高迁移率族蛋白B1对人T淋巴细胞免疫功能影响的体外研究

发布时间：2018-04-28 19:10

本文选题：高迁移族蛋白B1 + 免疫　；参考：《中国人民解放军军医进修学院》2005年硕士论文

【摘要】：研究背景:脓毒症的发生与发展反映了体内抗炎/促炎免疫平衡失控的过程,T细胞免疫活性由Th1优势向Th2优势漂移预示获得性免疫功能抑制,形成免疫麻痹,诱发多脏器功能障碍甚至死亡,而导致免疫状态呈现抗炎优势的确切原因尚待阐明。高迁移率族蛋白B1(HMGB1)作为“晚期”炎症因子介导了脓毒症发病过程,广泛参与炎症反应调节和脏器功能损害,研究其病理生理效应和致病途径将有助于深化对脓毒症发病机制的认识,并为免疫平衡的调控提供潜在的干预目标。本研究将观察HMGB1在体外对人T淋巴细胞免疫功能的影响。方法:分离健康人静脉血PBMC细胞,在含10%小牛血清的1640中调整细胞浓度2×10~6/ml接种于细胞培养板。以20μg/ml PHA作为非特异性刺激剂激活细胞体外增殖。实验一:rhHMGB1作用剂量1～1000ng/ml,刺激时间12～48小时。细胞培养72小时后,以MTT法检测细胞数量和细胞活力,观察HMGB1对T淋巴细胞增殖的影响。采用四色流式细胞术(FCM)分析CD3~+淋巴细胞CD4表达和胞内因子IFN-γ、IL-4分泌阳性(Th1、Th2)的比例。实验二:rhHMGB1作用剂量10～1000ng/ml,刺激时间12、48小时。在rhHMGB1刺激12、48小时后收集细胞与培养液上清。IL-2、IL-2Rα基因表达水平的测定采用半定量逆转录聚合酶链反应(RT-PCR)法分析。提取细胞总RNA,逆转录为cDNA后以IL-2、IL-2Rα链为目的基因、β-actin为内参行PCR扩增。琼脂糖凝胶电泳回收PCR产物,照片判定积分光度值。上清IL-2、sIL-2R蛋白含量用ELISA法检测。结果:(1)500ng/ml以上剂量HMGB1作用48小时后,淋巴细胞增殖显著抑制,低于这一剂量对增殖影响不显著。(2)不同HMGB1刺激时间和作用剂量对CD4~+T淋巴细胞和Th1亚群比例未造成明显改变,但发现HMGB1能时间一剂量依赖性增
[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- 纬 -IL-4. Experiment 2: rhHMGB1 at a dose of 10 ~ 1000 ng / ml, stimulation time was 12 ~ 48 hours. The expression of IL-2R 伪 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 伪 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.
【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392

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