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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 expression vector of recombinant adeno-associated virus (rAAV) carrying antisense Ⅱ-4 gene and then use it as an intervention in rat asthma model to explore its feasibility as a gene therapy program for asthma. Methods Recombinant adeno-associated virus (rAAV-asIL4) carrying antisense IL-4 gene was synthesized by cotransfecting 293T cells with plasmid pasIL-4, pXX2 and pXX680 by calcium phosphate precipitation method. Southem blot assay for titers of synthetic viruses. Rats in asthma group (group T) were treated with rAAV-asIL4 twice by airway administration on the first day and the fourteenth day. The normal rats (N group) and the asthma group (Group A) And rAAV-GFP intervention group (group C). After the last challenge, rats were sacrificed and the total number of nucleated cells and eosinophils in the BALF was counted. The levels of IL-4 and IgE in BALF were measured by ELISA, and the changes of IL-4 mRNA in lung tissue were detected by RT-PCR Pulmonary tissue for pathological examination. Results The total number of nucleated cells and the total number of Eos in BAIF were analyzed. The results showed that the two indexes in group T were lower than those in group A and C, but there was no significant difference between the three groups (P> 0.05). ELISA results of IL-4 and IgE in BALF and semi-quantitative RT-PCR analysis of IL-4 mRNA in lung tissue showed that the above indexes in T group were significantly lower than those in A and C groups (P <0.01). The pathological changes of lung in T group were lighter than those in A and C groups. Conclusion rAAV vector carrying antisense IL-4 gene may play a role in asthma intervention. rAAV-asIL4 may have some clinical application prospects.