自身免疫性感音神经性聋基因治疗的实验研究-东南大学学报医学版

自身免疫性感音神经性聋基因治疗的实验研究

单位：1. 南京医科大学第一附属医院耳鼻咽喉科暨耳鼻咽喉科学研究室, 江苏南京 210029;
2. 南京工业大学生物化学中心, 江苏南京 210009

分类号：R764.431; R-33

出版年·卷·期（页码）：2009·28·第二期（79-84）

摘要：

目的: 研究以重组复制缺陷型腺病毒为载体的Fas配体(Fas-Ligand,即FasL)基因和白细胞介素10(即IL-10)基因治疗实验性自身免疫性感音神经性聋。方法: 采用同种内耳组织抗原加弗氏佐剂免疫豚鼠,造成自身免疫性感音神经性聋的动物模型28只,按配对设计将其分为4组,通过鼓阶微量注射的方式,A组注入携带FasL基因的腺病毒(Ad-FasL),B组注入携带IL-10基因的腺病毒(Ad-IL-10),C组单纯注入腺病毒(携带绿色荧光蛋白,即Ad-GFP),D组注入等量的磷酸盐缓冲液。基因导入7 d后行听觉脑干诱发电位(ABR)测试,后取颞骨制作石蜡切片并行HE染色和光镜观察,每组各取两耳行螺旋韧带和基底膜透射电镜观察。每组各取3只(6耳)行免疫荧光和酶免疫组织化学试验,以检测基因产物表达和腺病毒转染情况。结果: 免疫组织化学显示携带目的基因的腺病毒可以转染血管纹、螺旋韧带、Corti器、螺旋神经节、蜗轴小血管周围及其耳蜗的骨壁等部位的细胞,并产生相应的蛋白产物(IL-10和FasL)。ABRⅢ波阈值的均值对比结果显示,A组和B组明显低于C组和D组。内耳组织的免疫炎性反应亦明显较C组减轻。结论: 重组复制缺陷型腺病毒可以携带目的基因转导入内耳,并在内耳表达基因产物,其抑制炎症反应的作用可有效地减轻自身免疫性感音神经性聋的内耳组织免疫炎性损伤和听觉功能障碍,有望成为治疗自身免疫性感音神经性聋新的方法和途径。

Objective To evaluate effects of transferring Fas ligand (FasL) gene and interleukin 10(IL-10) gene into inner ear for treatment of autoimmune sensorineural hearing loss(ASNHL).Methods The conspecific crude inner ear antigens (CIEAgs) were prepared and used to immunize guinea pigs with Freund's adjuvant that resulted ASNHL in 28 animals.We divided them into four groups by matched-pairs design.Through the way of scala tympani micro-injection,adenovirus vector containing FasL (Ad-FasL) gene were implanted in group A,while adenovirus vector containing IL-10(Ad-IL-10) gene were implanted in group B.Recombinant adenovirus carrying the gene for green fluorescent protein(Ad-GFP) were implanted in group C,and buffer phosphate were implanted in group D.7 days later,auditory brainstem response (ABR) thresholds were determined,temporal bone paraffin section was made and observed,the ultrastructure of spiral ligament and basilar membrane was observed with transmission electron microscope,and the immunohistochemistry tests were made for detecting adenovirus vector transfection with immunofluorescence and the gene product expressions with enzyme immunohistochemistry.Results Immunohistochemistry displayed that the adenovirus carrying purpose gene could transfer the psalterial cord,spiral ligament,Corti organ,spiral ganglion,cochlear axis vessels and cochlear bone paries.It could generate gene product (IL-10 or FasL) in same sites.Contrasting between each group,the mean ABR thresholds of group A and group B were lower than those of group C and group D,and the immunological inflammatory response were lighter in group A and group B than in group C and group D.Conclusion The adenovirus vector containing immunity regulating gene injecting into inner ear is one of feasible method for treating ASNHL.The adenovirus can transfer and genes can express its products in many parts of inner ear,and which can reduce the immunity damage and hearing functional impairment.We believe that it would become a hopeful new therapy and way for treating ASNHL.

参考文献：

[1] McCABE B F.Autoimmune sensorineural hearing loss[J].Ann Otol Rhinollmyrgol,1979,88:585-589.
[2] McCABE B F.Autoimmune inner ear disease: therapy[J].Am J Otol,1989,10:196-197.
[3] LALWANI A K,WALSH B J,REILLY P G,et al.Development of in vivo gene therapy for hearing disorders : introduction of adeno-associated virus into the guinea pig[J].Gene Ther,1996,3:588-592.
[4] RAPHAEL Y,FRISANCHO J C,ROESSLER B J.Adenoviral-mediated gene transfer into guinea pig cochlear cells in vivo[J].Neurosci Lett,1996,207:137-141.
[5] YAGI M,MAGAL E,SHENG Z,et al.Hair cell protection from aminoglycoside ototoxicity by adenovirus-mediated overexpression of glial cell line-derived neurotrophic factor[J].Hum Gene Ther,1999,20:813-823.
[6] 杨湘宁,钟启明.同种内耳抗原免疫致豚鼠感音神经性聋的实验研究[J].中华耳鼻咽喉科杂志,1992,27: 3-5.
[7] De KOZAK Y,THILLAYE-GOLDENBERG B,NAUD M C,et al.Inhibition of experimental autoimmune uveoretinitis by systemic and subconjunctival adenovirus-mediated transfer of the viral IL-10 gene[J].Clin Exp Immunol,2002,130:212-223.
[8] LIANG W,KARABEKIAN Z,XU Q H,et al.B-cell delivered gene transfer of human S-Ag-Ig fusion protein protects from experimental autoimmune uveitis[J].Clin Immunol,2006,118:35-41.
[9] 谭长强,钟启明,曹银成,等.在自身免疫性感音神经性聋发病机理中内淋巴囊作用的实验研究[J].耳鼻喉学报,1993,7: 153-157.
[10] 谭长强.特异性体液转移免疫致豚鼠先天性感音神经性聋实验研究 [J].东南大学学报:医学版,2007,26(5):327-332.

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