發(fā)布時(shí)間：2018-01-19 02:22

  本文關(guān)鍵詞： 沙眼衣原體 質(zhì)粒蛋白pORF5 絲裂原活化蛋白激酶 前炎癥細(xì)胞因子　出處：《南華大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：沙眼衣原體（Chlamydia trachomatis，Ct）是引起沙眼、尿道炎和盆腔炎等疾病的重要病原菌，炎癥反應(yīng)過度所導(dǎo)致的免疫病理損傷是Ct致病的重要環(huán)節(jié)，但其確切的致病機(jī)制還不甚明了。本研究試圖探討Ct質(zhì)粒蛋白pORF5能否誘導(dǎo)人單核細(xì)胞(THP-1)產(chǎn)生IL-8、IL-1β、TNF-α等前炎癥因子(CKs)，以及與Toll樣受體2（TLR2）和絲裂原活化蛋白激酶（MAPK）信號通路的關(guān)系，，為深入研究Ct的致病機(jī)制提供實(shí)驗(yàn)依據(jù)。 方法：將已構(gòu)建好的pGEX-6p-1/pORF5重組質(zhì)粒轉(zhuǎn)化XL1-blue大腸桿菌，IPTG誘導(dǎo)表達(dá)GST-pORF5融合蛋白，融合蛋白經(jīng)谷胱甘肽-瓊脂糖凝膠4B純化，純化的GST-pORF5融合蛋白再經(jīng)PreScission Protease切割GST標(biāo)簽制備不含GST的pORF5靶蛋白，BCA法測定pORF5靶蛋白濃度，SDS-PAGE分析和Western blotting鑒定靶蛋白。pORF5蛋白經(jīng)去內(nèi)毒素處理后，分別用2～8μg/mL的pORF5蛋白體外刺激經(jīng)PMA處理的THP-1細(xì)胞，并于0h、6h、12h、24h、36h收集細(xì)胞，ELISA檢測IL-8、IL-1β和TNF-α。Western blotting檢測p38、JNK和ERK1/2的活化情況；分別用不同濃度（1、10、30μM）的ERK1/2特異性抑制劑PD98059、p38特異性抑制劑SB203580及TLR2抗體預(yù)處理THP-1細(xì)胞后，再以6μg/mL pORF5蛋白刺激12h，分析其對IL-8、IL-1β和TNF-α產(chǎn)生的影響。 結(jié)果: (1) pGEX-6p-1/pORF5重組質(zhì)粒轉(zhuǎn)化XL1-blue大腸桿菌后，IPTG誘導(dǎo)表達(dá)一分子量約54KD的GST-pORF5融合蛋白，融合蛋白再經(jīng)PreScission Protease蛋白酶切割GST標(biāo)簽后獲得不含GST-tag的分子量約為28KD的pORF5蛋白，純度可達(dá)95%以上。 (2)不同濃度的pORF5蛋白能明顯誘導(dǎo)THP-1細(xì)胞產(chǎn)生IL-8、IL-1β和TNF-α，當(dāng)pORF5蛋白濃度為2μg/mL～6μg/mL，CKs的產(chǎn)生水平與pORF5蛋白呈明顯的劑量依賴性；當(dāng)pORF5濃度高于6μg/mL時(shí)，IL-8、IL-1β和TNF-α產(chǎn)生水平減少；用6μg/mL的pORF5蛋白刺激THP-1細(xì)胞6h時(shí)，TNF-α量達(dá)到高峰，持續(xù)到12h后TNF-α水平逐漸下降，12h時(shí)IL-1β和IL-8量達(dá)到高峰。 (3) Western blotting結(jié)果顯示，pORF5刺激THP-1細(xì)胞可活化ERK1/2/MAPK和p38/MAPK，其中ERK1/2途徑在蛋白刺激THP-1細(xì)胞后的15min磷酸化水平達(dá)到高峰，p38途徑在刺激細(xì)胞30min時(shí)磷酸化水平達(dá)到高峰，持續(xù)時(shí)間達(dá)60min以上，SAPK/JNK1/2未檢測到磷酸化。 (4)通過使用不同濃度的p38、ERK1/2抑制劑后，ELISA結(jié)果顯示IL-1β、IL-8和TNF-α產(chǎn)生水平均降低，呈劑量依賴性，在抑制劑濃度為30μM時(shí)，IL-8分別降至57%和58.8%，IL-1β分別降至22%和45%，TNF-α分別降至22.4%和16.3%。 (5) TLR2抗體預(yù)處理THP-1細(xì)胞30min后再用6μg/mL pORF5蛋白刺激THP-1細(xì)胞12h，IL-1β、IL-8和TNF-α產(chǎn)生水平與處理前相比，IL-1β、IL-8和TNF-α分別降低9.8%、32.7%和25.3%。 結(jié)論: (1) Ct pORF5蛋白可誘導(dǎo)THP-1細(xì)胞產(chǎn)生前炎癥CKs：IL-8、IL-1β和TNF-α； (2) pORF5蛋白可激活p38/MAPK和ERK1/2/MAPK通路，活化的p38/MAPK和ERK1/2/MAPK通路通過TLR2參與pORF5誘導(dǎo)前炎癥細(xì)胞因子IL-8、IL-1β和TNF-α的產(chǎn)生。
[Abstract]:Objective: Chlamydia trachomatisCtis is an important pathogen of trachoma, urethritis and pelvic inflammatory disease. The immune pathological damage caused by excessive inflammatory reaction is an important part of Ct pathogenicity. However, the exact pathogenicity of Ct plasmid protein pORF5 is still unclear. This study attempts to explore whether the Ct plasmid protein pORF5 can induce the production of IL-8 and IL-1 尾 in human mononuclear cells (THP-1). TNF- 偽 and other proinflammatory cytokines, and their relationship with Toll receptor 2 (TLR2) and mitogen-activated protein kinase (MAPK) signaling pathway. To provide experimental basis for further study of pathogenesis of Ct. Methods: the constructed pGEX-6p-1/pORF5 recombinant plasmid was transformed into XL1-blue Escherichia coli to induce the expression of GST-pORF5 fusion protein. The fusion protein was purified by glutathione-agarose gel 4B. The purified GST-pORF5 fusion protein was then digested with PreScission Protease to prepare pORF5 target protein without GST. The concentration of pORF5 target protein was determined by BCA method. SDS-PAGE and Western blotting were used to identify the target protein .pORF5 protein after endotoxin removal. The THP-1 cells treated with PMA were stimulated with 2 渭 g / mL of pORF5 protein in vitro, and the cells were collected at 0 h, 6 h, 12 h, 24 h and 36 h, respectively. IL-8 IL-1 尾 and TNF- 偽. Western blotting were detected by ELISA to detect the activation of p38 JNK and ERK1/2. PD98059, a specific inhibitor of ERK1/2, was used at different concentrations of 10 ~ 10 ~ (30 渭 M). THP-1 cells were pretreated with p38 specific inhibitor SB203580 and TLR2 antibody, and then stimulated with 6 渭 g / mL pORF5 protein for 12 h. Effects of IL-1 尾 and TNF- 偽. Results: 1) the recombinant plasmid of pGEX-6p-1/pORF5 was transformed into E. coli XL1-blue. IPTG induced the expression of a 54KD GST-pORF5 fusion protein. The fusion protein was then digested with PreScission Protease protease to obtain a 28KD pORF5 protein with a molecular weight of about 28KD without GST-tag. Purity can reach more than 95%. (2) different concentrations of pORF5 protein could induce the production of IL-8 IL-1 尾 and TNF- 偽 in THP-1 cells. When the concentration of pORF5 protein was 2 渭 g / mL ~ 6 渭 g / mL ~ (-1), the production level of CKs was in a dose-dependent manner. When the concentration of pORF5 was higher than 6 渭 g / mL, the production levels of IL-1 尾 and TNF- 偽 decreased. The level of TNF- 偽 in THP-1 cells reached the peak at 6 h after stimulation with 6 渭 g / mL pORF5 protein, and decreased gradually after 12 h. The levels of IL-1 尾 and IL-8 reached the peak at 12 h. The results of Western blotting showed that pORF5-stimulated THP-1 cells could activate ERK1/2/MAPK and p38 / MAPK. The phosphorylation level of ERK1/2 pathway reached the peak at 15 minutes after stimulation of THP-1 cells by protein and the phosphorylation level of p38 pathway reached the peak at 30 minutes after stimulation of THP-1 cells. No phosphorylation was detected in SAPKR / JNK1 / 2 for more than 60 min. The results of Elisa showed that the production of IL-1 尾 -IL-8 and TNF- 偽 decreased in a dose-dependent manner. At the concentration of 30 渭 M, IL-8 decreased to 57% and 58. 8 渭 M, respectively. IL-1 尾 decreased to 22% and 45, respectively. TNF- 偽 decreased to 22.4% and 16.3a, respectively. THP-1 cells were pretreated with TLR2 antibody for 30 minutes and then stimulated with 6 渭 g / mL pORF5 protein for 12 h. The production levels of IL-8 and TNF- 偽 were significantly lower than those before treatment. The levels of IL-1 尾 IL-8 and TNF- 偽 were decreased by 9.8% and 25.3%, respectively. Conclusion: Ct pORF5 protein could induce THP-1 cells to produce pro-inflammatory CKS: IL-8 IL-1 尾 and TNF- 偽. 2) pORF5 protein can activate p38 / MAPK and ERK1/2/MAPK pathway. The activated p38 / MAPK and ERK1/2/MAPK pathway is involved in the production of IL-8 IL-1 尾 and TNF- 偽 by TLR2.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R374

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