Math1诱导的哺乳动物的耳蜗再生毛细胞的形态学及功能学研究

发布时间：2018-08-15 17:27

【摘要】：目的转录因子Mathl诱导了终末支持细胞向新生毛细胞的转分化.1)本实验在描述支持细胞转分化为新生毛细胞的形态学变化过程的同时,通过使用正常毛细胞相关特异性标记物,在形态学方面对新生毛细胞和正常毛细胞进行了比较；2)本实验进一步阐述了在Mathl的作用下支持细胞转分化为毛细胞的转分化机制；3)本实验在功能学方面对新生毛细胞与发育中的正常毛细胞进行了比较,探讨其是否已经具备了正常毛细胞的功能性。方法本实验使用了正常出生后3天的新生SD大鼠耳蜗进行体外器官培养,实验组在培养的过程中通过腺病毒转染了Math1(作用基因)和EGFP(报告基因)两个基因(Ad5-EGFP-Mathl),对照组则只转染了EGFP(Ad5-EGFP).首先,在转染基因后不同的时间点对培养组织进行固定,通过免疫荧光染色技术标记毛细胞相关特异性蛋白来鉴定是否有新生毛细胞的产生,并描述了转分化的过程；其次,在耳蜗器官培养过程中,通过采用了Brdu掺入法,探讨了支持细胞向新生毛细胞的转分化机制；然后,通过检测新生毛细胞是否有摄取耳毒性药物新霉素, FM1-43的能力,来验证是否存在毛细胞特征性的机械性门控通道；最后,我们采用Patch Clamp技术,在不同的时间点对新生毛细胞的各种通道电流,动作电位以及特征性的机械门控通道电流等在不同时间变化以及相关的动力学分析,验证Math1诱导的新生毛细胞是否已经具备了正常毛细胞的功能。结果①实验组在Ad5-EGFP-Mathl转染耳蜗组织后的第三天,开始在小上皮嵴区域出现了MyosinVIIA阳性细胞,大上皮嵴区域则在转染五天后出现MyosinVIIA阳性细胞；钙缓冲蛋白,如Calbindin,Calmodulin,Calretinin和PMCA2作为毛细胞的相关特异性标记物,也能在异位新生毛细胞上表达；Phlloidin的染色展示了新生毛细胞顶部的簇状静纤毛样结构；在对照组的耳蜗的大、小上皮嵴区域则未出现这些现象；②Brdu掺入实验表明,支持细胞向新生毛细胞的转分化过程为直接转分化,不依赖于支持细胞的增殖；另外,Math1的表达水平是诱导毛细胞分化的关键因素；③1mM的耳毒性药物新霉素能够引起异位新生毛细胞的死亡；从Math1转染后5天,部分新生毛细胞开始有能力对FM1-43的摄取,间接说明毛细胞能通过机械门控通道摄取耳毒性药物及FM1-43；④Mathl转染后的2天,新生毛细胞开始表达钠电流,到第5天电流密度达最大,然后逐渐减小；对照组的支持细胞不表达钠电流；⑤Mathl转染后三天,新生毛细胞开始表达钙电流,电流密度随着时间逐渐增大；对照组支持细胞不表达钙电流；⑥Mathl转染后五天,新生毛细胞表达为快速失活的延迟性钾整流,然后,表现为持续性基本不失活的延迟性钾整流,且电流密度逐渐增大；而对照组的支持细胞仅表达持续不失活的幅度很小的延迟性钾整流；⑦Mathl转染后五天,新生毛细胞表达了典型的HCN电流,且随着时间电流密度逐渐减小,然后消失；⑧大多数的新生毛细胞上能记录到诱发的动作电位,极少数的能记录到自发的动作电位：⑨Mathl转染后7天,我们记录到了新生毛细胞的机械门控通道电流。结论①利用了腺病毒转基因方式成功的建立了新生大鼠耳蜗体外培养在Mathl诱导下产生异位新生毛细胞的模型；②新生毛细胞已经具备正常毛细胞样的形态,并且表达许多相同的特异性相关蛋白；③支持细胞转分化为毛细胞的机制为直接转分化,无需增殖后再转分化；④新生毛细胞有能力摄取耳毒性药物新霉素,FMl-43,间接表明机械门控通道在新生毛细胞纤毛上的存在；⑤机械门控通道电流的出现,在功能上标志了再生的毛细胞已经具备了毛细胞的特性；⑥各种通道电流,动作电位在新生毛细胞上的有序出现,模拟了发育中的正常毛细胞的各种离子通道电流有序出现的过程,证实了Mathl诱导的新生毛细胞是未成熟的、正在发育中的,具有功能的毛细胞。
[Abstract]:Objective Transcription factor Mathl induces the transformation of Sertoli cells into new hair cells. 1) In this study, the morphological changes of Sertoli cells into new hair cells were described and compared with those of normal hair cells by using specific markers related to normal hair cells. (3) We compared the function of new hair cells with that of normal hair cells in order to find out whether they have the function of normal hair cells.
Methods The cochlear organs of neonatal SD rats were cultured in vitro at 3 days after birth. The experimental group was transfected with Ad5-EGFP-Mathl and EGFP-Mathl by adenovirus. The control group was transfected with EGFP only. Immunofluorescence staining was used to identify whether there were new hair cells and to describe the process of transdifferentiation. Secondly, Brdu incorporation was used to explore the mechanism of the transformation of Sertoli cells into new hair cells. Then, we tested the ability of newborn hair cells to absorb the ototoxic drug neomycin, FM1-43, to verify the existence of the characteristic mechanical gated channels; finally, we used Patch Clamp technology, at different time points on the newborn hair cells of various channel currents, action potential and the characteristic mechanical gated pass. The changes of channel current at different time and related kinetic analysis verify that the new hair cells induced by Math1 have the function of normal hair cells.
Results (1) On the third day after cochlear tissue was transfected with Ad5-EGFP-Mathl, Myosin VIIA positive cells began to appear in the small epithelial ridge area, Myosin VIIA positive cells appeared in the large epithelial ridge area after transfection, and calcium buffering proteins such as Calbindin, Calmodulin, Calretinin and PMCA2 were used as specific markers of hair cells. Phlloidin staining showed a cluster of stationary cilia-like structures on the top of the newly formed hair cells; these phenomena did not occur in the large and small epithelial ridge areas of the control cochlea; (2) Brdu incorporation experiment showed that the transformation of the supporting cells into the new hair cells was direct, independent of the differentiation process. In addition, the expression level of Math1 was the key factor to induce hair cell differentiation; 3. 1 mM ototoxic drug neomycin could cause the death of ectopic hair cells; 5 days after Math1 transfection, some of the new hair cells began to have the ability to uptake FM1-43, which indirectly indicated that hair cells could be uptaken through a mechanical gated channel. Two days after Mathl transfection, the sodium current was expressed in the new hair cells, and the current density reached the maximum on the fifth day, then decreased gradually; the supporting cells in the control group did not express sodium current; _Three days after Mathl transfection, the calcium current began to express in the new hair cells, and the current density increased gradually with time in the control group; The Sertoli cells did not express calcium currents; _Five days after Mathl transfection, the new hair cells expressed delayed potassium rectifier which was rapidly inactivated, and then showed delayed potassium rectifier which was basically inactivated, and the current density increased gradually; while the control group only expressed delayed potassium rectifier which was not inactivated continuously. _Five days after Mathl transfection, HCN currents were expressed in the new hair cells, and the current density gradually decreased with time, then disappeared; _Most of the new hair cells could record evoked action potential, a few of them could record spontaneous action potential: _7 days after Mathl transfection, we recorded the mechanism of the new hair cells. Mechanical gated channel current.
Conclusion The model of ectopic hair cells in neonatal rat cochlea induced by Mattel was successfully established by adenovirus transgene. The new hair cells had normal hair cell-like morphology and expressed many identical specific related proteins. The mechanism of supporting cells transdifferentiation into hair cells was discussed. (4) Newborn hair cells have the ability to absorb the ototoxic drug neomycin, FMl-43, which indirectly indicates the existence of mechanical-gated channels on the cilia of newborn hair cells; (6) The appearance of mechanical-gated channel current indicates that the regenerated hair cells have the characteristics of hair cells. _The orderly appearance of various channel currents and action potentials on the new hair cells simulated the orderly appearance of various ion channel currents in the developing normal hair cells, and confirmed that the new hair cells induced by Mathl were immature, developing and functional hair cells.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R764

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