【作者】 秦贺；

【导师】 翟所强； 杨仕明；

【作者基本信息】 中国人民解放军军医进修学院 ， 耳鼻咽喉科学， 2010， 博士

【摘要】 感音神经性聋主要由耳蜗毛细胞或听觉神经病变引起。毛细胞位于耳蜗内,是高度特异性机械感受器,其功能障碍、损伤甚至缺失是耳蜗病变引起感音神经性聋的主要原因。相对于非哺乳动物,哺乳动物毛细胞损伤后不能自发再生,由此引起的听力损失难以恢复。应用干细胞进行细胞替代治疗是毛细胞缺失后恢复听力的一个主要治疗策略。骨髓间充质干细胞易于收集和增殖、能够自体移植、无临床应用伦理问题和免疫障碍,具有多潜能性,是目前进行干细胞替代治疗的主要干细胞来源。体外研究显示骨髓间充质干细胞在一定细胞蛋白作用下具有很强的可塑性,能够分化为神经元细胞类型。但是,骨髓间充质干细胞能否分化为内耳毛细胞或前体细胞,干细胞移植到内耳能否存活和分化,能否替代损伤的听毛细胞,这些都还不清楚。本课题对大鼠骨髓间充质干细胞体外定向诱导分化耳蜗毛细胞和骨髓间充质干细胞内耳导入正常和药物性聋耳蜗内的存活和分化情况进行了研究。本研究共分为两部分：第一部分骨髓间充质干细胞体外诱导分化为毛细胞样细胞目的：探讨骨髓间充质干细胞体外定向分化为耳蜗毛细胞的可行性。方法：1、体外分离培养骨髓间充质干细胞,观察不同换液方式对骨髓间充质干细胞纯化和增殖的影响；RT-PCR检测培养细胞表面分子表达；定向诱导培养细胞向成脂细胞、成骨细胞方向分化。2、采取不同细胞诱导因子定向诱导培养骨髓间充质干细胞地向分化为内耳毛细胞,培养后细胞进行免疫组化鉴定和扫描电镜观察。结果：1、24小时首次半量换液可使分离细胞在7天内迅速增殖铺满细胞培养皿,培养细胞表面分子SH2、CD31、CD44呈阳性表达,但不表达CD34,培养细胞可分别向脂肪细胞及成骨方向分化。2、体外骨髓间充质干细胞诱导后呈现神经干细胞样形态并表达其特异性标志Nestin,继续诱导分化表达内耳毛细胞特异性标志MyosinⅦa,电镜观察可见细胞表面长出微绒毛,类似毛细胞的静纤毛。结论：1、24小时首次半量换液培养有利于大鼠骨髓间充质干细胞的分离和纯化,培养细胞证实为骨髓间充质干细胞。2、骨髓间充质干细胞体外可定向诱导分化为内耳毛细胞样细胞。第二部分骨髓间充质干细胞内耳移植治疗药物性聋目的：1.探讨用于干细胞替代治疗研究的感音神经性聋动物模型的建立方法；2.观察骨髓间充质干细胞移植对正常耳蜗的影响；3.研究骨髓间充质干细胞移植到药物性聋耳蜗内的存活和分化情况。方法：1、应用不同剂量阿米卡星连续1周,通过听觉脑干反应阈值、耳蜗常规切片和扫描电镜观察,确定适合用于骨髓间充质干细胞移植的感音神经性聋大鼠动物模型。2、经鼓阶途径将骨髓间充质干细胞移植到正常听力大鼠耳蜗内,通过听觉脑干反应阈值、耳蜗常规切片观察骨髓间充质干细胞移植对耳蜗结构和功能的影响。3、经鼓阶途径将骨髓间充质干细胞移植到药物性聋大鼠耳蜗内,通过听觉脑干反应阈值、免疫组化和扫描电镜观察植入细胞对感音神经性聋听功能的影响及植入细胞在耳蜗内的分化情况。结果：1、应用阿米卡星按500mg·kg-1·d-1进行连续一周皮下注射,可造成大鼠听觉永久性阈移,3周后观察柯替器毛细胞缺失,支持细胞损伤,呈现立方上皮样结构。2、骨髓间充质干细胞鼓阶导入对正常大鼠听功能和耳蜗结构无明显影响,可在鼓阶和前庭阶内贴壁或游离存活至少4周。3、骨髓间充质干细胞移植到药物性聋动物耳蜗内可迁移到耳蜗基底膜处并具有听毛细胞特征,移植后8周听功能大多无明显改善。结论：1、应用阿米卡星可以建立起适合骨髓间充质干细胞替代治疗的理想动物模型。2、骨髓间充质干细胞移植适合进行耳蜗病变的替代治疗。3、骨髓间充质干细胞移植到药物性聋耳蜗内可以存活定位于基底膜外毛细胞区域,表现内耳听毛细胞特征。更多还原

【Abstract】 Sensorineural hearing loss represents dysfunction in the chchlea or auditory nerve. The most common site of lesion is the hair cell, a specialized mechanoreceptor located in the organ of Corti in mammals. For patients with sensorinueral hearing loss, the causes are dysfunction, injury, or death of the hair cell.In contrast to non-mammals, hair cc(?)s do not regenerate following hair cell death in mammals, so that it is difficult to restore hearing after hair cell injury or loss. Cell replacement therapy with stem cells represents a major strategy for restoring hearing following hair cell loss. Stem cells are self-renewing pluripotent cells that can give rise to highly specialized cells in the organism depending on the microenvironment in which the stem cells reside, to replace lost, damaged, or defective human cell.Bone marrow mesenchymal stem cells(MSCs), easy to collect and propagate, be used in autologous transplantation, few ethical and immunologic barriers to clinical applications, be multipotent, represent a promising stem cell source for stem cell replacement therapy. It has been demonstrated that MSCs have the competence of differentiating into neureons. However, it is not clear whether MSCs differentiate into hair cells or progenitors in vitro and MSCs transplanted into inner ear survive and migrate to the sites hair cells reside to replace the lost inner ear hair cells.Our experiment carried out a series of researches, including differentiating into cochlear sensory hair cells of bone marrow MSCs in vitro and the survival, migration and differentiation of MSCs transplantation into cochlea in normal and deafened rats. The present study can be divided into following two parts: Part one:Bone marrow MSCs differentiate into hair cell-like cells in vitroObjective:To explore the feasibility of directionally inducing bone marrow MSCs to cochlear sensory hair cells in vitro.Methods:1. Rat bone marrow cells were separated and expanded by adherence culture. The surface molecule expressions of cells were examined by RT-PCR. Multilineage differentiation capability of cells was examined by culturing cells under conditions favorable for adipogenic and osteogenic differentiation in vitro. 2. By using special cell growth factors, bone marrow MSCs were induced to cochlear sensory hair cells, the characteristic marks of which were detected by immunohistochemisty and scanning electron microscope.Results:1. The medium was firstly half changed after primary culture for 24 hours, which made the isolated cells expand fast and cover with cell culture dishes in 7 days. Cultured cells expressed SH2, CD31, CD44, but lacked expression of CD44.The cells were successfully induced to adipocytes and osteogenesis.2. Bone marrow MSCs could be induced to differentiate into neural stem cells, and expressed specific neural marker, such as Nestin. Following that, the cells expressed specific markers of cochlear inner ear hair cell in the other growth factors, such as MyosinⅦa. Scanning electron microscopy showed microvillus on the surface of cells, silimar to the stereocilia of inner ear hair cells.Conclusions:1. The method that medium was firstly half changed after primary culture for 24 hours is benefit for isolation and purity of rat bone marrow MSCs.The cells isolated in this experiment have biological characteristics of bone marrow MSCs. 2. Bone marrow MSCs can be induced to convert into inner ear hair cell-like cells.Part two:The treatment to drug deafness with inner ear implantation of bone marrow MSCsObjective:1. To explore the methods of the sensorineural hearing loss animal model for stem cells replacement therapy to regenerate hair cells.2. To investigate the impact on normal cochlea after bone marrow MSCs transplantation into cochlea through scala tympani (ST).3. To explore survival and differentiation of rat MSCs after transplantation into drug deafened rat cochlea.Methods:1. Rats were treated hypodermically with different dose of amikacin for one week. Auditory brainstem response (ABR), light microscope and scanning electron microscope were used to evaluate to establish a sensorineural hearing loss animal model for bone marrow MSCs transplantation.2. Bone marrow MSCs were transplated into cochlea of normal hearing rats via ST. The cochlear structures and auditory function were measured by ABR, immunohistochemisty and scanning electron microscope.3. The impact on auditory function was evaluated by ABR and the survival, migration and differentiation of engrafted MSCs were examined by immnofluoresence method and scanning electron microscopy after bone marrow MSCs were transplated into cochlea of drug deafened rats.Results:1. There were permanent threshold shift after rats were treated hypodermically with amikacin 500mg-kg-1·d-1 for one week. Three weeks after treatment, loss of cells in the organ of Corti from sensory hair cells to supporting cells and the cuboidal epithelium were observed.2. There were no significantly impact on the auditory function and cochlear structures of normal rats after MSCs transplantation into cochlea via ST. MSCs could survive at least 4 wekks, locating in ST and scala vestibular, adherence or dissociation. 3. After transplantation, Grafted MSCs were visualized in the basilar membrane of every turn of the cochlea, having the characteristics of inner ear hair cells. There were no significant hearing improvements in 8 weeks.Conclusions:1. This amikacin deafness animal model is promise of an ideal animal model for the further study for acoustic hair cell regeneration through bone marrow MSCs replacement therapy. 2. Bone marrow MSCs transplantation is a potential strategy for cochlear lesion. 3. Rat bone marrow MSCs can survive in the sites of the outer hair cells died and have the characteristics of the inner ear hair cells.更多还原