MYO7A基因缺陷的耳聋患者特异性iPS细胞的建立、基因修复及向毛细胞定向分化的研究

发布时间：2018-06-11 15:00

本文选题：耳聋 + 诱导多能干细胞　；参考：《浙江大学》2015年博士论文

【摘要】：耳聋是临床常见疾病,在耳聋人群中,由内耳毛细胞、听神经及各级听中枢不可逆损伤导致的感音神经性聋占重度耳聋的绝大部分。长期以来,对于感音神经性耳聋的治疗手段有助听器和人工耳蜗植入等,但这些方法并不能从根本上解决问题。人内耳耳蜗中只有约15000个毛细胞,外界声音的过度刺激、化疗、氨基糖苷类药物的副作用及年龄的增长都能够导致有功能的毛细胞数量减少。在人体内,毛细胞的不能再生直接导致听力衰退与耳聋。而毛细胞位于封闭的内耳环境中,加之其数量有限,这成为我们研究其损伤及再生机制的障碍。诱导多能干细胞(iPSCs)技术的兴起为临床研究和治疗耳聋提供了无限可能。这种技术能够对多种体细胞进行重编程,并将其诱导为与胚胎干细胞具有相同全能性的多能性细胞,只要给予一定的分化条件,这种细胞就能在体外诱导分化为几乎所有类型的细胞。这种技术直接规避了利用人胚胎干细胞进行研究的伦理问题；自体iPS细胞的产生也排除了免疫排斥的问题,使个体化医疗成为可能；构建的疾病来源的iPSC成为疾病研究及药物筛选的良好模型。目前已有多种疾病来源的人体细胞被重编程为iPS细胞,但耳聋来源的iPS细胞的建立未见报道。因此,本研究中采用诱导多能干细胞技术,完成了建立MYO7A突变的耳聋病人特异性iPS细胞株、向内耳毛细胞分化及基因矫正的研究。第一部分：首先,我们从正常人、MYO7A基因杂合双突变耳聋患者及其听力表现为正常但MYO7A基因单突变的父亲尿液中提取了尿液细胞,并将其诱导为iPS细胞；其次,我们对构建的三株iPS细胞全能性进行了检测分析。细胞形态、碱性磷酸酶染色、全能性基因表达、全能性标志蛋白免疫荧光、拟胚体形成及分化实验、畸胎瘤形成实验都表明,我们构建的三株iPS细胞均具有全能性。核型分析表明,我们构建的iPS细胞染色体结构正常,并未在诱导过程中受到破坏。最终结果表明,MYO7A突变的耳聋患者来源的尿液细胞与正常细胞一样,都能被重编程为具有全能性的诱导多能干细胞。第二部分：由于MYO7A在内耳耳蜗中只在毛细胞中表达,为了研究这一基因的突变对毛细胞功能有何影响及具体致聋机制,我们将正常人、MY07A突变耳聋患者及其父亲的特异iPS细胞向内耳祖细胞及进一步向内耳毛细胞诱导分化。在诱导过程中,我们采用细胞单层贴壁法,分两个阶段进行。向内耳祖细胞诱导阶段：通过添加FGF3和FGF10,使三株iPS细胞单层贴壁,诱导12天后,iPS细胞诱导分化为内耳神经样祖细胞和内耳上皮样祖细胞。早期内耳标志性基因表达检测和蛋白免疫荧光检测表明,三株iPS细胞成功分化为内耳祖细胞,并且各分化指标在三株细胞间没有显著性差异。这表明,无论是正常的还是MYO7A突变的iPS细胞,都能被诱导分化为内耳祖细胞,三株细胞的分化表现趋向一致。向毛细胞诱导阶段：我们将内耳上皮样祖细胞分离出来,添加EGF和维甲酸,贴壁诱导3周后,对分化的细胞进行毛细胞标志性基因表达检测及毛细胞标志性蛋白免疫荧光检测,结果表明正常人、MYO7A突变耳聋患者及其父亲来源的iPS细胞都能被诱导为毛细胞样细胞,且三株分化的毛细胞样细胞都表现了毛细胞特有的电生理功能。但是,具有MYO7A杂合双突变的分化的毛细胞对于FM1-43具有极低的内吞效率,电生理检测中IK1和ICa与正常iPS细胞来源的毛细胞样细胞相比显著偏大,且其静纤毛在聚拢成束中的表现与正常来源的毛细胞有明显区别；利用western blot对myosin7a蛋白表达检测表明,MY07A突变导致了一个缩短了的myosin7a蛋白的产生。以上结果表明,MY07A的突变是通过影响静纤毛束的功能来使毛细胞功能失常。第三部分：为了研究利用体外细胞株进行基因矫正、以作为临床应用的可行性,我们利用CRISPR-Cas9介导的同源重组技术,对MYO7A杂合双突变来源的iPS细胞的其中一个突变位点进行基因矫正。在经过初步流式分选之后,测序分析和酶切验证分析表明,有7.5%士1.3%的细胞被纯合矫正。之后我们对十个潜在脱靶位点进行PCR测序分析,未见有脱靶现象。全能性检测结果表明,矫正后的iPS细胞株仍具有iPS特异的全能性。这表明我们将杂合双突变中的其中一个突变位点矫正成功。将这一矫正后的细胞株向内耳毛细胞分化后,其在基因表达、蛋白表达等分化指标的表现与正常来源的iPS细胞分化的毛细胞样细胞表现一致,说明经过基因矫正操作后的iPS细胞仍能分化为内耳毛细胞。对这一iPS细胞株分化的毛细胞样细胞进行扫描电镜观察发现,静纤毛重新聚拢成束；Western blot检测表明,myosin7a蛋白条带与正常来源的毛细胞相同；分化的毛细胞中能够内吞FM1-43FX的比例恢复正常水平；电生理检测中,IK1和ICa恢复正常值。以上研究结果表明,我们成功将iPS细胞中MYO7A的一个突变位点进行了基因矫正,并且其分化为毛细胞的功能得到了修复。总之,本研究证明了耳聋来源的尿液细胞能够构建为iPS细胞,同时证明iPS细胞能向内耳毛细胞诱导分化。经过基因矫正的耳聋来源的iPS细胞在向毛细胞分化后,相关功能恢复正常,这为临床治疗耳聋提供了一个新的思路和途径。
[Abstract]:Deafness is a common clinical disease. In the deaf people, the sensorineural deafness caused by the inner ear hair cells, the auditory nerve and the irreversible damage of the auditory center at all levels accounts for most of the severe deafness. For a long time, the treatment methods for sensorineural deafness are hearing aids and artificial cochlear implants, but these methods can not be fundamentally solved. Problems. There are only about 15000 hair cells in the inner ear cochlea, overstimulation of external sound, chemotherapy, side effects of aminoglycosides and age, which can lead to a decrease in the number of functional hair cells. In the human body, the inability of hair cells to regenerate directly leads to hearing loss and deafness. In addition to its limited number, this has become an obstacle to our research on the mechanism of damage and regeneration. The rise of the induction of pluripotent stem cells (iPSCs) technology provides an infinite possibility for clinical research and treatment of deafness. This technique can reprogram a variety of somatic cells and induce it to be the same versatility with embryonic stem cells. As long as the cells are given certain differentiation conditions, the cells can be induced to differentiate into almost all types of cells in vitro. This technique directly avoids the ethical problems of using human embryonic stem cells for research; the production of autologous iPS cells also excludes the questions of immune rejection, making individualized medical treatment possible; the disease constructed. IPSC, a source of disease, has become a good model for disease research and drug screening. At present, the human cells of a variety of diseases have been reprogrammed into iPS cells, but the establishment of iPS cells from the source of deafness has not been reported. Therefore, in this study, the induction of pluripotent stem cell technology has been used to complete the specific iPS cell line of the deafness patients with the construction of MYO7A mutation. Study on the differentiation and gene correction of the inner ear hair cells. First part: first, we extracted the urine cells from the normal human, MYO7A gene heterozygous double mutant deafness and its hearing expression normal but single mutation of the MYO7A gene, and induced it to iPS cells; secondly, we have the total energy of the three iPS cells constructed. Cell morphology, alkaline phosphatase staining, omnipotent gene expression, omnipotent marker protein immunofluorescence, embryogenic body formation and differentiation experiments, and teratoma formation experiments showed that all of the three iPS cells constructed by us were omnipotent. Karyotype analysis showed that the chromosome structure of our iPS cells was normal and did not exist. The final result shows that the urine cells derived from the MYO7A mutant of the deafness can be reprogrammed into the omnipotent induced pluripotent stem cells, like normal cells. The second part: because MYO7A is only expressed in the hair cells in the inner ear cochlea, it is the study of the mutation of this gene to the function of hair cells. In the induction process, we use cell monolayer adherence to induce the differentiation of MY07A mutant deafness and their father's specific iPS cells to inner ear hair cells and further to the inner ear hair cells. In the induction process, we use the cell monolayer method in two stages. The induction stage to the inner ear progenitor cells: by adding FGF3 and FGF10 After 12 days, three iPS cells were induced to differentiate into inner ear nerve like progenitor cells and inner ear epithelioid progenitor cells. The early inner ear marker gene expression detection and protein immunofluorescence test showed that three iPS cells successfully differentiated into the inner ear progenitor cells, and the differentiation indexes were not significant difference between the three cells. This shows that both normal and MYO7A mutant iPS cells can be induced to differentiate into inner ear progenitor cells, and the differentiation of three cells tends to be consistent. Gene expression detection and hair cell marker protein immunofluorescence detection showed that normal people, MYO7A mutant deafness and their father's iPS cells could be induced to hair cell like cells, and the three differentiated hair cell like cells displayed the characteristic electrophysiological function of the hair cells. However, the MYO7A heterozygous double mutation was found. The hairy cells have very low endocytosis for FM1-43. In electrophysiological tests, IK1 and ICa are significantly larger than those of normal iPS cells, and the expression of its static cilia in the bundles is distincently different from that of normal hair cells. The detection of the expression of myosin7a protein by Western blot shows that MY07A mutation A shortened myosin7a protein was produced. The above results show that the mutation of MY07A is caused by the function of the cilium bundle to make the hair cell dysfunction. Third part: in order to study the use of an in vitro cell line for gene correction in order to be used as a clinical application, we use CRISPR-Cas9 mediated homologous recombination technology. Gene correction of one of the mutation sites of the MYO7A heterozygous double mutation source of iPS cells. After preliminary flow sorting, sequencing and enzymatic analysis showed that 7.5% of 1.3% of the cells were corrected by homozygosity. After that, we carried out PCR sequencing analysis of ten potential target loci and did not have a missing target. Omnipotent detection The results showed that the corrected iPS cell line still had iPS specific omnipotent. This showed that we corrected one of the mutation sites in the heterozygous double mutation. After the differentiation of the corrected cell line to the inner ear hair cells, the differentiation index of the gene expression and protein expression and the capillary differentiation of the normal iPS cells were observed. The cell like cells showed the same expression, indicating that the iPS cells after the gene correction could still differentiate into inner ear hair cells. The scanning electron microscopy of the hair cell like cells differentiated from the iPS cell line found that the static cilia was reassembled into bundles, and the Western blot detection showed that the myosin7a band was the same as the normal hair cells; In the hair cells, the ratio of endocytosis to FM1-43FX was restored to normal level; in electrophysiological tests, IK1 and ICa were restored to normal values. The above results showed that we successfully corrected a mutation of MYO7A in iPS cells and the function of its differentiation into hair cells was repaired. In conclusion, this study proved the source of deafness. The urine cells can be constructed as iPS cells and prove that the iPS cells can induce differentiation into the inner ear hair cells. The related functions of iPS cells, which are derived from the genetically modified deafness cells, are restored to normal after the differentiation of the hair cells. This provides a new way of thinking and path for the clinical treatment of deafness.
【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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