IL-10基因重组慢病毒载体不同途径导入内耳局部治疗自身免疫性感音神经性聋的实验研究
发布时间：2018-07-14 09:56
【摘要】： 目的: 探讨携带绿色荧光蛋白(Green Fluorescent Protein,GFP)的重组复制缺陷型慢病毒为载体的白细胞介素-10(Interleukin-10,IL-10)基因对实验性自身免疫性感音神经性聋(autoimmune sensorineural hearing loss, ASNHL)的治疗作用。同时了解内耳和中耳局部注射后慢病毒载体在内耳组织结构中的转染分布和治疗基因(即IL-10基因)局部表达情况。并通过三种不同注入途径的比较,探讨一种高效、安全和便捷的内耳基因导入方法和技术。? 方法: 采用纯化豚鼠内耳组织抗原(58kD),免疫豚鼠,造成自身免疫性感音神经性聋的动物模型36只。按配对设计,将动物分为六组:A、B、C组为实验组,注射重组治疗基因载体。A组鼓阶内注射,B组中耳腔途径(圆窗膜渗透),C组内淋巴囊局部注射。D、E、F为对照组,注射生理性外淋巴液。D组鼓阶内注射,E组中耳腔途径,F组内淋巴囊局部注射。内耳抗原免疫前后和基因治疗后(7天),采用ELISA试验测试血清特异性抗体水平。治疗前和治疗后(7天)行听性脑干诱发电位(ABR)测试,后各组分别各取4只动物行内耳光镜和荧光显微镜下观察。每组其余2只动物行酶免疫组织化学试验,以检测基因产物表达和慢病毒转染情况。 结果: 各组血清特异性抗体水平(A值的均值)比较:免疫后均较免疫前升高,差异有显著性;治疗前后则无显著性差异。治疗前后ABRⅢ波阈值的均值对比显示各实验组局部基因治疗后均降低。治疗后各实验组组间对比显示差异无显著。慢病毒主要转染部位:A组:血管纹、螺旋韧带、Corti器、螺旋神经节、蜗轴小血管周围;B组:螺旋神经节、螺旋韧带、Corti器、血管纹,蜗轴小血管周围前庭膜等;C组:内淋巴囊、内淋巴管、前庭囊斑、壶腹嵴等处。基因产物表达部位与转染部位基本相同。 结论: 采用纯化豚鼠内耳组织58kD抗原免疫豚鼠,成功造成自身免疫性感音神经性聋的动物模型。重组载体经不同途径均可转导入内耳,并在内耳表达基因产物,并可对ASNHL发挥有效的治疗作用。尤其是中耳腔给药途径甚为便捷和安全。
[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，
本文编号：2121283
[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，
本文编号：2121283
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