腺相关病毒介导胶质细胞源性神经营养因子转染牛眼虹膜色素上皮细胞

Adeno-associated virus mediated delivery and expression of GDNF cDNA in cultured bovine iris pigment epithelial cells

目的 探讨以腺相关病毒 (AAV)为载体对体外培养牛眼虹膜色素上皮细胞 (IPECs)进行胶质细胞源性神经营养因子 (GDNF)转染 ,获得高效表达GDNF转基因细胞的可行性。方法 采用狭隙杂交测定AAV GDNF的滴度 ,按感染复数 (MOI)为 50对传二代IPECs进行AAV GDNF转染 ,于含3 %胎牛血清DMEM培养基中连续不换液培养 4周 ,酶联免疫吸附测定 (ELISA)细胞培养上清液中GDNF的量。同样方法对传二代IPECs进行AAV介导的绿色荧光蛋白 (GFP)基因转染 ,于含 2 0 %胎牛血清DMEM培养基中进行常规细胞培养。荧光显微镜下每 2d计数 1次培养IPECs的GFP表达阳性率。结果 (1)AAV GFP转染后 ,IPECs生长正常 ,于转染后 4d部分IPECs开始出现GFP表达。GFP的表达在荧光显微镜 490nm波长激发光下呈黄绿色 ,弥漫于整个胞质。转染 8～ 10d后 ,IPECs中GFP的表达至高峰。 (2 )根据ELISA检测结果、经过计算 ,AAV GDNF转染IPECs的培养上清液中GDNF的含量为 (17.14± 1.10 ) μg/L。 结论 AAV GDNF能有效地转染体外培养的IPECs和表达GDNF。 (中华眼科杂志 ,2 0 0 4,40 :45 48)

Objectives To obtain transge nic bovine iris pigment epithelial cells ( IPECs ) by adeno-associated virus (AAV) mediated delivery of cDNA of glial cell-line derived neurotrophic factor (GDNF). Methods AAV-GDNF was titrated by slot blotting. Cultured bovine passage two IPECs were transfected using AAV-GDNF at dosage of MOI (multiplicity of infection)=50, then were cultured in DMEM medium complemented with 3% FBS for 4 weeks with no change of medium. The expression of GDNF in culture medium was examined using ELISA test. By using the same methods, cultured passage two IPECs were transfected with AAV-GFP (green fluorescent protein) at dosage of MOI=50, and then were cultured in DMEM medium complemented with 20% FBS. The expression of GFP in IPECs was examined using fluorescence microscope every 2 days after transfection. Results (1)GFP expression in IPECs could not be detected until 4 days after transfection. The positive GFP expression in IPECs reached fastigium in day 8 or 10. (2)According to the results of ELISA test, concentration of GDNF in the culture medium was (17.14±1.10) μg/L . Conclusions AAV-GDNF can effectively transfect cultured IPECs, and the transgenic cells show a high expression of GDNF. (Chin J Ophthalmol, 2004,40: 45-48)