人β神经生长因子基因转染体外培养猫角膜内皮细胞促进分裂再生的研究  

Proliferation and regeneration effects of constructed human β-nerve growth factor gene on transfected cat corneal endothelial cells in vitro

作　　者：李东侃[1] 王传富[1] 宋跃[3] 吕振华 

机构地区：[1]青岛大学医学院附属医院眼科,266003 [2]中心实验室 [3]汕头大学一香港中文大学联合汕头国际眼科中心

出　　处：《中华眼科杂志》2008年第4期337-342,共6页Chinese Journal of Ophthalmology

基　　金：国家自然科学基金资助项目（30572011）

摘　　要：目的探讨人β神经生长因子真核表达载体（pcDNA4-β-NGF）转染体外培养猫角膜内皮细胞并促进细胞分裂再生的机制，为将该基因应用于促进人角膜内皮细胞再生的研究打下基础。方法为实验研究。通过Effectene^TM脂质体介导将自行构建并经测序证实的人pcDNA4-β-NGF转染到体外培养的猫角膜内皮细胞中。采用分组对照的方法研究转染前后猫角膜内皮细胞分裂再生能力。转基因后48h通过逆转录聚合酶链反应（RT-PCR）和免疫组织化学染色方法分别在mRNA和蛋白水平检测人β神经生长因子（p-NGF）的表达。转基因后96h采用细胞四甲基偶氮唑盐染色（MTT）测量吸光度值、细胞有丝分裂指数、流式细胞仪检测G1期细胞比例及细胞损伤后愈合面积测量等方法检测目的基因对猫角膜内皮细胞增殖活性的作用。结果Effectene^TM脂质体可有效介导重组真核表达载体pcDNA4-β-NGF转染到经改良方法体外培养的猫角膜内皮细胞中，转染效率为11．3％，并促进该细胞表达β-NGF。正常对照组β-NGF／β-actin比值结果为3．14，加脂质体组为3．23，pcDNA4质粒转染组为3．21，pcDNA4-β-NGF重组质粒转染组为4．53。培养液、正常对照组、加脂质体组、pcDNA4质粒转染组及pcDNA4-β-NGF重组质粒转染组平均A值分别为0．178±0．007、0．482±0．033、0．488±0．017、0．520±0．021及0．623±0．041。正常对照组、加脂质体组、pcDNA4质粒转染组及pcDNA4．B．NGF重组质粒转染组细胞有丝分裂指数分别为3．3％、3．0％、3．1％及7．7％。正常对照组、加脂质体组、pcDNA4质粒转染组及pcDNA4-β-NGF重组质粒转染组G1期细胞比例分别为68．1％、51．6％、60．4％及87．9％。结论人B-NGF基因可通过Effectene^TM脂质体介导有效转染到体外培养的猫角膜内皮细胞中，并促进细胞分裂再生，为进一步将此基因转入人角膜内皮细胞的研究提�Objective To explore the mechanisms of proliferation and regeneration effects of a human nerve growth factor （β-NGF） expression vector （pcDNA4-β-NGF）on the transfected cat corneal endothelial cells in vitro. To provide a new method for long term cultivation of human corneal endothelial cells in vitro and to establish theoretical basis of gene therapy for corneal endothelial defects. Methods It was a experimental study. The human pcDNA4-β-NGF expression vector was constructed and transfected into cultured cat corneal endothelial cells by Effectene^TM lipofectine transfection technique. The expression of the reporter gene pcDNA4-β-LacZ expression was used to determine the transfection efficiency 48 hours after the transfection. RT-PCR and immunohistochemistry techniques were used to check the transient expression status at mRNA and protein levels in cat corneal endothelial cells. Mitotic index and methyl thiazolyl tetrazolium （MTT） value were measured and cell numbers at different stages of cell cycles were determined by flow cytometer 96 hours after transfection. An in vitro quantitative cat corneal endothelial cell traumatic model was established which was used for observing the effect of human β-NGF expression product on the DNA synthesis of cat endothelial cells and healing process of traumatized endothelial cells. Results A human nerve growth factor （β-NGF） expression vector （PeDNA4-β-NGF）was successfully constructed and confirmed by sequence analysis. Single layered pure cat corneal endothelial cells were obtained by a modified sliced tissue culture technique and confirmed by morphological analysis, neurone specific enolase immunohistochemistry study and transmission electronic microscope. EffecteneTM lipofectine mediated transfection efficiency of PeDNA4-β-NGF into cat corneal endothelial cells in vitro was 11.3%. The human β-NGF could be highly expressed in the transfected corneal endothelial cells at mRNA and protein levels. Mitotic index, MTr value and GI stage cell numbers, a

关 键 词：神经生长因子 转染 内皮 角膜 内皮细胞 再生 细胞 培养的 

分 类 号：R686[医药卫生—骨科学]