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

【摘要】： 目的：通過比較脂質(zhì)體轉(zhuǎn)染法與慢病毒介導(dǎo)法轉(zhuǎn)導(dǎo)小鼠精原干細(xì)胞,確立適宜小鼠精原干細(xì)胞的體外轉(zhuǎn)基因操作方法,獲得GFP-陽性精原干細(xì)胞后通過睪丸輸出小管顯微注射法進(jìn)行小鼠精原干細(xì)胞移植,以期觀察GFP陽性小鼠精原干細(xì)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(xì)胞系,比較了脂質(zhì)體介導(dǎo)法和慢病毒介導(dǎo)法轉(zhuǎn)染精原干細(xì)胞的效率。確立了適宜的方法,即通過脂質(zhì)體介導(dǎo),載體質(zhì)粒與慢病毒包裝質(zhì)粒pCMV、pMD. G共轉(zhuǎn)染293FT細(xì)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(biāo)記的病毒表達(dá)載體轉(zhuǎn)導(dǎo)入小鼠精原干細(xì)胞內(nèi),使其發(fā)出綠色熒光。收集GFP陽性精原干細(xì)胞后進(jìn)行體外培養(yǎng)與擴(kuò)增,于移植前消化成單細(xì)胞懸液,經(jīng)睪丸輸出小管顯微注射技術(shù),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(xì)胞在受體小鼠睪丸內(nèi)的存活情況。 結(jié)果：慢病毒可有效轉(zhuǎn)導(dǎo)小鼠精原干細(xì)胞,較普通脂質(zhì)體轉(zhuǎn)染法獲得更多GFP陽性細(xì)胞。移植術(shù)后30天熒光解剖顯微鏡下可見睪丸組織有散在光點(diǎn),但未見曲細(xì)精管發(fā)出明顯綠色熒光,睪丸組織冰凍切片內(nèi)可見少量綠色熒光蛋白表達(dá)陽性的精原干細(xì)胞沿著曲細(xì)精管基底膜生長增殖。 結(jié)論： 1、證實(shí)了慢病毒介導(dǎo)法是一種適宜小鼠精原干細(xì)胞的轉(zhuǎn)導(dǎo)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(yùn)用輸出小管顯微注射技術(shù)完成精原干細(xì)胞移植。 4、初步探討了ICR品系新生小鼠精原干細(xì)胞移植術(shù)后細(xì)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(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 孫源;陶科;涂炯炯;朱文兵;范立青;;小鼠精原干細(xì)胞體外培養(yǎng)體系的建立[J];中華男科學(xué)雜志;2008年08期

2 譚碧君;;慢病毒載體在制備轉(zhuǎn)基因動物中的最新進(jìn)展[J];上海畜牧獸醫(yī)通訊;2010年01期

3 張茨,王玲瓏,金化民,宋超;白消安誘導(dǎo)小鼠無精子癥模型[J];醫(yī)學(xué)新知雜志;2003年04期

，

本文編號：1629867