Tbx1基因在小鼠胚肾中的表达规律及其相互作用蛋白研究
发布时间：2018-11-26 08:34
【摘要】： 前言 TBX1 (GeneID:6899)基因定位于染色体22q11.21,胞内定位于细胞核,全称为T-box1,属于T-box转录因子家族,后者在种系发生和进化中趋于保守,因其成员均包含一个相同的DNA结合域,即T-box而得名。近期研究发现,TBX1基因可影响包括耳、心脏、甲状旁腺、胸腺、牙齿等多种组织和器官的发育,并在精神疾病的发病中扮演一定角色。 多项研究提示泌尿生殖系统发育缺陷与染色体22q11.2微缺失密切相关,具体的畸形可涉及肾脏、输尿管／膀胱以及尿道等,畸形发生率高达36%,显著高于自然人群。TBX1基因是定位于上述缺失关键区内的关键基因。围绕TBX1基因的研究提示,TBX1基因可能通过剂量依赖形式,影响SHH、FGF、WNT以及BMP等相关信号通路,进而调控包括肾脏、心脏、耳、胸腺、甲状旁腺在内的多种组织器官的发育。动物实验亦证实,Tbx1基因缺失或单倍剂量不足可导致类似于人类22q11.2微缺失综合征的表型。 迄今为止,围绕TBX1基因的研究主要集中于其与心脏畸形的关系,而该基因是否与泌尿生殖系统畸形的发生存在联系尚未见报道。在本研究中,我们以小鼠为对象,对Tbx1基因在肾脏发育过程中的表达进行了检测,之后,通过获取其相互作用蛋白和RNA干扰实验,对该蛋白以及其相互作用蛋白在发育中的角色进行进一步探索,以期初步揭示调控肾脏发育的分子网络。 材料与方法 一、实验材料 1、健康昆明小鼠 2、Western Blot相关试剂 3、RT-PCR实验相关试剂 4、免疫沉淀实验相关试剂 5、细胞培养及转染相关试剂 二、实验方法 1、取胚龄为E13.5d、E15.5d、E17.5d、E19.5d,新生鼠以及正常成年昆明小鼠的肾脏组织,抽提RNA,分析Tbx1基因在小鼠不同发育时间点的表达情况。 2、免疫沉淀获取Tbx1的相互作用蛋白并进行质谱分析,通过Western Blot对结果进行验证,半定量RT-PCR分析该基因的时间表达情况。 3、干扰Tbx1及其相互作用蛋白的表达以确认二者的相互影响。 实验结果 1、Tbx1基因的表达在胚鼠肾脏发育的第E15.5天达到第一个峰值,随后逐渐减弱,至出生时又剧烈增高,达到第二峰值。此外,该基因在小鼠成体肾脏中存在低水平表达。 2、在小鼠的胚肾组织中,Hoxd10与Tbx1存在蛋白之间的相互作用。 3、在小鼠成肌细胞系C2Cl2中,干扰Hoxd10可以使该基因及Tbx1的表达量下调；干扰Tbx1则可以使该基因及Hoxd10的表达下调。 结论 1、Tbx1基因的表达水平随小鼠肾脏的发育而剧烈变化,提示其可能在肾脏的发育过程中扮演重要角色,并可能在成体肾脏中发挥某种功能。 2、在小鼠的胚肾组织中,Tbx1蛋白可能与Hoxd10蛋白存在相互作用,共同调节肾脏的发育。 3、在蛋白水平,Tbx1与Hoxd10之间可能存在调节和反馈作用。
[Abstract]:The TBX1 (GeneID:6899) gene is located on chromosome 22q11.21, and is located in the nucleus, which is called T-box1. It belongs to the family of T-box transcription factors, which tends to be conserved in the genesis and evolution of the phylogeny. It is named because its members contain the same DNA associative domain, that is, T-box. Recent studies have found that TBX1 gene can affect the development of various tissues and organs, including ear, heart, parathyroid gland, thymus, teeth and so on, and play a role in the pathogenesis of mental diseases. Several studies have shown that the developmental defects of the genitourinary system are closely related to the microdeletion of chromosome 22q11.2. Specific deformities may involve kidney, ureter / bladder and urethra, and the incidence of deformities is as high as 36%. TBX1 gene is the key gene located in the key region of the above deletion. Studies on TBX1 gene suggest that the TBX1 gene may influence the development of various tissues and organs including kidney, heart, ear, thymus and parathyroid gland through dose-dependent forms, such as SHH,FGF,WNT and BMP signaling pathways. Animal experiments have also demonstrated that Tbx1 gene deletion or insufficient dose can lead to phenotypes similar to human 22q11.2 microdeletion syndrome. Up to now, the study of TBX1 gene is mainly focused on its relationship with cardiac malformation, and whether the gene is related to the occurrence of genitourinary malformation has not been reported. In this study, we detected the expression of Tbx1 gene during kidney development in mice, and then obtained its interaction protein and RNA interference. The role of the protein and its interacting protein in the development of kidney was further explored in order to reveal the molecular network of regulating renal development. Materials and methods: 1. Materials 1, 2 Western Blot related reagents of healthy Kunming mice, 4 of RT-PCR related reagents, 5 of immunoprecipitation related reagents. Cell culture and transfection reagent 2. Method 1. RNA, was extracted from the kidney tissues of newborn mice and normal Kunming mice at the age of E13.5d, E15.5dN, E17.5d, E19.5d. The expression of Tbx1 gene at different developmental time points in mice was analyzed. 2. The interaction protein of Tbx1 was obtained by immunoprecipitation and analyzed by mass spectrometry. The results were verified by Western Blot, and the time expression of the gene was analyzed by semi-quantitative RT-PCR. 3. Interfering with the expression of Tbx1 and its interacting proteins to confirm their interaction. The results showed that the expression of Tbx1 gene reached the first peak on the day 15.5 of embryonic mouse kidney development, then decreased gradually, and then increased sharply to the second peak at birth. In addition, there is a low level of expression of the gene in mouse adult kidney. 2, in mouse embryonic kidney tissue, Hoxd10 and Tbx1 exist protein interaction. 3. In murine myoblast cell line C2Cl2, interfering with Hoxd10 could down-regulate the expression of the gene and Tbx1, while interfering with Tbx1 could down-regulate the expression of the gene and Hoxd10. Conclusion 1 the expression level of Tbx1 gene changes dramatically with the development of mouse kidney, suggesting that Tbx1 gene may play an important role in the development of kidney and may play a certain function in adult kidney. 2. In mouse embryonic kidney, Tbx1 protein may interact with Hoxd10 protein to regulate kidney development. 3. At the protein level, there may be regulation and feedback between Tbx1 and Hoxd10.
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363
[Abstract]:The TBX1 (GeneID:6899) gene is located on chromosome 22q11.21, and is located in the nucleus, which is called T-box1. It belongs to the family of T-box transcription factors, which tends to be conserved in the genesis and evolution of the phylogeny. It is named because its members contain the same DNA associative domain, that is, T-box. Recent studies have found that TBX1 gene can affect the development of various tissues and organs, including ear, heart, parathyroid gland, thymus, teeth and so on, and play a role in the pathogenesis of mental diseases. Several studies have shown that the developmental defects of the genitourinary system are closely related to the microdeletion of chromosome 22q11.2. Specific deformities may involve kidney, ureter / bladder and urethra, and the incidence of deformities is as high as 36%. TBX1 gene is the key gene located in the key region of the above deletion. Studies on TBX1 gene suggest that the TBX1 gene may influence the development of various tissues and organs including kidney, heart, ear, thymus and parathyroid gland through dose-dependent forms, such as SHH,FGF,WNT and BMP signaling pathways. Animal experiments have also demonstrated that Tbx1 gene deletion or insufficient dose can lead to phenotypes similar to human 22q11.2 microdeletion syndrome. Up to now, the study of TBX1 gene is mainly focused on its relationship with cardiac malformation, and whether the gene is related to the occurrence of genitourinary malformation has not been reported. In this study, we detected the expression of Tbx1 gene during kidney development in mice, and then obtained its interaction protein and RNA interference. The role of the protein and its interacting protein in the development of kidney was further explored in order to reveal the molecular network of regulating renal development. Materials and methods: 1. Materials 1, 2 Western Blot related reagents of healthy Kunming mice, 4 of RT-PCR related reagents, 5 of immunoprecipitation related reagents. Cell culture and transfection reagent 2. Method 1. RNA, was extracted from the kidney tissues of newborn mice and normal Kunming mice at the age of E13.5d, E15.5dN, E17.5d, E19.5d. The expression of Tbx1 gene at different developmental time points in mice was analyzed. 2. The interaction protein of Tbx1 was obtained by immunoprecipitation and analyzed by mass spectrometry. The results were verified by Western Blot, and the time expression of the gene was analyzed by semi-quantitative RT-PCR. 3. Interfering with the expression of Tbx1 and its interacting proteins to confirm their interaction. The results showed that the expression of Tbx1 gene reached the first peak on the day 15.5 of embryonic mouse kidney development, then decreased gradually, and then increased sharply to the second peak at birth. In addition, there is a low level of expression of the gene in mouse adult kidney. 2, in mouse embryonic kidney tissue, Hoxd10 and Tbx1 exist protein interaction. 3. In murine myoblast cell line C2Cl2, interfering with Hoxd10 could down-regulate the expression of the gene and Tbx1, while interfering with Tbx1 could down-regulate the expression of the gene and Hoxd10. Conclusion 1 the expression level of Tbx1 gene changes dramatically with the development of mouse kidney, suggesting that Tbx1 gene may play an important role in the development of kidney and may play a certain function in adult kidney. 2. In mouse embryonic kidney, Tbx1 protein may interact with Hoxd10 protein to regulate kidney development. 3. At the protein level, there may be regulation and feedback between Tbx1 and Hoxd10.
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363
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