本文選題：ZymosanA + p38��； 參考：《南華大學(xué)》2012年碩士論文

【摘要】：目的：探討真菌多糖Zymosan A對(duì)其mRNA3’UTR含ARE的細(xì)胞因子（TNF-α、CXCL1、IL-10、IL-23a）的誘導(dǎo)表達(dá)，，研究p38對(duì)此類細(xì)胞因子mRNA穩(wěn)定性的調(diào)節(jié)作用及機(jī)制，為真菌的致病機(jī)制與治療研究提供新思路與新靶標(biāo)。方法：分離純化小鼠pMΦ，分別培養(yǎng)原代pMΦ及Raw264.7細(xì)胞株，提取蛋白樣品，采用western blot方法檢測(cè)Zymosan A誘導(dǎo)的MAPK信號(hào)通路中p38、ERK、JNK等激酶的磷酸化；用生物信息學(xué)方法分析Zymosan A誘導(dǎo)的細(xì)胞因子的mRNA3’UTR，尋找富含ARE可能存在mRNA轉(zhuǎn)錄后水平調(diào)控的細(xì)胞因子；采用real-time PCR 方法，檢測(cè)Zymosan A誘導(dǎo)的mRNA3’UTR含ARE的細(xì)胞因子（TNF-α、CXCL1、IL-10、IL-23a）的表達(dá)水平，及其相關(guān)細(xì)胞因子mRNA終止轉(zhuǎn)錄后的降解程度，觀察分析mRNA穩(wěn)定性；分別用p38抑制劑（SB202190）、ERK抑制劑（PD98059）、JNK抑制劑（SP600125）阻斷信號(hào)通路中的靶向蛋白激酶，real-timePCR方法檢測(cè)細(xì)胞因子（TNF-α、CXCL1、IL-10、IL-23a）mRNA穩(wěn)定性的改變；分別用p38抑制劑（SB202190）、ERK抑制劑（PD98059）、JNK抑制劑（SP600125）阻斷信號(hào)通路中的靶向蛋白激酶，提取蛋白樣品，采用western blot方法分別檢測(cè)CREB、ERK及c-Jun的磷酸化，鑒定抑制劑阻斷作用，同時(shí)檢測(cè)MK2的磷酸化，確定p38對(duì)MK2的調(diào)節(jié)作用；采用體外酶活實(shí)驗(yàn)，用Zymosan A處理細(xì)胞，提取蛋白樣品，加入λPP，30°C孵育5min，檢測(cè)TTP磷酸化，進(jìn)一步驗(yàn)證在Zymosan A誘導(dǎo)的細(xì)胞因子（TNF-α、CXCL1、IL-10、IL-23a）mRNA穩(wěn)定性調(diào)節(jié)中，p38通過(guò)對(duì)MK2及TTP的磷酸化發(fā)揮調(diào)節(jié)作用。 結(jié)果：Zymosan A能激活MAPK信號(hào)通路，磷酸化p38、ERK、JNK等蛋白激酶及RNA結(jié)合蛋白TTP，Zymosan A激活p38、ERK、JNK等蛋白激酶的最佳濃度為100μg/ml，同時(shí)，該濃度能有效誘導(dǎo)TNF-α、CXCL1、IL-10及IL-23a等細(xì)胞因子的表達(dá)。生物信息學(xué)分析表明上述細(xì)胞因子mRNA3’UTR富含ARE。Zymosan A誘導(dǎo)的ARE-細(xì)胞因子，在終止轉(zhuǎn)錄4h后，mRNA降解為50%-60%，mRNA穩(wěn)定性良好。p38抑制劑（SB202190）處理后，結(jié)果顯示p38抑制劑能夠有效的抑制其下游MK2及TTP的磷酸化，并使TNF-α、CXCL1、IL-10及IL-23a的mRNA含量在轉(zhuǎn)錄后明顯減少，降解為10%左右，表明其mRNA穩(wěn)定性降低。體外酶活實(shí)驗(yàn)檢測(cè)發(fā)現(xiàn)加入磷酸酯酶后，TTP磷酸化消失。ERK抑制劑（PD98059）處理后，結(jié)果顯示ERK磷酸化被明顯抑制，但TNF-α、CXCL1、IL-10及IL-23a的mRNA含量無(wú)變化，mRNA穩(wěn)定性無(wú)影響。JNK抑制劑（SP600125）處理后，結(jié)果顯示其下游轉(zhuǎn)錄因子c-Jun磷酸化被明顯抑制，但TNF-α、CXCL1、IL-10及IL-23a的mRNA含量無(wú)變化，mRNA穩(wěn)定性無(wú)影響。 結(jié)論： 1．Zymosan A能激活MAPK信號(hào)通路，磷酸化p38、ERK、JNK等蛋白激酶，并誘導(dǎo)TNF-α、CXCL1、IL-10、IL-23a等細(xì)胞因子的大量表達(dá)。 2．p38能夠增強(qiáng)Zymosan A誘導(dǎo)的TNF-α、CXCL1、IL-10及IL-23a的mRNA穩(wěn)定性。其作用機(jī)制為p38通過(guò)磷酸化MK2及TTP，使結(jié)合在mRNA3’UTR ARE的TTP游離，從而增強(qiáng)3’UTR富含ARE的細(xì)胞因子mRNA穩(wěn)定性。
[Abstract]:Objective: to investigate the induction and expression of mRNA3'UTR cytokine TNF- 偽 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- 偽 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- 偽 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 擄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- 偽 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- 偽 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- 偽 -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- 偽 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- 偽 CXCL1 IL-10 and mRNA of IL-23a had no effect on the stability of TNF- 偽 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- 偽, CXCL1, IL-10, IL-23a and so on. 2.p38 enhanced the mRNA stability of Zymosan A induced TNF- 偽 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.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R363

【共引文獻(xiàn)】

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