腺病毒载体介导的CTLA4-FasL基因转移联合应用供者脾细胞输注促进异基因小鼠皮肤移植耐受

Adenovirus-mediated CTLA4-FasL gene transfer combined with donor spleen cells improves induction of transplantation tolerance to the allogeneic skin allograft in mice

目的:探讨腺病毒介导CTLA4 -FasL基因转移联合应用供者脾细胞输注延长小鼠皮肤移植物存活的作用及其机制。方法:在移植的第0天,C5 7BL 6 (H 2 b,B6 )小鼠经尾静脉注射BALB c(H 2 d)小鼠脾细胞和5×10 9空斑形成单位 毫升(pfu ml)CTLA4 - FasL重组腺病毒(AdCTLA4 - FasL) ,同一天进行皮肤移植,每天观察移植物存活情况。耐受小鼠尾静脉取血,用酶联免疫吸附法(ELISA)测定CTLA4 FasL融合蛋白的体内表达;于第2 0天对受体小鼠进行迟发型超敏反应、单向混和淋巴细胞反应(MLR)、IL- 2逆转试验、过继转移实验和嵌合体检测等耐受状态的检测,以探讨耐受机制。结果:经尾静脉输注供体脾细胞及AdCTLA4 - FasL的B6小鼠,皮肤移植物的平均存活时间达(42 8±2 6 )天(n =6 ) ,而对照的未处理组,绿色荧光蛋白(EGFP)腺病毒处理组,重组腺病毒AdCTLA4Ig组和AdCTLA4 FasL处理组的平均存活时间为(10 .1±0 . 4 1)天(n =6 )、(10-.5±1.4 )天(n =6 )、(12 . 8±1. 1)天(n =6 )、(2 0 .0±2 .5 )天(n =6 ) ,差异具有显著意义(P <0 .-0 1) ;皮肤移植物长期存活的受体小鼠对供体抗原的低应答可以通过脾细胞被过继转移;DTH反应受到抑制、MLR活性特异性降低,且可以被外源性IL 2部分逆转。结论:供体脾细胞输注和腺病毒介导CTLA4 -

Objective:To investigate the potential effects and mechanisms of adenovirus-mediated CTLA4-FasL gene transfer combined with donor spleen cells in inducing transplantation tolerance using mice skin graft model.Methods:C57BL/6 mice(H-2 b,B6) received an injection of allogeneic spleen cells from BALB/c(H-2 d) and AdCTLA4-FasL at a dose of 5×10 9 pfu/ml via the tail vein,and then grafted with allogeneic skin from BALB/c mice.The survival of skin graft monitored by daily observation.The serum level of CTLA4-FasL was measured via ELISA.The twenty days after induction of tolerance,the tolerance mechanisms were investigated with delayed type hypersensitivity,mixed lymphocyte reaction,IL-2 reverse experiment,adoptive transfer and chimerism.Results:The survival of BALB/c allografts were prolonged significantly in B6 recipients receiving the allogeneic spleen cells plus AdCTLA4-FasL with a mean of (42.8±2.6 d, n =6),significantly longer than those of the untreated recipients(10.1±0.41 d, n =6),AdEGFP-treated recipients(10.5±1.4 d, n =6),AdCTLA4Ig-treated recipients(12.8±1.1 d, n =6) and AdCTLA4-FasL-treated recipients(20.0±2.5 d, n =6).Splenocytes of B6 recipients with long-term surviving skin allograft displayed donor-specific hypersponsiveness,which could be inhibited in the DTH and partly reversed in the presence of exogenous added IL-2 in MLR.Conclusion:Adenovirus-mediated CTLA4-FasL gene transfer renders prolonged therapeutic expression of CTLA4-FasL in B6 recipient mice,leading to long-term survival of skin allografts.The induced tolerance is donor-specific.Clonal anergy is the main mechanism of the tolerance.