【作者】 席琼；

【导师】 胡巢凤；

【作者基本信息】 暨南大学 ， 病理学与病理生理学， 2011， 硕士

【摘要】 目的受体相互作用蛋白2(receptor-interacting protein 2, RIP2)是一种丝氨酸/苏氨酸激酶,在信号转导途径中起关键作用,作为NOD样受体(NOD-like receptors, NLRs)信号通路的下游因子,衔接NLRs并激活NF-κB信号传导通路,在先天性免疫反应和清除病原体感染的炎症反应中起着重要作用。肠道细菌感染是临床实践中最常见的细菌感染之一,在其感染过程中可以被宿主NLRs识别,并通过RIP2信号分子激活细胞核内转录因子NF-κB的转录,并表达各种炎性细胞因子,从而引发宿主的多种防御机制。蛋白激酶-丝裂原活化蛋白激酶信号通路参与细胞的生长发育及细胞间各种功能等生理过程,其中p38MAPK主要参与炎症反应和应激反应。本研究将构建的RIP2基因转染至肠道细胞后,RT-PCR和Western blot检测观察mRNA转录水平和蛋白表达水平；将大肠杆菌感染已转染RIP2的SW480细胞,计数细胞胞内细菌数量变化；应用p38 MAPK通路抑制剂SB203580作用于各实验组细胞,计数胞内大肠杆菌活菌数的变化；并用Western blot法检测各实验组磷酸化p38表达水平,观察RIP2清除大肠杆菌的作用同p38MAPK信号通路的关系。方法本实验用阳离子聚合物JetPeiTM将人RIP2基因(pEGFP-C2-PIP2)转染至人结肠癌SW480细胞株,以转染空质粒(pEGFP-C2-PIP2)及未转染的SW480细胞株作为对照组,应用RT-PCR和Western blot法检测细胞外源性RIP2 mRNA和RIP2蛋白的表达；并用LB培养基培养大肠杆菌ATCC25922,收集细菌后用紫外分光光度计于600nm波长测定其OD值,将其绘制曲线后确定细菌浓度；按一定比例将活菌感染SW480细胞,分别在不同的时间点内(4h、24h、36h)用TritonX-100裂解细胞,裂解液涂布细菌平板培养,计数菌落并观察大肠杆菌菌落数的变化；用大肠杆菌刺激已转染RIP2、转染空质粒和未转染质粒的SW480细胞24小时,平板培养菌落观察3组组间大肠杆菌活菌数的变化；应用p38 MAPK通路抑制剂SB203580作用于实验组细胞,与未加抑制剂各组细胞相比较,计数胞内大肠杆菌活菌数的变化；用Western blot法观察转染RIP2及细菌刺激前后细胞内磷酸化p38蛋白表达量。结果与对照组相比,转染RIP2组其mRNA转录水平和蛋白表达水平均明显升高(P<0.01,P<0.05),表明RIP2表达质粒成功转染SW480细胞；大肠杆菌感染SW480细胞后,随着时间的推移,胞内的大肠杆菌数量也随之增长,说明大肠杆菌在SW480细胞内的生长呈增加趋势；将大肠杆菌刺激细胞24h后,转染RIP2细胞组的活菌数较转染空质粒组和未转染质粒组胞内活菌数数量明显减少,其差异有统计学意义(P<0.01),说明转染RIP2的SW480细胞能清除胞内大肠杆菌；而加入SB203580阻断p38 MAPK信号通路后,转染RIP2细胞组内活菌数量增加；并且转染RIP2组磷酸化p38蛋白表达量明显高于转染空质粒的对照组组,转染RIP2组间用大肠杆菌刺激后细胞磷酸化p38蛋白表达量高于未用细菌刺激的对照组,并且高于转染空质粒的对照组；而加入p38MAPK通路抑制剂SB203580后p38蛋白表达量减少。结论1.RIP2质粒成功转染SW480细胞,且mRNA转录水平较对照组明显增高(P<0.01),蛋白表达水平较对照组增高(P<0.05)；2.大肠杆菌感染SW480细胞后,细胞内活菌数量随时间的延长不断增加；而将RIP2转染SW480细胞后,胞内大肠杆菌数量明显减少,说明RIP2可清除胞内大肠杆菌。3. SB203580阻断p38 MAPK信号通路后,能逆转RIP2清除胞内大肠杆菌的作用；并且RIP2可诱导磷酸化p38 MAPK的表达,表明RIP2清除大肠杆菌作用可能与p38 MAPK信号通路有关；更多还原

【Abstract】 ObjectiveReceptor-interacting protein (RIP)-2 is a serine□threonine kinase containing a caspase recruitment domain (CARD) that is involved in the NOD-like receptor-signaling pathway, RIP2 is downstream of NLRs and activation of NF-κB signaling pathway, and play an important role in innate immune response and clear the pathogen infection in the inflammatory response. Intestinal infection is one of the most common bacterial infection, NLRs can be identified by the infected host, RIP2 signaling molecules activated by NF-κB nuclear transcripted and expressed inflammatory cytokines, triggering a variety of host defense mechanism. Mitogen-activated protein kinase (MAPK) signaling pathway participated in cell growth and function and other physiological processes simultaneously, including p38MAPK which mainly involved in inflammation and stress response. Our study will transfect RIP2 gene to intestinal cells, using RT-PCR and Western blot to test the level of mRNA and protein; using E. coli infected to transfected RIP2 cells, counting the number of intracellular bacteria; using p38MAPK pathway inhibitor SB203580 in the experimental group, counting the number of intracellular E. coli; detect by Western blot of phosphorylation of p38 in each experimental group, and test the expression level to observe the role of RIP2 in p38MAPK signaling pathway.MethodsRIP2 gene (pEGFP-C2-PIP2) were transfected into cell line SW480 by JetPeiTM. Western blot and RT-PCR analyses were then used to detect RIP2 protein and mRNA expression. These transfected and non-transfected cells were incubated with Escherichia coli(E coli) at 37℃. After 24 hours, viable intracellular bacteria were quantified by plating appropriate dilution on LB agar plates. Then transfected and non-transfected cells were treated with SB203580, the ResultsRT-PCR and Western blot analyses indicated that the expression of RIP2 mRNA and protein was significantly enhanced in SW480 cells transfected with the recombinant plasmid pEGFP-C2-PIP2 when compared with cells transfected with pEGFP-C2 and non-transfected cells. The pEGFP-C2-PIP2 transfected cells had the ability to clear invading E coli, and this antibacterial effect was inhibited by SB203580, the ability has decrease. after stimulation of bacteria, Western blot detected phosphorylation of p38 protein, the results showed that cells transfected with RIP2 of phosphorylated p38 protein was significantly higher than cells transfected with C2 group, and after stimulation with E. coli the p-p38 protein expression highest than the other group, further confirmed that RIP2 clear mechanism of intracellular E. coli may have connection to activation of p38MAPK signal pathway.Conclusion(1) Our results show RIP2 plasmid successfully transfected SW480 cells, mRNA transcription level was significantly higher than the control group (P<0.01) and the protein expression levels higher than control (P<0.05)(2) ATCC25922 in SW480 cells increased as time growing; in PIP2 transfected SW480 cells, the number of intracellular E. coli was significantly reduced compared with the control group, suggesting that RIP2 can clear intracellular E. coli.(3) After using p38MAPK inhibitor SB203580, we found it can also blok the function of RIP2; RIP2 can induce the expression of p38MAPK protein, suggesting that activation of p38MAPK may plays an important role in the role of RIP2 clearence effect.更多还原