阻断Kupffer细胞TIM-4功能对小鼠肝移植排斥反应的影响

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

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m RNA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-4mAb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i Treg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i Treg细胞的生成,致使宿主对移植物产生免疫耐受。

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-P65 and 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 shamoperated group, the liver graft showed significantly increased TIM-4 protein and m RNA levels at 1, 3, and 7days 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 m AB 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.