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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。
Objective: To construct rAAV2-PEDF adeno-associated virus and study the effect of high expression of pigment epithelium-derived factor (PEDF) gene on human retinal microvascular endothelial cells. Methods: The PEDF gene was cloned and recombined into the adeno-associated virus vector pSNAV. The obtained pSNAV-PEDF was transfected into BHK cells and screened with medium containing G418 to obtain the stable cell line transfected with pSNAV-PEDF. Recombinant 1 Type herpes simplex virus HSV1-rc / △ UL2 infected cells were packaged and purified to give high titer rAAV2-PEDF virus particles. According to the dose of 105v.g./cell, rAAV2-PEDF virus was transfected. Blank and negative controls were set respectively. GFP positive cells were observed under laser scanning confocal microscope. The expression of PEDF protein was detected by Western blotting. Boyden chamber method to observe the migration of cells, flow cytometry detection of apoptosis. Results: The rAAV2-PEDF was successfully constructed by PCR, restriction enzyme digestion and gene sequencing. 48 h after transfection, GFP positive cells were observed under confocal laser scanning microscope. The expression of PEDF in experimental group was significantly stronger than that in other groups. In rAAV2-PEDF group, the percentage of apoptotic cells was 2.10% ± 0.53% in rAAV2-GFP group, 3.40% ± 0.62% in rAAV2-GFP group and 1.60% ± 0.47% in rAAV2-PEDF group, No significant difference between the groups (P> 0.05). rAAV2-PEDF interfered with retinal microvascular endothelial cells under hypoxic conditions, the percentage of apoptotic cells in CoCl2 + rAAV2-PEDF group was 4.00% ± 0.55%, CoCl2 + rAAV2-GFP group was 6.10% ± 0.71%, CoCl2 + rAAV2-PEDF group was 40.00% ± 2.10%. There was significant difference between rAAV2-PEDF group and other two groups (P <0.05). Cell migration counts showed that 33.0 ± 2.7 in the normal control group, 35.0 ± 3.6 in the rAAV2-GFP group and 12.0 ± 2.1 in the rAAV2-PEDF group were significantly different from the other two groups (P <0.05) . Conclusion: rAAV2-PEDF was successfully constructed and PEDF was stably expressed after transfection into human retinal vascular endothelial cells. High expression of PEDF can significantly inhibit human retinal microvascular endothelial cell migration and induce apoptosis under hypoxic conditions.