DAPT调控大鼠耳蜗毛细胞分化的机制研究

发布时间：2018-08-15 12:31

【摘要】：传统观点认为，哺乳动物内耳毛细胞为终端有丝分裂细胞，损伤后不可再生，这是限制感音神经性耳聋治疗的瓶颈。近年来，国内外学者通过不断探索，发现了几种可以促使哺乳动物内耳毛细胞再生的新方法：促毛细胞再生因子的导入，干细胞植入及内耳相关microRNA因子的导入等。但这些技术仍处于实验研究阶段，关于毛细胞增多机制的阐明还待进一步探索。Notch通路是动物发育中重要的信号转导途径，参与了胚胎发育中多种细胞系定向分化的调控。最近研究认为，Notch信号通路调节的侧抑制参与哺乳动物内耳感觉祖细胞定向分化为毛细胞和支持细胞的过程。在Notch信号通路中，Notch受体的激活需要γ-分泌酶的水解。DAPT是一种实验中使用的γ-分泌酶抑制剂，它可阻止Notch受体的活化，使Notch信号途径中级联反应效应被抑制，导致其下游转录因子Hes1和Hes5的表达减少，从而使Hes1和Hes5对Math1表达的抑制减弱，间接增加了Math1的表达。可见，，DAPT可能参与促内耳毛细胞的发育过程。 MicroRNA是一种广泛存在于真核系统中的内源性非编码小RNA，从基因表达到基因调控的整个过程中都具有重要作用。目前发现，microRNA183家族在内耳毛细胞中高表达，且与毛细胞的分化和发育相关。microRNA主要通过其5’种子区域与靶向mRNA的3’非编码区域结合，从而阻断靶向mRNA的翻译而发挥作用。了解microRNA在不同情况下的表达谱是研究其具体功能的前提。 毛细胞的分化受多种因素的控制，本文主要观察DAPT对离体培养的大鼠耳蜗基底膜毛细胞分化的影响，并从Notch信号通路和microRNA表达水平两个方面探究毛细胞分化机制，试图从中找到对毛细胞增生具有促进/抑制作用的因素，用于成年鼠耳聋的治疗，进而将来用于临床，服务人类。 第一部分：DAPT对离体培养大鼠Corti’s器毛细胞再生的影响 目的观察DAPT对离体培养新生大鼠基底膜毛细胞生长的影响。 方法取新生（P0）SD大鼠耳蜗基底膜进行体外培养。采用自身对照方式，实验组基底膜加入含DAPT（终浓度5μM）的高糖培养基，对照组不加DAPT。培养5天(P5)后，用激光共聚焦显微镜观察毛细胞的生长情况，计量单位长度基底膜内毛细胞的数目，并用统计软件进行分析。 结果实验组基底膜毛细胞增多，对照组毛细胞数目基本不变，两组比较差异具有统计学意义。 结论新生大鼠基底膜体外培养成功；通过形态学观察和统计学分析发现，DAPT能够使离体培养的基底膜毛细胞数目增多。 第二部分：DAPT对调控耳蜗毛细胞分化因素的影响 目的从Notch信号通路和microRNA两方面探讨DAPT导致毛细胞增多的机制。 方法P0时采用RT-PCR检测Notch通路的存在与否；P5时采用qRT-PCR检测当Notch通路被DAPT阻断后下游基因的变化情况，同时对实验组和对照组的microRNA表达谱进行对比分析。 结果经DAPT处理后，实验组大鼠基底膜出现毛细胞增多，RT-PCR证实了出生第一天时尚有Notch通路的存在，qRT-PCR证实DAPT处理后，抑制毛细胞生成的Hes1和Hes5表达降低，而促进毛细胞生成的Math1表达升高。P5时，实验组和对照组相比，出现132种microRNA表达上调，36种microRNA表达下调；变化超过2倍的microRNA共有168种，占总检测数量的22%。microRNA292、microRNA124、microRNA18a、microRNA130b、microRNA99a、microRNA96及microRNA183的表达量在实验组高于对照组。 结论新生大鼠中Notch通路活性尚存，加入Notch通路阻断剂DAPT后，抑制毛细胞生成的Hes1和Hes5表达降低，而促进毛细胞生成的Math1表达升高。实验组内耳毛细胞增多后，microRNA表达谱系也发生变化，microRNA292、microRNA124、microRNA18a、microRNA130b、microRNA99a、microRNA96及microRNA183可能与大鼠内耳毛细胞分化相关。 第三部分：microRNA在新生和成年大鼠耳蜗中的表达 目的了解新生鼠和成年鼠内耳基底膜中microRNA的表达谱。 方法取新生大鼠和成年大鼠基底膜（各4只），灭酶方法提纯microRNA，采用TaqMan microRNAArray方法进行两组microRNA的差异分析。 结果新生鼠和成年鼠microRNA表达谱的比较：（1）新生鼠内耳有284种microRNA表达，成年鼠内耳有209种microRNA表达；（2）新生组和成年组相比较，成年组microRNA的总体表达趋势是下降的；（3）新生鼠microRNA183家族表达量高于成年鼠。 结论鼠类内耳中microRNA的表达谱广泛，并且其表达具有不同的时空模式。microRNA183家族在大鼠新生期基底膜中高表达，可能与此时毛细胞具有再生潜能有关。
[Abstract]:Traditionally, hair cells in the inner ear of mammals are terminal mitotic cells, which can not be regenerated after injury. This is a bottleneck in the treatment of sensorineural hearing loss. Stem cell transplantation and the introduction of microRNA factors related to the inner ear are still in the experimental stage, but the elucidation of the mechanism of hair cell proliferation remains to be further explored. Notch pathway is an important signal transduction pathway in animal development, which is involved in the regulation of directional differentiation of various cell lines in embryonic development. In Notch signaling pathway, the activation of Notch receptor requires the hydrolysis of gamma-secretase. DAPT is an inhibitor of gamma-secretase used in experiments, which blocks the activation of Notch receptor and enables Notch signaling pathway. Inhibition of cascade effect resulted in decreased expression of downstream transcription factors Hes1 and Hes5, which weakened the inhibition of Hes1 and Hes5 on the expression of Math1 and increased the expression of Math1 indirectly.
MicroRNA is an endogenous non-coding small RNA widely distributed in the eukaryotic system and plays an important role in the whole process from gene expression to gene regulation.It has been found that microRNA183 family is highly expressed in inner ear hair cells and is related to the differentiation and development of hair cells.MicroRNA is mainly targeted to mRNA through its 5'seed region. Understanding the expression profiles of microRNAs in different situations is a prerequisite for studying their specific functions.
The differentiation of hair cells is controlled by many factors. This paper mainly observes the effect of DAPT on the differentiation of rat cochlear basilar membrane hair cells in vitro, and explores the mechanism of hair cell differentiation from Notch signaling pathway and microRNA expression level, trying to find out the factors that can promote/inhibit the proliferation of hair cells for adult use. The treatment of mouse deafness will further serve the clinic and serve mankind.
Part one: the effect of DAPT on the regeneration of Corti 's hair cells in vitro.
Objective To observe the effect of DAPT on the growth of basement membrane hair cells of neonatal rats in vitro.
Methods Cochlear basement membrane of neonatal (P 0) SD rats was cultured in vitro. The basement membrane of the experimental group was added with high glucose medium containing DAPT (final concentration 5 mu M) and the control group without DAPT for 5 days (P5). The growth of hair cells was observed by laser confocal microscopy. The number of hair cells per unit length of basement membrane was measured. Statistical software was used for analysis.
Results The number of basement membrane hair cells increased in the experimental group and remained unchanged in the control group. The difference between the two groups was statistically significant.
Conclusion The basement membrane of neonatal rats was successfully cultured in vitro and DAPT could increase the number of basement membrane hair cells in vitro.
The second part: the effect of DAPT on the regulation of cochlear hair cell differentiation.
Objective to explore the mechanism of DAPT induced hair cell proliferation from Notch signaling pathway and microRNA two.
Methods The Notch pathway was detected by RT-PCR at P0, and the downstream gene was detected by qRT-PCR at P5 when the Notch pathway was blocked by DAPT.
Results After DAPT treatment, there were more hair cells in basement membrane of rats in the experimental group. RT-PCR confirmed the existence of Notch pathway on the first day of birth. QRT-PCR confirmed that after DAPT treatment, the expression of Hes1 and Hes5, which inhibited hair cell formation, was decreased, but the expression of Math1, which promoted hair cell formation, was increased. At P5, 132 kinds of MICR appeared in the experimental group and the control group. The expression of oRNA was up-regulated and 36 kinds of microRNAs were down-regulated; 168 kinds of microRNAs, accounting for 22% of the total detected amount, changed more than twice. The expression of microRNA 292, microRNA 124, microRNA 18a, microRNA 130b, microRNA 99a, microRNA 96 and microRNA 183 in the experimental group was higher than that in the control group.
Conclusion Notch pathway activity is still present in neonatal rats. When DAPT is added, the expression of Hes1 and Hes5, which inhibit hair cell formation, is decreased, and the expression of Math1, which promotes hair cell formation, is increased. The expression profiles of microRNA, including microRNA 292, microRNA 124, microRNA 18a, microRNA 130b and microRNA, are also changed after the increase of inner ear hair cells in experimental group. 99A, microRNA96 and microRNA183 may be associated with hair cell differentiation in inner ear of rats.
The third part: the expression of microRNA in the cochlea of newborn and adult rats.
Objective to investigate the expression profiles of microRNA in the inner ear basement membrane of neonatal rats and adult rats.
Methods MicroRNA was purified from basement membrane of neonatal rats and adult rats (4 rats each), and the differences between the two groups were analyzed by TaqMan microRNA Array method.
Results The microRNA expression profiles of newborn and adult rats were compared: (1) 284 kinds of microRNA were expressed in inner ear of newborn rats and 209 kinds of microRNA were expressed in inner ear of adult rats; (2) The overall expression trend of microRNA in newborn and adult rats was decreased; (3) The expression level of microRNA 183 family in newborn rats was higher than that in adult rats.
Conclusion The expression profiles of microRNA in rat inner ear are extensive and the expression patterns are different. The high expression of microRNA 183 family in rat basement membrane during neonatal period may be related to the regenerative potential of hair cells.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R764

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