【作者】 董伟；

【导师】 詹臻；

【作者基本信息】 南京中医药大学 ， 中西医结合基础， 2016， 博士

【摘要】 目的舌苔诊察是中医舌诊的重要组成,是舌诊客观化、现代化的重要内容。望舌苔可辨病邪深浅、邪正消长。舌黏膜上皮细胞的增殖、分化、凋亡、迁移与舌苔形成密切相关。大量研究表明舌黏膜上皮细胞的增殖/凋亡平衡是舌苔形成的重要机制之一。本文从临床病例和体外细胞实验两方面,分析EGF与胃癌患者舌苔形成的关联,探索舌苔形成的分子机理,以期为舌诊的临床应用提供客观依据和微观辨证基础。方法临床收集胃癌病例和健康人群,获得知情同意后调查一般人口学特征、生活习惯、舌诊、临床实验室检查等信息。采集外周静脉血,提取DNA,通过PCR-LDR法检测EGF的SNP(启动子区域的rs3756261、rs11568835、rs4444903,非同义外显子区域的 rs11568943、rs2237051、rs11569017,3’非翻译区的 rs3733625)。分离血清,ELISA检测血清EGF含量。分析EGF的SNP、生活习惯、胃癌病理分型、临床实验室检查、血清EGF含量与舌苔的相关性。选用无血清的细胞培养基体外培养人舌鳞状细胞癌细胞Tca-8113,模拟正常舌黏膜上皮细胞分裂、增殖、迁移时血清营养降低的状况,采用EGF、TGF-α处理细胞,通过MTT实验检测细胞增殖率,流式细胞术检测细胞周期,Annexin-FITC/PI双染检测Tca-8113细胞凋亡,Hoechst染色观察细胞核形态变化,琼脂糖电泳检测细胞DNA,实时定量PCR和western blot分别检测细胞mRNA和蛋白表达,主要包括Bcl-2、Bax、Fas、总 AKT、NF-κB p50、β-actin、HSP70,以研究细胞EGFR激活相关信号途径,探讨舌苔形成过程中凋亡/增殖平衡变化的分子机制。结果临床研究显示,胃癌患者与正常对照组舌苔构成比差异显著。胃癌组患者白厚苔比例最高,对照组白薄苔比例最高。胃癌组灰黑苔、厚苔、剥苔比例显著高于对照组。弥漫型胃癌患者中,EGF rs2237051 AG/GG基因型与AA基因型相比,厚苔或剥苔比例较薄苔明显增高;rs4444903携带GA/AA基因型与GG基因型相比,厚苔或剥苔比例较薄苔明显增高,灰黑苔较白苔明显增高;rs3733625携带GA/GG基因型与AA基因型相比,厚苔或剥苔比例较薄苔明显降低。胃癌患者血清EGF含量明显高于对照组。黄苔人群血清EGF含量高于白苔组,但无统计学意义(P>0.05)。苔质与血清EGF含量分析显示,胃癌组具有薄苔及厚苔者血清EGF表达水平都高于剥苔患者,对照组血清EGF表达趋势由高到低依次为厚苔、薄苔、剥苔,但结果无统计学意义(P>0.05)。生活习惯与苔色无明显关联。胃癌患者吃早餐可能会增加厚苔和剥苔的发生比例;饮食口味偏淡的胃癌患者厚苔及剥苔出现比例降低。对照组分析表明,吸烟及饮酒可能会增加厚苔发生比例。苔色的相关性研究显示,胃癌患者血清总蛋白水平异常与黄苔显著相关;对照组分析显示,血糖水平异常可能会提高灰黑苔比例。苔质的相关性研究表明,胃癌患者白细胞计数及淋巴细胞比例与苔厚变化相关;对照组血清总蛋白、谷氨酰转肽酶及血糖与舌苔厚度变化显著相关。无血清培养舌鳞癌细胞Tca-8113,MTT实验表明,细胞增殖停滞。流式细胞术检测细胞周期发现,Tca-8113细胞G0/G1期比例增高,S期比例降低。Annexin-FITC/PI染色、Hoechst染色、琼脂糖凝胶电泳观察,细胞凋亡率升高。荧光实时定量PCR、western blot检测显示,细胞Bcl-2/Bax比值呈现下降趋势,Fas表达增高,Akt和NF-κBp50在撤除血清12h表达水平下降,HSP70在撤血清24h后表达显著下降。EGF或TGF-α在48h内可以促进细胞增殖,增加S期细胞比例,促进细胞分裂,促进细胞迁移。细胞Bax、Fas表达均未受到显著抑制,细胞AKT和NF-κB p50蛋白表达增高。结论弥漫型胃癌患者EGF的rs2237051、rs4444903、rs3733625基因变异与舌苔形成相关。胃癌患者的生活习惯(早餐、饮食口味)与舌苔厚度变化有关,正常人群吸烟、饮酒的习惯与舌苔厚度变化有关。胃癌患者血清总蛋白水平异常与黄苔显著相关,白细胞计数及淋巴细胞比例与舌苔厚度变化相关;对照组血糖水平异常可能会提高灰黑苔比例,血清总蛋白、谷氨酰转肽酶及血糖与舌苔厚度变化显著相关。黄苔人群血清EGF含量略高于白苔组(P>0.05),胃癌组薄苔及厚苔患者血清EGF表达水平高于剥苔患者,对照组血清EGF表达趋势由高到低依次为厚苔、薄苔、剥苔(P>0.05)。无血清培养舌鳞癌细胞Tca-8113,细胞增殖停滞于G0/G1期,细胞凋亡率升高,Bcl-2、Fas介导的凋亡途径激活,细胞变形死亡。EGF或TGF-α可通过PI3K/AKT、NF-κB信号通路促进细胞增殖、分裂,促进细胞移行,引起增殖/凋亡平衡偏移。更多还原

【Abstract】 ObjectiveTongue coating,an essential part of tongue appearance in traditional Chinese medicine(TCM),is critical to the objectification and modernization of tongue diagnosis.TCM practitioners believe that the nature and location of disease can be differentiated by tongue coating observation.The formation of tongue coating is closely related to proliferation,differentiation,apoptosis and migration of tongue dorsum mucosal epithelial cells.It is generally accepted that equilibrium between proliferation and apoptosis of tongue dorsum mucosal epithelial cells is one of the formation mechanisms for tongue coating.In this study,we collected clinical samples and proceeded cell experiments in vitro to explore the relationship between EGF(epidermal growth factor)and tongue coating in gastrc cancer patients.By investigating the molecular mechanism of tongue coating,our research may help to provide objective basis for tongue diagnosis and microcosmic index for syndrome differentiation.MethodsGastric caner cases and healthy controls were recruited from Jiangsu Hospital of TCM.After an informed consent form was signed by every participant,information of demographic characteristics,living habits,tongue diagnosis and clinical examination parameters were colleted.DNA was extracted from peripheral venous blood,and EGF SNPs(rs11568943,rs2237051,rs11569017 in the non-synonymous exon region,rs3756261,rs11568835,rs4444903 in the promoter region,rs3733625 in the 3’untranslated region)were genotyped by PCR-LDR(polymerase chain reaction-ligation detection reaction).Serum EGF content was analized by ELISA(enzyme-linked immunosorbent assay).Association among EGF SNPs,EGF in serum,living habits,clinical examination parameters,pathological characteristics,and tongue coating formation was explored in our study.To imitate the normal environment of tongue mucosa epithelial cells,tongue squamous cell carcinoma cell line Tca-8113 was cultured in RPMI-1640 without serum and its basic functions,such as cell division,proliferation,survival and migration was investigated.In order to evaluate the effect of EGF or TGF-α on Tca-8113,proliferation rate was tested by MTT assay,and cell cycle was analized by flow cytometric analysis.Apoptosis was assessed by staining with annexin Vpropidium iodide or hoechst 33258.DNA fragmentation was detected by agarose gel electrophoresis.To explore the EGFR signal pathway induced by EGF or TGF-α(transforming growth factor-α)and to investigate molecular mechanism of proliferation-apoptosis balance of tongue coating formation,mRNA and protein expression,including Bcl-2,Bax,Fas,total AKT,NF-κB p50,β-actin and HSP70,were evaluated by real-time fluorescence quantitative polymerase chain reaction(FQ-PCR)and western blot test,respectively.ResultsClinical studies showed that there were statistically significant differences in distributions of tongue coating between gastric cancer(GC)cases and the controls.The percentage of white thick coating was the highest in GC group,while white thin coating was more popular in the controls.For GC patients,the percentages of grey,thick and exfoliative coating were higher than those in the controls.In diffuse type-gastric cancer cases,patients who carried GA/GG genotype of rs2237051 had an increased possibility of thick or exfoliative coating,compared with AA genotype.Patients with diffuse type GC who carried GA/AA genotype of rs4444903 had an increased possibility of thick,exfoliative or grey coating,compared with GG genotype.The thick or exfoliative coating had a more decreasing possibility in patients with diffuse type GC who carried GA/GG genotype of rs3733625 than AA genotype.The serum EGF concentrations in GC cases were obviously higher than those in the controls.GC cases with yellow coating had higher level of serum EGF than the patients with white coating(P>0.05).As to the relationship between tongue coating texture and serum EGF,patients with thin or thick coating had increasing serum EGF level compared with cases with exfoliative coating.The trend of serum EGF expression in the controls in descending order was thick coating,thin coating and exfoliative coating in sequence.There was no obvious correlation between coating color and lifestyle.However,our study showed that GC patients who often ate breakfast had an increased possibility of thick or exfoliative tongue coating,compared with thin coating.And the GC patients who did not prefer salty food had a decreasing possibility of thick or exfoliative tongue coating.As for the controls group,smoking or drinking might increase the probability of thick coating.This study revealed that of 141 GC patients with abnormal serum total protein(TP)level,32(22.7%)patients had yellow coating,while the controls with abnormal serum glucose had high proportion of grey coating.According to tongue coating texture analysis,white blood cell count or lymphocyte percent was associated with thickness of tongue coating for GC patients.For the controls,the level of serum TP,glutamyl transpeptidase,or serum glucose was related to the thickness of tongue coating.After tongue squamous cell carcinoma cell line Tca-8113 was cultivated in RPMI-1640 without serum in 12 hours,MTT assay showed that cell survival rate decreased prominently,while the apoptosis rate of Tca-8113 cells increased,which was detected by staining with annexin V-propidium iodide or hoechst 33258.The flow cytometric analysis revealed that the cells cycle was arrested at G0/G1 phase and the percentage of cells at S phase was decreasing.Real-time FQ-PCR and western blot test suggested that the expression of Fas increased,and the contents of AKT,NF-κB p50,HSP70,and the ratio of Bcl-2/Bax decreased in this study.The experiments in vitro indicated that EGF or TGF-α could promote cell proliferation,increase the proportion of Tca-8113 cells at S phase,stimulate cell division and migration.The expression of Bax and Fas was not inhibited obviously,whereas the level of AKT and NF-κB p50 increased.ConclusionsThe genotypes of rs2237051,rs4444903 and rs3733625 in EGF were related to tongue coating formation for patients with diffuse type GC.The lifestyles(eating breakfast and preference for salty food)of GC cases correlated with thickness of tongue coating,while the living habits,such as smoking and drinking,in the controls were related to thickness of tongue coating.For GC patients,abnormal serum TP level had closely relationship with yellow coating,and the white blood cell count or lymphocyte percent was associated with thickness of tongue coating.For the controls,abnormal serum glucose content could increase the percentage of grey coating,and the level of serum TP,glutamyl transpeptidase,or serum glucose had significant correlation with thickness of tongue coating.Subject with yellow coating had higher level of EGF than the participant with white coating,though there was no statistical significance.GC patients with thin or thick coating had increasing serum EGF contents compared with cases with peeling coating.According to the thickness of tongue coating,the order of serum EGF contents in the controls from high to low was thick coating,thin coating and peeling coating.When tongue squamous cell carcinoma cell line Tca-8113 was cultured in RPMI-1640 without serum,in vitro experiments indicated that the Tca-8113 cells were arrested in G0/G1 phase,apoptosis was mediated by Bcl-2 and Fas,and the apoptosis rate and dead cells increased.EGF or TGF-α could promote cell proliferation,division and migration through PI3K/AKT or NF-κB pathway,which influenced the balance of proliferation and apoptosis.更多还原