STAT6基因RNAi表达载体的构建及其沉默效应观察

发布时间：2018-01-04 05:09

本文关键词：STAT6基因RNAi表达载体的构建及其沉默效应观察　出处：《重庆医科大学》2010年硕士论文　论文类型：学位论文

更多相关文章： STAT6基因 STAT6蛋白 RNAi shRNA 哮喘

【摘要】： 哮喘是以气道高反应性和慢性气道炎症为特征的变态反应疾病。Th1/Th2平衡失调,即Th1功能下降,Th2功能亢进是目前比较公认的哮喘免疫学发病机制。由于特异性调控Th0细胞向Th2分化,STAT6基因成为治疗哮喘的靶基因。目前尚未报道STAT6基因的高效特异性的SiRNA序列,因此本研究选择STAT6基因作为RNA干扰(RNA interference,RNAi)的靶点,构建STAT6干扰质粒,为哮喘的治疗研究提供新思路。目的:构建针对STAT6基因的干扰质粒,并观察其对STAT6蛋白表达的抑制效果。方法:通过体外转录合成shRNA,与pIRES2 EGFP STAT6基因表达质粒共同转染COS7细胞,在荧光显微镜下观察干扰效果,同时从蛋白水平对干扰质粒的抑制效果进行验证。结果:将pIRES2 EGFP STAT6重组载体转染COS7细胞后,结果显示STAT6蛋白可以在细胞中高水平表达。与正常对照组相比,pGenesil3-STAT61和pGenesil3-STAT62对STAT6蛋白的抑制率分别是74.3%和85.8%,其中pGenesil3-STAT62的抑制作用更为显著。结论:成功构建并筛选到具有显著干扰效率的STAT6干扰质粒,为进一步研究STAT6在哮喘发病机制中的作用,同时为哮喘治疗提供新的思路。
[Abstract]:Asthma is an allergic disease characterized by airway hyperresponsiveness and chronic airway inflammation. Th1 / Th2 imbalance is called the decline of Th1 function. Hyperfunction of Th2 is recognized as the immunological mechanism of asthma, and the differentiation of Th0 cells into Th2 is regulated by specific regulation. STAT6 gene has become the target gene for the treatment of asthma. At present, the highly efficient and specific SiRNA sequence of STAT6 gene has not been reported. In this study, STAT6 gene was selected as the target of RNA interference RNA interference to construct STAT6 interference plasmid. To provide a new idea for the treatment of asthma. Aim: to construct the interference plasmid for STAT6 gene and observe its inhibitory effect on the expression of STAT6 protein. Methods: shRNAs were synthesized by transcription in vitro and transfected into COS7 cells with pIRES2 EGFP STAT6 gene expression plasmid. The interference effect was observed under fluorescence microscope and the inhibition effect of interference plasmid was verified from protein level. Results: the recombinant vector of pIRES2 EGFP STAT6 was transfected into COS7 cells. The results showed that STAT6 protein could be expressed at a high level in the cells compared with the normal control group. The inhibitory rates of pGenesil3-STAT61 and pGenesil3-STAT62 on STAT6 protein were 74.3% and 85.8%, respectively. The inhibitory effect of pGenesil3-STAT62 was more significant. Conclusion: STAT6 interference plasmids with significant interference efficiency were successfully constructed and screened to further study the role of STAT6 in the pathogenesis of asthma and to provide new ideas for the treatment of asthma.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R346

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