真核表达载体pcDNA3-CTLA4Ig转染小鼠对移植胰岛功能的影响  

Preventive effect of pcDNA3-CTLA4Ig plasmid transfection on islet allograft rejection in mice

作　　者：赵国华[1] 孙宏治[1] 钟鑫平[1] 张金祥 许国岩 赵宁[1] 程颖[1] 张佳林[1] 刘永锋[1] 

机构地区：[1]中国医科大学附属第一医院器官移植科 [2]解放军65735部队医院内科

出　　处：《世界华人消化杂志》2007年第14期1596-1601,共6页World Chinese Journal of Digestology

基　　金：辽宁省自然科学基金;No.20042096~~

摘　　要：目的:探讨真核表达载体pcDNA3-CTLA4Ig转染受体小鼠肌细胞后对胰岛移植后排斥反应的影响.方法:受体C57BL/6小鼠随机分为对照组、空白转染组和实验组(转染pcDNA3-CTLA4Ig).Western blot检测受体小鼠血清CTLA4-Ig表达,观察移植物存活时间,术后7 d取受体肾脏做HE染色及胰岛素免疫组化染色,采用微量全血~3H-胸腺嘧啶掺入法检测单个核细胞在刀豆蛋白A刺激下的增殖程度,流式细胞仪检测外周血T细胞亚群表达情况.结果:受体小鼠转染5 d后,Western blot检测到血清内有CTLA4Ig表达,转染效率为27.50%;实验组小鼠血糖维持正常时间明显延长(P<0.05):术后7 d,转染组单个核细胞在刀豆蛋白刺激下的增殖程度明显降低(P<0.05),CD4^+、CD8^+T淋巴细胞表达与对照组和空白转染组相比有显著差异差异(CD4^+:14.38%±0.84% vs 20.56%±0.68%,21.04%±1.14%,P<0.05:CD8^+:14.77%±0.92% vs 24.63%±1.30%,23.84%±1.21%,P<0.05),小鼠肾被膜下胰岛素免疫组化染色强度明显增高.结论:利用脂质体法可将pcDNA3-CTLA4Ig转染到活体小鼠肌细胞内并在小鼠体内表达CTLA4Ig,从而阻断B7/CD28共刺激信号途径,抑制胰岛移植术后排斥反应.AIM： To study the effect of pcDNA3-CTLA4Ig plasmid on islet allograft rejection in＇mice. METHODS： C57BL/6 mice were randomly divided into control group, blank group and experiment group （transfected with pcDNA3- CTLA4Ig plasmid）. The level of serum CTLA4Ig was detected by Western blot on day 5 after transfection, Blood glucose was examined after operation every other day. Immunohistochemical staining was used to determine the expression of insulin. 3H-thymidine incorporation was performed to detect the ability of T lymphocyte increment and the levels of CD4^＋ and CD8^＋ T lymphocytes were detected by flow cytometry on day 7 after transplantation. RESULTS： Five days after transfection, Western blot demonstrated serum expression of CTLA4Ig, and the transfection efficacy was 27.50%. Blood glucose maintained at the normal level for a longer period of time in the experiment group, and on day 7, the proliferation of lymphocytes was markedly decreased （P 〈 0.05）. The levels of CD4^＋ and CD8^＋ T lymphocytes were significantly lower in the experiment group than those in the control and blank group （CD4^＋ 14.38% ＋ 0.84% vs 20.56% ＋ 0.68%, 21.04% ＋ 1.14%, P 〈 0.05; CD8^＋ 14.77% ＋ 0.92% vs 24.63% ＋ 1.30%, 23.84% ＋ 1.21%, P 〈 0.05）, and the intensity of immunohistochemical staining was also stronger.CONCLUSION： CTLA4 gene can be transferred into mouse muscular cells by cation liposome and express its products in the serum, which leads to the inhibition of islet allograft rejection by the blockage of B7/CD28 signal pathway.

关 键 词：胰岛移植 排斥反应 共刺激信号 B7/CD28 

分 类 号：R657.5[医药卫生—外科学]