基因
编辑
食蟹猴异种肾
移植
手术
监测
初步
报道
杨树
第 14 卷 第 4 期2023 年 7 月Vol.14 No.4Jul.2023器官移植Organ Transplantation论著六基因编辑猪-食蟹猴异种肾移植围手术期监测初步报道杨树军卫浩许勇王恒恩宋翔宇贾志博彭江崔梦一杨博尧陈蕾佳郭爱桃 张小丽潘登科杜嘉祥尚攀峰孙圣坤【摘要】目的 探讨六基因编辑猪-非人灵长类动物异种肾移植模型的构建。方法 将人源化基因编辑猪(GTKO/4GalNT2KO/CMAHKO/hCD55/hCD46/hTBM)的肾脏移植给食蟹猴,观察受体存活情况及恢复血流灌注后肾脏情况。定期监测肾脏实质回声、血流变化及大小;进行血常规、肾功能检测及电解质检测;监测尿液、粪便及体质量动态变化。食蟹猴生命终点取移植肾、心脏、肝脏、脾脏、肺脏、盲肠行病理学检查。结果 受体术后 7 d 死亡。恢复血流后,肾脏灌注良好,器官质地柔软,色泽正常。受体生命终点时见肾脏腹侧有少量脓性分泌物附着,明显充血肿大,呈“大红肾”外观。术后随时间延长,肾实质回声增高,血流减少,皮质逐渐增厚,肾周及腹腔有少量积液。受体术后外周血红细胞、血红蛋白、白蛋白及血小板进行性下降,血清肌酐在术后 7 d升高至 308 mol/L,K+变化不大。术后即有淡黄色尿液排出,术后 3 h 内即恢复饮食及饮水并排淡黄色成形大便1 次。术后 1 d 内尿液颜色逐渐变淡红至恢复正常,尿常规检测结果相符。术后 2 d 晨起排褐色血便 2 次,量较多,予以奥美拉唑进行抑酸治疗,至术后 4 d 大便恢复正常。2-微球蛋白在术后 7 d 增至 0.75 mg/L。体质量增加 1.7 kg。尸检病理学检查发现移植肾间质水肿出血,大量淋巴细胞和巨噬细胞浸润,小动脉壁及动脉腔内淋巴细胞浸润,有动脉炎改变;盲肠间质有淋巴细胞浸润;脾脏组织有淤血表现;其余器官未见明显异常改变。结论 人源化基因编辑猪-非人灵长类动物异种肾移植模型构建获得初步成功,术后受体存活时间达 1 周。【关键词】异种移植;肾移植;基因编辑猪;食蟹猴;排斥反应;-1,3-半乳糖苷转移酶;-1,4-N-乙酰氨基半乳糖转移酶 2;补体调节蛋白【中图分类号】R617,Q78【文献标志码】A【文章编号】1674-7445(2023)04-0008-08【Abstract】Objective To investigate the establishment of a six-gene-edited pig-to-non-human primate kidney xenotransplantation model.Methods The kidney of humanized genetically-edited pig(GTKO/4GalNT2KO/CMAHKO/DOI:10.3969/j.issn.1674-7445.2023.04.008 基金项目:国家重点专项研发计划(2019YFA0110704)作者单位:730030 兰州,兰州大学第二医院泌尿外科 兰州大学第二临床医学院(杨树军、尚攀峰);青岛大学泌尿外科(卫浩);中国人民解放军总医院第三医学中心泌尿外科(许勇、王恒恩、孙圣坤),病理科(郭爱桃),检验科(张小丽);中国人民解放军总医院第四医学中心骨科医学部骨科研究所(宋翔宇、贾志博、彭江、崔梦一、杨博尧、陈蕾佳);中科奥格生物科技有限公司(潘登科、杜嘉祥)作者简介:杨树军(ORCID:0000-0002-4309-3695),博士研究生,研究方向为肾移植和泌尿系统肿瘤,Email:通信作者:尚攀峰(ORCID:0009-0008-4203-6585),主任医师,教授,博士研究生导师,研究方向为泌尿系统肿瘤和肾移植,Email:;孙圣坤(ORCID:0000-0002-6463-6425),主任医师,副教授,硕士研究生导师,研究方向为肾移植,Email:Preliminary report of perioperative monitoring of six-gene-edited pig-to-cynomolgus monkey kidney xenotransplantation Yang Shujun*,Wei Hao,Xu Yong,Wang Hengen,Song Xiangyu,Jia Zhibo,Peng Jiang,Cui Mengyi,Yang Boyao,Chen Leijia,Guo Aitao,Zhang Xiaoli,Pan Dengke,Du Jiaxiang,Shang Panfeng,Sun Shengkun.*Department of Urology,Second Hospital of Lanzhou University,the Second Clinical Medical College of Lanzhou University,Lanzhou 730030,ChinaCorresponding authors:Shang Panfeng,Email: Sun Shengkun,Email:522第 14 卷器官移植目前,中国的慢性肾衰竭患者逐渐增多,肾源短缺是肾移植手术最大的瓶颈1-3。基因编辑手段进行猪胚胎干细胞培育可以为异种移植提供肾脏来源,若能有效抑制排斥反应,异种移植应用于临床肾移植将成为可能1-6。2020 年,窦科峰院士团队报道多基因编辑猪异种器官移植在肾移植中获得成功,存活时间1 d3。近年来,基因编辑猪的改造和繁育技术不断成熟,且基因编辑猪-非人灵长类动物(non-human primate,NHP)异种肾移植模型在临床前研究已经获得长期存活,这为我们探索异种肾移植提供了理论指导7-12。异种移植最主要的障碍是天然抗原引起的排斥反应,凝血调节功能和生理功能也是需要考虑的问 题8-9,13-15。超急性排斥反应(hyperacute rejection,HAR)是由猪内皮细胞上存在的-1,3-半乳糖基转移酶(-1,3-galactosyltransferase,GalT)表位与受体内存在的天然抗体结合而引起的,天然抗体还可激活补体系统,进而引起机体迅速出现大量出血和血栓形成1,11。2002 年,Science报道 Lai 等16通过杂合突变体缺失的成纤维细胞进行基因编辑,使得 GalT 基因敲除(GalT gene-knockout,GTKO)小型猪在异种移植中的应用成功。该种方法可清除异种天然抗原表达,为克服异种移植 HAR 带来了希望。目前多种基因敲除以及不同类型基因片段的转入方法可以使得移植受体获得理想的存活时间3,10。本研究选择人源化基因编辑猪作为供体,食蟹猴作为受体,这是目前研究最多的基因编辑猪-NHP 异种肾移植模型。本文旨在构建基因编辑猪-NHP 异种移植模型,通过观察异种肾移植围手术期的变化,探索其临床前研究的应用前景和未来进入临床实验实现临床转化的可能。1 材料与方法1.1 实验动物受体为食蟹猴,编号 17101,5 周龄的雌性猴,体质量约 3.4 kg(图 1A),由中国人民解放军军事科学院军事医学研究院实验动物中心提供。供体为六基因编辑猪,编号 168,6 月龄,雄性,体质量 22.2 kg,由成都中科奥格生物科技有限公司提供。敲除了GalT 基因、-1,4-N-乙酰氨基半乳糖转移酶 2(-1,4 N-acetylg alactosaminyltransferase 2,4GalNT2)基hCD55/hCD46/hTBM)was transplanted into a cynomolgus monkey.The survival of the recipient and kidney condition after blood perfusion were observed.The parenchymal echo,blood flow changes,and size of the kidney were monitored on a regular basis.Routine blood test,kidney function test and electrolyte assessment were carried out.Dynamic changes of urine,feces and body mass were monitored.At the end of life,the transplant kidney,heart,liver,spleen,lung,and cecum were collected for pathological examination.Results The recipient died at postoperative 7 d.After blood flow was restored,the kidney was properly perfused,the organ was soft and the color was normal.At the end of the recipient s life,a slight amount of purulent secretion was attached to the ventral side of the kidney,with evident congestion and swelling,showing the appearance of red kidney.Postoperatively,the echo of renal parenchyma was increased,blood flow was decreased,the cortex was gradually thickened,and a slight amount of effusion surrounded the kidney and abdominal cavity over time.In the recipient,the amount of peripheral red blood cells,hemoglobin,albumin,and platelets was progressively decreased,and serum creatinine level was increased to 308 mol/L at postoperative 7 d,whereas the K+concentration did not significantly change.Light yellow urine was discharged immediately after surgery,diet and drinking water were resumed within postoperative 3 h,and light yellow and normal-shape stool was discharged.The reddish urine was gradually restored to normal color within postoperative 1 d,which were consistent with the results of the routine urine test.A large amount of brown bloody stool was discharged twice in the morning of 2 d after surgery.Omeprazole was given for acid suppression,and the stool returned to normal at postoperative 4 d.The 2-microglobulin level was increased to 0.75 mg/L at postoperative