【摘要】： 目的: 探討攜帶綠色熒光蛋白(Green Fluorescent Protein,GFP)的重組復(fù)制缺陷型慢病毒為載體的白細(xì)胞介素-10(Interleukin-10,IL-10)基因?qū)?shí)驗(yàn)性自身免疫性感音神經(jīng)性聾(autoimmune sensorineural hearing loss, ASNHL)的治療作用。同時了解內(nèi)耳和中耳局部注射后慢病毒載體在內(nèi)耳組織結(jié)構(gòu)中的轉(zhuǎn)染分布和治療基因(即IL-10基因)局部表達(dá)情況。并通過三種不同注入途徑的比較,探討一種高效、安全和便捷的內(nèi)耳基因?qū)敕椒ê图夹g(shù)。? 方法: 采用純化豚鼠內(nèi)耳組織抗原(58kD),免疫豚鼠,造成自身免疫性感音神經(jīng)性聾的動物模型36只。按配對設(shè)計,將動物分為六組:A、B、C組為實(shí)驗(yàn)組,注射重組治療基因載體。A組鼓階內(nèi)注射,B組中耳腔途徑(圓窗膜滲透),C組內(nèi)淋巴囊局部注射。D、E、F為對照組,注射生理性外淋巴液。D組鼓階內(nèi)注射,E組中耳腔途徑,F組內(nèi)淋巴囊局部注射。內(nèi)耳抗原免疫前后和基因治療后(7天),采用ELISA試驗(yàn)測試血清特異性抗體水平。治療前和治療后(7天)行聽性腦干誘發(fā)電位(ABR)測試,后各組分別各取4只動物行內(nèi)耳光鏡和熒光顯微鏡下觀察。每組其余2只動物行酶免疫組織化學(xué)試驗(yàn),以檢測基因產(chǎn)物表達(dá)和慢病毒轉(zhuǎn)染情況。 結(jié)果: 各組血清特異性抗體水平(A值的均值)比較:免疫后均較免疫前升高,差異有顯著性;治療前后則無顯著性差異。治療前后ABRⅢ波閾值的均值對比顯示各實(shí)驗(yàn)組局部基因治療后均降低。治療后各實(shí)驗(yàn)組組間對比顯示差異無顯著。慢病毒主要轉(zhuǎn)染部位:A組:血管紋、螺旋韌帶、Corti器、螺旋神經(jīng)節(jié)、蝸軸小血管周圍;B組:螺旋神經(jīng)節(jié)、螺旋韌帶、Corti器、血管紋,蝸軸小血管周圍前庭膜等;C組:內(nèi)淋巴囊、內(nèi)淋巴管、前庭囊斑、壺腹嵴等處。基因產(chǎn)物表達(dá)部位與轉(zhuǎn)染部位基本相同。 結(jié)論: 采用純化豚鼠內(nèi)耳組織58kD抗原免疫豚鼠,成功造成自身免疫性感音神經(jīng)性聾的動物模型。重組載體經(jīng)不同途徑均可轉(zhuǎn)導(dǎo)入內(nèi)耳,并在內(nèi)耳表達(dá)基因產(chǎn)物,并可對ASNHL發(fā)揮有效的治療作用。尤其是中耳腔給藥途徑甚為便捷和安全。
[Abstract]:Aim: to investigate the therapeutic effect of interleukin-10 (IL-10) gene carrying green fluorescent protein (GFP) on experimental autoimmune sensorineural hearing loss (autoimmune sensorineural hearing loss,). At the same time, the distribution of lentivirus vector transfection and local expression of therapeutic gene (IL-10 gene) in inner ear and middle ear were studied. Through the comparison of three different ways of injection, this paper discusses an efficient, safe and convenient method and technology of inner ear gene transfer. Methods: 36 guinea pigs with autoimmune sensorineural hearing loss were immunized with purified guinea pig inner ear tissue antigen (58kD). According to the pairing design, the animals were divided into six groups: control group: group C was injected with recombinant therapy gene carrier. Group A was injected into the drum stage of group B through the middle ear cavity (round window membrane osmosis) and group C was given local injection of. Physiological perilymph. Group D: intra drum injection: group E: middle ear cavity approach: group F: local injection of endolymphatic sac. Serum specific antibody levels were measured by Elisa before and after immunization with inner ear antigen and 7 days after gene therapy. Auditory brainstem evoked potentials (ABR) were measured before and after treatment (7 days). The other two animals in each group were tested for gene product expression and lentivirus transfection. Results: the level of serum specific antibody (A value) in each group was significantly higher than that before immunization, but there was no significant difference before and after treatment. The mean value of ABR 鈪，

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