血红素氧合酶-1基因治疗减缓移植物血管病及机制

Adenovirus-mediated heme oxygenase -1 gene therapy ameliorates transplant arteriosclerosis and the underlying mechanisms

目的观察血红素氧合酶-1（HO-1）基因治疗减缓同种移植物血管病的效果，探讨其机制。方法以BN—Lewis大鼠血管移植为对象，依据基因治疗方案分为4组：同系对照组、空白对照组、载体对照组、腺病毒介导的HO-1（AdHO-1）组。移植后2个月，观察各组移植物纤维化和内膜增生，检测T细胞（CD3＋）、B细胞（CD45RA）和巨噬细胞（CD68＋）浸润数量，逆转录．聚合酶链反应（RT—PCR）和Westernblot检测移植物HO-1基因和蛋白的表达，酶联免疫吸附试验（ELISA）法检测受体血清白细胞介素（IL）-10的浓度。结果同系对照组无移植物血管病表现，空白对照组和载体对照组大量纤维沉积，AdHO-1组纤维沉积轻微。血管内膜／（内膜＋中膜）百分比4组分别为7．6％、81．4％、85．9％、15．9％。每400倍视野浸润细胞数4组分别为T细胞（9．2±1．6、92．3±11．6、89．6±17．8、39．3±10．1）、B细胞（3．6±1．1、72．6±11．8、66．6±10．9、30．6±9．9）、巨噬细胞（7．5±1．2、78．5±21．7、72．5±19．8、34．5±18．7）。血清IL-10浓度4组分别为（50．2±20．1）、（40．2±11．1）、（38．6±19．3）、（481．2±69．1）ng／L。AdHO-1组与空白对照组和载体对照组间差异有统计学意义（P〈0．05）。AdHO．1基因治疗增高了移植血管HO．1基因和蛋白的表达。结论AdHO-1基因治疗减缓同种移植物血管病，移植物纤维化和内膜增生明显减轻。AdHO-1基因治疗下调了T细胞、B细胞和巨噬细胞在移植物中的浸润，增加了HO-1和IL-10的表达，IL-10-HO-1通路的活化可能是移植血管得到保护的重要原因。

Objective To observe the effect of adenovirns-mediated heme oxygenase-1 （AdHO-I） gene therapy on allograft transplant arteriosclerosis and to elucidate the underlying mechanisms. Methods Aorta transplants in BN-Lewis rats were used and divided into four groups： isograft group, control group, vector control group, and AdHO-1 group. The allograft fibrosis and neointimal proliferation were observed two months post transplant. The infiltrates of T cells （ CD3 ± ）, B cells （ CIM5RA）, and macrophages （CD68 ±） were detected. The gene and protein expression of heme oxygenase-1 （HO-1） was examined by using reverse transcription-polymerase chain reaction （RT-PCR） and Western blotting. Serum level of in- terleukin （IL）-10 was determined by enzyme-linked immunosorbent assay （ELISA）. Results No arteriosclerosis was found in the isograft group. Fibrosis was severe in control group and vector control group, but mild in AdHO-1 group. The ratio of intima/（ intima ± media） was 7.6%, 81.4%, 85.9% and 15.9% in isograft group, control group, vector control group, and AdHO-1 group, respectively. The infiltrates of T cells per view/400 magnification in isograft group, control group, vector control group, and AdHO-1 group were （9. 2 ± 1.6）, （92. 3 ± 11.6）, （89. 6 ± 17. 8） and （39. 3 ± 10. 1）, those of B cells were （3.6 ± 1.1 ）, （72. 6 ± tl. 8）, （66. 6 ± 10. 9） and （30. 6 ±9. 9）, and those of macrophages were （7. 5 ± 1.2）, （78. 5 ±21.7）, （72. 5 ± 19. 8） and （34. 5±18.7）, respectively. The serum level of IL-10 was （50. 2 ±20. 1 ）, （40. 2±1.1 ）, （38.6 ±19. 3） and （481.2 ±69. 1 ） ng/L in isograft group, control group, vector control group, and AdHO-1 group, respectively. A significant difference was observed between AdHO- 1 group and control group or vector control group （P 〈 0. 05 ）. AdHO-1 therapy Up-regulated the expression of mRNA and protein of HO-1. Conclusion AdHO-I gene therapy ameliorates allograft transplant arterio- sclerosis and fibrosis, and neointimal proliferation. AdHO-1 gene therapy down-regulates the infiltrates of T cells, B cells and macrophages, and increases the expression of HO-1 and IL-10. The IL-10-H0-1 pathway may play key roles in the protection of aorta allografts.