在人源化基因编辑猪-非人灵长类动物(NHPs)异种心脏腹腔异位移植实验中应用新型抗CD40单克隆抗体(anti-CD40)联合以他克莫司为主的免疫抑制方案，评价人源化基因修饰猪的基因编辑类型和免疫抑制方案的优化策略。

以雄性基因编辑(GTKO/hCD39/hCD55/hTBM)小型Bama猪为供体，以雄性食蟹猴为受体，构建人源化基因修饰猪-猴异种心脏腹腔异位移植模型。围术期采用anti-CD40联合吗替麦考酚酯、他克莫司、甲泼尼龙、抗CD20单克隆抗体以及抗胸腺细胞球蛋白等组成的新型免疫抑制方案，采用体外遥感心电监测设备实时监测移植心脏电信号活动情况。主要观察指标为血流重建后移植心脏冠状动脉血流、功能、心电信号变化、心肌酶变化以及受体免疫抑制状态和存活时间；次要目标为监测受体生理指标，包括血常规、肝肾功能、血清蛋白和电解质，并依据结果调整生命支持治疗方案。终末实验后留取移植心脏标本，采用HE染色及电镜观察移植心脏病理变化。

移植心脏血流重建后，器官颜色红润、质地柔软，恢复自主心律，心肌收缩有力。术后1周受体恢复良好，移植心脏冠状动脉灌注情况良好，心脏超声提示心肌收缩功能良好。受体血液学检查结果提示血清肌酸激酶及乳酸脱氢酶水平均一过性轻度升高，至术后第6天逐渐接近正常水平。术后1周受体血红蛋白、电解质和肝肾功能等指标均明显好转，一般状况好转。心脏移植后2周，心脏超声提示心肌肥厚明显。术后第20天，移植心脏心率由120~140次/分下降至50~80次/分，复查心脏超声提示吻合口血流通畅、冠状动脉灌注良好，心肌收缩乏力。考虑移植心脏功能明显减弱，终止实验后取出移植心脏，HE染色见心肌纤维增生、局部心肌细胞纤维化、间质水肿和极少量单核细胞浸润。电镜下可见心肌原纤维扭曲、肌间隙增宽，粗面内质网、线粒体和心肌肌丝结构破坏及心肌细胞线粒体囊性结构破坏。

成功建立人源化基因编辑猪-NHPs的异种心脏腹腔异位移植模型，应用新型anti-CD40联合他克莫司为主的免疫抑制方案，抑制了超急性排斥反应，延缓了急性排斥反应的发生，实现异种移植器官的较长时间存活。

To evaluate the optimal strategy of gene editing type and immunosuppressive regimen by applying a novel anti-CD40 monoclonal antibody (anti-CD40) in combination with tacrolimus-based immunosuppressive regimen in a humanized genome engineering porcine-non-human primates (NHPs) allogeneic cardiac ventral allograft experiment.

The heart of a humanized genome engineering (GTKO/hCD39/hCD55/hTBM) male Bama miniature pig was transplanted into the peritoneal cavity of a male cynomolgus to establish a cardiac xenotransplantation model. During the perioperative period, a new immunosuppressive regimens consisting of domestic anti-CD40 in combination with mycophenolate mofetil, tacrolimus, methylprednisolone, anti-CD20 monoclonal antibody and anti-thymocyte globulin were administered, and the electrical activity of the transplanted heart was monitored in real-time using an extracorporeal telemetry monitoring device. The main outcomes were coronary artery blood flow and function after revascularization, electrocardiographic signal changes and myocardial enzyme changes in the transplanted heart, and immunosuppression status and survival time of the recipient. The secondary outcomes were the physiological parameters of the recipient, including routine blood tests, liver and kidney function, serum protein and electrolytes, and life support therapy based on the results. When the xenograft suffered failure of function, the cardiac xenograft was removed and sent for pathological examination using HE staining and electron microscopy.

The transplanted heart showed a ruddy color and had a soft texture, autonomous heartbeat and strong myocardial contraction after revascularization. One week after transplantation, the transplanted heart had good coronary artery perfusion. The cardiac ultrasound indicated normal myocardial systolic function, and the condition of the recipient was normal. The levels of serum creatine kinase and lactic dehydrogenase in the recipient transiently increased after transplantation and decreased to normal levels at postoperative day 6. The hemoglobin, electrolytes and liver and kidney function of the recipient significantly improved at 1 week after transplantation. Cardiac ultrasound indicated significant myocardial hypertrophy of the cardiac xenograft at 2 weeks after transplantation. At 20 days after transplantation, the heartbeat of the xenograft decreased from 120-140 beats per minute to 50-80 beats per minute, the blood flow of the anastomotic site was normal, and the coronary artery was well-perfused, but the myocardium was debilitated in the systolic phase. The experiment was terminated at 20 days after transplantation because the function of the transplanted heart was significantly weakened. HE staining showed myocardial fiber hyperplasia, local myocardial fibrosis, interstitial edema and monocyte infiltration. Myofibril distortion, intermuscular space broadening, and structural damage to the rough endoplasmic reticulum, mitochondria and myocardial myofilament were observed under an electron microscope.

A humanized genome engineering porcine-NHPs model of intra-abdominal heterotopic cardiac xenotransplantation was successfully established. The application of a novel anti-CD40 monoclonal antibody with tacrolimus-based combination immunosuppressive regimens effectively inhibited hyperacute rejection, delayed the occurrence of acute rejection, and achieved longer survival of the xenograft organ.