重组AAV2-NTF2对人视网膜微血管内皮细胞中血管内皮生长因子表达的影响

Effect of rAAV2-NTF2 on vascular endothelial growth factor expression in human retinal capillary endothelial cells

目的观察核运输因子2(NTF2)在体外培养的人视网膜微血管内皮细胞(RCECs)中的表达,并探讨其高表达对RCECs中血管内皮生长因子(VEGF)表达的影响。方法取中山眼科中心眼库的人眼球,分离视网膜组织用以培养人RCECs,并用Ⅷ因子抗体免疫组织化学法鉴定。免疫组织化学法观察细胞中NTF2的表达。通过重组腺相关病毒(rAAV2)将外源性NTF2导入培养的RCECs内,rAAV2-NTF2和rAAV2-EGFP转染RCECs,未转染组为对照组。激光共焦显微镜下观察增强型绿色荧光蛋白(EGFP)阳性细胞,逆转录聚合酶链反应(RT-PCR)法检测各组NTF2、VEGFmRNA的表达变化,并进行定量分析。结果培养的RCECs呈典型铺路石样排列,荧光显微镜下可见Ⅷ因子阳性表达为绿色荧光,95%以上的活体细胞可摄取DiI标记的Ac-LDL,并在荧光显微镜下呈现红色荧光。RCECs可表达NTF2,主要位于细胞质内,富集于核周。rAAV2-EGFP转染后,激光共焦显微镜观察可见大量EGFP阳性细胞,实验组NTF2在mRNA水平表达明显强于GFP组和对照组(t=15.06,t=7.02,P<0.01),VEGF表达明显下降(t=7.40,t=14.08,P<0.01)。结论 rAAV2-NTF2转染视网膜内皮细胞后,NTF2可稳定高表达,VEGF表达下降,NTF2可能通过调节VEGF的表达参与新生血管的发生。

Background It is well-known that vascular endothelial growth factor（VEGF） is essential stimulator for neovascularization.Our previous study showed that the overexpression of nuclear transport factor 2（NTF2） is negatively correlated with diabetic retinopathy（DR）,but its mechanism remains unclear.Objective This study aimed to investigate the distribution of NTF2 in human retinal capillary endothelial cells（RCECs） in vitro and explore the effects of NTF2 on VEGF expression in human RCECs.Methods Human RCECs were isolated from the retina of healthy donor in Eye Bank of Zhongshan Eye Center and identified as endothelial cells by factor Ⅷ antibody.Expression and distribution of NTF2 were detected by immunocytochemistry under the fluorescence microscope.RCECs were transfected with rAAV2-NTF2,rAAV2-EGFP at passage 3,and untransfected cells were used as the control.The expression of NTF2 mRNA and VEGF in the cultured cells were analyzed by Reverse Transcription-Polymerase Chain Reaction.The use of eye of donor followed the Statement of Zhongshan Eye Center Ethic Committee.Results The cultured cells showed the cobblestone-like arrangement and presented the green fluorescence for Ⅷ-factor related antibody.95% living cells exhibited the red fluorescence after co-cultured with DiI-Ac-LDL.NTF2 was expressed mainly in perinuclear cytoplasm and EGFP positive cells were seen under laser confocal scanning microscopy after 48 hours of rAAV2-EGFP transfection.The level of NTF2 mRNA（ANTF2/Aβ-actin） in cultured cells was significantly elevated（t=15.06,t=7.02,P0.01） and that of VEGF mRNA（AVEGF/Aβ-actin） in cultured cells was considerably declined（t=7.40,t=14.08,P0.01） in rAAV2-NTF2 transfected group compared with rAAV2-GFP transfected group and control group.Conclusion These results indicate that NTF-2 might be involved in the processes of neovascularization by down-regulating the expression of VEGF.