羟基磷灰石纳米介导NT-3基因治疗豚鼠实验性感觉神经性耳聋的研究
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摘要
NT-3是神经系统发育和功能维持的重要物质，是内耳发育最基本的营养因子之一，并且对后天性损伤的神经元有保护和促进恢复的作用。众多研究已证实其对于内耳兴奋性损伤如缺血、噪声损伤，以及氨基苷类耳毒性损伤等各种因素造成的耳蜗损伤有一定的保护和促进恢复的作用。我们的前期研究已证实直接将NT-3蛋白通过微量渗透泵鼓阶灌注可减轻谷氨酸对豚鼠耳蜗螺旋神经节细胞兴奋性毒性损伤。基因克隆和基因转染技术的发展让内耳基因治疗前景广阔。目前常用的基因载体有病毒载体中的单纯疱疹病毒、腺病毒、腺病毒相关病毒载体和非病毒载体中的阳离子脂质体，我们的前期研究发现携带NT-3的重组腺病毒pAdeasy-NT-3对海人酸(KA)兴奋性毒性所造成的耳蜗形态学损害有明显的保护作用，但它们固有的一些缺陷如免疫原性和致癌性等让其在临床上的应用必将受到限制。随着纳米技术的发展，纳米颗粒的独特性质使其作为非病毒载体的应用逐渐受到重视，目前国内外尚无纳米颗粒内耳基因治疗的报道，我们首次尝试用羟基磷灰石纳米颗粒(HAT)做载体向靶细胞转染NT-3基因，研究体外细胞水平其在在小鼠螺旋神经节细胞的表达和对生长的影响，同时经外淋巴灌注途径向实验性神经性聋豚鼠内耳转染NT-3基因，研究其在活体状态下对耳蜗神经元的保护作用，为非病毒载体介导的内耳基因治疗提供实验依据。结果如下：
一、羟基磷灰石纳米颗粒神经营养素3转运载体的构建
1、重组质粒pEGFPC2-NT3构建
Neurotrophic factor-3(NT-3) play the important roles in nervous system development and functional maintenance.While NT-3 is the basic survival factor during the development of inner ear . It protects neurons and promotes recovery from being impaired neurons after birth. Most studies also have comfirmed that NT-3 could determinately preserve and promote its recovery from excitedly ototoxicity damage induced by ischemia noise injury and aminoglycosides ototoxicity. Prophase studies of our department had confirmed that NT-3 being poured straightly into tympanic scala with microdosis osmotic pump could lessen exitoxicity injury on guinea pig cochlea spiral ganglion cells induced by glutamate.Developments of gene cloning and gene transfection have broaded outlook of NT-3 gene therapy.Now gene vectors used frequently are herpes simplex virus ,adenovirus and adeno-associated virus that belong to viral vector,also including cation liposome belong to non-iral vector. Prophase studies of our department had confirmed that recombinated with NT-3(pAdeasy-NT-3) could protect morphologic injury on cochlea exitoxicity induced by kainic acid(KA).But adenovirus' natural characters can confine its clinical application.Peculiar properties of nanoparticles as non-viral vector have been gradually paid close attention.Now no report about nanopaticles on inner ear gene therapy. We first attempt to use hydroxyapatite nanoparticles(HAT) as NT-3 gene
一、羟基磷灰石纳米颗粒神经营养素3转运载体的构建
1、重组质粒pEGFPC2-NT3构建
Neurotrophic factor-3(NT-3) play the important roles in nervous system development and functional maintenance.While NT-3 is the basic survival factor during the development of inner ear . It protects neurons and promotes recovery from being impaired neurons after birth. Most studies also have comfirmed that NT-3 could determinately preserve and promote its recovery from excitedly ototoxicity damage induced by ischemia noise injury and aminoglycosides ototoxicity. Prophase studies of our department had confirmed that NT-3 being poured straightly into tympanic scala with microdosis osmotic pump could lessen exitoxicity injury on guinea pig cochlea spiral ganglion cells induced by glutamate.Developments of gene cloning and gene transfection have broaded outlook of NT-3 gene therapy.Now gene vectors used frequently are herpes simplex virus ,adenovirus and adeno-associated virus that belong to viral vector,also including cation liposome belong to non-iral vector. Prophase studies of our department had confirmed that recombinated with NT-3(pAdeasy-NT-3) could protect morphologic injury on cochlea exitoxicity induced by kainic acid(KA).But adenovirus' natural characters can confine its clinical application.Peculiar properties of nanoparticles as non-viral vector have been gradually paid close attention.Now no report about nanopaticles on inner ear gene therapy. We first attempt to use hydroxyapatite nanoparticles(HAT) as NT-3 gene
引文
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