腺相关病毒介导的增强型绿色荧光蛋白基因转染豚鼠巩膜成纤维细胞的实验研究  

Expression of enhanced green fluorescent protein gene in guinea pig fibroblasts mediated by adeno-associated virus

作　　者：鲍庆东 刘太祥 罗鑫 田祥 BAO Qingdong;LIU Taixiang;LUO Xin;TIAN Xiang(Department of Ophthalmology,Affiliated Hospital of Zunyi Medical University,Zunyi 563000,Guizhou Province,China)

机构地区：[1]遵义医科大学附属医院眼科,贵州省遵义市563000

出　　处：《眼科新进展》2020年第11期1029-1032,共4页Recent Advances in Ophthalmology

基　　金：国家自然科学基金资助(编号81660169)。

摘　　要：目的比较四种不同血清型腺相关病毒(adeno-associatedvirus,AAV)介导增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)基因转染豚鼠巩膜成纤维细胞(guinea pig scleral fibroblast,GSF)的转染效率及安全性,筛选合适的基因转染载体。方法原代分离培养GSF并鉴定,利用AAV2、AAV5、AAV8、AAV9介导EGFP基因并按不同感染复数(multiplicity of infection,MOI)10、100、1000、10000转染GSF,空白对照组加入空白培养基,转染后2 d、3 d、4 d、5 d荧光显微镜下观察GFP表达强度及细胞生长状态,流式细胞术检测细胞转染率及凋亡率,CCK-8法检测细胞活力。结果AAV感染GSF后2 d各组均可见弱荧光,荧光强度随时间延长及MOI值增大而增强,而AAV9-EGFP组全程弱荧光,空白对照组全程无荧光。转染后5 d,MOI=10000时各组荧光最强,基因转染率组间整体比较,差异具有统计学意义(P<0.05);组间两两比较,AAV8-EGFP组均高于其他各组,差异均有统计学意义(均为P<0.05)。各组Annexin V阳性凋亡率与空白对照组比较,差异均无统计学意义(均为P>0.05)。各组在450 nm波长处光密度值与空白对照组比较,差异均无统计学意义(均为P>0.05)。结论AAV8能有效地转染GSF,且对细胞凋亡、细胞活力无明显影响,是安全可行的。Objective Tocompare the transfection efficiency and safety of enhanced green fluorescent protein(EGFP)gene in guinea pig scleral fibroblasts(GSF)mediated by four different adeno-associated virus(AAV)to screen the suitable gene-transfection vectors.Methods GSF was isolated and cultured in the primary generation and identified.The EGFP gene was mediated by AAV2,AAV5,AAV8,and AAV9,and the GSF was transfected at different multiplicity of infection(MOI)(MOI 10,100,1000,10000),and blank transfected mediumwas added in blank control group.After staining 2 day,3 day,4 day,5 day,we observed the GFP expression intensity and cell growth status under a fluorescence microscope,flow cytometry to detect the cell transfection rate and apoptosis rate,and CCK-8 method to detect cell viability.Results Weak fluorescence was seen in each group 2 days after AAV infection with GSF,and the fluorescence intensity increased as time went by and the MOI value increased,while the AAV9-EGFP group had weak fluorescence throughout the whole process,and the blank control group had no fluorescence during the whole process.Five days after transfection,each group had the strongest fluorescence when MOI=10000,and the overall comparison of gene transfection rate between the groups showed statistically significant differences(P<0.05),and pairwise comparisons between the groups showed that the AAV8-EGFP group was higher than the other groups,and the differences were statistically significant(all P<0.05).Compared with the blank control group,the positive apoptosis rate of AnnexinV in each group was not statistically different(all P>0.05).There was no statistically significant difference in the optical density value of each group at 450 nm wavelength and the blank control group(all P>0.05).Conclusion The AAV8 can be steady and efficient to transfect GSF.It has no obvious effect on cell apoptosis and cell viability,which is safe and feasible.

关 键 词：腺相关病毒 增强型绿色荧光蛋白 豚鼠巩膜成纤维细胞 基因转染 

分 类 号：R778.1[医药卫生—眼科]