【摘要】 目的:构建pEGFP-HDGF真核表达质粒,转入胶质瘤U87细胞中,对其表达及生物学活性进行检测。方法:PCR扩增HDGF编码区序列,构建pEGFP-HDGF真核表达质粒,脂质体转染U87细胞,G418筛选稳定表达单克隆,荧光显微镜观察绿色荧光,RT-PCR及Westernblot检测融合蛋白在转染细胞中的整合及表达。通过U87细胞增殖实验对HDGF-GFP融合蛋白进行初步功能鉴定。结果:pEGFP-HDGF表达质粒转染U87细胞后,荧光显微镜下可见绿色荧光,RT-PCR、Westernblot结果证实在U87细胞中有HDGF-GFP融合蛋白的表达。细胞增殖实验的结果显示,稳定表达pEGFP-HDGF的U87细胞生长速率明显高于对照空载组细胞(P=0.006)。结论:pEGFP-HDGF真核表达质粒构建成功,且HDGF对于胶质瘤U87细胞株有着生长刺激作用,这将有助于胶质瘤发生发展的分子机制研究。更多还原

【Abstract】 Objective:To construct and identify pEGFP-HDGF over expression plasmid in U87 cells.Methods:HDGF fragment was amplified by RT-PCR method and then inserted into pEGFP-N1 vector.The level of HDGF was identified by RT-PCR and western blot after transfection of positive recombinant plasmid into glioma cell line U87.The biological activity of HDGF-GFP fusion protein was tested by the proliferation assay.Results:Fluorescence microscopy demonstrated that pEGFP-HDGF was successfully transfected into U87 cells.Over expression of HDGF was confirmed by RT-PCR and western blot.The proliferation rate of U87 cell transfected with pEGFP-HDGF plasmid was significantly higher than that of negative control.Conclusion:pEGFP-HDGF over expression plasmid with biological activity was successfully constructed and stablely transfected into U87 cells.更多还原