【作者】 袁先道；

【导师】 高志强； 赵春华； 杨华；

【作者基本信息】 中国协和医科大学 ， 耳鼻咽喉科学， 2009， 博士

【摘要】 内耳毛细胞对于维持听觉及平衡功能至关重要。但内耳细胞结构易受外伤、药物毒性、感染、遗传缺陷、年龄老化等多种因素的破坏,使毛细胞损伤,导致听觉及前庭功能障碍。目前人工耳蜗植入是这种重度感音神经性聋唯一可靠治疗方法,而相关研究表明通过干细胞替代耳蜗毛细胞也具有治疗感音神经性聋的可能性。前期研究指出,干细胞是一类具有未分化、全能或多能、并且具有能自我更新能力的细胞,其在适当的胞内基因调控,胞间联系及环境因素下具有分化为体内任何一种类型细胞的能力。脂肪间充质干细胞(AD-MSCs)在体外经诱导,能分化出类似神经元和神经胶质细胞,且具有类神经细胞的功能。本研究探讨通过利用人脂肪源性间充质干细胞(human adipose-derived mesenchymal stem cells,hAD-MSCs)体外定向诱导向耳蜗毛细胞方向分化,为干细胞耳蜗内移植治疗感音神经性聋提供新的细胞来源;并将人脂肪源性间充质干细胞移植入致聋小鼠耳蜗内,观察移植细胞的体内存活及移行转化情况。目的1、在体外验证将人脂肪源性间充质干细胞(human adipose-derived mesenchymalstem cells,hAD-MSCs)定向分化为耳蜗毛细胞的可行性。2、建立小鼠药物性耳聋模型;探讨耳蜗细胞移植途径;验证人脂肪间充质干细胞移植入药物致聋后的小鼠耳蜗内的存活,定植及分化结果。方法1、分离并培养扩增人脂肪MSCs,流式细胞仪进行细胞表型分析,及细胞周期分析。2、用特定的培养体系配合多种细胞因子定向诱导hAD-MSCs向神经祖细胞样细胞分化,培养后细胞进行免疫组化鉴定。3、将诱导后细胞与发育期鸡胚听泡细胞在体外进行共培养,以促使其向内耳毛细胞分化,通过免疫组化等方法对分化不同阶段的细胞特异性指标进行鉴定。4、联合应用硫酸卡那霉素及呋塞米快速制备药物性聋小鼠动物模型5、经外半规管途径及静脉途径向药物性耳聋小鼠耳蜗内移植入hAD-MSCs,通过组织免疫荧光及逆转录-聚合酶链式反应(RT-PCR)检测植入细胞的转化情况。结果1、体外hAD-MSCs诱导后呈现神经干祖细胞样的形态并表达其特异性标志Nestin,2、体外hAD-MSCs经诱导再与发育期鸡胚听泡细胞共培养后表达内耳毛细胞特异性标志Math1和MyosinⅦa。3、人脂肪间充质干细胞移植入药物致聋后的小鼠耳蜗内,观察到移植细胞能够在耳蜗内至少存活2周,耳蜗Rosenthal’s管及耳蜗感觉上皮可见少量植入细胞,耳蜗Corti器区植入细胞表达耳蜗毛细胞特异性抗体MyosinⅦa。小鼠耳蜗提取总cDNA,进行RT-PCR检测可见耳蜗有人特异性看家基因beta-actin及少量Math-1表达。结论1、hAD-MSCs在体外可定向诱导分化为具有内耳毛细胞特异性标志的内耳毛细胞样细胞。2、hAD-MSCs移植入药物性聋小鼠耳蜗内能够存活,移行定植于Rosenthal’s管及耳蜗感觉上皮,在Corti器区表达耳蜗毛细胞特异性抗体。3、经外半规管途径可安全有效地将hAD-MSCs定向移植入耳蜗内。更多还原

【Abstract】 The inner ear hair cells are essential for hearing and balance.When hair cells are lost through trauma,toxicity,infection,genetic disorders,or aging,that can lead to impaired hearing or disruption of vestibular reflexes.Cochlear hair cells are a terminally differentiated cell population that is crucial for hearing and have no possibility of spontaneous regeneration in mammals.Hearing impairment in humans is,in most cases, a direct consequence of hair-cell loss.Cochlear implants to restore some hearing after neurosensory hearing loss(NSHL) are,at present,the only therapy for these people.In contrast to this therapy,replacement of hair cells via stem cell therapies holds the promise for a cure.Stem cells are characterized as undifferentiated,toti-,pluri-or multipotent,and self-renewing cells that have the capacity to differentiate into any cell type of the body given appropriate intracellular gene regulation,intercellular communication,and environmental cues.Adipose-derived mesenchymal stem cells (ADMSCs) can be induced to exhibit several phenotypic,morphologic,and excitory characteristics consistent with developing neuronal and glial tissue.We therefore asked whether ADMSCs could generate cochlear sensory hair cells.The aim of our study is to induce human adipose-derived mesenchymal stem cells(hAD-MSCs) to differentiate to generate cochlear sensory hair cells,and to find a possible source of stem cells for cell therapy for NSHL.We further delivered hAD-MSCs into the cochlea of deafened mice and examined the survival,migration and location of transplanted cells within the cochlear.Objective 1.To explore the feasibility of directionally inducing human adipose-derived mesenchymal stem cells(hAD-MSCs) in vitro towards cochlear sensory hair cells.2.To examine the survival,migration,and differentiation of transplanted hADMSCs within the cochlear of drug deafened mice.Methods1.Mesenchymal stem cells from human adipose tissue were isolated,purified and cultured in vitro.2.HADMSCs were induced to neural progenitor-like cell by using special cultivate system with some cell factors,which were tested by immunofluorescence.3.The processed hADMSCs co-cultured with embryonic chick otic vesicle cells,were converted it into cochlear sensory hair cells.The characteristic marks of cochlear sensory hair cells were detected by immnofluoresence method.4.The combination of kanamycin sulfate and frusemide was used to build drug deafened mice model.5.HADMSCs were transplanted into cochlear of deafened mice through lateral semicircular canal,and were examined by immnofluoresence and reverse transcription polymerase chain reaction(RT-PCR).Results1.Morphologically,hAD-MSCs were induced to differentiate into neural progenitor cells and expressed specific neural marker.2.After being co-cultured with embryonic chick otic vesicle cells,hAD-MSCs expressed specific surface markers of cochlear inner ear hair cells,such as the transcription factors Math1(murine atonal homologue 1) and myosin VIIa.3.Small numbers of hADMSCs were capable of survival in the deafened mammalian cochlea for up to 2 weeks,without causing an inflammatory tissue response.A proportion of these cells was detected in the Rosenthal’s canal and cochlear sensory epithelium,evidenced by immunofluorescence detection of expression of MyosinVIIa. RT-PCR found that beta-actin and Math1 express of the experimental group was higher than that of the control group.Conclusion 1.HADMSCs can be directionally induced to differentiate towards hair cell-like cells in vitro given appropriate regulation and environmental cues.2.HADMSCs can survive for at least 2 weeks after transplanted into mice cochlear, and migrate into the Rosenthal’s canal and cochlear sensory epithelium with expression of characteristic marks of cochlear sensory hair cells.3.Through the lateral semicircular canal,hAD-MSCs can be transplanted into cochlear safely and effectively.更多还原