborn ICR mice spermatogonial stem cell survival situation after cell transplantation.更多还原