【作者】 许冬青；

【导师】 詹臻；

【作者基本信息】 南京中医药大学 ， 中西医结合基础， 2009， 博士

【摘要】 研究目的:舌象的变化能客观地反映出机体的正气盛衰、病邪深浅,是体内病变的一面镜子,因而舌诊在中医对疾病的诊断上起着重要作用。近年来,随着中医现代化、科学化和国际化的发展,中医得到了越来越多的肯定,并且发挥了越来越重要的作用,然而,如何使中医诊断和治疗客观化、科学化仍是进一步发扬光大祖国医学的关键问题。现代研究表明舌苔的厚薄与舌上皮细胞的增殖、分化及凋亡之间的平衡密切相关。舌黏膜上皮属于复层扁平上皮,舌上皮细胞在增殖、分化的同时,有一部分细胞发生了细胞凋亡。当舌上皮细胞增殖与分化、凋亡处于相对平衡状态时,舌苔表现为薄苔;而当细胞增殖占优势,细胞凋亡和分化相对占劣势时,舌上皮细胞增多,舌苔即变厚,反之则出现剥苔。临床观察发现肿瘤患者的舌苔、舌质变化较为明显,提示肿瘤在患者机体生长后对人体的影响。在肿瘤患者的舌苔变化与表皮生长因子(EGF)的关系研究中,我们观察到肿瘤患者的舌苔增厚明显较其它疾病患者及正常人显著,同时研究发现,肿瘤患者舌苔的变化与其唾液中EGF含量密切相关,舌苔增厚的肿瘤患者唾液中EGF含量明显增高。说明EGF与舌苔变化有着密切的关系。为了进一步分析EGF影响舌苔形成的机制,我们运用流式细胞术、Real Time-PCR、基因芯片等现代分子生物学技术,探讨了EGF对舌黏膜上皮细胞增殖、凋亡、黏附分子及EGF-R表达等的影响。由于黏附分子在组织形态发生、组织形成等方面具有重要的作用,推测其可能与舌苔变化密切相关。研究黏附分子在舌黏膜组织上的表达,可能有助于进一步在分子水平上阐明舌苔形成的机制。通过此项研究,可以:(1)分析EGF对舌黏膜上皮细胞EGF-R表达及细胞增殖的影响,进一步阐明EGF影响肿瘤病人舌苔变化的信号转导分子机制:(2)分析EGF与舌黏膜上皮细胞黏附分子、细胞增殖凋亡相关基因表达的关系;(3)深入地研究表皮生长因子在舌苔形成中的作用,从基因、分子水平上探讨舌苔变化的机制。(4)对不同舌苔类型的基因表达谱进行分析,筛选出差异表达基因,为进一步阐明舌苔形成的分子机制研究提供依据。研究方法:本课题从细胞模型、临床肿瘤患者两个角度来探讨EGF影响肿瘤患者舌苔形成的分子机制1、EGF对舌鳞癌Tca-8113细胞增殖、基因表达影响的研究(1)EGF对EGF-R表达以及细胞增殖影响的研究通过流式细胞术检测分析EGF对舌鳞癌Tca-8113细胞EGF-R表达及细胞周期的影响。(2)EGF对凋亡增殖相关基因Fas、c-myc表达影响的研究通过流式细胞术检测分析EGF对舌鳞癌Tca-8113细胞凋亡增殖相关基因Fas、c-myc表达的影响。(3)EGF对黏附分子表达影响的研究通过流式细胞术检测分析EGF对舌鳞癌Tca-8113细胞黏附分子CD29、CD54、CD106表达的影响。2、临床肿瘤患者不同舌苔类型基因表达差异的研究不同舌苔的组织标本均由江苏省口腔医院提供,参考《中医诊断学》舌诊标准,选择口腔舌鳞癌切除术患者,用无菌手术获取舌背粘膜相应正常的区域。不同舌苔病例分为以下5组:白薄苔组、白厚苔组、黄薄苔组、黄厚苔组、无苔组。对照组胎儿正常白薄苔(取自南京某医院妇产科引产的存活胎儿)。(1)EGF-R在肿瘤患者常见舌苔中表达水平的研究通过Real Time-PCR技术对不同舌苔类型舌黏膜上皮细胞EGF-R表达差异进行分析;(2)Fas在肿瘤患者常见舌苔中表达水平的研究通过Real Time-PCR技术对不同舌苔类型舌黏膜上皮细胞Fas表达差异进行分析;(3)c-myc、CD29在肿瘤患者常见舌苔中表达水平的研究应用HE染色观察不同舌苔类型舌粘膜组织结构,应用免疫组化SP法检测并分析舌癌病人舌粘膜上皮细胞c-myc、CD29的表达水平。(4)常见不同舌苔的表达谱基因芯片分析研究通过基因芯片技术检测分析不同舌苔类型之间舌黏膜上皮细胞差异表达的基因,并进行筛选:研究结果:1、EGF对舌鳞癌Tca-8113细胞增殖、基因表达影响的研究(1)EGF对EGF-R表达以及细胞增殖影响的研究①EGF对舌鳞癌Tca-8113细胞EGF-R表达影响结果分析:EGF作用3小时,5ng/ml组EGF-R表达增多,与对照组相比p＜0.05;EGF作用4小时,1ng/ml组、10ng/ml组EGF-R表达增多,与对照组相比p＜0.05。②EGF对舌鳞癌Tca-8113细胞细胞周期影响结果分析:EGF作用24小时,各剂量组细胞增殖活性均显著增强,表现为处于G0+G1期细胞比例降低,处于S期细胞比例增多,与对照组相比p＜0.01。(2)EGF对凋亡增殖相关基因Fas、c-myc表达影响的研究①EGF对舌鳞癌Tca-8113细胞Fas表达影响结果分析:EGF作用8小时,10ng/ml组细胞Fas表达增多,与对照组相比p＜0.05;EGF作用12小时,各剂量组Fas表达与对照组相比无显著性差异②EGF对舌鳞癌Tca-8113细胞c-myc表达影响结果分析:EGF作用8小时,10ng/ml组细胞c-myc表达增多,与对照组相比p＜0.05;EGF作用12小时,5ng/ml组、10ng/ml组细胞c-myc表达增多,与对照组相比p＜0.05。(3)EGF对黏附分子表达影响的研究结果①EGF对舌鳞癌Tca-8113细胞CD29表达影响结果分析:EGF作用8小时、12小时、16小时,5ng/ml组、10ng/ml组细胞CD29表达均增高,与对照组相比p＜0.05。②EGF对舌鳞癌Tca-8113细胞株CD54表达影响结果分析:EGF作用8小时,各剂量组细胞CD54表达均增高,与对照组相比p＜0.05;EGF作用12小时,5ng/ml组、10ng/ml组细胞CD54表达增高,与对照组相比p＜0.01;EGF作用16小时,各剂量组与对照组相比无显著性差异。③EGF对舌鳞癌Tca-8113细胞株CD106表达影响结果分析:EGF作用8小时、12小时、16小时,各剂量组细胞CD106表达与对照组相比均无显著性差异。2、临床肿瘤患者不同舌苔类型基因表达差异的研究结果(1)EGF-R在肿瘤患者常见舌苔中表达水平的研究不同舌苔舌黏膜上皮细胞EGF-R表达从高到低依次为:黄厚苔＞白薄苔＞黄薄苔＞无苔＞白厚苔,且均高于对照组:正常白薄苔(胎儿)组。(2)Fas在肿瘤患者常见舌苔中表达水平的研究不同舌苔舌黏膜上皮细胞Fas表达水平从高到低依次为:黄厚苔＞薄白苔＞剥苔＞薄黄苔＞白厚苔＞对照组。(3)c-myc、CD29在肿瘤患者常见舌苔中表达水平的研究①不同舌苔舌黏膜上皮细胞CD29表达水平从高到低依次为:白厚苔＞白薄苔＞黄厚苔＞无苔＞对照组＞黄薄苔②不同舌苔舌黏膜上皮细胞c-myc表达水平从高到低依次为:白厚苔、黄厚苔＞对照组＞白薄苔＞黄薄苔、无苔。(4)常见不同舌苔的表达谱基因芯片分析研究以Ratio大于2或小于0.5为标准,筛选出五种常见病理性舌苔的表达都有改变的共计180条基因,在此180条基因基础上进一步进行筛选,挑选出Ratio大于4或小于0.3的基因,其中表达下调的基因有42条,表达上调基因20条,上述基因表达变化主要与离子通道和运输蛋白基因、细胞周期蛋白类基因、细胞骨架和运动基因、细胞凋亡相关的蛋白基因、DNA结合、转录和转录因子基因、细胞受体基因、免疫相关基因、细胞信号和传递蛋白基因、代谢基因和蛋白翻译合成基因结论:1、EGF通过作用于舌黏膜上皮细胞的EGF-R,促进细胞增殖,同时诱导细胞表达更多的EGF-R,而使EGF的利用增多。2、EGF对舌黏膜上皮细胞凋亡基因Fas及增殖分化相关基因c-myc的表达都有促进作用,并以对c-myc的促进作用更为强烈。EGF可能一方面通过使舌粘膜上皮细胞Fas蛋白表达增高,促使舌粘膜上皮细胞凋亡;另一方面,又能促使c-myc持续高表达,使舌上皮细胞过度增殖,舌粘膜上皮细胞凋亡指数降低,进而形成病理性厚苔。3、EGF对舌黏膜上皮细胞表面黏附分子的表达有着重要的影响。EGF能明显促进舌黏膜上皮细胞CD29、CD54的表达,其中又以对CD29作用更为强烈,能使CD29持续高表达。EGF可能通过促进舌黏膜上皮细胞CD29、CD54的表达,介导细胞与细胞间、细胞与细胞外基质间的粘附,从而影响舌苔形成。4、通过基因芯片的研究,我们寻找到了部分可能在舌苔形成中或影响患者舌苔变化的相关基因,它们主要与离子通道和运输蛋白基因、细胞周期蛋白类基因、细胞骨架和运动基因、细胞凋亡相关的蛋白基因、DNA结合、转录和转录因子基因、细胞受体基因、免疫相关基因、细胞信号和传递蛋白基因、代谢基因和蛋白翻译合成基因有关。创新之处1、本研究内容获得两项国家自然科学基金资助(批准号:30070910,30472123),首创了EGF、EGF-R与舌苔变化关系这一新的研究领域,从分子、基因水平阐明舌苔形成的机制,为中医舌诊舌苔研究开拓了一条新途径。2、首次将中医的舌诊舌苔研究与黏附分子结合起来研究,初步揭示了粘附分子与舌苔变化之间有着密切的关系。更多还原

【Abstract】 Object:The change of tongue body could reflect the condition of vitality and pathogen in body,therefore tongue diagnosis plays an important role in the clinic of traditional Chinese medicine(TCM).In recent years,TCM gained more and more affirmation and showed important therapeutic effects as the development of modernization,scientizing and internationalization of TCM.However,objective and scientific diagnosis and treatment by TCM is the key problem for its development.Modern research shows that the thickness of tongue coating is closely related to the proliferation,differentiation and apoptosis of tongue epithelial cells.Tongue epithelium is stratified squamous epithelium.Some tongue epithelial cells apoptosis while they are proliferating and differeniation.When the proliferation,differentiation and apoptosis of tongue epithelial cells is in relative equilibrium state,The tongue coating is thin.When proliferation is dominant,apoptosis and differentiation is the relative disadvantage,the tongue epithelial cells increased,and the tongue coating is thickening.On the contrary the tongue coating is non-coating.In clinic tumor patients showed obvious changes of tongue coating and body,which indicates the influence of tumor growth on patients.Based the study on the relationship between tongue coating and epidermal growth factor(EGF) in tumor patients,we found that the tongue coating in tumor patient were thicker than that in healthy person or patient who got other diseases.In addition,changes of tongue coating are closely related to EGF content in saliva and tumor patients’ tongue coating became thicken as their EGF content in saliva increased.In conclusion,there is a strong relationship between changes of tongue coating and EGF.In order to further understand the mechanism of EGF influencing tongue coating,We use flow cytometry,Real Time-PCR,gene chips and other modern techniques of molecular biology,to discuss the influence of EGF on the tongue epithelial cell proliferation, apoptosis,adhesion molecules and EGF-R expression,etc.As adhesion molecules play an important role on the morphogenesis of organization,organizational formation,etc,the studies of adhesion molecules expression on the tongue epithelial cells may help to clarify the molecular mechanism of tongue coating formation.Through the study,it may:(1) Analyze the influence of EGF on the EGF-R expression and cell proliferation of tongue epithelial cells,and further clarify the molecular mechanism of signal transduction about EGF influencing tongue coating.(2) Analyze the relationship between EGF and adhesion molecules,cell proliferation and apoptosis-related gene expression.(3) Explore the mechanism of tongue coating changing from gene and molecular level.(4) Through gene expression profile analysis of different tongue coating types,selecte differentially expressed genes,which will provide the base for the further studies on the clarifying molecular mechanism of tongue coating formation.Methods:The study explore the molecular mechanism of EGF influencing tongue coating from two levels:cell models and clinic tumor patients.1 Studies of EGF influencing Tca-8113 proliferation and gene expression(1) Studies of EGF influencing cell cycle and EGF-R expressionThe expression of EGF-R and cell cycle of Tca-8113 were deteced by flow cytometry (FCM).(2) Studies of EGF influencing Fas and c-myc expressionThe expressions of Fas and c-myc of Tca-8113 were detected by FCM.(3) Studies of EGF influencing adhesion molecule expressionThe expressions of adhesion molecules CD29,CD54,CD106 of Tca-8113 were detected by FCM.2 Studies of the difference of gene expression in different tongue coating of tumor patients(1) The study of the difference of EGF-R expression in different tongue coating The mRNA of EGF-R was measured by real-time quantitative RT-PCR.(2) The study of the difference of Fas expression in different tongue coating The mRNA of Fas was measured by real-time quantitative RT-PCR.(3) Studies of the difference of CD29 and c-myc expression in different tongue coatingHE pigmentation was used to observe the common tongue;SP immunology and histochemistry pigmentation was used in the analysis of the gene expression of c-myc and CD29.(4) Detection of differentially expressed genes in common tongue coatingwe used gene chip technology to detect the differentially expressed genes in common tongue coating.Results:1 Studies of EGF influencing Tca-8113 proliferation and gene expression(1) Studies of EGF influencing cell cycle and EGF-R expression①EGF-R:After treated with EGF 3h,the EGF-R expression of 5ng/ml group increased(p＜0.05).After treated with EGF 4h,the EGF-R expressions of 1ng/ml group and 10ng/ml increased(p＜0.05).②cell cycle:After treated with EGF 24h,the cell proportion in G0+G1 phase decreased(p＜0.01),the cell proportion in S phase increased(p＜0.01).(2) Studies of EGF influencing Fas and c-myc expression①Fas:After treated with EGF 8h,the Fas expression of 10ng/ml group increased (p＜0.05).After treated with EGF 12h,there is no significant difference between the experiment group and control.②c-myc:After treated with EGF 8h,the c-myc expression of 10ng/ml group increased(p＜0.05).After treated with EGF 12h,the c-myc expression of 5ng/ml group and 10ng/ml group increased(p＜0.05).(3) Studies of EGF influencing adhesion molecule expression①CD29:After treated with EGF 8h、12h、16h,the CD29 expression of 5ng/ml group and 10ng/ml group increased respectively(p＜0.05). ②CD54:After treated with EGF 8h,the CD54 expression of every group increased(p＜0.05).After treated with EGF 12h,the CD54 expression of 5ng/ml group and 10ng/ml group increased(p＜0.01).After treated with EGF 16h,there is no significant difference between the experiment group and control.③CD106:After treated with EGF 8h、12h、16h,there is no significant difference between the experiment group and control.2 Studies of the effects EGF on the genes related with apoptosis and proliferation(1) The study of the difference of EGF-R expression in different tongue coatingThe the tendency from high to low of EGF-R mRNA expression was yellow thick coating,white thin coating,yellow thin coating,non-coating,white thick coating and fetal white thin coating.(2) The study of the difference of Fas expression in different tongue coatingThe the tendency from high to low of Fas mRNA expression was yellow thick coating,white thin coating,non-coating,yellow thin coating,white thick coating and fetal white thin coating.(3) Studies of the difference of CD29 and c-myc expression in different tongue coating①CD29:The tendency from high to low of CD29 expression was white thick coating, white thin coating,yellow thick coating,non-coating,fetal white thin coating,yellow thin coating.②c-myc:The tendency from high to low of c-myc expression was white thick coating, yellow thick coating,fetal white thin coating,white thin coating,yellow thin coating, and non-coating.(4) Detection of differentially expressed genes in common tongue coatingThe result indicated that 180 genes were differentially expressed with the standard of Ratio＞2 or Ratio＜0.5.Among the 180 genes,further investigation indicated that the expressions of 42 genes were down regulated while the expressions of 20 genes were up down regulated with the standard of Ratio＞4 or Ratio＜0.3.These.genes were mainly the ones of.ion channels and transport protein related genes,cell cycle related genes,cell framework and movement related genes,cell apoptosis related genes,DNA binding and transportion factors,cell receptors,immunity related genes,cell signal transduction protein,metabolize related genes,protein translation related genes.Conclusions:1、EGF can promote cell proliferation by combining EGF-R on target cell membrane, induce more EGF-R expression,thus increase the use of EGF.2、EGF could significantly increase the expressions of Fas and c-myc,and have a more strong role on c-myc expression.EGF may on the one hand,increase the expression of Fas which lead to the apoptosis of tongue epithelial cells,while on the other hand,continue stimulating tongue epithelial cells high expression of c-myc which leads to the excessive proliferation of tongue epithelial cells.So the tongue epithelial cell apoptosis index get lower,thus leads to the formation of pathological thick tongue coating.3、EGF have a significant impact on the expression of adhesion molecules of tongue epithelial cells.EGF may effect the formation of tongue coating by increasing expressions of CD29 and CD54.4、The formation of tongue coating may mainly related with ion channels and transport protein genes、cyclins genes,cytoskeletal genes and motility genes,apoptosis genes,DNA binding genes,transcription and transcription factor genes,cell receptor genes,the immune-related genes,cell signaling and transmission protein genes,metabolic genes and protein translation genes.Innovation:1 This study is supported by two National Natural Science Funds.For the first time,the changes in tongue coating were studied combining with EGF and EGF-R.It will help to clarify the mechanism of tongue coating formation from gene and molecular level and explore a new way for the research in tongue coating. 2、For the first time,the tongue coating were studied combining with adhesion molecules. This study revealed that adhesion molecule is closely related with the changes in tongue coating.更多还原