本文選題：高遷移族蛋白B1 + 免疫　； 參考：《中國人民解放軍軍醫(yī)進修學(xué)院》2005年碩士論文

【摘要】：研究背景:膿毒癥的發(fā)生與發(fā)展反映了體內(nèi)抗炎/促炎免疫平衡失控的過程,T細胞免疫活性由Th1優(yōu)勢向Th2優(yōu)勢漂移預(yù)示獲得性免疫功能抑制,形成免疫麻痹,誘發(fā)多臟器功能障礙甚至死亡,而導(dǎo)致免疫狀態(tài)呈現(xiàn)抗炎優(yōu)勢的確切原因尚待闡明。高遷移率族蛋白B1(HMGB1)作為“晚期”炎癥因子介導(dǎo)了膿毒癥發(fā)病過程,廣泛參與炎癥反應(yīng)調(diào)節(jié)和臟器功能損害,研究其病理生理效應(yīng)和致病途徑將有助于深化對膿毒癥發(fā)病機制的認識,并為免疫平衡的調(diào)控提供潛在的干預(yù)目標。本研究將觀察HMGB1在體外對人T淋巴細胞免疫功能的影響。 方法:分離健康人靜脈血PBMC細胞,在含10%小牛血清的1640中調(diào)整細胞濃度2×10~6/ml接種于細胞培養(yǎng)板。以20μg/ml PHA作為非特異性刺激劑激活細胞體外增殖。實驗一:rhHMGB1作用劑量1～1000ng/ml,刺激時間12～48小時。細胞培養(yǎng)72小時后,以MTT法檢測細胞數(shù)量和細胞活力,觀察HMGB1對T淋巴細胞增殖的影響。采用四色流式細胞術(shù)(FCM)分析CD3~+淋巴細胞CD4表達和胞內(nèi)因子IFN-γ、IL-4分泌陽性(Th1、Th2)的比例。實驗二:rhHMGB1作用劑量10～1000ng/ml,刺激時間12、48小時。在rhHMGB1刺激12、48小時后收集細胞與培養(yǎng)液上清。IL-2、IL-2Rα基因表達水平的測定采用半定量逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(RT-PCR)法分析。提取細胞總RNA,逆轉(zhuǎn)錄為cDNA后以IL-2、IL-2Rα鏈為目的基因、β-actin為內(nèi)參行PCR擴增。瓊脂糖凝膠電泳回收PCR產(chǎn)物,照片判定積分光度值。上清IL-2、sIL-2R蛋白含量用ELISA法檢測。 結(jié)果:(1)500ng/ml以上劑量HMGB1作用48小時后,淋巴細胞增殖顯著抑制,低于這一劑量對增殖影響不顯著。(2)不同HMGB1刺激時間和作用劑量對CD4~+T淋巴細胞和Th1亞群比例未造成明顯改變,但發(fā)現(xiàn)HMGB1能時間一劑量依賴性增
[Abstract]:Background: the occurrence and development of sepsis reflect the process of uncontrolled anti-inflammatory / pro-inflammatory immune balance in vivo. The shift of T cell immune activity from Th1 dominance to Th2 dominance indicates that acquired immune function is inhibited and immune paralysis is formed. Multiple organ dysfunction and even death were induced, and the exact reasons for the anti-inflammatory superiority of immune state remained to be elucidated. High mobility group protein B1 HMGB1, as a "late" inflammatory factor, mediates the pathogenesis of sepsis, and extensively participates in the regulation of inflammatory response and organ dysfunction. The study of its pathophysiological effects and pathogenetic pathways will help to deepen the understanding of the pathogenesis of sepsis and provide a potential intervention target for the regulation of immune balance. The aim of this study was to observe the effect of HMGB1 on the immune function of human T lymphocytes in vitro. Methods: PBMC cells were isolated from healthy human vein blood and inoculated on the cell culture plate in 1640 containing 10% calf serum. The cell concentration was adjusted by 2 脳 10~6/ml. Cell proliferation was activated by 20 渭 g/ml PHA as a nonspecific stimulant in vitro. Experiment 1: rhHMGB1 at a dose of 1 ~ 1000 ng / ml, stimulation time was 12 ~ 48 hours. After 72 hours of cell culture, the number and viability of T lymphocytes were detected by MTT assay, and the effect of HMGB1 on T lymphocyte proliferation was observed. Four color flow cytometry (FCM) was used to analyze the expression of CD4 in CD3 ~ ~ lymphocytes and the ratio of IL-4 secreted by IFN- 緯 -IL-4. Experiment 2: rhHMGB1 at a dose of 10 ~ 1000 ng / ml, stimulation time was 12 ~ 48 hours. The expression of IL-2R 偽 gene in the supernatant of cell and culture medium was detected by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Total RNAs were extracted, reverse transcripted to cDNA, IL-2R 偽 chain was used as target gene, 尾 -actin was used as internal reference for PCR amplification. PCR products were recovered by agarose gel electrophoresis. The content of IL-2sIL-2R in supernatant was detected by ELISA method. Results the proliferation of lymphocytes was significantly inhibited after 48 hours of exposure to HMGB1 at a dose of 500ng / ml or more than that of HMGB1. (2) the ratio of CD4T lymphocytes and Th1 subsets to CD4T lymphocytes and Th1 subsets was not significantly changed by different HMGB1 stimulation time and dose. But it was found that HMGB1 energy was increased in a dose-dependent manner.
【學(xué)位授予單位】：中國人民解放軍軍醫(yī)進修學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R392

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