【作者】 卢标清；

【导师】 李云英；

【作者基本信息】 广州中医药大学 ， 中医五官科学， 2009， 博士

【摘要】 一、研究目的与意义喉癌是头颈部最常见的恶性肿瘤之一,喉癌前病变是喉癌变的中心环节,加强对喉癌前病变的研究,不但可以积极预防喉癌的发生,还将加深对癌变机理的认识,推动肿瘤防治工作的发展。导师组前期研究提示:痰瘀是导致喉癌前病变发生发展的重要因素,临床采用活血化痰法有较好的防治作用,但对痰瘀在喉癌前病变发生发展过程中的作用机制尚不明了,对喉癌前病变的中医病因病机研究尚不深入。本项目拟从文献研究、临床调查及实验三个方面对喉癌前病变的病因病机进行初步的综合分析研究,探索喉癌前病变的基本中医证候特征,研究喉癌变过程中的中医证候特点及演变规律,从细胞分子水平识别其中有较高恶变潜质的中医证候类型,探讨痰瘀在喉癌前病变发生发展过程中的作用及分子生物学机制。研究成果将为中医药研究、治疗喉癌前病变及喉癌提供方向和思路,为开发治疗喉癌前病变的中药复方提供理论和实验依据,同时为喉癌前病变的辨证提供客观化、微量化的实验依据,深化中医对喉癌前病变的认识,这无疑具有重要的科学意义和实际价值。二、研究方法（一）临床研究1.文献病例研究利用CNKI中国期刊全文数据库（Web版）及中国生物医学文献数据库（CBMdisc）,应用SPSS11.5软件对文献中喉癌前病变的中医证候因素、四诊特点、治法方药等进行统计分析。2.临床调查病例研究采用临床流行病变调查方法,结合喉癌前病变临床调查表进行病例资料信息的采集。通过对88例喉癌前病变的四诊资料进行综合分析,同时取100例声带息肉及60例喉癌病例作病情进展对照研究,初步总结喉癌前病变的中医四诊特点及基本证候演变特点。3.临床治疗观察本研究拟比较活血化痰与益气法在喉癌前病变显微喉手术后应用的临床疗效,将34例喉癌前病变病例分为益气治疗组及活血化痰治疗组,对两组术后症状、体征等进行综合量化评分,比较两种治疗方法在改善喉癌前病变症状、体征及防止复发恶变方面的优劣。（二）实验研究细胞周期调控异常是肿瘤发生发展的一个重要前提,本项目利用免疫组化方法,检测痰瘀互结证喉癌前病变标本中细胞周期因子cyclin E、cyclin D1、P21、P16以及肿瘤抑制基因PTEN、P⁵³的表达,同时取痰浊凝聚证癌前病变标本及声带息肉组织、早期喉癌标本分别作证候对照及病情进展对照。三、研究结果（一）临床研究1.目前中医对喉癌前病变临床文献以病案报道、专家经验总结为主,在成人肥厚性喉炎的中医药辨证治疗方面较西医有一定优势,临床研究报道最多,基本上都主张从活血化痰开音方面对肥厚性喉炎进行治疗;对声带白斑、喉角化症及喉乳头状瘤则主张手术后应用中医药辨证治疗,报道的临床疗效是确切的,其中的一些临床经验,值得现在借鉴和参考。2.喉癌前病变中医四诊特点:喉癌前病变以声嘶、咽异物或痰粘感、咳嗽有痰、咽痛、胸闷不舒、讲话费力为主症,尚可有咽干、气短等症;舌象以淡红舌、暗舌、红舌为主;脉象以弦滑、弦细多见。喉癌前病变中医证候特点:喉癌前病变证候因素为痰瘀,以痰瘀互结为主要证候,痰证及血瘀证次之,尚可兼杂阴虚、气虚、热、毒等。3.中医证候演变特点:与声带息肉比较,喉癌前病变证候因素相对复杂,兼杂情况较多见（P＜0.05）;而与喉癌前病变比较,喉癌的证候因素更复杂,证候兼杂情况更普遍（P＜0.05）。从声带息肉→喉癌前病变→喉癌的病情发展变化过程中,证候演变存在一定规律:以痰瘀为主线,证候由单一向证候兼杂发展,最终出现痰瘀热毒、气阴两虚等多种证候因素兼杂的情况,证候的兼杂、复杂化及热毒的出现可能提示病邪深入,正气受损,预示病情的不良进展,值得临床注意。4.喉癌前病变发生发展过程中舌象变化特点:从声带息肉→喉癌前病变→喉癌,随着病情的发展变化,舌象可由正常的淡红舌为主,向暗红舌、红绛舌、紫舌等发展;舌苔由淡白、白腻向薄黄、黄腻到黄老厚腻变化发展;舌边瘀斑瘀点及舌下静脉曲张的情况更普遍;喉癌中少舌或无苔的出现率更高。这些预示病邪的深入,正气亏损,痰瘀热毒向纵深发展。提示当喉癌前病变患者舌质颜色加深,舌苔由薄变厚,出现绛舌或紫舌、裂纹舌,花剥苔时,应注意恶变倾向,应予重点密切随访及采取必要干预措施5.临床采用活血化痰法对喉癌前病变具有一定的干预治疗作用,可以在喉显微术后较快缓解症状、改善体征,延缓或减少复发（P＜0.05）。（二）实验研究1.P21^Waf1、cyclinD1、cyclin E、P16均主要表达于细胞核,在声带息肉中P21b^Waf1、P16表达染色着色深且阳性细胞密集;在喉癌前病变中P21^Waf1、P16蛋白表达染色变浅,阳性细胞减少,在喉癌中两蛋白表达染色浅,阳性细胞少,形成声带息肉→喉癌前病变→喉癌阳性强度逐渐递减的现象（P＜0.05）。而cyclin E、cyclinD1蛋白在声带息肉、喉癌前病变、喉癌中的表达则刚好相反,形成声带息肉→喉癌前病变→喉癌阳性强度逐渐增强的梯度现象（P＜0.05）。2.PTEN主要表达于细胞浆,P⁵³、PCNA主要表达于细胞核,在声带息肉中PTEN蛋白表达染色着色最深,且阳性细胞密集;在喉癌前病变中PTEN蛋白表达染色变浅,且深浅不一,阳性细胞减少,在喉癌中PTEN蛋白表达染色浅,阳性细胞少,形成声带息肉→喉癌前病变→喉癌阳性强度逐渐递减的现象（P＜0.01）。PCNA和P⁵³在声带息肉→喉癌前病变→喉癌呈表达上调现象（P＜0.05）。3.喉癌前病变P21^Waf1弱及中强阳性（+、++）表达率在痰瘀互结证和痰湿证组间差异无统计学意义（P＞0.05）,强阳性（+++）表达率组间差异有统计学意义（P＜0.05）。喉癌前病变cyclinD1蛋白表达各级阳性率在痰瘀互结证和痰湿证组间差异均无统计学意义（P＞0.05）。PCNA阳性表达率痰瘀互结证与痰证组组间差异有统计学意义（P＜0.05）。4.喉癌前病变cyclin E、P16蛋白表达各级阳性率在痰瘀互结证和痰湿证组间差异无统计学意义（P＞0.05）。四、研究结论（一）喉癌前病变的中医四诊及证候特点1.中医四诊特点:喉癌前病变以声嘶、咽异物或痰粘感、咳嗽有痰、咽痛、胸闷不舒、讲话费力为主症,尚可有咽干、气短等症;舌象以淡红舌、暗舌、红舌为主;脉象以弦滑、弦细多见。2.中医证候特点:喉癌前病变证候因素为痰瘀,以痰瘀互结为主要证候,痰证及血瘀证次之,尚可兼杂阴虚、气虚、热、毒等。3.临床采用活血化痰法对喉癌前病变具有一定的干预治疗作用,可以在喉显微术后较快缓解症状,延缓或减少复发（P＜0.05）。（二）喉癌前病变发展过程中证候及舌象演变初步规律1.中医证候演变特点:与声带息肉比较,喉癌前病变证候因素相对复杂,兼杂情况较多见（P＜0.05）;而与喉癌前病变比较,喉癌的证候因素更复杂,证候兼杂情况更普遍（P＜0.05）。从声带息肉→喉癌前病变→喉癌的病情发展变化过程中,证候演变存在一定规律:以痰瘀为主线,证候由单一向证候兼杂发展,最终出现痰瘀热毒、气阴两虚等多种证候因素兼杂的情况,证候的兼杂、复杂化及热毒的出现可能提示病邪深入,正气受损,预示病情的不良进展,值得临床注意。2.舌象变化特点:从声带息肉→喉癌前病变→喉癌,随着病情的发展变化,舌象可由正常的淡红舌为主,向暗红舌、红绛舌、紫舌等发展;舌苔由淡白、白腻向薄黄、黄腻到黄老厚腻变化发展;舌边瘀斑瘀点及舌下静脉曲张的情况更普遍;喉癌中少舌或无苔的出现率更高。这些预示病邪的深入,正气亏损,痰瘀热毒向纵深发展。提示当喉癌前病变患者舌质颜色加深,舌苔由薄变厚,出现绛舌或紫舌、裂纹舌,花剥苔时,应注意恶变倾向,应予重点密切随访及采取必要干预措施。（三）痰瘀在喉癌前病变发生发展中的作用及分子生物学机制1.从良性病变（声带息肉）→喉癌前病变→原位癌,相关的细胞周期因子、凋亡增殖因子有一个动态变化发展,这些相关基因蛋白可能对喉癌前病变的预后发展有一定标志物作用,可作为判断预后的较可靠指标。2.痰瘀在喉癌前病变发生发展中的作用可能为:以痰瘀互结为主线,在病情发展过程中,痰瘀互结,郁而化火,形成痰热、瘀毒等,同时痰瘀作为致病因子,攻伐、耗伤正气,导致气血阴阳失调,各种病机因素相互胶结,病情向纵深进一步发展,正虚邪实,阴阳气血失调,导致喉癌前病变向不良方向持续发展。3.痰瘀致喉癌变的分子生物学机制可能为通过影响细胞周期G1/S转换期正负调控因子而发挥作用,推测在痰瘀互结深入发展时,正性调控因子cyclinD1、cyclinE及PCNA高表达,负性调控因子P16、P21（WAF1/CIP1）低表达,导致细胞周期调控异常,肿瘤恶性增生而致喉癌发生,提示痰瘀型喉癌前病变恶变的风险可能相对较大,临床上对这类患者要加以重点随访追踪。（四）展望本项目从文献、临床、实验三个方面,从细胞周期调控角度探讨痰瘀与喉癌前病变发生发展的关系,识别喉癌前病变中有较高恶变潜质的中医证候类型,初步总结喉癌前病变的基本病因病机,从细胞分子生物学水平阐述喉痰瘀致喉癌变的机理,目前国内外未见系统研究报道,为今后进一步对喉癌前病变展开深入研究打下了较好的基础,对肿瘤的中医药防治工作有一定的参考价值。但由于研究时间有限,喉癌前病变病例较少,研究不可避免存在诸多不足之处,需要在今后的研究工作中不断补充修正。更多还原

【Abstract】 ObjectiveLaryngeal cancer is one common malignant tumour of the Head and Neck, Precancerous lesion is the center link of carcinogenesis,it is very important to study the molecular mechanism of carcinogenesis for the preventing and treatment of cancer.The preliminary study showed that Phlegm stagnation play a very important role in the process of laryngeal carcinogenesis,but its molecular mechanism is still unknown.To investigate the role and molecular mechanism of Phlegm stagnation in the process of laryngeal carcinogenesis.The project intends to study on laryngeal precancerous lesions from three aspects:the literature research,clinical investigation and experimental research.As to get a preliminary pathogenesis study of laryngeal precancerous lesions with comprehensive analysis,to explore the basic characteristics of Traditional Chinese Medicine syndrome of laryngeal precancerous lesions,and the characteristics and evolution changes of Traditional Chinese Medicine designate in the process of laryngeal carcinogenesis,as to identify the higher malignant potential Traditional Chinese Medicine syndrome type from molecular level,to explore the role and molecular biology mechanism of Phlegm stagnation syndrome in the process of laryngeal carcinogenesis.And to provide direction and ideas for research and treatment of laryngeal precancerous lesions and laryngeal cancer with Traditional Chinese Medicine.The study will accumulate date on study the pathogenic factors and pathogenesis of laryngeal precancerous lesions,and it provides a scientific evidence for the preventing and treatment of laryngeal precancerous lesions with Traditional Chinese Medicine,it undoubtedly has an important scientific significance and practical value.Methods Clinical Research1.The literature cases research use the CNKI（the Web version） and China biomedicine literature database（CBMdisc）,application software SPSS11.5 to analysis the laryngeal precancerous lesions in the literature of Traditional Chinese medicine,about the syndromes factors,the characteristics of four diagnostic methods and treatment prescription etc.2.The study of clinical investigation using the clinical popular survey method,combined with clinical information questionnaire of laryngeal precancerous lesions to collecte informations.To make a comprehensive analysis of the four diagnostic information of 88 cases of laryngeal precancerous lesions,simultaneously takes 100 vocal cord polyp cases and 60 laryngeal cancer cases as the progresses of diseases comparison research.A preliminary summary of laryngeal precancerous lesions of the characteristics of four diagnostic and the evolution characteristic of syndromes of Traditional Chinese medicine.3.The clinical observation research was to compare the therapeutic methods of Replenishing Qi and Activating blood circulation Resolving phlegm used in treating the laryngeal precancerous lesions.34 laryngeal precancerous lesions cases were divided into Replenishing Qi group and Activating blood circulation Resolving phlegm group,symptoms and signs such as a comprehensive quantitative score was observed,to compare therapeutic effect between Replenishing Qi group and Activating blood circulation Resolving phlegm group,as in improving symptoms and signs,and prevention recurrence of malignant aspects of laryngeal precancerous lesions.Experimental studyDefects in cell cycle regulation are common causes of the abnormal proliferation of cancer cells.The project use immunohistochemical method to detect expression of cyclin E,cyclin D1,P21,P16,and tumor suppressor gene PTEN,P53 in 50 cases of laryngeal precancerous lesions with Phlegm stagnation syndrome,simultaneously takes laryngeal precancerous lesions cases with Phlegm syndrome,the vocal cord polyp and the early laryngeal cancer specimens as control.ResultsClinical Research1.Judging from the literature,the current Traditional Chinese medicine study of laryngeal precancerous lesions is not deep enough,according to the clinical cases reported in the literature,the therapeutic methods is effective,accumulated some valuable clinical experience,and is worth for learning and reference.2.Phlegm stagnation syndrome is a main and important pathogenesis factor of laryngeal precancerous lesions,but not a single pathogenesis factor,there are Yin deficiency,Qi deficiency,heat or miscellaneous at the same time,as same as the previous literature study.Compared with vocal cord polyps and laryngeal cancer,Syndrome factors of laryngeal precancerous lesions is relatively complex（P＜0.05）.3.Laryngeal precancerous lesions have some common pathological characteristics:the sex ratio is extremely unbalanced,vast majority of men and aged over 40 years old,over-pronunciation,smoking may be the risk factors.Its main symptoms are hoarseness,almost 95%of cases have hoarseness,followed by foreign body sensation or sputum viscosity sensation in throat,dry or sore throat flu,less cough expectoration,there will be little difficulty swallowing or breathing difficulties.Dark tongue with spots ecchymosis,greasy moss,pulse string or thin slip mainly.It also supports Phlegm and blood stasis syndrome is the main pathogenesis factor of Laryngeal precancerous lesions.From vocal cord polyps,laryngeal precancerous lesions to laryngeal cancer,the cases showed an upward trend in miscellaneous syndromes（P＜0.05）.4.In terms of gender composition,age distribution in polyp of vocal cord, laryngeal precancerous lesions and laryngeal cancer was significant difference（P＜0.05）.The lead cause of vocal cord polyp may closely related to the excessive use of voice,while the risk factors of laryngeal precancerous lesions and laryngeal cancer may be long-term history of alcohol and smoking.The positive rate of treponemapallidum syphilisimmune body in cases of vocal cord polyps,laryngeal precancerous lesions and laryngeal cancer is 0%,1.92%,5%by statistics,compared with laryngeal cancer,the positive rate of treponemapallidum syphilisimmune body in laryngeal precancerous lesions cases was no significant inter-group,while compared with vocal cord polyps cases,the positive rates of treponemapallidum syphilisimmune body between group is significant,whether the occurrence of laryngeal cancer has certain relationship with treponemapallidum infection,it is worth discussing.The statistics shows the rate of carcinogenesis of laryngeal precancerous lesions is 2.63%,and the time for carcinogenesis of laryngeal precancerous lesions appeares to be 2-5 years.5.The therapeutic methods of Activate blood circulation and Resolving phlegm applied after laryngomicro-surgery on laryngeal precancerous lesions shows a certain therapeutic effect,such as to alleviate symptoms,improve the signs,slow down or reduce the recurrence（P＜0.05）etc. Experimental study1.P21^Waf1,cyclinD1,cyclin E,P16 are mainly expressed in cell nucleus, in vocal cord polyps cases,P21^Waf1,Pl6 protein expression dyeing coloration is deep,and the masculine cell is crowded.In laryngeal precancerous lesions P21^Waf1,P16 protein expression dyeing shoal,and the depth varies,the masculine cell reduces,positive cells decreased.From vocal cord polyps,laryngeal laryngeal precancerous lesions to laryngeal cancer,the positive intensity was gradually decreasing（P＜0.05）.Just on the contrary,the positive strength expression of cyclin E,cyclinD1 protein in vocal cord polyps,laryngeal precancerous lesions and laryngeal cancer is gradually increased（P＜0.05）.2.PTEN mainly expressed in cytoplasm,P53,PCNA expression in the nucleus mainly.PTEN protein expression in vocal cord polyps is deep dye staining and positive cell density.PTEN protein expression in laryngeal precancerous lesions is dyeing shoal and masculine cell reduces.PTEN protein expression in laryngeal cancer staining shallow,less-positive cells.From vocal cord polyps,laryngeal precancerous lesions to laryngeal cancer,positive intensity was gradually decreasing（P＜0.01）.The positive intensity expression of PCNA and P53 protein in vocal cord polyp,laryngeal precancerous lesions and laryngeal cancer showed a upward trend（P＜0.05）.3.In laryngeal precancerous lesions of Phlegm stagnation group and phlegm group,the positive rate of P21^Waf1protein expression was of no statistically significant（P=1.0000）,weak and strong masculine gende（+,++）is of the same（P＞0.05）.Expression of the strong masculine gender（+++）was significant（P=0.0228）.Positive rate of cyclinD1 protein expression levels in Laryngeal precancerous lesions with Phlegm stagnation or phlegm was of no statistically significant difference（P=1.0000,P=0.6618,P=0.6132）.4.In laryngeal precancerous lesions specimens with Phlegm stagnation or phlegm syndrome,all levels of masculine genders rate of cyclin E,P16 protein expression was of non-statistics significance（P＞0.05）.Conclusions1.Phlegm stagnation play an important role in the process of the occurrence and development of laryngeal precancerous lesions,it runs through all the process of carcinogenesis.Phlegm stagnation is one of the basic etiology and pathogenesis of laryngeal precancerous lesions.With the malignant developing of laryngeal precancerous lesions,the miscellaneous syndromes become more common and complex.The variety of syndromes and miscellaneous complex situation of laryngeal precancerous lesions may prompt its malignant transformation.Clinical use of Activate blood circulation Resolving phlegm should be a right direction.Tobacco and alcohol may be the risk factors of malignant transformation of laryngeal precancerous lesions.2.From benign lesions（vocal polyps）to laryngeal precancerous lesions to laryngeal carcinoma in situ,there is a dynamic changes of the cell cycle-related factor,apoptosis factor.P21^Waf1,Pl6 expression in vocal cord polyp was of the deepest dye staining and positive cell density,In laryngeal precancerous lesions,P21^Waf1,P16 protein expression staining shallow and positive cells decrease.In laryngeal cancer the proteins expression was superficial staining and positive cells less.From vocal cord polyps,laryngeal precancerous lesions to laryngeal cancer,positive rate was of gradually decreasing intensity.And the expression of cyclin E,cyclinD1 protein in vocal cord polyps,laryngeal precancerous lesions and laryngeal cancer is just on the contrary,the positive gradient strength is gradually increased （P＜0.05）.The relevant gene protein may be the prognosis markers of laryngeal precancerous lesions,can be used to as a more reliable indicator of carcinogenesis.3.As to strong positive（+++）expression of P21 in laryngeal precancerous lesions,there were significant differences between group with Phlegm stagnation and phlegm syndrome.It shows that laryngeal precancerous lesions with Phlegm stagnation syndrome was of the higher malignant potential,while group with Phlegm syndrome was of less malignant potential.Suggesting that need to strengthening monitoring and intervention of laryngeal precancerous lesions with Phlegm stagnation syndrome.4.Tongue can reflect healthy condition of the body,The tongue from normal pale red to dark red indicates the process of pathogenic fire and phlegm coagulation with stagnation.Tongue fur from whitish to thin greasy yellow to yellow,indicating the progress of disease.When color of patients’ tongue with laryngeal precancerous lesions deepened,thickens thinly,presents deep red tongue,purple tongue,crack tongue,or yellow greasy moss,attention should be given to the tendency of malignant transformation,and it is necessary to follow-up closely and take preventing interventions.5.The study on laryngeal precancerous lesions exploring the relationship between the occurrence of laryngeal cancer and Phlegm stagnation from perspective of cell cycle regulation,identification which Syndrome type of laryngeal precancerous lesions is of higher malignant potential.To get a initial summary of the basic pathogenesis of laryngeal precancerous lesions,exploring the molecular mechanism of carcinogenesis from the level of molecular biology.At present,there was no systematic research domestic and foreign about these,the project will build a good foundation for cancer prevention and treatment with Chinese medicine,and have some reference value for future study on laryngeal precancerous lesions.Due to few research time and cases of laryngeal precancerous lesions,there are many unavoidable deficiencies,it needs for further research work.更多还原