阻断Kupffer细胞TIM-4功能对小鼠肝移植排斥反应的影响以及相关机制研究

发布时间：2018-03-28 12:33

  本文选题：T细胞免疫球蛋白黏蛋白4　切入点：Kupffer细胞　出处：《重庆医科大学》2017年硕士论文

【摘要】：目的:探讨阻断Kupffer细胞(KCs)TIM-4对诱导小鼠原位肝移植术后调节性T细胞(i Treg)的产生以及相关机制的研究。方法:1.小鼠原位肝移植术后KCs TIM-4的表达以及阻断TIM-4对肝移植急性排斥反应的影响采用改良的Kamada“二袖套管”法建立C57BL/6→C3H小鼠原位肝移植急性排斥反应模型。在未处理小鼠中,小鼠仅做原位肝移植而未做任何处理(LT组),设sham为对照组(仅开腹手术暴露门静脉)。免疫组化检测LT组和sham组移植术后1d、2d、3d受体肝组织中KCs的活化情况。分离受体KCs,Western blot和RT-PCR检测移植术后1d、3d、7d KCs TIM-4蛋白以及m RNA表达情况。在处理小鼠中,实验动物随机分为3组:sham组,小鼠开腹手术暴露门静脉,并经门静脉注入1m L PBS;control m Ab组,于供体冷血TIM-4 m Ab组,于供体冷血期经门静脉注入含抗TIM-4抗体(0.35mg/只)的1m L PBS。各组均于术后3d经尾静脉再次分别注入上述液体。小鼠于移植后7d麻醉下开腹穿刺取腹主动脉血0.5m L,然后处死小鼠,取下中叶肝组织,余固定包埋。提取各组肝脏KCs,激光共聚焦检测KCs TIM-4阻断情况;全自动生化分析仪检测各组小鼠血清AST、ALT、TBIL;ELISA和Western blot检测各组小鼠肝组织匀浆TNF-α、IFN-γ、CCL2、CXCL2表达水平;TUNEL法检测各组肝组织细胞凋亡情况;Western blot检测各组KCs p-P65和p-P38蛋白表达水平。2.阻断KCs TIM-4对初始CD4+T细胞分化以及IL-4/STAT6信号通路的影响分离模型LT组KCs,流氏分选出TIM-4+KCs(预先加或不加0.5mg/L TIM-4 m Ab处理)。分离和纯化WT C3H小鼠脾脏初始CD4+T细胞。将上述两种细胞按1:1比例进行共培养3d。处理分为三组:control组,作为对照组,不予以处理;control m Ab组,预先加入0.5mg/L control m Ab处理;TIM-4 m Ab组,预先加入0.5mg/L TIM-4 m Ab处理。收集各组T细胞以及上清液。CFSE法检测各组CD4+T细胞增殖情况;ELISA检测各组上清IL-4、IL-6、IL-13表达水平;流氏细胞术检测各组CD4+CD25+Foxp3+T细胞的产生情况。分离模型LT组KCs,流氏分选出TIM-4+和TIM-4-KCs,并按1:1比例与初始CD4+T细胞共培养3d,分为三组:control组,作为对照组,仅初始CD4+T细胞单独培养;TIM-4+组,TIM-4+KCs与CD4+T细胞共培养组;TIM-4-组,TIM-4-KCs与CD4+T细胞共培养组。Western blot分析T细胞p-STAT6蛋白表达情况;上述各组用0.5mg/L TIM-4m Ab+/-处理,Western blot分析T细胞p-STAT6蛋白表达情况;在TIM-4+组中,用0.5mg/L TIM-4 m Ab+/-和IL-4+/-处理,Western blot分析T细胞p-STAT6蛋白表达情况。3.阻断TIM-4+KCs TIM-4功能诱导的i Treg对肝移植排斥反应以及小鼠生存率的影响分离模型LT组KCs,流氏分选出TIM-4+KCs(预先用TIM-4 m Ab处理)与初始CD4+T细胞进行共培养,3d后获取CD4+CD25+Foxp3+Treg细胞,调整i Treg细胞浓度1×106个/m L,将所得细胞于供体冷血期经门静脉注入i Treg组,sham组以及LT组注入相应的PBS作为对照。部分小鼠于移植后7d麻醉下开腹穿刺取腹主动脉血0.5m L,然后处死小鼠,取下中叶肝组织,余固定包埋。全自动生化分析仪检测各组小鼠血清AST、ALT、TBIL;HE检测各组小鼠肝组织病理变化情况;余下各组小鼠作为观察生存期之用,观察期以小鼠死亡为终点,并做Log-Rank生存资料分析。结果:1.(1)小鼠移植术后肝组织KCs的活化数随着时间变化逐渐增多。术后1d、3d、7d KCs TIM-4蛋白相对表达水平分别为0.31±0.04、0.86±0.05、0.77±0.03,明显高于sham组的0.11±0.03(P0.05),m RNA相对表达水平分别1.96±0.07、5.25±0.23、4.02±0.17,显著高于sham组的0.98±0.03(P0.05)。(2)在处理小鼠分组中,激光共聚焦检测KCs TIM-4表达情况发现,TIM-4 m Ab组TIM-4荧光表达强度明显低于control m Ab组(P0.05)。术后7d,与sham组比较,control m Ab组血清肝功AST、ALT、TBIL以及肝组织匀浆炎症因子TNF-α、IFN-γ、CCL2、CXCL2水平明显增高(P0.05),而TIM-4 m Ab组上述指标水平明显低于control m Ab组(P0.05)。Western blot检测肝脏组织炎症因子蛋白表达和上述结果一致。各组肝组织细胞凋亡指数(AI),TIM-4 m Ab组AI值(11.04±2.28)明显低于TIM-4 m Ab组(29.23±2.56)(P0.05)。TIM-4 m Ab组p-P65以及p-P38蛋白表达水平分别为0.82±0.23、0.54±0.10,明显低于control m Ab组的1.39±0.27、1.07±0.23(P0.05)。2.(1)KCs与CD4+T细胞按共培养发现,control、control m Ab以及TIM-4 m Ab组CD4+T细胞的增殖率分别为(32.3±1.2)%、(31.1±1.4)%、(16.9±0.5)%。ELISA检测各组上清IL-4、IL-6、IL-13分泌水平发现,阻断TIM-4+KCs TIM-4可明显减少上述炎症因子的分泌(P0.05)。流式检测各组CD4+CD25+Foxp3+T细胞产生情况,control组、control m Ab组以及TIM-4 m Ab组CD25、Foxp3双阳性细胞分别为(12.8±0.3)%、(13.3±0.5)%、(28.1±0.4)%,后者明显高于前两组(P0.05)。(2)TIM-4+组T细胞p-STAT6相对蛋白表达水平(1.75±0.06)明显高于TIM-4-组(0.60±0.06)(P0.05)。然而TIM-4+KCs与CD4+T细胞共培养时,加入或未加入TIM-4 m Ab的p-STAT6蛋白表达分别为2.29±0.25、1.30±0.11,两者比较差异有统计学意义(P0.05),此现象在TIM-4-组间比较无统计学上的差异(P0.05)。另外,向培养液中加入IL-4(0.25mg/L)发现,阻断KCs TIM-4的表达,外源性加入IL-4可明显增高CD4+T细胞p-STAT6蛋白的表达(P0.05)。3.(1)LT组肝功指标明显高于sham组,差异具有统计学意义(P0.05),而i Treg组肝功指标明显好转(P0.05)。肝组织病理学检查,i Treg组RAI平均得分为3.97±0.67,明显低于LT组8.47±0.90(P0.05)。(2)i Treg组小鼠平均存活时间(55.7±5.5)d明显长于LT组平均存活时间(14.5±3.7)d(P0.05)。结论:1.小鼠移植术后,活化的KCs TIM-4的表达水平逐渐增高。阻断KCs TIM-4可能通过影响NF-κB以及MAPK通路减少炎症因子的释放,改善肝组织排斥反应的损伤。2.阻断KCs TIM-4通过抑制IL-4/STAT6信号通路促进初始CD4+T细胞向i Treg细胞的分化。3阻断TIM-4+KCs TIM-4功能诱导的i Treg细胞可明显改善急性排斥反应损伤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〃杈惧己搴︽槑鏄句綆浜巆ontrol m Ab缁，

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