体外诱导骨髓间充质干细胞向内耳毛细胞分化的实验研究

发布时间：2018-06-08 00:33

本文选题：骨髓间充质干细胞 + Corti氏器　；参考：《南昌大学》2010年硕士论文

【摘要】： 研究背景和目的: 感音神经性耳聋是耳鼻喉科常见疾病之一,据第39届世界卫生大会资料表明全球共有聋人及听力损伤者达四亿两千万人,各种因素如病毒、细菌感染、噪音、耳毒性药物等均可导致感音神经性耳聋,研究证实其病理变化主要是耳蜗毛细胞及其与其相连的螺旋神经节神经元的缺失,因此如果能够找到某种方法修复缺失的毛细胞或螺旋神经节神经元,就有希望治愈感音神经性耳聋。细胞移植是未来治疗感音神经性耳聋的方法之一,在其他医学领域,已经从临床和实验上证实移植干细胞可以治疗某种疾病,如移植神经干细胞可以治疗帕金森氏病,移植心肌干细胞可以治疗心肌梗死等,近年来研究发现哺乳动物如鼠内耳前庭器官和耳蜗也存在干细胞或高增殖细胞,体外能分化成为毛细胞样细胞及神经元,为干细胞移植治疗感音神经性聋带来了希望,此外,日本学者将大鼠神经干细胞移植入用氨基酸甙类抗生素或噪音致聋的鼠耳蜗,观察到神经干细胞在耳蜗内存活,可以分化为少量的毛细胞及神经元。也有学者发观将神经干细胞移植入耳蜗能改善耳蜗缺血导致的听力下降,这些研究成果表明,移植干细胞是治疗感音神经性耳聋的希望途径,由于内耳干细胞或神经干细胞存在于内耳或中枢神经系统,未来临床应用中取材困难,影响其使用价值,所以必须找一个取材方便、并且能够分化为内耳毛细胞的干细胞来源。本课题拟从SD大鼠中提取骨髓间充质干细胞(MSCs),并将其与耳蜗Corti氏器进行共培养,探讨骨髓间充质干细胞在体外培养的条件下,是否能够分化为内耳毛细胞,为将来利用MSCs移植治疗感音神经性耳聋的临床应用提供理论依据。研究内容和方法: 1.骨髓间充质干细胞的纯化和鉴定:采用贴壁法分离、培养、纯化SD大鼠MSCs,在倒置相差显微镜下观察细胞形态,数量的变化,同时对培养的细胞进行CD29、CD34、CD45、CD90、CD117免疫细胞化学鉴定,收集第四代MSCs用于共培养实验。 2.耳蜗Corti氏器细胞的分离。取生后1-3天内的SD大鼠乳鼠20只,断头处死,无菌条件下,取出耳蜗,在解剖显微镜下,去除螺旋韧带和蜗轴,得到Corti氏器。再加入2ml 0.25%胰蛋白酶(含EDTA 1mmol/L)置入37℃孵箱8min进行消化,中止消化并离心后弃去上清液,接种于细胞培养瓶中培养。 3.分化细胞的基因表达分析:分两组:空白对照组及共培养组。空白对照组是指取第四代MSCs,按(5×104cells/ml)密度接种于6孔板,不与耳蜗Corti氏器细胞共培养,滴加有EGF/bFGF/IGF-1以及N2和B27的DMEM/F12(1:1)无血清培养液,隔日换液一次,连续14天,再收集培养细胞。共培养组是指取第四代MSCs,按上述培养条件,与耳蜗Corti氏器细胞共培养14天,再收集分化细胞,将收集的两组细胞分别提取总RNA,用于RT-PCR鉴定。 4.分化细胞的免疫细胞化学鉴定:用毛细胞的标志物(Myosin7A、Math1、Calretinin)对分化细胞进行免疫细胞化学鉴定。结果: 1.培养第四代的MSCs为均一的“纺锤形”,呈漩涡样生长,细胞荧光化学染色为CD29(+)、CD90(+)、CD117(+)、CD34(-)CD45(-),符合大鼠MSCs的特征。 2. MSCs与耳蜗Corti氏器细胞共培养2周后行免疫细胞化学鉴定示分化细胞能表达毛细胞的特异性标志物Myosin7A、Math1、Calretinin,其中Myosin7A是目前认为特异性比较高的毛细胞标志物,Math1在毛细胞形成早期即有表达一直到成熟期,Calretinin为前庭毛细胞早期特异性分子标志物。 3.空白对照组未见毛细胞标志物mRNA的表达。共培养组可见在Marker为500bp、750bp的电泳条带之间,出现扩增片段长度为624bp的Myosin7A电泳条带以及扩增片段长度为545bp的Math1电泳条带。结论: 1.体外已成功将骨髓间充质干细胞诱导分化为具有内耳毛细胞分子特征的毛细胞样细胞。 2.耳蜗Corti氏器细胞可能分泌多种调节因子,诱导骨髓间充质干细胞向毛细胞分化。
[Abstract]:Research background and purpose:
Sensorineural deafness is one of the common diseases in the Department of ENT. According to the data of the thirty-ninth World Health conference, there are four hundred and twenty million deaf and hearing impaired people in the world. Various factors such as virus, bacterial infection, noise and ototoxic drugs can lead to sensorineural deafness. The study confirms that the pathological changes are mainly cochlear hair cells. It is expected to cure sensorineural deafness by finding some way to repair missing hair cells or spiral ganglion neurons. Cell transplantation is one of the ways to treat sensorineural deafness in the future. In other medical fields, it has been proved from clinical and experimental evidence. The transplanted stem cells can treat some diseases, such as the transplanted neural stem cells can treat Parkinson's disease and the transplantation of myocardial stem cells can treat myocardial infarction. In recent years, it has been found that the mammalian vestibule organs and cochlea also have stem cells or high proliferating cells, which can differentiate into hair cell like cells and neurons in vitro. In addition, Japanese scholars have transplanted neural stem cells into the cochlea of rats with amino acid glucoside or noise deafness to observe the survival of neural stem cells in the cochlea, which can differentiate into a small amount of hair cells and Shen Jing Yuan. The cochlea can improve hearing loss caused by cochlear ischemia. These results suggest that transplanted stem cells are a promising approach for the treatment of sensorineural deafness. As inner ear stem cells or neural stem cells exist in the inner ear or the central nervous system, it is difficult to use material in future clinical application, so a material must be found. The stem cells can be differentiated into stem cells from the inner ear hair cells. This topic is to extract bone marrow mesenchymal stem cells (MSCs) from SD rats and co culture with the cochlear Corti's organ to explore whether bone marrow mesenchymal stem cells can differentiate into inner ear hair cells in vitro, and to treat the sense sound with MSCs transplantation in the future. The clinical application of neurogenic deafness provides a theoretical basis.
Research contents and methods:
1. the purification and identification of bone marrow mesenchymal stem cells: isolation, culture and purification of MSCs in SD rats by adhesion method. The cell morphology and quantity were observed under the inverted phase contrast microscope. At the same time, the cultured cells were identified by CD29, CD34, CD45, CD90, CD117 immunocytochemical identification, and fourth generation MSCs were collected for co culture experiment.
2. the isolation of Corti's cell in the cochlea. 20 rats of SD rats were killed in 1-3 days after birth. Under aseptic conditions, the cochlea was removed. Under the anatomic microscope, the spiral ligaments and the cochlear axis were removed and the Corti's organs were removed. Then 2ml 0.25% trypsin (including EDTA 1mmol/L) was added to the 37 centigrade incubator for digestion, discontinuation of digestion and centrifugation. The supernatant was inoculated into the cell culture bottle.
The gene expression analysis of 3. differentiated cells was divided into two groups: blank control group and co culture group. The blank control group was treated with fourth generations of MSCs, inoculated on 6 orifice plates at the density of (5 x 104cells/ml), not coculture with the cochlear Corti's organ cells, and DMEM/ F12 (1:1) serum-free culture medium with EGF/bFGF/IGF-1 and N2 and B27, once every other day, for 14 days for a continuous period, The culture cells were collected again. The co culture group was fourth generation of MSCs. According to the above conditions, the cochlear Corti's organ cells were co cultured for 14 days, and then the differentiated cells were collected. The total RNA of the collected two groups of cells were extracted for the identification of RT-PCR.
4. immunocytochemical identification of differentiated cells: immunocytochemical identification of differentiated cells with Myosin7A (Math1, Calretinin).
Result:
1. the MSCs of the fourth generation is a homogeneous "spindle shaped", which is whirlpool like growth. The cell fluorescent chemical staining is CD29 (+), CD90 (+), CD117 (+), CD34 (-) CD45 (-), which conforms to the characteristics of rat MSCs.
2. MSCs and cochlear Corti's organ cells were co cultured for 2 weeks after immunocytochemical identification, indicating that differentiation cells can express the specific markers of hair cells, Myosin7A, Math1, Calretinin, of which Myosin7A is a highly specific marker of hair cells, Math1 has been expressed in the early stage of hair cell form to maturity, Calretinin is Early specific molecular markers of vestibular hair cells.
3. the blank control group had no expression of the hair cell marker mRNA. In the co culture group, there was a Myosin7A strip with the length of 624bp and the Math1 electrophoresis strip with the length of 545bp in the Marker 500bp and 750bp bands.
Conclusion:
1. in vitro, bone marrow mesenchymal stem cells have been successfully induced to differentiate into hair cell like cells with inner ear hair cells.
2. cochlear Corti cells may secrete various regulatory factors and induce bone marrow mesenchymal stem cells to differentiate into hair cells.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R764

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