腺相关病毒介导绿色荧光蛋白基因对体外培养IPE细胞的转染及毒性

Study on recombinant adeno-associated virus-mediated transfection of green fluorescent protein gene and the influence of adeno-associated virus to iris pigment epithelial cells in vitro

目的 研究腺相关病毒 (recombinantadeno associatedvirus ,rAAV)载体介导的绿色荧光蛋白基因 (greenfluorescentprotein ,GFP)对体外培养虹膜色素上皮细胞 (irispig mentepithelial,IPE)的转染和病毒载体对IPE细胞的毒性 ,为rAAV携带目的基因治疗视网膜色素变性提供理论依据。方法 酶辅助的显微分离方法体外培养IPE细胞并鉴定。按转染倍数 (multipleofinfection ,MOI)为 10 3 、10 4、10 5、10 6转染已培养 3d的传二代IPE细胞 ,流式细胞仪检测rAAV GFP对IPE的转染效率。MTT法检测rAAV GFP对IPE细胞的毒性。结果 rAAV GFP对IPE的转染效率 ,MOI =10 3 时为 2 6 .7%、MOI =10 4时为 5 3.6 %、MOI =10 5时为 6 0 .2 %、MOI =10 6时是 6 3.7% .AAV GFP转染IPE细胞后 ,细胞生长正常 ,MTT法显示各转染组与未转染组的OD值无统计学差异。结论rAAV GFP对体外培养的IPE细胞转染效率可达 6 0 %以上 ,而且腺相关病毒对细胞生长无明显抑制 。

Objective To evaluate the efficiency of recombinant adeno associated virus(rAAV) mediated transfection of green fluorescent protein(GFP) gene and the influence of rAAV to iris pigment epithelial(IPE) cells in vitro and the possibility of rAAV as a vector in the gene therapy of retinitis pigmentosa(RP). Methods IPE cells were cultured and identified by immunocytochemical staining(AE1/AE3 and S 100). According to various multiples of infection (MOI=10 3,10 4,10 5,10 6), rAAV GFP was added to the second generation IPE cells cultured for three days. The transfection efficiency of rAAV GFP to IPE cells was evaluated by flow cytometer. The effect of rAAV vector on IPE cells' growth was determined with assay. Results The transfection efficiency of rAAV GFP to IPE cells was 26.7%(MOI=10 3), 53.6% (MOI=10 4),60.2%(MOI=10 5),63.7% (MOI=10 6). MTT method showed that the OD records among transfected group (with various MOI) and untransfected group had no statistical difference. Conclusion The efficiency of rAAV mediated transfection of GFP gene to IPE cells is above 60%, rAAV vector has no inhibitory effect to IPE cells in vitro. rAAV is a safe vector in gene therapy of RP.