调控Tim-3通路在免疫性肝损伤大鼠中对Th17细胞的影响  

Effect of regulation of Tim-3 pathway on Th17 cells in rats with immunological hepatic injury

作　　者：张文[1] 张蓓[1] 李秀梅[1] 沈若武[1] 丛蓓蓓[1] 杨丽华[1] 孙东君 王建伟[1] 

机构地区：[1]青岛大学医学院免疫教研室,山东青岛266071

出　　处：《中国实验动物学报》2015年第1期7-12,共6页Acta Laboratorium Animalis Scientia Sinica

基　　金：国家自然科学基金资助项目(编号:81072398);山东省科技发展计划(编号:2009GG10002044);山东省自然科学基金资助(编号:ZR2014CM037)

摘　　要：目的建立免疫性肝损伤模型,探讨Tim-3通路在阻断或激活时对Th17细胞的影响。方法大鼠尾静脉注射刀豆蛋白A(Con A)8周,建立免疫性肝损伤模型。无菌取脾脏制备脾淋巴细胞,取外周血分离血清后于-20℃保存,取肝脏组织放入4%的多聚甲醛中固定备用。在96孔板上将脾淋巴细胞平均分成5组,即阻断实验组、阻断对照组、激活实验组、激活对照组和Con A对照组。用Tim-3的单克隆抗体即Anti-Tim-3来阻断Tim-3通路;用Tim-3的重组配体galectin-9来激活Tim-3通路,37℃、5%CO2培养箱中培养72 h,收集细胞上清液于-20℃保存。生化分析法测血清中ALT、AST和ALB的表达;HE染色观察肝脏组织的病理变化;免疫组化法测肝脏组织中IL-17A和ROR-γt蛋白的表达;ELISA法测细胞上清液中IL-17A及IL-6的表达;实时定量PCR测各组脾淋巴细胞ROR-γt mRNA的表达。结果与对照组相比,模型组HE染色可见明显的炎性细胞浸润、肝脏组织损伤及较多的假小叶,免疫组化可见IL-17A和ROR-γt蛋白的表达明显升高;与阻断对照组相比,阻断实验组中IL-17A和IL-6的水平升高(P<0.05);与激活对照组相比,激活实验组中IL-17A和IL-6的水平降低(P<0.05);实时定量PCR显示与阻断对照组相比,阻断实验组中ROR-γt mRNA的表达明显增加(P<0.05)。结论免疫性肝损伤的发病与Th17细胞密切相关,免疫调控Tim-3通路可通过影响Th17细胞效应,进而参与免疫性肝损伤的发病机制。Objective To establish a rat model of immunological hepatic injury and to explore the effect of bloc- king or activating Tim-3 pathway on Thl7 cells in the rats. Methods Forty SPF male Wistar rats （body weight 180 ＋20 g） were used in this study. The rat model of inamunological hepatic injury was established by injecting 12.5 mg/kg con- canavalin A （Con A） through the tail vein once a week for eight weeks. Splenic lymphocytes were isolated. Serum was sep- arated and stored in -20~C. Liver tissue samples were fixed in 4% paraformaldehyde for further study.; The splenic lym- phocytes were divided into 5 groups ： blocking group, blocking control group, activating group, activating control group and ConA control group. The Tim-3 pathway was blocked by anti-Tim-3 while activated by recombinant galectin-9, and cultured 72 h under 37~C , 5% CO2 environment. The cell culture supernatants were saved at -20℃ for later use. . Biochemical a- nalysis was performed to determine the expression of serum ALT, AST and ALB. Morphological changes of the liver were examined by his topathology using HE staining. Immunohistochemical staining was used to detect the expression of IL-17A and ROR-γt proteins in the liver tissues. Enzyme-linked immunosorbent assay （ELISA） was used to assess the IL-17A and IL-6 in the cell supernatants while real time PCR was applied to determine the expression of ROR-γt mRNA in splenic lym- phocytes. Results Compared with the control group, inflammatory cell infiltration, hepatic tissue injury and many pseud- olobules were easily found in the liver tissue, and immunohistochemical examination showed significantly increased levels of IL-17A and ROR-γt proteins in the liver tissues of the model group. The expressions of IL-17A and IL-6 in the blocking group were increased than that of the blocking control group （ P 〈 0.05 ） , while the levels of IL-17A and IL-6 in the activa- ting group were lower when compared with that of the activating control group （ P 〈 0.05 ）. Compared wit

关 键 词：免疫性肝损伤 Tim-3通路 TH17细胞 大鼠 

分 类 号：Q95-33[生物学—动物学] R392.12[医药卫生—免疫学]