【摘要】：目的：體外培養(yǎng)小鼠精原干細(xì)胞(mouse spermatogonial stem cells,mSSCs),并通過全反式視黃酸(ATRA, all-trans retinoic acid)進(jìn)行誘導(dǎo)分化,觀察小鼠精原干細(xì)胞在誘導(dǎo)中的變化情況,檢測Stra8、mina53、Oct-4、GCNF在mSSCs體外分化中的表達(dá),有助于研究精原干細(xì)胞體外分化的最佳條件和分子生物學(xué)特性,從精原干細(xì)胞體外分化的角度理解和認(rèn)識此過程基因表達(dá)調(diào)控機制 方法：本研究體外分離培養(yǎng)6-8天BABL/C小鼠睪丸的SSCs,進(jìn)行形態(tài)學(xué)鑒定,并通過含10% FBS、ATRA濃度為1.0×10-5mol/L的L-DMEM培養(yǎng)基對精原干細(xì)胞進(jìn)行體外誘導(dǎo)分化,觀察mSSCs加入誘導(dǎo)劑后的形態(tài)學(xué)變化,用間接免疫熒光技術(shù)從c-kit、Oct-4、GCNF三個標(biāo)志物表達(dá)情況觀察誘導(dǎo)分化情況,通過RT-PCR檢測mSSCs誘導(dǎo)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的表達(dá)狀況,以及與mSSCs體外分化的相關(guān)情況。 結(jié)果：1、形態(tài)學(xué)變化顯示ATRA使mSSCs向精母細(xì)胞方向發(fā)生了分化,并通過間接免疫熒光反應(yīng)進(jìn)行鑒定；2、Stra8在mSSCs誘導(dǎo)分化2 h、12 h、3 d、5 d的水平高于成年小鼠和未誘導(dǎo)分化的水平,其中12h的表達(dá)水平最高,3d、5d時水平低于2 h,各對照組間差異具有統(tǒng)計學(xué)顯著性；mina53的表達(dá)在誘導(dǎo)后增加,其中誘導(dǎo)2 h、12h時水平高于3d、5d及成年小鼠和未誘導(dǎo)mSSCs的水平,但誘導(dǎo)3d、5d組與成年小鼠組差異不具統(tǒng)計學(xué)顯著性。3、Oct-4在1nSSCs誘導(dǎo)前呈表達(dá)水平最高,誘導(dǎo)2h、12h后下降,3d組和5d組均為陰性。4、GCNF的表達(dá)在誘導(dǎo)2h后表達(dá)開始增強,3d時表達(dá)水平最高,而5d時表達(dá)為陰性,在誘導(dǎo)組與成年小鼠睪丸之間表達(dá)差異具有顯著統(tǒng)計學(xué)意義。 結(jié)論：(1)ATRA在體外誘導(dǎo)mSSCs分化成功。(2)Stra8基因在ATRA誘導(dǎo)mSSCs體外分化中可能發(fā)揮了啟動減數(shù)分裂的作用,但Stra8、mina53基因在SSCs分化中的表達(dá)是否具有相關(guān)性是不能確定的。(3)在ATRA誘導(dǎo)mSSCs體外分化過程中,GCNF和Oct-4的表達(dá)呈負(fù)相關(guān)。
[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.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R329

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