【摘要】：目的用刀豆蛋白A(Con A)建立免疫性肝損傷模型,探討Tim-3和Th17細胞相關(guān)因子在發(fā)病過程中的動態(tài)變化及意義;靶向干預Tim-3通路后探討其對Th17細胞的影響。方法60只Wistar雄性大鼠隨機分為兩組,即模型組(50只)和對照組(10只)。模型組大鼠尾靜脈注射12.5mg/kg Con A,每周1次連續(xù)8周。對照組大鼠尾靜脈注射無菌PBS,每周1次連續(xù)8周。隨機從模型組中取10只大鼠,在第0周(0w)、第2周(2w)、第4周(4w)、第6周(6w)、第8周(8w)時對大鼠實施體外心尖取血1.5 ml,分離血清后于-20℃?zhèn)溆谩?周后隨機從模型組中取出20只大鼠,酒精浸泡20分鐘后無菌取脾制備脾淋巴細胞。在脾淋巴細胞中加入Tim-3的單克隆抗體來阻斷Tim-3通路;用Tim-3的重組配體來激活Tim-3通路,37℃、5%CO2培養(yǎng)箱中培養(yǎng)72h,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;ELISA法測血清中Tim-3、IL-17A、IL-6、TGF-β和IL-23的表達及上清液中IL-17A和IL-6的表達;免疫組化法測肝臟組織中Tim-3、IL-17A和ROR-γt蛋白的表達;實時定量PCR測各組脾淋巴細胞ROR-γt m RNA的表達。結(jié)果與對照組相比,模型組大鼠的ALT及AST水平均顯著升高(P0.05),ALB水平顯著下降(P0.05)。在模型組的不同階段中,HE染色檢測肝組織可見4w時開始偶見假小葉,6w時量明顯增多,而8w時發(fā)現(xiàn)有明顯的炎性細胞和肝臟損傷,鏡下假小葉較多見;ELISA法檢測相應(yīng)的細胞因子可見IL-17A、IL-6、IL-23的動態(tài)趨勢是先升高,4w后開始降低(P0.05);Tim-3的動態(tài)趨勢則是先降低,4w以后開始升高(P0.05);免疫組化檢測相應(yīng)的蛋白可見4w時IL-17A和ROR-γt蛋白的水平明顯升高,而Tim-3蛋白的水平明顯降低。靶向干預后,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;實時定量PCR顯示與阻斷對照組相比,阻斷實驗組中ROR-γt m RNA的水平明顯增加(P0.05)。結(jié)論Tim-3及Th17細胞參與了免疫性肝損傷的發(fā)生發(fā)展,其水平的變化可以在一定程度上反應(yīng)病情的嚴重程度,可望為免疫性肝損傷發(fā)生機制提供新的線索;免疫調(diào)控Tim-3通路可通過影響Th17細胞效應(yīng),進而影響肝損傷的程度,以期為臨床治療提供新的依據(jù)。
[Abstract]:Objective to establish an immunological liver injury model with concanavalin A (Con A), to investigate the dynamic changes and significance of Tim-3 and Th17 cytokines in the pathogenesis of liver injury, and to explore the effect of targeted intervention of Tim-3 pathway on Th17 cells. Methods 60 male Wistar rats were randomly divided into two groups: model group (50 rats) and control group (10 rats). Rats in the model group were injected with 12.5mg/kg Con A once a week for 8 weeks. Rats in control group were injected with sterile PBS, once a week for 8 weeks. Ten rats were randomly selected from the model group and were given extracorporeal apical blood for 1.5 ml, at week 0 (0w), week 2 (2w), week 4 (4w), week 6 (6w) and week 8 (8w). 20 rats were randomly removed from the model group at -20 鈩，

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