人毛乳头细胞CCDC72基因的表达及启动子活性分析
发布时间：2018-12-08 15:03
【摘要】： 毛囊是毛发的主体,是一个典型的再生系统,从胚胎期发生后就伴随个体一生并周而复始地循环。毛发具有许多有益的生物学功能和重要的社会心理学作用,因毛发生长异常而发生的少毛或多毛现象不仅影响毛发生理功能的正常发挥,而且使患者承受极大的心理压力,影响他们的健康和生活质量。因此,弄清毛发胚胎期发生、生长周期调控机制及毛囊生长期基因的表达变化,开发有效治疗毛发疾病的药物是目前毛囊生物学研究的热点。 位于毛囊基底部的毛乳头是一个可诱导毛囊生长的结构,它能发送或接受信号,从而影响毛囊的发育和周期性生长。毛乳头细胞是组成毛乳头的唯一细胞,是胚胎期诱导毛囊形成和成熟毛囊完成周期性循环的主要调节细胞。凝集性生长是毛乳头细胞的重要生物学特性之一,凝集性生长与毛乳头细胞的功能和分化状态有关。但是毛乳头细胞凝集性生长的分子机制不明,同时凝集性生长状态下毛乳头细胞所表达的基因在毛囊发育和毛囊周期性生长调控中的作用也少有研究。 凝集性生长状态的DPC仍然保持诱导毛囊发生的能力。为了探索哪些基因在DPC凝集性生长状态下表达,筛选与DPC凝集生长相关的基因,宋志强等通过抑制性消减杂交技术建立了人类DPC消减cDNA文库,并从凝集生长状态的DPC筛选出一批表达上调和表达下调基因。CCDC72即是通过上述方法筛选出的与DPC凝集性生长相关基因,它首次从CD34+造血干细胞/祖细胞( CD34+ hematopoietic stem/progenitor cells,HSPC)中被分离鉴定,但功能不清。王继文等通过RNA干扰技术使CCDC72基因在DPC内的表达下降或消失,结果显示CCDC72表达阴性后的DPC生长速度明显减慢并失去凝集性生长特性,细胞形态也发生了显著的改变。夏汝山等发现CCDC72重组蛋白能促进高传代DPC的增殖及DNA的合成。这些结果表明CCDC72对调节DPC生长及其分化至关重要,是毛囊生长期维持DPC处于功能状态的重要基因之一。基于以上认识,本课题拟对CCDC72基因在DPC中的基因表达、蛋白定位和启动子活性进行实验研究。 1、两步酶消化法是一种快速、简单、经济的分离培养毛乳头细胞的方法。我们首先从人头皮毛囊分离培养人毛乳头细胞,这些细胞具有周期性凝集性生长特性,而且这种特性随着传代次数的增多而逐步丧失。 2、利用细胞免疫荧光和还原型线粒体特异性荧光探针MitoTracker Red CM-H2X Ros,结合激光共聚焦显微镜技术发现CCDC72蛋白位于胞浆内,可能定位于线粒体上。通过荧光半定量Real-time PCR发现随着DPCs传代数的增加,CCDC72基因mRNA的表达呈下降趋势,1～3代DPCs中CCDC72mRNA的表达稳定且显著高于4～8代,4～6代DPCs中CCDC72mRNA的表达又显著高于第8代。 3、运用在线软件Cister对CCDC72基因5’端2000bp进行启动子特征分析表明其5’侧翼区的主要特点是在-200～-1bp及-1000～-600bp两段区域间顺式作用元件呈高频分布。根据上述结果从5’端逐步缺失,成功构建4个5’端不等,3’端平齐的启动子荧光素酶报告基因载体,分别转染第3代和第8代的DPCs中,采用双荧光素酶报告系统对荧光素酶表达量进行检测。结果显示:在第3代DPCs中,这四个不同长度启动子重组pGL3-Basic载体荧光素酶活性较pGL3-Basic空载体明显增强,pGL3-B-604的荧光素酶活性最强。在第8代DPCs中,4个重组pGL3-Basic载体荧光素酶活性均较第3代DPCs的明显降低,pGL3-B-1924荧光素酶活性与pGL3-Basic空载体无差异,pGL3-B-204、pGL3-B-604、pGL3-B-1004均显著高于pGL3-Basic空载体,pGL3-B-204的荧光素酶活性最强,可能是最小核心启动子所在区域,维持了基础转录活性。在第3代DPCs中,pGL3-B-604的荧光素酶活性最强,而在第8代DPCs中,pGL3-B-604的荧光素酶活性明显降低,提示DPCs在传代过程中,可能是通过-600～-200bp之间的调控元件发挥转录调控的作用。
[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.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R346
本文编号：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.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R346
【参考文献】
相关期刊论文 前10条
1 伍津津,刘荣卿,唐书谦,钟白玉,叶庆佾;正常人毛乳头细胞和真皮鞘细胞的生长特性观察[J];第三军医大学学报;2000年12期
2 宋志强,郝飞,杨希川,杨卫兵,程波,麦跃,叶庆佾;人毛乳头细胞凝集性生长差异表达基因cDNA文库的构建[J];第三军医大学学报;2003年11期
3 麦跃,刘荣卿,伍津津,程波,唐书谦,钟白玉;毛乳头细胞凝集性生长对诱导毛囊样结构形成能力的影响[J];第三军医大学学报;2004年13期
4 郭晓静;郝飞;姜晓勇;宋志强;;人毛乳头细胞在不同传代时期某些细胞因子表达的变化[J];第三军医大学学报;2006年21期
5 宋志强,郝飞,杨卫兵,王继文,邹锋;Screening and analysis of differentially expressed genes of dermal papillae cells with aggregative behavior[J];Journal of Medical Colleges of PLA;2004年01期
6 宋志强,郝飞,钟白玉,杨卫兵,麦跃;体外长期培养人毛乳头细胞生长特性[J];临床皮肤科杂志;2003年05期
7 夏汝山;郝飞;尹锐;宋志强;邹锋;钟白玉;;HSPCO16重组蛋白对毛乳头细胞增殖的影响[J];中国皮肤性病学杂志;2007年01期
8 夏汝山;郝飞;牟芝蓉;杨希川;宋志强;钟白玉;;凝集性生长毛乳头细胞蛋白质组的差异分析[J];中国皮肤性病学杂志;2008年05期
9 伍津津,刘荣卿,叶庆佾,钟白玉,唐书谦;毛乳头细胞高效培养方法探索[J];中华皮肤科杂志;1997年06期
10 刘荣卿,唐书谦,钟白玉,叶庆佾,伍津津;人毛乳头细胞体外培养生长曲线观察[J];中华皮肤科杂志;2001年01期
，本文编号：2368550
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2368550.html
最近更新
教材专著
