人Nanog基因的克隆及其在CHO-K1 细胞中的表达和Nanog对Smad3，Wnt3和c-Jun的调控

发布时间：2018-02-02 07:23

本文关键词： Nanog 肿瘤胚胎干细胞 Smad3 Wnt3　出处：《西北农林科技大学》2010年硕士论文　论文类型：学位论文

【摘要】：Nanog基因并非只表达在胚胎干细胞中,在多种肿瘤细胞中也检测到了Nanog基因的表达,这就提示我们,肿瘤细胞中高水平表达的Nanog蛋白是被重新异常激活的,那么这种激活对于肿瘤的发生尤其是对于一些肿瘤因子的作用值得探讨。 Smad3是TGF-β信号通路家族中重要一个信号分子,参与多种肿瘤信号通路,Smad3也可以和其他因子相互作用,Smad3家族的协同作用在TGF-β信号通路中的作用是关键的。Wnt3是另外一个重要的细胞因子,主要参与Wnt信号通路并参与其他多种信号通路,而且在肿瘤中也有表达。Boyer预测Nanog在Wnt3上游8kb之内可能有结合位点。为了推测Nanog在肿瘤发生中的作用,有必要探明Nanog与Smad3和Wnt3之间的作用。实验方法:首先建立体外高表达Nanog的CHO细胞系,还有转入空载体pcDNA3.1的对照细胞系,为随后的荧光报告检测做准备。筛选基因,设计Smad3,Wnt3以及内参的实时定量引物,提取高表达Nanog的293f细胞总RNA,反转录后检测高表达Nanog的细胞中相应的Smad3和Wnt3是否也在高表达。确定以上基因相对于照细胞表达明显升高之后,设计其上游调控序列的引物,确定载体构建成功后,将其克隆入PGL-3-basic荧光报告载体中。共转染CHO或293细胞,并检测其荧光表达的变化,从而研究Nanog对这些基因的调控效果。结果:荧光定量PCR检测到高表达Nanog的同时,293f细胞也在同时高表达上述被测基因,提示Nanog对这些基因具有调控作用,同时荧光报告基因结果显示调控不是直接的。应该是通过其他途径进行的。结论:成功构建了体外高表达Nanog的CHO细胞系,为将来进行体外高表达模型提供了方便的工具,而且通过实时PCR和荧光报告基因技术,验证了Nanog对上述基因具有调控关系,但是何种调控通路还需深入研究。
[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.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R346

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