喉癌前病变中医证候分布特点及细胞周期调控机制研究
|
摘要
目的：
喉癌是头颈部恶性肿瘤中最常见的疾病之一,仍严重威胁人类的生命和健康。喉癌前病变是喉癌变的中心环节,它既可以恶化发展转化为癌,也可以良性逆转为正常细胞。早期发现并进行干预,可以防止其向恶性肿瘤发展。因此,加强对喉癌前病变的研究,深化对癌变机理的认识,已成为头颈肿瘤防治工作的前沿和重点之一。
目前研究认为,细胞周期调控受正负两大类因子的调节,正负两类因子之间的平衡被打破,就可能导致肿瘤的发生,细胞周期调控异常是肿瘤发生发展的一个重要前提。这与中医认为疾病是人体阴阳平衡失调所致具有相似之处。祖国医学理论认为痰瘀是导致肿瘤发生发展的重要因素,现代中医临床及导师的前期临床研究均发现采用活血化痰法对喉癌前病变具有较好的治疗作用。但喉癌前病变的中医基本病因病机,及痰瘀在喉癌前病变的发生发展中的作用机制,目前尚缺乏科学的支持与验证。
本研究拟在文献研究的基础上,在临床方面对喉癌前病变的中医四诊特征及基本证型分布特点进行研究,为喉前期病变的中医临床辨治提供科学依据,同时也为中医证候的客观化、量化研究提供新的研究途径。在实验方面对喉癌前过程及喉癌前病变痰瘀证的细胞周期调控机制进行研究,深化对喉癌前病变发生发展及痰瘀致癌的机理认识,确立具有较高恶变潜质的中医证候类型。从而为临床上采用活血化痰法阻断逆转喉癌前病变提供理论支撑与实验依据,为中医药对癌前病变的防治提供思路与方向。方法：
1.文献研究
通过古文献数据库(湖南电子音像出版社的《中华医典》及广东省中医院重点专科中医古籍文献数据库)及现代文献数据库(中国生物医学文献数据库CBMdisc、维普中文生物医学期刊数据库VIP Information、中国知网数据库CNKI、美国NCBI的PubMed、Medline数据库)的查阅挖掘,将相关的古文条文及现代文献报道分类归纳,总结出喉癌前病变的古代在病因病机、辩证分析、施治方法,现代在流行病学、分类诊断、治疗方法等方面的特点与进展。
2.临床调研
本研究拟通过运用循证医学、临床流行病学等现代科研方法学结合传统医学进行喉前期病变的临床病例的证候信息采集。通过建立流行病学调查表,对214例喉癌前病变的四诊资料集流行病学资料进行采集,采用Epidata3.1软件包建立数据库,双人双输的办法进行数据录入及核对,应用SPSS17.0软件对数据进行统计分析,初步得出喉癌前病变的证候诊断重要指标、证型类型及分布相关情况及流行病学特点。
3.实验研究
本研究拟利用流式细胞术,检测痰瘀证的喉癌前病变标本中细胞周期因子CyclinD1、Cyclin E、p21、p16蛋白的表达情况,将喉癌前病变阴虚证标本、声带息肉及早期喉癌标本作为证候对照及病情进展对照研究。通过对声带息肉→喉癌前病变→早期喉癌、喉癌前病变、喉癌的不同病理类型及分级组织中的细胞周期因子动态变化的分析,及对痰瘀证、阴虚证癌前病变组织中细胞周期因子表达情况的对比分析,明确喉癌变过程及痰瘀证喉癌前病变的细胞周期调控机制。
结果：
1.临床研究
(1)发病因素：喉癌前病变患者中有长期过度用嗓发音占74.8%、有长期吸烟史者占43.9%,有饮酒习惯者占21.5%,饮食不节者占27.6%,熬夜者占9.3%,感冒者占5.1%。发病因素在三种病理类型中无显著性差异(P值均大于0.05)。
(2)性别构成：慢性肥厚性喉炎(非典型增生)中男性60例,女性92例；喉乳头状瘤中男性22例,女性10例；喉角化症(声带白斑)中男性20例,女性10例。三组的男女性别比为0.65：1、2.2：1及2：1,后两者男性均多于女性,性别构成组间差异有统计学意义(P<0.05)。
(3)常见症状体征频数：出现频率大于10%的症状依次有：声音嘶哑(声嘶轻度、声嘶中度)、咽异物感、咽干灼热、多言后喉痛、痛处不移、咽痒咳嗽、痰多质粘、清嗓习惯。体征出现频率大于10%的依次有声带闭合欠佳、声带充血肥厚、肿物表面光滑、肿物结节状或乳头状、肿物白斑样改变。舌苔脉象出现频率大于10%的依次有舌质暗或有瘀点、舌苔腻、脉弦、舌苔白、脉滑、舌质红、舌苔黄、舌苔薄、舌胖有齿印、脉细、脉沉。
(4)证候聚类分析：根据计算机聚类分析的结果,结合专家意见认为,应聚为4类：工类：声音嘶哑、舌质红、舌苔黄、舌苔腻、脉滑、肿物结节状或乳头状、声带闭合不好。Ⅰ类：清嗓习惯、多言后喉痛、痛处不移、咽异物感、舌胖有齿印、舌质暗或有瘀点、脉沉、声带充血肥厚、肿物光滑。Ⅲ类：声嘶轻、痰多质粘、咽痒咳嗽、舌苔白、脉弦、声带闭合尚好。Ⅳ类：声嘶中重度、咽干灼热、舌苔薄、脉细、肿物白斑样改变。
(5)证候诊断的重要指标按主症、次症、舌苔脉象、局部体征依次如下：
①湿热困结证主症：声嘶；舌苔脉象：舌质红、舌苔腻、舌苔黄、脉滑；局部体征：声带充血肥厚、声带闭合不好、肿物结节状或乳头状。
②气虚痰瘀证主症：声嘶；次症：清嗓习惯、咽异物感、多言后喉痛、痛处不移；舌苔脉象：舌质暗或有瘀点、舌苔腻、舌苔白、脉弦、脉滑、脉沉；局部体征：声带充血肥厚、肿物光滑。
③痰浊结聚证主症：声嘶(轻)；次症：痰多质粘、咽瘁咳嗽；舌苔脉象：舌苔白、脉弦；局部体征：声带充血肥厚、肿物表面光滑、声带闭合尚好。
④阴虚火旺证主症：声嘶(中重度)；次症：咽干灼热、咽异物感；舌苔脉象：舌质红、舌苔白、舌苔薄、脉细；局部体征：声带充血肥厚、肿物白斑样改变。
(6)证型分布情况：Ⅰ类：湿热互结型；Ⅱ类：气虚痰瘀型；Ⅲ类：痰浊困结型；Ⅳ类：阴虚火旺型。其中湿热互结型49例,占22.9%；气虚痰瘀型87例,占40.7%；痰浊困结型23例,占10.7%；阴虚火旺型55例,占25.7%。
(7)证候分布的相关因素：
①中医证候与性别的关系：在湿热困结证中,男性所占比率较大(67.3%),而在气虚痰瘀证、痰浊结聚证、阴虚火旺证中,女性所占比率较大(分别为58.6%、65.2%、54.5%),统计学上有显著差异(P<0.05)。
②中医证候与年龄的关系：喉癌前病变的中年组患者中,气虚痰瘀证占较大比率(42.9%),其他证型相对分散；青年组及老年组各证型均相对分散,统计学上均无意义(P>0.05)。
③中医证候与病程的关系：喉癌前病变各证候间的平均发病时间(28.17～37.67个月)相差不大,病程时间呈非正态分布,采用秩和检验,P>0.05,病程在各证候间的差异无统计学意义。
④中医证候与病理类型的关系：慢性肥厚性喉炎、喉乳头状瘤多见气虚痰瘀证(分别占40.1%、56.3%),喉白斑角化病多见阴虚火旺证(占33.3%),但各证候间的构成比差异无统计学意义(P>0.05)。
⑤中医证型与病因的关系：吸烟、饮酒在湿热困结、气虚痰瘀这两种证型中多见,且有统计学意义(P<0.05)。饮食不节、熬夜在湿热困结证、气虚痰瘀证中多见；用声过度、感冒在气虚痰瘀证、阴虚火旺证中多见,但均没有统计学意义(P>0.05)。
⑥中医证型与局部体征的关系：肿物表面光滑多见于气虚痰瘀证中(44.9%),肿物呈结节状或乳头状的多见于湿热困结证(47.1%),肿物白斑样改变多见于阴虚火旺证(68.8%),而且统计学上有显著差异(P=0.001<0.05),因“肿物粗糙质脆易出血”例数较少(3例),暂不做分析。
⑦中医证型与声嘶程度的关系：声嘶程度根据VHI-10问卷调查表中的嗓音障碍指数得分而来,各个中医证型均集中在轻度异常与中度异常之间(轻度异常的共50例,中度异常的共150例,正常的共6例,严重失常的共8例),没有统计学意义(P>0.05)。
(8)复发率与恶变率
本研究对其中137例喉癌前病变进行了随访,共有15例发生复发,4例为声带白斑,4例为喉乳头状瘤,7例为声带息肉伴轻度～中度不典型增生。本研究得到喉癌前病变的复发率为10.95%。其中有6例发生恶变,4例为声带白斑,1例为喉乳头状瘤,1例为声带鳞状上皮中度不典型增生。恶变者均有多年吸烟史饮酒史。本研究得到的喉癌前病变的恶变率为4.38%。
2.实验研究
(1) CyclinD1的阳性表达率形成声带息肉→喉癌前病变→喉癌阳性强度逐渐增强的梯度现象(分别为13.04%、20.29%、42.42%),但总体上荧光强度较弱；三组分别比较均有统计学差异(P<0.05)。在喉癌前病变组中,CyclinD1在病理分级中的轻度不典型增生、中度不典型增生、重度不典型增生的的阳性率表达分别为13.95%、28.57%、31.58%,呈上升趋势,但统计学上没有意义(P>0.05)。CyclinD1在不同病理类型中上皮不典型增生(慢性肥厚性喉炎)、喉乳头状瘤、喉角化症的阳性率表达分别为19.51%、21.05%、22.22%,但三组间差异无统计学意义(P>0.05)。在喉癌组中,不同病理类型高分化、中分化、低分化的CyclinD1的阳性率表达分别为40.00%、41.67%、43.75%；不同病理分级中原位癌、浸润癌的CyclinD1的阳性率表达分别为35.71%、47.37%,两者均呈上升趋势,但统计学上无显著性差异(P>0.05)。
(2) CyclinE的阳性表达率形成声带息肉→喉癌前病变→喉癌阳性强度逐渐增强的梯度现象(分别为26.09%、43.48%、93.94%)；且荧光强度较强,统计学上有显著差异(P<0.001)。在喉癌前病变组中,CyclinDl在病理类型中上皮不典型增生(慢性肥厚性喉炎)、喉乳头状瘤、喉角化症的阳性率表达分别为36.59%、52.63%、55.56%,呈上升趋势,但三组比较统计学上亦无意义(P>0.05)。病理分级中轻度不典型增生、中度不典型增生、重度不典型增生的三组中CyclinE的阳性率表达分别为25.58%、57.14%、78.95%,也呈上升趋势,统计学上有显著差异(P<0.001)。在喉癌组中,不同病理类型高分化、中分化、低分化的三组中CyclinE的阳性率表达与不同病理分级原位癌、浸润癌中的阳性率表达均较高(80%-100%),且呈上升趋势,但统计学上均无显著差异(P>0.05)。
(3)P16的阳性表达率形成声带息肉→喉癌前病变→喉癌阳性强度逐渐递减的现象(分别为60.87%、40.58%、15.15%),统计学上有显著性差异(P<0.001)。在喉癌前病变组中,P16在病理分级中轻度不典型增生、中度不典型增生、重度不典型增生的阳性率表达分别为51.16%、42.86%、15.78%,呈逐步下降趋势,统计学上有显著差异(P<0.05)；在病理类型中上皮不典型增生(慢性肥厚性喉炎)、喉乳头状瘤、喉角化症中的阳性率表达分别为41.46%、42.11%、33.33%,三组间差异无统计学意义(P>0.05)。在喉癌组中,不同病理类型高分化、中分化、低分化的P16的阳性率表达分别为20.00%、25.00%、12.50%,不同病理分级原位癌、浸润癌中P16的阳性率表达分别为14.29%%、15.79%,两者在统计学上均无显著性差异(P>0.05)。
(4)P21的阳性表达率形成声带息肉→喉癌前病变→喉癌阳性强度逐渐递减的现象(分别为47.83%、26.09%、24.24%)。但没有统计学意义(P>0.05)。在喉癌前病变组中,P21在病理类型上皮不典型增生(慢性肥厚性喉炎)、喉乳头状瘤、喉角化症中的阳性率表达分别为26.83%、26.32%、22.22%；在病理分级中轻度不典型增生、中度不典型增生、重度不典型增生的P21的阳性率表达分别为27.91%、28.57%、21.05%,呈下降趋势,但两者在统计学上均无差异(P>0.05)。在喉癌组中,不同病理类型高分化、中分化、低分化的P21的阳性率表达相差不大(分别为20.00%、25.00%、18.75%),不同病理分级原位癌、浸润癌中P21的阳性率表达分别为21.43%、21.05%,统计学上亦均无差异(P>0.05)。
(5)CyclinD1、CyclinE在痰瘀证中的阳性率表达相对较高(分别为23.40%、46.81%),在阴虚证中的阳性率表达相对较低(分别为9.09%、36.36%),但两两比较无统计学意义(P>0.05)。P16在痰瘀证中的阳性率表达相对较低(34.04%),在阴虚证中的阳性表达相对较高(45.0%),但两组间差异仍无统计学意义(P>0.05)。然而P21在痰瘀证与阴虚证中的阳性率表达则无明显差异(分别为27.66%、23.0%),P>0.05。
结论：
1.喉癌前病变的流行病学特点
(1)喉癌前病变的危险因素依次有用嗓不当、吸烟、饮酒、饮食不节、熬夜、感冒等。
(2)喉癌前病变的性别构成在喉乳头状瘤与喉白斑角化病中男女之比约为2：1；在慢性肥厚性喉炎中的比例则为0.65：1。
(3)喉癌前病变的复发率为10.95%,喉癌前病变的恶变率为4.38%。
2.喉癌前病变的中医病因病机、证候特征及分布特点
(1)中医的病因病机主要有：外因为吸烟喝酒、外感之湿热毒邪；内因则为饮食不节损伤脾胃致痰浊结聚,过度用声、熬夜致劳累过度致正气虚损、气阴两伤。
(2)喉癌前病变的中医证型按由大至小的比例分布情况,主要分为气虚痰瘀型、阴虚火旺型、湿热互结型、痰浊困结型,共4类。其证候诊断的重要指标特点可见结果部分内容(略)。
(3)中医证型分布相关性分析中,男性多见湿热困结证,女性多见气虚痰瘀证、痰浊结聚证、阴虚火旺证；吸烟史、饮酒史多见于湿热困结型和气虚痰瘀型两种证型；喉癌前病变的局部体征对中医证型分布有显著差异(略)。病理类型、年龄、病程、声嘶程度对喉癌前病变的中医证型分布无明显影响。
3.喉癌变过程及中医证型的细胞周期调控机制
(1) CyclinD1、CyclinE在声带息肉→喉癌前病变→喉癌组、喉癌前病变的轻度不典型增生→中度不典型增生→重度不典型增生组中的表达均呈现逐渐增强的梯度现象,统计学上特别是第一组均有显著差异,提示正性调控因子CyclinDl及CyclinE的过度表达,导致细胞周期加快,肿瘤恶性增生。CyclinDl及CyclinE在喉癌的不同分化类型及病理分级中,尚未呈现明显差异,可能与标本量少有关。总体上CyclinE在恶性程度高的病变组织中的表达较CyclinDl高,CyclinE是否比CyclinDl在G1/S限制点上发挥更大的促进作用,仍需进一步深入研究。
(2)P16、P21在声带息肉→喉癌前病变→喉癌组、喉癌前病变的轻度不典型增生→中度不典型增生→重度不典型增生组中,阳性率表达均呈逐渐减弱的梯度现象,但只有P16在统计学上有显著性差异。提示P16作为抑癌基因,在抑制CDK4/CDK6介导的Rb磷酸化,阻止不正常细胞在G1/S的进程上起重要的作用。而在喉癌的不同分化类型、病理分级中,P16、P21的阳性率表达均较低,但组间比较仍无显著差异,仍考虑与样本量有限相关。
(3) CyclinD1、CyclinE在痰瘀证中的阳性率表达相对较高,在阴虚证中的阳性率表达相对较低；P16在痰瘀证中的阳性率表达相对较低,在阴虚证中的阳性表达相对较高；P21在痰瘀证与阴虚证中的阳性率表达则相反。但以上表达的组间差异均无统计学意义。提示在痰瘀致病的过程中,正性调控因子CyclinD1、CyclinE的高表达,负性调控因子P16的低表达导致细胞周期调控异常可能是其分子生物学机制,但仍需进一步扩大样本量深入研究以验证。
4.不足与展望
虽然本研究仍存在许多不足,如研究资料有限,包括样本量不够、非多中心研究；研究设计有所欠缺,包括临床调研表内容及实验研究标本的采集方法选择等。现有的研究结果暂未能为中医辩证及其相关细胞分子生物作用机制提供明确依据,仍必须继续在今后的临床及实验实践中逐步修改和完善。
Objective
Laryngeal cancer is one of the most common diseases in the head and neck cancer remains a serious threat to human life and health. Laryngeal precancerous lesions is the central link of becoming laryngeal cancer. It not only can worsen development transformed into cancer, but also can be benign reversal of the normal cells. Early detection and intervention can prevent its development to malignancy. Therefore, the strengthening study of laryngeal precancerous lesions, to deepen the understanding of cancer mechanism, has become one of the forefront and key of head and neck cancer prevention and control work.
The preliminary study suggested that cell cycle regulation was adjusted by two major factors of positive and negative types. The balance between them once became broken, it could lead to tumorigenesis. This had similarities with diseases caused by imbalance of human yin and yang in TCM. Abnormal cell cycle regulation was an important prerequisite for tumor development, and phlegm and blood stasis was an important factor leading to tumor development. Pre-clinical study of modern TCM and my tutor had found better therapeutic effects of laryngeal precancerous lesions with blood circulation and phlegm resolving method. But the TCM etiology and pathogenesis of Laryngeal precancerous lesions, and the mechanism of phlegm and blood stasis in the development of laryngeal precancerous lesions remained the lack of scientific support and validation currently.
Basing on the literature research, This research will investigate the characteristics of the four diagnostic methods and distribution of the types of syndrome on laryngeal precancerous lesions in the clinical aspects. We will also study the cell cycle regulation mechanisms in process of laryngeal precancerous lesions and Phlegm and Blood Stasis of it In the experimental aspects. The results will provide ideas and direction for the medicine in prevention and treatment of precancerous lesions, and the theoretical support and experimental basis for blood circulation and phlegm resolving method of laryngeal precancerous lesions in clinic.
Methods
1. Literature research
Searching through ancient literature database such as Chinese Medical Code from Hunan Electronic Audio and Video Publishing House, and Chinese ancient literature database from the Guangdong Provincial Hospital, and meanwhile through modern literature database such as Chinese biology and medicine database (CBM),Chinese biomedical journals in the database (VIP Information),Chinese national knowledge infrastructure database (CNKI) and PubMed or Medline database of American national center for biotechnology information (NCBI).We summarized the relevant provisions of the classical and modern literature, and summed up the etiology and pathogenesis of laryngeal precancerous lesions in the ancient, dialectical analysis of the characteristics and treatment methods, modern epidemiology, classification, diagnosis, treatment methods and progress.
2. Clinical research
This study collected clinical cases of the syndrome information of laryngeal precancerous lesions through the use of evidence-based medicine, clinical epidemiology, and other modern research methodology combined with TCM. Through the establishment of an epidemiological questionnaire, the four diagnostic information and epidemiological data of214cases of laryngeal precancerous lesions were collected. With Epidata3.1packages to establish a database for data entry and verification, SPSS17.0statistical software for data analysis, the result preliminary draw the important indicators of syndrome diagnosis of laryngeal precancerous lesions, syndrome distribution and epidemiological features.
3. Experimental research
In this study, using flow cytometry, we will detect expressions of cell cycle factors (Cyclin Dl, Cyclin E, p21and p16protein) on specimens of phlegm and blood stasis type of laryngeal precancerous lesions, Yin deficiency type of the Laryngeal precancerous lesions, vocal cord polyps and early laryngeal cancer as the control groups. Through analysis of dynamic changes of the cell cycle factor in vocal cord polyps→laryngeal precancerous lesions→early laryngeal cancer, and different histological types and pathological classification of laryngeal precancerous lesions and laryngeal cancer, It will be made clear that cell cycle control mechanisms in the process of laryngeal cancer and phlegm and blood stasis type of laryngeal precancerous lesions.
Results
1. Clinical research
(1) Pathogenic factors:In group of laryngeal precancerous lesions, long-term excessive use of voice pronunciation accounted for74.8%, long history of smoking accounted for43.9%, drinking habits accounted for21.5%, improper diet accounted for27.6%, staying up too late to sleep accounted for9.3%, getting cold accounted for5.1%. Among the three pathological types, there is no significant difference.
(2) The gender composition:There are60male cases and92female cases of chronic hypertrophic laryngitis (atypical hyperplasia),22male cases and10female cases of laryngeal papilloma,20male cases and10female cases of laryngeal keratosis (vocal cord leukoplakia), The sex ratio of the three groups are as follows:0.65:1,2.2:1and2:1, and there are more males than females in the latter two groups, the gender composition between the groups has difference which has statistic significance (P <0.05).
(3)The frequency of common signs and symptoms:The frequency of the symptoms which occurred greater than10%were:hoarseness (mild hoarseness, moderate hoarseness), foreign body sensation of pharynx, dry and burning throat, sore throat after over saying, and the pain did not move, itchy throat and cough, profuse sticky phlegm, preference of clearing the throat. The frequency of the signs which occurred greater than10%were poor vocal cord closure, vocal cord congestion and hypertrophy, smooth tumor surface, nodular、papillary or leukoplakia change of the tumor. The frequency of the tongue coating and pulse which present greater than10%were dark tongue or with Petechiae, greasy tongue coating, wiry pulse, white tongue coating, slippery pulse, red tongue, yellow tongue coating, thin tongue coating, fat tongue with teeth marks, thready pulse, deep pulse.
(4) Syndrome cluster analysis:According to the results of computer cluster analysis, and combined with expert opinion, it should be clustered into four categories:Class I:hoarseness, red tongue, yellow tongue coating, greasy tongue coating, slippery pulse, nodular or papillary tumor, poor vocal cord closure. Class Ⅱ:clearing throat habits, sore throat after over saying, and the pain did not move, foreign body sensation of pharynx, fat tongue with teeth marks, dark tongue or with petechia, deep pulse, vocal cords congestion and hypertrophy, smooth tumor surface. Class Ⅲ:mild hoarseness, profuse sticky phlegm, cough, itchy throat, white tongue coating, wiry pulse, normal vocal cord closure. Class IV: severe hoarseness, dry and burning throat, thin tongue coating, thready pulse, and tumor leukoplakia-like change.
(5) The important index of syndrome diagnosis according to main symptom, secondary symptom, tongue coating and pulse, local signs in turn are as follows:
①Syndrome of damp-heat stagnation main symptoms:hoarseness; tongue coating and pulse:red tongue, greasy tongue coating, yellow tongue coating, slippery pulse; local signs:vocal cords congestion and hypertrophy, poor vocal cord closure, nodular or papillary tumor.
②Syndrome of qi deficiency and phlegm stasis main symptoms: hoarseness; secondary symptoms:preference of clearing voice, foreign body sensation of pharynx, sore throat after over saying, and the pain did not move; tongue coating and pulse:dark tongue or with petechia, greasy tongue coating, white tongue coating, wiry pulse, slippery pulse, deep pulse; local signs:vocal cords congestion and hypertrophy, smooth tumor surface.
③Syndrome of turbid phlegm accumulation main symptoms: hoarseness(mild); secondary symptoms:profuse sticky phlegm, itchy throat and cough; tongue coating and pulse:white tongue coating, wiry pulse; local signs:congestive hypertrophy of the vocal cords, smooth tumor surface, normal vocal cords closure
④Syndrome of hyperactivity of fire due to yin deficiency main symptoms:hoarseness(severe):dry and burning throat, foreign body sensation of pharynx; tongue coating and pulse:red tongue, white tongue coating, thin tongue coating, thready pulse; local signs:congestive hypertrophy of the vocal cords, nodular papillary or leukoplakia change of the tumor.
(6) Syndrome distribution:Class Ⅰ:syndrome of damp-heat stagnation; Class Ⅱ:syndrome of qi deficiency and phlegm stasis; Class Ⅲ:syndrome of turbid phlegm accumulation; Class Ⅳ:syndrome of hyperactivity of fire due to yin deficiency. among which syndrome of damp-heat stagnation are49cases, accounting for22.9%; syndrome of qi deficiency and phlegm stasis are87cases, accounting for40.7%; syndrome of turbid phlegm accumulation are23cases,10.7%; syndrome of hyperactivity of fire due to yin deficiency are55cases, accounting for25.7%.
(7) The syndrome distribution factors as follows:
①The relationship between the Traditional Chinese Medicine syndrome and gender:Male accounted for a large percentage (67.3%) for the syndrome of damp-heat stagnation, while the female accounted for a large percentage (respectively58.6%and65.2%,54.5%) for the syndrome of qi deficiency and phlegm stasis, turbid phlegm accumulation, hyperactivity of fire due to yin deficiency of which has statistic significant differences (P<0.05).
②The relationship between the Traditional Chinese Medicine syndrome and age:Qi deficiency and phlegm and blood stasis account for a large percentage (42.9%) of the laryngeal precancerous lesions of the middle-aged patient,other syndromes are relatively dispersed; each type in the youth group and older group were relatively scattered, it has no statistic significance (P>0.05).
③The relationship between the Traditional Chinese Medicine syndrome and duration:Difference between the average onset time of syndrome types of laryngeal precancerous lesions (28.17to37.67) was small, and it has no statistic significance (P>0.05)
④The relationship between the Traditional Chinese Medicine syndrome and pathological types:Qi deficiency and phlegm and blood stasis type was common in chronic hypertrophic laryngitis and laryngeal papilloma (accounting for40.1%、56.3%). Hyperactivity of fire due to yin deficiency type was common in laryngeal keratosis(accounting for33.3%). But it has no statistic significance (P>0.05)
⑤The relationship between the Traditional Chinese Medicine syndrome and pathogenic factors:History of smoking and drinking were common in damp-heat stagnation and Qi deficiency and phlegm and blood stasis types, and i t had statistic significance (P<0.05). Improper diet and staying up too late to sleep were common in Qi deficiency and phlegm and blood stasis types, and long-term excessive use of voice and getting cold were common in Qi deficiency and phlegm and hyperactivity of fire due to yin deficiency types, but those had no statistic significance (P>0.05)
⑥The relationship between the Traditional Chinese Medicine syndrome and local signs:Smooth tumor surface was related to Qi deficiency and phlegm and blood stasis type (44.9%), nodular or papillary tumorn related to damp-heat stagnation (47.1%), leukoplakia change of the tumor related to hyperactivity of fire due to yin deficiency types (68.8%).They had statistic significance (P<0.05)
⑦The relationship between the Traditional Chinese Medicine syndrome and degree of hoarseness:The hoarseness degree of voice came from the score of disability index in VHI-10questionnaire. There were50cases of mildly abnormal patients,150cases of moderately abnormal cases, six of the normal cases, eight of severe disorders cases, which had not statistically significant (P>0.05).
(8) Rate of recurrence and malignant transformation:In this study,137cases of laryngeal precancerous lesions were followed up. There were15cases of recurrence, four cases of vocal cord leukoplakia, four cases of laryngeal papilloma,7cases of vocal cord polyps with mild to moderate dysplasia. Recurrence rate of laryngeal precancerous lesions was10.95%. Among those, six cases of mal ignant transformation, four cases of vocal cord leukoplakia, one cases of laryngeal papilloma, and one case of squamous moderate atypical hyperplasia in vocal cord. Malignancy cases have years of history of smoking and drinking history. Malignant transformation rate of laryngeal precancerous lesions was4.38%.
2. Experimental research
(1) Expressions of Cyclin D1had a progressive enhancement from vocal cord polyps, laryngeal precancerous lesions to Laryngeal cancer(the percentage as follows:13.04%、20.29%、42.42%). However, all of the fluorescence intensity are weak. There were statistical differences in pairwise comparisons (P<0.05). The result of mild dysplasia, moderate dysplasia and severe dysplasia in laryngeal precancerous lesions had an up trend of positive expressions, which were13.95%、28.57%、31.58%, but there was no significant difference (P>0.05). Expressions of Cyclin D1 in epithelium atypical hyperplasia of different types(chronic hypertrophic laryngitis), laryngeal papilloma and laryngeal keratosis were19.51%,21.05%,22.22%, respectively. There was no statistical significance among the three (P>0.05). The positive expression rates of Cyclin D1in well differentiated was40.00%, moderately differentiated41.67%and poorly differentiated43.75%in different pathologic types of laryngeal carcinoma, and carcinoma in situ was35.71%, invasive carcinoma47.37%in different pathological grade of laryngeal carcinoma. Both showed an increasing tendency, without statistical significances (P>0.05)
(2)Expressions of Cyclin E had a Progressive enhancement from vocal cord polyps, Laryngeal precancerous lesions to Laryngeal cancer(the percentage as follows:26.09%、43.48%、93.94%). All of the fluorescence intensity are strong. There were statistical differences in pairwise comparisons (P<0.001). The result of mild dysplasia, moderate dysplasia and severe dysplasia in laryngeal precancerous lesions had an up trend of positive expressions, which were36.59%、52.63%、55.56%, but there was also no significant difference (P>0.05). Expressions of Cyclin D1in epithelium atypical hyperplasia of different types(chronic hypertrophic laryngitis), laryngeal papilloma and laryngeal keratosis were25.58%,57.14%and78.95%, respectively. It showed an increasing tendency, and there was statistical significance among the three (P<0.001).The positive expression rates of Cyclin Dl in different pathologic types and grade of laryngeal carcinoma were high (80%～100%), a rising trend. But there were no statistical significance (P>0.05)
(3) Expressions of P16had a diminishing gradient phenomenon from vocal cord polyps, laryngeal precancerous lesions to Laryngeal cancer (the percentage as follows:60.87%、40.58%、15.15%). There were statistical differences in pairwise comparisons (P<0.05). The result of mild dysplasia, moderate dysplasia and severe dysplasia in Laryngeal precancerous lesions had an downward trend of positive expressions, which were51.16%、42.86%、15.78%, and had significant difference (P<0.05). Expressions of P16in epithelium atypical hyperplasia of chronic hypertrophic laryngitis, laryngeal papilloma and laryngeal keratosis were41.46%、42.11%、33.33%, respectively. There was no statistical significance (P>0.05). The positive expression rates of P16in high, mediate and low differentiated laryngeal carcinoma were20.00%、25.00%,12.50%, and in different pathological grade of laryngeal carcinoma, carcinoma in situ35.71%and invasive carcinoma47.37%.Both showed no statistical significances (P>0.05)
(4) Expressions of P21had a diminishing gradient phenomenon from vocal cord polyps, laryngeal precancerous lesions to Laryngeal cancer (the percentage as follows:47.83%、26.09%、24.24%). But there were no statistical differences in pairwise comparisons (P>0.05).The result of mild dysplasia, moderate dysplasia and severe dysplasia in Laryngeal precancerous lesions had an downward trend of positive expressions, which were27.91%、28.57%、21.05%.Expressions of P21in epithelium atypical hyperplasia of chronic hypertrophic laryngitis, laryngeal papilloma and laryngeal keratosis were26.83%、26.32%、22.22%, respectively. Both had no significant difference (P<0.05). The positive expression rates of P21in high, mediate and low differentiated laryngeal carcinoma were20.00%、25.00%、18.75%, and in different pathological grade of laryngeal carcinoma, carcinoma in situ21.43%and invasive carcinoma21.05%.Both showed no statistical significances (P>0.05)
(5) The positive rates of CyclinDl and CyclinE expression in phlegm and blood stasis type of laryngeal precancerous lesions were relatively high(23.40%and46.81%respectively), while in Yin Deficiency type relatively low(9.09%and36.36%respectively). There was no statistical significance (P>0.05). The positive rates of P16expression in phlegm and blood stasis type of laryngeal precancerous lesions were relatively low (34.04%), while in the Yin deficiency type relatively high(45.0%). Still, no statistical significances showed (P>0.05). However, the positive rate of expression of P21in the phlegm and blood stasis type and Yin Deficiency type was opposite (27.66%and23.0%respectively), and had no statistical significances (P>0.05)
Conclusion
1. The epidemiological characteristics of laryngeal precancerous lesions
(1)The risk factors of laryngeal precancerous lesions were followed by improper using of voice, smoking, drinking, improper diet, staying up late, colds.
(2) The sex ratio of man and women in laryngeal papilloma and laryngeal keratosis were2.2:1and2:1, while0.65:1in chronic hypertrophic laryngitis.
(3) Recurrence rate of laryngeal precancerous lesions was10.95%, and the malignant transformation rate was4.38%.
2. TCM pathogenesis, syndrome distribution and epidemiological features
(1)TCM pathogenesis as follows:The outside pathogenesis was damp-heat stagnation and toxic evil due to smoking, drinking and Exopathy. The Internal ones were phlegm stagnation from spleen injury due to improper diet, and deficiency of vital qi and yin due to staying up too late and long-term excessive use of voice.
(2) In syndrome distribution according to the rate from large to small, there were four types as follows:damp-heat stagnation syndrome, qi deficiency and phlegm stasis syndrome, turbid phlegm accumulation syndrome, hyperactivity of fire due to yin deficiency syndrome. The important index of syndrome diagnosis can be found in the part of results (abbreviated).
(3)Factors related to the syndrome distribution as follows:male common in the syndrome of damp-heat stagnation type, while the female common in the syndrome of qi deficiency and phlegm stasis, turbid phlegm accumulation, hyperactivity of fire due to yin deficiency types;history of smoking and drinking common in damp-heat stagnation and Qi deficiency and phlegm and blood stasis types. There were significant differences in the local signs of TCM syndromes (abbreviated). Pathological type, age, duration of disease, hoarseness extent had no significant effect for TCM syndrome distribution of laryngeal precancerous lesions.
3. mechanisms of cell cycle control
(1) The expressions of CyclinDl and CyclinE in vocal cord polyps→laryngeal precancerous lesions→laryngeal cancer, and in mild dysplasia→moderate dysplasia→severe dysplasia groups of Laryngeal precancerous lesions showed a gradual increase of the gradient phenomenon. Statistically, there were significant differences, especially the first group.It prompted that the over-expression of the positive regulatory factor of CyclinDl and CyclinE leaded cell cycle to accelerate the malignant proliferation. Expressions of CyclinDl and CyclinE in different types and pathologic grading of laryngeal cancer, did not show obvious differences, which may be related to the less specimen. Generally, expression of CyclinE in the highly malignant lesions was higher than CyclinD1.CyclinE would play a greater role in promoting the restriction point of G1/S than CyclinD1, which still needs further study.
(2) The expressions of P16and P21in vocal cord polyps→laryngeal precancerous lesions→laryngeal cancer, and in mild dysplasia→moderate dysplasia→severe dysplasia groups of Laryngeal precancerous lesions showed a diminishing gradient phenomenon. But only the group of P16showed significant difference statistically. It prompted that the tumor suppressor gene of P16played an important role in the inhibition of Rb phosphorylation mediated by CDK4/CDK6to prevent the process of abnormal cells in G1/S. In different types and tumor grades of laryngeal cancer, positive rates of P16and p21expression was lower. But no significant difference was between the two groups, still related to the limited sample size.
(3)The positive rates of CyclinD1and CyclinE expression in phlegm and blood stasis type of laryngeal precancerous lesions were relatively high, while in Yin Deficiency type relatively low. The positive rates of P16expression in phlegm and blood stasis type of laryngeal precancerous lesions were relatively low, while in the Yin deficiency type relatively high. Comparison of P16, the positive rate of expression of P21in the phlegm and blood stasis type and Yin Deficiency type was opposite. But the above expressions between the two groups showed no significant difference.It prompted that in the process of disease caused by phlegm and blood stasis, high expression of positive regulatory factor of CyclinD1and CyclinE, and low expression of negative regulatory factor P16leaded to abnormal cell cycle regulation might be the molecular biology mechanism. However, it needs to be verified by further in-depth study of expanding the sample size.
Deficiencies and Prospects
The study still had many deficiencies, such as limitted research information including insufficient sample size and non-multi-center study, and the insufficient study design in both clinical research table content and the selection of specimen collection method in experimental study. There was no any clear basis for Chinese Medicine of the cellular and molecular biological mechanism from the research results by now. The research study should be gradually modified and improved in the future clinical and experimental practice.
喉癌是头颈部恶性肿瘤中最常见的疾病之一,仍严重威胁人类的生命和健康。喉癌前病变是喉癌变的中心环节,它既可以恶化发展转化为癌,也可以良性逆转为正常细胞。早期发现并进行干预,可以防止其向恶性肿瘤发展。因此,加强对喉癌前病变的研究,深化对癌变机理的认识,已成为头颈肿瘤防治工作的前沿和重点之一。
目前研究认为,细胞周期调控受正负两大类因子的调节,正负两类因子之间的平衡被打破,就可能导致肿瘤的发生,细胞周期调控异常是肿瘤发生发展的一个重要前提。这与中医认为疾病是人体阴阳平衡失调所致具有相似之处。祖国医学理论认为痰瘀是导致肿瘤发生发展的重要因素,现代中医临床及导师的前期临床研究均发现采用活血化痰法对喉癌前病变具有较好的治疗作用。但喉癌前病变的中医基本病因病机,及痰瘀在喉癌前病变的发生发展中的作用机制,目前尚缺乏科学的支持与验证。
本研究拟在文献研究的基础上,在临床方面对喉癌前病变的中医四诊特征及基本证型分布特点进行研究,为喉前期病变的中医临床辨治提供科学依据,同时也为中医证候的客观化、量化研究提供新的研究途径。在实验方面对喉癌前过程及喉癌前病变痰瘀证的细胞周期调控机制进行研究,深化对喉癌前病变发生发展及痰瘀致癌的机理认识,确立具有较高恶变潜质的中医证候类型。从而为临床上采用活血化痰法阻断逆转喉癌前病变提供理论支撑与实验依据,为中医药对癌前病变的防治提供思路与方向。方法：
1.文献研究
通过古文献数据库(湖南电子音像出版社的《中华医典》及广东省中医院重点专科中医古籍文献数据库)及现代文献数据库(中国生物医学文献数据库CBMdisc、维普中文生物医学期刊数据库VIP Information、中国知网数据库CNKI、美国NCBI的PubMed、Medline数据库)的查阅挖掘,将相关的古文条文及现代文献报道分类归纳,总结出喉癌前病变的古代在病因病机、辩证分析、施治方法,现代在流行病学、分类诊断、治疗方法等方面的特点与进展。
2.临床调研
本研究拟通过运用循证医学、临床流行病学等现代科研方法学结合传统医学进行喉前期病变的临床病例的证候信息采集。通过建立流行病学调查表,对214例喉癌前病变的四诊资料集流行病学资料进行采集,采用Epidata3.1软件包建立数据库,双人双输的办法进行数据录入及核对,应用SPSS17.0软件对数据进行统计分析,初步得出喉癌前病变的证候诊断重要指标、证型类型及分布相关情况及流行病学特点。
3.实验研究
本研究拟利用流式细胞术,检测痰瘀证的喉癌前病变标本中细胞周期因子CyclinD1、Cyclin E、p21、p16蛋白的表达情况,将喉癌前病变阴虚证标本、声带息肉及早期喉癌标本作为证候对照及病情进展对照研究。通过对声带息肉→喉癌前病变→早期喉癌、喉癌前病变、喉癌的不同病理类型及分级组织中的细胞周期因子动态变化的分析,及对痰瘀证、阴虚证癌前病变组织中细胞周期因子表达情况的对比分析,明确喉癌变过程及痰瘀证喉癌前病变的细胞周期调控机制。
结果：
1.临床研究
(1)发病因素：喉癌前病变患者中有长期过度用嗓发音占74.8%、有长期吸烟史者占43.9%,有饮酒习惯者占21.5%,饮食不节者占27.6%,熬夜者占9.3%,感冒者占5.1%。发病因素在三种病理类型中无显著性差异(P值均大于0.05)。
(2)性别构成：慢性肥厚性喉炎(非典型增生)中男性60例,女性92例；喉乳头状瘤中男性22例,女性10例；喉角化症(声带白斑)中男性20例,女性10例。三组的男女性别比为0.65：1、2.2：1及2：1,后两者男性均多于女性,性别构成组间差异有统计学意义(P<0.05)。
(3)常见症状体征频数：出现频率大于10%的症状依次有：声音嘶哑(声嘶轻度、声嘶中度)、咽异物感、咽干灼热、多言后喉痛、痛处不移、咽痒咳嗽、痰多质粘、清嗓习惯。体征出现频率大于10%的依次有声带闭合欠佳、声带充血肥厚、肿物表面光滑、肿物结节状或乳头状、肿物白斑样改变。舌苔脉象出现频率大于10%的依次有舌质暗或有瘀点、舌苔腻、脉弦、舌苔白、脉滑、舌质红、舌苔黄、舌苔薄、舌胖有齿印、脉细、脉沉。
(4)证候聚类分析：根据计算机聚类分析的结果,结合专家意见认为,应聚为4类：工类：声音嘶哑、舌质红、舌苔黄、舌苔腻、脉滑、肿物结节状或乳头状、声带闭合不好。Ⅰ类：清嗓习惯、多言后喉痛、痛处不移、咽异物感、舌胖有齿印、舌质暗或有瘀点、脉沉、声带充血肥厚、肿物光滑。Ⅲ类：声嘶轻、痰多质粘、咽痒咳嗽、舌苔白、脉弦、声带闭合尚好。Ⅳ类：声嘶中重度、咽干灼热、舌苔薄、脉细、肿物白斑样改变。
(5)证候诊断的重要指标按主症、次症、舌苔脉象、局部体征依次如下：
①湿热困结证主症：声嘶；舌苔脉象：舌质红、舌苔腻、舌苔黄、脉滑；局部体征：声带充血肥厚、声带闭合不好、肿物结节状或乳头状。
②气虚痰瘀证主症：声嘶；次症：清嗓习惯、咽异物感、多言后喉痛、痛处不移；舌苔脉象：舌质暗或有瘀点、舌苔腻、舌苔白、脉弦、脉滑、脉沉；局部体征：声带充血肥厚、肿物光滑。
③痰浊结聚证主症：声嘶(轻)；次症：痰多质粘、咽瘁咳嗽；舌苔脉象：舌苔白、脉弦；局部体征：声带充血肥厚、肿物表面光滑、声带闭合尚好。
④阴虚火旺证主症：声嘶(中重度)；次症：咽干灼热、咽异物感；舌苔脉象：舌质红、舌苔白、舌苔薄、脉细；局部体征：声带充血肥厚、肿物白斑样改变。
(6)证型分布情况：Ⅰ类：湿热互结型；Ⅱ类：气虚痰瘀型；Ⅲ类：痰浊困结型；Ⅳ类：阴虚火旺型。其中湿热互结型49例,占22.9%；气虚痰瘀型87例,占40.7%；痰浊困结型23例,占10.7%；阴虚火旺型55例,占25.7%。
(7)证候分布的相关因素：
①中医证候与性别的关系：在湿热困结证中,男性所占比率较大(67.3%),而在气虚痰瘀证、痰浊结聚证、阴虚火旺证中,女性所占比率较大(分别为58.6%、65.2%、54.5%),统计学上有显著差异(P<0.05)。
②中医证候与年龄的关系：喉癌前病变的中年组患者中,气虚痰瘀证占较大比率(42.9%),其他证型相对分散；青年组及老年组各证型均相对分散,统计学上均无意义(P>0.05)。
③中医证候与病程的关系：喉癌前病变各证候间的平均发病时间(28.17～37.67个月)相差不大,病程时间呈非正态分布,采用秩和检验,P>0.05,病程在各证候间的差异无统计学意义。
④中医证候与病理类型的关系：慢性肥厚性喉炎、喉乳头状瘤多见气虚痰瘀证(分别占40.1%、56.3%),喉白斑角化病多见阴虚火旺证(占33.3%),但各证候间的构成比差异无统计学意义(P>0.05)。
⑤中医证型与病因的关系：吸烟、饮酒在湿热困结、气虚痰瘀这两种证型中多见,且有统计学意义(P<0.05)。饮食不节、熬夜在湿热困结证、气虚痰瘀证中多见；用声过度、感冒在气虚痰瘀证、阴虚火旺证中多见,但均没有统计学意义(P>0.05)。
⑥中医证型与局部体征的关系：肿物表面光滑多见于气虚痰瘀证中(44.9%),肿物呈结节状或乳头状的多见于湿热困结证(47.1%),肿物白斑样改变多见于阴虚火旺证(68.8%),而且统计学上有显著差异(P=0.001<0.05),因“肿物粗糙质脆易出血”例数较少(3例),暂不做分析。
⑦中医证型与声嘶程度的关系：声嘶程度根据VHI-10问卷调查表中的嗓音障碍指数得分而来,各个中医证型均集中在轻度异常与中度异常之间(轻度异常的共50例,中度异常的共150例,正常的共6例,严重失常的共8例),没有统计学意义(P>0.05)。
(8)复发率与恶变率
本研究对其中137例喉癌前病变进行了随访,共有15例发生复发,4例为声带白斑,4例为喉乳头状瘤,7例为声带息肉伴轻度～中度不典型增生。本研究得到喉癌前病变的复发率为10.95%。其中有6例发生恶变,4例为声带白斑,1例为喉乳头状瘤,1例为声带鳞状上皮中度不典型增生。恶变者均有多年吸烟史饮酒史。本研究得到的喉癌前病变的恶变率为4.38%。
2.实验研究
(1) CyclinD1的阳性表达率形成声带息肉→喉癌前病变→喉癌阳性强度逐渐增强的梯度现象(分别为13.04%、20.29%、42.42%),但总体上荧光强度较弱；三组分别比较均有统计学差异(P<0.05)。在喉癌前病变组中,CyclinD1在病理分级中的轻度不典型增生、中度不典型增生、重度不典型增生的的阳性率表达分别为13.95%、28.57%、31.58%,呈上升趋势,但统计学上没有意义(P>0.05)。CyclinD1在不同病理类型中上皮不典型增生(慢性肥厚性喉炎)、喉乳头状瘤、喉角化症的阳性率表达分别为19.51%、21.05%、22.22%,但三组间差异无统计学意义(P>0.05)。在喉癌组中,不同病理类型高分化、中分化、低分化的CyclinD1的阳性率表达分别为40.00%、41.67%、43.75%；不同病理分级中原位癌、浸润癌的CyclinD1的阳性率表达分别为35.71%、47.37%,两者均呈上升趋势,但统计学上无显著性差异(P>0.05)。
(2) CyclinE的阳性表达率形成声带息肉→喉癌前病变→喉癌阳性强度逐渐增强的梯度现象(分别为26.09%、43.48%、93.94%)；且荧光强度较强,统计学上有显著差异(P<0.001)。在喉癌前病变组中,CyclinDl在病理类型中上皮不典型增生(慢性肥厚性喉炎)、喉乳头状瘤、喉角化症的阳性率表达分别为36.59%、52.63%、55.56%,呈上升趋势,但三组比较统计学上亦无意义(P>0.05)。病理分级中轻度不典型增生、中度不典型增生、重度不典型增生的三组中CyclinE的阳性率表达分别为25.58%、57.14%、78.95%,也呈上升趋势,统计学上有显著差异(P<0.001)。在喉癌组中,不同病理类型高分化、中分化、低分化的三组中CyclinE的阳性率表达与不同病理分级原位癌、浸润癌中的阳性率表达均较高(80%-100%),且呈上升趋势,但统计学上均无显著差异(P>0.05)。
(3)P16的阳性表达率形成声带息肉→喉癌前病变→喉癌阳性强度逐渐递减的现象(分别为60.87%、40.58%、15.15%),统计学上有显著性差异(P<0.001)。在喉癌前病变组中,P16在病理分级中轻度不典型增生、中度不典型增生、重度不典型增生的阳性率表达分别为51.16%、42.86%、15.78%,呈逐步下降趋势,统计学上有显著差异(P<0.05)；在病理类型中上皮不典型增生(慢性肥厚性喉炎)、喉乳头状瘤、喉角化症中的阳性率表达分别为41.46%、42.11%、33.33%,三组间差异无统计学意义(P>0.05)。在喉癌组中,不同病理类型高分化、中分化、低分化的P16的阳性率表达分别为20.00%、25.00%、12.50%,不同病理分级原位癌、浸润癌中P16的阳性率表达分别为14.29%%、15.79%,两者在统计学上均无显著性差异(P>0.05)。
(4)P21的阳性表达率形成声带息肉→喉癌前病变→喉癌阳性强度逐渐递减的现象(分别为47.83%、26.09%、24.24%)。但没有统计学意义(P>0.05)。在喉癌前病变组中,P21在病理类型上皮不典型增生(慢性肥厚性喉炎)、喉乳头状瘤、喉角化症中的阳性率表达分别为26.83%、26.32%、22.22%；在病理分级中轻度不典型增生、中度不典型增生、重度不典型增生的P21的阳性率表达分别为27.91%、28.57%、21.05%,呈下降趋势,但两者在统计学上均无差异(P>0.05)。在喉癌组中,不同病理类型高分化、中分化、低分化的P21的阳性率表达相差不大(分别为20.00%、25.00%、18.75%),不同病理分级原位癌、浸润癌中P21的阳性率表达分别为21.43%、21.05%,统计学上亦均无差异(P>0.05)。
(5)CyclinD1、CyclinE在痰瘀证中的阳性率表达相对较高(分别为23.40%、46.81%),在阴虚证中的阳性率表达相对较低(分别为9.09%、36.36%),但两两比较无统计学意义(P>0.05)。P16在痰瘀证中的阳性率表达相对较低(34.04%),在阴虚证中的阳性表达相对较高(45.0%),但两组间差异仍无统计学意义(P>0.05)。然而P21在痰瘀证与阴虚证中的阳性率表达则无明显差异(分别为27.66%、23.0%),P>0.05。
结论：
1.喉癌前病变的流行病学特点
(1)喉癌前病变的危险因素依次有用嗓不当、吸烟、饮酒、饮食不节、熬夜、感冒等。
(2)喉癌前病变的性别构成在喉乳头状瘤与喉白斑角化病中男女之比约为2：1；在慢性肥厚性喉炎中的比例则为0.65：1。
(3)喉癌前病变的复发率为10.95%,喉癌前病变的恶变率为4.38%。
2.喉癌前病变的中医病因病机、证候特征及分布特点
(1)中医的病因病机主要有：外因为吸烟喝酒、外感之湿热毒邪；内因则为饮食不节损伤脾胃致痰浊结聚,过度用声、熬夜致劳累过度致正气虚损、气阴两伤。
(2)喉癌前病变的中医证型按由大至小的比例分布情况,主要分为气虚痰瘀型、阴虚火旺型、湿热互结型、痰浊困结型,共4类。其证候诊断的重要指标特点可见结果部分内容(略)。
(3)中医证型分布相关性分析中,男性多见湿热困结证,女性多见气虚痰瘀证、痰浊结聚证、阴虚火旺证；吸烟史、饮酒史多见于湿热困结型和气虚痰瘀型两种证型；喉癌前病变的局部体征对中医证型分布有显著差异(略)。病理类型、年龄、病程、声嘶程度对喉癌前病变的中医证型分布无明显影响。
3.喉癌变过程及中医证型的细胞周期调控机制
(1) CyclinD1、CyclinE在声带息肉→喉癌前病变→喉癌组、喉癌前病变的轻度不典型增生→中度不典型增生→重度不典型增生组中的表达均呈现逐渐增强的梯度现象,统计学上特别是第一组均有显著差异,提示正性调控因子CyclinDl及CyclinE的过度表达,导致细胞周期加快,肿瘤恶性增生。CyclinDl及CyclinE在喉癌的不同分化类型及病理分级中,尚未呈现明显差异,可能与标本量少有关。总体上CyclinE在恶性程度高的病变组织中的表达较CyclinDl高,CyclinE是否比CyclinDl在G1/S限制点上发挥更大的促进作用,仍需进一步深入研究。
(2)P16、P21在声带息肉→喉癌前病变→喉癌组、喉癌前病变的轻度不典型增生→中度不典型增生→重度不典型增生组中,阳性率表达均呈逐渐减弱的梯度现象,但只有P16在统计学上有显著性差异。提示P16作为抑癌基因,在抑制CDK4/CDK6介导的Rb磷酸化,阻止不正常细胞在G1/S的进程上起重要的作用。而在喉癌的不同分化类型、病理分级中,P16、P21的阳性率表达均较低,但组间比较仍无显著差异,仍考虑与样本量有限相关。
(3) CyclinD1、CyclinE在痰瘀证中的阳性率表达相对较高,在阴虚证中的阳性率表达相对较低；P16在痰瘀证中的阳性率表达相对较低,在阴虚证中的阳性表达相对较高；P21在痰瘀证与阴虚证中的阳性率表达则相反。但以上表达的组间差异均无统计学意义。提示在痰瘀致病的过程中,正性调控因子CyclinD1、CyclinE的高表达,负性调控因子P16的低表达导致细胞周期调控异常可能是其分子生物学机制,但仍需进一步扩大样本量深入研究以验证。
4.不足与展望
虽然本研究仍存在许多不足,如研究资料有限,包括样本量不够、非多中心研究；研究设计有所欠缺,包括临床调研表内容及实验研究标本的采集方法选择等。现有的研究结果暂未能为中医辩证及其相关细胞分子生物作用机制提供明确依据,仍必须继续在今后的临床及实验实践中逐步修改和完善。
Objective
Laryngeal cancer is one of the most common diseases in the head and neck cancer remains a serious threat to human life and health. Laryngeal precancerous lesions is the central link of becoming laryngeal cancer. It not only can worsen development transformed into cancer, but also can be benign reversal of the normal cells. Early detection and intervention can prevent its development to malignancy. Therefore, the strengthening study of laryngeal precancerous lesions, to deepen the understanding of cancer mechanism, has become one of the forefront and key of head and neck cancer prevention and control work.
The preliminary study suggested that cell cycle regulation was adjusted by two major factors of positive and negative types. The balance between them once became broken, it could lead to tumorigenesis. This had similarities with diseases caused by imbalance of human yin and yang in TCM. Abnormal cell cycle regulation was an important prerequisite for tumor development, and phlegm and blood stasis was an important factor leading to tumor development. Pre-clinical study of modern TCM and my tutor had found better therapeutic effects of laryngeal precancerous lesions with blood circulation and phlegm resolving method. But the TCM etiology and pathogenesis of Laryngeal precancerous lesions, and the mechanism of phlegm and blood stasis in the development of laryngeal precancerous lesions remained the lack of scientific support and validation currently.
Basing on the literature research, This research will investigate the characteristics of the four diagnostic methods and distribution of the types of syndrome on laryngeal precancerous lesions in the clinical aspects. We will also study the cell cycle regulation mechanisms in process of laryngeal precancerous lesions and Phlegm and Blood Stasis of it In the experimental aspects. The results will provide ideas and direction for the medicine in prevention and treatment of precancerous lesions, and the theoretical support and experimental basis for blood circulation and phlegm resolving method of laryngeal precancerous lesions in clinic.
Methods
1. Literature research
Searching through ancient literature database such as Chinese Medical Code from Hunan Electronic Audio and Video Publishing House, and Chinese ancient literature database from the Guangdong Provincial Hospital, and meanwhile through modern literature database such as Chinese biology and medicine database (CBM),Chinese biomedical journals in the database (VIP Information),Chinese national knowledge infrastructure database (CNKI) and PubMed or Medline database of American national center for biotechnology information (NCBI).We summarized the relevant provisions of the classical and modern literature, and summed up the etiology and pathogenesis of laryngeal precancerous lesions in the ancient, dialectical analysis of the characteristics and treatment methods, modern epidemiology, classification, diagnosis, treatment methods and progress.
2. Clinical research
This study collected clinical cases of the syndrome information of laryngeal precancerous lesions through the use of evidence-based medicine, clinical epidemiology, and other modern research methodology combined with TCM. Through the establishment of an epidemiological questionnaire, the four diagnostic information and epidemiological data of214cases of laryngeal precancerous lesions were collected. With Epidata3.1packages to establish a database for data entry and verification, SPSS17.0statistical software for data analysis, the result preliminary draw the important indicators of syndrome diagnosis of laryngeal precancerous lesions, syndrome distribution and epidemiological features.
3. Experimental research
In this study, using flow cytometry, we will detect expressions of cell cycle factors (Cyclin Dl, Cyclin E, p21and p16protein) on specimens of phlegm and blood stasis type of laryngeal precancerous lesions, Yin deficiency type of the Laryngeal precancerous lesions, vocal cord polyps and early laryngeal cancer as the control groups. Through analysis of dynamic changes of the cell cycle factor in vocal cord polyps→laryngeal precancerous lesions→early laryngeal cancer, and different histological types and pathological classification of laryngeal precancerous lesions and laryngeal cancer, It will be made clear that cell cycle control mechanisms in the process of laryngeal cancer and phlegm and blood stasis type of laryngeal precancerous lesions.
Results
1. Clinical research
(1) Pathogenic factors:In group of laryngeal precancerous lesions, long-term excessive use of voice pronunciation accounted for74.8%, long history of smoking accounted for43.9%, drinking habits accounted for21.5%, improper diet accounted for27.6%, staying up too late to sleep accounted for9.3%, getting cold accounted for5.1%. Among the three pathological types, there is no significant difference.
(2) The gender composition:There are60male cases and92female cases of chronic hypertrophic laryngitis (atypical hyperplasia),22male cases and10female cases of laryngeal papilloma,20male cases and10female cases of laryngeal keratosis (vocal cord leukoplakia), The sex ratio of the three groups are as follows:0.65:1,2.2:1and2:1, and there are more males than females in the latter two groups, the gender composition between the groups has difference which has statistic significance (P <0.05).
(3)The frequency of common signs and symptoms:The frequency of the symptoms which occurred greater than10%were:hoarseness (mild hoarseness, moderate hoarseness), foreign body sensation of pharynx, dry and burning throat, sore throat after over saying, and the pain did not move, itchy throat and cough, profuse sticky phlegm, preference of clearing the throat. The frequency of the signs which occurred greater than10%were poor vocal cord closure, vocal cord congestion and hypertrophy, smooth tumor surface, nodular、papillary or leukoplakia change of the tumor. The frequency of the tongue coating and pulse which present greater than10%were dark tongue or with Petechiae, greasy tongue coating, wiry pulse, white tongue coating, slippery pulse, red tongue, yellow tongue coating, thin tongue coating, fat tongue with teeth marks, thready pulse, deep pulse.
(4) Syndrome cluster analysis:According to the results of computer cluster analysis, and combined with expert opinion, it should be clustered into four categories:Class I:hoarseness, red tongue, yellow tongue coating, greasy tongue coating, slippery pulse, nodular or papillary tumor, poor vocal cord closure. Class Ⅱ:clearing throat habits, sore throat after over saying, and the pain did not move, foreign body sensation of pharynx, fat tongue with teeth marks, dark tongue or with petechia, deep pulse, vocal cords congestion and hypertrophy, smooth tumor surface. Class Ⅲ:mild hoarseness, profuse sticky phlegm, cough, itchy throat, white tongue coating, wiry pulse, normal vocal cord closure. Class IV: severe hoarseness, dry and burning throat, thin tongue coating, thready pulse, and tumor leukoplakia-like change.
(5) The important index of syndrome diagnosis according to main symptom, secondary symptom, tongue coating and pulse, local signs in turn are as follows:
①Syndrome of damp-heat stagnation main symptoms:hoarseness; tongue coating and pulse:red tongue, greasy tongue coating, yellow tongue coating, slippery pulse; local signs:vocal cords congestion and hypertrophy, poor vocal cord closure, nodular or papillary tumor.
②Syndrome of qi deficiency and phlegm stasis main symptoms: hoarseness; secondary symptoms:preference of clearing voice, foreign body sensation of pharynx, sore throat after over saying, and the pain did not move; tongue coating and pulse:dark tongue or with petechia, greasy tongue coating, white tongue coating, wiry pulse, slippery pulse, deep pulse; local signs:vocal cords congestion and hypertrophy, smooth tumor surface.
③Syndrome of turbid phlegm accumulation main symptoms: hoarseness(mild); secondary symptoms:profuse sticky phlegm, itchy throat and cough; tongue coating and pulse:white tongue coating, wiry pulse; local signs:congestive hypertrophy of the vocal cords, smooth tumor surface, normal vocal cords closure
④Syndrome of hyperactivity of fire due to yin deficiency main symptoms:hoarseness(severe):dry and burning throat, foreign body sensation of pharynx; tongue coating and pulse:red tongue, white tongue coating, thin tongue coating, thready pulse; local signs:congestive hypertrophy of the vocal cords, nodular papillary or leukoplakia change of the tumor.
(6) Syndrome distribution:Class Ⅰ:syndrome of damp-heat stagnation; Class Ⅱ:syndrome of qi deficiency and phlegm stasis; Class Ⅲ:syndrome of turbid phlegm accumulation; Class Ⅳ:syndrome of hyperactivity of fire due to yin deficiency. among which syndrome of damp-heat stagnation are49cases, accounting for22.9%; syndrome of qi deficiency and phlegm stasis are87cases, accounting for40.7%; syndrome of turbid phlegm accumulation are23cases,10.7%; syndrome of hyperactivity of fire due to yin deficiency are55cases, accounting for25.7%.
(7) The syndrome distribution factors as follows:
①The relationship between the Traditional Chinese Medicine syndrome and gender:Male accounted for a large percentage (67.3%) for the syndrome of damp-heat stagnation, while the female accounted for a large percentage (respectively58.6%and65.2%,54.5%) for the syndrome of qi deficiency and phlegm stasis, turbid phlegm accumulation, hyperactivity of fire due to yin deficiency of which has statistic significant differences (P<0.05).
②The relationship between the Traditional Chinese Medicine syndrome and age:Qi deficiency and phlegm and blood stasis account for a large percentage (42.9%) of the laryngeal precancerous lesions of the middle-aged patient,other syndromes are relatively dispersed; each type in the youth group and older group were relatively scattered, it has no statistic significance (P>0.05).
③The relationship between the Traditional Chinese Medicine syndrome and duration:Difference between the average onset time of syndrome types of laryngeal precancerous lesions (28.17to37.67) was small, and it has no statistic significance (P>0.05)
④The relationship between the Traditional Chinese Medicine syndrome and pathological types:Qi deficiency and phlegm and blood stasis type was common in chronic hypertrophic laryngitis and laryngeal papilloma (accounting for40.1%、56.3%). Hyperactivity of fire due to yin deficiency type was common in laryngeal keratosis(accounting for33.3%). But it has no statistic significance (P>0.05)
⑤The relationship between the Traditional Chinese Medicine syndrome and pathogenic factors:History of smoking and drinking were common in damp-heat stagnation and Qi deficiency and phlegm and blood stasis types, and i t had statistic significance (P<0.05). Improper diet and staying up too late to sleep were common in Qi deficiency and phlegm and blood stasis types, and long-term excessive use of voice and getting cold were common in Qi deficiency and phlegm and hyperactivity of fire due to yin deficiency types, but those had no statistic significance (P>0.05)
⑥The relationship between the Traditional Chinese Medicine syndrome and local signs:Smooth tumor surface was related to Qi deficiency and phlegm and blood stasis type (44.9%), nodular or papillary tumorn related to damp-heat stagnation (47.1%), leukoplakia change of the tumor related to hyperactivity of fire due to yin deficiency types (68.8%).They had statistic significance (P<0.05)
⑦The relationship between the Traditional Chinese Medicine syndrome and degree of hoarseness:The hoarseness degree of voice came from the score of disability index in VHI-10questionnaire. There were50cases of mildly abnormal patients,150cases of moderately abnormal cases, six of the normal cases, eight of severe disorders cases, which had not statistically significant (P>0.05).
(8) Rate of recurrence and malignant transformation:In this study,137cases of laryngeal precancerous lesions were followed up. There were15cases of recurrence, four cases of vocal cord leukoplakia, four cases of laryngeal papilloma,7cases of vocal cord polyps with mild to moderate dysplasia. Recurrence rate of laryngeal precancerous lesions was10.95%. Among those, six cases of mal ignant transformation, four cases of vocal cord leukoplakia, one cases of laryngeal papilloma, and one case of squamous moderate atypical hyperplasia in vocal cord. Malignancy cases have years of history of smoking and drinking history. Malignant transformation rate of laryngeal precancerous lesions was4.38%.
2. Experimental research
(1) Expressions of Cyclin D1had a progressive enhancement from vocal cord polyps, laryngeal precancerous lesions to Laryngeal cancer(the percentage as follows:13.04%、20.29%、42.42%). However, all of the fluorescence intensity are weak. There were statistical differences in pairwise comparisons (P<0.05). The result of mild dysplasia, moderate dysplasia and severe dysplasia in laryngeal precancerous lesions had an up trend of positive expressions, which were13.95%、28.57%、31.58%, but there was no significant difference (P>0.05). Expressions of Cyclin D1 in epithelium atypical hyperplasia of different types(chronic hypertrophic laryngitis), laryngeal papilloma and laryngeal keratosis were19.51%,21.05%,22.22%, respectively. There was no statistical significance among the three (P>0.05). The positive expression rates of Cyclin D1in well differentiated was40.00%, moderately differentiated41.67%and poorly differentiated43.75%in different pathologic types of laryngeal carcinoma, and carcinoma in situ was35.71%, invasive carcinoma47.37%in different pathological grade of laryngeal carcinoma. Both showed an increasing tendency, without statistical significances (P>0.05)
(2)Expressions of Cyclin E had a Progressive enhancement from vocal cord polyps, Laryngeal precancerous lesions to Laryngeal cancer(the percentage as follows:26.09%、43.48%、93.94%). All of the fluorescence intensity are strong. There were statistical differences in pairwise comparisons (P<0.001). The result of mild dysplasia, moderate dysplasia and severe dysplasia in laryngeal precancerous lesions had an up trend of positive expressions, which were36.59%、52.63%、55.56%, but there was also no significant difference (P>0.05). Expressions of Cyclin D1in epithelium atypical hyperplasia of different types(chronic hypertrophic laryngitis), laryngeal papilloma and laryngeal keratosis were25.58%,57.14%and78.95%, respectively. It showed an increasing tendency, and there was statistical significance among the three (P<0.001).The positive expression rates of Cyclin Dl in different pathologic types and grade of laryngeal carcinoma were high (80%～100%), a rising trend. But there were no statistical significance (P>0.05)
(3) Expressions of P16had a diminishing gradient phenomenon from vocal cord polyps, laryngeal precancerous lesions to Laryngeal cancer (the percentage as follows:60.87%、40.58%、15.15%). There were statistical differences in pairwise comparisons (P<0.05). The result of mild dysplasia, moderate dysplasia and severe dysplasia in Laryngeal precancerous lesions had an downward trend of positive expressions, which were51.16%、42.86%、15.78%, and had significant difference (P<0.05). Expressions of P16in epithelium atypical hyperplasia of chronic hypertrophic laryngitis, laryngeal papilloma and laryngeal keratosis were41.46%、42.11%、33.33%, respectively. There was no statistical significance (P>0.05). The positive expression rates of P16in high, mediate and low differentiated laryngeal carcinoma were20.00%、25.00%,12.50%, and in different pathological grade of laryngeal carcinoma, carcinoma in situ35.71%and invasive carcinoma47.37%.Both showed no statistical significances (P>0.05)
(4) Expressions of P21had a diminishing gradient phenomenon from vocal cord polyps, laryngeal precancerous lesions to Laryngeal cancer (the percentage as follows:47.83%、26.09%、24.24%). But there were no statistical differences in pairwise comparisons (P>0.05).The result of mild dysplasia, moderate dysplasia and severe dysplasia in Laryngeal precancerous lesions had an downward trend of positive expressions, which were27.91%、28.57%、21.05%.Expressions of P21in epithelium atypical hyperplasia of chronic hypertrophic laryngitis, laryngeal papilloma and laryngeal keratosis were26.83%、26.32%、22.22%, respectively. Both had no significant difference (P<0.05). The positive expression rates of P21in high, mediate and low differentiated laryngeal carcinoma were20.00%、25.00%、18.75%, and in different pathological grade of laryngeal carcinoma, carcinoma in situ21.43%and invasive carcinoma21.05%.Both showed no statistical significances (P>0.05)
(5) The positive rates of CyclinDl and CyclinE expression in phlegm and blood stasis type of laryngeal precancerous lesions were relatively high(23.40%and46.81%respectively), while in Yin Deficiency type relatively low(9.09%and36.36%respectively). There was no statistical significance (P>0.05). The positive rates of P16expression in phlegm and blood stasis type of laryngeal precancerous lesions were relatively low (34.04%), while in the Yin deficiency type relatively high(45.0%). Still, no statistical significances showed (P>0.05). However, the positive rate of expression of P21in the phlegm and blood stasis type and Yin Deficiency type was opposite (27.66%and23.0%respectively), and had no statistical significances (P>0.05)
Conclusion
1. The epidemiological characteristics of laryngeal precancerous lesions
(1)The risk factors of laryngeal precancerous lesions were followed by improper using of voice, smoking, drinking, improper diet, staying up late, colds.
(2) The sex ratio of man and women in laryngeal papilloma and laryngeal keratosis were2.2:1and2:1, while0.65:1in chronic hypertrophic laryngitis.
(3) Recurrence rate of laryngeal precancerous lesions was10.95%, and the malignant transformation rate was4.38%.
2. TCM pathogenesis, syndrome distribution and epidemiological features
(1)TCM pathogenesis as follows:The outside pathogenesis was damp-heat stagnation and toxic evil due to smoking, drinking and Exopathy. The Internal ones were phlegm stagnation from spleen injury due to improper diet, and deficiency of vital qi and yin due to staying up too late and long-term excessive use of voice.
(2) In syndrome distribution according to the rate from large to small, there were four types as follows:damp-heat stagnation syndrome, qi deficiency and phlegm stasis syndrome, turbid phlegm accumulation syndrome, hyperactivity of fire due to yin deficiency syndrome. The important index of syndrome diagnosis can be found in the part of results (abbreviated).
(3)Factors related to the syndrome distribution as follows:male common in the syndrome of damp-heat stagnation type, while the female common in the syndrome of qi deficiency and phlegm stasis, turbid phlegm accumulation, hyperactivity of fire due to yin deficiency types;history of smoking and drinking common in damp-heat stagnation and Qi deficiency and phlegm and blood stasis types. There were significant differences in the local signs of TCM syndromes (abbreviated). Pathological type, age, duration of disease, hoarseness extent had no significant effect for TCM syndrome distribution of laryngeal precancerous lesions.
3. mechanisms of cell cycle control
(1) The expressions of CyclinDl and CyclinE in vocal cord polyps→laryngeal precancerous lesions→laryngeal cancer, and in mild dysplasia→moderate dysplasia→severe dysplasia groups of Laryngeal precancerous lesions showed a gradual increase of the gradient phenomenon. Statistically, there were significant differences, especially the first group.It prompted that the over-expression of the positive regulatory factor of CyclinDl and CyclinE leaded cell cycle to accelerate the malignant proliferation. Expressions of CyclinDl and CyclinE in different types and pathologic grading of laryngeal cancer, did not show obvious differences, which may be related to the less specimen. Generally, expression of CyclinE in the highly malignant lesions was higher than CyclinD1.CyclinE would play a greater role in promoting the restriction point of G1/S than CyclinD1, which still needs further study.
(2) The expressions of P16and P21in vocal cord polyps→laryngeal precancerous lesions→laryngeal cancer, and in mild dysplasia→moderate dysplasia→severe dysplasia groups of Laryngeal precancerous lesions showed a diminishing gradient phenomenon. But only the group of P16showed significant difference statistically. It prompted that the tumor suppressor gene of P16played an important role in the inhibition of Rb phosphorylation mediated by CDK4/CDK6to prevent the process of abnormal cells in G1/S. In different types and tumor grades of laryngeal cancer, positive rates of P16and p21expression was lower. But no significant difference was between the two groups, still related to the limited sample size.
(3)The positive rates of CyclinD1and CyclinE expression in phlegm and blood stasis type of laryngeal precancerous lesions were relatively high, while in Yin Deficiency type relatively low. The positive rates of P16expression in phlegm and blood stasis type of laryngeal precancerous lesions were relatively low, while in the Yin deficiency type relatively high. Comparison of P16, the positive rate of expression of P21in the phlegm and blood stasis type and Yin Deficiency type was opposite. But the above expressions between the two groups showed no significant difference.It prompted that in the process of disease caused by phlegm and blood stasis, high expression of positive regulatory factor of CyclinD1and CyclinE, and low expression of negative regulatory factor P16leaded to abnormal cell cycle regulation might be the molecular biology mechanism. However, it needs to be verified by further in-depth study of expanding the sample size.
Deficiencies and Prospects
The study still had many deficiencies, such as limitted research information including insufficient sample size and non-multi-center study, and the insufficient study design in both clinical research table content and the selection of specimen collection method in experimental study. There was no any clear basis for Chinese Medicine of the cellular and molecular biological mechanism from the research results by now. The research study should be gradually modified and improved in the future clinical and experimental practice.
引文
[1]林明雄,焦华梅,王发渭.化痰祛瘀法治疗恶性肿瘤探讨[J].中华中医药杂志2005,20(6)：369.
[2]高静东,太加斌,张彦博.痰瘀相关与肿瘤[J].浙江中医药大学学报.2006,30(4)：333-334.
[3]曾永蕾.从痰瘀论治肿瘤探析[J].中医药临床杂志2007,19(4)：326-328.
[4]罗克强,张小伯,王娜亚,等.喉显微手术及中西医结合治疗喉癌前病变临床观察[J].中国中西医结合耳鼻咽喉科杂志2005,13(5)：262-264.
[5]李云英.中医古籍论喉喑的辨治[J].广州中医药大学学报,1999,16(4)：325-327
[6]李云英,陈海,邓时贵,等.“金喉雾化剂”治疗慢性喉炎的疗效评价及计算机声图谱分析[J].中国中西医结合耳鼻咽喉科杂志1999,7(3)：114-117.
[7]曾益新主编.肿瘤学[M].北京：人民卫生出版社,2006.7(第二版)：151-177.
[8]Sandal T, Molecular aspects of themammalian cell cycle and cancer.2002,7:73-81
[9]Dobashi Y, Jiang S X, et al. Diver sity in expression and prognostic significance of G1/S cyclins in human primary lung carcinomas [J]. Journal of Pathology,2003, 199(2):208-220.
[10]邹向阳,李连宏.细胞周期调控与肿瘤[J].国际遗传学杂志,2006,29(1)：70-73.
[11]王陈应,等.中医临床大全·五官科分册[M],北京：北京科学技术出版社,1991.12.
[12]王德鉴.中医耳鼻咽喉口腔科学[M].北京：人民卫生出版社,1994,第一版：466-467.
[13]王德鉴.中医耳鼻咽喉口腔科学[M].北京：人民卫生出版社,1994,第一版：467.
[14]蔡福养,王永钦主编.嗓音病的中医论治[M].北京：人民卫生出版社,1988,第一版：156.
[15]何宗德等主编.现代中医耳鼻喉口齿科学[M].合肥：安徽科学技术出版社,1986,192、285.
[16]鹿道温主编.中西医临床耳鼻咽喉科学[M].北京：中国中医药出版社,1998
[17]王德鉴主编.中国医学百科全书·中医耳鼻咽喉口腔科学[M].上海：上海科学技术出版社,1985：65
[18]王永钦主编.中医药学高级丛书·中医耳鼻咽喉口腔科学[M].北京：人民卫生出版社,2001,第一版：745.
[19]程爵棠.中医喉科精义[M].北京：学苑出版社,1993,第一版：201.
[20]严道南,陈小宁主编.干祖望中医五官科经验集[M].江苏科学技术出版社,1992,10：112
[21]余养居,张守杰等主编.中西医结合嗓音病学[M].上海：知识出版社,1996,第一版：444.
[22]郑吕雄,忻耀杰,李春芳.中医中药治疗喉白斑[J].上海中医药杂志,2003.1,37(1)：41-42.
[23]李春芳,忻耀杰,张弢.喉白斑和喉乳头状瘤的中医治疗[J].中国中西医结合耳鼻咽喉科杂志,2006,14(1)：43-44.
[24]王辉.中医药治疗喉白斑临床心得[J].甘肃科技,2007.12,23(12)：217,215.
[25]刘树春.中医药治疗喉白斑6例[J].中华中西医临床杂志,2003,3(12)：1526-1527.
[26]李朝晖,郭晓东,宣永华.中西医结合治疗喉白斑病临床观察[J].中国中西医结合耳鼻咽喉科杂志,2005,13(3)：170-171.
[27]杨旭,尉瑞,袁晓辉.等.熊大经预防声带白斑术后复发的经验[J].上海中医药杂志,2007,41(11)：12-13.
[28]李蕾.李淑良教授治疗喉良性肿瘤的经验[J].中华中医药杂志.2008,23(7)：597-598.
[29]姚松苗,丁志红.自拟益气清音汤治疗慢性喉炎46例疗效浅析[J].中国中西医结合耳鼻咽喉科杂志.1998,6(4)：208.
[30]万爱珍.二阴煎加减治疗慢性喉炎20例[J].实用中西医结合临床.2002,2(6)：26-27.
[31]徐泳,俞军.化痰活血汤治疗慢性肥厚性喉炎临床研究[J].辽宁中医杂志,2000,27(9)：401-402.
[32]林丹娜,郭雄伟.化痰逐瘀汤治疗慢性肥厚性喉炎57例[J].新中医,2006,38(1)：63.
[33]曹建国,朱敏君,,金杰,等.喉癌前期病变的中西医结合治疗分析[J].上海医药,2004,25(3)：126-127.
[34]曹建国,朱敏君,金杰,等.喉癌前期病变的中西医结合治疗分析[J].上海医药.2004,25(3)：126-127.
[35]徐静.干祖望教授对嗓音疾病的辨治[J].辽宁中医杂志,1987,(7)：5-7.
[36]刘昕,刘桂英.中西医结合治疗喉白斑[J].基层医学论坛,2010,14(1)：66-67.
[37]罗克强,张小伯,王娜亚,等.喉显微手术及中西医结合治疗喉癌前病变临床观察[J].中国中西医结合耳鼻咽喉科杂志,2005,13(5)：262-264.
[38]蔡纪堂,柴峰.慢性肥厚性喉炎辨治体会[J].中国中医药信息杂志,2003,10(9)：66.
[39]黄志强.中西医结合治疗慢性肥厚性喉炎疗效观察(附42例报告)[J].医学理论与实践,2008,21(7)：811-81
[40]刘永革,马学忠.加味会厌逐瘀汤治疗慢性肥厚性喉炎129例[J].中国民间疗法.2004,12(3)：47-48.
[41]王济生,赵春霞,乔自军.活血开音汤治疗肥厚性喉炎42例[J].四川中医,1999；17(5)：47.
[42]庄金梅,白帆,林淑琴.活血化瘀法治疗肥厚性喉炎200例疗效观察[J].中国中西医结合耳鼻咽喉科杂志,2000,8(1)：30-31.
[43]余养居,张守杰,胡素琴,主编.中西医结合嗓音病学[M].上海：知识出版社,1996,第版：453.
[44]蔡福养,王永钦主编.嗓音病的中医论治[M].北京：人民卫生出版社,1988,第一版：156.
[45]郭毅.温肾活血法治喉瘖[J].四川中医,1994；(2)：50-51.
[46]冯纬纭.中药超声雾化配合微波治疗慢性喉炎疗效观察[J].广西中医药,2001；24(6)：36.
[47]郭志正,孙月梅.中药超声雾化吸入治疗慢性喉炎50例[J].中国中西医结合耳鼻咽喉科杂志,2000：8(4)：192.
[48]陈国琴.针刺治疗喉瘖32例疗效分析[J].针灸临床杂志,2003；19(11)：31-32.
[49]皮健.针刺人迎、水突穴治疗歌者声带肥厚如例的疗效观察[J].中国针灸,1987；(3)：17.
[50]邵素霞.针灸治疗喉瘖38例[J].河南中医,2003；23(9)：69.
[51]崔允孟.耳针治疗声嘶症150例疗效观察[J].中级医刊,1993；18(10)：52-53.
[52]张安福.消炎止痛膏治疗慢性喉炎效佳[J].四川中医,1989；(1)封3.
[53]马玉杰,白力学,纪建芹.中药透入治疗慢性咽喉炎35例[J].中国民间疗法,1998；6(5)：7.
[54]詹光宗,中药离子直流电导入治疗急慢性咽喉炎[J].中国针灸,2001,21(8)：489.
[55]杨华,仲彦.推拿法治疗慢性喉炎18例[J].中国民间疗法,1997；(5)：33.
[56]金平林.鱼际隔蒜泥敷贴为主治疗喉瘖26例[J].中国针灸,2001；21(10)：592.
[57]宋南吕.耳压配合中药治疗喉瘖30例[J].江西中医药,1997；28(3)：40.
[58]李云英.中医古籍论喉瘖的辨治[J].广州中医药大学学报,1999,16(4)：325-327.
[59]李云英,陈海,邓时贵,等.“金喉雾化剂”治疗慢性喉炎的疗效评价及计算机声图谱分析[J].中国中西医结合耳鼻咽喉科杂志1999,7(3)：114-117.
[60]邓时贵,欧润妹,李云英,等.雾化吸入活血化痰中药对实验动物气管分泌和微循环的影响[J].广州中医药大学学报.1999,16(4)：311-313.
[61]彭桂原,李云英.喉癌前病变气滞血瘀证与VEGF的相关性研究[J].新中医,2007,39(7)：43-44.
[62]乔宗海,徐洁洁,程雷,主编.现代喉部疾病诊断与治疗[M].南京：东南大学出版社,1999,第一版：178-183.
[63]Ferlito A, Carbone A, DeSanto LW. " Early" cancer of the larynx:the concept as defined by clinicians, pathologists and biologists[J]. Ann Otol Rhinol Laryngol,1996, 106:245-250.
[64]田勇泉主编.耳鼻咽喉一头颈外科学[M].北京：人民卫生出版社,2004.6(第6版)：215-220.
[65]屠规益,吕春梅.耳鼻咽喉头颈外科医师与喉癌术后发声功能重建.听力学及言语疾病杂志,2010,18(1)：1.
[66]王鹏,刘宝国,李树玲.全喉切除术后发音重建术研究进展.中华临床床医师杂志(电子版).2009,3(9)：82-86
[67]黄选兆,汪吉宝主编.实用耳鼻咽喉科学[M].北京：人民卫生出版社,2005.7：501-502.
[68]韩德民,Robert T主编.嗓音医学[M].人民卫生出版社.2007第一版：198-199.
[69]韩德民,Robert T主编.嗓音医学[M].人民卫生出版社.2007第一版：199.
[70]王军,韩德民,叶京英,等.应用C02激光治疗可疑喉癌前病变[J].耳鼻咽喉-头颈外科,2001,8(4)：217-219.
[71]Talamini R, Bosetti C, Lavecchia C, et al. Combined effect of tobacco and alcohol on laryngeal cancer risk:a case-control study [J]. Cancer Causes Control,2002,13:957-964.
[72]Riedel F, Goessler U, Hormann K. Alcohol-related diseases of the mouth and throat[J]. Best Pract Res Clin Gastroenterol,2003,17(4):543-55.
[73]李惠萍,崔秀娟,关超,等.喉癌前病变100例分析[J].中华耳鼻咽喉科杂志,1995,30(5)：302-304.
[74]Mork J, Lie AK, Glattre E, et al. Human papillomavirus infectionas a risk factor for squamous-cell carcinoma of the head and neck[J].New English Med.2001,344:1125-1131.
[75]Judd R, Zaki SR, Coffied LM, et al. Human papillomavirus type 6 detected by the polymerase chain reaction in invasive sionasal papillary squamous cell carcino mas[J]. Arch Pathol Labmed,1991;115:1150-1153.
[76]李晓明,卜国弦,郭晓峰.喉癌的病因学研究进展[J].国外医学耳鼻咽喉科分册,1998,12(1)：16.
[77]Shaw H, Ballantyne J, Groves J. Scoth-Brown's diseases of the ear, nose and throat[M]. London:Butterworth,1979:109-122.
[78]张小伯.发声显微外科的发展与临床应用.中华耳鼻咽喉科杂志,2004,39：253-256.
[79]Mozota Ortiz JR. Cancer of the vocal cord, early diagnosis by stroboscop[J]y. Acta Otorhinolaryngol Esp,1998;28:91-104.
[80]宋发全,张芩娜,王建亭,等.声带癌前病变及癌变动态喉镜追踪观察[J].中华耳鼻咽喉科杂志,2000；35(2)：144-146.
[81]Zariwala M, Schmlu S, Pialoz M, et al. P53 gene mutations in oropharyngeal carcinomas:A comparison of solitary and mutiple primary tumours and lymph node metastases[J]. Cancer.1994,56(6):807.
[82]Hall PA, Ray A, Lemoine NR, et al.P53 immunostaining as a marker of mali gnant disease in diagnostic cytopathology[J]. Lancet,1991,338:513.
[83]周光耀,林代诚,梁传余,等.P53蛋白在喉上皮癌前病变及癌组织中的表达及其意义[J].华西医大学报,1999,30(3)：265-267.
[84]肖健云,罗均利,陶正德,等.P53蛋白在喉癌及癌旁病变组织中的表达[J].中国耳鼻咽喉颅底外科杂志,1995,1(1)：47-49.
[85]邓志宏,黄维国,邱建华等.血管生成及细胞凋亡与喉黏膜上皮癌变的关系[J].第四军医大学学报,1999,20(1)：17-20.
[86]Feinmesser R, et al. Flow cytometer analysis of DNA content in laryngeal cancer[J]. Laryngology Otology,1998,104:485.
[87]周剑勇,梁素霞.喉癌前病变DNA含量测定及细胞周期分析的意义[J].广东医学.2001,22(3)：201-202.
[88]Yuan Y, Chi F, et al. Significance of ceramide and DNA ploidy in laryngeal carcinogenesis[J]. ORL Otorhinolaryngol Relat Spec.2007,69(5):283-288.
[89]Guillermo J Tearney, Mark E Brezinski, Brett E Bouma, et al. In vivo endoscopic optical biopsy with optical coherence tomogyaphy[J]. Science,1997,276(27):2037-2039.
[90]Ralph S Dacosta, Brian C Wilson, Norman E Marcon. New optical technologies for earlier endoscopic diagnosis of premalignant gastrointestinal lesions[J]. Journal of Gastroenterology and Hepatology,2002,17(suppl):85-104.
[91]Kulapadituarom B et al. Ann Otol Rhinol Laryngol,2001,110:45-52.
[92]张小伯,刘丹丹,杨大章,等.早期喉癌与声带癌前病变的诊断和微创治疗[J].中华耳鼻咽喉科杂志,1999,34(6)：337-339
[93]顾建森,李秋珍,张艳萍.喉癌前病变早期干预治疗[J].南京铁路医学院学报,2000,19(2)：129-130.
[94]Ricci G, Molini E. Retrospective stuffy on precancerous laryngeal lesions:long-term follow-up[J].Acta Otorhinolaryngologica Italica,2003,23 (5):362-367.
[95]杨桂芬,曾勇,管俊华.喉癌前病变临床疗效观察——附27例报告[J].肿瘤预防与治疗,2008,21(2)：185,189.
[96]宋磊,张涛源.喉乳头状瘤治疗新进展[J].山西医药杂志,2007,36(1)：49-50.
[97]王新华,孙敬武.小儿喉乳头状瘤的研究进展[J].听力学及言语疾病杂志,2008,16(4)：347-348.
[98]Benjamin BN, et al. Ann Otol Rhinol Lanyngol,1988;97:376-380
[99]Healy GB, et al. N Eng J Med,1988:319:401-407.
[100]Leventhal GB, et al. N Eng J Med,1991;325:613-617.
[101]Mc cabe BF, et al. Ann Otol Rhinol Lanyngol,1987:96:158-162.
[102]Deunas LV, et al. J Lanyngol Otol,1997;111:134-140.
[103]张小伯,刘丹丹,杨大章,等.早期喉癌与声带癌前病变的诊断和微创治疗[J].中华耳鼻咽喉科杂志,1999；34(6)：337-339.
[104]骆云珍,钱林荣,李海同.水分离技术在声带白斑剥脱术中的应用[J].中国中西医结合耳鼻咽喉科杂志,2008,16(3)：213-214.
[105]王军,韩德民,叶京英,等.应用C02激光治疗可疑喉癌前病变[J].耳鼻咽喉-头颈外科,2001,8(4)：217-219.
[106]顾建森,李秋珍,张艳萍.喉癌前病变早期干预治疗[J].南京铁道医学院学报,2000,19(2)：129-130.
[107]吕秋萍,孙敬武,胡燕明,等.激光治疗喉癌前病变[J].中国耳鼻咽喉头颈外科.2008,15(12)：661-664.
[108]Devaiah AK, Shapshay SM, Desai U, et al. Surgical utility of a new carbondioxide laser fiber:functional and histological stud[J]. Laryngoscope,2005,115(8):1463-1468.
[109]冯卫文,黄桂球,黄家英,等.显微喉镜下钬激光治疗喉癌前病变36例观察[J].暨南大学学报(医学版),2009,30(2)：215-216.
[110]孙广滨,周水淼,王昶,等Diomed-25半导体激光在治疗喉癌前病变中的应用[J].山东大学基础医学院学报,2003,17(3)：158-159.
[111]孙广滨,周水淼,温武,等.半导体激光治疗声带白斑30例临床分析[J].临床耳鼻咽喉科杂志,2004,18(3)：141-143.
[112]杨哗,白薇琦.Nd-YAG激光结合内窥镜在喉癌前病变手术治疗中的应用[J].现代实用医学,2007,19(11)：909-910.
[113]刁玉华,张玉兰,王玮.纤维喉镜下微波凝集治疗喉角化病的临床研究[J].中国中西医结合耳鼻咽喉科杂志,2002,10(5)：226-228.
[114]刘锋综.微波在耳鼻喉科的应用[J].国外医学耳鼻喉科学分册,1993,17(4)：222-225.
[115]蔡冰国,王雪峰.喉癌前病变的诊断和治疗[J].社区医学杂志,2008,6(9)：38-39.
[116]Malczewsk M. Clinical applications of photodynamic therapy in treatment of laryngeal lesions[J]. Otolaryngol,1999,53(6):671-675.
[117]Sibata CH, Colussi VC, Oleinick NL, et al. Photodynamic therapy in oncology [J]. Expert Opin Pharmacother,2001,2:917-927.
[118]刘蓓,梁建平,朱习平,等.显微支撑喉镜下射频治疗早期声门癌及癌前病变[J].广西医学,2007,29(9),1331-1333.
[119]张庆丰,刘得龙,张悦,等.等离子射频治疗早期声门型喉癌的初步研究[J].中华耳鼻咽喉头颈外科杂志.2011,46(01)：164-165.
[120]Patel RS, MacKenzie K. Powered laryngeal shavers and laryngeal papillomatosis: a preliminary report[J]. CAin Otolaryngol,2000,25:358.
[121]Flint PW. Powered surgical instruments for laryngeal surgery [J]. Otolaryngol Head Neck surg,2000,122:263.
[122]Parsons DS, Bothwell MR. Powered instrument papiloma excision:an alternative to laser therapy for recurrent respiratory papilloma[J]. Laryngoseope,2001. 111:1494.
[123]钱晓云,俞晨杰,沈晓辉.支撑喉镜下吸引旋切结合射频治疗喉癌前病变的疗效评价[J].中国微创外科杂志,2008,8(2)：104-106.
[124]Maran A. Head and neck surgery:An ercyciopaedia of ignorance[J]. Laryngol Otol, 1990,104:529.
[125]邹向阳,李连宏.细胞周期调控与肿瘤[J].国际遗传学杂志,2006,29(1)：70-73.
[126]邓琳.Cyclin D1的多态性与肿瘤[J].国外医学：肿瘤学分册,2001,28(3)：163-164.
[127]陈万军,王天铎.细胞周期蛋白循环索在喉咽癌生物学行为中的意义[J].临床耳鼻咽喉科杂志,2005,19(3)：250-252.
[128]付志婕,潘新良,雷大鹏,等.细胞周期索D1和p16在喉咽癌中的表达及临床意义[J].临床耳鼻咽喉头颈外科杂志,2007,21(9)：406-409.
[129]Zhang H. Life without kinase:cyclin E prootes DNA repliction licensing and beyond[J].Mol Cell.2007,25(2):175-176.
[130]Yamanouchi H, Furihata M, Fujita J, et al. Expression of cyclin E and cyclin D1 in non-small cell lung Cancers[J]. Lung Cancer,2001,31(1):3-8.
[131]Pagano M, Pepperkok R, Verde F. Cyclin A is required at two points in the human cell cycle[J]. EMBOJ,1992,11(5):961.
[132]Yasmeen A, Berdel W E, Serve H, et al. E-and A-type cyclins as markers for cancer diagnosis and prognosis[J]. Expert Rev Mol Diagn,2003,3(5):617-633.
[133]王建波,高福平,张秀兰,等.喉鳞状细胞癌中Cyclin A、B1表达的临床意义[J].中国肿瘤临床与康复,2002,9(4)：63-65.
[134]Fraczek M, Wozniak Z, Ramsey D, et al. Expression patterns of Cyclin E, Cyclin A and CDC25 phosphatases in laryngeal carcinogenesis[J]. Eur Arch Otorhinolaryngol,2007, 264 (8):923-928.
[135]Rodriguez-Pinilla M, Rodriguez-Peralto JL, Hitt R, et al. Cyelin A as a predictive factor for chemotherapy respOnse in advanced head and neck cancer[J]. Clin Cancer Res, 2004,10(24):8486-8492.
[136]Stark GR, Taylor WR. Control of the G2/Mtransition[J]. Mol Bioteehnol,2006,32(3): 227.
[137]Nurse P. Ordering S phase and M phase in the cell cycle [J]. Cell,1994,79:547.
[138]Yew PR. Ubiquitin mediated proteolysis of vertebrate Gl-and S-phase regulator [J]. J Cell Physiol,2001,187:1.
[139]Dong Y, Sui L, Watanabe Y, et al. Clinical relevance of Cyclin B1 overexpression in laryngeal squamous cell carcinoma[J]. Cancer Lett,2002,177(1):13-19.
[140]王建波,高福平,张秀兰,等.喉鳞状细胞癌中Cyclin A、BI表达的临床意义[J].中国肿瘤临床与康复,2002,9(4)：63-65.
[141]陈万军,李梅,王天铎,等.CDK4与下咽癌病变的相关性及其生物学行为的初步研究[J].山东大学基础医学院学报,2005,19(3)：149-152.
[142]李秀敏.细胞周期、染色质和癌：基于肿瘤机制的治疗[J].国外医学：药学分册,2004,31(2)：75-77.
[143]Zhang H. Life without kinase:cyclin E prootes DNA repliction licensing and beyond[J].Mol Cell.2007,25(2):175-176.
[144]蔡扬,何园,卢虹,等.口腔鳞状细胞癌cyclin E及P21Waf 1蛋白定量检测及意义[J].中国肿瘤临床,2008,35(3)：142-144.
[145]Shapiro GI, Edwards CD, Rollins BJ. The physiology of p16(INK4A)-mediated G1 proliferative arest[J]. Cell Biochem Biophys,2000,33(2):189-197.
[146]Jares P, Nadal A, Fem6ndez PL, et al. Disregulation of p16MTSI/CDK41 protein and mRNA expression is associated with gene alteations in squamous cell carcinoma of the larynx[J]. Int J Cancer,1999,81(5):705-71I.
[147]付志婕,潘新良,雷大鹏,等.细胞周期索D1和p16在喉咽癌中的表达及临床意义[J].临床耳鼻咽喉头颈外科杂志,2007,21(9)：406-409.
[148]温树信,王斌全,陈彦球,等.抑癌基因p15、Rb在喉鳞状细胞癌中表达的研究[J].临床耳鼻咽喉科杂志,2003,17(9)：543-544.
[149]李福才,李英慧,赵旭,等.喉癌中p15、p16基因纯合缺失与EGFR基因扩增相关性研究[J].遗传学报,2004,31(2)：109-113.
[150]Eddy AA. Expression of genes that poremote renal interstitial fibrosisin rats with proteinuria[J]. Kidney Int,1996,54:149-154.
[151]Sofia G, Speroni J, Podhajcer OL, et al. p21 differentially regulates DNA replication and DNA-repair-associated processes afterUV irradiation [J]. J Cell Sci,2008,121(19): 3271-3282.
[152]王建波,王金泉,娄卫华等.细胞周期蛋白Dl和P21WAF1/CIP1在喉癌癌变过程中的表达及其意义[J].中国癌症杂志,2000,10(1)：59-61.
[153]李晖,江孝清,周绪红。Skp2、p27蛋白在喉鳞状细胞癌中的表达及意义[J].临床耳鼻咽喉科杂志,2005,19(9)：409-411.
[154]金晓杰,钱敏飞,许雁平,等.喉鳞状细胞癌手术切缘分子水平亚健康与预后关系研究[J].临床耳鼻咽喉科杂志,2006,20(24)：1111-1113.
[155]孔萌萌,中丽娟,金克炜.p57kip2与肿瘤[J].国外医学：肿瘤学分册,2005,32(2)：100-102.
[156]Fan GK, Xu F, Yang B, et al. P57kip2 expression is related to carci, nogenesis and tumor pmgresslon in laryngeal tissues[J]. Acta Otolaryngol,2006,126(3):301-305.
[157]杨氏麟,著.嗓音医学基础与临床[M].辽宁科学技术出版社.2001：370-371.
[158]陆美萍,徐洁洁.用声不当或过度致发声障碍的基础与临床研究[J].听力学及言语疾病杂志.2004,12(4)：276-277.
[159]李慧萍,崔秀娟.喉癌前病变100例分析[J].中华耳鼻咽喉科杂志,1995,30(5)：302-304.
[160]Yang GH, Ma JM, Liu N, et al. Smoking and passive smoking in Chinese,2002[J]. Chin J Epidemiol,2005,26(2):77-83.
[161]陈爱俊.绝大多数喉癌患者有长期吸烟史[J].中国乡村医药.2009,16(1)：15.
[162]Jacques B. Head and neck cacer:Multimodality Management[M]. Springer Science Business Media.2011,9:238.
[163]韩德民,Robert TS.主编.嗓音医学[M].人民卫生出版社,2007,第一版：199.
[164]韩德民,Robert TS.主编.嗓音医学[M].人民卫生出版社,2007,第一版：199.
[165]李慧萍,崔秀娟.喉癌前病变100例分析[J].中华耳鼻咽喉科杂志,1995,30(5)：302-304.
[166]Hashibe M, Sankaranarayanan R, Thomas G, et al. Alcohol drinking, body mass index and the risk of oral leukoplakia in an Indian opulation[J]. Cancer.2000,88:129-134.
[167]Gupta PC, Hebert JR, Bhonsle RB, et al. Dietary factors in oral leukoplakia and submucous fibrotlis in a population-based case control study in Gujarat, India[J]. Oral Dis 1998,4:200-6.
[168]Gupta PC, Hebert JR, Bhonsle RB, et al. Influence of dietary factors on oral precancerous lesions in a population-based case-control study in Kerala, India[J]. Cancer.1999,85:1885-93.
[169]Flanders W D, et al. Work-related risk factors for laxyngeal cancer. Am J Epidemiol 1984,119:23.
[170]Olsen J, Sabroe S. Occupational causes for laryngeal cancer. J Epldemiol. Community Health.1984; 38:117.
[171]张克,费声重,陈哲,等.辽宁省工业城市大气污染与喉癌发病率的关系[J].中华耳鼻咽喉科杂志,1990,25(4)：240-242.
[172]孙克敏,肖立峰,周彬,等.喉癌患者血清微量元素的研究[J].中国眼耳鼻喉科杂志,1998,3(3)：94-96+85.
[173]智铁铮.喉癌前病变国外医学·耳鼻咽喉科分册[J].1988,12：13.
[174]谢汝华,闵华庆,曾宗渊,等.喉癌与性激素受体及手术前后血清性激素水平的研究[J].癌症.1994,(5)：56-59.
[175]Robin. Expression of Androgen Receptor and Estrogen Receptor in Glottic and Supraglottic Carcinoma of Larynx[J]. Cervical Cancer,2002, (7):165-168.
[176]Plch J, Par J, Navratilova J, et al. Long term follow-up study of laryngeal precancer[J]. Auris Nasus Larynx,1998,25:407-412.
[177]王正敏,陆书昌,主编.现代耳鼻咽喉科学[M].北京：人民军事出版社,2001,第一版：952.
[178]李惠萍,崔秀娟,关超,等.喉癌前病变100例分析[J].中华耳鼻咽喉科杂志,1995,30(5)：302-304.
[179]孙广滨,周水淼,王昶,等Diomed-25半导体激光在治疗喉癌前病变中的应用[J].山东大学基础医学院学报,2003,17(3)：158-159.
[180]黄选兆,汪吉宝,主编.实用耳鼻咽喉科学[M].北京：人民卫生出版社,1998,第一版：501-502.
[181]彭桂原,李云英.喉癌前病变气滞血瘀证与VEGF的相关性研究[J].新中医,2007,39(7)：43-44.
[182]Yamanouchi H, Furihata M, Fujita J, et al. Expression of cyclin E and cyclin D1 in non-small cell lung Cancers[J]. Lung Cancer,2001,31(1):3-8.
[183]胡立胜,周强.中医临床研究设计与SAS编程统计分析[M].北京：学苑出版社,2004：68.
[184]徐静.干祖望教授对嗓音疾病的辨治[J].辽宁中医杂志,1987；(7)：5-7.
[185]王琦.王琦男科学[M].第2版.郑州：河南科学技术出版社,2007：3：79.
[186]王莉,何裕民.男女体质特点及其异同的研究——附2967例流调资料的模糊识别分析[J].中国中医基础医学杂志.1998,2(2)：7-9.
[187]张晓英,郭利平.冠心病心绞痛中医证型分布在性别上的差异性[J].浙江中医杂志.2009,44(6)：406-407.
[188]陈志强,吕立国.整体辨证、局部辨证与微观辨证——对现代中医辨证论治体系的思考[J].中国中西医结合杂志.2006,(12)：1076-1080.
[189]干祖望著.干氏耳鼻咽喉口腔科学[M].江苏科学技术出版.1999第1版：28-30.
[190]李云英.中医古籍论喉喑的辨治[J].广州中医药大学学报,1999,16(4)：325-327.
[191]李云英,陈海,邓时贵,等.“金喉雾化剂”治疗慢性喉炎的疗效评价及计算机声图谱分析[J].中国中西医结合耳鼻咽喉科杂志1999,7(3)：114-117.
[192]邓时贵,欧润妹,李云英,等.雾化吸入活血化痰中药对实验动物气管分泌和微循环的影响[J].广州中医药大学学报.1999,16(4)：311-313.
[193]彭桂原,李云英.喉癌前病变气滞血瘀证与VEGF的相关性研究[J].新中医,2007,39(7)：43-44.
[194]杜芸王小玲吴国祥,等.流式细胞术对食管癌组织中四种细胞周期调控蛋白的定量检测及意义[J].中华肿瘤杂志.2004,26(10)：612-614.
[195]Gillett CE, Lee AHS, Millis RR, et al. Cyclin D1 and associated proteins inmammary ductal carcinoma in situ and atypical ductal hyperplasia[J]. J Pathol,1998,184(4): 396.
[196]sherr CJ, Roberts JM. Inhibitorsofmammalian G1 cyclin dependent kinase[J]. Genes Dev,1995,9(10):1149.
[197]Donnellan R, Cherty R. Cyclin D1 and human neoplasia[J]. Mol Pathol,1998,51(1): 1.
[198]刘馨莲,殷舞,李淑蓉,等.细胞周期蛋白在喉癌组织中的表达及意义.实用癌症杂志.2011,26(6)：585-587+595
[199]Donnelan R, Kleinschmidt I, Chetty R, et al. Cyclin E immunoexpression in breast ductal carcinoma:pathologic correlations and prognostic implications[J]. Hum Pathol, 2001,32(1):89.
[200]Kim H K, Park I A, Heo D S, et al. Cyclin E overexpression as an independent risk factor of visceral relapse in breastcancer[J]. Eur J Surg Oncol,2001,27(5):464.
[201]Georgieva J, Sinha P, Schadendorf D, et al. Expression of cyclins and eyclin dependent kinases in human benign and malignant melanocytic lesion[J]. Clin Pathol,2001,54(3): 229.
[202]Reika I, Takashi K, Yoichi A. Association of P16 homozygous deletions with clinicopathologic characteristics and EGFR/RAS/53 mutations in lung adenocarcinoma colloid[J]. Clin Cancer Res,2008,14:3746-3753.
[203]Semczuk A, Miturski R, Skomra D, et al. Expression of the cell. cyle regulatory proteins (pRb, cyclin D1, pI6INK4A and cdk4) in human endometrial cancer:correlation with clinicopathological features[J]. Arch Gynecol Obstet,2004,269(2):104-110.
[204]Ivanova TA, Golovina DA, Zavalishina LE, et al. Up-regulation of expression and lack of 5 CpG island hypermethylation of pl6ink4a in HPV-positive cervical carcinomas [J]. BMC Cancer,2007,14(7):47.
[205]马玉泉,杨晓光,刘辉,等.p21,p27基因多态性对食管癌的发生及淋巴结转移的影响[J].第四军医大学学报.2008,29(17)：1600-1603.
[206]高蔚,金锋.大肠癌中p21 ras表达与血管新生的临床病理意义[J].中国医科大学学报.2007,36(4)：455-457.
[207]冉志华,冯樱,邱德凯,等.重组细小病毒H-1(rhH1/p21)对胃癌细胞HGC27生长抑制作用的研究[J].肿瘤.2007,27(4)：251-255.
[208]王黎明,鹿彩莲,纪祥瑞,等.细胞周期调控相关蛋白P53、P16、P21和Rb在子宫内膜癌的表达及意义[J].中国妇产科临床.2001,2(5)：292-294
[209]Weinstein IB, Santella RM, Perera F. Molecular biology and molecular epidemiology of cancer. In:greenwald P, Kramer BS, Weed DL(eds)Cancer Prevention and Control[J]. Bethesda:Marcel-Dekker,1995:83-110.
[210]Yespa SH. Overview of carcinogenesis:past, present and future[J]. Carcinogenesis.2000,21:341-345.
[211]Hanahan D, Weinberg RA. The hallmarks of cancer[J]. Cell 2000,100:57-70.
[212]Yuspa SH, Poirier MC. Chemical carcinogenesis:from animal models to molecular models in one decade[J]. Adv Cancer Res.1988,50:25-70.
[213]王建波,王金泉,娄卫华.等.细胞周期蛋白D1和P21WAF1/CIP1在喉癌癌变过程中表达及其意义[J].中国癌症杂志.2000,10(1)：59-61.
[214]陈万军,李梅,王兴武.等.细胞周期调控因子与下咽癌生物学行为相关性初步研究[J].中国肿瘤临床,2007,34(1)：26-29.
[215]蔡扬,何园,卢虹,等.口腔鳞状细胞癌cyclin E及P21wWaf1蛋白定量检测及意义[J].中国肿瘤临床.2008,35(3)：142-144.
[216]Zhang H. Life without kinase:cyclin E prootes DNA repliction licensing and beyond[J].Mol Cell.2007,25(2):175-176.
[217]Yamanouchi H, Furihata M, Fujita J, et al. Expression of cyclin E and cyclin D1 in non-small cell lung Cancers[J]. Lung Cancer,2001,31 (1):3-8.
[218]Hu YX, Watanabe H, Ohtsubo, et al. Frequent loss of p16 expression and its correlation with clinicopathological parameters in pancreatic carcinoma[J]. Clin Cancer Res,1997, 3:1473-1477.
[219]Arap W, Nishikama R, Furnari FB, et al. Replaement of the p16/CDKN2 gene suppresses human glioma cell growth[J]. Cancer Res,1995,55:1351.
[220]OHTANI N, YAMA KOSHI K, TA KA HASHI A, et al. The p16 IN K4a-RB pathway:molecular link between cellular senescence and tumor suppression [J]. J Med Invest,2004,51:146-153.
[221]GALLO O, SANTUCCI M, FRANCHI A. Cumulative prognostic value of pl6/CDKN2 and p53 oncoprotein expression in premalignant laryngeal lesions[J].J Natl Cancer Inst.1997, 89:1161-1163.
[222]李叶青,顾其华,李运良,等.口腔鳞癌K-ras基因测序分析及p21蛋白的表达[J].海南医学院学报.2004,10(4)：217-219.
[223]JangTJ, KangMS, KimH, et al. Increased Expression of Cyclin D1, Cyclin E and p21Cip1 Associated with Decreased Expression of p27Kipl in Chemically Induced Rat Mammary Carcinogenesis[J].Jpn. Cancer Res.2000,12 (91):1222-1232.
[224]Coverley D, Laman H, Laskey RA. Distinct roles for cyclin-E and A during DNA relication complex assembly and activation[J]. Nat CellBiol,2002,4(7):523-528.
[225]LodenM, StighalLM, Nielsen NH, et al. The cyclin-D1 high and cyclin-E high subgroup s of breast cancer:separate pathways in tumorgenesis based on pattern of genetic aberrations and inactivation of the pRb node[J]. Oncogene.2002,21 (30):4680-4690.
[226]冯红,于卫芳,张立玮,等.贲门癌变过程中Survivin和CyclinE蛋白的表达及相关性研究[J].中国综合临床.2007,23(1)：52-54.
[227]Shintani S, Mihara M, Nakahara Y, et al. Expression of cell cycle control proteins in normal epithelium, premalignant and malignant lesions of oral cavity[J]. Oral Oncol.2002,38(3):235-243.
[228]姜文奇,张晓实,朱孝峰,等.肿瘤生物治疗学[M].广州：广东科技出版社.2006：4.
[229]曾益新 主编.肿瘤学[M].第2版.北京：人民卫生出版社,2003：11-12.
[2]高静东,太加斌,张彦博.痰瘀相关与肿瘤[J].浙江中医药大学学报.2006,30(4)：333-334.
[3]曾永蕾.从痰瘀论治肿瘤探析[J].中医药临床杂志2007,19(4)：326-328.
[4]罗克强,张小伯,王娜亚,等.喉显微手术及中西医结合治疗喉癌前病变临床观察[J].中国中西医结合耳鼻咽喉科杂志2005,13(5)：262-264.
[5]李云英.中医古籍论喉喑的辨治[J].广州中医药大学学报,1999,16(4)：325-327
[6]李云英,陈海,邓时贵,等.“金喉雾化剂”治疗慢性喉炎的疗效评价及计算机声图谱分析[J].中国中西医结合耳鼻咽喉科杂志1999,7(3)：114-117.
[7]曾益新主编.肿瘤学[M].北京：人民卫生出版社,2006.7(第二版)：151-177.
[8]Sandal T, Molecular aspects of themammalian cell cycle and cancer.2002,7:73-81
[9]Dobashi Y, Jiang S X, et al. Diver sity in expression and prognostic significance of G1/S cyclins in human primary lung carcinomas [J]. Journal of Pathology,2003, 199(2):208-220.
[10]邹向阳,李连宏.细胞周期调控与肿瘤[J].国际遗传学杂志,2006,29(1)：70-73.
[11]王陈应,等.中医临床大全·五官科分册[M],北京：北京科学技术出版社,1991.12.
[12]王德鉴.中医耳鼻咽喉口腔科学[M].北京：人民卫生出版社,1994,第一版：466-467.
[13]王德鉴.中医耳鼻咽喉口腔科学[M].北京：人民卫生出版社,1994,第一版：467.
[14]蔡福养,王永钦主编.嗓音病的中医论治[M].北京：人民卫生出版社,1988,第一版：156.
[15]何宗德等主编.现代中医耳鼻喉口齿科学[M].合肥：安徽科学技术出版社,1986,192、285.
[16]鹿道温主编.中西医临床耳鼻咽喉科学[M].北京：中国中医药出版社,1998
[17]王德鉴主编.中国医学百科全书·中医耳鼻咽喉口腔科学[M].上海：上海科学技术出版社,1985：65
[18]王永钦主编.中医药学高级丛书·中医耳鼻咽喉口腔科学[M].北京：人民卫生出版社,2001,第一版：745.
[19]程爵棠.中医喉科精义[M].北京：学苑出版社,1993,第一版：201.
[20]严道南,陈小宁主编.干祖望中医五官科经验集[M].江苏科学技术出版社,1992,10：112
[21]余养居,张守杰等主编.中西医结合嗓音病学[M].上海：知识出版社,1996,第一版：444.
[22]郑吕雄,忻耀杰,李春芳.中医中药治疗喉白斑[J].上海中医药杂志,2003.1,37(1)：41-42.
[23]李春芳,忻耀杰,张弢.喉白斑和喉乳头状瘤的中医治疗[J].中国中西医结合耳鼻咽喉科杂志,2006,14(1)：43-44.
[24]王辉.中医药治疗喉白斑临床心得[J].甘肃科技,2007.12,23(12)：217,215.
[25]刘树春.中医药治疗喉白斑6例[J].中华中西医临床杂志,2003,3(12)：1526-1527.
[26]李朝晖,郭晓东,宣永华.中西医结合治疗喉白斑病临床观察[J].中国中西医结合耳鼻咽喉科杂志,2005,13(3)：170-171.
[27]杨旭,尉瑞,袁晓辉.等.熊大经预防声带白斑术后复发的经验[J].上海中医药杂志,2007,41(11)：12-13.
[28]李蕾.李淑良教授治疗喉良性肿瘤的经验[J].中华中医药杂志.2008,23(7)：597-598.
[29]姚松苗,丁志红.自拟益气清音汤治疗慢性喉炎46例疗效浅析[J].中国中西医结合耳鼻咽喉科杂志.1998,6(4)：208.
[30]万爱珍.二阴煎加减治疗慢性喉炎20例[J].实用中西医结合临床.2002,2(6)：26-27.
[31]徐泳,俞军.化痰活血汤治疗慢性肥厚性喉炎临床研究[J].辽宁中医杂志,2000,27(9)：401-402.
[32]林丹娜,郭雄伟.化痰逐瘀汤治疗慢性肥厚性喉炎57例[J].新中医,2006,38(1)：63.
[33]曹建国,朱敏君,,金杰,等.喉癌前期病变的中西医结合治疗分析[J].上海医药,2004,25(3)：126-127.
[34]曹建国,朱敏君,金杰,等.喉癌前期病变的中西医结合治疗分析[J].上海医药.2004,25(3)：126-127.
[35]徐静.干祖望教授对嗓音疾病的辨治[J].辽宁中医杂志,1987,(7)：5-7.
[36]刘昕,刘桂英.中西医结合治疗喉白斑[J].基层医学论坛,2010,14(1)：66-67.
[37]罗克强,张小伯,王娜亚,等.喉显微手术及中西医结合治疗喉癌前病变临床观察[J].中国中西医结合耳鼻咽喉科杂志,2005,13(5)：262-264.
[38]蔡纪堂,柴峰.慢性肥厚性喉炎辨治体会[J].中国中医药信息杂志,2003,10(9)：66.
[39]黄志强.中西医结合治疗慢性肥厚性喉炎疗效观察(附42例报告)[J].医学理论与实践,2008,21(7)：811-81
[40]刘永革,马学忠.加味会厌逐瘀汤治疗慢性肥厚性喉炎129例[J].中国民间疗法.2004,12(3)：47-48.
[41]王济生,赵春霞,乔自军.活血开音汤治疗肥厚性喉炎42例[J].四川中医,1999；17(5)：47.
[42]庄金梅,白帆,林淑琴.活血化瘀法治疗肥厚性喉炎200例疗效观察[J].中国中西医结合耳鼻咽喉科杂志,2000,8(1)：30-31.
[43]余养居,张守杰,胡素琴,主编.中西医结合嗓音病学[M].上海：知识出版社,1996,第版：453.
[44]蔡福养,王永钦主编.嗓音病的中医论治[M].北京：人民卫生出版社,1988,第一版：156.
[45]郭毅.温肾活血法治喉瘖[J].四川中医,1994；(2)：50-51.
[46]冯纬纭.中药超声雾化配合微波治疗慢性喉炎疗效观察[J].广西中医药,2001；24(6)：36.
[47]郭志正,孙月梅.中药超声雾化吸入治疗慢性喉炎50例[J].中国中西医结合耳鼻咽喉科杂志,2000：8(4)：192.
[48]陈国琴.针刺治疗喉瘖32例疗效分析[J].针灸临床杂志,2003；19(11)：31-32.
[49]皮健.针刺人迎、水突穴治疗歌者声带肥厚如例的疗效观察[J].中国针灸,1987；(3)：17.
[50]邵素霞.针灸治疗喉瘖38例[J].河南中医,2003；23(9)：69.
[51]崔允孟.耳针治疗声嘶症150例疗效观察[J].中级医刊,1993；18(10)：52-53.
[52]张安福.消炎止痛膏治疗慢性喉炎效佳[J].四川中医,1989；(1)封3.
[53]马玉杰,白力学,纪建芹.中药透入治疗慢性咽喉炎35例[J].中国民间疗法,1998；6(5)：7.
[54]詹光宗,中药离子直流电导入治疗急慢性咽喉炎[J].中国针灸,2001,21(8)：489.
[55]杨华,仲彦.推拿法治疗慢性喉炎18例[J].中国民间疗法,1997；(5)：33.
[56]金平林.鱼际隔蒜泥敷贴为主治疗喉瘖26例[J].中国针灸,2001；21(10)：592.
[57]宋南吕.耳压配合中药治疗喉瘖30例[J].江西中医药,1997；28(3)：40.
[58]李云英.中医古籍论喉瘖的辨治[J].广州中医药大学学报,1999,16(4)：325-327.
[59]李云英,陈海,邓时贵,等.“金喉雾化剂”治疗慢性喉炎的疗效评价及计算机声图谱分析[J].中国中西医结合耳鼻咽喉科杂志1999,7(3)：114-117.
[60]邓时贵,欧润妹,李云英,等.雾化吸入活血化痰中药对实验动物气管分泌和微循环的影响[J].广州中医药大学学报.1999,16(4)：311-313.
[61]彭桂原,李云英.喉癌前病变气滞血瘀证与VEGF的相关性研究[J].新中医,2007,39(7)：43-44.
[62]乔宗海,徐洁洁,程雷,主编.现代喉部疾病诊断与治疗[M].南京：东南大学出版社,1999,第一版：178-183.
[63]Ferlito A, Carbone A, DeSanto LW. " Early" cancer of the larynx:the concept as defined by clinicians, pathologists and biologists[J]. Ann Otol Rhinol Laryngol,1996, 106:245-250.
[64]田勇泉主编.耳鼻咽喉一头颈外科学[M].北京：人民卫生出版社,2004.6(第6版)：215-220.
[65]屠规益,吕春梅.耳鼻咽喉头颈外科医师与喉癌术后发声功能重建.听力学及言语疾病杂志,2010,18(1)：1.
[66]王鹏,刘宝国,李树玲.全喉切除术后发音重建术研究进展.中华临床床医师杂志(电子版).2009,3(9)：82-86
[67]黄选兆,汪吉宝主编.实用耳鼻咽喉科学[M].北京：人民卫生出版社,2005.7：501-502.
[68]韩德民,Robert T主编.嗓音医学[M].人民卫生出版社.2007第一版：198-199.
[69]韩德民,Robert T主编.嗓音医学[M].人民卫生出版社.2007第一版：199.
[70]王军,韩德民,叶京英,等.应用C02激光治疗可疑喉癌前病变[J].耳鼻咽喉-头颈外科,2001,8(4)：217-219.
[71]Talamini R, Bosetti C, Lavecchia C, et al. Combined effect of tobacco and alcohol on laryngeal cancer risk:a case-control study [J]. Cancer Causes Control,2002,13:957-964.
[72]Riedel F, Goessler U, Hormann K. Alcohol-related diseases of the mouth and throat[J]. Best Pract Res Clin Gastroenterol,2003,17(4):543-55.
[73]李惠萍,崔秀娟,关超,等.喉癌前病变100例分析[J].中华耳鼻咽喉科杂志,1995,30(5)：302-304.
[74]Mork J, Lie AK, Glattre E, et al. Human papillomavirus infectionas a risk factor for squamous-cell carcinoma of the head and neck[J].New English Med.2001,344:1125-1131.
[75]Judd R, Zaki SR, Coffied LM, et al. Human papillomavirus type 6 detected by the polymerase chain reaction in invasive sionasal papillary squamous cell carcino mas[J]. Arch Pathol Labmed,1991;115:1150-1153.
[76]李晓明,卜国弦,郭晓峰.喉癌的病因学研究进展[J].国外医学耳鼻咽喉科分册,1998,12(1)：16.
[77]Shaw H, Ballantyne J, Groves J. Scoth-Brown's diseases of the ear, nose and throat[M]. London:Butterworth,1979:109-122.
[78]张小伯.发声显微外科的发展与临床应用.中华耳鼻咽喉科杂志,2004,39：253-256.
[79]Mozota Ortiz JR. Cancer of the vocal cord, early diagnosis by stroboscop[J]y. Acta Otorhinolaryngol Esp,1998;28:91-104.
[80]宋发全,张芩娜,王建亭,等.声带癌前病变及癌变动态喉镜追踪观察[J].中华耳鼻咽喉科杂志,2000；35(2)：144-146.
[81]Zariwala M, Schmlu S, Pialoz M, et al. P53 gene mutations in oropharyngeal carcinomas:A comparison of solitary and mutiple primary tumours and lymph node metastases[J]. Cancer.1994,56(6):807.
[82]Hall PA, Ray A, Lemoine NR, et al.P53 immunostaining as a marker of mali gnant disease in diagnostic cytopathology[J]. Lancet,1991,338:513.
[83]周光耀,林代诚,梁传余,等.P53蛋白在喉上皮癌前病变及癌组织中的表达及其意义[J].华西医大学报,1999,30(3)：265-267.
[84]肖健云,罗均利,陶正德,等.P53蛋白在喉癌及癌旁病变组织中的表达[J].中国耳鼻咽喉颅底外科杂志,1995,1(1)：47-49.
[85]邓志宏,黄维国,邱建华等.血管生成及细胞凋亡与喉黏膜上皮癌变的关系[J].第四军医大学学报,1999,20(1)：17-20.
[86]Feinmesser R, et al. Flow cytometer analysis of DNA content in laryngeal cancer[J]. Laryngology Otology,1998,104:485.
[87]周剑勇,梁素霞.喉癌前病变DNA含量测定及细胞周期分析的意义[J].广东医学.2001,22(3)：201-202.
[88]Yuan Y, Chi F, et al. Significance of ceramide and DNA ploidy in laryngeal carcinogenesis[J]. ORL Otorhinolaryngol Relat Spec.2007,69(5):283-288.
[89]Guillermo J Tearney, Mark E Brezinski, Brett E Bouma, et al. In vivo endoscopic optical biopsy with optical coherence tomogyaphy[J]. Science,1997,276(27):2037-2039.
[90]Ralph S Dacosta, Brian C Wilson, Norman E Marcon. New optical technologies for earlier endoscopic diagnosis of premalignant gastrointestinal lesions[J]. Journal of Gastroenterology and Hepatology,2002,17(suppl):85-104.
[91]Kulapadituarom B et al. Ann Otol Rhinol Laryngol,2001,110:45-52.
[92]张小伯,刘丹丹,杨大章,等.早期喉癌与声带癌前病变的诊断和微创治疗[J].中华耳鼻咽喉科杂志,1999,34(6)：337-339
[93]顾建森,李秋珍,张艳萍.喉癌前病变早期干预治疗[J].南京铁路医学院学报,2000,19(2)：129-130.
[94]Ricci G, Molini E. Retrospective stuffy on precancerous laryngeal lesions:long-term follow-up[J].Acta Otorhinolaryngologica Italica,2003,23 (5):362-367.
[95]杨桂芬,曾勇,管俊华.喉癌前病变临床疗效观察——附27例报告[J].肿瘤预防与治疗,2008,21(2)：185,189.
[96]宋磊,张涛源.喉乳头状瘤治疗新进展[J].山西医药杂志,2007,36(1)：49-50.
[97]王新华,孙敬武.小儿喉乳头状瘤的研究进展[J].听力学及言语疾病杂志,2008,16(4)：347-348.
[98]Benjamin BN, et al. Ann Otol Rhinol Lanyngol,1988;97:376-380
[99]Healy GB, et al. N Eng J Med,1988:319:401-407.
[100]Leventhal GB, et al. N Eng J Med,1991;325:613-617.
[101]Mc cabe BF, et al. Ann Otol Rhinol Lanyngol,1987:96:158-162.
[102]Deunas LV, et al. J Lanyngol Otol,1997;111:134-140.
[103]张小伯,刘丹丹,杨大章,等.早期喉癌与声带癌前病变的诊断和微创治疗[J].中华耳鼻咽喉科杂志,1999；34(6)：337-339.
[104]骆云珍,钱林荣,李海同.水分离技术在声带白斑剥脱术中的应用[J].中国中西医结合耳鼻咽喉科杂志,2008,16(3)：213-214.
[105]王军,韩德民,叶京英,等.应用C02激光治疗可疑喉癌前病变[J].耳鼻咽喉-头颈外科,2001,8(4)：217-219.
[106]顾建森,李秋珍,张艳萍.喉癌前病变早期干预治疗[J].南京铁道医学院学报,2000,19(2)：129-130.
[107]吕秋萍,孙敬武,胡燕明,等.激光治疗喉癌前病变[J].中国耳鼻咽喉头颈外科.2008,15(12)：661-664.
[108]Devaiah AK, Shapshay SM, Desai U, et al. Surgical utility of a new carbondioxide laser fiber:functional and histological stud[J]. Laryngoscope,2005,115(8):1463-1468.
[109]冯卫文,黄桂球,黄家英,等.显微喉镜下钬激光治疗喉癌前病变36例观察[J].暨南大学学报(医学版),2009,30(2)：215-216.
[110]孙广滨,周水淼,王昶,等Diomed-25半导体激光在治疗喉癌前病变中的应用[J].山东大学基础医学院学报,2003,17(3)：158-159.
[111]孙广滨,周水淼,温武,等.半导体激光治疗声带白斑30例临床分析[J].临床耳鼻咽喉科杂志,2004,18(3)：141-143.
[112]杨哗,白薇琦.Nd-YAG激光结合内窥镜在喉癌前病变手术治疗中的应用[J].现代实用医学,2007,19(11)：909-910.
[113]刁玉华,张玉兰,王玮.纤维喉镜下微波凝集治疗喉角化病的临床研究[J].中国中西医结合耳鼻咽喉科杂志,2002,10(5)：226-228.
[114]刘锋综.微波在耳鼻喉科的应用[J].国外医学耳鼻喉科学分册,1993,17(4)：222-225.
[115]蔡冰国,王雪峰.喉癌前病变的诊断和治疗[J].社区医学杂志,2008,6(9)：38-39.
[116]Malczewsk M. Clinical applications of photodynamic therapy in treatment of laryngeal lesions[J]. Otolaryngol,1999,53(6):671-675.
[117]Sibata CH, Colussi VC, Oleinick NL, et al. Photodynamic therapy in oncology [J]. Expert Opin Pharmacother,2001,2:917-927.
[118]刘蓓,梁建平,朱习平,等.显微支撑喉镜下射频治疗早期声门癌及癌前病变[J].广西医学,2007,29(9),1331-1333.
[119]张庆丰,刘得龙,张悦,等.等离子射频治疗早期声门型喉癌的初步研究[J].中华耳鼻咽喉头颈外科杂志.2011,46(01)：164-165.
[120]Patel RS, MacKenzie K. Powered laryngeal shavers and laryngeal papillomatosis: a preliminary report[J]. CAin Otolaryngol,2000,25:358.
[121]Flint PW. Powered surgical instruments for laryngeal surgery [J]. Otolaryngol Head Neck surg,2000,122:263.
[122]Parsons DS, Bothwell MR. Powered instrument papiloma excision:an alternative to laser therapy for recurrent respiratory papilloma[J]. Laryngoseope,2001. 111:1494.
[123]钱晓云,俞晨杰,沈晓辉.支撑喉镜下吸引旋切结合射频治疗喉癌前病变的疗效评价[J].中国微创外科杂志,2008,8(2)：104-106.
[124]Maran A. Head and neck surgery:An ercyciopaedia of ignorance[J]. Laryngol Otol, 1990,104:529.
[125]邹向阳,李连宏.细胞周期调控与肿瘤[J].国际遗传学杂志,2006,29(1)：70-73.
[126]邓琳.Cyclin D1的多态性与肿瘤[J].国外医学：肿瘤学分册,2001,28(3)：163-164.
[127]陈万军,王天铎.细胞周期蛋白循环索在喉咽癌生物学行为中的意义[J].临床耳鼻咽喉科杂志,2005,19(3)：250-252.
[128]付志婕,潘新良,雷大鹏,等.细胞周期索D1和p16在喉咽癌中的表达及临床意义[J].临床耳鼻咽喉头颈外科杂志,2007,21(9)：406-409.
[129]Zhang H. Life without kinase:cyclin E prootes DNA repliction licensing and beyond[J].Mol Cell.2007,25(2):175-176.
[130]Yamanouchi H, Furihata M, Fujita J, et al. Expression of cyclin E and cyclin D1 in non-small cell lung Cancers[J]. Lung Cancer,2001,31(1):3-8.
[131]Pagano M, Pepperkok R, Verde F. Cyclin A is required at two points in the human cell cycle[J]. EMBOJ,1992,11(5):961.
[132]Yasmeen A, Berdel W E, Serve H, et al. E-and A-type cyclins as markers for cancer diagnosis and prognosis[J]. Expert Rev Mol Diagn,2003,3(5):617-633.
[133]王建波,高福平,张秀兰,等.喉鳞状细胞癌中Cyclin A、B1表达的临床意义[J].中国肿瘤临床与康复,2002,9(4)：63-65.
[134]Fraczek M, Wozniak Z, Ramsey D, et al. Expression patterns of Cyclin E, Cyclin A and CDC25 phosphatases in laryngeal carcinogenesis[J]. Eur Arch Otorhinolaryngol,2007, 264 (8):923-928.
[135]Rodriguez-Pinilla M, Rodriguez-Peralto JL, Hitt R, et al. Cyelin A as a predictive factor for chemotherapy respOnse in advanced head and neck cancer[J]. Clin Cancer Res, 2004,10(24):8486-8492.
[136]Stark GR, Taylor WR. Control of the G2/Mtransition[J]. Mol Bioteehnol,2006,32(3): 227.
[137]Nurse P. Ordering S phase and M phase in the cell cycle [J]. Cell,1994,79:547.
[138]Yew PR. Ubiquitin mediated proteolysis of vertebrate Gl-and S-phase regulator [J]. J Cell Physiol,2001,187:1.
[139]Dong Y, Sui L, Watanabe Y, et al. Clinical relevance of Cyclin B1 overexpression in laryngeal squamous cell carcinoma[J]. Cancer Lett,2002,177(1):13-19.
[140]王建波,高福平,张秀兰,等.喉鳞状细胞癌中Cyclin A、BI表达的临床意义[J].中国肿瘤临床与康复,2002,9(4)：63-65.
[141]陈万军,李梅,王天铎,等.CDK4与下咽癌病变的相关性及其生物学行为的初步研究[J].山东大学基础医学院学报,2005,19(3)：149-152.
[142]李秀敏.细胞周期、染色质和癌：基于肿瘤机制的治疗[J].国外医学：药学分册,2004,31(2)：75-77.
[143]Zhang H. Life without kinase:cyclin E prootes DNA repliction licensing and beyond[J].Mol Cell.2007,25(2):175-176.
[144]蔡扬,何园,卢虹,等.口腔鳞状细胞癌cyclin E及P21Waf 1蛋白定量检测及意义[J].中国肿瘤临床,2008,35(3)：142-144.
[145]Shapiro GI, Edwards CD, Rollins BJ. The physiology of p16(INK4A)-mediated G1 proliferative arest[J]. Cell Biochem Biophys,2000,33(2):189-197.
[146]Jares P, Nadal A, Fem6ndez PL, et al. Disregulation of p16MTSI/CDK41 protein and mRNA expression is associated with gene alteations in squamous cell carcinoma of the larynx[J]. Int J Cancer,1999,81(5):705-71I.
[147]付志婕,潘新良,雷大鹏,等.细胞周期索D1和p16在喉咽癌中的表达及临床意义[J].临床耳鼻咽喉头颈外科杂志,2007,21(9)：406-409.
[148]温树信,王斌全,陈彦球,等.抑癌基因p15、Rb在喉鳞状细胞癌中表达的研究[J].临床耳鼻咽喉科杂志,2003,17(9)：543-544.
[149]李福才,李英慧,赵旭,等.喉癌中p15、p16基因纯合缺失与EGFR基因扩增相关性研究[J].遗传学报,2004,31(2)：109-113.
[150]Eddy AA. Expression of genes that poremote renal interstitial fibrosisin rats with proteinuria[J]. Kidney Int,1996,54:149-154.
[151]Sofia G, Speroni J, Podhajcer OL, et al. p21 differentially regulates DNA replication and DNA-repair-associated processes afterUV irradiation [J]. J Cell Sci,2008,121(19): 3271-3282.
[152]王建波,王金泉,娄卫华等.细胞周期蛋白Dl和P21WAF1/CIP1在喉癌癌变过程中的表达及其意义[J].中国癌症杂志,2000,10(1)：59-61.
[153]李晖,江孝清,周绪红。Skp2、p27蛋白在喉鳞状细胞癌中的表达及意义[J].临床耳鼻咽喉科杂志,2005,19(9)：409-411.
[154]金晓杰,钱敏飞,许雁平,等.喉鳞状细胞癌手术切缘分子水平亚健康与预后关系研究[J].临床耳鼻咽喉科杂志,2006,20(24)：1111-1113.
[155]孔萌萌,中丽娟,金克炜.p57kip2与肿瘤[J].国外医学：肿瘤学分册,2005,32(2)：100-102.
[156]Fan GK, Xu F, Yang B, et al. P57kip2 expression is related to carci, nogenesis and tumor pmgresslon in laryngeal tissues[J]. Acta Otolaryngol,2006,126(3):301-305.
[157]杨氏麟,著.嗓音医学基础与临床[M].辽宁科学技术出版社.2001：370-371.
[158]陆美萍,徐洁洁.用声不当或过度致发声障碍的基础与临床研究[J].听力学及言语疾病杂志.2004,12(4)：276-277.
[159]李慧萍,崔秀娟.喉癌前病变100例分析[J].中华耳鼻咽喉科杂志,1995,30(5)：302-304.
[160]Yang GH, Ma JM, Liu N, et al. Smoking and passive smoking in Chinese,2002[J]. Chin J Epidemiol,2005,26(2):77-83.
[161]陈爱俊.绝大多数喉癌患者有长期吸烟史[J].中国乡村医药.2009,16(1)：15.
[162]Jacques B. Head and neck cacer:Multimodality Management[M]. Springer Science Business Media.2011,9:238.
[163]韩德民,Robert TS.主编.嗓音医学[M].人民卫生出版社,2007,第一版：199.
[164]韩德民,Robert TS.主编.嗓音医学[M].人民卫生出版社,2007,第一版：199.
[165]李慧萍,崔秀娟.喉癌前病变100例分析[J].中华耳鼻咽喉科杂志,1995,30(5)：302-304.
[166]Hashibe M, Sankaranarayanan R, Thomas G, et al. Alcohol drinking, body mass index and the risk of oral leukoplakia in an Indian opulation[J]. Cancer.2000,88:129-134.
[167]Gupta PC, Hebert JR, Bhonsle RB, et al. Dietary factors in oral leukoplakia and submucous fibrotlis in a population-based case control study in Gujarat, India[J]. Oral Dis 1998,4:200-6.
[168]Gupta PC, Hebert JR, Bhonsle RB, et al. Influence of dietary factors on oral precancerous lesions in a population-based case-control study in Kerala, India[J]. Cancer.1999,85:1885-93.
[169]Flanders W D, et al. Work-related risk factors for laxyngeal cancer. Am J Epidemiol 1984,119:23.
[170]Olsen J, Sabroe S. Occupational causes for laryngeal cancer. J Epldemiol. Community Health.1984; 38:117.
[171]张克,费声重,陈哲,等.辽宁省工业城市大气污染与喉癌发病率的关系[J].中华耳鼻咽喉科杂志,1990,25(4)：240-242.
[172]孙克敏,肖立峰,周彬,等.喉癌患者血清微量元素的研究[J].中国眼耳鼻喉科杂志,1998,3(3)：94-96+85.
[173]智铁铮.喉癌前病变国外医学·耳鼻咽喉科分册[J].1988,12：13.
[174]谢汝华,闵华庆,曾宗渊,等.喉癌与性激素受体及手术前后血清性激素水平的研究[J].癌症.1994,(5)：56-59.
[175]Robin. Expression of Androgen Receptor and Estrogen Receptor in Glottic and Supraglottic Carcinoma of Larynx[J]. Cervical Cancer,2002, (7):165-168.
[176]Plch J, Par J, Navratilova J, et al. Long term follow-up study of laryngeal precancer[J]. Auris Nasus Larynx,1998,25:407-412.
[177]王正敏,陆书昌,主编.现代耳鼻咽喉科学[M].北京：人民军事出版社,2001,第一版：952.
[178]李惠萍,崔秀娟,关超,等.喉癌前病变100例分析[J].中华耳鼻咽喉科杂志,1995,30(5)：302-304.
[179]孙广滨,周水淼,王昶,等Diomed-25半导体激光在治疗喉癌前病变中的应用[J].山东大学基础医学院学报,2003,17(3)：158-159.
[180]黄选兆,汪吉宝,主编.实用耳鼻咽喉科学[M].北京：人民卫生出版社,1998,第一版：501-502.
[181]彭桂原,李云英.喉癌前病变气滞血瘀证与VEGF的相关性研究[J].新中医,2007,39(7)：43-44.
[182]Yamanouchi H, Furihata M, Fujita J, et al. Expression of cyclin E and cyclin D1 in non-small cell lung Cancers[J]. Lung Cancer,2001,31(1):3-8.
[183]胡立胜,周强.中医临床研究设计与SAS编程统计分析[M].北京：学苑出版社,2004：68.
[184]徐静.干祖望教授对嗓音疾病的辨治[J].辽宁中医杂志,1987；(7)：5-7.
[185]王琦.王琦男科学[M].第2版.郑州：河南科学技术出版社,2007：3：79.
[186]王莉,何裕民.男女体质特点及其异同的研究——附2967例流调资料的模糊识别分析[J].中国中医基础医学杂志.1998,2(2)：7-9.
[187]张晓英,郭利平.冠心病心绞痛中医证型分布在性别上的差异性[J].浙江中医杂志.2009,44(6)：406-407.
[188]陈志强,吕立国.整体辨证、局部辨证与微观辨证——对现代中医辨证论治体系的思考[J].中国中西医结合杂志.2006,(12)：1076-1080.
[189]干祖望著.干氏耳鼻咽喉口腔科学[M].江苏科学技术出版.1999第1版：28-30.
[190]李云英.中医古籍论喉喑的辨治[J].广州中医药大学学报,1999,16(4)：325-327.
[191]李云英,陈海,邓时贵,等.“金喉雾化剂”治疗慢性喉炎的疗效评价及计算机声图谱分析[J].中国中西医结合耳鼻咽喉科杂志1999,7(3)：114-117.
[192]邓时贵,欧润妹,李云英,等.雾化吸入活血化痰中药对实验动物气管分泌和微循环的影响[J].广州中医药大学学报.1999,16(4)：311-313.
[193]彭桂原,李云英.喉癌前病变气滞血瘀证与VEGF的相关性研究[J].新中医,2007,39(7)：43-44.
[194]杜芸王小玲吴国祥,等.流式细胞术对食管癌组织中四种细胞周期调控蛋白的定量检测及意义[J].中华肿瘤杂志.2004,26(10)：612-614.
[195]Gillett CE, Lee AHS, Millis RR, et al. Cyclin D1 and associated proteins inmammary ductal carcinoma in situ and atypical ductal hyperplasia[J]. J Pathol,1998,184(4): 396.
[196]sherr CJ, Roberts JM. Inhibitorsofmammalian G1 cyclin dependent kinase[J]. Genes Dev,1995,9(10):1149.
[197]Donnellan R, Cherty R. Cyclin D1 and human neoplasia[J]. Mol Pathol,1998,51(1): 1.
[198]刘馨莲,殷舞,李淑蓉,等.细胞周期蛋白在喉癌组织中的表达及意义.实用癌症杂志.2011,26(6)：585-587+595
[199]Donnelan R, Kleinschmidt I, Chetty R, et al. Cyclin E immunoexpression in breast ductal carcinoma:pathologic correlations and prognostic implications[J]. Hum Pathol, 2001,32(1):89.
[200]Kim H K, Park I A, Heo D S, et al. Cyclin E overexpression as an independent risk factor of visceral relapse in breastcancer[J]. Eur J Surg Oncol,2001,27(5):464.
[201]Georgieva J, Sinha P, Schadendorf D, et al. Expression of cyclins and eyclin dependent kinases in human benign and malignant melanocytic lesion[J]. Clin Pathol,2001,54(3): 229.
[202]Reika I, Takashi K, Yoichi A. Association of P16 homozygous deletions with clinicopathologic characteristics and EGFR/RAS/53 mutations in lung adenocarcinoma colloid[J]. Clin Cancer Res,2008,14:3746-3753.
[203]Semczuk A, Miturski R, Skomra D, et al. Expression of the cell. cyle regulatory proteins (pRb, cyclin D1, pI6INK4A and cdk4) in human endometrial cancer:correlation with clinicopathological features[J]. Arch Gynecol Obstet,2004,269(2):104-110.
[204]Ivanova TA, Golovina DA, Zavalishina LE, et al. Up-regulation of expression and lack of 5 CpG island hypermethylation of pl6ink4a in HPV-positive cervical carcinomas [J]. BMC Cancer,2007,14(7):47.
[205]马玉泉,杨晓光,刘辉,等.p21,p27基因多态性对食管癌的发生及淋巴结转移的影响[J].第四军医大学学报.2008,29(17)：1600-1603.
[206]高蔚,金锋.大肠癌中p21 ras表达与血管新生的临床病理意义[J].中国医科大学学报.2007,36(4)：455-457.
[207]冉志华,冯樱,邱德凯,等.重组细小病毒H-1(rhH1/p21)对胃癌细胞HGC27生长抑制作用的研究[J].肿瘤.2007,27(4)：251-255.
[208]王黎明,鹿彩莲,纪祥瑞,等.细胞周期调控相关蛋白P53、P16、P21和Rb在子宫内膜癌的表达及意义[J].中国妇产科临床.2001,2(5)：292-294
[209]Weinstein IB, Santella RM, Perera F. Molecular biology and molecular epidemiology of cancer. In:greenwald P, Kramer BS, Weed DL(eds)Cancer Prevention and Control[J]. Bethesda:Marcel-Dekker,1995:83-110.
[210]Yespa SH. Overview of carcinogenesis:past, present and future[J]. Carcinogenesis.2000,21:341-345.
[211]Hanahan D, Weinberg RA. The hallmarks of cancer[J]. Cell 2000,100:57-70.
[212]Yuspa SH, Poirier MC. Chemical carcinogenesis:from animal models to molecular models in one decade[J]. Adv Cancer Res.1988,50:25-70.
[213]王建波,王金泉,娄卫华.等.细胞周期蛋白D1和P21WAF1/CIP1在喉癌癌变过程中表达及其意义[J].中国癌症杂志.2000,10(1)：59-61.
[214]陈万军,李梅,王兴武.等.细胞周期调控因子与下咽癌生物学行为相关性初步研究[J].中国肿瘤临床,2007,34(1)：26-29.
[215]蔡扬,何园,卢虹,等.口腔鳞状细胞癌cyclin E及P21wWaf1蛋白定量检测及意义[J].中国肿瘤临床.2008,35(3)：142-144.
[216]Zhang H. Life without kinase:cyclin E prootes DNA repliction licensing and beyond[J].Mol Cell.2007,25(2):175-176.
[217]Yamanouchi H, Furihata M, Fujita J, et al. Expression of cyclin E and cyclin D1 in non-small cell lung Cancers[J]. Lung Cancer,2001,31 (1):3-8.
[218]Hu YX, Watanabe H, Ohtsubo, et al. Frequent loss of p16 expression and its correlation with clinicopathological parameters in pancreatic carcinoma[J]. Clin Cancer Res,1997, 3:1473-1477.
[219]Arap W, Nishikama R, Furnari FB, et al. Replaement of the p16/CDKN2 gene suppresses human glioma cell growth[J]. Cancer Res,1995,55:1351.
[220]OHTANI N, YAMA KOSHI K, TA KA HASHI A, et al. The p16 IN K4a-RB pathway:molecular link between cellular senescence and tumor suppression [J]. J Med Invest,2004,51:146-153.
[221]GALLO O, SANTUCCI M, FRANCHI A. Cumulative prognostic value of pl6/CDKN2 and p53 oncoprotein expression in premalignant laryngeal lesions[J].J Natl Cancer Inst.1997, 89:1161-1163.
[222]李叶青,顾其华,李运良,等.口腔鳞癌K-ras基因测序分析及p21蛋白的表达[J].海南医学院学报.2004,10(4)：217-219.
[223]JangTJ, KangMS, KimH, et al. Increased Expression of Cyclin D1, Cyclin E and p21Cip1 Associated with Decreased Expression of p27Kipl in Chemically Induced Rat Mammary Carcinogenesis[J].Jpn. Cancer Res.2000,12 (91):1222-1232.
[224]Coverley D, Laman H, Laskey RA. Distinct roles for cyclin-E and A during DNA relication complex assembly and activation[J]. Nat CellBiol,2002,4(7):523-528.
[225]LodenM, StighalLM, Nielsen NH, et al. The cyclin-D1 high and cyclin-E high subgroup s of breast cancer:separate pathways in tumorgenesis based on pattern of genetic aberrations and inactivation of the pRb node[J]. Oncogene.2002,21 (30):4680-4690.
[226]冯红,于卫芳,张立玮,等.贲门癌变过程中Survivin和CyclinE蛋白的表达及相关性研究[J].中国综合临床.2007,23(1)：52-54.
[227]Shintani S, Mihara M, Nakahara Y, et al. Expression of cell cycle control proteins in normal epithelium, premalignant and malignant lesions of oral cavity[J]. Oral Oncol.2002,38(3):235-243.
[228]姜文奇,张晓实,朱孝峰,等.肿瘤生物治疗学[M].广州：广东科技出版社.2006：4.
[229]曾益新 主编.肿瘤学[M].第2版.北京：人民卫生出版社,2003：11-12.