纯化内耳P0蛋白诱发听神经脱髓鞘的实验研究

Correlation between P0 protein purified from the inner ear and auditory neuropathy

目的用从豚鼠内耳组织中纯化的P0蛋白免疫大鼠,观察实验动物听神经髓鞘变化,推测P0蛋白在听神经病的发生发展中的作用。方法采用SDS-PAGE从内耳组织中分离、纯化P0蛋白,用Western Blot法鉴定纯化的P0蛋白。用纯化的P0蛋白作为抗原免疫大鼠,观察其听性脑干反应阈、耳声发射的变化、动物血清IgG水平以及听神经的髓鞘在光镜电镜下的形态学变化,免疫组织化学技术观察P0蛋白在听神经上的分布。结果免疫后实验组大鼠听力学显示听神经病的特征:耳声发射正常,而听性脑干反应严重异常;实验组血清IgG水平显著升高。电镜下可见听神经的脱髓鞘病变,免疫组织化学技术显示P0分布在蜗神经纤维的髓鞘上。结论P0蛋白作为抗原能诱发蜗神经的脱髓鞘病变,可能是听神经病的重要发病机制之一。

Objective To purify P0 protein from guinea pigs＇ inner ears by preparative SDS-PAGE and to study the correlation between P0 protein and auditory neuropathy. Methods A mixture of membranous proteins of the inner ears was separated by preparative SDS-PAGE. The corresponding band at 30 000 was cut and electrally eluted. The collected proteins were identified by Western blot assay. A group of 20 healthy Sprague-Dawley rots were immunized with P0 protein emulsified in complete Freund＇s adjuvant, and another 20 Sprague-Dawley rats immtmized with complete Freunds adjuvant only were used as controls. The rats＇ hearing thresholds, serum IgG level and morphological changes in the cochlear nerve were investigated. The distribution of P0 protein in the cochlear nerve was detected by immunohistochemical technique. Results The purity of the protein was demonstinted by a single band at the 30 000 site in SDS-PAGE, which was identified as P0 protein by＇western blot analysis assay. About 29.4% of P0-immtmized rats showed increased heating thresholds and elevated IgG level（ P 〈 0.05）, and the demyelination of the cochlear nerve was observed by an electron microscope. P0 protein was distributed in the myelin sheath of the cochlear nerve. Condusions P0 protein from guinea pigs＇ inner ears can be successfully purified by preparative SDS-PAGE and an animal model of demyelination induced by P0 protein was successfully established, which is very usefnl to research the mechanism of auditory neuropathy.