3种腺相关病毒介导的绿色荧光蛋白对西藏小型猪胚胎成纤维细胞的转染效率

Efficiency of three adeno-associated viruses for transfecting enhanced green fluorescent protein in Tibet minipig fetal fibroblasts

目的比较3种不同血清型腺相关病毒(AAV)介导的绿色荧光蛋白对西藏小型猪胚胎成纤维细胞的转染效率。方法原代分离培养西藏小型猪胚胎成纤维细胞并进行形态学鉴定,按照感染复数(MOI)为103、104、105转染传代两次的猪胚胎成纤维细胞,流式细胞学检测不同血清型AAV对西藏小型猪胚胎成纤维细胞的转染效率,并在倒置显微镜下观察转染后绿色荧光的表达强度;同时,应用MTT方法检测不同血清型AAV对西藏小型猪胚胎成纤维细胞的毒性。结果腺相关病毒AAV2-EGFP对PFF的转染效率随MOI值的增加而增高,MOI为103、104、105时,转染效率分别为(33.68±1.18)%、(50.80±2.59)%和(60.08±1.08)%,而其他两种血清型病毒感染效率均低于AAV2-EGFP。3种病毒转染PFF过程中,细胞生长正常,MTT显示各个转染组与对照组(未转染组)在D570处吸光度无显著差异。结论AAV2血清型载体对体外培养的猪胚胎成纤维细胞的感染效率显著高于其他几种血清型,AAV8和AAV9对PFF感染能力极差,筛选AAV2作为西藏小型猪基因打靶的最适病毒载体;同时,3类血清型病毒载体对猪胚胎成纤维细胞均无明显细胞毒性。

Objective To compare the efficiency of three different serotypes of adeno-associated virus （AAV） in mediating the transfection of enhanced green fluorescent protein （EGFP） in Tibet minipig fetal fibroblasts （PFFs）. Methods Three recombinant AAV of different serotypes encoding EGFP were constructed and transfected into primary cultured PFFs at the multiplicity of infection （MOI） ranging from 10^3 to 10^5. The expression rates of EGFP in the PFFs were assessed 72 h after the infection by flow cytometry, and the transfected PFFs were observed under inverted fluorescence microscope. The toxicity of AAVs to PFFs was analyzed using MTT assay. Results The transfection efficiency of AAV2-EGFP increased with MOI. At the MOI of 10^3, the transfection efficiency of AAV2-EGFP was （33.68±1.18）%, which increased to （50.80±2.59）% at the MOI of 10^4 but without obvious further increase at the MOI of 10s. The other two serotypes of the virus （AAV8 and AAV9） showed no obvious changes in the infection efficiency at any MOIs. The transfection efficiency of AAV8 was （8.3±0.02）% and that of AAV9 was （2.20±1.02）% at the MOI of 10^5. Transfection with the 3 viruses caused no adverse effects on the normal cell growth of the PFFs. Conclusions AAV2 has a significantly higher infection rate in cultured PFFs than AAV8 and AAV9, and the latter two have a rather tow infection efficiency. All the three AAVs have no cell toxicity to the PFFs.