[重组1型和2型腺相关病毒在视网膜细胞中的转导效率]

[Transduction efficacy of recombinant type 1 and type 2 adeno-associated virus in the retinal cells].

作者信息

Wu Ji-hong, Zhang Sheng-hai, Liu Yan, Chen Xia-fang, Xu Ping, Wu Xiao-bing, Huang Qian

机构信息

Central Experimental Laboratory, First People's Hospital, Shanghai Jiaotong University, Shanghai 200080, China.

出版信息

Zhonghua Yi Xue Za Zhi. 2006 Oct 31;86(40):2841-6.

PMID:17200021

Abstract

OBJECTIVE

To investigate the transduction and gene expression of the recombinant adeno-associated viruses (rAAV) of the serotypes 1 and 2 in the retinal cells.

METHODS

rAAV vectors of type 1 and type 2 encoding EGFP were infected into the cultured retinal pigmentary epithelium (RPE) cells of the line CRL-2302 and primarily cultured retinal neural cells from normal SD rats, and primarily cultured RPE cells from an adult cornea donor. The cultured RPE cells transduced by rAAV2-EGFP or rAAV2/1-EGFP were harvested at the 7 th and 14 th day after infection to be detected by fluorescence-activated cell sorter. The onset of EGFP gene expression and EGFP positive rate were detected by flow cytometry and fluorescence microscopy. Then, rAAV2/1-EGFP and rAAV2-EGFP were injected into the subretinal spaces of 32 SD rats to investigate the onset of EGFP fluorescence and its distribution in the fundus in vivo via fluorescence stereoscope. HE staining and immunohistochemistry were used to observe the infected cell type and immune response in the retina.

RESULTS

The percentage of EGFP positive cells and mean intensity of EGFP fluorescence in the cells transduced by rAAV2-EGFP 7 and 14 days after transduction were 13.50% +/- 1.70% and 15.60% +/- 0.82%, and 2.75 +/- 0.12 and 3.80 +/- 0.72 respectively; and the EGFP positive cells and mean intensity of EGFP fluorescence in the cells transduced by rAAV2/1-EGFP were 1.09% +/- 0.5% and 1.98% +/- 0.45%, and 1.12 +/- 0.09 and 1.75 +/- 0.2 respectively. The EGFP fluorescence area in the retina were (5389 +/- 211) microm(2), (9832 +/- 364) microm(2), (14 454 +/- 446) microm(2), (20 528 +/- 648) microm(2), and (20 264 +/- 683) microm(2) respectively 3, 7, 14, and 75 days, and 4 month after transduction by rAAV2-EGFP in vivo; In the rat retina transduced by rAAV2/1-EGFP, the EGFP fluorescence areas were (9666 +/- 348) microm(2), (12 160 +/- 439) microm(2), (19 794 +/- 621) microm(2), (26 172 +/- 923) microm(2), and (26 022 +/- 965) microm(2) respectively 3, 7, 14, and 75 days, and 4 month after infection.

CONCLUSION

rAAV2 efficiently transduces retinal cells both in vitro and in vivo. rAAV2/1 is a more effective gene-transferring vector to be used in retinal cells in vivo than rAAV2.

摘要

目的

研究1型和2型重组腺相关病毒（rAAV）在视网膜细胞中的转导及基因表达情况。

方法

将编码增强绿色荧光蛋白（EGFP）的1型和2型rAAV载体分别感染培养的CRL-2302系视网膜色素上皮（RPE）细胞、正常SD大鼠原代培养的视网膜神经细胞以及成年角膜供体的原代培养RPE细胞。在感染后第7天和第14天收获经rAAV2-EGFP或rAAV2/1-EGFP转导的培养RPE细胞，采用荧光激活细胞分选仪进行检测。通过流式细胞术和荧光显微镜检测EGFP基因表达的起始时间及EGFP阳性率。随后，将rAAV2/1-EGFP和rAAV2-EGFP注射到32只SD大鼠的视网膜下间隙，通过荧光立体显微镜观察体内EGFP荧光的起始情况及其在眼底的分布。采用苏木精-伊红（HE）染色和免疫组织化学方法观察视网膜中的感染细胞类型及免疫反应。

结果

rAAV2-EGFP转导后7天和14天，EGFP阳性细胞百分比及细胞内EGFP荧光平均强度分别为13.50%±1.70%和15.60%±0.82%，以及2.75±0.12和3.80±0.72；rAAV2/1-EGFP转导的细胞中，EGFP阳性细胞百分比及细胞内EGFP荧光平均强度分别为1.09%±0.5%和1.98%±0.45%，以及1.12±0.09和1.75±0.2。体内经rAAV2-EGFP转导后3天、7天、14天、75天和4个月时，视网膜中EGFP荧光面积分别为(5389±211)μm²、(9832±364)μm²、(14454±446)μm²、(20528±648)μm²和(20264±683)μm²；在经rAAV2/1-EGFP转导的大鼠视网膜中，感染后3天、7天、14天、75天和4个月时，EGFP荧光面积分别为(9666±348)μm²、(12160±439)μm²、(19794±621)μm²、(26172±923)μm²和(26022±965)μm²。