腺伴随病毒介导绿色荧光蛋白基因对体外培养的鼠视网膜神经节细胞转染的实验研究  

Experimental study on transfecting green fluorescence protein gene mediated by recombinant adeno-associated virus to retinal ganglion cells of rats in vitro

作　　者：李海燕[1] 赵家良[1] 张华[1] 

机构地区：[1]中国协和医科大学中国医学科学院北京协和医院眼科

出　　处：《眼科新进展》2006年第10期737-739,共3页Recent Advances in Ophthalmology

摘　　要：目的评价重组腺伴随病毒载体介导的绿色荧光蛋白基因(recombinant adeno-associated virus-mediated green fluorescence protein,rAAV-GFP)对体外培养的鼠视网膜神经节细胞(retinal ganglion cells ,RGCs)的转染效率和安全性。方法取出生24 h内的SD乳鼠的视网膜神经上皮层,行视网膜神经细胞的体外混合培养;培养2 d后,按转染倍数(multiple oninfection,MOI)为10、102、103、104、105对细胞进行转染;5 d后行流式细胞仪检测细胞表达绿荧光的比率,并应用PE-Thy1 .1抗体标记RGCs ,评价不同MOI的rAAV-GFP对RGCs的转染效率。应用MTT比色方法检测各组细胞的生长活性。结果MOI为10、102、103、104、105时,对混合培养的全体细胞的转染效率分别为6·4 %、15.6 %、20.2 %、45.1 %和65.8 %,其中对RGCs的转染效率分别为11.7 %、22.5 %、27.7 %、52.0 %和75.3 %;rAAV-GFP转染RGCs后,细胞生长状态正常;MTT比色结果显示,各转染细胞和未转染细胞的光吸收值无显著性差异(P>0 .05)。结论 rAAV作为体外培养的SD乳鼠RGCs转染的载体是安全可行的,且对细胞生长无明显影响。Objective To evaluate the efficiency and safety of transfecting green fluorescence protein gene mediated by recombinant adeno-associated virus（rAAV-GFP） to retinal ganglion cells （RGCs） of rats. Methods The retina of neonatal Sprague-Dawley rats （ postnatal within 24 hours） was dissected into cells suspension with trypsin digestion ,and the cells suspension were cultured without purification. Two days later, the cells were transfected by rAAVGFP by adding virus suspension into the cell culture medium according to various multiples on infection （MOI） （ MOI =10、10^2、10^3、10^4、10^5 ）. The transfecting efficiency of rAAV-GFP to RGCs was evaluated by flow cytometer 5 days after the transfection. MTT was added to each well, then the survival cells were evaluated by the optical density （OD） values with ELISA-reader. Results When MOI was 10、10^2、10^3、10^4、and 10^5, the transducting efficiency of rAAV-GFP to the total cells was 6.4%, 15.6%,20.2%, 45. 1%and 65.8% respectively; And the transducting efficiency of rAAV-GFP to RGCs was 11.7 %, 22.5 %,27.7 %, 52.0 % and 75.3% respectively. MTT colortmetric assay showed that the Or） values of tmnsfected cells with various MOI and untransfected cells had no statistical difference （P〉0.05）.Conclusion rAAV as the transfecting vector has a satisfying safety for RGCs of rats in vitro and has no inhibitory effect to the RGCs.

关 键 词：重组腺伴随病毒载体 绿色荧光蛋白 视网膜神经节细胞 细胞转染 

分 类 号：R774.1[医药卫生—眼科] Q959.836[医药卫生—临床医学]