羟基磷灰石纳米颗粒介导NT-3基因对豚鼠兴奋毒性损伤耳蜗的保护作用

目的:利用羟基磷灰石纳米颗粒(hydroxyapatite nanoparticle, HAT)携带构建的神经营养因子-3 (neurotrophic factor-3,NT-3)-绿色荧光蛋白基因(enhancement type Green fluorescent protein C2, pEGFPC2),通过耳蜗灌注方法转染兴奋毒性损伤后的豚鼠耳蜗螺旋神经节细胞(spinal ganglion cells,SGCs),观察pEGFPC2-NT3的表达及其对耳蜗螺旋神经节细胞的保护作用.方法:构建携带绿色荧光蛋白报告基因的重组质粒pEGFPC2-NT3.通过耳蜗灌注海人酸(kainic acid,KA)建立豚鼠耳蜗兴奋性损伤模型,在给KA 1周后利用HAT携带重组质粒进行耳蜗灌注以转染耳蜗螺旋神经节细胞,免疫组化法观察转染后1周NT-3的表达及4周后电镜下螺旋神经节细胞形态学变化,同时观察对听觉脑干诱发电位(auditory brain-stem response,ABR)的影响.结果:成功构建豚鼠耳蜗兴奋损伤模型.灌注重组质粒后1周免疫组化法观察到螺旋神经节细胞胞浆内NT-3蛋白表达.4周后电镜下螺旋神经节细胞形态学损害减轻,ABR检测听功能较兴奋毒性损害后有恢复.结论:在豚鼠耳蜗灌注KA造成耳蜗兴奋性损伤后第7天,经耳蜗鼓阶转染羟基磷灰石纳米颗粒介导的NT-3基因仍可减轻KA对耳蜗螺旋神经节细胞的兴奋性毒性损伤.

Protective effect of NT-3 gene mediated by hydroxyapatite nanoparticleon the cochlea of guinea pigs injured by excitotoxicity

OBJECTIVE: To transfect the recombinant plasmid enhancement type green fluorescent protein C2- neurotrophic factor-3 (pEGFPC2-NT3) into the spinal ganglion cells(SGCs) of guinea pigs' cochlea injured by the excitotoxicity of hydroxyapatite particle (HAT), to inject the recombinant plasmid pEGFPC2-NT3 into the guinea pigs' cochlea, and to observe the expression of pEGFPC2-NT3 and the protective effect of pEGFPC2-NT3 on SGCs of the cochlea in guinea pigs. METHODS: The recombinant plasmid pEGFPC2-NT3 with gene-green fluorescent protein was established. Kanic acid (KA) was injected into guinea pigs'cochleae and the excitotoxicity model was established. After a week the recombinant plasmid was transferred into SGCs of guinea pigs'cochlea treated with HAT. The following week the expression of NT-3 was examined by the immunohistochemical method, and the morphology of SGNs was observed under the electronic microscope after 4 weeks, in the mean time the changes of auditory brain-stem response (ABR) were examined. RESULTS: The excitotoxicity models were established successfully. NT-3 expression in the intracytoplasm of SGNs was observed by the immunohistochemical method 1 week after the injection, the morphologic damages of SGNs lessened under the electronic microscope after 4 weeks. ABR was partly restored, compared with ABR after the injury of the excitotoxicity. CONCLUSION: On the 7th day, NT3 gene transferred by HAT through the scala tympani can lessen the excitotoxicity of SGCs after KA was injected into the guinea pigs cochlea.