本文選題：骨髓間充質(zhì)干細(xì)胞 + Corti氏器��； 參考：《南昌大學(xué)》2010年碩士論文

【摘要】： 研究背景和目的: 感音神經(jīng)性耳聾是耳鼻喉科常見疾病之一,據(jù)第39屆世界衛(wèi)生大會(huì)資料表明全球共有聾人及聽力損傷者達(dá)四億兩千萬(wàn)人,各種因素如病毒、細(xì)菌感染、噪音、耳毒性藥物等均可導(dǎo)致感音神經(jīng)性耳聾,研究證實(shí)其病理變化主要是耳蝸毛細(xì)胞及其與其相連的螺旋神經(jīng)節(jié)神經(jīng)元的缺失,因此如果能夠找到某種方法修復(fù)缺失的毛細(xì)胞或螺旋神經(jīng)節(jié)神經(jīng)元,就有希望治愈感音神經(jīng)性耳聾。細(xì)胞移植是未來治療感音神經(jīng)性耳聾的方法之一,在其他醫(yī)學(xué)領(lǐng)域,已經(jīng)從臨床和實(shí)驗(yàn)上證實(shí)移植干細(xì)胞可以治療某種疾病,如移植神經(jīng)干細(xì)胞可以治療帕金森氏病,移植心肌干細(xì)胞可以治療心肌梗死等,近年來研究發(fā)現(xiàn)哺乳動(dòng)物如鼠內(nèi)耳前庭器官和耳蝸也存在干細(xì)胞或高增殖細(xì)胞,體外能分化成為毛細(xì)胞樣細(xì)胞及神經(jīng)元,為干細(xì)胞移植治療感音神經(jīng)性聾帶來了希望,此外,日本學(xué)者將大鼠神經(jīng)干細(xì)胞移植入用氨基酸甙類抗生素或噪音致聾的鼠耳蝸,觀察到神經(jīng)干細(xì)胞在耳蝸內(nèi)存活,可以分化為少量的毛細(xì)胞及神經(jīng)元。也有學(xué)者發(fā)觀將神經(jīng)干細(xì)胞移植入耳蝸能改善耳蝸缺血導(dǎo)致的聽力下降,這些研究成果表明,移植干細(xì)胞是治療感音神經(jīng)性耳聾的希望途徑,由于內(nèi)耳干細(xì)胞或神經(jīng)干細(xì)胞存在于內(nèi)耳或中樞神經(jīng)系統(tǒng),未來臨床應(yīng)用中取材困難,影響其使用價(jià)值,所以必須找一個(gè)取材方便、并且能夠分化為內(nèi)耳毛細(xì)胞的干細(xì)胞來源。本課題擬從SD大鼠中提取骨髓間充質(zhì)干細(xì)胞(MSCs),并將其與耳蝸Corti氏器進(jìn)行共培養(yǎng),探討骨髓間充質(zhì)干細(xì)胞在體外培養(yǎng)的條件下,是否能夠分化為內(nèi)耳毛細(xì)胞,為將來利用MSCs移植治療感音神經(jīng)性耳聾的臨床應(yīng)用提供理論依據(jù)。 研究?jī)?nèi)容和方法: 1.骨髓間充質(zhì)干細(xì)胞的純化和鑒定:采用貼壁法分離、培養(yǎng)、純化SD大鼠MSCs,在倒置相差顯微鏡下觀察細(xì)胞形態(tài),數(shù)量的變化,同時(shí)對(duì)培養(yǎng)的細(xì)胞進(jìn)行CD29、CD34、CD45、CD90、CD117免疫細(xì)胞化學(xué)鑒定,收集第四代MSCs用于共培養(yǎng)實(shí)驗(yàn)。 2.耳蝸Corti氏器細(xì)胞的分離。取生后1-3天內(nèi)的SD大鼠乳鼠20只,斷頭處死,無菌條件下,取出耳蝸,在解剖顯微鏡下,去除螺旋韌帶和蝸軸,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進(jìn)行消化,中止消化并離心后棄去上清液,接種于細(xì)胞培養(yǎng)瓶中培養(yǎng)。 3.分化細(xì)胞的基因表達(dá)分析:分兩組:空白對(duì)照組及共培養(yǎng)組。空白對(duì)照組是指取第四代MSCs,按(5×104cells/ml)密度接種于6孔板,不與耳蝸Corti氏器細(xì)胞共培養(yǎng),滴加有EGF/bFGF/IGF-1以及N2和B27的DMEM/F12(1:1)無血清培養(yǎng)液,隔日換液一次,連續(xù)14天,再收集培養(yǎng)細(xì)胞。共培養(yǎng)組是指取第四代MSCs,按上述培養(yǎng)條件,與耳蝸Corti氏器細(xì)胞共培養(yǎng)14天,再收集分化細(xì)胞,將收集的兩組細(xì)胞分別提取總RNA,用于RT-PCR鑒定。 4.分化細(xì)胞的免疫細(xì)胞化學(xué)鑒定:用毛細(xì)胞的標(biāo)志物(Myosin7A、Math1、Calretinin)對(duì)分化細(xì)胞進(jìn)行免疫細(xì)胞化學(xué)鑒定。 結(jié)果: 1.培養(yǎng)第四代的MSCs為均一的“紡錘形”,呈漩渦樣生長(zhǎng),細(xì)胞熒光化學(xué)染色為CD29(+)、CD90(+)、CD117(+)、CD34(-)CD45(-),符合大鼠MSCs的特征。 2. MSCs與耳蝸Corti氏器細(xì)胞共培養(yǎng)2周后行免疫細(xì)胞化學(xué)鑒定示分化細(xì)胞能表達(dá)毛細(xì)胞的特異性標(biāo)志物Myosin7A、Math1、Calretinin,其中Myosin7A是目前認(rèn)為特異性比較高的毛細(xì)胞標(biāo)志物,Math1在毛細(xì)胞形成早期即有表達(dá)一直到成熟期,Calretinin為前庭毛細(xì)胞早期特異性分子標(biāo)志物。 3.空白對(duì)照組未見毛細(xì)胞標(biāo)志物mRNA的表達(dá)。共培養(yǎng)組可見在Marker為500bp、750bp的電泳條帶之間,出現(xiàn)擴(kuò)增片段長(zhǎng)度為624bp的Myosin7A電泳條帶以及擴(kuò)增片段長(zhǎng)度為545bp的Math1電泳條帶。 結(jié)論: 1.體外已成功將骨髓間充質(zhì)干細(xì)胞誘導(dǎo)分化為具有內(nèi)耳毛細(xì)胞分子特征的毛細(xì)胞樣細(xì)胞。 2.耳蝸Corti氏器細(xì)胞可能分泌多種調(diào)節(jié)因子,誘導(dǎo)骨髓間充質(zhì)干細(xì)胞向毛細(xì)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.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R764

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