Oct-4在精原干细胞定向诱导分化中的表达及其甲基化的研究

发布时间：2018-02-28 05:50

  本文关键词： Oct-4 精原干细胞 分化 甲基化　出处：《石河子大学》2010年硕士论文　论文类型：学位论文

【摘要】：目的：体外分离培养小鼠精原干细胞(mouse spermatogonial stem cells,mSSCs),并通过干细胞因子(SCF, stem cell factor)诱导其向精母细胞方向分化,观察小鼠精原干细胞在诱导分化前后Oct-4的表达情况,并检测其甲基化状态,为进一步研究Oct-4在mSSCs分化中的作用以及探讨表观遗传修饰调节对干细胞分化的影响提供研究思路。 方法：采用与支持细胞共培养法,分离培养6～8天BABL/C小鼠的SSCs,培养48h时用含10%FBS和50ng/mL SCF的L-DMEM培养基对mSSCs进行诱导分化,观察nSSCs加入诱导剂前后的形态学变化,用c-kit、GCNF的表达鉴定mSSCs是否发生分化。分别采用RT-PCR和间接免疫荧光染色技术,从mRNA和蛋白水平检测Oct-4在mSSCs诱导分化前和分化后2d、5d的表达情况,运用甲基化特异性PCR检测Oct-4在mSSCs诱导分化前和分化后2d、5d的甲基化状态。 结果：1、形态学观察mSSCs细胞核较大,细胞质少,SCF诱导培养后,随着培养时间的延长,细胞克隆逐渐增长缓慢,并出现衰退、凋亡的细胞；2、间接免疫荧光染色显示,mSSCs表达c-kit,不表达GCNF,但诱导5d后不表达c-kit,表达GCNF;3、间接免疫荧光染色和RT-PCR显示,Oct-4在mSSCs诱导前表达水平最高,诱导2d后下降,5d后几乎不表达；4、甲基化特异性PCR显示,Oct-4在nSSCs诱导前未发生甲基化改变,诱导2d检测到其甲基化状态改变,诱导5d甲基化水平增高。 结论：(1)mSSCs在诱导分化前后的形态及c-kit,GCNF表达的变化,提示其用SCF诱导后发生了分化；(2)Oct-4在mSSCs诱导前表达水平最高,用SCF诱导后表达下调乃至不表达；(3)Oct-4在mSSCs诱导分化前后有甲基化状态的改变,且甲基化水平与表达呈负相关。
[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.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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