输注转染IL-2基因同源短发夹型RNA的淋巴细胞抑制大鼠肝移植急性排斥反应  

The inhibitory effect of infusion of lymphocytes transfected with IL-2 shRNA mediated by pGenesil-2 vector plasmid on acute rejection in rat liver transplantation

作　　者：鄢涛[1,3] 王森[2] 龚建平[1] 彭勇[1] 刘作金[1] 石毓君[1] 刘鸿翔[1] 

机构地区：[1]重庆医科大学附属第二医院肝胆外科,400016 [2]德阳市人民医院内分泌科 [3]德阳市人民医院急诊科,618000

出　　处：《中华器官移植杂志》2008年第11期661-665,共5页Chinese Journal of Organ Transplantation

基　　金：国家自然科学基金（30500473和30401693）;重庆市卫生局科研项目基金（渝卫03-2-065）

摘　　要：目的探讨输注转染白细胞介素2（IL-2）基因同源短发夹型RNA（shRNA）的受者淋巴细胞对大鼠肝移植后急性排斥反应的影响。方法构建针对大鼠活化T淋巴细胞IL-2基因编码区的shRNA表达质粒，体外转染受者（BN大鼠）淋巴细胞。以Lewis大鼠为供者、BN大鼠为受者，进行肝移植，干扰组的受者于移植术中经门静脉回输转染IL-2-shRNA的BN大鼠淋巴细胞；环孢素A组（CsA组）的受者移植术中经门静脉输入生理盐水，肝移植后应用CsA肌肉注射；生理盐水对照组的受者移植术中经门静脉输入生理盐水；空载体对照组的受者移植术中经门静脉输入转染空载体的BN大鼠淋巴细胞。术后第7天，处死BN大鼠，取移植肝组织，观察组织和细胞超微结构改变情况以及肝细胞凋亡情况，同时测定肝功能指标、肝组织中IL-2基因的表达以及血清IL-2、肿瘤坏死因子α（TNF-α）和γ干扰素（IFN-γ）水平。计算各组受者1周存活率。结果移植后第7天，生理盐水对照组和空载体对照组的移植肝组织中可见中、重度急性排斥反应改变，而干扰组和CsA组病理切片中无或仅有轻度急性排斥反应征象。干扰组凋亡肝细胞数为（23±3．2）／mm2，CsA组为（26±4．0）／mm2，均明显低于生理盐水对照组和空载体对照组（P〈0．05）。干扰组和CsA组的IL-2mRNA表达明显低于生理盐水对照组和空载体对照组（P〈0．05）；干扰组和CsA组的血清IL-2、TNFrn和IFN-γ水平较空载体对照组和生理盐水对照组分别有不同程度降低（P〈O．05）。干扰组和CsA组的丙氨酸转氨酶和总胆红素均明显低于空载体对照组和生理盐水对照组（P〈0．05），而白蛋白则高于空载体对照组和生理盐水对照组（P〈0．05）。干扰组受者1周存活率为87．5％（7／8），明显高于生理盐水对照组的37．5％（3／8）和空载体对照组的37．5％（3／8，P〈OObjective To investigate the inhibitory effects of RNA silencing via plasmid- mediated interleukin-2 shRNA on acute rejection of rat liver transplantation. Methods pGenesil-2-IL- 2-shRNA vector plasmid expressing IL-2-shRNA was constructed, and transfeeted into freshly isolated rat lymphocytes with cationic liposome. At 24 h post-transfection, the production of IL-2 in the supernatant was measured by ELISA, and the change in the IL-2 mRNA levels was detected by using RT-PCR. The 32 rats were randomly divided into 4 groups., contrast group in which recipients were injected by the branch of portal vein with the BN rat lymphocytes （1 ml, 2 × 10^6/ml） carrying pGensil-2 in advance after anastomosis of infrahepatic veins, rejection group with isotonic Na chloride 1 ml, interference group with the lymphoeytes carrying pGenesil-2-IL-2-shRNA, and CsA group with isotonic Na chloride during operation and Cyelosporine A （3.0 mg ·kg-1· d-1 ） was intramuscularly injected daily for 7 days on the day 0 postoperatively. Recipients were sacrificed on 7th day postoperatively, and liver tissues and blood samples were collected. Recipient survival rate, histopathological and ultrastruetural characteristics were observed. Apoptosis cells in the graft were measured by using TUNEL. IL-2 mRNA expression in liver graft was detected by RT-PCR. Serum levels of IL-2, TNF-α, IFN-γ, ALT, TBIL, and Alb were also determined. Results （1） pGenesil-2- IL-2-shRNA vector plasmid was successfully constructed and identified with digestion and sequencing. At 24th h post transfection, the results of ELISA and semi-quantitative RT-PCR assay indicated that the inhibition ratio was about 82 %, compared with the control （P〈0. 05）. （2） On the 7th day postoperatively, the results of IL-2 rnRNA expression in liver graft, the serum levels of IL-2, TNF-α, IFN-γ, ALT, TBil, ALB, and the change of histopathological and ultrastructural characteristics, the detection of apoptosis cells in graft, all indicated that injection of lymphocyte

关 键 词：RNA干扰 白细胞介素2 转染 肝移植 移植物排斥 

分 类 号：R686[医药卫生—骨科学]