骨髓间充质干细胞治疗药物性聋的基础研究

发布时间：2018-07-10 04:01

本文选题：听觉丧失 + 感音神经性　；参考：《中国人民解放军军医进修学院》2010年博士论文

【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。
[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.
【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R764.43

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