本文關(guān)鍵詞： Nanog 腫瘤 胚胎干細(xì)胞 Smad3 Wnt3　出處：《西北農(nóng)林科技大學(xué)》2010年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：Nanog基因并非只表達(dá)在胚胎干細(xì)胞中,在多種腫瘤細(xì)胞中也檢測(cè)到了Nanog基因的表達(dá),這就提示我們,腫瘤細(xì)胞中高水平表達(dá)的Nanog蛋白是被重新異常激活的,那么這種激活對(duì)于腫瘤的發(fā)生尤其是對(duì)于一些腫瘤因子的作用值得探討。 Smad3是TGF-β信號(hào)通路家族中重要一個(gè)信號(hào)分子,參與多種腫瘤信號(hào)通路,Smad3也可以和其他因子相互作用,Smad3家族的協(xié)同作用在TGF-β信號(hào)通路中的作用是關(guān)鍵的。Wnt3是另外一個(gè)重要的細(xì)胞因子,主要參與Wnt信號(hào)通路并參與其他多種信號(hào)通路,而且在腫瘤中也有表達(dá)。Boyer預(yù)測(cè)Nanog在Wnt3上游8kb之內(nèi)可能有結(jié)合位點(diǎn)。為了推測(cè)Nanog在腫瘤發(fā)生中的作用,有必要探明Nanog與Smad3和Wnt3之間的作用。 實(shí)驗(yàn)方法:首先建立體外高表達(dá)Nanog的CHO細(xì)胞系,還有轉(zhuǎn)入空載體pcDNA3.1的對(duì)照細(xì)胞系,為隨后的熒光報(bào)告檢測(cè)做準(zhǔn)備。 篩選基因,設(shè)計(jì)Smad3,Wnt3以及內(nèi)參的實(shí)時(shí)定量引物,提取高表達(dá)Nanog的293f細(xì)胞總RNA,反轉(zhuǎn)錄后檢測(cè)高表達(dá)Nanog的細(xì)胞中相應(yīng)的Smad3和Wnt3是否也在高表達(dá)。 確定以上基因相對(duì)于照細(xì)胞表達(dá)明顯升高之后,設(shè)計(jì)其上游調(diào)控序列的引物,確定載體構(gòu)建成功后,將其克隆入PGL-3-basic熒光報(bào)告載體中。 共轉(zhuǎn)染CHO或293細(xì)胞,并檢測(cè)其熒光表達(dá)的變化,從而研究Nanog對(duì)這些基因的調(diào)控效果。 結(jié)果:熒光定量PCR檢測(cè)到高表達(dá)Nanog的同時(shí),293f細(xì)胞也在同時(shí)高表達(dá)上述被測(cè)基因,提示Nanog對(duì)這些基因具有調(diào)控作用,同時(shí)熒光報(bào)告基因結(jié)果顯示調(diào)控不是直接的。應(yīng)該是通過(guò)其他途徑進(jìn)行的。 結(jié)論:成功構(gòu)建了體外高表達(dá)Nanog的CHO細(xì)胞系,為將來(lái)進(jìn)行體外高表達(dá)模型提供了方便的工具,而且通過(guò)實(shí)時(shí)PCR和熒光報(bào)告基因技術(shù),驗(yàn)證了Nanog對(duì)上述基因具有調(diào)控關(guān)系,但是何種調(diào)控通路還需深入研究。
[Abstract]:Nanog gene is not only expressed in embryonic stem cells, but also detected in many tumor cells, suggesting that the high expression of Nanog protein in tumor cells is reactivated abnormally. The role of this activation in tumorigenesis, especially for some tumor factors, is worth exploring. Smad3 is an important signal molecule in TGF- 尾 signaling pathway family. Smad3 may also interact with other factors. The role of Smad3 family in TGF- 尾 signaling pathway is crucial. Wnt3 is another important cytokine. In order to speculate the role of Nanog in tumorigenesis, Nanog may have binding sites in 8kb upstream of Wnt3. It is necessary to find out the role of Nanog with Smad3 and Wnt3. Methods: a CHO cell line with high expression of Nanog in vitro and a control cell line transferred to empty vector pcDNA3.1 were established to prepare for the subsequent detection of fluorescence report. Screening genes, designing real-time quantitative primers for Smad3 Wnt3 and internal reference, extracting the total RNAs of 293f cells with high expression of Nanog, and detecting whether the corresponding Smad3 and Wnt3 are also overexpression in the cells with high expression of Nanog after reverse transcription. After confirming that the expression of the above gene was significantly higher than that of the irradiated cells, the primer of its upstream regulatory sequence was designed. After the successful construction of the vector, the gene was cloned into the PGL-3-basic fluorescent report vector. The effect of Nanog on the regulation of these genes was studied by co-transfection of CHO or 293 cells and detection of their fluorescence expression. Results: the high expression of Nanog was detected by fluorescence quantitative PCR, and the expression of these genes was also high in T293f cells, suggesting that Nanog has a regulatory effect on these genes. At the same time, fluorescent report gene results show that regulation is not direct. It should be done through other channels. Conclusion: the CHO cell line with high expression of Nanog in vitro was successfully constructed, which provides a convenient tool for future hyperexpression model in vitro. Moreover, real-time PCR and fluorescence reporter gene technique are used to verify the regulatory relationship of Nanog to these genes. But what kind of regulatory pathway needs to be further studied.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R346

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 杜麗麗;林戈;盧光t;;4種全能性基因轉(zhuǎn)入人胚胎成纖維細(xì)胞誘導(dǎo)多能性干細(xì)胞系的建立及其鑒定(英文)[J];中南大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2009年12期

2 ;Stem cell pluripotency and transcription factor Oct4[J];Cell Research;2002年Z2期

3 ;Identification of two distinct transactivation domains in the pluripotency sustaining factor nanog[J];Cell Research;2003年06期

，

本文編號(hào)：1483970