人源化六基因编辑猪红细胞治疗恒河猴急性失血性休克的疗效观察  

Humanized six-gene edited porcine erythrocytes in treatment of acute hemorrhagic shock in rhesus monkeys

作　　者：贾志博 崔梦一 王恺 杜嘉祥 董阳辉 宋翔宇 陈蕾佳 杨博尧 陈胜峰 彭熙为 许文静[2] 潘登科 彭江[2] JIA Zhibo;CUI Mengyi;WANG Kai;DU Jiaxiang;DONG Yanghui;SONG Xiangyu;CHEN Leijia;YANG Boyao;CHEN Shengfeng;PENG Xiwei;XU Wenjing;PAN Dengke;PENG Jiang(Hebei North College,Zhangjiakou 075000,Hebei Province,China;Institute of Orthopaedic Medicine Department,All-Army Orthopaedic Battle Trauma Key Laboratory,Beijing Key Laboratory of Orthopaedic Regenerative Medicine,the Fourth Medical Center,Chinese PLA General Hospital,Beijing 100853,China;Chinese PLA Medical School,Beijing 100853,China;Graduate School,Guizhou Medical University,Guiyang 550025,Guizhou Province,China;Chengdu Zhongke Aoge Bio-technology Co.Ltd.,Chengdu 610041,Sichuan Province,China)

机构地区：[1]河北北方学院,河北张家口075000 [2]解放军总医院第四医学中心,骨科医学部研究所,全军骨科战创伤重点实验室,骨科再生医学北京市重点实验室,北京100853 [3]解放军医学院,北京100853 [4]贵州医科大学研究生院,贵州贵阳550025 [5]成都中科奥格生物科技有限公司,四川成都610041

出　　处：《解放军医学院学报》2024年第4期397-404,共8页Academic Journal of Chinese PLA Medical School

基　　金：国家重点研发计划(2019YFA0110704)。

摘　　要：背景临床上血液制品短缺是一项世界难题,猪红细胞与人类红细胞有诸多相似之处,有望解决这一难题。目的将人源化基因编辑猪(GTKO/β4GalNT2KO/CMAHKO/hCD55/hCD46/hTBM)的红细胞输注至非人灵长类动物恒河猴体内,探讨异种输血的可能性。方法采用盐水介质交叉配血法,检测六基因编辑猪红细胞(6GE-pRBC)与恒河猴的血液凝集情况。通过IgG、IgM结合实验和补体介导的细胞毒性实验验证异种输血安全性,从恒河猴体内抽取25%血容量血液后输注等量的异硫氰酸荧光素标记6GE-pRBC,通过流式细胞分析术监测在其体内的存活状态,并定期采集受体血液样本验证异种输血的有效性,观察6GE-pRBC输注治疗失血性休克恒河猴的可能性。结果6GE-pRBC与恒河猴主次侧交叉配血试验均为阴性;其基因表型鉴定符合预期结果;恒河猴血清与6GE-pRBC的IgG、IgM结合实验结果显示IgG(3.35%)、IgM(4.13%)表达量较低;6GE-pRBC的补体介导细胞毒作用(CDC)显著低于野生猪红细胞(P<0.0001)。输血后60 h 6GEpRBC在恒河猴体内仍存活;血细胞比容失血后为28.6%,60 h升高至35%;血红蛋白含量失血后为92 g/L,60 h升高至116 g/L并维持于失血前正常水平。结论6-GE-pRBC输注给恒河猴在一定时间内能有效地纠正恒河猴休克症状,且未发现免疫排斥反应。随着基因编辑技术不断创新,基因编辑猪红细胞有望替代人红细胞治疗人类急性失血性休克。Background Clinical shortage of blood products is a world problem.Porcine erythrocytes,with many similarities as human erythrocytes,are expected to solve this problem.Objective To explore the possibility of xenotransfusion of humanized geneedited porcine(GTKO/β4GalNT2KO/CMAHKO/hCD55/hCD46/hTBM)erythrocytes into rhesus monkeys.Methods Blood agglutination of six gene-edited porcine erythrocytes(6GE-pRBC)with rhesus monkeys was detected by saline-mediated crossmatching method.The safety of xenotransfusion was verified by IgG and IgM binding assay and complement-mediated cytotoxicity assay.An equal amount of fluorescein isothiocyanate(FITC)-labeled 6GE-pRBC was transfused into rhesus monkeys after 25%of the blood volume was withdrawn from the monkeys,and the survival status in their bodies was monitored by flow cytometry,and the validity of the xenotransfusion was verified by collecting the recipient's blood samples at regular intervals.The possibility of 6GE-pRBC transfusion for the treatment of hemorrhagic shock in rhesus monkeys was observed.Results The primary and secondary crossmatch blood tests of 6GE-pRBC with rhesus monkeys were negative.The six-gene porcine genotyping conformed to the expected results.The results of IgG and IgM binding assay of rhesus monkey serum with 6GE-pRBC showed low expression of IgG(3.35%)and IgM(4.13%).The complement-mediated cytotoxicity(CDC)of 6GE-pRBC was significantly lower than that of wild porcine erythrocytes(P<0.0001).The 6-GE-pRBC survived in rhesus monkeys until 60 h after blood transfusion.Erythrocyte pressure volume was 28.6%after blood loss,and then increased to 35%at 60 h.The hemoglobin content was 92 g/L after blood loss,and then increased to 116 g/L at 60 h and was maintained at the normal pre-blood loss level.Conclusion 6-GE-pRBC infusion to rhesus monkeys is effective in correcting the symptoms of shock in rhesus monkeys within a certain period of time,and no immune rejection is found.With the continuous innovation of gene editing technology,gene edited porcine ery

关 键 词：异种输血 人源化编辑猪 猪红细胞 恒河猴 免疫排斥反应 

分 类 号：R617[医药卫生—外科学] R-332[医药卫生—临床医学]