17-β-雌二醇对HMGB1过表达THP1细胞的细胞周期进程的影响  

The Effects of 17-β-estradiol on cell-cycle progression in HMGB1 overexpressed THP1 cells

作　　者：黄青松[1] 牛志国[1,2] 袁理想[1,3] 李焕焕[1,3] 宋向凤[1,2] 

机构地区：[1]新乡医学院微生物学教研室,新乡453003 [2]新乡医学院免疫学研究中心,新乡453003 [3]新乡医学院,临床医学三年级学生新乡453003

出　　处：《中国免疫学杂志》2011年第7期620-624,629,共6页Chinese Journal of Immunology

基　　金：河南省教育厅基金项目(No.200510472009);新乡医学院大学生科研创新计划项目(No.2010005)

摘　　要：目的:研究17-β-雌二醇联合HMGB1的过表达对人单核细胞系THP1细胞细胞周期和NF-κB转录因子的影响。方法:血清饥饿法同步化细胞周期后,利用脂质体法将pFLAG-CMV-HMGB1转染THP1细胞;10-9mol/L17-β-雌二醇刺激同步化THP1细胞16、30小时后,流式细胞术检测细胞周期分布,RT-qPCR检测Cyclin A、Cyclin D1的mRNA表达水平,免疫印迹检测HMGB1蛋白的表达;荧光素酶报告基因检测NF-κB的活性。结果:HMGB1过表达的THP1细胞中HMGB1蛋白的表达水平比正常THP1细胞要高3.7倍,血清饥饿法成功将THP1细胞周期同步化,同步化后G0/G1期细胞百分数由53%上升到78%;17-β-雌二醇刺激HMGB1过表达细胞30小时后出现Cyclin AmRNA表达上升为原来的7.9倍,而Cyclin D1 mRNA表达明显下降,G1期细胞百分比由53.11%下降为33.33%,S期细胞百分比由35.43%上升为53.91%,此改变在刺激后30小时达到最大值;NF-κB活性在17-β-雌二醇刺激HMGB1过表达细胞明显升高,且时间上提前于细胞周期动力学改变。结论:雌激素对THP1细胞周期调节依赖HMGB1,NF-κB可能参与其中。Objective：To research the effect of 17-β-estradiol allying with HMGB1 overexpression on the cell cycle and NF-κB signal pathway of human monocytes lines THP1 cells.Methods：The HMGB-1 expression vector was transfected into THP1 cells after cell cycle synchronization by serum starvation;Synchronized THP1 cells were stimulated by 10-9 mol/L 17-β-estradiol for 16 h and 30 h,the cell cycle phase distribution was analyzed by flow cytometry,and the mRNA expression level of Cyclin A and Cyclin D1 were analyzed by RT-qPCR,and the expression level of HMGB-1 protein was analyzed by western blot,and the activity of NF-κB was analyzed by luciferase reporter gene.Results：The HMGB1 protein level in the transfected cells increased 3.7-fold compared to the normal THP1 cells,and THP1 cells were synchronized by serum starvation successfully and the proportion of cells in the G1 phase increases from 53% to 78% after synchronization;After HMGB1 overexpression cells stimulated by 17-β-estradiol for 30h,mRNA level of Cyclin A increased 7.9-fold compared to the control group while Cyclin D1 mRNA level decreased by 41%,as well as the proportion of cells in the G1 phase decreased from 53.11% to 33.33% and that in the S phase increased from 35.43% to 53.91%,and this difference arrived to the maximum at 30h after stimulation;The activity of NFκB in HMGB1 overexpressed cells increased obviously after stimulated by estrogen,and this effection is ahead of the change of cell cycle kinetics.Conclusion：The regulation of estrogen to THP1 cell cycle is dependent on HMGB1,and NF-κB appears to be involved in this procedure.

关 键 词：17-Β-雌二醇 HMGB1蛋白 细胞周期 THP1细胞 

分 类 号：R392.9[医药卫生—免疫学]