【摘要】：傳統(tǒng)觀點認為，哺乳動物內(nèi)耳毛細胞為終端有絲分裂細胞，損傷后不可再生，這是限制感音神經(jīng)性耳聾治療的瓶頸。近年來，國內(nèi)外學者通過不斷探索，發(fā)現(xiàn)了幾種可以促使哺乳動物內(nèi)耳毛細胞再生的新方法：促毛細胞再生因子的導入，干細胞植入及內(nèi)耳相關microRNA因子的導入等。但這些技術仍處于實驗研究階段，關于毛細胞增多機制的闡明還待進一步探索。Notch通路是動物發(fā)育中重要的信號轉(zhuǎn)導途徑，參與了胚胎發(fā)育中多種細胞系定向分化的調(diào)控。最近研究認為，Notch信號通路調(diào)節(jié)的側(cè)抑制參與哺乳動物內(nèi)耳感覺祖細胞定向分化為毛細胞和支持細胞的過程。在Notch信號通路中，Notch受體的激活需要γ-分泌酶的水解。DAPT是一種實驗中使用的γ-分泌酶抑制劑，它可阻止Notch受體的活化，使Notch信號途徑中級聯(lián)反應效應被抑制，導致其下游轉(zhuǎn)錄因子Hes1和Hes5的表達減少，從而使Hes1和Hes5對Math1表達的抑制減弱，間接增加了Math1的表達�？梢�，，DAPT可能參與促內(nèi)耳毛細胞的發(fā)育過程。 MicroRNA是一種廣泛存在于真核系統(tǒng)中的內(nèi)源性非編碼小RNA，從基因表達到基因調(diào)控的整個過程中都具有重要作用。目前發(fā)現(xiàn)，microRNA183家族在內(nèi)耳毛細胞中高表達，且與毛細胞的分化和發(fā)育相關。microRNA主要通過其5’種子區(qū)域與靶向mRNA的3’非編碼區(qū)域結(jié)合，從而阻斷靶向mRNA的翻譯而發(fā)揮作用。了解microRNA在不同情況下的表達譜是研究其具體功能的前提。 毛細胞的分化受多種因素的控制，本文主要觀察DAPT對離體培養(yǎng)的大鼠耳蝸基底膜毛細胞分化的影響，并從Notch信號通路和microRNA表達水平兩個方面探究毛細胞分化機制，試圖從中找到對毛細胞增生具有促進/抑制作用的因素，用于成年鼠耳聾的治療，進而將來用于臨床，服務人類。 第一部分：DAPT對離體培養(yǎng)大鼠Corti’s器毛細胞再生的影響 目的觀察DAPT對離體培養(yǎng)新生大鼠基底膜毛細胞生長的影響。 方法取新生（P0）SD大鼠耳蝸基底膜進行體外培養(yǎng)。采用自身對照方式，實驗組基底膜加入含DAPT（終濃度5μM）的高糖培養(yǎng)基，對照組不加DAPT。培養(yǎng)5天(P5)后，用激光共聚焦顯微鏡觀察毛細胞的生長情況，計量單位長度基底膜內(nèi)毛細胞的數(shù)目，并用統(tǒng)計軟件進行分析。 結(jié)果實驗組基底膜毛細胞增多，對照組毛細胞數(shù)目基本不變，兩組比較差異具有統(tǒng)計學意義。 結(jié)論新生大鼠基底膜體外培養(yǎng)成功；通過形態(tài)學觀察和統(tǒng)計學分析發(fā)現(xiàn)，DAPT能夠使離體培養(yǎng)的基底膜毛細胞數(shù)目增多。 第二部分：DAPT對調(diào)控耳蝸毛細胞分化因素的影響 目的從Notch信號通路和microRNA兩方面探討DAPT導致毛細胞增多的機制。 方法P0時采用RT-PCR檢測Notch通路的存在與否；P5時采用qRT-PCR檢測當Notch通路被DAPT阻斷后下游基因的變化情況，同時對實驗組和對照組的microRNA表達譜進行對比分析。 結(jié)果經(jīng)DAPT處理后，實驗組大鼠基底膜出現(xiàn)毛細胞增多，RT-PCR證實了出生第一天時尚有Notch通路的存在，qRT-PCR證實DAPT處理后，抑制毛細胞生成的Hes1和Hes5表達降低，而促進毛細胞生成的Math1表達升高。P5時，實驗組和對照組相比，出現(xiàn)132種microRNA表達上調(diào)，36種microRNA表達下調(diào)；變化超過2倍的microRNA共有168種，占總檢測數(shù)量的22%。microRNA292、microRNA124、microRNA18a、microRNA130b、microRNA99a、microRNA96及microRNA183的表達量在實驗組高于對照組。 結(jié)論新生大鼠中Notch通路活性尚存，加入Notch通路阻斷劑DAPT后，抑制毛細胞生成的Hes1和Hes5表達降低，而促進毛細胞生成的Math1表達升高。實驗組內(nèi)耳毛細胞增多后，microRNA表達譜系也發(fā)生變化，microRNA292、microRNA124、microRNA18a、microRNA130b、microRNA99a、microRNA96及microRNA183可能與大鼠內(nèi)耳毛細胞分化相關。 第三部分：microRNA在新生和成年大鼠耳蝸中的表達 目的了解新生鼠和成年鼠內(nèi)耳基底膜中microRNA的表達譜。 方法取新生大鼠和成年大鼠基底膜（各4只），滅酶方法提純microRNA，采用TaqMan microRNAArray方法進行兩組microRNA的差異分析。 結(jié)果新生鼠和成年鼠microRNA表達譜的比較：（1）新生鼠內(nèi)耳有284種microRNA表達，成年鼠內(nèi)耳有209種microRNA表達；（2）新生組和成年組相比較，成年組microRNA的總體表達趨勢是下降的；（3）新生鼠microRNA183家族表達量高于成年鼠。 結(jié)論鼠類內(nèi)耳中microRNA的表達譜廣泛，并且其表達具有不同的時空模式。microRNA183家族在大鼠新生期基底膜中高表達，可能與此時毛細胞具有再生潛能有關。
[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.
【學位授予單位】：南京醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R764

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