【作者】 杨珊珊；

【导师】 王传富；

【作者基本信息】 青岛大学 ， 眼科学， 2012， 博士

【摘要】 目的：通过腺相关病毒.(AAV)载体介导人β-神经生长因子(β-NGF)基因和人血小板源性生长因子基因B(PDGF-B)联合转染体外培养的猫角膜内皮细胞,探讨其对该细胞生物学效应的影响。方法：体外培养的猫角膜内皮细胞,通过细胞形态学观察、神经特异烯醇化酶(NSE)单克隆抗体免疫荧光染色鉴定细胞的种类及纯度。构建AAV载体,利用AAV载体介导绿色荧光蛋白(GFP)基因转染猫角膜内皮细胞,根据其阳性表达率测定转染效率。AAV-β-NGF和AAV-PDGF-B分别和联合转染猫角膜内皮细胞后,Real-Time PCR及Western blot分别在mRNA和蛋白水平检测转染24h、48h、72h后β-NGF和PDGF-B的表达变化。AAV-β-NGF和AAV-PDGF-B分别和联合转染猫角膜内皮细胞72小时后,通过MTT检测细胞增殖能力的改变,流式细胞仪测定处于G1期的细胞比例,检测外源基因对细胞周期的影响。划痕实验观察AAV-β-NGF和AAV-PDGF-B对细胞迁移能力的影响。结果：猫角膜内皮细胞体外培养可形成完整的细胞单层,经形态学观察、NSE免疫荧光染色证明为纯净的猫角膜内皮细胞。AAV介导的绿色荧光蛋白基因转染猫角膜内皮细胞72h后,荧光显微镜下观察可见细胞内清晰的绿色荧光,测得转染效率可达67.8%。AAV-β-NGF和AAV-PDGF-B分别和联合转染猫内皮细胞后,Real-Time PCR和Western blot结果显示,转染后各时间点猫角膜内皮细胞中β-NGF和PDGF-B的mRNA和蛋白表达随时间延长不断增高,与对照组相比差异有统计学意义(P<0.05)。联合转染组中β-NGF和PDGF-B在不同时间点的表达与单独转染组差别无统计学意义(P>0.05)。MTT结果显示,AAV-β-NGF和AAV-PDGF-B分别和联合转染猫角膜内皮细胞后增殖能力较对照组增强,联合转染组更为明显,差别均有统计学意义(P<0.05)。流式细胞仪检测AAV-β-NGF和AAV-PDGF-B转染组细胞周期中G1期细胞比例增加,联合转染组增加更为明显。划痕实验表明,AAV-β-NGF和AAV-PDGF-B转染后可以增强细胞迁徙能力,联合转染组效果更为明显,差异具有统计学意义(P<0.05)结论：AAV可有效介导人β-NGF基因和PDGF-B基因转染体外培养的猫角膜内皮细胞,并在猫角膜内皮细胞中持续稳定表达。AAV-β-NGF和AAV-PDGF-B分别和联合转染猫角膜内皮细胞后可以增强细胞的增殖和迁移能力,联合转染的效果更加明显。更多还原

【Abstract】 PURPOSE:To research the biological effect of co-transfection of adeno-associated virus (AAV) mediated human nerve growth fector-B (β-NGF) and human platelet derived growth factor B (PDGF-B) in cat corneal endothelial cells in vitro.METHODS:Cat corneal endothelial cells were cultured in vitro. The type and purity of the endothelial cells were further confirmed by morphological analysis and NSE immunofluorescence study. After forming the adeno-associated virus vector, cat corneal endothelial cells were transfected by AAV mediated green fluorescent protein (GFP) gene. Then the efficiency of transfection was evaluated according to positive expression of GFP. Humanβ-NGF gene and human PDGF-B gene were transfected into cat corneal endothelial cells in vitro by AAV vector. Real-Time PCR and Western blot were used to check the expression status ofβ-NGF and PDGF-B on mRNA and protein level in cat corneal endothelial cells at 24 hours,48 hours and 72 hours after respective transfection and co-transfection. MTT was used to detect the proliferation ability of cells, and flow cytometry was used to detect cell number at different stages of cell cycles at 72 hours after respective transfection and co-transfection of AAV-β-NGF and AAV-PDGF-B. By cell scratch test, the effect of cell spreading ability in cat corneal endothelial cells after transfection was detected.RESULTS:Cat corneal endothelial cells could be rapidly cultivated to form pure single layer. The type and purity of cells were further confirmed by morphological analysis and NSE immunofluorescence study. Cat corneal endothelial cells displayed green fluorescence clearly under fluorescence microscope in 72 hours after transfection of AAV mediated GFP gene. The efficiency of transfection reached 67.8%. The results of Real-Time PCR and Western blot showed that the expression ofβ-NGF and PDGF-B increased with time extended. Compared with the control groups, the difference had statistical significance (P< 0.05). There were no significant difference (P>0.05) between the expression ofβ-NGF and PDGF-B in co-transfection groups and respective transfection groups at 24 hours,48 hours,72 hours. The results of MTT showed that the proliferation ability of cells increased after the transfection of AAV-β-NGF and AAV-PDGF-B, moreover co-transfection groups increased obviously. There were statistical difference(P<0.05)between the each transfection groups and the control groups. The results of flow cytometry showed that the cell number at Gl stage increased after the transfection of AAV-B-NGF and AAV-PDGF-B, and the co-transfection groups increased more obviously than others. The results of cell scratch test showed the effect of cell spreading ability in cat comeal endothelial cells was increased after the transfection of AAV-β-NGF and AAV-PDGF-B, especially co-transfection groups. The cell spreading ability of each groups had statistical difference (P<0.05) between the transfection groups and the control groups.CONCLUSIONS:Humanβ-NGF gene and human PDGF-B gene could be effectively transfected into cat corneal endothelial cells in vitro by AAV vector, and expressed stably. Co-transfection of AAV-B-NGF and AAV-PDGF-B could increase the ability of proliferation and spreading in cat corneal endothelial cells.更多还原