【摘要】：目的建立免疫性肝損傷模型,探討Tim-3通路在阻斷或激活時(shí)對(duì)Th17細(xì)胞的影響。方法大鼠尾靜脈注射刀豆蛋白A(Con A)8周,建立免疫性肝損傷模型。無(wú)菌取脾臟制備脾淋巴細(xì)胞,取外周血分離血清后于-20℃保存,取肝臟組織放入4%的多聚甲醛中固定備用。在96孔板上將脾淋巴細(xì)胞平均分成5組,即阻斷實(shí)驗(yàn)組、阻斷對(duì)照組、激活實(shí)驗(yàn)組、激活對(duì)照組和Con A對(duì)照組。用Tim-3的單克隆抗體即Anti-Tim-3來(lái)阻斷Tim-3通路;用Tim-3的重組配體galectin-9來(lái)激活Tim-3通路,37℃、5%CO2培養(yǎng)箱中培養(yǎng)72 h,收集細(xì)胞上清液于-20℃保存。生化分析法測(cè)血清中ALT、AST和ALB的表達(dá);HE染色觀察肝臟組織的病理變化;免疫組化法測(cè)肝臟組織中IL-17A和ROR-γt蛋白的表達(dá);ELISA法測(cè)細(xì)胞上清液中IL-17A及IL-6的表達(dá);實(shí)時(shí)定量PCR測(cè)各組脾淋巴細(xì)胞ROR-γt mRNA的表達(dá)。結(jié)果與對(duì)照組相比,模型組HE染色可見(jiàn)明顯的炎性細(xì)胞浸潤(rùn)、肝臟組織損傷及較多的假小葉,免疫組化可見(jiàn)IL-17A和ROR-γt蛋白的表達(dá)明顯升高;與阻斷對(duì)照組相比,阻斷實(shí)驗(yàn)組中IL-17A和IL-6的水平升高(P0.05);與激活對(duì)照組相比,激活實(shí)驗(yàn)組中IL-17A和IL-6的水平降低(P0.05);實(shí)時(shí)定量PCR顯示與阻斷對(duì)照組相比,阻斷實(shí)驗(yàn)組中ROR-γt mRNA的表達(dá)明顯增加(P0.05)。結(jié)論免疫性肝損傷的發(fā)病與Th17細(xì)胞密切相關(guān),免疫調(diào)控Tim-3通路可通過(guò)影響Th17細(xì)胞效應(yīng),進(jìn)而參與免疫性肝損傷的發(fā)病機(jī)制。
[Abstract]:Objective to establish an immune liver injury model and to investigate the effect of Tim-3 pathway on Th17 cells during blocking or activating. Methods the immune liver injury model was established by injection of concanavalin A (Con A) into rat tail vein for 8 weeks. Spleen lymphocytes were prepared from sterile spleen, serum was isolated from peripheral blood and preserved at -20 鈩，

本文編號(hào)：2262783