NGB基因转染拮抗庆大霉素耳毒性作用的实验研究

Experimental Study of NGB Gene Transfected into Guinea Pigs against Gentamicin Ototoxicity

任毅;周琦;唐安洲;谭颂华;方勤;谢利红;尹时华;

1:广西医科大学附属第一医院耳鼻咽喉头颈外科

摘要
目的验证阳离子脂质体介导脑红蛋白(neuroglobin,NGB)基因转染对庆大霉素致豚鼠耳毒性的保护作用。方法将ABR反应阈均不超过40dB SPL的120只健康花色豚鼠随机分为5组,每组24只:Ⅰ组:空白对照组;Ⅱ组:人工外淋巴液对照组(经左耳注入人工外淋巴液);Ⅲ组:人工外淋巴液实验组(经左耳注入人工外淋巴液后肌肉注射庆大霉素);Ⅳ组:空质粒转染组(经左耳注入空质粒pEGFP-C1后肌肉注射庆大霉素);Ⅴ组:NGB基因转染组(经左耳注入pEGFP-NGB后肌肉注射庆大霉素),庆大霉素均经后腿肌肉注射120mg.kg-1.d-1,共给药14天。停止给药后喂养14天,各组均行ABR检测,耳蜗基底膜铺片、免疫组化观察各组豚鼠耳蜗基底膜毛细胞形态学及NGB蛋白表达的变化。结果给药后Ⅰ组ABR反应阈平均为37.22dB SPL(左耳)和36.94dB SPL(右耳),Ⅱ组阈值平均为37.22dB SPL(左耳)和37.50dB SPL(右耳),Ⅲ组阈值平均为119.44dB SPL(左耳)和122.22dB SPL(右耳);Ⅳ组阈值平均为119.72dB SPL(左耳)和120.83dB SPL(右耳);Ⅴ组阈值平均为83.89dB SPL(左耳)和100.56dB SPL(右耳)。Ⅴ组ABR反应阈较Ⅰ组和Ⅱ组显著升高(P<0.05),较Ⅲ组和Ⅳ组显著降低(P<0.05)。Ⅴ组中手术耳ABR反应阈较非手术耳降低(P<0.05)。耳蜗基底膜铺片示Ⅰ组和Ⅱ组内外毛细胞排列整齐,无缺失,Ⅲ组和Ⅳ组内外毛细胞极少量残存,其中ABR阈值大于135dB SPL的豚鼠耳蜗毛细胞几乎消失殆尽,Ⅴ组毛细胞部分缺失,且主要是外毛细胞;免疫组织化学染色示Ⅴ组耳蜗毛细胞NGB蛋白表达量较其余各组均显著增高(P<0.05),其余各组几乎均未见明显阳性表达。结论本研究成功验证了阳离子脂质体介导NGB基因转染对庆大霉素致豚鼠耳毒性具有有效的保护作用。
关键词
脑红蛋白;基因转染;阳离子脂质体;耳蜗;庆大霉素
基金项目(Foundation):
国家自然科学基金(30860312);; 广西自然科学基金(桂科自0728134)
作者
任毅;周琦;唐安洲;谭颂华;方勤;谢利红;尹时华;
参考文献

1 Burmester T,Weich B,Reinhardt S,et al.A vertebrate glo-bin expressed in the brain[J].Nature,2000,407:520.

2 Garry DJ,Mammen PP.Neuroprotection and the role of neu-roglobin[J].Lancet,2003,362:342.

3 Wystub S,Laufs T,Schmidt M,et al.Localization of neuro-globin protein in the mouse brain[J].Neurosci Lett,2003,346:114.

4 Pesce A,Bolognesi M,Bocedi A,et al.Neuroglobin and cyto-globin.Fresh blood for the vertebrate globin family[J].EM-BO Rep,2002,3:1 146.

5尹时华,龚树生,鄢开胜,等.重组小鼠质粒pEGFP-NGB的构建和在培养神经胶质细胞中表达的实验研究[J].中国病理生理杂志,2005,21:1 388.

6 Izumikawa M,Minoda R,Kawamoto K,et al.Auditory hair cell replacement and hearing improvement by Atoh1gene ther-apy in deaf mammals[J].Nat Med,2005,11:271.

7 Staecker H,Praetorius M,Baker K,et al.Vestibular hair cell regeneration and restoration of balance function induced by math1gene transfer[J].Otol Neurotol,2007,28:223.

8 Ladrech S,Wang J,Simonneau L,et al.Macrophage contri-bution to the response of the rat organ of Corti to amikacin[J].Neurosci Res,2007,85:1 970.

9 Wareing M,Mhatre AN,Pettis R,et al.Cationic liposome mediated transgene expression in the guinea pig cochlea[J].Hear Res,1999,128:61.

10 Zhu N,Liggitt D,Liu Y,et al.Systemic gene expression af-ter intravenous DNA delivery into adult mice[J].Science,1993,261:209.

11 Hsiao M,Tse V,Carmel J,et al.Stabilization of cationic li-posome plasmid DNA complexes by polyamines and poly(ethylene glycol)phospholipid conjugates for efficient in vivo gene delivery[J].FEBS Lett,1997,400:233.

12李昱,王爱梅.氨基糖甙类抗生素耳毒性的研究进展[J].医学综述,2008,14:459.

13 Vallone B,Nienhaus K,Matthes A,et al.The structure of carbonmonoxy neuroglobin reveals a heme-sliding mecha-nism for control of ligand affinity[J].Proc Natl Acad Sci USA,2004,101:17 351.