p38参与调节Zymosan A诱导的相关细胞因子mRNA稳定性研究

发布时间：2018-04-27 05:41

  本文选题：ZymosanA + p38　； 参考：《南华大学》2012年硕士论文

【摘要】：目的：探讨真菌多糖Zymosan A对其mRNA3’UTR含ARE的细胞因子（TNF-α、CXCL1、IL-10、IL-23a）的诱导表达，，研究p38对此类细胞因子mRNA稳定性的调节作用及机制，为真菌的致病机制与治疗研究提供新思路与新靶标。方法：分离纯化小鼠pMΦ，分别培养原代pMΦ及Raw264.7细胞株，提取蛋白样品，采用western blot方法检测Zymosan A诱导的MAPK信号通路中p38、ERK、JNK等激酶的磷酸化；用生物信息学方法分析Zymosan A诱导的细胞因子的mRNA3’UTR，寻找富含ARE可能存在mRNA转录后水平调控的细胞因子；采用real-time PCR 方法，检测Zymosan A诱导的mRNA3’UTR含ARE的细胞因子（TNF-α、CXCL1、IL-10、IL-23a）的表达水平，及其相关细胞因子mRNA终止转录后的降解程度，观察分析mRNA稳定性；分别用p38抑制剂（SB202190）、ERK抑制剂（PD98059）、JNK抑制剂（SP600125）阻断信号通路中的靶向蛋白激酶，real-timePCR方法检测细胞因子（TNF-α、CXCL1、IL-10、IL-23a）mRNA稳定性的改变；分别用p38抑制剂（SB202190）、ERK抑制剂（PD98059）、JNK抑制剂（SP600125）阻断信号通路中的靶向蛋白激酶，提取蛋白样品，采用western blot方法分别检测CREB、ERK及c-Jun的磷酸化，鉴定抑制剂阻断作用，同时检测MK2的磷酸化，确定p38对MK2的调节作用；采用体外酶活实验，用Zymosan A处理细胞，提取蛋白样品，加入λPP，30°C孵育5min，检测TTP磷酸化，进一步验证在Zymosan A诱导的细胞因子（TNF-α、CXCL1、IL-10、IL-23a）mRNA稳定性调节中，p38通过对MK2及TTP的磷酸化发挥调节作用。 结果：Zymosan A能激活MAPK信号通路，磷酸化p38、ERK、JNK等蛋白激酶及RNA结合蛋白TTP，Zymosan A激活p38、ERK、JNK等蛋白激酶的最佳浓度为100μg/ml，同时，该浓度能有效诱导TNF-α、CXCL1、IL-10及IL-23a等细胞因子的表达。生物信息学分析表明上述细胞因子mRNA3’UTR富含ARE。Zymosan A诱导的ARE-细胞因子，在终止转录4h后，mRNA降解为50%-60%，mRNA稳定性良好。p38抑制剂（SB202190）处理后，结果显示p38抑制剂能够有效的抑制其下游MK2及TTP的磷酸化，并使TNF-α、CXCL1、IL-10及IL-23a的mRNA含量在转录后明显减少，降解为10%左右，表明其mRNA稳定性降低。体外酶活实验检测发现加入磷酸酯酶后，TTP磷酸化消失。ERK抑制剂（PD98059）处理后，结果显示ERK磷酸化被明显抑制，但TNF-α、CXCL1、IL-10及IL-23a的mRNA含量无变化，mRNA稳定性无影响。JNK抑制剂（SP600125）处理后，结果显示其下游转录因子c-Jun磷酸化被明显抑制，但TNF-α、CXCL1、IL-10及IL-23a的mRNA含量无变化，mRNA稳定性无影响。 结论： 1．Zymosan A能激活MAPK信号通路，磷酸化p38、ERK、JNK等蛋白激酶，并诱导TNF-α、CXCL1、IL-10、IL-23a等细胞因子的大量表达。 2．p38能够增强Zymosan A诱导的TNF-α、CXCL1、IL-10及IL-23a的mRNA稳定性。其作用机制为p38通过磷酸化MK2及TTP，使结合在mRNA3’UTR ARE的TTP游离，从而增强3’UTR富含ARE的细胞因子mRNA稳定性。
[Abstract]:Objective: to investigate the induction and expression of mRNA3'UTR cytokine TNF- 伪 CXCL1hIL-10hIL-23a by fungal polysaccharide Zymosan A, and to study the regulatory effect and mechanism of p38 on the stability of mRNA, and to provide new ideas and targets for the study of pathogenic mechanism and treatment of fungi. Methods: mouse PM 桅 and Raw264.7 cell lines were isolated and purified, and protein samples were extracted. The phosphorylation of p38 ERKN JNK and other kinases in MAPK signaling pathway induced by Zymosan A was detected by western blot method. Bioinformatics method was used to analyze the mRNA3UTRs of cytokines induced by Zymosan A and to search for cytokines rich in ARE which may exist in the regulation of mRNA posttranscriptional level. Real-time PCR Methods: the expression level of TNF- 伪 CXCL1CXCL1 IL-10 IL-23a in mRNA3'UTR induced by Zymosan A and the degree of degradation of mRNA were detected, and the stability of mRNA was analyzed. P38 inhibitor SB20190 / ERK inhibitor PD98059 / JNK inhibitor SP600125) was used to detect the stability of cytokine TNF- 伪 CXCL1 / IL-10 / IL-23aI mRNA by real-time PCR. P38 inhibitor SB20190 / ERK inhibitor PD98059JNK inhibitor (SP600125) was used to block the target protein kinase in the signal pathway, and protein samples were extracted. The phosphorylation of CREBERK and c-Jun was detected by western blot method, and the blocking effect of the inhibitor was evaluated, and the phosphorylation of MK2 was also detected. The regulation of p38 on MK2 was determined, the cells were treated with Zymosan A in vitro, the protein samples were extracted and incubated with 位 PP30 掳C for 5 min to detect the phosphorylation of TTP. It was further demonstrated that p38 regulates the stability of Zymosan A induced cytokine TNF- 伪 CXCL1, IL-10 and IL-23aI mRNA by phosphorylation of MK2 and TTP. Results the MAPK signaling pathway was activated by 1: Zymosan A. the optimal concentration of phosphorylated protein kinases such as p38 ERKN JNK and RNA binding protein TTPN A was 100 渭 g / ml. At the same time, the expression of TNF- 伪 CXCL1IL-10, IL-23a and other cytokines were effectively induced. Bioinformatics analysis showed that the above cytokine mRNA3'UTR was rich in ARE- cytokines induced by ARE.Zymosan A, and was degraded into 50-60m mRNA after termination of transcription for 4 hours, and treated with p38 inhibitor SB20190. The results showed that p38 inhibitor could effectively inhibit the phosphorylation of MK2 and TTP downstream, and decrease the mRNA content of TNF- 伪 -CXCL1, IL-10 and IL-23a after transcription and degrade to about 10%, indicating that the stability of mRNA was decreased. The results of enzyme activity test in vitro showed that the phosphorylation of TTP disappeared after addition of phosphatase, ERK inhibitor PD98059), the results showed that the phosphorylation of ERK was significantly inhibited, but the mRNA content of TNF- 伪 CXCL1 IL-10 and IL-23a had no effect on the stability of mRNA. The results showed that the phosphorylation of the downstream transcription factor c-Jun was significantly inhibited, but the content of TNF- 伪 CXCL1 IL-10 and mRNA of IL-23a had no effect on the stability of TNF- 伪 CXCL1 IL-10 and mRNA. Conclusion: 1.Zymosan A activated the MAPK signaling pathway, phosphorylated protein kinase such as p38 ERK, and induced the expression of cytokines such as TNF- 伪, CXCL1, IL-10, IL-23a and so on. 2.p38 enhanced the mRNA stability of Zymosan A induced TNF- 伪 CXCL1 IL-10 and IL-23a. The mechanism is that p38 dissociates the TTP binding to mRNA3'UTR ARE by phosphorylation of MK2 and TTP, thus enhancing the stability of mRNA, which is rich in ARE in 3'UTR.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

【共引文献】

相关期刊论文 前7条

1 杨乐莹;李国华;;DC-SIGN的研究进展[J];江西医药;2012年04期

2 张彦洁;许春娣;周同;;上皮细胞转分化、抗原提呈及区室化免疫调节[J];现代免疫学;2011年06期

3 李晓;周同;陈楠;;天然免疫分子在糖基化异常IgA致肾小球足细胞损伤中的免疫调节作用[J];生命科学;2010年12期

4 张彦洁;钟文伟;刘伟;许春娣;夏振炜;周同;;免疫系统区室化与上皮细胞局部微环境中的免疫调节作用[J];生命科学;2011年08期

5 张彦洁;王俊青;任建敏;余熠;杨芬;许春娣;周同;;上皮细胞转分化及其表观遗传学调控[J];生命科学;2012年02期

6 任建敏;张彦洁;王俊青;吴菁;刘昕

本文编号：1809461