腺病毒介导slit2基因转染人视网膜色素上皮细胞对RF/6A细胞迁移及管腔形成的影响

Retinal pigment epithelial cells with adenovirus-slit2 transfectin improve migration and tube formation in choroidal vascular endothedial cells in vitro

目的观察共培养模型下,腺病毒介导的slit2基因转染人视网膜色素上皮细胞(RPE)对猴脉络膜视网膜血管内皮细胞(RF/6A)迁移及管腔形成的影响。方法用腺病毒介导的slit2基因(Ad-slit2)转染RPE细胞,同时设空载体腺病毒转染组(Ad-null)及空白对照组。转染24 h及48 h后,采用RT-PCR及Western blot法分别检测RPE细胞中slit2、VEGF的mRNA及蛋白表达情况;ELISA法检测RPE细胞培养基上清液中VEGF的分泌水平;建立RPE&RF/6A共培养模型,采用Transwell小室法及Matrigel胶体外三维成型法分别检测过表达slit2基因的RPE细胞对RF/6A细胞迁移能力及管腔形成能力的影响,并计算迁移数目及管腔形成长度、管腔数。结果 Ad-slit2转染24、48 h后,RPE细胞在基因及蛋白水平表达slit2、VEGF均有明显增高;ELISA结果显示过表达slit2基因的RPE细胞其培养基上清液中分泌型VEGF水平增高;在RPE&RF/6A共培养下,空白对照组、Ad-null转染24 h组、Ad-slit2转染24 h组、Ad-null转染48 h组、Ad-slit2转染48 h组中,RF/6A细胞迁移数目分别是(40.6±4.5)、(36.7±3.5)、(67.7±4.2)、(36.0±3.6)、(79.7±3.5);管腔形成长度分别是(3.95±0.31)、(4.53±0.26)、(6.04±0.34)、(4.09±0.18)、(7.27±0.45)mm,管腔数分别是(8.7±3.2)、(8.9±3.6)、(15.9±4.2)、(8.3±2.4)、(23.3±4.5)。Ad-slit2转染24 h及48 h组与空白对照组比较,差异具有统学意义(P<0.05)。结论腺病毒介导的slit2基因转染RPE细胞可促进RF/6A细胞的迁移及管腔形成。RPE细胞中的slit2可能参与脉络膜新生血管形成过程。

Objective To determine the effect of retinal pigment epithelial （RPE） cells with adenovirus-slit2 （Ad-slit2） transfeetion on the migration and tube formation of choroidal vascular endothelial （RF/6A） cells in a co-culture system. Methods After RPE cells were transfected with ad-slit2 or ad-null for 24 and 48 h, the expression of slit2 and vascular endothelial growth factor （VEGF） at mRNA and protein levels was quantified by RT-PCR and Western blotting, and the level of secreted VEGF protein from the media of RPE cells were investigated by ELISA. Cocuhure models （RPE＆RF/6A） were used to observe the effects of RPE cells transfection with ad-slit2 on the migration and tube formation of RF/6A by Transwell chamber test and Matrigel invasion assay respectively. Results After ad-slit2 transfected to RPE cells for 24 and 48 h, the expression of slit2 and VEGF at mRNA and protein levels was obviously increased. At the same time, the level of secreted VEGF protein from the media of RPE cells was also elevated. In the coeulture system, the number of RF/6A migrated was 40.6 ±4.5, 36.7± 3.5, 67.7± 4.2, 36.0± 3.6 and 79.7± 3.5 in the blank control group, ad-null for 24 h group, ad-slit2 for 48 h group, ad-null for 48 h and ad-slit2 for 48 h group respectively, the total length of tube formation of RF/6A was 3.95±0.31,4.53 ±0.26, 6.04 ±0.34, 4.09±0.18, 7.27 ±0.45 mm respectively, and the number of tube was 8.7 ± 3.2, 8.9 ±3.6, 15.9± 4.2,8.3 ±2.4, and 23.3 ±4.5 respectively. There were significant differences in the above indexes between the ad-slit2 for 24 h group, ad-slit 2 for 48 h group with blank control group respectively （ P 〈 0.05 ）.Conclusion RPE cells with ad-slit2 transfection promote the migration and tube formation of RF/6A in vitro Slit2 gene in the RPE cells may take part in the formation process of choroidal neovascularization.