玻璃体注射腺相关病毒2和慢病毒转染大鼠视网膜的比较

Comparison of adeno-associated viral 2 vector and lentiviral vector transfection in rat retina after intravitreal injection

目的通过玻璃体注射重组腺相关病毒2载体(r AAV2)和慢病毒载体(LV),比较两者转染成年大鼠视网膜的异同。方法实验组大鼠60只,每组12只玻璃体内分别注射重组腺相关病毒2载体-增强绿色荧光蛋白(r AAV2-EGFP)、重组腺相关病毒2载体-神经突起素-增强绿色荧光蛋白(r AAV2-neuritin-EGFP)、慢病毒载体-红色荧光蛋白(LV-RFP)和慢病毒载体-神经突起素-红色荧光蛋白(LV-neuritin-RFP),对照组注射等量的生理盐水,4周后取材,采用免疫荧光和CTB-FITC检测2种病毒载体在视网膜中转染的细胞及转染率,然后分别采用Real-time PCR和Western blotting检测神经突起素(neuritin)在视网膜中的表达变化。结果 r AAV2能够转染约70%视网膜节细胞(RGCs),LV主要转染色素上皮细胞,RGCs转染率仅为30%;r AAV2-neuritin-EGFP组神经突起素mRNA表达约是r AAV2-EGFP组和对照组的16倍,蛋白表达约为3倍;LV-neuritin-RFP组神经突起素mRNA表达约是LVRFP组和对照组的5.5倍,蛋白表达约为1.7倍。结论 r AAV2玻璃体注射后,转染大部分RGCs且神经突起素表达量高于LV,LV主要转染色素上皮细胞,提示基因治疗眼科疾病和损伤时,涉及到转染RGCs时应采用r AAV2载体,转染色素上皮时宜采用LV载体。

Objective To compare the transfection of recombinant adeno-associated viral 2 vector（ r AAV2） with lentiviral vector（ LV） in rat retina after intravitreal injection,in order to supply experimental basis of vector choice for gene therapy of ophthalmic diseases and injuries. Methods Twelve rats each group of sixty in the experimental groups were accepted r AAV2-EGFP, r AAV2-neuritin-EGFP, LV-RFP or LV-neuritin-RFP by intravitreal injection respectively,whereas rats in the control group,normal saline. Four weeks later,retinas of all rats were taken for observation. The immunohistochemical staining and CTB-FITC were used to determine the cell types and percentage of transfection. Realtime PCR and Western blotting were used to detect the expression of mRNA and protein of neuritin in the retina. Results The majority of retinal ganglion cells（ RGCs） with a percentage of 70% were transfected by r AAV2,whereas LV transfected pigment epithelium and only 30% of RGCs. The expression of neuritin mRNA and protein upregulated 16-flod and 3-fold respectively in the r AAV2-neuritin-EGFP group compared with the r AAV2-EGFP group and the control group,whereas the expression of neuritin mRNA and protein upregulated 5. 5-flod and 1. 7-fold in the LV-neuritin-RFP group compared with the LV-RFP group and the control group. Conclusion After intravitreal injections,r AAV2 but not LV could provide majority RGCs transduction and more overexpression of neuritin,LV mainly transfect the pigment epithelium.The results suggest that when RGCs need to be transfected,we should choose r AAV2,and when pigment epithelium need to be transfected,we should choose LV in the gene therapy of ophthalmic diseases and injuries.