抗凋亡基因bcl-X_L转染视网膜感光细胞的研究

Transfection of bcl-X_L into retinal photoreceptors in vitro

目的 :探讨介导抗凋亡基因bcl-XL 转染视网膜感光细胞的有效方法。方法 :采用视网膜神经胶质细胞条件培养液体外培养的视网膜感光细胞 ;用脂质体LIPOFECTAMINETM2 0 0 0介导 pEGFP -C3 -bcl-XL 转染至感光细胞 ;采用滴度为 6 5× 10 12 pfu/L的重组腺病毒rAd -gfp -bcl-XL 转染感光细胞 ;在荧光显微镜下观察感光细胞中的绿色荧光蛋白表达情况来比较脂质体、重组腺病毒转染法的转染率 ;用免疫组化比较这两种方法转染的细胞中Bcl-XL蛋白水平。结果 :脂质体、重组腺病毒转染法的转染率分别为 0 1%、 60 % ,免疫组化示重组腺病毒转染的细胞的Bcl -XL 蛋白水平较脂质体转染的细胞高。结论 :在视网膜感光细胞的基因转染中 ,腺病毒介导的目的基因bcl-XL 的转染率及表达明显高过脂质体法 ,重组腺病毒介导的基因转染法是视网膜变性性疾病基因治疗的一种较理想方法。

Objective:To investigate an optimal gene transfer method for retinal photoreceptors. Methods:pEGFP-C3-bcl-X L was transfected into retinal photoreceptors by using LIPOFECTAMINE TM2000 (LF2000),and 6.5×10 12pfu/mL rAd-gfp-bcl-X L was used to transfect retinal photoreceptors.48h after transfection,the positive photoreceptors with green fluorescence were numbered under fluorescence microscopy and the transfection efficiency was calculated.The expression level of Bcl-X L protein in the transfected photoreceptors was assessed by immunohistochemistry assay. Results:The transfection efficiency of LF2000 mediated and rAd-mediated method was 0.1% and 60%,respectively.A significant different expression of Bcl-X L protein was observed between lipofectin-transfecting and rAd-transfecting neurons. Conclusions:The transfection efficiency of rAd is higher than that of LF2000 in cultured retinal photoreceptors,and rAd is an optimal gene transfer method for gene therapy in retinal degenerative diseases.