ZafA基因沉默对烟曲霉菌生长毒力的影响.doc

ZafA基因沉默对烟曲霉菌生长、毒力的影响 中文摘要 目的 通过RNA干扰技术,探讨ZafA基因在烟曲霉菌生长、毒力中的重要性,为肺曲 霉病新的治疗方法提供理论依据。 方法 用化学合成法合成siRNA,通过电转染技术将siRNA转入烟曲霉菌孢子,提取总 mRNA,进行RT-PCR,观察ZafA基因mRNA表达变化。将经过不同处理的烟曲霉菌孢子通 过鼻内接种于麻醉后的免疫力低下小鼠,通过G试验、观察小鼠死亡率、肺部病理改 变等,了解曲霉菌在体内的生长、毒力情况,并将肺组织于沙氏琼脂培养基中培养, 确定其感染的特异性及真菌的生长情况。 结果 (1)电转染后, siRNA干预组烟曲霉菌ZafA基因mRNA表达水平与18srRNA内参 的比值为0.533±0.026,较错义链对照组1.098±0.027和PBS对照组1.082±0.012明 显降低(P0.05); (2)转染后siRNA干预组烟曲霉菌在沙氏琼脂培养基上生长较错义 链对照组及PBS对照组明显受限(P0.05);(3)小鼠免疫指标:IgG造模前后分别为 0.69±0.04、0.33±0.07;IgA 0.71±0.05、0.51±0.06;IgM 1.41±0.08、1.04± 0.10(P0.05);(4)G试验检测1-3-β-D葡聚糖的含量,ZafA基因siRNA干预组较 其余两对照组明显降低(P0.05);(5)体内实验显示siRNA干预组死亡2只,死亡率 为6.3%,错义链对照组死亡16只,死亡率为53.3%,PBS对照组死亡15只,死亡率为50%。 肺部病理切片显示siRNA干预组肺部损伤较错义链对照组及PBS对照组明显减轻。生存 分析提示siRNA干预组小鼠的生存时间较错义链对照组及PBS对照组明显延长。肺组织 沙氏琼脂培养基培养示siRNA干预组未见真菌生长,错义链对照组及PBS对照组均有烟 曲霉菌生长。 结论 ZafA基因是烟曲霉菌体内生长和毒力的必须基因,RNA干扰技术可有效抑制该 基因的表达,使其生长及毒力受限,有望成为肺部真菌感染的有效治疗方法。 关键词 烟曲霉菌 RNA干扰 电转染 毒力IAbstract Objective Through RNAi ,investigating ZafA gene contributions to aspergillus fumigatus’ virulence and growth,to find out a new method to treat pulmonary aspergillosis Methods Useing chemical synthesis the siRNA,transform aspergillus fumigatus with synthetic siRNA by electroporation. Extract the total mRNA of aspergillus fumigatusPerforming RT-PCR,observe the change in mRNA level and analyze the gene whether or 7 -1 not be silenced.Infect the immunosuppressed BALB/c mouse intranasally with 2×10 cm aspergillus fumigatus, thought G-test result 、observe the mouce’ mortality and the lung pathological section change, to learn the aspergillus aspergillus growth and virulence.Put the lung tissue in culture Results 1After electroporation, the siRNA intervention group aspergillus fumigatus’s ZafA gene mRNA ZafA/18srRNA is 0.533±0.026, compare with the missense chain group 1.098±0.027 and the PBS contrast group 1.082±0.012 is obviously reduced P0.05; 2 The siRNA intervention group aspergillus fumigatus grows on culture medium compare