發(fā)布時間：2018-07-10 04:01

  本文選題：聽覺喪失 + 感音神經(jīng)性　； 參考：《中國人民解放軍軍醫(yī)進修學(xué)院》2010年博士論文

【摘要】： 感音神經(jīng)性聾主要由耳蝸毛細胞或聽覺神經(jīng)病變引起。毛細胞位于耳蝸內(nèi),是高度特異性機械感受器,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。相對于非哺乳動物,哺乳動物毛細胞損傷后不能自發(fā)再生,由此引起的聽力損失難以恢復(fù)。應(yīng)用干細胞進行細胞替代治療是毛細胞缺失后恢復(fù)聽力的一個主要治療策略。骨髓間充質(zhì)干細胞易于收集和增殖、能夠自體移植、無臨床應(yīng)用倫理問題和免疫障礙,具有多潛能性,是目前進行干細胞替代治療的主要干細胞來源。體外研究顯示骨髓間充質(zhì)干細胞在一定細胞蛋白作用下具有很強的可塑性,能夠分化為神經(jīng)元細胞類型。但是,骨髓間充質(zhì)干細胞能否分化為內(nèi)耳毛細胞或前體細胞,干細胞移植到內(nèi)耳能否存活和分化,能否替代損傷的聽毛細胞,這些都還不清楚。 本課題對大鼠骨髓間充質(zhì)干細胞體外定向誘導(dǎo)分化耳蝸毛細胞和骨髓間充質(zhì)干細胞內(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)干細胞體外誘導(dǎo)分化為毛細胞樣細胞 目的：探討骨髓間充質(zhì)干細胞體外定向分化為耳蝸毛細胞的可行性。 方法：1、體外分離培養(yǎng)骨髓間充質(zhì)干細胞,觀察不同換液方式對骨髓間充質(zhì)干細胞純化和增殖的影響；RT-PCR檢測培養(yǎng)細胞表面分子表達；定向誘導(dǎo)培養(yǎng)細胞向成脂細胞、成骨細胞方向分化。2、采取不同細胞誘導(dǎo)因子定向誘導(dǎo)培養(yǎng)骨髓間充質(zhì)干細胞地向分化為內(nèi)耳毛細胞,培養(yǎng)后細胞進行免疫組化鑒定和掃描電鏡觀察。 結(jié)果：1、24小時首次半量換液可使分離細胞在7天內(nèi)迅速增殖鋪滿細胞培養(yǎng)皿,培養(yǎng)細胞表面分子SH2、CD31、CD44呈陽性表達,但不表達CD34,培養(yǎng)細胞可分別向脂肪細胞及成骨方向分化。2、體外骨髓間充質(zhì)干細胞誘導(dǎo)后呈現(xiàn)神經(jīng)干細胞樣形態(tài)并表達其特異性標(biāo)志Nestin,繼續(xù)誘導(dǎo)分化表達內(nèi)耳毛細胞特異性標(biāo)志MyosinⅦa,電鏡觀察可見細胞表面長出微絨毛,類似毛細胞的靜纖毛。 結(jié)論：1、24小時首次半量換液培養(yǎng)有利于大鼠骨髓間充質(zhì)干細胞的分離和純化,培養(yǎng)細胞證實為骨髓間充質(zhì)干細胞。2、骨髓間充質(zhì)干細胞體外可定向誘導(dǎo)分化為內(nèi)耳毛細胞樣細胞。 第二部分骨髓間充質(zhì)干細胞內(nèi)耳移植治療藥物性聾 目的：1.探討用于干細胞替代治療研究的感音神經(jīng)性聾動物模型的建立方法；2.觀察骨髓間充質(zhì)干細胞移植對正常耳蝸的影響；3.研究骨髓間充質(zhì)干細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,通過聽覺腦干反應(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)干細胞移植的感音神經(jīng)性聾大鼠動物模型。2、經(jīng)鼓階途徑將骨髓間充質(zhì)干細胞移植到正常聽力大鼠耳蝸內(nèi),通過聽覺腦干反應(yīng)閾值、耳蝸常規(guī)切片觀察骨髓間充質(zhì)干細胞移植對耳蝸結(jié)構(gòu)和功能的影響。3、經(jīng)鼓階途徑將骨髓間充質(zhì)干細胞移植到藥物性聾大鼠耳蝸內(nèi),通過聽覺腦干反應(yīng)閾值、免疫組化和掃描電鏡觀察植入細胞對感音神經(jīng)性聾聽功能的影響及植入細胞在耳蝸內(nèi)的分化情況。 結(jié)果：1、應(yīng)用阿米卡星按500mg·kg-1·d-1進行連續(xù)一周皮下注射,可造成大鼠聽覺永久性閾移,3周后觀察柯替器毛細胞缺失,支持細胞損傷,呈現(xiàn)立方上皮樣結(jié)構(gòu)。2、骨髓間充質(zhì)干細胞鼓階導(dǎo)入對正常大鼠聽功能和耳蝸結(jié)構(gòu)無明顯影響,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)干細胞移植到藥物性聾動物耳蝸內(nèi)可遷移到耳蝸基底膜處并具有聽毛細胞特征,移植后8周聽功能大多無明顯改善。 結(jié)論：1、應(yīng)用阿米卡星可以建立起適合骨髓間充質(zhì)干細胞替代治療的理想動物模型。2、骨髓間充質(zhì)干細胞移植適合進行耳蝸病變的替代治療。3、骨髓間充質(zhì)干細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胞區(qū)域,表現(xiàn)內(nèi)耳聽毛細胞特征。
[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】：中國人民解放軍軍醫(yī)進修學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R764.43

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