【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對Th17細胞的影響。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。無菌取脾臟制備脾淋巴細胞,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孔板上將脾淋巴細胞平均分成5組,即阻斷實驗組、阻斷對照組、激活實驗組、激活對照組和Con A對照組。用Tim-3的單克隆抗體即Anti-Tim-3來阻斷Tim-3通路;用Tim-3的重組配體galectin-9來激活Tim-3通路,37℃、5%CO2培養(yǎng)箱中培養(yǎng)72 h,收集細胞上清液于-20℃保存。生化分析法測血清中ALT、AST和ALB的表達;HE染色觀察肝臟組織的病理變化;免疫組化法測肝臟組織中IL-17A和ROR-γt蛋白的表達;ELISA法測細胞上清液中IL-17A及IL-6的表達;實時定量PCR測各組脾淋巴細胞ROR-γt mRNA的表達。結果與對照組相比,模型組HE染色可見明顯的炎性細胞浸潤、肝臟組織損傷及較多的假小葉,免疫組化可見IL-17A和ROR-γt蛋白的表達明顯升高;與阻斷對照組相比,阻斷實驗組中IL-17A和IL-6的水平升高(P0.05);與激活對照組相比,激活實驗組中IL-17A和IL-6的水平降低(P0.05);實時定量PCR顯示與阻斷對照組相比,阻斷實驗組中ROR-γt mRNA的表達明顯增加(P0.05)。結論免疫性肝損傷的發(fā)病與Th17細胞密切相關,免疫調(diào)控Tim-3通路可通過影響Th17細胞效應,進而參與免疫性肝損傷的發(fā)病機制。
[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，

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