腺病毒载体介导的ICOSIg融合基因对实验性自身免疫性心肌炎作用的研究

Effects of adenovirus-mediated gene transfer of ICOSIg fusion protein on experimental autoimmune myocarditis in Lewis rats

目的探讨腺病毒载体介导的 ICOSIg 基因治疗对实验性自身免疫性心肌炎(EAM)的作用。方法将人 ICOS 胞外域与免疫球蛋白 IgGFc 段融合,构建 ICOSIg 融合基因的腺病毒表达载体 p-Adeno-ICOSIg。Pac I 消化腺病毒载体后转染 HEK 293细胞,生产表达 ICOSIg 融合蛋白的腺病毒;构建增强型绿色荧光蛋白腺病毒作为对照。皮下注射猪心肌肌球蛋白诱导 Lewis 大鼠 EAM 模型,随机分为4组,分别于免疫当天(第0天)( A组,n=15)和第14天(B 组,n=15)通过股静脉注射ICOSIg 重组腺病毒,观察共刺激分子融合蛋白对 T 细胞激活阶段和炎症阶段的作甩,C 组(n=10)和D组(n=10)分别在第0天和第14天注射表达增强型绿色荧光蛋白(EGFP)的重组腺病毒。另设 E组(n=10)未接受免疫的正常对照组。免疫第28天,超声心动图检测后处死动物。HE 染色检测心肌炎症浸润程度。Western blot 检测心肌 ICOS、ICOSL、B7-1及 B7-2蛋白表达。实时定量 RT-PCR 定量心肌 IL-2、IL-4和 IFN-γ mRNA 水平。结果第28天 B 组大鼠心功能指标(短轴缩短率:40.6±6.2比26.2±4.6,20.8±5.6,21.7±9.6)、心肌炎症程度较对照组显著改善。Western blot 显示 B组 ICOS、ICOSL 及 B7-1蛋白表达显著下调,各组间 B7-2蛋白表达差异未见统计学意义。B 组大鼠心肌组织 IFN-γ mRNA 表达降低(19.8±7.9比66.6±4.5,79.6±5.9,80.9±5.1),IL-4 mRNA 表达增加(42.3±8.6比19.7±3.8,22.3±9.0,28.3±2.2),IL-2表达各组间无显著差异,因此 B 组 IFN-γ/IL-4比值显著低于 A、C 及 D 组(0.47±0.36比2.91±0.22,1.89±0.42,2.32±0.83),表明 Th细胞因子平衡向 Th2方向偏离。结论炎症高峰期以 ICOSIg 阻断共刺激分子通路能够减轻 EAM 心肌组织炎症,改善大鼠心脏功能。其机制可能是下调心肌组织局部 ICOS、ICOSL 和 B7-1表达及对炎症性细胞因子的抑制作用。

Objective To explore the effects of adenovirus vector-mediated gene transfer of ICOSIg fusion protein on experimental autoimmune myocardifis （EAM） in Lewis rats. Methods Expression vector containing ICOSIg （p-Adeno-ICOSIg） was constructed by fusion of human ICOS and IgGFc segment. Adenovirus vector was digested by Pac i enzyme and transfected into HEK 293 cells. Adenovirus expressing ICOSIg was produced. EGFP was constructed into adenovirus vector and used as control. EAM was induced in Lewis rats by injection of porcine cardiac myosin. All immunized Lewis rats were divided into 4 groups. Group A （n = 15） and B （n = 15） received adenovirus containing ICOSIg on day 0 and day 14 respectively to study the effects of costimulatory molecules gene therapy on T cell activation and inflammation; group C （n = 10） and group D （n = 10） received adenovirus containing EGFP on day 0 and day 14 respectively as controls. Group E （ n = 10） was normal controls that did not receive immunization. On day 25, all rats were killed after echecardiography examination. Histopathological examination was performed to observe myocardial inflammation. Protein levels of ICOS,ICOSL,BT-1 and B7-2 were detected by Western blot. INF-γ, IL-2 and IL-4 mRNA were determined by realtime RT-PCR. Results On day 28, cardiac function was significantly improved and myocardial inflammation significantly attenuated in group B compared to group A, C and D （ all P 〈 0. 05 ）. B7-1 expression at protein level was significantly lower in group B than that of group C （ P 〈 0. 05 ）. ICOS and ICOSL expressions at protein level were significantly decreased in both group A and B compared with group C and D （ P 〈 0. 05 ）. IFN-γ mRNA level significantly decreased and IL-4 mRNA significantly increased in group A and B compared to group C and D （ P 〈 0. 05 ）. Conclusions Blockade of costimulatory pathway with gene therapy of ICOSIg alleviated autoimmune inflammatory damage and improved cardiac function in Lewis rats with EAM. Down-regulated costimulatory molecules in the myocardium and reduced inflammatory cytokine secretion might be responsible for the beneficial effects of ICOSIg in this model.