發(fā)布時(shí)間：2018-03-11 06:45

  本文選題：精原干細(xì)胞　切入點(diǎn)：原癌基因c-src　出處：《南昌大學(xué)》2006年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：探索及完善精原干細(xì)胞的分離、鑒定和體外培養(yǎng)技術(shù)；研究原癌基因c-src對體外培養(yǎng)的精原干細(xì)胞存活及與凋亡密切相關(guān)的caspase-3表達(dá)的影響。 方法：非連續(xù)密度梯度離心及差速貼壁法分離精原干細(xì)胞；c-Kit和TERT細(xì)胞免疫組織化學(xué)鑒定精原干細(xì)胞；用MTT比色法檢測精原干細(xì)胞的存活及增殖情況，觀察AS-src對精原干細(xì)胞存活的劑量性影響及AS-src對精原干細(xì)胞存活的時(shí)間性影響；用RT-PCR檢測c-src mRNA和caspase-3 mRNA的表達(dá)情況。 結(jié)果：非連續(xù)密度梯度及差速貼壁法分離精原干細(xì)胞，臺盼藍(lán)染色計(jì)數(shù)細(xì)胞存活率分別為92.5％和92.1％；分離后所獲取的細(xì)胞c-Kit和TERT免疫組織化學(xué)陽性細(xì)胞數(shù)平均值分別為90.8±1.0％和91.7±1.2％；精原干細(xì)胞在含10％NBS的DMEM中培養(yǎng)96h時(shí)增殖達(dá)高峰；10μmol／l AS-src作用12h后精原干細(xì)胞存活率下降(P＜0.05)，于24h，48h，，72h持續(xù)下降(P＜0.01)；與S-src和空白對照組相比，AS-src組c-src mRNA水平明顯下調(diào)，而caspase-3 mRNA表達(dá)水平明顯上調(diào)。 結(jié)論：非連續(xù)密度梯度結(jié)合差速貼壁法是分離精原干細(xì)胞的有效方法；以c-Kit及TERT作為marker分子通過免疫組織化學(xué)檢測為精原干細(xì)胞的鑒定提供較為充分的依據(jù)；精原干細(xì)胞可以在含10％NBS的DMEM培養(yǎng)基中短期增殖；c-Src可以促進(jìn)精原干細(xì)胞的存活；c-Src可能通過PI-3K／PKB途徑，導(dǎo)致Caspase-3磷酸化從而抑制精原干細(xì)胞的凋亡。
[Abstract]:Aim: to study the effects of proto-oncogene c-src on the survival of spermatogonial stem cells and the expression of caspase-3, which is closely related to apoptosis, and to explore and improve the isolation, identification and in vitro culture of spermatogonial stem cells. Methods: spermatogonial stem cells were isolated by discontinuous density gradient centrifugation and differential adherent method, c Kit and TERT cells were identified by immunohistochemistry, survival and proliferation of spermatogonial stem cells were detected by MTT colorimetry. To observe the dose effect of AS-src on the survival of spermatogonial stem cells and the effect of AS-src on the survival time of spermatogonial stem cells, and to detect the expression of c-src mRNA and caspase-3 mRNA by RT-PCR. Results: spermatogonial stem cells were isolated by discontinuous density gradient and differential adhesion. The cell survival rate of Trypan blue staining was 92.5% and 92.1, the average number of c-Kit and TERT immunocytochemical positive cells were 90.8 鹵1.0% and 91.7 鹵1.2, respectively. The proliferation of spermatogonial stem cells reached the peak after 96 h culture in DMEM containing 10NBS. After treated with 10 渭 mol/l AS-src for 12 h, the survival rate of spermatogonial stem cells decreased significantly (P < 0.05), and the level of c-src mRNA in AS-src group decreased significantly compared with S-src group and control group. The expression of caspase-3 mRNA was up-regulated. Conclusion: discontinuous density gradient method combined with differential adherent method is an effective method for isolation of spermatogonial stem cells, c-Kit and TERT as marker molecules provide sufficient basis for identification of spermatogonial stem cells by immunohistochemistry. The short-term proliferation of spermatogonial stem cells in DMEM medium containing 10NBS may promote the survival of spermatogonial stem cells through PI-3K/PKB pathway, which may lead to Caspase-3 phosphorylation and inhibit the apoptosis of spermatogonial stem cells.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R321

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 張學(xué)明,賴良學(xué),李德雪,李子義,文興豪,杜惜明,岳占碰,王延釗;小鼠精原細(xì)胞的分離和純化[J];解剖學(xué)報(bào);2000年03期

2 楊俊玲,徐斯凡;原癌基因在生精細(xì)胞中的表達(dá)及其作用[J];中華男科學(xué)雜志;2005年07期

3 李學(xué)軍;反義核酸藥物的研究現(xiàn)狀[J];生理科學(xué)進(jìn)展;2000年02期

4 王曉燕,譚德勇,周翔,昝瑞光;原癌基因c-myc、c-H-ras、c-sis在小鼠卵巢和睪丸中的轉(zhuǎn)錄表達(dá)研究[J];遺傳;1998年05期

本文編號：1597026