【作者】 张军峰；

【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】 目的舌诊在中医临床疾病诊断中起着重要作用,其中舌苔(包括苔色和苔质)在辨证中的价值最大,舌苔形成的原理及其客观化研究是中医现代化基础研究中的一个重大的关键性课题。舌苔的形成与舌粘膜上皮细胞的分裂、增殖、分化、迁移和脱落密切相关,与凋亡相关基因的表达差异密切相关。因此,本文从临床样本和实验动物两个方面,研究了不同舌苔粘膜组织的基因表达谱,探索EGF和TGF-α对舌背粘膜上皮细胞增殖、细胞周期分布和凋亡、信号途径相关分子的影响,为更深入研究舌苔形成的分子机制奠定了基础。方法临床收集舌鳞癌患者术后癌周正常舌背粘膜,分为薄白苔、白厚苔、黄薄苔、黄厚苔、剥苔等5组,以胎儿薄白苔对照,利用基因芯片技术联合实时荧光定量PCR研究不同舌苔的基因表达谱。取临床舌鳞癌患者术后癌周正常舌背粘膜组织作为对照,健康的大鼠、小鼠和兔处死后取舌背粘膜组织,dispaseⅡ和胰蛋白酶处理分离舌背粘膜上皮细胞,体外10%血清的RPMI-1640培养基培养不同时段,流式细胞术检测细胞凋亡和细胞周期。分析人和实验动物舌背粘膜上皮细胞的细胞行为相似性,研究EGF对小鼠、大鼠、兔的细胞周期分布和凋亡影响。在无血清培养基K-SFM中,初步研究了EGF和TGF-α调控舌苔形成的差异机制,利用MTT法研究大鼠舌背粘膜上皮细胞增殖活性,流式细胞术研究细胞周期分布和凋亡,细胞免疫组织化学法研究CK4、p38-MAP kinase、c-fos、c-myc和NF-κB的表达水平。结果基因表达谱结果显示,与胎儿薄白苔相比,60个基因在5种常见舌苔中同时表达上调或下调,包括21个上调基因和39个下调基因,这些基因均与舌背粘膜上皮细胞的增殖、分化、迁移、凋亡和脱落相关,参与舌背粘膜上皮的损伤、修复和重建。人、大鼠、小鼠、兔的舌背粘膜上皮细胞周期分布和凋亡比较显示,大鼠舌背粘膜上皮凋亡趋势与人最为接近。利用含血清培养基培养大鼠、小鼠、兔的舌背粘膜上皮细胞,结果表明:EGF对小鼠和兔舌背粘膜上皮细胞均表现为抗凋亡作用,但不显示浓度依赖性;此外,EGF和TGF-α可以促进大鼠舌背粘膜上皮细胞凋亡发生,且细胞周期分布不一致。利用含不同浓度EGF和TGF-α的无血清培养基K-SFM体外培养大鼠舌背粘膜上皮细胞,随着时间推移,细胞增殖活性逐渐下降,与细胞凋亡率变化基本一致。细胞凋亡率具有浓度依赖性,随着EGF和TGF-α的浓度升高,细胞凋亡率表现为下降趋势。EGF和TGF-α可以降低CK4的表达水平,其中1μg/L和10μg/L浓度表达水平下降最明显,而5μg/L EGF和TGF-α对CK4表达水平下降较少;EGF和TGF-α可以上调p38-MAP kinase、c-myc和c-fos表达水平,却下调NF-κB表达水平;不同浓度EGF和TGF-α对相关分子表达影响具有相同的趋势,但相同浓度作用后相关分子表达量不同。EGF上调大鼠舌背粘膜上皮细胞p38-MAP kinase和c-myc的作用强于TGF-α,而EGF上调c-fos的作用弱于TGF-α;EGF下调NF-κB p65和p50的作用不如TGF-α更明显,从阳性目标平均光密度值看,NF-κB p50的表达水平高于NF-κBp65。结论在参与舌苔形成的基因中,EGF和TGF-α是两个关键分子,虽然它们可以竞争性结合共同的细胞受体EGF-R,但对细胞周期分布和凋亡的影响不同,提示它们在舌苔形成中的功能不同。以大鼠舌背粘膜上皮细胞作为体外细胞模型,研究EGF和TGF-α对p38-MAPkinase、c-myc、c-fos和NF-κB等信号通路分子的表达水平,结果显示EGF和TGF-α对这些分子表达水平的变化趋势基本一致,但表达水平明显不同。因此,我们推测:在正常人群和不同疾病患者的舌苔形成过程中,EGF和TGF-α是影响舌苔变化的两个关键分子,可能通过多种信号通路调控舌背粘膜上皮细胞的增殖、分化、迁移、角化成熟和凋亡脱落。更多还原

【Abstract】 ObjectiveGlossoscopy plays a key role in diagnostics of traditional Chinese medicine(TCM) and is regarded as an important method in TCM.Thus,the tongue picture has been investigated in clinical and experimental studies.Tongue coating is an important content of glossocopy.The mechanism and objectivity of tongue coating is becoming one of the key problems in the modernizational study of TCM.Histogenetic study of tongue coating showed that the formation of tongue coating was closely related to the epidermis of tongue dorsum.Many studies revealed that the formation of tongue coating was closely related to apoptosis-related genes.Therefore,we collected clinical samples and laboratory animals to investigate the mechanism of the formation of tongue coating,cDNA microarray analysis and real-time PCR were used to identify novel genes in lingual epithelial cells to gain new insights on the molecular biological mechanism of tongue coatings.Cell culture in vitro, flow cytometric analysis,MTT method and immunohistochemistry were employed to investigate the cell cycle distribution and apoptosis,cell proliferation and expressed level of several key molecules in signal pathways.MethodsClinical samples were obtained from 22 patients with histopathologically defined tongue dorsum carcinoma and need surgical removal.The total 22 selected typical samples were separated into 5 groups:5 white thin coatings 5 white thick coatings,5 yellow thick coatings,4 yellow thin coatings,and 3 non-coatings.Four fetal samples covered with white thin coating were regarded as the control.Five cDNA microarrays containing 4096 human cDNA clones were used to estimate the differences in gene expression in the lingual tissues.The epithelia obtained from healthy rats,mice and rabbits were investigated with normal epithelium adjacent to lingual squamous cell carcinoma as control.DispaseⅡand trypsin were combined to digest the lingual dorsum epidermis and then,the epithelia were incubated in RPMI-1640 under different period of time.Flow cytometric analysis was used to analyze apoptosis and cell cycle changes.In addition,the effect of EGF on cell cycle and apoptosis in the lingual dorsal epithelial cells obtained from rats,mice and rabbits was investigated in vitro.To investigate the effect of EGF and TGF-αon modulating the formating of tongue coating,MTT method was used to evaluate the activities of cell proliferation,and flow cytometric analysis to analyze the cell cycle distribution and apoptosis in the lingual dorsal epithelial cells obtained from rats in K-SFM.And immunohistochemistry was employed to investigate the expressed level of several key molecules in signal pathways,such as CK4, p38-MAP kinase,c-fos,c-myc and NF-κB.ResultsThe differential expression in the five kinds of common tongue coatings vs.the fetal white thin coating from the blast analyses revealed that 60 synchronous differentially expressed genes in five kinds of common tongue coatings were identified in cDNA microarray results,of which 21 genes were up-regulated and 39 genes were down-regulated. They were all correlated with proliferation,differentiation and apoptosis of the lingual mucous membrane epithelia.The characteristic of apoptosis and cell cycle changes in the lingual dorsum epithelia from rats,mice,rabbits and human were compared,and the results showed that rats could be the ideal model animal to investigate the apoptosis of tongue coatings.The results of cell cycle distribution and apoptosis in 10%RPMI-1640 revealed that EGF could inhibit the apoptosis of the epithelia from mice and rabbits,but the effect of anti-apoptosis didn’t show dose-dependent.In contrast,EGF and TGF-αcould promote the apoptosis of the epithelia from rats in 10%RPMI-1640.EGF and TGF-αcould decrease the activity of cell proliferation dose-dependently in K-SFM,consistenting with apoptosis change,which may be attribute to cell cycle change. The results of immunohistochemistry showed that EGF and TGF-αcould affect the expressed level of CK4,p38-MAP kinase,c-fos,c-myc and NF-κB.EGF and TGF-αcould inhibit the expressed level of CK4.It was noted that both 1μg/L and 10μg/L EGF and TGF-αmay significantly inhibit the expressed level of CK4,but the effect of moderate concentration(5μg/L) was slight.In addition,we foud that EGF and TGF-αcould increase the expressed level of p38-MAP kinase,c-myc and c-fos,but decrease the expressed level of NF-κB p50 and p65.The tendency of the effect on p38-MAP kinase,c-myc,c-fos and NF-κB was similar to that in EGF and TGF-αwhereas the intensity of the effect was different.The effect of EGF on p38-MAP kinase and c-myc was stronger than that of TGF-α,on the contrary,the effect of EGF on c-fos and NF-κB was weaker than that of TGF-α.ConclusionsThe result showed that EGF and TGF-αwere the two key molecules in the formation of tongue coating.EGF and TGF-αcould competitively integrate the same EGF-receptor (EGF-R),but they had different effect on the cell cycle distribution and apoptosis, indicating that EGF and TGF-αmay play distinct roles in the formation of tongue coating. Therefore,the advancement of investigation on the signal pathways of EGF and TGF-αwas very important to understand the molecular mechanism of the formation of tongue coating.The lingual dorsum epithelia obtained from healthy rats was used as the cell model to investigated that the expressed levels of CK4,p38-MAP kinase,c-fos,c-myc and NF-κB were affected by EGF and TGF-α.The cell immunohistochemistry result also evidenced that EGF and TGF-αmay play distinct roles in the formation of tongue coating.Thereby, we infered that EGF and TGF-αwere the two key molecules in the formation of tongue coating,but they play distinct roles in the formation of tongue coating of healthy and various diseases populations.EGF and TGF-αmay attach themselves to multifarious signal pathways to modulating the formation of tongue coating.更多还原