摘要
目的 观察碱性成纤维细胞生长因子 (basicfibroblastgrowthfactor,bFGF)双顺反子真核表达载体在豚鼠耳蜗中的表达 ,及噪声损伤后对毛细胞的保护作用。方法 利用脑炎心肌炎病毒的内部核糖体进入位点 (internalribosomeentrysite,IRES) ,构建人bFGF与增强型绿色荧光蛋白 (enhancegreenfluorescenceprotein ,EGFP)基因真核表达载体pIRES bFGF EGFP。采用硬脂胺 (stearylamine ,SA)脂质体介导bFGF基因转染豚鼠内耳 ,噪声暴露即刻通过圆窗向豚鼠耳蜗注入含有bFGF基因作为治疗基因的真核表达载体 ;或在噪声暴露前 7d ,作为保护因子转导转染豚鼠耳蜗。结果 构建的pIRES bFGF EGFP在转染 2 4h后开始表达 ,48h达到最高 ,表达时间可持续 1个月。治疗组在噪声后听性脑干反应平均阈值均低于噪声组 ,在保护组 :pIRES bFGF EGFP对耳蜗毛细胞有明显的保护作用。结论本研究成功地构建了pIRES bFGF EGFP真核共表达载体 ,具有bFGF、EGFP的双重活性 。
Objective To construct basic fibroblast growth factor(bFGR) and enhance green fluorescence protein(EGFP) fusion gene eukaryotic expression vector internal ribosome entry site (pIRES) bFGF GFP and to evaluate the effect of transduction bFGF gene on noise induced hearing loss in inner ear hair cells of guinea pigs Methods Human bFGF cDNA was inserted into mammalian expressed plasmid pIRES EGFP The recombinant expression plasmid pIRES bFGF EGFP was transfected into inner ear of guinea pigs, using lipofectin method The transduced bFGF gene was mediated by SA lipidsome IRES bFGF GFP was administered into the round window as rescue agent at the same time of noise exposure or as a protective agent 7 days before Results SA liposome mediated bFGF expressed at a high level in the cochlea of guinea pigs, and in the rescue group, a significant lower hearing thresholds was displayed pIRES bFGF EGFP could protect hair cells It demonstrated that pIRES bFGF GFP could protect the inner ear both structurally and functionally bFGF/EGFP gene could be transcripted and translated into inner ear hair cells of guinea pig bFGF/EGFP gene could express a specific protein The recombinant bFGF/EGFP had significant protective effect as well as manifestation of autonomous fluorescence Conclusion bFGF/EGFP fusion protein not only expressed in hair cells of guinea pig but also showed significant bFGF activity and autonomous fluorescence IRES induced exogenous gene could enter the hair cells
出处
《中华耳鼻咽喉科杂志》
CAS
CSCD
北大核心
2003年第1期21-23,I002,共4页
Chinese Journal of Otorhinolaryngology
基金
国家自然科学基金 (30 1710 0 5 )
北京自然科学基金(70 2 2 0 36)
