小鼠精原干细胞体外分化及相关基因表达的研究

发布时间：2018-08-12 09:51

【摘要】：目的：体外培养小鼠精原干细胞(mouse spermatogonial stem cells,mSSCs),并通过全反式视黄酸(ATRA, all-trans retinoic acid)进行诱导分化,观察小鼠精原干细胞在诱导中的变化情况,检测Stra8、mina53、Oct-4、GCNF在mSSCs体外分化中的表达,有助于研究精原干细胞体外分化的最佳条件和分子生物学特性,从精原干细胞体外分化的角度理解和认识此过程基因表达调控机制 方法：本研究体外分离培养6-8天BABL/C小鼠睾丸的SSCs,进行形态学鉴定,并通过含10% FBS、ATRA浓度为1.0×10-5mol/L的L-DMEM培养基对精原干细胞进行体外诱导分化,观察mSSCs加入诱导剂后的形态学变化,用间接免疫荧光技术从c-kit、Oct-4、GCNF三个标志物表达情况观察诱导分化情况,通过RT-PCR检测mSSCs诱导分化前后(0h,2h,12h,3d,5d) Stra8、mina53、Oct-4、GCNF的mRNA的水平,初步了解Stra8、mina53、Oct-4、GCNF的表达状况,以及与mSSCs体外分化的相关情况。 结果：1、形态学变化显示ATRA使mSSCs向精母细胞方向发生了分化,并通过间接免疫荧光反应进行鉴定；2、Stra8在mSSCs诱导分化2 h、12 h、3 d、5 d的水平高于成年小鼠和未诱导分化的水平,其中12h的表达水平最高,3d、5d时水平低于2 h,各对照组间差异具有统计学显著性；mina53的表达在诱导后增加,其中诱导2 h、12h时水平高于3d、5d及成年小鼠和未诱导mSSCs的水平,但诱导3d、5d组与成年小鼠组差异不具统计学显著性。3、Oct-4在1nSSCs诱导前呈表达水平最高,诱导2h、12h后下降,3d组和5d组均为阴性。4、GCNF的表达在诱导2h后表达开始增强,3d时表达水平最高,而5d时表达为阴性,在诱导组与成年小鼠睾丸之间表达差异具有显著统计学意义。 结论：(1)ATRA在体外诱导mSSCs分化成功。(2)Stra8基因在ATRA诱导mSSCs体外分化中可能发挥了启动减数分裂的作用,但Stra8、mina53基因在SSCs分化中的表达是否具有相关性是不能确定的。(3)在ATRA诱导mSSCs体外分化过程中,GCNF和Oct-4的表达呈负相关。
[Abstract]:Objective: to culture mouse spermatogonial stem cells (mouse spermatogonial stem cells) and differentiate them by ATRA (all-trans retinoic acid), observe the changes of mouse spermatogonial stem cells during induction, and detect the expression of Stra8mina53 Oct-4GCNF in the differentiation of mSSCs in vitro. Contributing to the study of the optimal conditions for differentiation of spermatogonial stem cells in vitro and the molecular biological characteristics, To understand and understand the regulation mechanism of gene expression in spermatogonial stem cells from the perspective of differentiation in vitro. In this study, the testis of BABL/C mice were isolated and cultured for 6 to 8 days in vitro. The differentiation of spermatogonial stem cells was induced by L-DMEM medium with 10% FBS ATRA concentration of 1.0 脳 10-5mol/L in vitro. The morphological changes of spermatogonial stem cells were observed after mSSCs was added to the inducer, and the differentiation was observed by indirect immunofluorescence technique from the expression of c-kitt-4 and GCNF markers. The level of mRNA of mSSCs was detected by RT-PCR before and after differentiation induced by mSSCs (0 h2h, 12h, 3d, 5d), and the expression of mSSCs in vitro and the expression of Oct-4C were also preliminarily studied. The results showed that the expression of GCNF was related to the differentiation of mSSCs in vitro. Results the morphological changes of ATRA showed that mSSCs differentiated into spermatocytes by ATRA, and the level of mSSCs induced differentiation was higher than that of adult mice and uninduced differentiation at 2 h, 12 h and 3 d after mSSCs induction by indirect immunofluorescence reaction. The highest expression level at 12 h was lower than 2 h at 5 d after induction. There was a statistically significant increase in the expression of mina53 in each control group after induction, and the level at 12 h after induction was higher than that at 12 h after induction, and the level of mSSCs was higher in adult mice than that in adult mice and uninduced mSSCs at 12 h after induction. However, there was no significant difference between the 3 d group and the adult mouse group. The expression level of Oct-4 was the highest before 1nSSCs induction. The negative expression of GCNF in the 3d group and the 5d group was the highest at the beginning of 3d after 2 h of induction, and the highest level was observed at the beginning of 3d after 2 hours of induction, and the expression of GCNF was the highest in the 5d group and the 5d group. On the 5th day, the expression was negative, and there was significant difference between the induction group and the adult mouse testis. Conclusion: (1) ATRA can induce the differentiation of mSSCs in vitro. (2) Stra8 gene may play a role in the initiation of meiosis in ATRA induced mSSCs differentiation in vitro. However, the correlation between the expression of Stra8mina53 gene in SSCs differentiation is uncertain. (3) in the process of mSSCs differentiation induced by ATRA, the expression of GCNF and Oct-4 is negatively correlated.
【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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