多基因编辑猪-猴心脏、肝脏、肾脏移植临床前研究初步报道

目的探讨目前国际上基因改造程度最大的基因编辑猪在临床前异种器官移植中的应用前景。方法将1只猪内源性逆转录病毒(PERV)敲除联合3种主要异种抗原基因敲除以及抑制补体活化、调节凝血紊乱、抗炎抗吞噬的9种人源化基因转入猪(PERV-KO/3-KO/9-TG)作为供体,获取其心脏、肝脏和肾脏,分别移植给3只恒河猴受体,建立猪-猴异种器官移植临床前研究模型。观察血流重建后各移植物的功能状态并总结受体存活情况;监测移植物的血流动力学情况;比较各器官移植受体的血液学指标变化;观察移植物组织病理学表现。结果血流重建后各移植器官颜色红润、质地柔软、血流灌注状态良好。术后1 d,移植心脏、肝脏和肾脏均表现为动、静脉血流状态充盈,灌注情况良好。心脏、肝脏和肾脏移植受体的术后存活时间分别为7 d、26 d和1 d。心脏移植受体术后1 d肌酸激酶、肌酸激酶同工酶以及乳酸脱氢酶水平均升高,至术后6 d逐渐恢复至接近正常水平。术后7 d各项指标均急剧升高。肝脏移植受体术后2 d天冬氨酸转氨酶水平升高,术后10 d转氨酶基本恢复正常,但总胆红素持续升高。术后12 d天冬氨酸转氨酶和丙氨酸转氨酶水平均出现升高,至术后15 d达到高峰。肾脏移植受体术后1 d出现轻微蛋白尿,后因突发严重心律失常死亡。组织病理学显示心脏和肾脏移植物组织结构接近正常,移植肝脏表现为片状坏死,肝组织结构出现紊乱,并伴有炎症损伤、间质出血和血栓性微血管病形成。结论PERV-KO/3-KO/9-TG猪在克服超急性排斥反应、缓解体液性排斥反应及凝血紊乱方面具有一定优势,但其能否作为临床异种器官移植潜在供体需进一步评估。

Preliminary report of preclinical trial of multi-genome engineering pig-to-macaque heart,liver and kidney transplantation

Objective To investigate the application prospect of the most extensive genome engineering pig internationally in preclinical xenotransplantation.Methods Porcine endogenous retrovirus(PERV)knockout combined with 3 major heterologous antigen gene knockouts and 9 humanized genes for inhibition of complement activation,regulation of coagulation disorders,anti-inflammatory and anti-phagocytosis were transferred into a pig(PERV-KO/3-KO/9-TG)as a donor,and the heart,liver and kidney were obtained and transplanted to 3 Rhesus macaque recipients respectively to establish a preclinical research model of pig-to-Rhesus macaque xenotransplantation.The functional status of xenografts after blood flow reconstruction was observed and the survival of recipients was summarized.The hemodynamics of xenografts were monitored.The change of hematological indexes of each recipient was compared.The histopathological manifestation of xenografts was observed.Results After the blood flow was reconstructed,all xenografts showed ruddy color,soft texture and good perfusion.The transplant heart,liver and kidney showed full arterial and venous blood flow and good perfusion at 1 d after operation.The postoperative survival time of heart,liver,and kidney transplant recipients was 7,26,and 1 d,respectively.The levels of creatine kinase,creatine kinase isoenzyme,and lactate dehydrogenase increased in heart transplant recipient at 1 d after operation,and gradually recovered to near normal levels at 6 d after operation.All indexes increased sharply at 7 d after operation.The level of aspartate aminotransferase increased in liver transplant recipients at 2 d after operation,and the alanine aminotransferase basically returned to normal at 10 d after operation,but the total bilirubin continued to increase.Both aspartate aminotransferase and alanine aminotransferase increased at 12 d after operation,and reached a peak at 15 d after operation.The kidney transplant recipient developed mild proteinuria at 1 d after operation,and died of sudden severe arrhythmia.Histopathology showed that the tissue structure of cardiac and renal xenografts was close to normal,and liver xenografts presented with patchy necrosis,the liver tissue structure was disordered,accompanied by inflammatory damage,interstitial hemorrhage and thrombotic microangiopathy.Conclusions PERV-KO/3-KO/9-TG pig shows advantages in overcoming hyperacute rejection,mitigating humoral rejection and coagulation dysregulation.However,whether it can be used as potential donor for clinical xenotransplantation needs further evaluation.