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目的:用从内耳组织中纯化的P0蛋白免疫豚鼠,建立P0蛋白诱发的自身免疫性内耳病动物模型,研究其在自身免疫性内耳病中的作用。方法:采用制备性SDS-PAGE从内耳组织中分离、纯化P0蛋白。以纯化的豚鼠内耳P0蛋白作为抗原免疫豚鼠,观察其听性脑干反应阈,血清中抗体水平和内耳形态学的改变,并用免疫组织化学法确定P0蛋白在耳蜗的分布情况。结果:SDS-PAGE结果显示纯化的蛋白质只在分子量为30 000的位置上出现单一的蛋白染色带,Western blot结果示该蛋白即为P0蛋白。免疫后有22%豚鼠的听性脑干反应阈升高,对照组动物无变化。实验组血清IgG显著升高(F=6.48,P<0.01),反应阈提高豚鼠的螺旋神经节细胞均有不同程度的数目减少,蜗轴小血管周围有炎性细胞浸润。P0蛋白在耳蜗仅分布于螺旋神经节、蜗轴神经纤维的髓鞘上。结论:用制备性SDS-PAGE能够成功地从内耳纯化P0蛋白,用于自身免疫性内耳病的研究。P0蛋白在部分豚鼠能够诱发自身免疫性内耳病,可能是自身免疫性内耳病的自身抗原之一。
OBJECTIVE: To establish an animal model of autoimmune inner ear disease induced by P0 protein by immunizing guinea pigs with P0 protein purified from inner ear tissue and investigate its role in autoimmune inner ear disease. Methods: The P0 protein was isolated and purified from inner ear tissue by preparative SDS-PAGE. The purified guinea pig inner ear P0 protein was used as antigen to immunize guinea pigs. The threshold of auditory brainstem response, serum antibody level and inner ear morphological changes were observed. The distribution of P0 protein in cochlea was determined by immunohistochemistry. Results: SDS-PAGE showed that the purified protein showed a single protein band only at a molecular weight of 30 000. Western blot showed that the protein was P0 protein. After immunization, 22% of guinea pigs had higher threshold of auditory brainstem response and no change in the control animals. Serum IgG was significantly increased in the experimental group (F = 6.48, P <0.01). The response threshold increased the number of spiral ganglion cells in guinea pigs reduced to varying degrees, inflammatory cell infiltration around the small vessels in the mitral axis. P0 protein in the cochlea is only distributed in the spiral ganglion, the nerve fibers of the myelin sheath. CONCLUSION: The P0 protein can be successfully purified from the inner ear by preparative SDS-PAGE for the study of autoimmune inner ear disease. P0 protein in some guinea pigs can induce autoimmune inner ear disease, autoimmune inner ear disease may be one of the autoantigens.