人源iPSC中致聋基因MYO15A的修正对诱导获得的毛细胞样细胞的形态和功能的影响

发布时间：2018-11-22 13:30

【摘要】：耳聋和听力障碍是全球性重大健康问题之一。耳是人类重要的听觉以及平衡器官,内耳毛细胞是听觉转换的关键感觉细胞。当声波经过的时候,毛细胞通过其表面静纤毛的摆动,将声波信号转换为电信号,并由神经传给大脑,从而产生听觉。内耳毛细胞的损伤或丢失是诱发耳聋和听力丧失的主要原因之一。噪音、衰老、基因突变或药物滥用(尤其是氨基糖苷类抗生素)都可能会导致内耳毛细胞损伤或丧失。在哺乳动物中,包括人类,内耳毛细胞一旦遭到破坏,将无法再生,这将导致永久性耳聋。研究耳聋的发病机理,探索如何在哺乳动物(以至于人类)中再生正常内耳毛细胞,这将为治疗耳聋或听力丧失提供帮助。诱导多能干细胞(iPSC)是一类性状极其类似于胚胎干细胞(ESC)的多能干细胞。规避伦理问题,分化能力的全能性,无限的扩增能力,获得病人特异iPSC的能力以及自体移植不存在免疫排斥的效应,这些种种优势都使iPSC在再生医学,疾病模型以及药物筛选等领域拥有广阔的应用前景。基因编辑技术的出现,尤其是CRISPR/Cas9技术的快速发展,使人类能够随心所欲的改造或是修正基因。近几年来内耳毛细胞再生研究的突破性进展,使哺乳动物毛细胞再生方法日趋成熟。本研究中,通过iPSC技术,我们建立了致聋基因MY015A突变的iPSC细胞系；通过CRISPR/Cas9技术,我们将MYO15A突变iPSC细胞系中的MYO15A基因进行了修正；通过将iPSC体外诱导分化毛细胞,我们证明了MYO15A的基因校正逆转了由MYO15A突变导致的毛细胞样细胞的形态和功能缺陷。第一部分：通过逆转录病毒,我们将来源于MYO15A突变耳聋病人(MY015A c.4642GA, c.8374GA),病人父亲(MYO15A c.8374GA)以及一个听力正常女孩的成纤维细胞诱导成了iPSC(分别为M-/-iPSC, M+/-iPSC和M+/+iPSC)。三株iPSC都表现出与ESC类似的特性,例如AP阳性,表达多能干细胞特异性标志蛋白OCT4,SOX2, NANOG, SSEA4, TRA-1-60以及TRA-1-81,具有体内外三胚层分化的潜能。同时对这三株iPSC的核型进行了检测,结果显示,在经过重编程的漫长过程后,三株细胞系都保持了正常的核型。第二部分：通过单层贴壁诱导法,我们将三株iPSC诱导成了听祖细胞,诱导获得的听祖细胞表达听相关特异性标志蛋白与基因。通过分离听上皮祖细胞,并与鸡胚椭圆囊基质细胞共培养,我们成功获得了三株细胞的毛细胞样细胞。诱导获得的毛细胞样细胞表达毛细胞特异标志基因与蛋白,能够特异摄取FM1-43染料,具有与内耳毛细胞类似的钾电流和钙电流,并且在这些细胞的表面存在毛细胞特异的静纤毛样结构。但同时也观察到了MY015A突变对M-/-ipSC诱导获得的毛细胞样细胞结构和功能的影响。M-/-iPSC诱导获得的毛细胞样细胞与M+/+iPSC和M+/-iPSC诱导获得的毛细胞样细胞相比表现出肌动蛋白丝结构的紊乱以及显著缩短的静纤毛样结构。第三部分：利用CRISPR-Cas9介导的同源重组技术,我们对M-/-iPSC中MY015A c.4642GA突变位点进行了校正。我们设计并构建了打靶载体pX330-maxGFP-sgRNA4,同源重组模板ssODN以及pUC 19-MYO15Asynonymous用于基因校正实验。通过流式细胞分选以及Sanger测序,我们成功筛选获得了基因校正的MC/-iPSC细胞系。MC/-iPSC维持正常细胞核型,表达多能干细胞标志基因与蛋白,且具有体内外分化的全能性。第四部分：为了了解MYO15A c.4642GA位点的基因校正对M-/-iPSC诱导获得的毛细胞样细胞的结构与功能的影响,我们将基因校正的MC/iPSCs诱导听祖细胞,并进一步诱导为内耳毛细胞样细胞。与M+/+iPSC和M+/-iPSC相似,MC/iPSC分化的听祖细胞,表达听相关基因与蛋白,分化的毛细胞样细胞,表达毛细胞特异性标记基因与蛋白。MC/-iPSC诱导获得的毛细胞样细胞具有与M+/+iPSC和M+/iPSC诱导获得的毛细胞样细胞相似的电压依赖电流,具有正常的肌动蛋白丝组织,正常的静纤毛长度,这与M-/-iPSC诱导获得的毛细胞样细胞的表现(肌动蛋白丝结构的紊乱以及显著缩短的静纤毛样结构)具有显著差异。我们的研究表明MY015A的基因校正逆转了由MYO15A突变导致的毛细胞样细胞的形态和功能缺陷。总之,本研究通过对致聋基因MYO15A突变的iPSC细胞系的基因校正与内耳毛细胞的诱导分化成功证明了从人iPSC诱导获得毛细胞样细胞的可行性,同时也证明了基因校正能有效逆转MY015A突变导致的毛细胞样细胞的形态和功能缺陷。
[Abstract]:Deafness and hearing impairment are one of the major health problems in the world. Ear is the most important auditory and balanced organ of human, and the inner ear hair cell is the key sensory cell of the auditory transition. when the sound wave passes, the hair cell converts the sound wave signal into an electric signal through the swinging of the surface static cilia, and transmits the sound wave signal to the brain, so as to generate the hearing. The damage or loss of hair cells in the inner ear is one of the main causes of deafness and hearing loss. Noise, aging, gene mutations, or drug abuse, in particular, aminosugars and antibiotics, may result in damage or loss of the inner ear hair cells. In mammals, including humans, the inner ear hair cells will not be regenerated once they are destroyed, which will lead to permanent deafness. To study the pathogenesis of deafness and to explore how to regenerate the normal inner ear hair cells in a mammal (such as a human), which will help to treat deafness or hearing loss. The induction of pluripotent stem cells (iPSC) is a class of pluripotent stem cells that are very similar to embryonic stem cells (ESCs). To avoid the ethical problem, the ability of differentiation, the ability of the infinite amplification, the ability of obtaining the specific iPSC of the patient and the effect of no immune rejection in the autotransplantation, all these advantages make the iSC have a wide application prospect in the fields of regenerative medicine, disease model and drug screening. The emergence of gene editing technology, in particular the rapid development of the CRISPR/ Cas9 technology, can make human being able to transform or modify genes as they can. In recent years, the development of the regeneration of hair cells in the inner ear has made the regeneration of the hair cells of the mammalian hair more mature. In this study, we set up an iPSC cell line for the mutation of the deafness gene MY015A through the iPSC technique; through the CRISPR/ Cas9 technique, we corrected the MYO15A gene in the MYO15A mutant iPSC cell line; and by inducing the iPSC in vitro to induce the differentiated hair cell, We demonstrated that the gene correction of MYO15A reversed the morphological and functional deficiencies of the hair cell-like cells resulting from the MYO15A mutation. Part One: By retrovirals, we have induced an iPSC (M-/-iPSC, M +/-iPSC, and M +/ + iPSC, respectively) from the MYO15A mutant deafness patient (MY015A c. 4642GA, c. 8374GA), the patient's father (MYO15A c. 8374GA), and a normal hearing girl. Sanzhu iPSCs exhibit similar characteristics as ESC, such as AP-positive, expression of multipotent stem cell-specific marker proteins OCT4, SOX2, NNOG, SSEA4, TRA-1-60, and TRA-1-81, with the potential for ectodermal differentiation in vivo. At the same time, the karyotype of Sanzhu iPSCs was tested, and the results showed that after the long process of reprogramming, the normal karyotype of Sanzhu cell line was maintained. The second part: Through the single-layer malapposition method, Sanzhu iPSC is induced to be an auditory progenitor cell, and the expression of the obtained auditory progenitor cells is induced to the relevant specific marker protein and gene. We successfully obtained the hair cell-like cells of Sanzhu cells by separating and co-culturing the epithelial progenitor cells and co-culturing with the matrix cells of the chick embryo. The induced hair cell-like cells express the specific marker gene and the protein of the hair cell, can specifically capture the FM1-43 dye, have similar potassium current and calcium current to the hair cell of the inner ear, and have a specific static cilia-like structure on the surface of the cells. However, the effect of the MY015A mutation on the structure and function of the hair cell-like cells induced by M-/-ipSC was also observed. The M-/-iPSC-induced hair cell-like cells exhibited a disorder of the actin filament structure as compared to the hair cell-like cells obtained by the M +/ + iPSC and M +/-iPSC-induced hair cell-like cells, as well as a significantly reduced static cilia-like structure. The third part: Using the homologous recombination technique mediated by CRISPR-Cas9, we corrected the mutation site of MY015A c. 4642GA in M-/-iPSC. We designed and constructed the target vector pX330-maxGFP-sgRNA4, the homologous recombination template ssODN and pUC 19-MYO15Asyncymous for gene correction experiments. By flow cytometry and Sanger sequencing, we successfully screened the MC/-iPSC cell line that obtained the gene correction. The MC/-iPSC maintains the normal cell nucleus type, expresses the multipotent stem cell marker gene and the protein, and has the function of external differentiation in vivo. In the fourth part, in order to understand the effect of gene correction on the structure and function of the hair cell-like cells induced by M-/-iPSC, the gene-corrected MC/ iPSCs were induced to the progenitor cells and further induced to the inner ear hair cell-like cells. Similar to M +/ + iPSC and M +/-iPSC, the MC/ iPSC-differentiated auditory progenitor cells express the related genes and proteins, differentiated hair cell-like cells, and express hair cell-specific marker genes and proteins. The MC/-iPSC-induced hair cell-like cells had a similar voltage-dependent current to the hair cell-like cells obtained by the M +/ + iPSC and M +/ iPSC-induced hair cell-like cells, with normal actin filament tissue, normal cilia length, This has a significant difference with the expression of the hair cell-like cells obtained by the M-/-iPSC-induced hair cell-like cells (the disorder of the actin filament structure and the significantly shortened static cilia-like structure). Our study showed that the gene correction of MY015A reversed the morphological and functional defects of hair cell-like cells caused by the mutation of MYO15A. In conclusion, this study successfully demonstrated the feasibility of induction of hair cell-like cells from human iPSC by gene correction of the iPSC cell line that has been mutated to the deafness gene MYO15A and the induction differentiation of the inner ear hair cell. It also proved that the gene correction can effectively reverse the morphological and functional defects of hair cell-like cells caused by the mutation of MY015A.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R764.43

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