NGB基因转染拮抗庆大霉素耳毒性作用的实验研究

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。

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

Objective To study the protective effects of cationic liposome-mediated NGB gene transfected into deaf guinea pig cochlea induced by gentamicin. Methods 120 healthy guinea pigs(ABR threshod ≤40 dB SPL) were employed in this experiment and divided randomly into five groups(24 each group):groupⅠ: blank control group;groupⅡ: artificial perilymph control group(artificial perilymph was injected into left ear);group Ⅲ: artificial perilymph experimental group(artificial perilymph was injected into left ear and then gentamicin administration);group IV: empty plasmid transfection group(empty plasmid pEGFP-C1 was injected into left ear and then gentamicin administration);group V: NGB gene transfection group(pEGFP-NGB was injected into left ear and then gentamicin administration).Gentamicin was injected with 120 mg kg-1 d-1 for 14 days,then observed the animals for 14 days.Auditory brainstem response(ABR),stretched preparation technique,immunohistochemistry were utilized to evaluate the auditory function,the morphological changes of the hair cells on basement membrane in cochlear and the expression of NGB protein of each group. Results Average ABR thresholds in all the groups: groupⅠ:37.22 dB SPL(left ear) and 36.94 dB SPL(right ear);groupⅡ:37.22 dB SPL(left ear) and 37.50 dB SPL(right ear);group Ⅲ:119.44 dB SPL(left ear) and 122.22 dB SPL(right ear);group IV: 119.72 dB SPL(left ear) and 120.83dB SPL(right ear);group V: 83.89dB SPL(left ear) and 100.56 dB SPL(right ear).The stretching of cochlear basement membrane showed that both the inside and outside hair cells in groupⅠand groupⅡarranged neatly and had no defects.In group Ⅲand group IV,the hair cells almost vanished corresponding with the ABR threshold(more than 135 dB SPL).Some hair cells in group V were missing,damaged lightly,and mainly on the outer hair cells;Immunohistochemistry showed that NGB protein expressed on cochlear hair cells of NGB gene transfection group increased significantly(P<0.05),and all the other groups nearly had no obvious positive expression. Conclusion This study verified that cationic liposome-mediated NGB gene transfection could provide effective protection to the guinea pigs treated with gentamicin in a certain degree.