腺相关病毒载体介导人β-NGF和PDGF-B基因联合转染猫角膜内皮细胞的实验研究
发布时间：2019-03-06 16:26
【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(P0.05)。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(P0.05)。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(P0.05)。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(P0.05) 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。
[Abstract]:Objective: to co-transfect human 尾-nerve growth factor (尾-NGF) gene and human platelet-derived growth factor B (PDGF-B) gene into cat corneal endothelial cells by adeno-associated virus. (AAV) vector. To investigate the biological effects of these cells. Methods: cat corneal endothelial cells were cultured in vitro. The types and purity of cultured cat corneal endothelial cells were identified by morphological observation and immunofluorescence staining with monoclonal antibody against neuro-specific enolase (NSE). AAV vector was constructed and transfected into cat corneal endothelial cells by AAV vector mediated green fluorescent protein (GFP) gene. The transfection efficiency was measured according to its positive expression rate. AAV-尾-NGF and AAV-PDGF-B were transfected into cat corneal endothelial cells respectively and co-transfected with AAV-尾-NGF and AAV-PDGF-B. The expression of 尾-NGF and PDGF-B were detected at mRNA and protein levels for 24 h, 48 h and 72 h after transfection of Real-Time PCR and Western blot, respectively. AAV-尾-NGF and AAV-PDGF-B were transfected into cat corneal endothelial cells for 72 hours, respectively. The cell proliferation ability was detected by MTT, the proportion of cells in G1 phase was measured by flow cytometry, and the effect of exogenous genes on cell cycle was detected. The effects of AAV- 尾-NGF and AAV-PDGF-B on cell migration were observed by scratch assay. Results: cat corneal endothelial cells were cultured in vitro to form intact monolayers. Morphological observation and NSE immunofluorescence staining showed that they were pure cat corneal endothelial cells. AAV-mediated green fluorescent protein gene was transfected into cat corneal endothelial cells for 72 hours. A clear green fluorescence was observed under fluorescence microscope, and the transfection efficiency was 67.8%. After AAV-尾-NGF and AAV-PDGF-B were co-transfected with cat endothelial cells, the results of Real-Time PCR and Western blot showed that the transfection efficiency was 67.8%. The expression of mRNA and protein of 尾-NGF and PDGF-B in cat corneal endothelial cells increased with time after transfection, and the difference was statistically significant compared with the control group (P0.05). There was no significant difference in the expression of 尾-NGF and PDGF-B between the combined transfection group and the single transfection group at different time points (P0.05). The proliferation of cat corneal endothelial cells transfected with AAV- 尾-NGF and AAV-PDGF-B was significantly higher than that of the control group, and the difference was statistically significant (P0.05). The proportion of G1 phase cells in cell cycle of AAV- 尾-NGF and AAV-PDGF-B transfected group was increased by flow cytometry, especially in combined transfection group. Scratch test showed that AAV- 尾-NGF and AAV-PDGF-B could enhance the ability of cell migration after transfection, and the effect of combined transfection group was more obvious. The difference was statistically significant (P0.05) conclusion: AAV can effectively mediate the transfection of human 尾-NGF gene and PDGF-B gene into cat corneal endothelial cells in vitro. AAV-尾-NGF and AAV-PDGF-B could enhance the proliferation and migration of cat corneal endothelial cells, and the co-transfection of AAV-尾-AAV and AAV-尾-AAV could enhance the proliferation and migration of cat corneal endothelial cells.
【学位授予单位】：青岛大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R77
本文编号：2435695
[Abstract]:Objective: to co-transfect human 尾-nerve growth factor (尾-NGF) gene and human platelet-derived growth factor B (PDGF-B) gene into cat corneal endothelial cells by adeno-associated virus. (AAV) vector. To investigate the biological effects of these cells. Methods: cat corneal endothelial cells were cultured in vitro. The types and purity of cultured cat corneal endothelial cells were identified by morphological observation and immunofluorescence staining with monoclonal antibody against neuro-specific enolase (NSE). AAV vector was constructed and transfected into cat corneal endothelial cells by AAV vector mediated green fluorescent protein (GFP) gene. The transfection efficiency was measured according to its positive expression rate. AAV-尾-NGF and AAV-PDGF-B were transfected into cat corneal endothelial cells respectively and co-transfected with AAV-尾-NGF and AAV-PDGF-B. The expression of 尾-NGF and PDGF-B were detected at mRNA and protein levels for 24 h, 48 h and 72 h after transfection of Real-Time PCR and Western blot, respectively. AAV-尾-NGF and AAV-PDGF-B were transfected into cat corneal endothelial cells for 72 hours, respectively. The cell proliferation ability was detected by MTT, the proportion of cells in G1 phase was measured by flow cytometry, and the effect of exogenous genes on cell cycle was detected. The effects of AAV- 尾-NGF and AAV-PDGF-B on cell migration were observed by scratch assay. Results: cat corneal endothelial cells were cultured in vitro to form intact monolayers. Morphological observation and NSE immunofluorescence staining showed that they were pure cat corneal endothelial cells. AAV-mediated green fluorescent protein gene was transfected into cat corneal endothelial cells for 72 hours. A clear green fluorescence was observed under fluorescence microscope, and the transfection efficiency was 67.8%. After AAV-尾-NGF and AAV-PDGF-B were co-transfected with cat endothelial cells, the results of Real-Time PCR and Western blot showed that the transfection efficiency was 67.8%. The expression of mRNA and protein of 尾-NGF and PDGF-B in cat corneal endothelial cells increased with time after transfection, and the difference was statistically significant compared with the control group (P0.05). There was no significant difference in the expression of 尾-NGF and PDGF-B between the combined transfection group and the single transfection group at different time points (P0.05). The proliferation of cat corneal endothelial cells transfected with AAV- 尾-NGF and AAV-PDGF-B was significantly higher than that of the control group, and the difference was statistically significant (P0.05). The proportion of G1 phase cells in cell cycle of AAV- 尾-NGF and AAV-PDGF-B transfected group was increased by flow cytometry, especially in combined transfection group. Scratch test showed that AAV- 尾-NGF and AAV-PDGF-B could enhance the ability of cell migration after transfection, and the effect of combined transfection group was more obvious. The difference was statistically significant (P0.05) conclusion: AAV can effectively mediate the transfection of human 尾-NGF gene and PDGF-B gene into cat corneal endothelial cells in vitro. AAV-尾-NGF and AAV-PDGF-B could enhance the proliferation and migration of cat corneal endothelial cells, and the co-transfection of AAV-尾-AAV and AAV-尾-AAV could enhance the proliferation and migration of cat corneal endothelial cells.
【学位授予单位】：青岛大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R77
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