本文選題：納米顆粒　切入點：Atoh1基因　出處：《中國人民解放軍醫(yī)學(xué)院》2015年博士論文

【摘要】：自1994年,Fujiyoshi研究小組成功制備了中樞神經(jīng)系統(tǒng)的髓鞘堿性蛋白基因純合突變導(dǎo)致小鼠感音神經(jīng)性耳聾的模型以來,圍繞感音神經(jīng)性耳聾基因治療的研究逐漸展開。感音神經(jīng)性耳聾主要是由聽覺感受器官…耳蝸的變性引起的。對人類顳骨解剖學(xué)研究發(fā)現(xiàn),感音神經(jīng)性耳聾可由耳蝸多種細胞類型的變性引起,其中,承擔將聲音信號轉(zhuǎn)換為電信號的毛細胞病變,是目前針對感音神經(jīng)性耳聾研究的核心問題之一。以往,研究者們認為在哺乳動物體內(nèi),耳蝸毛細胞損傷變性后是不可再生的。近年來，有研究發(fā)現(xiàn)將包含毛細胞分化的關(guān)鍵基因---Atohl/Mathl基因的質(zhì)粒導(dǎo)入體外培養(yǎng)的Corti器中,可以產(chǎn)生異位的毛細胞,本研究小組前期,利用腺病毒作為載體將Atoh1基因成功介導(dǎo)轉(zhuǎn)染至噪聲性聾的豚鼠耳蝸內(nèi),發(fā)現(xiàn)噪聲損傷的豚鼠耳蝸過表達Atoh1基因,可使受損耳蝸內(nèi)產(chǎn)生大量新的內(nèi)外毛細胞纖毛簇,促進聽功能的恢復(fù)。雖然腺病毒基因載體有較高的基因轉(zhuǎn)染效率,但是也有很明顯的缺點,其介導(dǎo)的基因轉(zhuǎn)染為瞬時轉(zhuǎn)染且有很強的宿主免疫原性,且缺乏細胞特異性,難以通過倫理委員會的審查應(yīng)用于臨床。此外,與腫瘤的基因治療研究目的不同的是,針對于腫瘤細胞的治療研究多是以殺細胞為目的,可以使用病毒載體,而耳科學(xué)研究多以促進細胞再生或修復(fù)為目的,病毒載體的缺點大大限制了其進一步應(yīng)用。因此,為了篩選出適用于耳科研究的非病毒載體,本研究在研究小組前期工作的基礎(chǔ)上,研發(fā)合成新的納米基因載體，，并利用其將包含Atoh1基因的質(zhì)粒導(dǎo)入健康及噪聲致聾的豚鼠耳蝸內(nèi),運用聽功能檢測和形態(tài)學(xué)檢測方法對新載體轉(zhuǎn)導(dǎo)基因的效率及生物學(xué)效應(yīng)進行了初步研究。本研究第一部分,利用透明質(zhì)酸(Hyaluronic Acid, HA)對聚乙烯亞胺(Polyetherimide, PEI)納米顆粒進行修飾,獲得基于透明質(zhì)酸修飾的聚乙烯亞胺納米顆�；蜉d體(HA-PEI),利用其介導(dǎo)Atoh1-EGFP基因質(zhì)粒,通過完整圓窗膜滲透途徑對聽力正常豚鼠及噪聲性聾豚鼠進行耳蝸內(nèi)轉(zhuǎn)導(dǎo),觀察其在豚鼠耳蝸內(nèi)的轉(zhuǎn)基因能力及生物學(xué)特性。第二部分,利用前期合成的HA-PEI納米基因載體介導(dǎo)Atoh11基因轉(zhuǎn)染正常聽力豚鼠體外分離的毛細胞,并采用活細胞成像系統(tǒng)動態(tài)觀察HA-PEI-DNA納米基因復(fù)合體進入毛細胞的方式、分布及時間。第三部分,在本研究小組前期研究基礎(chǔ)上, 轉(zhuǎn)入HA-PEI-DNA納米基因復(fù)合體,觀察其對豚鼠噪聲性耳聾的治療和預(yù)防作用。綜合以上研究結(jié)果,為感音神經(jīng)性耳聾基因治療,特別是對于適用于聽覺系統(tǒng)的非病毒轉(zhuǎn)基因載體研發(fā)進行初步的研究。
[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.
【學(xué)位授予單位】：中國人民解放軍醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R764.43

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