联合转移人HT和DAF基因对模拟异种移植超急性排斥反应的抑制作用  

Research of the transgenic mice co-expressing human [alpha] (1,2)-fucosyltransferase gene and decay accelerating factor to avoid xenograft hyperacute rejection

作　　者：刘秉乾[1] 王广有[1] 李胜芝[1] 张志宏[1] 李光三[2] 张立霞[2] 马腾骧[1] 

机构地区：[1]天津市泌尿外科研究所 [2]中国科学院遗传与发育生物学研究所

出　　处：《中华器官移植杂志》2007年第9期517-521,共5页Chinese Journal of Organ Transplantation

基　　金：天津市科技发展计划项目重点资助(043803411);天津医科大学博士课题创新基金

摘　　要：目的探讨联合转移人α1,2-岩藻糖苷转移酶(HT)和衰变加速因子(DAF)基因对模拟异种移植超急性排斥反应的抑制作用。方法通过显微注射制备转人HT及DAF基因的子代小鼠,以聚合酶链反应和流式细胞仪筛选出人HT和(或)DAF基因整合与表达阳性的子代转基因小鼠。以同时表达人HT和DAF基因的小鼠为实验组,单一表达人HT或DAF基因的小鼠为HT对照组和DAF对照组,取其心脏,采用改良的Langendorff心脏灌注装置灌注心脏,先以KH液逆向灌注,30 min后再以含15%(体积分数)人AB血清的Krebs-Henseleit液(KH液)灌注。记录不同灌注时间的心率、左心室收缩压及左心室舒张末压,计算左心室获得压,以心率与左心室获得压的乘积作为心脏左心室收缩功能的指标,免疫组化法观察灌注后的心脏血管内皮细胞和组织中IgM与C3c的沉积情况。结果KH液灌注过程中,各组间心脏做功能力的差异无统计学意义;KH液中加入人血清后,HT对照组和DAF对照组的心脏做功能力明显下降,在灌注60 min时心脏做功分别为最大值的27%和23%,平均做功时间分别为118 min和89 min,普通小鼠心脏在灌注后50 min时停止搏动,而实验组的心脏做功能力下降缓慢,至60 min时心脏做功维持在最大值的67%以上,平均做功时间为225 min,其心脏做功能力与搏动时间高于HT对照组和DAF对照组(P<0.05)。免疫组化染色显示,实验组小鼠心脏血管内皮细胞与组织中未见IgM和C3c沉积,HT对照组可见C3c少量沉积,DAF对照组可见IgM沉积和C3c少量沉积,普通小鼠心脏可见IgM和C3c沉积。结论小鼠联合转移人HT和DAF基因后,其心脏在人血清灌注下的免疫损伤明显减轻,这可能对抵御异种移植超急性排斥反应有一定帮助。Objective To investigate whether the isolated perfused hearts from transgenic mice co-expressing the human [alpha] （1,2）-fucosyltransferase （HT） and decay accelerating factor （DAF） can be provided more effective protection against xenograft hyperacute rejection. Methods Transgenic mice co-expressing the human HT and DAF were produced by co-microinjection of transgene constructs for human HT and DAF. Integration and expression of human HT and DAF transgenes were tested by ploymerase chain reaction （PCR） and flow cytometry （FCM）. The transgenic mice coexpressing human HT/DAF were used as experimental groups, and the transgenic mice expressing human HT or DAF alone as control groups. The hearts of the mice were perfused ex vivo with 15 human plasma by using a modified Langendoff apparatus. After perfusion of hearts with Krebs-Henseleit （KH） buffer for 30 min, pooled normal human AB plasma was added continuously to the buffer reservoir to reach a final concentration of approximately 15 %, after which perfusion was continued. Heart rate and force of contractions were monitored and heart work was calculated as the product of heart rate and force. Immunohistochemistry was used to detect the deposition of IgM and C3c in the hearts. Results The heart work was similar when the hearts were perfused with KH buffer. Heart work was deteriorated rapidly in HT and DAF control groups, falling below 27 % and 23 % of the initial level within 60 min of the addition of human plasma respectively. In contrast, the hearts from experimental mice maintained greater than 67% of their initial work rate within 60 min after the addition of human plasma. The deposition of IgM and C3c was not found on the surface of endothelial cells in the experimental groups. There was a little C3c in HT control group, and a considerable amount IgM and a little C3c in DAF control group. Conclusion Functional studies with hearts from the double transgenic mice showed that the co-expression of HT and DAF markedly increased their resist

关 键 词：转基因 小鼠 抗原 嗜异性 心室功能 左 

分 类 号：R617[医药卫生—外科学]