重组腺相关病毒载体介导CTLA4Ig转染延长同种大鼠心脏移植存活时间

Prolonged cardiac allograft survival in rats by systemic injection of adeno associated viral vectors containing CTLA4Ig gene

目的研究重组腺相关病毒载体介导CTLA4Ig(rAAV-CTLA4Ig)全身转染对大鼠同种心脏移植的影响.方法以BN大鼠为供者,Lewis大鼠为受者,建立心脏移植模型.实验分为两组.对照组:供、受者不给予任何处理;转染组:受者于心脏移植前30d,通过尾静脉全身注射1×109斑点形成单位(PFU)的rAAV-CTLA4Ig.观察移植心存活时间;ELISA法检测血清CTLA4Ig、干扰素γ(IFN γ)和白细胞介素4(IL-4)水平;免疫组织化学法(SABC法)检测移植心组织中CD4+和CD8+T细胞的浸润.对移植心存活时间明显延长的受者,用其脾细胞与供者脾细胞进行混合淋巴细胞培养,观察受者对供者的免疫反应状态.结果转染组移植心存活时间较对照组明显延长(P＜0.05);转染组血清CTLA4Ig蛋白一直维持在26.67～35.47 mg/L,移植后转染组血清IFN-γ水平下调,血清IL-4水平上调(P＜0.05);对照组出现典型的急性排斥反应表现,转染组心肌组织基本正常,间质内无炎性细胞浸润或血管外周及心肌间质内有局灶性炎性细胞浸润,未见坏死:对照组移植心组织中浸润的CD4+和CD8+T细胞数量明显高于转染组(P＜0.01);转染组受者对供者的脾细胞增殖反应明显低于对照组(P＜0.05).结论重组腺相关病毒载体可以介导CTLA4Ig基因的持续表达,通过全身途径转染受者可以明显延长同种移植心的存活时间,降低受者对供者的免疫反应性.

Objective To investigate the expression levels of the transfected gene and the survival times of the allografts in cardiac recipients who were systemically administered with recombinant adeno associated viral vectors containing CTLA4Ig. Methods Hearts from BN rats were transplanted into the abdomen of Lewis recipients to set up heart transplant model. The experiment was divided into 2 groups: Group 1: n =8, control, no treatment; Group 2: n =10, rAAV CTLA4Ig was injected via a recipient vein 30 days before transplantation. The level of serum CTLA4Ig was detected by enzyme linked immunosorbent assay (ELISA) analysis. After transplantation, the level of serum interferon γ and interleukin 4 in the recipients was determined by ELISA analysis also. The infiltrating CD4 + and CD8 + T cells in transplanted hearts were detected by immunohistochemistry assay. In the recipients with a prolonged survival of the transplanted hearts, the mixed lymphocyte culture of the recipients’ splenic cells and donors’ splenic cells was done to observe the immunity. Results The serum level of CTLA4Ig was detectable at 14th day after injection of recombinant adeno associated viral vectors, peaked ( 26.67 ~ 35.47 mg/L) at 30th day after gene transfection and maintained at this level afterwards. The survival time of the allografts in Group 2 was significantly prolonged as compared with that in Group 1 ( P < 0.05 ). In addition, down regulation of interferon γ and up regulation of interleukin 4 were observed in the Group 2 ( P < 0.05 ). The infiltrating CD4 +and CD8 + T cells in the hearts of group 2 were less than in group 1 ( P < 0.05 ). Conclusion The adeno associated virus mediated CTLA4Ig gene transfer into rats by systemic administration resulted in an efficient and stable expression of CTLA4Ig in vivo, and remarkable prolongation of cardiac allograft survival, which may be mediated by inhibition of CD28 associated signal transduction.