HA-PEI纳米颗粒介导Atoh1基因转导豚鼠耳蜗细胞的研究

发布时间：2018-04-07 14:58

本文选题：纳米颗粒　切入点：Atoh1基因　出处：《中国人民解放军医学院》2015年博士论文

【摘要】：自1994年,Fujiyoshi研究小组成功制备了中枢神经系统的髓鞘碱性蛋白基因纯合突变导致小鼠感音神经性耳聋的模型以来,围绕感音神经性耳聋基因治疗的研究逐渐展开。感音神经性耳聋主要是由听觉感受器官…耳蜗的变性引起的。对人类颞骨解剖学研究发现,感音神经性耳聋可由耳蜗多种细胞类型的变性引起,其中,承担将声音信号转换为电信号的毛细胞病变,是目前针对感音神经性耳聋研究的核心问题之一。以往,研究者们认为在哺乳动物体内,耳蜗毛细胞损伤变性后是不可再生的。近年来，有研究发现将包含毛细胞分化的关键基因---Atohl/Mathl基因的质粒导入体外培养的Corti器中,可以产生异位的毛细胞,本研究小组前期,利用腺病毒作为载体将Atoh1基因成功介导转染至噪声性聋的豚鼠耳蜗内,发现噪声损伤的豚鼠耳蜗过表达Atoh1基因,可使受损耳蜗内产生大量新的内外毛细胞纤毛簇,促进听功能的恢复。虽然腺病毒基因载体有较高的基因转染效率,但是也有很明显的缺点,其介导的基因转染为瞬时转染且有很强的宿主免疫原性,且缺乏细胞特异性,难以通过伦理委员会的审查应用于临床。此外,与肿瘤的基因治疗研究目的不同的是,针对于肿瘤细胞的治疗研究多是以杀细胞为目的,可以使用病毒载体,而耳科学研究多以促进细胞再生或修复为目的,病毒载体的缺点大大限制了其进一步应用。因此,为了筛选出适用于耳科研究的非病毒载体,本研究在研究小组前期工作的基础上,研发合成新的纳米基因载体，，并利用其将包含Atoh1基因的质粒导入健康及噪声致聋的豚鼠耳蜗内,运用听功能检测和形态学检测方法对新载体转导基因的效率及生物学效应进行了初步研究。本研究第一部分,利用透明质酸(Hyaluronic Acid, HA)对聚乙烯亚胺(Polyetherimide, PEI)纳米颗粒进行修饰,获得基于透明质酸修饰的聚乙烯亚胺纳米颗粒基因载体(HA-PEI),利用其介导Atoh1-EGFP基因质粒,通过完整圆窗膜渗透途径对听力正常豚鼠及噪声性聋豚鼠进行耳蜗内转导,观察其在豚鼠耳蜗内的转基因能力及生物学特性。第二部分,利用前期合成的HA-PEI纳米基因载体介导Atoh11基因转染正常听力豚鼠体外分离的毛细胞,并采用活细胞成像系统动态观察HA-PEI-DNA纳米基因复合体进入毛细胞的方式、分布及时间。第三部分,在本研究小组前期研究基础上, 转入HA-PEI-DNA纳米基因复合体,观察其对豚鼠噪声性耳聋的治疗和预防作用。综合以上研究结果,为感音神经性耳聋基因治疗,特别是对于适用于听觉系统的非病毒转基因载体研发进行初步的研究。
[Abstract]:Since 1994 Fujiyoshi research group successfully prepared the mouse model of sensorineural deafness caused by homozygous mutation of myelin basic protein gene in central nervous system, the research on gene therapy of sensorineural deafness has been carried out gradually.Sensorineural hearing loss is mainly caused by auditory organs.Degeneration of the cochlea.Anatomical studies of human temporal bone have shown that sensorineural deafness can be caused by degeneration of many types of cells in the cochlea, in which hair cell lesions are responsible for converting sound signals into electrical signals.It is one of the core problems in the research of sensorineural hearing loss.Previously, researchers believed that in mammals, cochlear hair cells were damaged and denatured as non-regenerative.In recent years, it has been found that the plasmids containing the key gene of hair cell differentiation, namely, Atohl / Mathl gene, can be transferred into the Corti organ cultured in vitro to produce ectopic hair cells.Atoh1 gene was successfully transfected into noise induced deafness guinea pig cochlea by adenovirus. It was found that noise damaged guinea pig cochlea over-expressed Atoh1 gene, resulting in a large number of new inner and outer hair cell ciliated clusters in damaged cochlea.Promote the recovery of auditory function.Although adenovirus gene vector has high efficiency of gene transfection, it also has obvious disadvantages. Its mediated gene transfection is transient transfection, strong host immunogenicity, and lack of cell specificity.It is difficult to apply to the clinic through the examination of ethics committee.In addition, unlike the purpose of gene therapy for cancer, most of the research on cancer cell therapy is aimed at killing cells and using viral vectors, while otoscience is mostly aimed at promoting cell regeneration or repair.The disadvantages of virus vector greatly limit its further application.Therefore, in order to screen non-viral vectors suitable for ear research, a new nano-gene vector was developed and synthesized on the basis of the previous work of the research team.The plasmid containing Atoh1 gene was introduced into the cochlea of healthy and noise-induced deafness guinea pigs. The efficiency and biological effects of the new vector transduction gene were studied by means of auditory function test and morphological test.In the first part of this study, hyaluronic acid (HA) was used to modify polyethylene imine Polyetherimide (PEI) nanoparticles to obtain HA-PEIN gene vector based on hyaluronic acid modification.Cochlear transduction was carried out in normal hearing guinea pigs and noise-induced deafness guinea pigs through a complete round window membrane osmotic pathway. The transgenic ability and biological characteristics in the cochlea of guinea pigs were observed.In the second part, Atoh11 gene was transfected into hair cells isolated from normal hearing guinea pigs in vitro by HA-PEI gene vector, and the way of HA-PEI-DNA nanogene complex entering hair cells was observed dynamically by living cell imaging system.Distribution and timeIn the third part, on the basis of the previous study of this research group, HA-PEI-DNA nanogene complex was transferred to observe its therapeutic and preventive effects on noise-induced deafness in guinea pigs.Based on the above results, a preliminary study on gene therapy of sensorineural deafness, especially the development of non-viral transgenic vectors suitable for hearing system was carried out.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R764.43

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