小鼠精原干细胞转导与移植的初步研究

发布时间：2018-03-18 13:56

本文选题：精原干细胞　切入点：精子发生　出处：《中南大学》2010年硕士论文　论文类型：学位论文

【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。
[Abstract]:Objective: to establish an in vitro transgenic method for murine spermatogonial stem cells by comparing liposome transfection and lentivirus mediated transduction of mouse spermatogonial stem cells. GFP-positive spermatogonial stem cells were obtained by microinjection of testicular output tubules in order to observe the survival of spermatogonial stem cells of GFP positive mice in the testis of recipient mice. Methods: the efficiency of transfection of spermatogonial stem cells by liposome mediated method and lentivirus-mediated method was compared with that of newly established ICR strain of mouse spermatogonial stem cell line, and the appropriate method was established, which was mediated by liposome. Vector plasmid and lentivirus packaging plasmid pCMVPMD.G were co-transfected into 293FT cells to prepare lentivirus-GFPG and detect viral titer. The virus expression vector labeled with green fluorescent protein (GFP) was transferred into mouse spermatogonial stem cells by lentivirus. GFP positive spermatogonial stem cells were collected for culture and amplification in vitro, digested into single cell suspension before transplantation, and microinjected through testicular output tubules. GFP positive spermatogonial stem cells in the testis of recipient mice were observed by allograft transplantation in sterile mice treated with Baoxian intraperitoneal injection. Results: lentivirus could effectively transduction mouse spermatogonial stem cells, and more GFP positive cells were obtained than normal liposome transfection methods. 30 days after transplantation, there were scattered light spots in testis under fluorescence anatomical microscope. However, there was no obvious green fluorescence from seminiferous tubule, and a few spermatogonial stem cells with positive expression of green fluorescent protein could be seen in frozen sections of testis along the basal membrane of seminiferous tubule. Conclusion:. 1. It is confirmed that lentivirus mediated method is a suitable transduction method for mouse spermatogonial stem cells. 2. The mouse model of sterility receptor by intraperitoneal injection of Baoxuan was established. 3. Spermatogonial stem cell transplantation was successfully completed by microinjection of output tubules. 4. The survival of spermatogonial stem cells after transplantation of spermatogonial stem cells in ICR newborn mice was preliminarily studied.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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