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  本文選題：大鼠精原干細胞 + c-raf��； 參考：《南昌大學》2007年碩士論文

【摘要】： 目的:完善大鼠精原干細胞的分離和體外培養(yǎng)技術;研究c-raf對精原干細胞體外培養(yǎng)的存活作用及機制。 方法:非連續(xù)密度梯度離心及差速貼壁法分離精原干細胞;c-kit細胞免疫組織化學鑒定精原干細胞;基因測序和SeqMan、檢驗精原干細胞擴增DNA和c-raf的同源性;MTT比色法檢測體外培養(yǎng)精原干細胞的存活及增殖情況,觀察c-raf AON對精原干細胞體外培養(yǎng)存活的量效關系及c-raf AON對精原干細胞體外培養(yǎng)存活的時效關系;RT-PCR檢測c-raf mRNA和caspase-3 mRNA的表達情況;TUNEL檢測c-raf AON對精原干細胞凋亡的影響。 結果:非連續(xù)密度梯度離心及差速貼壁法分離的精原干細胞,經(jīng)臺盼藍染色計數(shù)其存活率分別為92.3%和91.5%;分離后的精原干細胞經(jīng)c-kit鑒定其純度為90.1%;精原干細胞在含10%NBS的DMEM中培養(yǎng)120h時增殖達高峰,在無10%NBS的DMEM中培養(yǎng)其存活率持續(xù)下降;精原干細胞擴增DNA和c-raf的同源性為98%;15μmol/L c-raf AON作用12h后精原干細胞存活率下降(P0.05),于18h后存活率持續(xù)下降(P0.05);與對照組相比c-raf AON組的c-raf mRNA水平明顯下調(P0.05),而caspase-3 mRNA表達水平明顯上調(P0.05);c-raf AON組凋亡指數(shù)(40.5%)明顯大于對照組(30.2%)和錯義組(29.4%)的凋亡指數(shù)(P0.05)。 結論:非連續(xù)密度梯度離心結合差速貼壁法是分離精原干細胞的有效方法;c-kit可作為鑒定精原干細胞的分子標志物;精原干細胞在含10%NBS的DMEM培養(yǎng)基中短期增殖;c-raf在九天的雄性SD大鼠的精原干細胞上表達;c-raf可能通過抑制凋亡而促進精原干細胞存活;c-raf可能通過下調capspase-3的表達而抑制精原干細胞凋亡。
[Abstract]:Aim: to improve the isolation and in vitro culture of rat spermatogonial stem cells and to study the survival effect and mechanism of c-raf on spermatogonial stem cells in vitro. Methods: spermatogonial stem cells were isolated by discontinuous density gradient centrifugation and differential adherent method. The survival and proliferation of spermatogonial stem cells in vitro were detected by DNA sequencing and SeqMan. the homology of DNA and c-raf was detected by MTT colorimetry. To observe the dose-effect relationship of c-raf AON on the survival of spermatogonial stem cells in vitro and the time-dependent relationship of c-raf AON to the survival of spermatogonial stem cells in vitro, the expressions of c-raf mRNA and caspase-3 mRNA were detected by RT-PCR. Tunel was used to detect the effect of c-raf AON on apoptosis of spermatogonial stem cells. Results: spermatogonial stem cells were isolated by discontinuous density gradient centrifugation and differential adhesion. The survival rate of spermatogonial stem cells was 92.3% and 91.5% by trypan blue staining, the purity of spermatogonial stem cells was 90.1% by c-kit, the proliferation of spermatogonial stem cells reached the peak after 120 h culture in DMEM containing 10%NBS, and the survival rate of spermatogonial stem cells cultured in DMEM without 10%NBS continued to decline. The homology of amplified DNA and c-raf of spermatogonial stem cells was 980.15 渭 mol/L c-raf AON, the survival rate of spermatogonial stem cells decreased after 12h, the survival rate of spermatogonial stem cells continued to decrease after 18h, and the c-raf mRNA level of c-raf AON group was significantly lower than that of control group, but the expression level of caspase-3 mRNA was lower than that of control group. The apoptotic index of P0.05 AON group was significantly higher than that of control group (30.2%) and missense group (29.444). Conclusion: discontinuous density gradient centrifugation combined with differential adhesion is an effective method for isolation of spermatogonial stem cells. C-kit can be used as a molecular marker for identification of spermatogonial stem cells. Short-term proliferation of spermatogonial stem cells on DMEM medium containing 10%NBS c-raf expression of c-raf on spermatogonial stem cells of nine-day male SD rats may inhibit the survival of spermatogonial stem cells by inhibiting apoptosis. C-raf may inhibit the expression of capspase-3 by down-regulating the expression of capspase-3. Apoptosis of spermatogonial stem cells.
【學位授予單位】：南昌大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R329

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本文編號：1809436