【作者】 杨阳；

【导师】 孔维佳；

【作者基本信息】 华中科技大学 ， 耳鼻咽喉科学， 2008， 博士

【摘要】 第一部分病毒与非病毒载体转导大鼠耳蜗组织细胞的研究目的通过病毒与非病毒载体对胎鼠耳边缘细胞和耳蜗组织转染情况的研究,选择出合适的内耳基因治疗载体。方法分别用质粒(pEGFP-N1)、腺病毒(rAd5-EGFP)以及腺相关病毒(rAAV2- EGFP)载体转导原代培养的新生Wistar大鼠(≤72小时)耳边缘细胞或经圆窗膜将病毒液注入鼓阶外淋巴液,通过激光共聚焦显微镜、流式细胞仪、CCK-8、免疫组化(SP法)、免疫组化(ApopTag过氧化物酶凋亡检测)、Westernblot及ABR检测方法对三种载体加以比较。结果三种载体对原代培养的大鼠耳边缘细胞的转染研究发现,腺病毒的转染效率最高,而腺相关病毒转染对细胞活性影响最小。rAAV2和rAd5对大鼠耳蜗组织的感染实验发现,腺病毒和腺相关病毒携带的增强型绿色荧光蛋白可在多种耳蜗组织中及转染对侧耳蜗组织表达,且并不引起明显的耳蜗组织细胞凋亡。rAAV2携带的EGFP基因在第90天时仍可检测到大量表达。rAd5携带的EGFP基因在第30天时表达明显减弱。结论病毒载体相对于非病毒载体以其高的转导效率、低细胞毒性在对原代培养的胎鼠耳边缘细胞的转导中表现出明显优势。在病毒载体对大鼠耳蜗组织感染研究方面,腺病毒载体的优势在于其携带的基因表达迅速而高效,而腺相关病毒载体则以其长表达时程,低组织毒性见长。第二部分重组病毒rAAV2-IRES-EGFP/MnSOD的构建及其表达目的构建含有大鼠源性锰超氧化物歧化酶(Manganese superoxide dismutase, MnSOD)基因的重组病毒rAAV2-IRES-EGFP/MnSOD,并检测其在大鼠耳血管纹边缘细胞和耳蜗组织内的表达。方法将大鼠心肌组织来源的SOD基因克隆入融合表达载体pEGFP-N1中,再以限制性酶切方法克隆入真核表达载体pSNAV2.0-IRES-EGFP中,最后将其包装为rAAV2- IRES-EGFP/MnSOD病毒颗粒,用激光共聚焦显微镜、流式细胞仪、PCR及Westernblot检测MnSOD基因的表达。结果真核表达载体pEGFP-N1/MnSOD和pSNAV2.0-IRES-EGFP/MnSOD经酶切、PCR和测序鉴定均检测到MnSOD基因的完整序列,被rAAV2-IRES-EGFP/MnSOD感染的耳边缘细胞和内耳组织中MnSOD蛋白的表达均高于空白对照耳。结论成功构建了大鼠源性MnSOD基因重组病毒rAAV2-IRES-EGFP/MnSOD,并使其在大鼠耳边缘细胞和大鼠耳蜗组织中成功表达,为抗氧化基因治疗内耳疾病的研究奠定了基础。第三部分MnSOD基因转染对拟老化大鼠内耳的干预性保护目的探讨锰超氧化物歧化酶(manganese superoxide dismutase, MnSOD)基因内耳注射对氨基糖甙类抗生素造成的拟老化大鼠内耳损伤的干预性保护作用。方法在半乳糖注射的第6周(注射氨基糖甙类抗生素之前2周)将rAAV2-IRES- EGFP/MnSOD病毒液经圆窗膜注入拟老化大鼠的耳蜗外淋巴,通过免疫组化(ApopTag过氧化物酶凋亡检测)、Westernblot及MnSOD活性检测的方法观察MnSOD基因在对抗内耳氧化应激损伤中的干预性保护作用。结果MnSOD基因的干预性注射,感染有效地减少了氧化应激引起的拟老化大鼠内耳细胞凋亡,有效地提升了内耳中MnSOD的活性,并在一定程度上缓解了氨基糖甙类抗生素造成的拟老化大鼠的听力损失。结论外源性MnSOD基因在内耳过表达能部分对抗氨基糖甙类抗生素对拟老化大鼠内耳的损伤。更多还原

【Abstract】 PartⅠStudy of gene transfer into rat cochlea using different vectors in vivo and in vitroObjective Plasmids,recombinant adenoviruses (rAds), and recombinant adeno- associated viruses (rAAVs) are frequently used vectors,each with distinctive characteristics that may be advantageous in cochlear gene therapy. Although the general advantages and disadvantages of these vectors have been evaluated, the existing information is incomplete and no data are available on the delivery of transgenes to primary cultured marginal cells from the stria vascularis using nonviral and viral vectors. Methods In this study,a plasmid vector,rAd5,and rAAV2 were used to transfect rat cochlear marginal cells of stria vascularis by injection into the perilymph through the round window membrane,and the transfection efficiency, target tissue accessibility, cell/ tissue toxicity, time course of expression, and effect on hearing were evaluated in vivo and in vitro. Results We found that rAd5 had high transfection efficiency and low cytotoxicity in vitro, and rAAV2 produced the long-time stable expression and had the lowest vector-related ototoxicity in vivo. Conclusions rAd5 was more suitable for transgenic research of stria vascularis cells in vitro,while rAAV2 was a better vector for cochlear tissue transfection in vivo. PartⅡConstruction and expression of rAAV2-IRES-EGFP/ MnSOD in vivo and in vitroObjective Construct recombinant adeno-associated viral vector 2-IRES-EGFP/ MnSOD (rAAV2-IRES-EGFP/MnSOD) and make it express in primary cultured marginal cells of the stria vascularis and cochlea tissue of the rats. Methods The aimed segments were obtained from rat myocardial tissue,which were inserted into a eukaryotic expression plasmid pEGFP-N1/MnSOD or pSNAV2.0-IRES-EGFP/MnSOD, then the production of recombinant adeno-associated viral vector 2-IRES-EGFP/MnSOD (rAAV2-IRES-EGFP/ MnSOD) particles were made.The expression of interested gene was detected by co-Focus Fluorescence microscopy, fluorescence activated call sorting (FACS),PCR and Westernblot. Results Rat MnSOD cDNA of full sequence was detected in eukaryotic expression plasmid pEGFP-N1/MnSOD and pSNAV2.0-IRES-EGFP/MnSOD. MnSOD protein detected in the marginal cells and cochlea tissue after transfection was higher than the control. Conclusion Recombinant virus, rAAV2-IRES-EGFP/MnSOD have been successfully constructed and expressed in marginal cells and cochlea tissue of the rat.This research paved the way for antioxidant gene therapy of inner ear dieases. PartⅢProtection of cochlea from aminoglycoside ototoxicity by Manganese superoxide dismutase gene in aging ratsObjective To determine the feasibility of manganese superoxide dismutase (MnSOD) gene therapy for protecting the inner ear against aminoglycoside- induced oxidative stress in aging rats. Methods The viral particles of recombinant adeno-associated viral vector 2-IRES-EGFP/MnSOD(rAAV2-IRES-EGFP/MnSOD) were injected into the perilymph through the round window membrane on the 6th week.To observe the feasibility of MnSOD gene therapy for protecting the cochlear function with the methods of Westernblot, ApopTag peroxidase apoptosis detection, MnSOD activity detection and effect on hearing. Results The apoptosis of cochlear tissue was partly reduced, the MnSOD activity is increased, and the hearing threshold is decreased after rAAV2-IRES-EGFP/MnSOD inner injection,compared with saline injection group. Conclusion MnSOD could play a partly role to treat oxidative damage of inner ear in aging rats.With the heredity baxkground of mtDNA 4834 bp common deletion, aminoglycoside ototoxicity can not be resist compeletly by antioxidant gene therapy.更多还原