重组色素上皮衍生因子对人视网膜微血管内皮细胞迁移及凋亡的影响

Effect of rAAV2-PEDF on migration and apoptosis of human retinal capillary endothelial cells

目的:构建rAAV2-PEDF腺相关病毒,研究色素上皮衍生因子(PEDF)基因高表达对人视网膜微血管内皮细胞的作用。方法:将PEDF基因克隆、重组到腺相关病毒载体pSNAV,得到的pSNAV-PEDF后转染BHK细胞,用含G418的培养基进行筛选,得到稳定转染pSNAV-PEDF的生产细胞系,用重组1型单纯疱疹病毒HSV1-rc/△ UL2感染细胞进行病毒包装,纯化后得到高滴度rAAV2-PEDF病毒颗粒。按照105v.g./cell的剂量进行rAAV2-PEDF病毒转染,分别设空白和阴性对照,激光共聚焦显微镜下观察GFP阳性细胞,Western blotting检测PEDF蛋白表达。Boyden小室法观察细胞迁移情况,流式细胞术检测细胞凋亡情况。结果:通过PCR、酶切及基因测序的方法,证实rAAV2-PEDF构建成功。转染病毒48h后,激光共聚焦显微镜下观察可见GFP阳性细胞,Western blotting检测,实验组PEDF表达明显强于其它组。rAAV2-PEDF干预正常氧条件下人视网膜微血管内皮细胞,正常对照组凋亡细胞比例为2.10%±0.53%,rAAV2-GFP组为3.40%±0.62%,rAAV2-PEDF组为1.60%±0.47%,各组间无显著差异(P>0.05)。rAAV2-PEDF干预低氧条件下人视网膜微血管内皮细胞,单纯CoCl2组凋亡细胞比例为4.00%±0.55%,CoCl2+rAAV2-GFP组为6.10%±0.71%,CoCl2+rAAV2-PEDF组为40.00%±2.10%。rAAV2-PEDF组与其它2组相比,有显著差异(P<0.05)。细胞迁移计数显示,正常对照组为33.0±2.7,rAAV2-GFP组为35.0±3.6,rAAV2-PEDF组为12.0±2.1,rAAV2-PEDF组与其它2组相比,有显著差异(P<0.05)。结论:成功构建了rAAV2-PEDF,转染人视网膜血管内皮细胞后PEDF可稳定表达。PEDF高表达可显著抑制人视网膜微血管内皮细胞迁移并可在低氧条件下诱导其凋亡。

AIM： To construct a rAAV2-pigment epithelium-derived factor （PEDF） vector and to explore the effect of rAAV2-PEDF on the migration and apoptosis of human retinal capillary endothelial cells （HRCECs）. METHODS： PEDF gene was cloned into an adeno-associated virus vector pSNAV. The recombinant pSNAV-PEDF was transfected into BHK cells. The G418 resistant cells were selected and infected with recombinant HSV which packaged the recombinant pSNAV-PEDF. The high-titer rAAV2-PEDF was obtained by purification. After transfected the rAAV2-PEDF into HRCECs, the protein expression of PEDF were detected by Western blotting, the migration of HRCECs was assessed by a boyden chamber, and the apoptosis of HRCECs was analyzed by flow cytometry. RESULTS： The evaluation results of PCR, enzyme digestion, and DNA sequencing showed that the rAAV2-PEDF was constructed correctly. The GFP positive cells were observed under laser scanning confocal microscope 48 h after rAAV2-GFP transfection. The expression level of PEDF protein was higher in experimental group than that in control group. The number of migrated cells was 33.0±2.7 in normal control group, 35.0±3.6 in rAAV2-GFP treatment group, and 12.0±2.1 in rAAV2-PEDF treatment group （P〈0.05）. In normoxic condition, the apoptotic cells were 2.10%±0.53% in control group, 3.40%±0.62% in rAAV2-GFP group and 1.60%±0.47% in rAAV2-PEDF group （P〉0.05）. In hypoxic condition, the apoptotic cells were 4.00%±0.55% in CoCl2 treatment group, 6.10%±0.71% in CoCl2＋rAAV2-GFP treated group and 40.00%±2.10% in CoCl2＋rAAV2-PEDF treatment group （P〈0.05）. CONCLUSION： rAAV2-PEDF is successfully constructed and PEDF gene is stably expressed in HRCECs after rAAV2-PEDF transfection. Over-expression of PEDF inhibits the migration of HRCECs and induces the cell apoptosis obviously in hypoxia.