AD.Egr-Smad7定向基因治疗放射性肺纤维化的体内实验研究

Effects of gene therapy with replication-defective adenovirus ericlosing Egr-1 promoter and Smad7 cDNA on irradiation-induced pulmonary fibrosis: experiment with mice

目的通过体内实验研究，观察AD．Egr-Smad7防治C57BL小鼠放射性肺纤维化的疗效，探讨其作用机制。方法将Egr-1基因启动子的放射敏感元件和Smad7 cDNA包装到复制缺陷型腺病毒内，制备成AD．Egr-Smad7。288只小鼠随机分成6组，分别为空白对照组，单纯放射组（每日照射2次，每次8Gy，两次照射间隔8h）、AD．Egr-Smad7组，AD．Egr-Smad7放射组，病毒载体组，病毒载体放射组，各组又分为放射后0、1、2、4、8及12周小组，每小组小鼠8只。小鼠气管内给AD．Egr-Smad7、病毒载体，24h后单次全胸放射。用ELISA法检测肺组织内TGF-81、CTGF、Ⅰ型及Ⅲ型胶原含量；碱水解法测定羟脯氨酸含量；光镜下病理组织学做肺泡炎及肺纤维化镜下评分。结果γ射线照射激活Egr-1启动子调控腺病毒介导的外源性Smad7在C57BL小鼠肺内放射后1～4周内有降低TGF-B、Ⅰ型、Ⅲ型胶原及羟脯氨酸含量的作用，与对照组相比差异有统计学意义（均P〈0．01）；光镜下病理组织学改变及肺纤维化评分也证明放射激活Egr-1启动子调控外源性Smad7有降低放射后12周时肺纤维化的作用。结论放射前单次给药，经放射线诱导Egr-1启动子靶向性调控外源性Smad7基因表达，4周内在一定程度上存在阻断TGF-β信号传导通路的作用，12周时病理结果显示有定向阻断放射性肺纤维化的作用。但降低病毒载体的免疫源性及解决外源性基因持续作用问题需要进一步研究。

Objective To investigate the effects of gene therapy with replication - defective adenovirus enclosing Egr-1 promoter and Smad7 cDNA on irradiation-induced pulmonary fibrosis. Methods The recombinant replication-defective adenovirus AD. Egr-Smad7 enclosing Egr-1 promoter and Smad7 cDNA was constructed. 288 C57BL mice were randomly divided into 6 groups： AD. Egr-Smad7 group （ Group RA, receiving intratracheal instillation of AD. Egr-Smad7 of the dose of 109 pfu/0. 1 ml） , AD. Egr-Smad7 ＋ radioation group （ Group RAR, receiving intratracheal instillation of AD. Egr-Smad7 of the dose of 109 pfu/ 0.1 ml and then radiation to the chest 14 h later）, replication-defective adenovirus group （Group AV, receiving intratracheal instillation of replication-defective adenovirus of the dose of 10^9 pfu/0. 1 ml ）, replication-defective adenovirus ＋ irradiation group （Group AVR, receiving intratraeheal instillation of replication-defective adenovirus of the dose of 109 pfu/0.1 ml and then radiation to the chest 14 h later）, blank control group （Group C） , and pure irradiation group （Group R） , each group was re-divided into 6 subgroups of 8 mice to be observed 0, 1,2, 4, 8, and 12 weeks after the treatment. The mice were killed at different time points and their lungs were taken out. The levels of type Ⅰ collagen, type Ⅲ collagen, connective tissue growth factor （ CTGF）, and transforming growth factor-β1 （ TGF-β1 ） were detected by ELISA. The level of hydroxyproline was examined by alkaline hydrolysis method. The lung tissues were stained with HE to undergo pathological examination. Results The TGF-β1 levels of the irradiation groups all increased, peaking in the second week （ all P 〈 0.05 ） , all significantly higher than those of Group C. However, the TGF-β1 levels at different time points of Group RAR were all significantly lower then those of the other irradiation groups. The CTGF levels of different groups at different time points were all significantly higher than than of Group C （ P 〈 0.05 or P 〈 0.01 ）, and the CTGF levels of Groups RA and AV were decreased to almost normal 12 weeks after the irradiation. The levels of type Ⅰ collagen and type Ⅲ collagen of the 1 and 2-week subgroups of Group RAR were significantly lower than those of Group C （ all P 〈 0.01 ）, then gradually increased, and were slightly higher than those Group C 12 weeks later. The levels of type Ⅰ collagen and type Ⅲ collagen at different time points of the other groups were all significantly higher than those of Group C （ P 〈 0.05 or P 〈 0.01 ）. However, The levels of type Ⅰ collagen at different time points of Group RAR were all lower than those of the other groups except Group C, and the levels of type Ⅲ collagen in the first to eighth weeks after irradiation of Group RAR were all lower than those of the other groups except Group C. The hydroxyproline level of the Ⅰ and 2 week subgroups of Group RAR were significantly lower than those of Group C （ all P 〈 0.01 ）, and then gradually increased. The hydroxyproline levels of the other irradiation groups all gradually increased significantly, peaking at the 12 th week （ all P 〈 0.01 ）. 1- 2 weeks after irradiation Groups RAR, RA, and AV showed remarkable pulmonary congestion changes, even more remarkable then those in Group R, 8 - 12 week later, fibrosis changes were found in Group R and AVR, and 12 weeks later the histological structure of lung of Group AV, RAR, and RA returned almost normal. Conclusion Radioactive rays induce Egr-1 promoter to regulated the expression of exogenous Smad7 gene that blocks the signal transduction of rrGF-β. Thus use of AD. Egr-Smad7 may become a novel strategy of gene therapy in prevention and treatment of pulmonary fibrosis.