Studies Of The Molecular Mechanism Of EGF Influencing Tongue Coating

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.