DA至Lewis大鼠肝移植急性排斥反应模型的建立:技术改良分析

Construction of a rat DA to Lewis orthotopic liver transplantation model:Technical improvement

背景:国际上研究肝移植免疫耐受的基础动物模型是大鼠肝移植急性排斥反应模型,国际上公认的肝移植急性排斥反应模型鼠种配对方式为DA至Lewis大鼠、DA至BN大鼠及BN至Lewis大鼠,但由于鼠种缺乏和操作技术有待成熟的原因,国内较少引用以上鼠种配对方式进行该模型的建立。目的:课题组在大量SD大鼠肝移植模型建立训练的基础上,采用DA大鼠为供体,Lewis大鼠为受体,摸索DA至Lewis大鼠肝移植急排模型建立技巧和经验。方法:通过改良二袖套法,以雄性DA大鼠为供体,雄性Lewis大鼠为受体,建立原位肝移植动物模型60只,受体大鼠术前1d和术后1周内饲喂治疗剂量的他克莫司,1周后半量递减并停药,记录移植手术时间,观察受体大鼠的术后生存状况、手术成功率及生存期,分别于术后7,14,21,28d处死受体大鼠,获取肝组织标本,苏木精-伊红染色,观察肝脏大体和镜下的病理学变化,进行急性排斥反应评分。结果与结论:供肝冷缺血时间30～60min,供体手术时间(18.5±4.0)min,供肝修整时间(7±3)min,受体手术时间(35.0±7.3)min,无肝期为(13.0±3.0)min,手术成功率为98%,1周存活率为91.6%。术后2周随着他克莫司撤药,受体大鼠迅速发生急性排斥反应,于术后14～28d死亡,平均生存时间为(20.85±0.71)d,中位生存时间为21d。实验建立DA至Lewis大鼠肝移植急性排斥反应动物模型需要以大量SD大鼠肝移植训练为基础进行,通过对二袖套法技术的改良和围手术期短期应用他克莫司有助于该模型的稳定建立。

BACKGROUND：The acute rejection model of rat liver transplantation is the basic for studying the immunologic tolerance of animals,and the accepted matching pattern was DA to Lewis rats,DA to BN rats,and BN to Lewis rats.The above mentioned matching methods are few in China due to insufficient rat species and immature operation technology.OBJECTIVE：To establish rat（DA to Lewis） orthotopic liver transplantation model and to explore the skill and experience for establishing this model.METHODS：A total of 60 rats were established orthotopic liver transplantation models using Kamada two-cuff technique.Male DA rats served as donors,and male Lewis rats were used as recipients.Therapeutic amount of tacrolimus was administrated to recipients during 1 day before operation and 1 week after operation,which was continuously half dose decreasing every day began from eighth day post-operation and stopped administration by thirteenth day post-operation.The operation time was recorded,and the survival status,success rate and survival time was observed.All rats were sacrificed at 7,14,21 and 28 days after operation.The gross and histopathologic changes of liver tissues were observed.And rejection activity index of liver tissues was scored.RESULTS AND CONCLUSION：The cold ischemia time of donor liver was 30-60 minutes,the donor operation time,graft truing time,recipient operation time,and anhepatic period were（18.5±4.0） minutes,（7±3） minutes,（35.0±7.3） minutes,（13.0±3.0） minutes,respectively.The operation success rate and one week survival rate were 98% and 91.6%,respectively.Acute rejection occurred rapidly 2 weeks after liver transplantation due to tacrolimus discontinuation.Recipient＇s dead time distributed from 14 to 28 days after the operation and mean survival time were（20.85±0.71）days,and the median survival time was 21 days respectively.Based on improvements in Kamada two-cuff technique and short-term tacrolimus application on perioperative period,DA to Lewis rat acute rejection orthotopic liver transplantation models were stably established.