Ins2细胞世系的追踪及相关表达研究

发布时间：2018-07-12 08:50

本文选题：Ins2-cre世系细胞 + Wnt/β-catenin信号　；参考：《重庆医科大学》2017年硕士论文

【摘要】：研究背景:糖尿病(Diabetes mellitus,DM)现已成为仅次于肿瘤和心血管的第三大致死率较高的疾病,是在成年人中颇为常见的一种慢性代谢性疾病。糖尿病以胰岛素缺乏为主要症状。而在口腔医学领域,糖尿病与口腔疾病已成为公认的互相影响的疾病之一。胰岛素因其与机体血糖水平的密切关系而受到了广泛的关注。胰岛素是由胰腺内的胰岛β细胞受内源性或外源性物质的刺激而分泌的一种蛋白质激素。胰岛素是人体内唯一降低血糖的激素,在生长发育中发挥着重要的作用。近年来,为了研究胰岛素相关基因在胰岛素合成及分泌中的作用,研究人员构建了包括Tg(Ins2-cre)25Mgn在内多种转基因小鼠,同时在研究中发现ins2-cre不仅在胰腺中有所表达,在其他与内分泌调节有关的组织中也有所表达,这些发现均表明,ins2-cre的表达不仅仅局限于胰腺的β细胞内,可能还存在于多种组织器官内。然而到目前为止,对胰岛素基因调控下的细胞世系在动物体内的整体分布仍然缺乏全面具体的研究。细胞世系追踪(cell lineage tracing)是一种对动物体内特定细胞进行追踪的技术,在在动物发育早期对细胞进行特殊标记,经过一段时间后,通过检测标记物来研究被标记细胞的迁徙或者改变的一种新兴的生物学技术。此技术具有简单直观的优点,可以在保证细胞存活的前提下对细胞进行鉴定和示踪,已成为现今研究细胞发育学的重要工具之一。利用Cre/Loxp系统标记细胞,使特定的细胞群体及其子代都携带有该报告蛋白,可以很好的研究特定细胞群体的迁徙分化。在本实验中,我们将利用Cre/Loxp位点特异性重组系统在小鼠体内追踪Ins2表达细胞的世系图谱,及利用该系统研究Wnt/β-catenin信号通路在Ins2细胞群体的迁移及命运决定过程的作用;同时探索这些细胞迁徙部位的胰岛素存在状态。目的:获得Ins2-cre世系细胞在小鼠体内的分布图谱,探索Wnt/β-catenin信号通路在Ins2-cre世系细胞迁徙和发育过程中的作用,检测Ins2-cre世系细胞迁徙部位的胰岛素存在状态,检测颌下腺在应激状态下胰岛素的存在及分泌状况。方法:1.利用Cre/loxp系统标记细胞并观察Ins2-cre世系细胞的迁徙位点及时间;2.通过分析β-catenin条件性敲除小鼠,研究Wnt/β-catenin信号通路在Ins2世系细胞迁徙和分化过程中的作用3.用免疫组化和免疫荧光等方法检测在Ins2-cre世系细胞内的胰岛素存在状态;4.建立小鼠高血糖应激状态的动物模型,运用免疫荧光和实时定量聚合酶链反应(Quantificational Real-time Polymerase Chain Reaction,q RT-PCR)等方法检测颌下腺在应激反应的条件下胰岛素的分泌状况。结果:1.成功观察到在Ins2-cre世系细胞在成年小鼠体内的迁徙部位:主要有胰腺,颌下腺,大脑;2.通过交配和筛选得到β-catenin功能完全缺失的小鼠。成功观察到在Ins2-cre世系细胞在β-catenin功能完全缺失的小鼠体内出现的起始时间:在小鼠体内可从胚胎第9天开始出现,直至成年;其体内的ins2-cre世系细胞的迁徙位点增加,主要有胰腺,颌下腺,嘴巴,上颚,舌体,牙齿及垂体。3.Ins2-cre世系细胞内存在有表达胰岛素阳性的细胞。4.成功建立高血糖应激状态的小鼠动物模型,检测到颌下腺在应激状态下有胰岛素存在。结论:Ins2世系细胞在小鼠体内可迁徙至多个器官组织,在这些组织中可能有胰岛素的存在;Wnt/β-catenin信号通路会减少Ins2世系细胞的迁徙位点;颌下腺在应激状态下可能会分泌胰岛素以维持机体的稳态。
[Abstract]:Background: Diabetes mellitus (DM) is now the third most fatal disease after tumor and cardiovascular disease. It is a common chronic metabolic disease among adults. Diabetes is the main symptom of insulin deficiency. In the field of oral medicine, diabetes and oral diseases have become recognized as one of the other diseases. Insulin is a protein hormone secreted by the pancreatic islet beta cells stimulated by endogenous or exogenous substances in the pancreas. Insulin is the only hormone in the human body to reduce blood sugar and plays an important role in the growth and development of the pancreas. In recent years, in order to study the role of insulin related genes in the synthesis and secretion of insulin, researchers have constructed a variety of transgenic mice, including Tg (Ins2-cre) 25Mgn. In the study, it was found that ins2-cre was not only expressed in the pancreas, but also expressed in other tissues related to internal secretion regulation. It has been shown that the expression of ins2-cre is not limited to the beta cells of the pancreas and may exist in a variety of tissues and organs. However, to date, the overall distribution of the cell lineage in the animals under the regulation of insulin gene is still lack of a comprehensive and specific study. The cell lineage tracing is a kind of animal body. A technique in which specific cells are traced in the early stages of animal development. After a period of time, a new biological technique is used to study the migration or change of labeled cells by detection of markers. This technique has a simple and intuitive advantage and can be used to ensure cell survival on the premise of cell survival. Identification and tracing have become one of the most important tools in the study of cell development today. Using Cre/Loxp system to mark cells so that a specific cell population and its progeny are carried with this reporter protein, it is very good to study the migration and differentiation of specific cell populations. In this experiment, we will use the Cre/Loxp specific recombination system in this experiment. The mice in vivo traced the lineage map of Ins2 expression cells, and used this system to study the role of Wnt/ beta -catenin signaling pathway in the migration of Ins2 cells and the fate determination process, and also to explore the status of insulin in the migratory parts of these cells. Objective: to obtain the distribution map of Ins2-cre lineage cells in mice and explore Wnt/ beta -ca. The role of tenin signaling pathway in the migration and development of Ins2-cre lineage cells, detecting the status of insulin in the migratory parts of the Ins2-cre lineage, detecting the existence and secretion of insulin in the stress state of the submandibular gland. Methods: 1. using the Cre/loxp system to mark the cells and observe the migration sites and time of the Ins2-cre lineage cells. 2. by analyzing beta -catenin conditional knockout mice, the role of Wnt/ beta -catenin signaling pathway in the migration and differentiation of Ins2 lineage cells was examined by immunohistochemical and immunofluorescence methods to detect the state of insulin in the Ins2-cre lineage cells; 4. the animal model of the stress state of hyperglycemia in mice was established and immunofluorescence was used. And real-time quantitative polymerase chain reaction (Quantificational Real-time Polymerase Chain Reaction, Q RT-PCR) and other methods to detect the secretion of insulin under the stress response of the submandibular gland. Results: 1. the migration sites of the Ins2-cre lineage cells in the adult mice were successfully observed: the main pancreas, submandibular gland, and the brain; 2. pass. Copulation and screening of mice with complete deletion of beta -catenin function. The onset time of the Ins2-cre lineage in mice with complete deletion of the beta -catenin function was observed. In mice, the mice could begin to appear from the ninth day of the embryo to adulthood; the migration sites of the ins2-cre lineage cells in the body increased, mainly with the pancreas, and the jaw. In the cells of the lower glands, the mouth, the jaws, the tongue, the teeth and the pituitary.3.Ins2-cre cells, there are insulin positive cells,.4., which successfully establish the mice model of hyperglycemic stress, and detect the presence of insulin in the stress state of the submandibular glands. Conclusion: the Ins2 lineage cells can migrate to many organ tissues in the mice. These Insulin may exist in the tissue; the Wnt/ beta -catenin signaling pathway reduces the migration site of the Ins2 lineage cells; the submandibular gland may secrete insulin to maintain the body's homeostasis under stress.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1;R78

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