Tbx1基因在小鼠胚腎中的表達規(guī)律及其相互作用蛋白研究
發(fā)布時間：2018-11-26 08:34
【摘要】： 前言 TBX1 (GeneID:6899)基因定位于染色體22q11.21,胞內定位于細胞核,全稱為T-box1,屬于T-box轉錄因子家族,后者在種系發(fā)生和進化中趨于保守,因其成員均包含一個相同的DNA結合域,即T-box而得名。近期研究發(fā)現(xiàn),TBX1基因可影響包括耳、心臟、甲狀旁腺、胸腺、牙齒等多種組織和器官的發(fā)育,并在精神疾病的發(fā)病中扮演一定角色。 多項研究提示泌尿生殖系統(tǒng)發(fā)育缺陷與染色體22q11.2微缺失密切相關,具體的畸形可涉及腎臟、輸尿管／膀胱以及尿道等,畸形發(fā)生率高達36%,顯著高于自然人群。TBX1基因是定位于上述缺失關鍵區(qū)內的關鍵基因。圍繞TBX1基因的研究提示,TBX1基因可能通過劑量依賴形式,影響SHH、FGF、WNT以及BMP等相關信號通路,進而調控包括腎臟、心臟、耳、胸腺、甲狀旁腺在內的多種組織器官的發(fā)育。動物實驗亦證實,Tbx1基因缺失或單倍劑量不足可導致類似于人類22q11.2微缺失綜合征的表型。 迄今為止,圍繞TBX1基因的研究主要集中于其與心臟畸形的關系,而該基因是否與泌尿生殖系統(tǒng)畸形的發(fā)生存在聯(lián)系尚未見報道。在本研究中,我們以小鼠為對象,對Tbx1基因在腎臟發(fā)育過程中的表達進行了檢測,之后,通過獲取其相互作用蛋白和RNA干擾實驗,對該蛋白以及其相互作用蛋白在發(fā)育中的角色進行進一步探索,以期初步揭示調控腎臟發(fā)育的分子網(wǎng)絡。 材料與方法 一、實驗材料 1、健康昆明小鼠 2、Western Blot相關試劑 3、RT-PCR實驗相關試劑 4、免疫沉淀實驗相關試劑 5、細胞培養(yǎng)及轉染相關試劑 二、實驗方法 1、取胚齡為E13.5d、E15.5d、E17.5d、E19.5d,新生鼠以及正常成年昆明小鼠的腎臟組織,抽提RNA,分析Tbx1基因在小鼠不同發(fā)育時間點的表達情況。 2、免疫沉淀獲取Tbx1的相互作用蛋白并進行質譜分析,通過Western Blot對結果進行驗證,半定量RT-PCR分析該基因的時間表達情況。 3、干擾Tbx1及其相互作用蛋白的表達以確認二者的相互影響。 實驗結果 1、Tbx1基因的表達在胚鼠腎臟發(fā)育的第E15.5天達到第一個峰值,隨后逐漸減弱,至出生時又劇烈增高,達到第二峰值。此外,該基因在小鼠成體腎臟中存在低水平表達。 2、在小鼠的胚腎組織中,Hoxd10與Tbx1存在蛋白之間的相互作用。 3、在小鼠成肌細胞系C2Cl2中,干擾Hoxd10可以使該基因及Tbx1的表達量下調；干擾Tbx1則可以使該基因及Hoxd10的表達下調。 結論 1、Tbx1基因的表達水平隨小鼠腎臟的發(fā)育而劇烈變化,提示其可能在腎臟的發(fā)育過程中扮演重要角色,并可能在成體腎臟中發(fā)揮某種功能。 2、在小鼠的胚腎組織中,Tbx1蛋白可能與Hoxd10蛋白存在相互作用,共同調節(jié)腎臟的發(fā)育。 3、在蛋白水平,Tbx1與Hoxd10之間可能存在調節(jié)和反饋作用。
[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.
【學位授予單位】：中國醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：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.
【學位授予單位】：中國醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R363
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