凋亡抑制分子SNX7在异种肝脏移植中抑制凋亡、促进移植肝损伤修复的功能及机制研究

Xenotransplantation that uses organs of pigs is an important way to solve the shortage of donor. However, the great interspecific differences between pigs and primates induce serious injuries and repair disorders of xenografts, resulting in poor graft function and even recipients death. The applicants have successfully implemented the first domestic liver xenotransplantation with α-1,3-galactosyltransferase gene-knockout (GTKO) pigs as donors and primates as recipients, analyzed the differences between preoperative and postoperative xenogenic livers on genes expression and found that an apoptosis inhibitory gene——SNX7 specific expressed in livers was apparently reduced. Further studies show that SNX7 might be the key molecule involved in the repair of injured xenogenic livers and deletion of its expression leads to apoptosis of liver cells and losing repairing ability, but the exact mechanism is not clear. This project intends to use GTKO pig-Tibetan macaque liver xenotransplantation model as the object of the study, and prove for the first time that loss of SNX7 expression is the key molecule mechanism, mediating injuries and repairing disorders of xenogenic livers. If that goes as expected, the applicant would look for the upstream genes which are regulating the expression of SNX7, and search the downstream key molecules affected by the loss of SNX7 expression, engaged to construct a signal pathway molecular network which regulates repair of xenogenic livers, with SNX7 as a Key node and provide new targets and new ideas for promoting the xenograft liver repair.

以猪为供体的器官移植是解决同种供体短缺的重要方法之一。但猪与灵长类动物种间差异大，异种肝移植术后存在严重移植肝损伤及修复障碍，导致术后早期移植肝失功、受体死亡。申请者成功实施国内首例以α-1,3-半乳糖苷转移酶基因敲除（GTKO）猪为供体的异种肝移植实验，并利用芯片技术分析移植前后移植肝的差异基因表达谱，发现肝脏特异表达的凋亡抑制基因SNX7出现显著下调。进一步研究表明SNX7可能是参与异种移植肝损伤修复的关键分子，其表达缺失导致肝细胞凋亡，使移植肝失去修复能力，但机制不清。因此，本项目拟以GTKO猪—藏酋猴异种辅助性肝移植模型为研究对象，证明SNX7表达缺失是介导移植肝损伤和损伤修复障碍的关键分子，向上游寻找抑制SNX7表达的关键调控基因，向下游寻找SNX7缺失后受影响的关键分子，以期构建以SNX7为中心的调控异种移植肝损伤修复的分子网络，为促进异种移植肝修复提供新靶点和新思路。

以猪为供体的器官移植是解决同种供体短缺的重要方法之一。但异种肝移植术后存在严重移植肝损伤及修复障碍，导致术后早期移植肝失功、受体死亡。课题组在前期实施的α-1,3-半乳糖苷转移酶基因敲除（GTKO）猪为供体的异种肝移植实验基础上，并利用芯片技术分析移植前后移植肝的差异基因表达谱，发现肝脏特异表达的凋亡抑制基因SNX7出现显著下调。SNX7可能是参与异种移植肝损伤修复的关键分子，其调控机制不清。本项目后期继续实施了6例GTKO猪-猴异种肝移植，完善了供受体配对筛选方案，修正了免疫抑制方案，分析了术后受体血小板急剧丢失的可能原因，术后移植肝胆汁分泌和猪白蛋白合成情况。在此基础上，利用高通量芯片技术，对比分析移植术前和术后移植肝的基因差异表达谱，发现922个基因表达上调，1113个基因表达下调。同时，我们利用qPCR、western blot、以及免疫组化分别检测了SNX7在术前、开放血流1h、术后移植肝组织中的表达变化，结果证实SNX7术后表达下调。同时，我们利用人源的PBMC细胞和我们建立的永生化GTKO细胞裂解物进行混合培养，模拟异种免疫杀伤效应，挑选出免疫相关基因进行qPCR检测验证，结果显示大部分关键基因变化与测序结果一致，提示我们的体外细胞培养模型一定程度上能够反应体内情况。体外培养模型发现SNX7在刺激0h、6h、12h、24h后出现明显下调。SNX7下调后，肝细胞发生自发性凋亡，SNX7上调组上清中AST、BU含量明显低于对照组，ALB含量无明显变化，细胞凋亡率明显低于对照组。同时，SNX7上调组cleaved-Caspase3表达量明显降低，抑制肝细胞凋亡。加入Caspase-3抑制剂后，实验组凋亡率明显低于对照组。ENCODE 分析：P53、STAT1 和c-Myc 编码蛋白可与SNX7 基因启动子结合，调控SNX7的表达。c-Myc在移植后发生了上调，而STAT1和P53无明显变化。组化染色显示c-Myc在移植后发生了上调。SNX7的表达量与c-Myc呈负相关，c-Myc可能是抑制SNX7表达的上游基因。因此，本项目证明了SNX7表达缺失是介导移植肝损伤和损伤修复障碍的关键分子，以SNX7为中心的分子调控网络可能成为异种移植肝损伤预防和治疗的新靶点。