纯化内耳P0蛋白诱发实验性自身免疫性内耳病动物模型

Animal model of experimental autoimmune inner ear disease induced by P0 protein purified from inner ear

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 purify P0 protein from guinea pig＇s inner ear by preparative SDS-PAGE and to study the possible role it may play in the etiology of autoimmune inner ear disease. Method： A mixture of membraneous proteins of inner ear was separeted by preparative SDS-PAGE. The corresponding band at 30 000 was cut and electrally eluted. The protein collected was identified by analytical SDS-PAGE and Western blot assay. A group of 20 guinea pigs were immunized with P0 protein emulsified in complete Freund＇s adjuvant,another 10 guinea pigs were immunized with complete Freund＇s adjuvant only as control. The guinea pigs＇ hearing thresholds,serum IgG level and morphological changes in the inner ear were investigated. The distribution of P0 protein in the cochlear was detected by immunohistochemical technique. Result：The purity of the protein was demonstrated by a single band at the 30000 site in SDS-PAGE, which was identified as P0 protein by western blot analysis assay. About 17.5% P0-immunized guinea pigs showed increased hearing thresholds, elevated IgG level（ F = 6.48, P 〈0.01）, as well as a decreased number of spiral ganglion cells and inflammatory cell infiltration in the cochlear nerve region. The P0 protein is distributed in the cochlear nerve and spiral ganglion only. Concision：P0 protein from guinea pig＇s inner ear can be successfully purified by preparative SDS-PAGE and an animal model of experimental autoimmune inner ear disease induced by P0 protein was successfully established.