| 目的：研究重组腺相关病毒载体介导的绿荧光蛋白（AAV-GFP）对体外培养的S-D乳鼠视网膜神经细胞的转染，为AAV携带目的基因用于青光眼视神经保护治疗提供理论依据。
方法：取出生1～3天的S-D乳鼠的视网膜神经上皮层，采用胰酶消化制成细胞悬液，行视网膜神经细胞的体外混合培养；通过NSE单克隆抗体和Thy1.1抗体进行鉴定；培养3d后，按转染倍数（MOI）为100、101、102、103转染视网膜神经细胞；转染后每天应用倒置荧光显微镜观察视网膜神经细胞表达GFP表达阳性的情况；4d后行流式细胞仪检测细胞表达绿荧光的比率，评价不同转染倍数的AAV-GFP对视网膜神经细胞的转染效率。
结果：转染倍数（MOI）为100、101、102、103时，转染效率分别为1.3％、10.3％、23.6％、21.8％。
结论：本研究首次证实，AAV-GFP对体外培养的S-D乳鼠视网膜神经细胞具有一定的转染效率，应用腺相关病毒载体携带目的基因用于青光眼的视神经保护治疗是可行的。
【关键词】腺相关病毒 绿荧光蛋白 视网膜神经细胞 体外培养 转染
Transduction of green fluorescent protein gene by recombinant adeno-associated virus to nerve cells of retina in vitro
LI Hai-yan,ZHAO Jia-liang, ZHANG Hua. .Department of Ophthalmology, Peking Union Medical College Hospital, Peking Union Medical College,Chinese Academy of Medical Sciences.Beijing 100730,China
[Abstract]
Objective To evaluate the efficiency of recombinant adeno-associated virus (AAV)-mediated transduction of green fluorescent protein (GFP) to nerve cells of retina in vitro.
Methods The neurosensory retina of Spague-Dawley rats within postnatal 1 to 3 days were dissociated into cell suspension with 0.25% trypsin digestion. The cells were identified with immunohistochemistry method using anti-rat-NSE monoclonal antibody and with flow cytometry using anti rat-Thy1.1 monoclonal antibody. After 3 days, according to various multiples of infection ( MOI=100、101、102、103 ) , AAV-GFP was added to the cultured cells. Afer 4 days,The transfection efficiency of AAV-GFP to nerve cells of retina was evaluated by flow cytometer.
Results The transfection efficiency of AAV-GFP to nerve cells of retina was 1.3% (MOI=100 )、10.3% (MOI=101 )、23.6% (MOI=102 )、21.8% (MOI=103 )。
Conclusion Adeno-associated virus is a effective vector for cultured nerve cells of retina.
【Key words】 Adeno-associated virus(AAV); green fluorescent protein (GFP) ; nerve cells of retina; in vitro; transfection |
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