南方医科大学学报

南方医科大学学报 ›› 2017, Vol. 37 ›› Issue (04): 451-.

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阻断Kupffer细胞TIM-4功能对小鼠肝移植排斥反应的影响

李学强，李旭宏，段世刚，徐雪松，刘一鸣，李金政，龚建平，吴皓   

  • 出版日期:2017-04-20 发布日期:2017-04-20

Effect of inhibiting TIM-4 function in Kupffer cells on liver graft rejection in mice

  • Online:2017-04-20 Published:2017-04-20

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摘要： 目的探讨阻断Kupffer细胞（KCs）TIM-4功能对诱导小鼠原位肝移植术后免疫耐受的产生以及相关机制的研究。方法 建立小鼠原位肝移植急性排斥反应模型，随机分为Sham 组、Control mAb组、TIM-4 mAb组，术后组化检测KCs活化，提取 KCs，Western blot和PCR检测肝脏TIM-4表达，检测血清谷草转氨酶、谷丙转氨酶、总胆红素，ELISA检测肝组织匀浆肿瘤坏死 因子α、干扰素γ、CC族趋化因子2，激光共聚焦检测KCs TIM-4表达，苏木精伊红染色（HE）染色观察肝组织排斥反应，Western blot检测肿瘤坏死因子α、干扰素γ、CC族趋化因子2、p-P65、p-P38表达，流式检测CD25+Foxp3+T细胞的产生。氯膦酸二钠脂质 体（CL）可耗竭肝脏KCs，用CL处理移植模型，HE染色观察肝组织排斥反应。结果肝移植术后KCs的活化随着时间变化逐渐 增加，术后1、3、7 d TIM-4蛋白以及mRNA表达水平明显高于Sham组（P<0.05），术后7 d 谷草转氨酶、谷丙转氨酶、总胆红素、 肿瘤坏死因子α、干扰素γ、CC族趋化因子2水平高于Sham组（P<0.05），HE染色TIM-4 mAb组小鼠肝病理改变排斥活动指数平 均得分为2.67±0.75，集中于轻度排斥反应，明显低于Control mAb组病理改变排斥活动指数得分8.17±0.69（P<0.05），且TIM-4 mAb组小鼠平均存活时间53.8±6.4 d明显长于Control mAb组平均存活时间14.5±2.9 d（P<0.05）。CL处理供体小鼠，HE染色 CL+TIM-4 mAb组和CL+Control mAb组病理改变排斥活动指数平均得分分别为8.01±0.64、7.93±0.56，两者比较差异无统计学 意义（P>0.05）。Western blot 检测TIM-4 mAb 组KCs p-P65 以及p-P38 蛋白，明显低于Control mAb 组（P<0.05）。流式示 TIM-4 mAb组肝脏iTreg细胞明显增多。结论阻断KCs TIM-4可能通过核转录因子κB以及分裂原激活的蛋白激酶通路减少 移植后排斥反应发生，诱导iTreg细胞的生成，致使宿主对移植物产生免疫耐受。

Abstract: Objective To investigate the effects of inhibiting TIM-4 function in Kupffer cells (KCs) on liver graft rejection in mice and explore the underlying mechanism. Methods Mouse models of orthotopic liver transplantation were treated with a control mAb group and TIM-4 mAb. The activated KCs were assayed with immunohistochemistry after operation. The expression of TIM-4 in KCs were assayed with Western blotting and RT-PCR and the levels of AST, ALT, TBIL, TNF-α, IFN-γ and CCL2 were assayed detected. The expression of TIM-4 in KCs was observed with laser confocal microscopy. HE staining was used to observe the microstructure of the liver tissues, and the number of CD25 + Foxp3 + T cells was determined using with flow cytometry; the proteins levels of p-P65and p-P38 were assayed with Western blotting. The donor mice were treated with clodronate liposomes to destroy the KCs in the liver before transplantation, and the liver grafts were examined for graft rejection. Results The number of activated KCs in the liver graft increased progressively over time. Compared with the sham-operated group, the liver graft showed significantly increased TIM-4 protein and mRNA levels at 1, 3, and 7 days after transplantation (P<0.05) and increased levels of AST, ALT, TBIL, TNF-α, IFN-γ and CCL2 at 7 days (P< 0.05). The graft in TIM-4 mAb group showed mild pathological changes with a mean RAI score of 2.67±0.75, which was significantly lower than that in control mAb group (P<0.05). The mean survival time of the recipient mice was 53.8±6.4 days in TIM-4 mAb group, significantly longer than that in the control mAB group (14.5±2.9 days, P<0.05). Donor treatment with clodronate liposomes resulted in comparable RAI scores in TIM-4 mAb and control mAb groups (8.01±0.64 vs 7.93±0.56, P>0.05). The protein levels of p-P65 and p-P38 in TIM-4 mAb group were significantly lower than those in control mAb group (P<0.05), and CD25+Foxp3+T cells in the liver graft increased significantly in TIM-4 mAb group. Conclusion Inhibition of TIM-4 function in KCs reduces the production of inflammatory factors after liver transplantation possibly by inhibiting the NF-κB and MAPK signaling pathways and promoting the proliferation of Foxp3+Treg cells to induce allograft tolerance.