目的探讨阻断Kupffer细胞(Kupffer cells,KCs)T细胞免疫球蛋白黏蛋白4(T-cell immunoglobulin and mucin-domain-containing molecule 4,TIM-4)诱导小鼠原位肝移植术后i Treg细胞的产生及其相关机制。方法建立小鼠原位肝移植急性排斥反...目的探讨阻断Kupffer细胞(Kupffer cells,KCs)T细胞免疫球蛋白黏蛋白4(T-cell immunoglobulin and mucin-domain-containing molecule 4,TIM-4)诱导小鼠原位肝移植术后i Treg细胞的产生及其相关机制。方法建立小鼠原位肝移植急性排斥反应模型,分为sham组、control m Ab组、TIM-4 m Ab组。流式细胞仪检测KCs活化,Western blot检测肝脏TIM-4表达,TUNEL检测肝细胞凋亡指数(apoptotic index,AI)。提取KCs,激光共聚焦检测KCs TIM-4表达,将KCs和提取的初始CD4+T细胞进行共培养,激光共聚焦检测Kupffer细胞TIM-4表达,分为control组、control m Ab组以及TIM-4 m Ab组;CFSE检测CD4+T细胞增殖,流式细胞仪检测CD4+CD25+Foxp3+T细胞,ELISA检测IL-4、IL-6、IL-13水平,Western blot检测STAT6表达。建立小鼠移植模型,分为3组:i Treg组移植模型注入i Treg细胞(预先用TIM-4 m Ab处理的TIM-4+KCs与初始CD4+T细胞共培养所诱导),sham组和单纯肝移植组(LT)注入相应的PBS作为对照,检测各组AST、ALT、TBIL水平,HE染色评价肝组织排斥活动指数(rejective activity index,RAI),观察小鼠生存率。结果肝移植术后24、48、72 h KCs的活化分别为15.9%、21.6%、28.3%,术后1、3、7 d TIM-4蛋白表达水平明显高于sham组(P<0.05);control m Ab组AI值(29.23±2.56)明显高于sham组和TIM-4 m Ab组[(0.40±0.49),(11.04±2.28),P<0.05]。KCs与CD4+T细胞共培养后,control、control m Ab以及TIM-4 m Ab组CD4+T细胞的增殖率分别为32.5%、31.6%、17.2%,CD4+CD25+Foxp3+T细胞分化分别为12.4%、13.9%、28.1%,共培养上清液TIM-4 m Ab组IL-4、IL-6、IL-13分泌水平明显低于control m Ab(P<0.05),且加入TIM-4 m Ab明显降低了与TIM-4+KCs共培养的CD4+T细胞p-STAT6蛋白表达(P<0.05),外源性加入IL-4可明显增高CD4+T细胞p-STAT6蛋白的表达(P<0.05)。移植模型注入i Treg细胞,i Tregz组肝功指标明显好转,与LT组比较差异有统计学意义(P<0.05)。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),P<0.05],且i Treg组小鼠平均存活时间延长。结论阻断KCs TIM-4能够抑制初始CD4+T细胞IL-4/STAT6信号通路的激活,从而诱导更多i Treg细胞的生成,致使宿主对移植物产生免疫耐受。展开更多
To investigate the distribution characteristics and linkage disequilibrium of T cell immunoglobulin domain and mucin domain protein 4 （TIM4） promoter polymorphisms in asthma patients of Chinese Han population, the p...To investigate the distribution characteristics and linkage disequilibrium of T cell immunoglobulin domain and mucin domain protein 4 （TIM4） promoter polymorphisms in asthma patients of Chinese Han population, the promoter region of TIM4 was re-sequenced by PCR-sequencing, and linkage disequilibrium was analyzed by SHEsis software. Four single nucleotide polymor- phisms （SNPs） in the promoter region of TIM4 were detected, including two new SNPs （at positions -1609, -153） and two reported SNPs （rs6874202, rs6882076）. The frequency distribution of rs6882076 was different among different races （P〈0.05）. In addition, linkage disequilibrium among the SNPs of the promoter region of TIM4 was found and GGTG was the predominant haplotype. There were four SNPs in the promoter region of TIM4 in asthma patients of Chinese Han population, which were in linkage disequilibrium.展开更多
文摘目的探讨阻断Kupffer细胞(Kupffer cells,KCs)T细胞免疫球蛋白黏蛋白4(T-cell immunoglobulin and mucin-domain-containing molecule 4,TIM-4)诱导小鼠原位肝移植术后i Treg细胞的产生及其相关机制。方法建立小鼠原位肝移植急性排斥反应模型,分为sham组、control m Ab组、TIM-4 m Ab组。流式细胞仪检测KCs活化,Western blot检测肝脏TIM-4表达,TUNEL检测肝细胞凋亡指数(apoptotic index,AI)。提取KCs,激光共聚焦检测KCs TIM-4表达,将KCs和提取的初始CD4+T细胞进行共培养,激光共聚焦检测Kupffer细胞TIM-4表达,分为control组、control m Ab组以及TIM-4 m Ab组;CFSE检测CD4+T细胞增殖,流式细胞仪检测CD4+CD25+Foxp3+T细胞,ELISA检测IL-4、IL-6、IL-13水平,Western blot检测STAT6表达。建立小鼠移植模型,分为3组:i Treg组移植模型注入i Treg细胞(预先用TIM-4 m Ab处理的TIM-4+KCs与初始CD4+T细胞共培养所诱导),sham组和单纯肝移植组(LT)注入相应的PBS作为对照,检测各组AST、ALT、TBIL水平,HE染色评价肝组织排斥活动指数(rejective activity index,RAI),观察小鼠生存率。结果肝移植术后24、48、72 h KCs的活化分别为15.9%、21.6%、28.3%,术后1、3、7 d TIM-4蛋白表达水平明显高于sham组(P<0.05);control m Ab组AI值(29.23±2.56)明显高于sham组和TIM-4 m Ab组[(0.40±0.49),(11.04±2.28),P<0.05]。KCs与CD4+T细胞共培养后,control、control m Ab以及TIM-4 m Ab组CD4+T细胞的增殖率分别为32.5%、31.6%、17.2%,CD4+CD25+Foxp3+T细胞分化分别为12.4%、13.9%、28.1%,共培养上清液TIM-4 m Ab组IL-4、IL-6、IL-13分泌水平明显低于control m Ab(P<0.05),且加入TIM-4 m Ab明显降低了与TIM-4+KCs共培养的CD4+T细胞p-STAT6蛋白表达(P<0.05),外源性加入IL-4可明显增高CD4+T细胞p-STAT6蛋白的表达(P<0.05)。移植模型注入i Treg细胞,i Tregz组肝功指标明显好转,与LT组比较差异有统计学意义(P<0.05)。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),P<0.05],且i Treg组小鼠平均存活时间延长。结论阻断KCs TIM-4能够抑制初始CD4+T细胞IL-4/STAT6信号通路的激活,从而诱导更多i Treg细胞的生成,致使宿主对移植物产生免疫耐受。
基金the National Natural Sciences Foundation of China (No. 30672008)
文摘To investigate the distribution characteristics and linkage disequilibrium of T cell immunoglobulin domain and mucin domain protein 4 （TIM4） promoter polymorphisms in asthma patients of Chinese Han population, the promoter region of TIM4 was re-sequenced by PCR-sequencing, and linkage disequilibrium was analyzed by SHEsis software. Four single nucleotide polymor- phisms （SNPs） in the promoter region of TIM4 were detected, including two new SNPs （at positions -1609, -153） and two reported SNPs （rs6874202, rs6882076）. The frequency distribution of rs6882076 was different among different races （P〈0.05）. In addition, linkage disequilibrium among the SNPs of the promoter region of TIM4 was found and GGTG was the predominant haplotype. There were four SNPs in the promoter region of TIM4 in asthma patients of Chinese Han population, which were in linkage disequilibrium.
