人血小板源性生长因子B基因转染对猫角膜内皮细胞增殖的影响

Effect of recombinant human platelet-derived growth factor B gene transfection on cat corneal endothelial cell proliferation

背景：许多研究显示外源性的人血小板源性生长B基因的转染可促进创伤的愈合。目的：用重组人血小板源性生长因子B基因真核表达载体及重组人血小板源性生长因子B基因腺相关病毒转染和感染猫角膜内皮细胞，观察其对猫角膜内皮细胞增殖的影响。设计、时间及地点：分组对照观察细胞基因工程研究，于2007—01／11在青岛大学医学院附属医院中心实验室完成。材料：健康足月幼猫，体质量250-300g，雌雄不限。重组人血小板源性生长因子B基因真核表达载体及重组人血小板源性生长因子B基因腺相关病毒由青岛大学医学院附属医院中心实验室自行构建。方法：常规显微镜下撕取内皮培养法，培养出原代猫角膜内皮细胞，传2代的细胞用于实验。脂质体法将重组人血小板源性生长因子B基因真核表达载体转染到猫角膜内皮细胞中，重组人血小板源性生长因子B基因腺相关病毒采用直接感染法。实验分正常对照组、质粒对照组、重组质粒组、腺相关病毒对照组和重组腺相关病毒组。转基因24h和48h后收集细胞，Trizol试剂提取其总RNA。主要观察指标：通过荧光定量聚合酶链反应检测目的基因在细胞中的表达。四甲基偶氮唑盐法检测转基因48h后细胞的增殖活性。结果：①撕取内皮法可方便获取到原代猫角膜内皮细胞。②自行设计的针对目的基因及内参基因的探针引物经扩增后电泳验证及序列测定证明特异性高。③转基因24h和48h后荧光定量聚合酶链反应检测人血小板源性生长因子B基因mRNA发现：正常对照组、真核表达质粒对照组和腺相关病毒对照组间差异无显著性意义（P〉0．05）；重组人血小板源性生长因子B基因真核表达质粒转染组和重组人血小板源性生长因子B基因腺相关病毒实验组与正常对照组相比差异显著（P〈0．05）。④四甲基偶氮唑盐法检测显示重组质粒转染和重组病毒感染均可促进细胞增殖，真核表达质粒对照组和腺相关病毒对照组与正常对照组相比差异无显著性意义（P〉0．05）。结论：重组人血小板源性生长因子B基因真核表达载体及重组人血小板源性生长因子B基因腺相关病毒可在角膜内皮细胞中高效表达并具有促进细胞增殖的活性。

BACKGROUND： Many studies show that ectogenic human platelet-derived growth factor B gene transfection can effectively promote the wound healing. OBJECTIVE： To transfer the cat corneal endothelial cells with the recombinant human platelet-derived growth factor B gene eukaryotic expression vector and recombinant human platelet-derived growth factor B gene adeno-associated virus and to observe the effect of the transfection on the proliferation activity of cat corneal endothelial cells. DESIGN, TIME AND SETTING： The controlled cell gene engineering study was done at the Central Laboratory of the Affiliated Hospital of Qingdao University Medical College between January 2007 and November 2007. MATERIALS： Healthy full-term young cats, weighing 250-300 g, without sex restriction. The recombinant human platelet-derived growth factor B gene eukaryotic expression vector and recombinant human platelet-derived growth factor B gene adeno-associated virus were constructed by the Central Laboratory of the Affiliated Hospital of Qingdao University Medical College. METHODS： The endothelium of cat cornea was torn under the microscope and rapidly cultivated in DMEM medium to form single layer corneal endothelial cells and the passage 2 endothelial cells were used in this experiment. The recombinant human platelet-derived growth factor B gene eukaryotic expression vector was transferred into cat corneal endothelial cells by EffecteneTM lipofectine, and recombinant human platelet-derived growth factor B gene adeno-associated virus was transferred into cat corneal endothelial cells directly. Five groups were as following： blank control group, eukaryotic expression vector control group, recombinant eukaryotic expression vector group, adeno-associated virus control group and recombinant adeno-associated virus group. At 24 hours and 48 hours after transfection, the total RNA was extracted from the corneal endothelial cells by Trizol. MAIN OUTCOME MEASURES： The expression of the target gene detected by fluorescence quantitative polymerase chain reaction; proliferation activity of the transfected corneal endothelial cells detected at 48 hours by MTr assay. RESULTS： The torn endothelium culture technique could rapidly cultivate single layer cat corneal endothelial cells. The self-designed primers for the target gene and reference gene were efficient and special confirmed through electrophoresis analysis and DNA sequencing. At 24 hours and 48 hours after transfection, the human platelet-derived growth factor B gene mRNA detected by fluorescence quantitative polymerase chain reaction showed： there were no significant differences between blank control group, eukaryotic expression vector control group, and adeno-associated virus control group （P 〉 0.05）; there were significant differences between blank control group and recombinant eukaryotic expression vector group, recombinant adeno-associated virus group （P 〈 0.05）. MTT assay showed both the recombinant eukaryotic expression vector and recombinant adeno-associated virus with human platelet-derived growth factor B gene could promote the proliferation activity of cat corneal endothelial cells. There was no significant difference between eukaryotic expression vector control group and adeno-associated virus control group （P 〉 0.05）. CONCLUSION： The recombinant eukaryotic expression vector and recombinant adeno-associated virus both with human platelet-derived growth factor B gene can express in cat corneal endothelial cells, which will benefit the proliferation activity of the cells.