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目的:包装携载神经营养素(NT4)与活性依赖性神经营养因子-9(ADNF-9)融合基因的重组腺相关病毒rAAV-NT4-ADNF-9,并观察该重组病毒对体外培养的耳蜗组织的转染。方法:使用pSSHG-CMV-NT4-ADNF-9、pFG140和pAAV/Ad三种质粒共转染293包装细胞,制备ADNF-9重组腺相关病毒(AAV),应用斑点杂交实验检测重组病毒滴度;体外分离培养新生SD大鼠的耳蜗毛细胞;将重组病毒感染体外培养的新生SD大鼠耳蜗毛细胞,于24h后提取组织行RT-PCR以检测rAAV-NT4-ADNF-9对耳蜗的转染。结果:应用斑点杂交实验检测重组病毒滴度为2×1016cfu/L,表明成功构建了重组AAV载体。成功分离培养新生SD大鼠的耳蜗毛细胞后,经RT-PCR反应,琼脂糖凝胶电泳检测结果显示NT4-ADNF-9在耳蜗组织中得到表达。结论:构建的重组病毒rAAV-NT4-ADNF-9在体外可成功转染耳蜗组织,用于基因治疗的研究。
OBJECTIVE: To package recombinant adeno-associated virus rAAV-NT4-ADNF-9 carrying neurotrophic factor (NT4) and active-dependent neurotrophic factor-9 (ADNF-9) fusion gene and observe the effect of the recombinant virus on cultured cochlear tissue Transfection. METHODS: 293 cells were co-transfected with pSSHG-CMV-NT4-ADNF-9, pFG140 and pAAV / Ad plasmids to prepare ADNF-9 recombinant adeno-associated virus (AAV), and the titer of recombinant adenovirus was detected by dot blot hybridization. The cochlear hair cells of neonatal SD rats were isolated and cultured in vitro. The recombinant virus was used to infect the cochlear hair cells of neonatal SD rats. RT-PCR was used to detect the transfection of rAAV-NT4-ADNF-9 . Results: The titer of recombinant virus was 2 × 10 16 cfu / L by dot blot hybridization assay, indicating that recombinant AAV vector was successfully constructed. After successfully isolated and cultured cochlear hair cells of neonatal SD rats, the expression of NT4-ADNF-9 was detected in the cochlea by RT-PCR and agarose gel electrophoresis. Conclusion: The recombinant virus rAAV-NT4-ADNF-9 can be successfully transfected into cochlear tissue in vitro for the study of gene therapy.