重组腺相关病毒rAAVNT4ADNF9构建及其对体外培养大鼠耳.docVIP

重组腺相关病毒rAAVNT4ADNF9构建及其对体外培养大鼠耳 　 作者：祝康 郑国玺 韦俊荣 许珉 杨广笑 王全颖 【摘要】 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，pGF140和pAAV/Ad 3种质粒共转染293包装细胞，制备ADNF9重组腺相关病毒（recombinate adenoassociated virus, rAAV），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24 h后提取组织行反转录聚合酶链式反应（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2×1013 cfu/ml，表明成功地构建了重组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在耳蜗组织得到了表达。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。 【关键词】 重组腺相关病毒； 活性依赖性神经营养因子9； 耳蜗毛细胞； 反转录聚合酶链式反应 　　［Abstract］ Objective: To construct an universal recombinate adenoassociated virus(AAV), rAAVNT4ADNF9, and to detect the ability of transfection of the rAAV vector into cochlea in vitro.Methods： pSSVHGCMVADNF9 plasmid was introduced into 293 cell by method of Ca3(PO4)2 using three plasmids of pSSHGCMVNT4ADNF9,pGF140 and pAAV/Ad. The recombinate adenoassociated virus(rAAV) was harvested, and the titrations of the rAAV concentrated was detected by dotblot test. To isolate and culture the cochlear hair cell of SD rats newly born in vitro. The rAAVNT4ADNF9 was added to the medium while plating. Cochlears were collected 24 h after cultivation for RTPCR to detect the transfection of rAAVNT4ADNF9. Results： The titration of rAAV stock produced 2×1013 total particles/ml, which showed that rAAVNT4ADNF9 was constructed successfully. The cochlear hair cell of SD rats newly born was isolated and cultured in vitro successfully. It certified that rAAVNT4ADNF9 was able to transfect into cochlear and express secretory NT4ADNF9 peptide by RTPCR. Conclusion： The rAAV vector constructed in this paper, rAAVNT4ADNF9, can transfer into cochlear cultured in vitro, which layed a foundation of further research for gene therapy. 　　［Key words］ recombinate adeno-associated virus；ADNF9；cochlear hair cell；RTPCR 　　感音神经性耳聋是耳鼻喉科的常见病、多发病之一，严重影响人类的健康和生活质量［1］。