E-cadherin在急性噪声损伤中的分子机制研究

发布时间：2018-05-01 03:24

本文选题：噪声性聋 + E-粘钙蛋白　；参考：《中国人民解放军医学院》2014年博士论文

【摘要】：E-cadherin蛋白(CDH1)是一种钙依赖性的上皮细胞间的粘附蛋白，在耳蜗感觉细胞和支持细胞中几乎均有表达。它在Corti器的发育和结构及功能的维持中有重要作用。前期研究表明噪声暴露后E-cadherin在大鼠耳蜗感觉上皮组织中的表达量增加。但目前还不清楚噪声暴露导致E-cadherin的增加是由Corti氏器中的哪种细胞引起的以及其表达量增加的作用是什么。因此，有必要对E-cadherin在噪声损伤中的作用作进一步深入研究。本研究综合运用诱导性条件基因敲除，显微分离，，激光共聚焦，免疫透射电镜等细胞分子生物学技术，探索E-cadherin分子在急性噪声损伤中的作用。实验分为以下三个部分：第一部分E-cadherin条件转基因小鼠模型的建立本部分实验的目的是获得仅在内外毛细胞或外毛细胞中将E-cadherin基因敲除的条件转基因敲除小鼠。通过应用Cre/loxP技术，将B6.129-Cdh1tm2Kem/J小鼠与Tg(Atoh1-cre/Esr1*)14Fsh/J小鼠和Prestin-CreERT2小鼠进行杂交配对和筛选。上述两种Cre小鼠为诱导性的Cre系统，只有与Tamoxifen相结合时才能启动Cre重组酶的活性。因此通过药物诱导可以对E-cadherin基因的敲除实现时间和空间两个方面的控制。本实验中首先将配对后所得的两种条件转基因小鼠：Cdh1-loxP/Atoh1-cre小鼠和Cdh1-loxP/Prestin-CreERT2小鼠进行基因分型，初步筛选获得基因型Cdh1-loxP为纯和表达的转基因小鼠。同时进一步通过免疫荧光组织染色法检测所得小鼠在Tamoxifen诱导后Cre重组酶的表达活性，判断小鼠的Cre/loxP重组酶是否工作正常。最后使用脑干诱发电位法对条件转基因小鼠的听力水平进行检测，为下一步噪声暴露实验提供听阈水平的基线。实验筛选得到了符合后续实验要求的条件转基因小鼠Cdh1-loxP (+/+) Prestin-CreERT2(+/-)，为后续研究噪声暴露后E-cadherin在耳蜗内的表达变化和结构改变提供了动物模型。第二部分高纯度内耳细胞显微分离技术的建立本部分实验的目的是建立一种样本中RNA保存完好的毛细胞富集组织(Haircell-enriched tissue)的提取方法。耳蜗病变分子生物学研究的成功依赖于高品质耳蜗样本的收集。由于耳蜗结构的复杂性，从哺乳动物耳蜗中分离感觉细胞富集的样本一直是一个挑战。本研究采用显微解剖技术分离感觉细胞富集的耳蜗组织样本，使感觉细胞的纯度显著提高。同已前方法中获得的感觉上皮组织相比，新方法制备的样品中含有了一个相对稳定感觉细胞/支持细胞的比例，并且样品中的RNA保存完好。使用该显微解剖方法，我们能够收集小鼠耳蜗中分离的三种组织类型：感觉细胞富集组织，外毛细胞富集组织和内毛细胞富集组织。所有样本可以用于相关的下游分析，包括qRT-PCR，微阵列和RNA测序等。第三部分E-cadherin在急性噪声损伤中的分子机制本部分实验的目的是研究E-cadherin在急性噪声损伤中围绕外毛细胞（OHC）所发生的变化。分析该分子在外毛细胞富集组织中RNA转录水平的改变及在Corti氏器的位置结构变化，初步理解E-cadherin在急性噪声损伤中的分子机制。实验利用条件转基因小鼠模型研究外毛细胞在cdh1基因被敲除后E-cadherin在网状层中的变化，使用外毛细胞富集组织分离方法开展相关细胞粘附分子qRT-PCR检测，应用3D重建和免疫透射电镜技术观察网状层外毛细胞与Deiters细胞之间E-cadherin连接的位置结构。结论如下：1.急性噪声损伤后，外毛细胞周围E-cadherin的表达增加是由Deiters细胞引起的；2. Deiters细胞中的E-cadherin通过指状突连接网状层；3.急性噪声损伤后，Deiters细胞之间通过过量表达E-cadherin封闭由毛细胞凋亡后的空缺，维持网状层的完整性。
[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.

【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R764

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