人肝再生增强因子对体外单个核细胞PKCα-NF-кB-第三军医大学学报.DOCVIP

人肝再生增强因子对体外单个核细胞PKCα-NF-кB 及IL-2的影响 王新国，刘杞，孙航（重庆医科大学：第二附属医院、重庆医科大学病毒性肝炎研究所、教育部感染性疾病分子生物学重点实验室，重庆 400010） 摘要： 目的 观察人肝再生增强因子（human Augmenter of liver regeneration，hALR） 对人外周血单个核细胞（Peripheral blood mononuclear cell，PBMC）PKCα-NF-кB及IL-2的影响，以期探讨hALR免疫抑制机制。方法 用梯度离心法分离正常人PBMC，分成正常对照组（N）、ConA刺激组（ConA组）和hALR干预组（ConA+hALR组）。ConA组用5μg/mlConA刺激细胞增殖和分泌细胞因子IL-2；hALR干预组用 30μg/mlhALR对ConA刺激进行干预。于细胞培养0、8、16、32、40h时，用MTT法测定细胞增殖程度，Weston blot检测细胞内信号通路PKCα-NF-кB表达含量,Fluo-3AM装载检测胞内钙离子浓度；用双抗夹心酶联法检测上清细胞因子IL-2含量。结果 ConA组细胞增殖逐渐加强，16h时具有统计学意义（Ρ0.05），PKCα、NF-кB表达上调及上清IL-2含量增加趋势一致，在16h达到高峰（Ρ0.05）。ALR干预组细胞没有增殖，PKCα-NF-кB表达及上清IL-2含量最高峰后移，在32h。每个时间点各组细胞内钙离子没有差别。结论 ALR可能通过抑制PKCα-NF-кB通路抑制IL-2的产生速度抑制免疫。 关键词：人肝再生增强因子；外周血单个核细胞；PKCα-NF-кB；IL-2 中图法分类号：R392．1 文献标识码：A The effect of human Augmenter of liver regeneration on PKCα-NF-кB and IL-2 for Mononuclear cell in vitro WANG Xin-guo，LIU Qi，SUN Hang(Institute for Viral Hepatitis Chongqing University of Medical Sciences ,Key Laboratories for Molecular Biology of Infectious Diseases affiliated to Ministry of Education of China , Second Affiliated Hospital, Chongqing University of Medical Sciences，Chongqing 400010,China) Abstract Objective To observe the effect of human Augmenter of liver regeneration(hALR) on PKCαNF-кB and IL-2 for human peripheral blood mononuclear cells(PBMC) and to evaluate the relevant mechanism of hALR’s immunosuppression. Methods PBMCs were isolated from healthy volunteers by gradient centrifugation and were divided into Normal control group(N),ConA stimulated group(ConA group) and hALR interference group(ConA+hALR group). Cells in the ConA group were stimulated with ConA (5μg/ml) to proliferate and to secret IL-2. Stimulation of ConA in the hALR interference group was interfered by hALR at the level of 30μg/ml. After cells were cultured for a series of time of 0,8,16,32 and 40h, the proliferation of cells stimulated with ConA was asserted by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazol