本文選題：噪聲性聾 + E-粘鈣蛋白��； 參考：《中國人民解放軍醫(yī)學(xué)院》2014年博士論文

【摘要】：E-cadherin蛋白(CDH1)是一種鈣依賴性的上皮細(xì)胞間的粘附蛋白，在耳蝸感覺細(xì)胞和支持細(xì)胞中幾乎均有表達(dá)。它在Corti器的發(fā)育和結(jié)構(gòu)及功能的維持中有重要作用。前期研究表明噪聲暴露后E-cadherin在大鼠耳蝸感覺上皮組織中的表達(dá)量增加。但目前還不清楚噪聲暴露導(dǎo)致E-cadherin的增加是由Corti氏器中的哪種細(xì)胞引起的以及其表達(dá)量增加的作用是什么。因此，有必要對E-cadherin在噪聲損傷中的作用作進(jìn)一步深入研究。本研究綜合運用誘導(dǎo)性條件基因敲除，顯微分離，，激光共聚焦，免疫透射電鏡等細(xì)胞分子生物學(xué)技術(shù)，探索E-cadherin分子在急性噪聲損傷中的作用。實驗分為以下三個部分： 第一部分E-cadherin條件轉(zhuǎn)基因小鼠模型的建立 本部分實驗的目的是獲得僅在內(nèi)外毛細(xì)胞或外毛細(xì)胞中將E-cadherin基因敲除的條件轉(zhuǎn)基因敲除小鼠。通過應(yīng)用Cre/loxP技術(shù)，將B6.129-Cdh1tm2Kem/J小鼠與Tg(Atoh1-cre/Esr1*)14Fsh/J小鼠和Prestin-CreERT2小鼠進(jìn)行雜交配對和篩選。上述兩種Cre小鼠為誘導(dǎo)性的Cre系統(tǒng)，只有與Tamoxifen相結(jié)合時才能啟動Cre重組酶的活性。因此通過藥物誘導(dǎo)可以對E-cadherin基因的敲除實現(xiàn)時間和空間兩個方面的控制。本實驗中首先將配對后所得的兩種條件轉(zhuǎn)基因小鼠：Cdh1-loxP/Atoh1-cre小鼠和Cdh1-loxP/Prestin-CreERT2小鼠進(jìn)行基因分型，初步篩選獲得基因型Cdh1-loxP為純和表達(dá)的轉(zhuǎn)基因小鼠。同時進(jìn)一步通過免疫熒光組織染色法檢測所得小鼠在Tamoxifen誘導(dǎo)后Cre重組酶的表達(dá)活性，判斷小鼠的Cre/loxP重組酶是否工作正常。最后使用腦干誘發(fā)電位法對條件轉(zhuǎn)基因小鼠的聽力水平進(jìn)行檢測，為下一步噪聲暴露實驗提供聽閾水平的基線。實驗篩選得到了符合后續(xù)實驗要求的條件轉(zhuǎn)基因小鼠Cdh1-loxP (+/+) Prestin-CreERT2(+/-)，為后續(xù)研究噪聲暴露后E-cadherin在耳蝸內(nèi)的表達(dá)變化和結(jié)構(gòu)改變提供了動物模型。 第二部分高純度內(nèi)耳細(xì)胞顯微分離技術(shù)的建立 本部分實驗的目的是建立一種樣本中RNA保存完好的毛細(xì)胞富集組織(Haircell-enriched tissue)的提取方法。耳蝸病變分子生物學(xué)研究的成功依賴于高品質(zhì)耳蝸樣本的收集。由于耳蝸結(jié)構(gòu)的復(fù)雜性，從哺乳動物耳蝸中分離感覺細(xì)胞富集的樣本一直是一個挑戰(zhàn)。本研究采用顯微解剖技術(shù)分離感覺細(xì)胞富集的耳蝸組織樣本，使感覺細(xì)胞的純度顯著提高。同已前方法中獲得的感覺上皮組織相比，新方法制備的樣品中含有了一個相對穩(wěn)定感覺細(xì)胞/支持細(xì)胞的比例，并且樣品中的RNA保存完好。使用該顯微解剖方法，我們能夠收集小鼠耳蝸中分離的三種組織類型：感覺細(xì)胞富集組織，外毛細(xì)胞富集組織和內(nèi)毛細(xì)胞富集組織。所有樣本可以用于相關(guān)的下游分析，包括qRT-PCR，微陣列和RNA測序等。 第三部分E-cadherin在急性噪聲損傷中的分子機(jī)制 本部分實驗的目的是研究E-cadherin在急性噪聲損傷中圍繞外毛細(xì)胞（OHC）所發(fā)生的變化。分析該分子在外毛細(xì)胞富集組織中RNA轉(zhuǎn)錄水平的改變及在Corti氏器的位置結(jié)構(gòu)變化，初步理解E-cadherin在急性噪聲損傷中的分子機(jī)制。實驗利用條件轉(zhuǎn)基因小鼠模型研究外毛細(xì)胞在cdh1基因被敲除后E-cadherin在網(wǎng)狀層中的變化，使用外毛細(xì)胞富集組織分離方法開展相關(guān)細(xì)胞粘附分子qRT-PCR檢測，應(yīng)用3D重建和免疫透射電鏡技術(shù)觀察網(wǎng)狀層外毛細(xì)胞與Deiters細(xì)胞之間E-cadherin連接的位置結(jié)構(gòu)。結(jié)論如下：1.急性噪聲損傷后，外毛細(xì)胞周圍E-cadherin的表達(dá)增加是由Deiters細(xì)胞引起的；2. Deiters細(xì)胞中的E-cadherin通過指狀突連接網(wǎng)狀層；3.急性噪聲損傷后，Deiters細(xì)胞之間通過過量表達(dá)E-cadherin封閉由毛細(xì)胞凋亡后的空缺，維持網(wǎng)狀層的完整性。
[Abstract]:E-cadherin protein (CDH1) is a calcium dependent epithelial cell adhesion protein that is almost expressed in the cochlear sensory and supporting cells. It plays an important role in the development of Corti and the maintenance of its structure and function. The previous study showed that the expression of E-cadherin in the sensory epithelium of the cochlea of rats increased after exposure to noise. But it is not clear what the increase of E-cadherin caused by noise exposure is caused by which cell in the Corti's organ and the effect of its increase in expression. Therefore, it is necessary to further study the role of E-cadherin in noise damage. The role of E-cadherin molecules in acute noise injury was explored by confocal, immuno transmission electron microscopy and other cellular and molecular biological techniques. The experiment is divided into three parts:
Part one establishment of transgenic mice with conditional E-cadherin condition
The purpose of this part of the experiment is to obtain the conditional transgenic knockout mice that only knock out the E-cadherin gene in internal and external hair cells or outer hair cells. Through the application of Cre/loxP technology, the B6.129-Cdh1tm2Kem/J mice and Tg (Atoh1-cre/Esr1*) 14Fsh/J mice and Prestin-CreERT2 mice were hybridized and screened. The above two Cre mice were induced. The conductivity of the Cre system, only when combined with the Tamoxifen, can initiate the activity of the Cre recombinant enzyme. Therefore, the E-cadherin gene knockout can be controlled by two aspects of time and space by drug induction. In this experiment, the first two conditional transgenic mice obtained after pairing: Cdh1-loxP/Atoh1-cre mice and Cdh1-loxP/Prestin- CreERT2 mice were genotyping and screened to obtain genetically modified Cdh1-loxP as pure and expressed transgenic mice. At the same time, the expression of Cre recombinant enzyme in the mice induced by Tamoxifen was detected by immunofluorescence staining, and the Cre/loxP recombinant enzyme of the mice was normal. Finally, the brainstem evoked potential was used. The method was used to detect the hearing level of the conditional transgenic mice and provide the baseline of the hearing threshold level for the next step of the noise exposure experiment. The experimental screening obtained the Cdh1-loxP (+ / +) Prestin-CreERT2 (+ / -) of the conditional transgenic mice which met the requirements of the follow-up experiment. The expression changes and structural changes of E-cadherin in the cochlea after the subsequent study of the noise exposure were changed and the structure of the cochlea was changed. Changes provide animal models.
The second part is the establishment of high purity inner ear cell microdissection technology.
The purpose of this part of the experiment is to establish a method for the extraction of well preserved hair cell enriched tissue (Haircell-enriched tissue) in a sample. The success of the molecular biology of cochlear lesions depends on the collection of high quality cochlear samples. Because of the complexity of the cochlear structure, the samples from the mammalian cochlea are separated from the samples of the RNA. This study has been a challenge. This study uses microanatomy to separate the samples of the cochlear tissue enriched by sensory cells to make the purity of the sensory cells significantly improved. Compared with the sensory epithelial tissue obtained in the previous method, the sample prepared by the new method contains a relatively stable sensory cell / support cell ratio, and the RN in the sample. A is well preserved. Using this microdissection, we can collect three types of tissue isolated from the mouse cochlea: sensory cell enrichment tissue, outer hair cell enrichment tissue and inner hair cell enrichment tissue. All samples can be used for the related downstream analysis, including qRT-PCR, microarrays, and RNA sequencing.
The third part is about the molecular mechanism of E-cadherin in acute noise injury.
The purpose of this part is to study the changes of E-cadherin in acute noise injury around the outer hair cell (OHC). The change of RNA transcriptional level in the enrichment tissue of the outer hair cell and the change of the position and structure in the Corti's organ are analyzed. The molecular mechanism of E-cadherin in the acute noise damage is preliminarily understood. The transgenic mouse model studied the changes of E-cadherin in the reticular layer after the CDH1 gene was knocked out of the outer hair cells. The cell adhesion molecule qRT-PCR detection was carried out by the enrichment tissue separation method of outer hair cells. 3D reconstruction and transmission electron microscopy were used to observe the E-cadherin connection between the reticular outer capillary cells and the Deiters cells. The results are as follows: 1. after acute noise injury, the increase of the expression of E-cadherin around the outer hair cells is caused by Deiters cells; the E-cadherin in the 2. Deiters cells is connected to the reticular layer through the finger process; 3. after the acute noise injury, the Deiters cells are overexpressed by E-cadherin to seal the vacancy after the apoptosis of the hair cells. Hold the integrity of the reticulate layer.

【學(xué)位授予單位】：中國人民解放軍醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R764

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