本文選題：T細(xì)胞免疫球蛋白黏蛋白4　切入點(diǎn)：Kupffer細(xì)胞　出處：《重慶醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:探討阻斷Kupffer細(xì)胞(KCs)TIM-4對(duì)誘導(dǎo)小鼠原位肝移植術(shù)后調(diào)節(jié)性T細(xì)胞(i Treg)的產(chǎn)生以及相關(guān)機(jī)制的研究。方法:1.小鼠原位肝移植術(shù)后KCs TIM-4的表達(dá)以及阻斷TIM-4對(duì)肝移植急性排斥反應(yīng)的影響采用改良的Kamada“二袖套管”法建立C57BL/6→C3H小鼠原位肝移植急性排斥反應(yīng)模型。在未處理小鼠中,小鼠僅做原位肝移植而未做任何處理(LT組),設(shè)sham為對(duì)照組(僅開腹手術(shù)暴露門靜脈)。免疫組化檢測(cè)LT組和sham組移植術(shù)后1d、2d、3d受體肝組織中KCs的活化情況。分離受體KCs,Western blot和RT-PCR檢測(cè)移植術(shù)后1d、3d、7d KCs TIM-4蛋白以及m RNA表達(dá)情況。在處理小鼠中,實(shí)驗(yàn)動(dòng)物隨機(jī)分為3組:sham組,小鼠開腹手術(shù)暴露門靜脈,并經(jīng)門靜脈注入1m L PBS;control m Ab組,于供體冷血TIM-4 m Ab組,于供體冷血期經(jīng)門靜脈注入含抗TIM-4抗體(0.35mg/只)的1m L PBS。各組均于術(shù)后3d經(jīng)尾靜脈再次分別注入上述液體。小鼠于移植后7d麻醉下開腹穿刺取腹主動(dòng)脈血0.5m L,然后處死小鼠,取下中葉肝組織,余固定包埋。提取各組肝臟KCs,激光共聚焦檢測(cè)KCs TIM-4阻斷情況;全自動(dòng)生化分析儀檢測(cè)各組小鼠血清AST、ALT、TBIL;ELISA和Western blot檢測(cè)各組小鼠肝組織勻漿TNF-α、IFN-γ、CCL2、CXCL2表達(dá)水平;TUNEL法檢測(cè)各組肝組織細(xì)胞凋亡情況;Western blot檢測(cè)各組KCs p-P65和p-P38蛋白表達(dá)水平。2.阻斷KCs TIM-4對(duì)初始CD4+T細(xì)胞分化以及IL-4/STAT6信號(hào)通路的影響分離模型LT組KCs,流氏分選出TIM-4+KCs(預(yù)先加或不加0.5mg/L TIM-4 m Ab處理)。分離和純化WT C3H小鼠脾臟初始CD4+T細(xì)胞。將上述兩種細(xì)胞按1:1比例進(jìn)行共培養(yǎng)3d。處理分為三組:control組,作為對(duì)照組,不予以處理;control m Ab組,預(yù)先加入0.5mg/L control m Ab處理;TIM-4 m Ab組,預(yù)先加入0.5mg/L TIM-4 m Ab處理。收集各組T細(xì)胞以及上清液。CFSE法檢測(cè)各組CD4+T細(xì)胞增殖情況;ELISA檢測(cè)各組上清IL-4、IL-6、IL-13表達(dá)水平;流氏細(xì)胞術(shù)檢測(cè)各組CD4+CD25+Foxp3+T細(xì)胞的產(chǎn)生情況。分離模型LT組KCs,流氏分選出TIM-4+和TIM-4-KCs,并按1:1比例與初始CD4+T細(xì)胞共培養(yǎng)3d,分為三組:control組,作為對(duì)照組,僅初始CD4+T細(xì)胞單獨(dú)培養(yǎng);TIM-4+組,TIM-4+KCs與CD4+T細(xì)胞共培養(yǎng)組;TIM-4-組,TIM-4-KCs與CD4+T細(xì)胞共培養(yǎng)組。Western blot分析T細(xì)胞p-STAT6蛋白表達(dá)情況;上述各組用0.5mg/L TIM-4m Ab+/-處理,Western blot分析T細(xì)胞p-STAT6蛋白表達(dá)情況;在TIM-4+組中,用0.5mg/L TIM-4 m Ab+/-和IL-4+/-處理,Western blot分析T細(xì)胞p-STAT6蛋白表達(dá)情況。3.阻斷TIM-4+KCs TIM-4功能誘導(dǎo)的i Treg對(duì)肝移植排斥反應(yīng)以及小鼠生存率的影響分離模型LT組KCs,流氏分選出TIM-4+KCs(預(yù)先用TIM-4 m Ab處理)與初始CD4+T細(xì)胞進(jìn)行共培養(yǎng),3d后獲取CD4+CD25+Foxp3+Treg細(xì)胞,調(diào)整i Treg細(xì)胞濃度1×106個(gè)/m L,將所得細(xì)胞于供體冷血期經(jīng)門靜脈注入i Treg組,sham組以及LT組注入相應(yīng)的PBS作為對(duì)照。部分小鼠于移植后7d麻醉下開腹穿刺取腹主動(dòng)脈血0.5m L,然后處死小鼠,取下中葉肝組織,余固定包埋。全自動(dòng)生化分析儀檢測(cè)各組小鼠血清AST、ALT、TBIL;HE檢測(cè)各組小鼠肝組織病理變化情況;余下各組小鼠作為觀察生存期之用,觀察期以小鼠死亡為終點(diǎn),并做Log-Rank生存資料分析。結(jié)果:1.(1)小鼠移植術(shù)后肝組織KCs的活化數(shù)隨著時(shí)間變化逐漸增多。術(shù)后1d、3d、7d KCs TIM-4蛋白相對(duì)表達(dá)水平分別為0.31±0.04、0.86±0.05、0.77±0.03,明顯高于sham組的0.11±0.03(P0.05),m RNA相對(duì)表達(dá)水平分別1.96±0.07、5.25±0.23、4.02±0.17,顯著高于sham組的0.98±0.03(P0.05)。(2)在處理小鼠分組中,激光共聚焦檢測(cè)KCs TIM-4表達(dá)情況發(fā)現(xiàn),TIM-4 m Ab組TIM-4熒光表達(dá)強(qiáng)度明顯低于control m Ab組(P0.05)。術(shù)后7d,與sham組比較,control m Ab組血清肝功AST、ALT、TBIL以及肝組織勻漿炎癥因子TNF-α、IFN-γ、CCL2、CXCL2水平明顯增高(P0.05),而TIM-4 m Ab組上述指標(biāo)水平明顯低于control m Ab組(P0.05)。Western blot檢測(cè)肝臟組織炎癥因子蛋白表達(dá)和上述結(jié)果一致。各組肝組織細(xì)胞凋亡指數(shù)(AI),TIM-4 m Ab組AI值(11.04±2.28)明顯低于TIM-4 m Ab組(29.23±2.56)(P0.05)。TIM-4 m Ab組p-P65以及p-P38蛋白表達(dá)水平分別為0.82±0.23、0.54±0.10,明顯低于control m Ab組的1.39±0.27、1.07±0.23(P0.05)。2.(1)KCs與CD4+T細(xì)胞按共培養(yǎng)發(fā)現(xiàn),control、control m Ab以及TIM-4 m Ab組CD4+T細(xì)胞的增殖率分別為(32.3±1.2)%、(31.1±1.4)%、(16.9±0.5)%。ELISA檢測(cè)各組上清IL-4、IL-6、IL-13分泌水平發(fā)現(xiàn),阻斷TIM-4+KCs TIM-4可明顯減少上述炎癥因子的分泌(P0.05)。流式檢測(cè)各組CD4+CD25+Foxp3+T細(xì)胞產(chǎn)生情況,control組、control m Ab組以及TIM-4 m Ab組CD25、Foxp3雙陽性細(xì)胞分別為(12.8±0.3)%、(13.3±0.5)%、(28.1±0.4)%,后者明顯高于前兩組(P0.05)。(2)TIM-4+組T細(xì)胞p-STAT6相對(duì)蛋白表達(dá)水平(1.75±0.06)明顯高于TIM-4-組(0.60±0.06)(P0.05)。然而TIM-4+KCs與CD4+T細(xì)胞共培養(yǎng)時(shí),加入或未加入TIM-4 m Ab的p-STAT6蛋白表達(dá)分別為2.29±0.25、1.30±0.11,兩者比較差異有統(tǒng)計(jì)學(xué)意義(P0.05),此現(xiàn)象在TIM-4-組間比較無統(tǒng)計(jì)學(xué)上的差異(P0.05)。另外,向培養(yǎng)液中加入IL-4(0.25mg/L)發(fā)現(xiàn),阻斷KCs TIM-4的表達(dá),外源性加入IL-4可明顯增高CD4+T細(xì)胞p-STAT6蛋白的表達(dá)(P0.05)。3.(1)LT組肝功指標(biāo)明顯高于sham組,差異具有統(tǒng)計(jì)學(xué)意義(P0.05),而i Treg組肝功指標(biāo)明顯好轉(zhuǎn)(P0.05)。肝組織病理學(xué)檢查,i Treg組RAI平均得分為3.97±0.67,明顯低于LT組8.47±0.90(P0.05)。(2)i Treg組小鼠平均存活時(shí)間(55.7±5.5)d明顯長(zhǎng)于LT組平均存活時(shí)間(14.5±3.7)d(P0.05)。結(jié)論:1.小鼠移植術(shù)后,活化的KCs TIM-4的表達(dá)水平逐漸增高。阻斷KCs TIM-4可能通過影響NF-κB以及MAPK通路減少炎癥因子的釋放,改善肝組織排斥反應(yīng)的損傷。2.阻斷KCs TIM-4通過抑制IL-4/STAT6信號(hào)通路促進(jìn)初始CD4+T細(xì)胞向i Treg細(xì)胞的分化。3阻斷TIM-4+KCs TIM-4功能誘導(dǎo)的i Treg細(xì)胞可明顯改善急性排斥反應(yīng)損傷以及提高小鼠生存率。
[Abstract]:Objective: To investigate the blocking of Kupffer cells (KCs) on TIM-4 induced mouse orthotopic liver transplantation regulatory T cells (I Treg) on the production and the related mechanism. Methods: 1. mice after orthotopic liver transplantation KCs the expression of TIM-4 and the blocking effect of TIM-4 on acute rejection after liver transplantation by modified Kamada two cuff "method to establish the C57BL/6 and C3H mice orthotopic liver transplantation model of acute rejection in untreated mice, mice only orthotopic liver transplantation without any treatment (LT group), sham group (only laparotomy exposed portal vein). Immunohistochemical detection of LT transplantation group and sham group after 1D, 2D, activation of 3D receptor in the liver tissue of KCs. Blot and Western receptor KCs separation, RT-PCR detection after transplantation of 1D, 3D, 7d KCs TIM-4 and m RNA protein expression. In mice, the experimental animal were randomly divided into 3 groups: Group sham, mice open Surgical exposure of portal vein, and portal vein injection of 1m L PBS; control m Ab group, TIM-4 M group Ab from the donor blood from the donor, cold period injected through the portal vein with anti TIM-4 antibody (0.35mg/) 1m L PBS. was observed after 3D through tail vein once again were injected into the liquid in mice. 7d after transplantation anesthesia abdominal puncture and abdominal aortic blood 0.5m L, then the mice were killed, the liver tissue under the middle, more than fixed embedding. Extracted from liver KCs, confocal laser detection KCs TIM-4 blocking; the mice serum AST detection, automatic biochemical analyzer, ALT, TBIL; liver tissue homogenates were detected by TNF-. Mice ELISA and Western blot IFN- gamma, CCL2, CXCL2 expression level; detection of apoptotic cells in liver tissues were detected by TUNEL method; Western blot KCs p-P65 and p-P38 protein expression level of.2. KCs TIM-4 CD4+T on the initial block of cell differentiation and IL-4/STAT6 signaling The effect of LT pathway separation model group KCs's flow sorting of TIM-4+KCs (with or without 0.5mg/L TIM-4 m Ab in advance). The separation and purification of WT C3H mouse spleen CD4+T cells co cultured with 3D.. The initial treatment were divided into three groups of the two kinds of cells according to the ratio of 1:1: control group as the control group, no to deal with; control m Ab 0.5mg/L Control M group, pretreatment with Ab treatment; TIM-4 m Ab 0.5mg/L TIM-4 M group, pretreatment with Ab treatment. T cells were collected and supernatant were detected by.CFSE CD4+T cell proliferation; ELISA IL-4 IL-6, was detected, the expression level of IL-13; production flow cytometry detected CD4+CD25+Foxp3+T cells. Separation model LT group KCs, TIM-4+ and TIM-4-KCs's flow sorting out, and according to the proportion of 1:1 and initial CD4+T cells co cultured with 3D, divided into three groups: group control, as control group, cultured alone and only the initial CD4+T cells; TIM-4 + group, group TIM-4+KCs were co cultured with CD4+T cells; TIM-4- group,.Western group and blot T analysis of the expression of p-STAT6 protein in TIM-4-KCs cells and CD4+T cells were co cultured with 0.5mg/L TIM-4m; the group Ab+/-, Western blot analysis of T cells p-STAT6 protein expression; in group TIM-4+, 0.5mg/L TIM-4 m Ab+/- and IL-4+/- Western. Blot analysis of T cells p-STAT6 protein expression of.3. blocked TIM-4+KCs induced I TIM-4 Treg rejection and influence the survival rate of mice in liver transplantation model of separation of group LT KCs, TIM-4+KCs's flow separation (TIM-4 m pre Ab treatment) were co cultured with naive CD4+T cells, 3D CD4+CD25+Foxp3+Treg cells to obtain, adjust the I Treg the cell concentration of 1 * 106 /m L, the cells from the donor blood through the portal vein injection of I Treg group, sham group and LT group were injected with corresponding PBS as control. Part of the mouse to move After planting 7d anesthesia abdominal puncture and abdominal aortic blood 0.5m L, then the mice were killed, the liver tissue under the middle, more than fixed embedding. The mice serum AST detection, automatic biochemical analyzer ALT, TBIL; detection of pathological changes in liver tissues of mice HE; for the remaining mice in each group to observe the survival time for the observation period in mice death as the end point, and Log-Rank analysis of survival data. Results: 1. (1) mice after transplantation the number of activation of liver KCs increased gradually with the change of time. After 1D, 3D, 7d KCs and TIM-4 protein expression levels were 0.31 + 0.04,0.86 + 0.05,0.77 + 0.03, significantly higher than that of sham group 0.11 + 0.03 (P0.05), m RNA relative expression levels were 1.96 + 0.07,5.25 + 0.23,4.02 + 0.17, sham group was significantly higher than that of the 0.98 + 0.03 (P0.05). (2) in the treated mice group, laser confocal detection of KCs expression of TIM-4 TIM-4 m Ab TIM-4 found that group. 鍏夎〃杈懼己搴︽槑鏄句綆浜巆ontrol m Ab緇，

本文編號(hào)：1676344