本文關(guān)鍵詞： Oct-4 精原干細(xì)胞 分化 甲基化　出處：《石河子大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：體外分離培養(yǎng)小鼠精原干細(xì)胞(mouse spermatogonial stem cells,mSSCs),并通過干細(xì)胞因子(SCF, stem cell factor)誘導(dǎo)其向精母細(xì)胞方向分化,觀察小鼠精原干細(xì)胞在誘導(dǎo)分化前后Oct-4的表達(dá)情況,并檢測其甲基化狀態(tài),為進(jìn)一步研究Oct-4在mSSCs分化中的作用以及探討表觀遺傳修飾調(diào)節(jié)對干細(xì)胞分化的影響提供研究思路。 方法：采用與支持細(xì)胞共培養(yǎng)法,分離培養(yǎng)6～8天BABL/C小鼠的SSCs,培養(yǎng)48h時(shí)用含10%FBS和50ng/mL SCF的L-DMEM培養(yǎng)基對mSSCs進(jìn)行誘導(dǎo)分化,觀察nSSCs加入誘導(dǎo)劑前后的形態(tài)學(xué)變化,用c-kit、GCNF的表達(dá)鑒定mSSCs是否發(fā)生分化。分別采用RT-PCR和間接免疫熒光染色技術(shù),從mRNA和蛋白水平檢測Oct-4在mSSCs誘導(dǎo)分化前和分化后2d、5d的表達(dá)情況,運(yùn)用甲基化特異性PCR檢測Oct-4在mSSCs誘導(dǎo)分化前和分化后2d、5d的甲基化狀態(tài)。 結(jié)果：1、形態(tài)學(xué)觀察mSSCs細(xì)胞核較大,細(xì)胞質(zhì)少,SCF誘導(dǎo)培養(yǎng)后,隨著培養(yǎng)時(shí)間的延長,細(xì)胞克隆逐漸增長緩慢,并出現(xiàn)衰退、凋亡的細(xì)胞；2、間接免疫熒光染色顯示,mSSCs表達(dá)c-kit,不表達(dá)GCNF,但誘導(dǎo)5d后不表達(dá)c-kit,表達(dá)GCNF;3、間接免疫熒光染色和RT-PCR顯示,Oct-4在mSSCs誘導(dǎo)前表達(dá)水平最高,誘導(dǎo)2d后下降,5d后幾乎不表達(dá)；4、甲基化特異性PCR顯示,Oct-4在nSSCs誘導(dǎo)前未發(fā)生甲基化改變,誘導(dǎo)2d檢測到其甲基化狀態(tài)改變,誘導(dǎo)5d甲基化水平增高。 結(jié)論：(1)mSSCs在誘導(dǎo)分化前后的形態(tài)及c-kit,GCNF表達(dá)的變化,提示其用SCF誘導(dǎo)后發(fā)生了分化；(2)Oct-4在mSSCs誘導(dǎo)前表達(dá)水平最高,用SCF誘導(dǎo)后表達(dá)下調(diào)乃至不表達(dá)；(3)Oct-4在mSSCs誘導(dǎo)分化前后有甲基化狀態(tài)的改變,且甲基化水平與表達(dá)呈負(fù)相關(guān)。
[Abstract]:Objective: to isolate and culture mouse spermatogonial stem cells from mouse spermatogonial stem cells in vitro, and to investigate the expression of Oct-4 in mouse spermatogonial stem cells before and after differentiation by inducing the differentiation of mouse spermatogonial stem cells into spermatocytes by stem cell factor. The methylation status of Oct-4 in mSSCs differentiation and the effect of epigenetic modification on stem cell differentiation were studied. Methods: SSCs of BABL/C mice were isolated and cultured with Sertoli cells for 6 days. After 48 hours of culture, L-DMEM medium containing 10s and 50ng / mL SCF was used to induce the differentiation of mSSCs, and the morphological changes before and after the addition of nSSCs were observed. RT-PCR and indirect immunofluorescence staining techniques were used to detect the expression of Oct-4 before and at 2 days after mSSCs differentiation at the level of mRNA and protein. Methylation-specific PCR was used to detect the methylation status of Oct-4 before and 2 days after mSSCs differentiation. Results: the morphology of mSSCs showed that the nucleus was larger and the cytoplasm was less. With the prolongation of the culture time, the cell clone gradually increased slowly and appeared to decline. In apoptotic cells, indirect immunofluorescence staining showed that mSSCs expressed c-kitand not GCNF.However, after 5 days of induction, they did not express c-kitand GCNF3. Indirect immunofluorescence staining and RT-PCR showed that Oct-4 expressed the highest level before mSSCs induction. The methylation specific PCR showed that there was no methylation change before nSSCs induction, but the methylation state was detected on the 2nd day after induction, and the methylation level increased at 5 days after induction. Conclusion the morphological changes and the expression of c-kitarine GCNF before and after the differentiation of MSCs suggested that the differentiation of MSCs induced by SCF had the highest expression level before and after the induction of mSSCs. After induction of SCF, there was a change of methylation state before and after induction of mSSCs, and the level of methylation was negatively correlated with the expression.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329

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