本文選題：精原干細胞　切入點：精子發(fā)生　出處：《中南大學》2010年碩士論文　論文類型：學位論文

【摘要】： 目的：通過比較脂質體轉染法與慢病毒介導法轉導小鼠精原干細胞,確立適宜小鼠精原干細胞的體外轉基因操作方法,獲得GFP-陽性精原干細胞后通過睪丸輸出小管顯微注射法進行小鼠精原干細胞移植,以期觀察GFP陽性小鼠精原干細胞在受體小鼠睪丸中的存活情況。 方法：利用本所已建立的ICR品系新生小鼠精原干細胞系,比較了脂質體介導法和慢病毒介導法轉染精原干細胞的效率。確立了適宜的方法,即通過脂質體介導,載體質粒與慢病毒包裝質粒pCMV、pMD. G共轉染293FT細胞,制備慢病毒(Lentivirus-GFP),檢測病毒滴度。通過慢病毒將帶有綠色熒光蛋白(GFP)標記的病毒表達載體轉導入小鼠精原干細胞內,使其發(fā)出綠色熒光。收集GFP陽性精原干細胞后進行體外培養(yǎng)與擴增,于移植前消化成單細胞懸液,經(jīng)睪丸輸出小管顯微注射技術,對白消安腹腔注射處理的不育小鼠行同種異體移植,觀察GFP陽性精原干細胞在受體小鼠睪丸內的存活情況。 結果：慢病毒可有效轉導小鼠精原干細胞,較普通脂質體轉染法獲得更多GFP陽性細胞。移植術后30天熒光解剖顯微鏡下可見睪丸組織有散在光點,但未見曲細精管發(fā)出明顯綠色熒光,睪丸組織冰凍切片內可見少量綠色熒光蛋白表達陽性的精原干細胞沿著曲細精管基底膜生長增殖。 結論： 1、證實了慢病毒介導法是一種適宜小鼠精原干細胞的轉導方法。 2、建立了白消安腹腔注射法不育受體小鼠模型。 3、成功運用輸出小管顯微注射技術完成精原干細胞移植。 4、初步探討了ICR品系新生小鼠精原干細胞移植術后細胞存活情況。
[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.
【學位授予單位】：中南大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R329

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本文編號：1629867