反义白介素4重组腺相关病毒载体气道给药对哮喘大鼠的影响

Effects of recombinant adeno-associated virus-antisense IL-4 on asthmatic rats by airway delivery

目的构建携带反义Ⅱ-4基因的重组腺相关病毒(rAAV)真核表达载体,并以此干预大鼠哮喘模型,探讨其作为哮喘基因治疗方案的可行性。方法磷酸钙沉淀法将质粒pasIL-4、pXX2及pXX680共转染293T细胞合成携带反义IL-4基因的重组腺相关病毒(rAAV-asIL4)。Southem blot法检测合成病毒的滴度。rAAV-asIL4在动物实验开始的第1天和第14天2次通过气道给药干预哮喘大鼠(T组),实验中同时设立大鼠正常组(N组)、哮喘组(A组)以及rAAV-GFP干预组(C组)。末次激发后处理大鼠,计数肺泡灌洗液(BALF)中有核细胞以及嗜酸性细胞总数,ELISA检测BALF中IL-4和IgE蛋白量,RT-PCR检测肺组织IL-4 mRNA变化,取肺组织作病理学检查。结果分析BAIF中有核细胞细胞总数和Eos总数,结果表明:T组该2项指标较A组和C组有降低趋势,但这3组间差异无统计学意义(P>0．05)。BALF中IL-4、IgE的ELISA检测结果以及肺组织IL-4 mRNA RT-PCR半定量检测结果表明:T组的上述指标水平较A组和C组均明显降低(P<0．01)。T组大鼠肺组织病理学改变较A组和C组轻。结论携带反义IL-4基因的rAAV载体,对于哮喘的干预具有一定的作用。rAAV-asIL4可能具有一定临床应用前景。

Objective To construct eukaryotic antisense IL-4 expressing vector of recombinant adeno-associated virus eukaryotic expression vector bearing antiseuse IL-4 （ rAAV-asIL4）, and explore effects of this vector on asthmatic rats, for investigating rAAV-asIL4 potential use in gene therapy for asthma. Methods 293T cells were transfected with pasllA, pXX2 and pXX680 using the calcium phosphate method to construct rAAV-asIL4. Southern blot was performed to detect the titer of rAAV-asIL4. 30 SD rats were randomized into four groups： normal group （group N）, asthma group （group A）, rAAV-asIL4 treated group （group T）, and rAAV-GFP treated group （group C）. The rots of group T were intratracheally administered rAAV-asIL4 on day 1 and day 14. rAAV- GFP was used as a control. The nucleated cells and eosinophils in bronchoalveolar lavage fluids （BALF） were counted. The production of IL-4 and IgE in BALF were measured using ELISA kits respectively. IL-4 mRNA in lungs was determined with RT-PCR. Histological sections of lungs were evaluated by HE staining. Results Although the number of nucleated cells and eosinophils were decreased in group T compared with group A and group C, there was no significantly difference among them, In rats administered with rAAV-asIL4, IL-4 mRNA in lung tissues, IL-4 protein and IgE in BALF were significantly decreased compared with those of group A and group C. The pulmonary histopathological changes were less in group T compared with group A and group C . Conclusion The results of this study provide evidence for the potential utility of rAAV-asIL4 as an approach to gene therapy for asthma.