人毛乳頭細胞CCDC72基因的表達及啟動子活性分析
發(fā)布時間：2018-12-08 15:03
【摘要】： 毛囊是毛發(fā)的主體,是一個典型的再生系統(tǒng),從胚胎期發(fā)生后就伴隨個體一生并周而復始地循環(huán)。毛發(fā)具有許多有益的生物學功能和重要的社會心理學作用,因毛發(fā)生長異常而發(fā)生的少毛或多毛現(xiàn)象不僅影響毛發(fā)生理功能的正常發(fā)揮,而且使患者承受極大的心理壓力,影響他們的健康和生活質(zhì)量。因此,弄清毛發(fā)胚胎期發(fā)生、生長周期調(diào)控機制及毛囊生長期基因的表達變化,開發(fā)有效治療毛發(fā)疾病的藥物是目前毛囊生物學研究的熱點。 位于毛囊基底部的毛乳頭是一個可誘導毛囊生長的結構,它能發(fā)送或接受信號,從而影響毛囊的發(fā)育和周期性生長。毛乳頭細胞是組成毛乳頭的唯一細胞,是胚胎期誘導毛囊形成和成熟毛囊完成周期性循環(huán)的主要調(diào)節(jié)細胞。凝集性生長是毛乳頭細胞的重要生物學特性之一,凝集性生長與毛乳頭細胞的功能和分化狀態(tài)有關。但是毛乳頭細胞凝集性生長的分子機制不明,同時凝集性生長狀態(tài)下毛乳頭細胞所表達的基因在毛囊發(fā)育和毛囊周期性生長調(diào)控中的作用也少有研究。 凝集性生長狀態(tài)的DPC仍然保持誘導毛囊發(fā)生的能力。為了探索哪些基因在DPC凝集性生長狀態(tài)下表達,篩選與DPC凝集生長相關的基因,宋志強等通過抑制性消減雜交技術建立了人類DPC消減cDNA文庫,并從凝集生長狀態(tài)的DPC篩選出一批表達上調(diào)和表達下調(diào)基因。CCDC72即是通過上述方法篩選出的與DPC凝集性生長相關基因,它首次從CD34+造血干細胞/祖細胞( CD34+ hematopoietic stem/progenitor cells,HSPC)中被分離鑒定,但功能不清。王繼文等通過RNA干擾技術使CCDC72基因在DPC內(nèi)的表達下降或消失,結果顯示CCDC72表達陰性后的DPC生長速度明顯減慢并失去凝集性生長特性,細胞形態(tài)也發(fā)生了顯著的改變。夏汝山等發(fā)現(xiàn)CCDC72重組蛋白能促進高傳代DPC的增殖及DNA的合成。這些結果表明CCDC72對調(diào)節(jié)DPC生長及其分化至關重要,是毛囊生長期維持DPC處于功能狀態(tài)的重要基因之一�；谝陨险J識,本課題擬對CCDC72基因在DPC中的基因表達、蛋白定位和啟動子活性進行實驗研究。 1、兩步酶消化法是一種快速、簡單、經(jīng)濟的分離培養(yǎng)毛乳頭細胞的方法。我們首先從人頭皮毛囊分離培養(yǎng)人毛乳頭細胞,這些細胞具有周期性凝集性生長特性,而且這種特性隨著傳代次數(shù)的增多而逐步喪失。 2、利用細胞免疫熒光和還原型線粒體特異性熒光探針MitoTracker Red CM-H2X Ros,結合激光共聚焦顯微鏡技術發(fā)現(xiàn)CCDC72蛋白位于胞漿內(nèi),可能定位于線粒體上。通過熒光半定量Real-time PCR發(fā)現(xiàn)隨著DPCs傳代數(shù)的增加,CCDC72基因mRNA的表達呈下降趨勢,1～3代DPCs中CCDC72mRNA的表達穩(wěn)定且顯著高于4～8代,4～6代DPCs中CCDC72mRNA的表達又顯著高于第8代。 3、運用在線軟件Cister對CCDC72基因5’端2000bp進行啟動子特征分析表明其5’側翼區(qū)的主要特點是在-200～-1bp及-1000～-600bp兩段區(qū)域間順式作用元件呈高頻分布。根據(jù)上述結果從5’端逐步缺失,成功構建4個5’端不等,3’端平齊的啟動子熒光素酶報告基因載體,分別轉(zhuǎn)染第3代和第8代的DPCs中,采用雙熒光素酶報告系統(tǒng)對熒光素酶表達量進行檢測。結果顯示:在第3代DPCs中,這四個不同長度啟動子重組pGL3-Basic載體熒光素酶活性較pGL3-Basic空載體明顯增強,pGL3-B-604的熒光素酶活性最強。在第8代DPCs中,4個重組pGL3-Basic載體熒光素酶活性均較第3代DPCs的明顯降低,pGL3-B-1924熒光素酶活性與pGL3-Basic空載體無差異,pGL3-B-204、pGL3-B-604、pGL3-B-1004均顯著高于pGL3-Basic空載體,pGL3-B-204的熒光素酶活性最強,可能是最小核心啟動子所在區(qū)域,維持了基礎轉(zhuǎn)錄活性。在第3代DPCs中,pGL3-B-604的熒光素酶活性最強,而在第8代DPCs中,pGL3-B-604的熒光素酶活性明顯降低,提示DPCs在傳代過程中,可能是通過-600～-200bp之間的調(diào)控元件發(fā)揮轉(zhuǎn)錄調(diào)控的作用。
[Abstract]:The hair follicle is the main body of the hair. It is a typical regeneration system, which is followed by the individual's life and cycle back and forth from the embryo stage. The hair has a plurality of beneficial biological functions and important social psychology functions, and the phenomenon that the hair growth is abnormal can not only affect the normal play of the physiological function of the hair, but also cause the patient to bear great psychological pressure and influence the health and the quality of life of the hair. Therefore, it is the focus of the present study on the biological research of the hair follicle in order to find out the changes of the development of the hair, the regulation mechanism of the growth cycle and the expression of the gene in the growth phase of the hair follicle. The hair papilla located at the bottom of the hair follicle is a structure that can induce the growth of the hair follicle, which can transmit or receive signals, thereby affecting the development and cycle of the hair follicle. Sex growth. The hair papilla cells are the only cells that make up the dermal papilla. It is the main modulation of the periodic cycle of the induction of the formation of the hair follicle and the maturation of the mature hair follicle during the embryo period. The agglutinative growth is one of the important biological characteristics of the dermal papilla cells, the function and the differentiation of the agglutinative growth and the dermal papilla cells. However, the molecular mechanism of the agglutinative growth of the papilla cells is unknown, and the expression of the hair papilla cells in the agglutinative growth state has little effect on the development of the hair follicle and the regulation of the periodic growth of the hair follicle. There was a study. The DPC of the agglutinative growth state was still induced In order to explore which genes were expressed in DPC agglutinative growth state, to screen the gene related to DPC agglutination growth, Song Zhiqiang and so on, the human DPC reduced cDNA library was established by suppression subtractive hybridization, and a batch of expression up-regulation was selected from DPC in the aggregated growth state. and an expression down-regulation gene. The CCDC72 is a DPC agglutinative growth-related gene selected by the method, and is first separated from the CD34 + hematopoietic stem cell/ progenitor cell (CD34 + haematopinitic stem/ promotor cells, HSPC) for the first time. The expression of CCD72 gene in DPC decreased or disappeared by RNA interference technique, and the results showed that the growth rate of DPC after the expression of CCD72 was significantly reduced and the agglutinative growth was lost, and the cell morphology was also increased. Significant changes have been made. CCDC72 recombinant protein has been found to promote the proliferation of high-passaged DPC by CCDC72 recombinant protein. The results show that CCD72 is critical to the regulation of DPC growth and its differentiation, and it is the function of DPC in the growth period of the hair follicle. Based on the above recognition, the gene expression, protein localization and promoter activity of CCDC72 gene in DPC are proposed. The two-step enzymatic digestion method is a rapid, simple and economical method. The method of culturing the dermal papilla cells. We first isolate the cultured human dermal papilla cells from the human scalp hair follicle, these cells have periodic agglutinative growth characteristics, and this characteristic will follow the transmission The CDC72 protein was found by using the fluorescence and reduced mitochondrial-specific fluorescence probe Mitogen Red CM-H2X Ros in combination with the laser confocal microscope technique. The expression of CCDC72 mRNA in CCDC72 was stable and significantly higher than that of CCDC72 in 4 ~ 6 passages. The expression of the mRNA was significantly higher than that of the 8th generation. The analysis of the promoter characteristics of the 5 'end 2000bp of the CCDC72 gene by the on-line software Cister showed that the main characteristics of the 5' flanking region were from -200 to -1bp and -1000 to -60. The two-stage cis-acting element in the 0bp region is distributed in high frequency. According to the above results, the 5 'end is gradually deleted, and four 5'-terminal unequal and 3 '-end-level promoter luciferase reporter gene vectors are successfully constructed, in that third generation, the luciferase activity of the recombinant pGL3-basic vector of the four different length promoter is obviously enhanced than that of the pGL3-basic empty vector, The luciferase activity of pGL3-B-604 is the strongest. In the 8th generation, the luciferase activity of the four recombinant pGL3-Basic vectors is significantly lower than that of the third generation, and the luciferase activity of pGL3-B-1924 is not different from the pGL3-Basic empty vector, and pGL3-B-204, pGL3-B-604 and pGL3-B-1004 are all significantly higher than that of the pGL3-Basic empty vector, and the luciferase of the pGL3-B-204 The most active, probably the least In the region of the core promoter, the basal transcriptional activity was maintained. In the 3rd generation, the luciferase activity of pGL3-B-604 was the strongest, while in the 8th generation, the luciferase activity of pGL3-B-604 was significantly reduced, suggesting that in the passage, the luciferase activity of pGL3-B-604 may be through -600 ~ -20.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R346
本文編號：2368550
[Abstract]:The hair follicle is the main body of the hair. It is a typical regeneration system, which is followed by the individual's life and cycle back and forth from the embryo stage. The hair has a plurality of beneficial biological functions and important social psychology functions, and the phenomenon that the hair growth is abnormal can not only affect the normal play of the physiological function of the hair, but also cause the patient to bear great psychological pressure and influence the health and the quality of life of the hair. Therefore, it is the focus of the present study on the biological research of the hair follicle in order to find out the changes of the development of the hair, the regulation mechanism of the growth cycle and the expression of the gene in the growth phase of the hair follicle. The hair papilla located at the bottom of the hair follicle is a structure that can induce the growth of the hair follicle, which can transmit or receive signals, thereby affecting the development and cycle of the hair follicle. Sex growth. The hair papilla cells are the only cells that make up the dermal papilla. It is the main modulation of the periodic cycle of the induction of the formation of the hair follicle and the maturation of the mature hair follicle during the embryo period. The agglutinative growth is one of the important biological characteristics of the dermal papilla cells, the function and the differentiation of the agglutinative growth and the dermal papilla cells. However, the molecular mechanism of the agglutinative growth of the papilla cells is unknown, and the expression of the hair papilla cells in the agglutinative growth state has little effect on the development of the hair follicle and the regulation of the periodic growth of the hair follicle. There was a study. The DPC of the agglutinative growth state was still induced In order to explore which genes were expressed in DPC agglutinative growth state, to screen the gene related to DPC agglutination growth, Song Zhiqiang and so on, the human DPC reduced cDNA library was established by suppression subtractive hybridization, and a batch of expression up-regulation was selected from DPC in the aggregated growth state. and an expression down-regulation gene. The CCDC72 is a DPC agglutinative growth-related gene selected by the method, and is first separated from the CD34 + hematopoietic stem cell/ progenitor cell (CD34 + haematopinitic stem/ promotor cells, HSPC) for the first time. The expression of CCD72 gene in DPC decreased or disappeared by RNA interference technique, and the results showed that the growth rate of DPC after the expression of CCD72 was significantly reduced and the agglutinative growth was lost, and the cell morphology was also increased. Significant changes have been made. CCDC72 recombinant protein has been found to promote the proliferation of high-passaged DPC by CCDC72 recombinant protein. The results show that CCD72 is critical to the regulation of DPC growth and its differentiation, and it is the function of DPC in the growth period of the hair follicle. Based on the above recognition, the gene expression, protein localization and promoter activity of CCDC72 gene in DPC are proposed. The two-step enzymatic digestion method is a rapid, simple and economical method. The method of culturing the dermal papilla cells. We first isolate the cultured human dermal papilla cells from the human scalp hair follicle, these cells have periodic agglutinative growth characteristics, and this characteristic will follow the transmission The CDC72 protein was found by using the fluorescence and reduced mitochondrial-specific fluorescence probe Mitogen Red CM-H2X Ros in combination with the laser confocal microscope technique. The expression of CCDC72 mRNA in CCDC72 was stable and significantly higher than that of CCDC72 in 4 ~ 6 passages. The expression of the mRNA was significantly higher than that of the 8th generation. The analysis of the promoter characteristics of the 5 'end 2000bp of the CCDC72 gene by the on-line software Cister showed that the main characteristics of the 5' flanking region were from -200 to -1bp and -1000 to -60. The two-stage cis-acting element in the 0bp region is distributed in high frequency. According to the above results, the 5 'end is gradually deleted, and four 5'-terminal unequal and 3 '-end-level promoter luciferase reporter gene vectors are successfully constructed, in that third generation, the luciferase activity of the recombinant pGL3-basic vector of the four different length promoter is obviously enhanced than that of the pGL3-basic empty vector, The luciferase activity of pGL3-B-604 is the strongest. In the 8th generation, the luciferase activity of the four recombinant pGL3-Basic vectors is significantly lower than that of the third generation, and the luciferase activity of pGL3-B-1924 is not different from the pGL3-Basic empty vector, and pGL3-B-204, pGL3-B-604 and pGL3-B-1004 are all significantly higher than that of the pGL3-Basic empty vector, and the luciferase of the pGL3-B-204 The most active, probably the least In the region of the core promoter, the basal transcriptional activity was maintained. In the 3rd generation, the luciferase activity of pGL3-B-604 was the strongest, while in the 8th generation, the luciferase activity of pGL3-B-604 was significantly reduced, suggesting that in the passage, the luciferase activity of pGL3-B-604 may be through -600 ~ -20.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R346
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