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目的 构建真核表达载体pIRES2 EGFP NT3,并观察其在豚鼠耳蜗成纤维细胞中的表达及分布。方法 用RT PCR法 ,从人肝脏组织中克隆NT3 cDNA基因 ,构建真核表达载体 pIRES2 EGFP NT3,并经PCR及EcoRI/BamHI双酶切鉴定。用脂质体介导的方法 ,将用真核表达载体 pIRES2 EGFP NT3瞬时转染豚鼠原代耳蜗成纤维细胞。转染 4 8h后 ,分别在荧光显微镜下及用免疫组化法观察转染细胞中NT 3的表达。结果 人NT 3cDNA的PCR产物约为 74 4bp。序列测定结果用BLAST软件进行同源性比较 ,同源性为 10 0 %。经EcoRI/BamHI双酶切证实 ,NT 3cDNA已成功地克隆到真核表达载体pIRES2 EGFP中。用脂质体介导法 ,将 pIRES2 EGFP NT3瞬时转染豚鼠原代耳蜗成纤维细胞 ,在荧光显微镜下观察到呈绿色荧光的成纤维细胞。NT 3免疫组化染色结果显示 ,转染NT 3基因的成纤维细胞胞浆呈棕黄色着色 ;而转染空载体的成纤维细胞免疫组化染色呈阴性。结论 成功地构建真核表达载体pIRES2 EGFP NT3,并在豚鼠耳蜗原代成纤维细胞中表达 ,为NT 3基因转染治疗致聋豚鼠耳蜗动物试验奠定了基础
Objective To construct eukaryotic expression vector pIRES2 EGFP NT3 and observe its expression and distribution in cochlear fibroblasts of guinea pigs. Methods The cDNA of NT3 cDNA was cloned from human liver tissue by RT PCR. The eukaryotic expression vector pIRES2 EGFP NT3 was constructed and identified by PCR and EcoRI / BamHI digestion. In a liposome-mediated approach, guinea pig primary cochlear fibroblasts will be transiently transfected with the eukaryotic expression vector pIRES2 EGFP NT3. After 48 h of transfection, the expression of NT 3 in transfected cells was observed under fluorescence microscope and immunohistochemistry respectively. Results The PCR product of human NT3 cDNA was approximately 74 4bp. Sequence analysis results using BLAST software homology comparison, the homology was 10%. The result of double digestion with EcoRI / BamHI confirmed that NT 3 cDNA was successfully cloned into eukaryotic expression vector pIRES2 EGFP. The pIRES2 EGFP NT3 was transiently transfected into primary cochlear fibroblasts using liposome-mediated method, and the green fluorescent fibroblasts were observed under a fluorescence microscope. The results of NT 3 immunohistochemical staining showed that the cytoplasm of NT 3 gene transfected fibroblasts showed brownish yellow staining while the empty vector transfected fibroblasts were negative for immunohistochemistry staining. Conclusion The eukaryotic expression vector pIRES2 EGFP NT3 was successfully constructed and expressed in the cochlear primary fibroblasts of guinea pigs, which laid the foundation for the experimental study on deafness-induced cochlear animals in NT 3 gene transfection