常见舌苔上皮细胞凋亡及其相关基因表达变化规律研究

【作者】 李明；

【导师】 吴正治；

【作者基本信息】 湖北中医学院 ， 中西医结合基础， 2003， 硕士

【摘要】 目的 舌诊是指导中医辨证论治的重要诊断依据，舌苔是临床察舌的主要内容。近年来的研究表明，表皮等复层扁平上皮的角质形成细胞在由基底层向棘层、颗粒层、角质层生长、分化过程中发生了细胞凋亡，而构成舌苔的主体即舌粘膜上皮细胞也属于复层扁平上皮。因此，我们从舌苔上皮细胞凋亡及其相关基因表达的角度出发，在mRNA和蛋白质两个水平探讨常见舌苔形成的机理，以促进舌诊的客观化、微观化和现代化。 方法 将观察对象根据有无各系统器质性疾病及舌苔表现，分为正常薄白苔、病理薄苔、厚苔、剥苔组，每例用消毒丝瓜络用力刮取舌苔后离心、洗涤、涂片。 采用TUNEL法检测舌苔涂片中舌粘膜上皮细胞的凋亡情况。 运用原位分子杂交技术和CMIAS真彩色病理图像分析系统检测舌上皮细胞中TGF-β3、bax、fas、bcl-2 mRNA的转录水平。 应用免疫组织化学LDP法和图像分析技术检测舌上皮细胞中TGF-β3、Bax、Fas、Bcl-2蛋白的翻译水平。 结果 TUNEL法检测结果显示：四种常见舌苔其舌上皮细胞都可见细胞凋亡发生，剥苔组的凋亡指数高于其他各组，而厚苔组的凋亡指数低于其他各组(均为P＜0.05)。厚苔组与剥苔组凋亡指数差异有非常显著性意义(P＜0.01)。 原位杂交和免疫组化检测结果表明：与正常组比较，厚苔组TGF-β3、bax基因表达和蛋白产物显著降低，剥苔组bax、fas表达水平显著升高而TGF-β3表达水平显著降低(均为P＜0.05)。厚苔组与剥苔组bax表达水平差异有非常显著性(均为P＜0.01)。 结论 舌上皮细胞在增殖、分化的过程中有一部分细胞发生了细胞凋亡，舌苔厚度与舌上皮细胞增殖、分化和凋亡的动态平衡有关。舌苔厚度变化伴随有凋亡相关基因表达的变化：在上皮细胞中起促进凋亡作用的基因TGF-β3、fas、bax表达上调时，细胞凋亡增加，舌苔变薄；反之则舌苔变厚。更多还原

【Abstract】 ObjectiveGlossocopy is an important diagnosis basis of traditional Chinese medicine, and the research of tongue coating is an important content of it. In recent years, a lot of study revealed that keratinocytes of stratified squamous epithelium such as in skin undergone apoptosis, during the process of proliferation and differentiation from basal layer, prickle cell layer, granular layer to cuticle. The main constitution of the tongue coating namely the tongue mycoderma epithelium is also belong to the stratified squamous epithelium. The aim of the study was to investigate whether apoptosis may contribute to the changes of tongue coating’ s thickness and whether certain apoptosis-related genes such as TGF-beta 3, bax, fas, bcl-2 take part in this process, thus to determine the mechanisms of tongue coating’ s change.MethodsObjects of observation were divided into four groups namely normal white and thin coating tongue, pathological thin fur, thick fur and peeling fur. Scraping tongue coating with sterilized vegetable sponge, then centrifugalizing, washing and smearing.Terminal deoxynucleotidyl transferase-mediate deoxyuridine triphosphate nick end labeling technique (TUNEL) was used to identify apoptotic nuclei in smears.Smears were examined by in situ hybridization and image analysis for the expression of mRNAs associated with promoting (TGF-beta 3, bax, fas) or preventing (bcl-2) apoptosis.These smears from all the cases also were stained with immunohistochemical technique for the expression of TGF-beat 3, Bax, Fas, and Bcl~2 proteins.ResultsApoptotic changes were seen in all smears by means of the TUNEL method. Apoptotic index of the peeling fur was higher than any of the others, while the thick fur’ s is the lowest.The results observed by in situ hybridization and immunohistochemistry revealed that TGF-beta 3, bax mRNAs and proteins expression were significant lower in thick fur as compared with normal control’ s .There was a significant increase in bax, fas mRNAs and proteins expression in peeling fur, acccompaning with a significant decrease in TGF-beta 3 expression.ConclusionDuring the process of proliferation and terminal differentiation, some stratified squamous epithelial cells of tongue-coating undergone apoptosis, and there was a certain relationship between the thickness of the tongue coating and the apoptotic index. The higher expression level of bax, fas may contribute to promote the apoptosis process involved in the terminal differentiation program of tongue epidermal keratinocytes.更多还原