转录共激活因子p100蛋白对转录因子STAT1和STAT6基因转录活性的调控作用

发布时间：2018-02-21 18:47

本文关键词： P100蛋白 STAT1 STAT6 免疫共沉淀虫荧光素酶　出处：《天津医科大学》2009年硕士论文　论文类型：学位论文

【摘要】： 研究目的:细胞内信号转导的转录响应的基本机制就是信号转导途径最终激活转录因子,并使相应的基因表达和造成细胞行为的改变。JAK/STAT信号转导途径是胞内重要的信号通路之一,其在细胞生成、分化、凋亡等生物学方面有重要的意义,而其参与的调控需要多种转录激活因子的参与。p100蛋白首先作为EBNA2(Epstein-Barr virus nuclear antigen 2,EB病毒细胞核抗原2)的转录调控激活因子被发现,研究表明p100蛋白是STAT6,STAT5的转录共激活因子,能增强其转录活性。STAT1与STAT6同属于STAT家族,两者存在高度相似性,p100是否也是STAT1的共激活因子呢?本课题对此作了研究,同时进一步明确p100蛋白与STAT6在活细胞内结合共定位情况,为进一步研究p100蛋白的功能提供依据。方法:本课题分三大部分进行,第一部分是研究p100蛋白与STAT1或STAT6的相互结合作用。该部分从两方面研究p100蛋白与STAT1或STAT6的相互结合作用。其一是采用GST pull down技术来研究p100蛋白与STAT1或STAT6的体外结合作用,其二采用免疫共沉淀(CO-IP)研究p100蛋白与STAT1或STAT6的体内结合作用;第二部分是采用虫荧光素酶报告基因检测p100蛋白及其片段调控STAT1或STAT6的转录活性;第三部分是观察p100蛋白及其片段与STAT6的定位观察。首先构建以绿色或红色荧光蛋白为报告基因的重组质粒pEGFP-CI-STAT6和pERFP-CI-STAT6,其次转染入HeLa细胞,经刺激后在激光共聚焦显微镜下观察融合蛋白的运动变化。结果:①p100蛋白,p100-SN能够与STAT6结合,但p100-TSN不能与STAT6结合。p100不能与STAT1结合。②虫荧光素酶检测到p100蛋白,p100-SN能够增强STAT6的基因转录活性,但p100-TSN不能够增强STAT6的基因转录活性。p100不能够增强STAT1的基因转录活性。③成功构建质粒pEGFP-CI-STAT1和pERFP-CI-STAT6,并能够在HeLa细胞中实现表达。在激光共聚焦显微镜下观察到p100蛋白,p100-SN蛋白能够在IL-4刺激下与STAT6共同进入核内,而p100-TSN不能相应的进入核内。结论①p100蛋白及其SN片段能够与STAT6结合,并能够增强STAT6的转录活性,而且呈剂量依赖性,TSN片段不能与STAT6结合,亦不能影响STAT6基因转录活性;p100蛋白不能与STAT1结合,且不能影响其转录活性。②成功构建重组质粒pEGFP-CI-STAT6和pERFP-CI-STAT6,并实现其在细胞中的表达。在激光共聚焦显微镜下观察在活细胞状态下蛋白的运动情况,在IL-4刺激下,STAT6和p100蛋白能够进入核内,p100-SN和STAT6也在同样的条件下进入核内,提示它们在细胞内存在共定位。而在IL-4刺激下,p100-TSN不能相应的进入细胞核内,提示与STAT6不存在共定位。③在IL-4长时间刺激下,可见到STAT6在细胞核内聚集成颗粒状物质,具体原因与机制有待进一步研究。
[Abstract]:Objective: the basic mechanism of transcriptional response to intracellular signal transduction is that signal transduction pathways ultimately activate transcription factors. It also makes the corresponding gene expression and changes in cell behavior. JAK / stat signal transduction pathway is one of the important intracellular signaling pathways, which plays an important role in cell generation, differentiation, apoptosis and other biological aspects. The regulation of p100 requires the involvement of many transcriptional activators. P100 protein is first found as the transcription activator of EBNA2(Epstein-Barr virus nuclear antigen 2 Epstein-Barr virus (Epstein-Barr virus Nuclear Antigen 2). Studies have shown that p100 protein is the transcription co-activator of STAT6 and STAT5. STAT1 can enhance its transcriptional activity. STAT1 and STAT6 belong to the STAT family. Is there a high similarity between them? is p100 also a co-activator of STAT1? In this study, the co-localization of p100 protein and STAT6 in living cells was further determined, which provided the basis for further study of the function of p100 protein. Methods: the subject was divided into three parts. The first part is to study the interaction between p100 protein and STAT1 or STAT6. In this part, the interaction between p100 protein and STAT1 or STAT6 is studied from two aspects. One is to use GST pull down technique to study the in vitro binding of p100 protein to STAT1 or STAT6. The binding of p100 protein to STAT1 or STAT6 was studied by co-immunoprecipitation in vivo, and the transcriptional activity of STAT1 or STAT6 was detected by luciferase reporter gene. The third part was to observe the localization of p100 protein and its fragment with STAT6. Firstly, the recombinant plasmids pEGFP-CI-STAT6 and pERFP-CI-STAT6 with green or red fluorescent protein as reporter gene were constructed, and then transfected into HeLa cells. The motor changes of fusion protein were observed under confocal laser microscope after stimulation. Results p100-SN could bind to STAT6, but p100-TSN could not bind to STAT6. P100 could not bind to STAT1. 2 luciferase assay showed that p100-SN could enhance the transcriptional activity of STAT6. However, p100-TSN could not enhance the transcriptional activity of STAT6. P100 could not enhance the transcriptional activity of STAT1. 3. The plasmids pEGFP-CI-STAT1 and pERFP-CI-STAT6 were successfully constructed and expressed in HeLa cells. P100 protein p100-SN eggs were observed under confocal laser microscope. White can enter the nucleus together with STAT6 under the stimulation of IL-4. However, p100-TSN could not enter the nucleus. Conclusion p100 protein and its SN fragment can bind to STAT6 and enhance the transcriptional activity of STAT6. Moreover, in a dose-dependent manner, the p100 protein can neither bind to STAT6 nor affect the transcriptional activity of STAT6 gene. The recombinant plasmids pEGFP-CI-STAT6 and pERFP-CI-STAT6 were successfully constructed, and their expression was realized. The movement of proteins in living cells was observed under confocal laser microscope. Under the same conditions, STAT6 and p100 proteins could enter the nucleus under the same conditions, suggesting that they were co-located in the cell, but under the stimulation of IL-4, p100-TSN could not enter the nucleus. It is suggested that there is no co-localization with STAT6. 3. Under the stimulation of IL-4 for a long time, it can be seen that STAT6 aggregates into granular substance in the nucleus. The specific reason and mechanism need to be further studied.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R346

【共引文献】