本文選題：聽(tīng)覺(jué)喪失 + 感音神經(jīng)性　； 參考：《中國(guó)人民解放軍軍醫(yī)進(jìn)修學(xué)院》2010年博士論文

【摘要】： 感音神經(jīng)性聾主要由耳蝸毛細(xì)胞或聽(tīng)覺(jué)神經(jīng)病變引起。毛細(xì)胞位于耳蝸內(nèi),是高度特異性機(jī)械感受器,其功能障礙、損傷甚至缺失是耳蝸病變引起感音神經(jīng)性聾的主要原因。相對(duì)于非哺乳動(dòng)物,哺乳動(dòng)物毛細(xì)胞損傷后不能自發(fā)再生,由此引起的聽(tīng)力損失難以恢復(fù)。應(yīng)用干細(xì)胞進(jìn)行細(xì)胞替代治療是毛細(xì)胞缺失后恢復(fù)聽(tīng)力的一個(gè)主要治療策略。骨髓間充質(zhì)干細(xì)胞易于收集和增殖、能夠自體移植、無(wú)臨床應(yīng)用倫理問(wèn)題和免疫障礙,具有多潛能性,是目前進(jìn)行干細(xì)胞替代治療的主要干細(xì)胞來(lái)源。體外研究顯示骨髓間充質(zhì)干細(xì)胞在一定細(xì)胞蛋白作用下具有很強(qiáng)的可塑性,能夠分化為神經(jīng)元細(xì)胞類型。但是,骨髓間充質(zhì)干細(xì)胞能否分化為內(nèi)耳毛細(xì)胞或前體細(xì)胞,干細(xì)胞移植到內(nèi)耳能否存活和分化,能否替代損傷的聽(tīng)毛細(xì)胞,這些都還不清楚。 本課題對(duì)大鼠骨髓間充質(zhì)干細(xì)胞體外定向誘導(dǎo)分化耳蝸毛細(xì)胞和骨髓間充質(zhì)干細(xì)胞內(nèi)耳導(dǎo)入正常和藥物性聾耳蝸內(nèi)的存活和分化情況進(jìn)行了研究。本研究共分為兩部分： 第一部分骨髓間充質(zhì)干細(xì)胞體外誘導(dǎo)分化為毛細(xì)胞樣細(xì)胞 目的：探討骨髓間充質(zhì)干細(xì)胞體外定向分化為耳蝸毛細(xì)胞的可行性。 方法：1、體外分離培養(yǎng)骨髓間充質(zhì)干細(xì)胞,觀察不同換液方式對(duì)骨髓間充質(zhì)干細(xì)胞純化和增殖的影響；RT-PCR檢測(cè)培養(yǎng)細(xì)胞表面分子表達(dá)；定向誘導(dǎo)培養(yǎng)細(xì)胞向成脂細(xì)胞、成骨細(xì)胞方向分化。2、采取不同細(xì)胞誘導(dǎo)因子定向誘導(dǎo)培養(yǎng)骨髓間充質(zhì)干細(xì)胞地向分化為內(nèi)耳毛細(xì)胞,培養(yǎng)后細(xì)胞進(jìn)行免疫組化鑒定和掃描電鏡觀察。 結(jié)果：1、24小時(shí)首次半量換液可使分離細(xì)胞在7天內(nèi)迅速增殖鋪滿細(xì)胞培養(yǎng)皿,培養(yǎng)細(xì)胞表面分子SH2、CD31、CD44呈陽(yáng)性表達(dá),但不表達(dá)CD34,培養(yǎng)細(xì)胞可分別向脂肪細(xì)胞及成骨方向分化。2、體外骨髓間充質(zhì)干細(xì)胞誘導(dǎo)后呈現(xiàn)神經(jīng)干細(xì)胞樣形態(tài)并表達(dá)其特異性標(biāo)志Nestin,繼續(xù)誘導(dǎo)分化表達(dá)內(nèi)耳毛細(xì)胞特異性標(biāo)志MyosinⅦa,電鏡觀察可見(jiàn)細(xì)胞表面長(zhǎng)出微絨毛,類似毛細(xì)胞的靜纖毛。 結(jié)論：1、24小時(shí)首次半量換液培養(yǎng)有利于大鼠骨髓間充質(zhì)干細(xì)胞的分離和純化,培養(yǎng)細(xì)胞證實(shí)為骨髓間充質(zhì)干細(xì)胞。2、骨髓間充質(zhì)干細(xì)胞體外可定向誘導(dǎo)分化為內(nèi)耳毛細(xì)胞樣細(xì)胞。 第二部分骨髓間充質(zhì)干細(xì)胞內(nèi)耳移植治療藥物性聾 目的：1.探討用于干細(xì)胞替代治療研究的感音神經(jīng)性聾動(dòng)物模型的建立方法；2.觀察骨髓間充質(zhì)干細(xì)胞移植對(duì)正常耳蝸的影響；3.研究骨髓間充質(zhì)干細(xì)胞移植到藥物性聾耳蝸內(nèi)的存活和分化情況。 方法：1、應(yīng)用不同劑量阿米卡星連續(xù)1周,通過(guò)聽(tīng)覺(jué)腦干反應(yīng)閾值、耳蝸常規(guī)切片和掃描電鏡觀察,確定適合用于骨髓間充質(zhì)干細(xì)胞移植的感音神經(jīng)性聾大鼠動(dòng)物模型。2、經(jīng)鼓階途徑將骨髓間充質(zhì)干細(xì)胞移植到正常聽(tīng)力大鼠耳蝸內(nèi),通過(guò)聽(tīng)覺(jué)腦干反應(yīng)閾值、耳蝸常規(guī)切片觀察骨髓間充質(zhì)干細(xì)胞移植對(duì)耳蝸結(jié)構(gòu)和功能的影響。3、經(jīng)鼓階途徑將骨髓間充質(zhì)干細(xì)胞移植到藥物性聾大鼠耳蝸內(nèi),通過(guò)聽(tīng)覺(jué)腦干反應(yīng)閾值、免疫組化和掃描電鏡觀察植入細(xì)胞對(duì)感音神經(jīng)性聾聽(tīng)功能的影響及植入細(xì)胞在耳蝸內(nèi)的分化情況。 結(jié)果：1、應(yīng)用阿米卡星按500mg·kg-1·d-1進(jìn)行連續(xù)一周皮下注射,可造成大鼠聽(tīng)覺(jué)永久性閾移,3周后觀察柯替器毛細(xì)胞缺失,支持細(xì)胞損傷,呈現(xiàn)立方上皮樣結(jié)構(gòu)。2、骨髓間充質(zhì)干細(xì)胞鼓階導(dǎo)入對(duì)正常大鼠聽(tīng)功能和耳蝸結(jié)構(gòu)無(wú)明顯影響,可在鼓階和前庭階內(nèi)貼壁或游離存活至少4周。3、骨髓間充質(zhì)干細(xì)胞移植到藥物性聾動(dòng)物耳蝸內(nèi)可遷移到耳蝸基底膜處并具有聽(tīng)毛細(xì)胞特征,移植后8周聽(tīng)功能大多無(wú)明顯改善。 結(jié)論：1、應(yīng)用阿米卡星可以建立起適合骨髓間充質(zhì)干細(xì)胞替代治療的理想動(dòng)物模型。2、骨髓間充質(zhì)干細(xì)胞移植適合進(jìn)行耳蝸病變的替代治療。3、骨髓間充質(zhì)干細(xì)胞移植到藥物性聾耳蝸內(nèi)可以存活定位于基底膜外毛細(xì)胞區(qū)域,表現(xiàn)內(nèi)耳聽(tīng)毛細(xì)胞特征。
[Abstract]:Sensorineural deafness is mainly caused by cochlear hair cells or auditory neuropathy. Hair cells are located in the cochlea, a highly specific mechanoreceptor, and their dysfunction, damage and even loss are the main causes of sensorineural deafness caused by cochlear lesions. It is difficult to recover hearing loss. Cell replacement therapy using stem cells is a major treatment strategy for hearing loss after hair cell loss. Bone marrow mesenchymal stem cells are easy to collect and proliferate, can be transplanted in autologous transplantation, have no clinical ethical problems and immune disorders, and have multiple potential. It is currently used as a substitute therapy for stem cells. In vitro studies have shown that bone marrow mesenchymal stem cells have strong plasticity under the action of certain cell proteins and can differentiate into neuronal cell types. However, whether bone marrow mesenchymal stem cells can differentiate into inner ear hair cells or precursor cells, whether stem cells transplant to the inner ear can survive and differentiate, can substitute for damage. The injured auditory hair cells are not yet clear.
In this study, the survival and differentiation of normal and drug-induced deafness cochlear induced by bone marrow mesenchymal stem cells (MSCs) and bone marrow mesenchymal stem cells (MSCs) were induced in vitro. This study was divided into two parts.
The first part is the differentiation of bone marrow mesenchymal stem cells into hair cell like cells in vitro.
Objective: To investigate the feasibility of directional differentiation of bone marrow mesenchymal stem cells into cochlear hair cells in vitro.
Methods: 1, bone marrow mesenchymal stem cells were isolated and cultured in vitro, and the effects of different ways of exchanging liquid on the purification and proliferation of bone marrow mesenchymal stem cells were observed. RT-PCR was used to detect the expression of surface molecules on the cultured cells; directed induced culture cells to adipocyte, osteoblast differentiation.2, and extraction of different cell inducible factors to induce the culture of bone marrow Mesenchymal stem cells were differentiated into inner ear hair cells. After culture, the cells were identified by immunohistochemistry and scanning electron microscopy.
Results: the first half of the 1,24 hour solution could rapidly proliferate and spread the cell culture dish in 7 days. The cell surface molecules SH2, CD31, and CD44 were expressed positive, but the CD34 was not expressed. The cultured cells could differentiate to the adipocytes and the osteogenic direction of.2 respectively. After the induction of bone marrow mesenchymal stem cells in vitro, the neural stem cell like morphology was presented. The specific marker Nestin was expressed, and the differentiation and expression of inner ear hair cell specific markers Myosin VII a, and microvilli on the surface of the cells, similar to the static cilia of hair cells.
Conclusion: the first half volume of 1,24 hour culture is beneficial to the isolation and purification of bone marrow mesenchymal stem cells in rats. The cultured cells are confirmed to be bone marrow mesenchymal stem cells (.2), and bone marrow mesenchymal stem cells can be induced to differentiate into inner ear hair cell like cells in vitro.
The second part is the transplantation of bone marrow mesenchymal stem cells into inner ear for drug deafness.
Objective: 1. to explore the establishment of an animal model of sensorineural hearing loss for stem cell replacement therapy; (2.) to observe the effect of bone marrow mesenchymal stem cell transplantation on normal cochlea, and 3. to study the survival and differentiation of bone marrow mesenchymal stem cells transplanted into drug-induced deafness cochlea.
Methods: 1, using different doses of Amikacin for 1 weeks, the auditory brainstem response threshold, cochlear routine section and scanning electron microscopy were used to determine the.2 model of sensorineural deafness rat model suitable for bone marrow mesenchymal stem cells transplantation, and the bone marrow mesenchymal stem cells were transplanted into the cochlea of normal hearing rats through the drum step. The threshold of auditory brainstem response, the effect of bone marrow mesenchymal stem cells transplantation on the structure and function of the cochlea.3, the bone marrow mesenchymal stem cells were transplanted into the cochlea of drug-induced deafness rats. The auditory brainstem response threshold, immunohistochemistry and scanning electron microscopy were used to observe the auditory function of the sensorineural hearing loss. The influence of energy and the differentiation of implanted cells in the cochlea.
Results: 1, using Amikacin kg-1 / D-1 for one week subcutaneously subcutaneous injection, it could cause the permanent auditory threshold shift of the rat. After 3 weeks, the hair cell loss of the cot apparatus was observed, the support cell injury, the cubic epithelioid structure.2, and the drums of bone marrow mesenchymal stem cells had no obvious effect on the auditory function and cochlear structure of normal rats. The bone marrow mesenchymal stem cells transplanted into the cochlear cochlea of the drug-induced deafness could migrate to the cochlear basement membrane and have the characteristics of the auditory hair cell in the cochlea of drug-induced deafness animals. Most of the auditory functions were not obviously improved at 8 weeks after the transplantation in the drums and vestibule orders for at least 4 weeks of.3.
Conclusions: 1, Amikacin can establish an ideal animal model.2 for bone marrow mesenchymal stem cell replacement therapy. Bone marrow mesenchymal stem cells transplantation is suitable for the replacement of cochlear lesions for.3. Bone marrow mesenchymal stem cells can be transplanted into the drug-induced deafness cochlea and can be located in the outer layer of the basal membrane and the inner ear. Auditory hair cell characteristics.
【學(xué)位授予單位】：中國(guó)人民解放軍軍醫(yī)進(jìn)修學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R764.43

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