高迁移率蛋白族B1通过调控p53表达抑制T细胞增殖  

High mobility group B1 inhibits T cell proliferation by regulating the expression of p53 protein

作　　者：贾敏[1,2] 张卉[3] 童亚林[1,2] 姚咏明[3] 

机构地区：[1]广西师范大学生命科学学院,广西桂林541002 [2]解放军第181医院烧伤整形中心,广西桂林541002 [3]解放军总医院第一附属医院全军创伤修复与组织再生重点实验室暨皮肤损伤修复与组织再生北京市重点实验室,北京100048

出　　处：《感染．炎症．修复》2016年第3期143-148,共6页Infection Inflammation Repair

基　　金：国家自然科学基金资助项目(81372054;81501698;81130035);国家重点基础研究发展计划项目(2012CB518102);广西科学研究与技术开发计划项目(1140003A-39)

摘　　要：目的:探讨高迁移率蛋白族B1(HMGB1)对T细胞的增殖抑制作用及其分子机制。方法:将培养的Jurkat细胞分为对照组、HMGB1(100 ng/ml)12、24、48 h组,HMGB1组加入HMGB1培养相应时间;将细胞分为对照组、HMGB1 10、100、1 000 ng/ml组,予不同浓度HMGB1培养24 h;采用细胞计数试剂盒(CCK-8)法检测各组细胞增殖率,采用实时荧光定量-聚合酶链反应(RT-PCR)和Western blot法检测各组细胞中p53m RNA、磷酸化p53及p53总蛋白的表达水平。将载有p53 sh RNA、p53 m RNA表达序列及空白质料的病毒转染入Jurkat细胞中,并选择100 ng/ml HMGB1刺激24 h,采用同样方法检测细胞增殖率。最后,将细胞分为正常组、HMGB1组、SB203580[p38丝裂原活化蛋白激酶(p38MAPK)抑制剂]+HMGB1组进行相应处理。收集各组细胞,RT-PCR和Western blot法检测p53 m RNA、磷酸化p53及p53总蛋白的表达水平。结果:HMGB1(100ng/ml)刺激细胞24、48 h后细胞增殖率降低,较正常组差异有显著性(P<0.05);100、1 000 ng/ml HMGB1刺激细胞24 h后,增殖率明显下降,与正常组相比差异具有统计学意义(P<0.05)。HMGB1(100 ng/ml)刺激细胞后,p53 m RNA、磷酸化及总蛋白表达水平在24、48 h逐步上升,且有明显统计学意义(P<0.05);HMGB1刺激24 h,10、100 ng/ml HMGB1刺激组细胞p53 m RNA、p53磷酸化及总蛋白表达水平较正常组显著上升,但1 000 ng/ml HMGB1没有明显变化;在不同转染组中,予HMGB1刺激细胞,空载组增殖率降低,而p53 sh RNA表达组细胞增殖趋于正常,p53 m RNA过表达组增殖率较空载组下降更为明显。Jurkat细胞在p38 MAPK阻断剂和HMGB1共同作用后,p53 m RNA、磷酸化及总蛋白表达水平比HMGB1刺激组明显下降(P<0.05)。结论:胞外HMGB1可通过诱导p38 MAPK信号通路激活p53蛋白,抑制T细胞增殖。Objective：To investigate the inhibitory effect of high mobility group box-1 protein （HMGB1） on T cell proliferation and the regulation mechanisms thereof. Methods： Cultured Jurkat cells were divided into 4 groups： control group, HMGB1 treatment （100 ng/ml） for 12, 24 and 48 hours groups. Cells were also divided into control group, 10, 100 and 1 000 ng/ml HMGB1 treated groups, and then stimulated for 24 hours. Proliferation rates of cells were measured using cell counting kit （CCK-8）. The expression levels of p53 mRNA, phosphorylated p53 and p53 protein were determined by real-time polymerase chain reaction （RT-PCR） and Western blot respectively. Jurkat cells were transfected with lentivirus containing p53 shRNA, p53 mRNA sequence expressing plasmids or control vector. The cells were afterwards stimulated with HMGB1 （100 ng/ml）for 24 hours, and subjected to cell proliferation assay with the same methods mentioned above. In addition, Jurkat cells were divided into 3 groups： control group, HMGB1 group and HMGBl＋SB203580 group, and stimulated with indicated reagents for 24 hours. The expression levels of p53 mRNA; phosphorylated p53 and p53 protein were detected by RT-PCR and Western blot. Results： Compared to the control group, the proliferation rates of cells were decreased after HMGB 1 （100 ng/ml） stimulation for 24 and 48 hours （P〈0.05）. In dose-devendent stimulations, nroliferation rates were reduced in treatment with 100 or 1 000 ng/ml HMGB1 groups （P〈0.05）. The expression levels of p53 mRNA, phosphorylated p53 and p53 protein increased in cells after stimulation with 100 ng/ml HMGB 1 for 24 and 48 hours （P〈0.05）. The expression levels increased obviously in 10 and 100 ng/ml HMGB1 treated cells 24 hours later, but the level was not changed significantly in HMGB1 1 000 ng/ml group. After transfection, the proliferation rates of vector-expressing cells decreased obviously after HMGB1 stimulation. The proliferation of p53 shRNA-expressing cells was basically no

关 键 词：高迁移率族蛋白B1 P53 P38丝裂原活化蛋白激酶 T细胞增殖 

分 类 号：R392.12[医药卫生—免疫学]