本文選題：高遷移族蛋白B1 + 免疫�。� 參考：《中國(guó)人民解放軍軍醫(yī)進(jìn)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(xì)胞免疫活性由Th1優(yōu)勢(shì)向Th2優(yōu)勢(shì)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(shì)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(duì)膿毒癥發(fā)病機(jī)制的認(rèn)識(shí),并為免疫平衡的調(diào)控提供潛在的干預(yù)目標(biāo)。本研究將觀察HMGB1在體外對(duì)人T淋巴細(xì)胞免疫功能的影響。 方法:分離健康人靜脈血PBMC細(xì)胞,在含10%小牛血清的1640中調(diào)整細(xì)胞濃度2×10~6/ml接種于細(xì)胞培養(yǎng)板。以20μg/ml PHA作為非特異性刺激劑激活細(xì)胞體外增殖。實(shí)驗(yàn)一:rhHMGB1作用劑量1～1000ng/ml,刺激時(shí)間12～48小時(shí)。細(xì)胞培養(yǎng)72小時(shí)后,以MTT法檢測(cè)細(xì)胞數(shù)量和細(xì)胞活力,觀察HMGB1對(duì)T淋巴細(xì)胞增殖的影響。采用四色流式細(xì)胞術(shù)(FCM)分析CD3~+淋巴細(xì)胞CD4表達(dá)和胞內(nèi)因子IFN-γ、IL-4分泌陽性(Th1、Th2)的比例。實(shí)驗(yàn)二:rhHMGB1作用劑量10～1000ng/ml,刺激時(shí)間12、48小時(shí)。在rhHMGB1刺激12、48小時(shí)后收集細(xì)胞與培養(yǎng)液上清。IL-2、IL-2Rα基因表達(dá)水平的測(cè)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(xì)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(kuò)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(cè)。 結(jié)果:(1)500ng/ml以上劑量HMGB1作用48小時(shí)后,淋巴細(xì)胞增殖顯著抑制,低于這一劑量對(duì)增殖影響不顯著。(2)不同HMGB1刺激時(shí)間和作用劑量對(duì)CD4~+T淋巴細(xì)胞和Th1亞群比例未造成明顯改變,但發(fā)現(xiàn)HMGB1能時(shí)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(guó)人民解放軍軍醫(yī)進(jìn)修學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R392

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