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　論文類型：學(xué)位論文

【摘要】：Nanog基因并非只表達在胚胎干細(xì)胞中,在多種腫瘤細(xì)胞中也檢測到了Nanog基因的表達,這就提示我們,腫瘤細(xì)胞中高水平表達的Nanog蛋白是被重新異常激活的,那么這種激活對于腫瘤的發(fā)生尤其是對于一些腫瘤因子的作用值得探討。 Smad3是TGF-β信號通路家族中重要一個信號分子,參與多種腫瘤信號通路,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信號通路中的作用是關(guān)鍵的。Wnt3是另外一個重要的細(xì)胞因子,主要參與Wnt信號通路并參與其他多種信號通路,而且在腫瘤中也有表達。Boyer預(yù)測Nanog在Wnt3上游8kb之內(nèi)可能有結(jié)合位點。為了推測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。 實驗方法:首先建立體外高表達Nanog的CHO細(xì)胞系,還有轉(zhuǎn)入空載體pcDNA3.1的對照細(xì)胞系,為隨后的熒光報告檢測做準(zhǔn)備。 篩選基因,設(shè)計Smad3,Wnt3以及內(nèi)參的實時定量引物,提取高表達Nanog的293f細(xì)胞總RNA,反轉(zhuǎn)錄后檢測高表達Nanog的細(xì)胞中相應(yīng)的Smad3和Wnt3是否也在高表達。 確定以上基因相對于照細(xì)胞表達明顯升高之后,設(shè)計其上游調(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熒光報告載體中。 共轉(zhuǎn)染CHO或293細(xì)胞,并檢測其熒光表達的變化,從而研究Nanog對這些基因的調(diào)控效果。 結(jié)果:熒光定量PCR檢測到高表達Nanog的同時,293f細(xì)胞也在同時高表達上述被測基因,提示Nanog對這些基因具有調(diào)控作用,同時熒光報告基因結(jié)果顯示調(diào)控不是直接的。應(yīng)該是通過其他途徑進行的。 結(jié)論:成功構(gòu)建了體外高表達Nanog的CHO細(xì)胞系,為將來進行體外高表達模型提供了方便的工具,而且通過實時PCR和熒光報告基因技術(shù),驗證了Nanog對上述基因具有調(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)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R346

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本文編號：1483970