【作者】 李华；

【导师】 李云英；

【作者基本信息】 广州中医药大学 ， 中医五官， 2011， 博士

【摘要】 背景嗓音医学(Phonetics)是耳鼻咽喉科学和语言病理学共同关注的一个新型的学科分支,治疗的对象包括嗓音疾病、言语疾病和语言疾病。近10余年,随着嗓音医学的发展,越来越受到临床医生的重视,逐渐由单纯的科研手段扩展到定量分析临床研究喉发声功能及嗓音疾病的一种手段。由于生活、工作节奏加快,不同生理阶段(变声期、更年期)、内分泌、精神情志因素、环境空气粉尘等因素影响,声嘶的患者越来越多,而研究发现声带息肉和声带小结是引起声嘶的最常见原因。喉癌是头颈部最常见的恶性肿瘤之一,近年来其发病率呈上升趋势,尽管经手术、放化疗等治疗,其死亡率和复发率仍然很高,严重威胁人的生命。由声带良性病变发展到喉癌是逐步演变的过程,因此对喉癌前病变的潜在恶变的预测显得尤为重要。喉癌前的病变有一种基本的病理特征是喉黏膜上皮的异型增生,因此,研究喉上皮从正常状态向恶性转化过程中细胞增殖的情况,可以反映喉上皮增生病变的程度。转化生长因子与肿瘤的发生、发展及肿瘤细胞增殖、分化、凋亡有着密切关系。近年来关于转化生长因子β1 (Transforming Growth factor-β1、TGFβ1)与肿瘤关系的研究较活跃。TGFβ1为多效生长因子,对喉癌则表现为抑制作用；细胞周期相关抗原Ki67 (Ki-67 Antigen)是增殖细胞核内的一种非蛋白性核抗原,是目前最好检测细胞增殖活性的指标。关于TGFβ1、Ki67在声带良性增生病变(包括良性病变及癌前病变)方面研究及二者相关性研究较少。本研究试图寻找嗓音指标与声带病理的相关性,筛选有效的嗓音参数及免疫组化指标,为判病情、预后提供依据。目的1.从分子水平探讨嗓音疾病的发病机制；2.为嗓音疾病的定量、判断嗓音障碍程度提供客观诊断依据——客观化、科学化、数据化评估；3.试图建立嗓音客观多参数评估模式；4.探寻嗓音疾病病理、喉功能检查之间的内在联系；5.为判断疾病的转归及预后提供依据——排查喉癌前病变；6.为术后复查、随访提供观察及疗效评价依据,防止复发。方法按纳入、排除标准随机选取2008年12月至2010年10月的声带病理切片,依病理结果分为三组：声带息肉组27例,轻度不典型增生组38例,中度不典型增生组11例。年龄：18岁-65岁(包括18及65岁),平均为50.28岁；性别：男36例,女40例；病程：1-123个月,12月以内35例,12-60月29例,60月以上12例。分别做组织病理学研究及临床研究。基础研究方面,按常规制备病理切片,用二步法EnVision检测系统分别行TGFβ1、Ki67免疫组化染色,固定图像采集条件、按阳性细胞所占比例的平均数与细胞染色强弱的等级分数相结合的半定量H积分评分方法,分别记录TGFβ1、Ki67在三组表达的H积分。为便于统计,本研究者将H积分进一步分为4个级别：0(0分,阴性),1(卜100分,弱阳性),2(10卜200,中等阳性),3(201-200,强阳性)。分别作TGFβ1、Ki67在各病理分组H积分表达分析,以及各病理分组内TGFβ1、Ki67的相关性分析。临床研究方面,固定同等检查条件,进行术前及术后嗓音声学参数(基频微扰Jitter、振幅微扰Shimmer、标准化噪声能量NNE、谐噪比HNR、最大声时MPT、S/Z比)、电声门图参数(Jitter、Shimmer、NNE、HNR、接触率微扰CQP、接触率CQ)、频闪喉镜主观形态评估参数(粘膜波、闭合相)、医生心理听觉评估参数(总嘶哑声G、粗糙声R、气息声B)录入。其中粘膜波、闭合相两参数分为3个等级：0为完全失功能,1为部分失功能,2为功能正常。G、R、B三型又分为4个等级：0为正常,1为轻度,2为中度,3为重度。行各病理分组术前、术后检查参数对比,及各病理分组内每种检查参数相关性分析。结果1.组织病理学研究方面(1)Ki67在不同病理分组中的表达各组阳性率对比均差异显著(p<0.001)。息肉组,阳性率中以弱阳性为多,达到18.5%；轻度非典型增生组中,阳性率也以弱阳性为主,达到57.9%；中度非典型增生组,阳性率则以强阳性为主,达到36.4%。不同病理分组中,Ki67表达阳性率随病变程度的加重呈逐渐增高趋势。尤其是息肉组与轻度非典型组阳性率差别明显。经卡方检验,差异有统计学意义(p<0.05)。(2) TGFβ1在不同病理分组中的表达各组阳性率对比均差异显著(p<0.001)。息肉组,阳性率中以中等阳性为多,达到40.7%；轻度非典型增生组中,阳性率也以中等阳性为主,达到63.2%；中度非典型增生组,阳性率则以弱阳性为主,达到45.5%。不同病理分组中,TGFβ1表达阳性率随病变程度的加重亦呈逐渐增高趋势,且差异显著。经卡方检验,有统计学意义(p<0.001)。(3)各组分别分析Ki67与TGFβ1表达的相关性①息肉组：Ki67表达与TGFβ1表达呈正相关,相关系数无统计学意义；r=0.227,p=0.255；②轻度非典组：Ki67表达与TGFβ1表达呈负相关,相关系数无统计学意义；r=-0.216,p=0.193；③中度非典组：Ki67表达与TGFβ1表达呈显著负相关,相关系数有统计学意义,r=-0.760,p=0.007。(4)不同病理分组年龄、性别、病程分布特点中度非典型增生组年龄与轻度非典型组比较年龄明显偏大,有统计学差异(p<0.05)。各病理分组在病程、性别方面比较均无明显差异。经卡方检验,P均大于0.5,无统计学意义。2.临床研究方面(1)不同病理分组嗓音声学测试结果比较(术前)在三组间计算机声学各项结果对比无统计学意义(P>0.05)。(2)不同病理分组与嗓音声学测试结果比较(术后)三组间轻度非典型组与息肉组比较,术后息肉组S/Z明确优于轻度非典型增生组,有统计学意义(p<0.05)。(3)手术前后嗓音声学测试结果比较术后计算机语音分析各项参数除S/Z外均有明显改善,差异显著,尤其是在改善Jitter、Shimmer、NNE方面差异非常显著(p≤0.001)。(4)不同病理分组手术前后电声门图测试结果比较Shimmer、CQP手术前后对比经秩和检验,差异非常显著(分别为p<0.003、p<0.002)。Jitter、HNR手术前后配对t检验,差异亦非常显著(分别为p=0.001、p<0.05),说明手术后电声门图各参数均较术前明显改善。(5)不同病理分组手术前后频闪喉镜主观形态评估结果比较术后粘膜波明显改善,声门闭合情况较术前明显好转。统计学意义显著(P<0.001)。(6)不同病理分组手术前后心理听觉评估比较息肉组G及轻度非典型增生组B术后明显改善,经术前后对比有显著统计学意义(P≤0.001)；轻度非典型增生组G术前后比较亦统计学意义明显(P<0.005)。息肉组R、B及中度非典型增生组G均较术前也明显改善,有统计学意义(均P<0.05)。(7)嗓音声学测试与电声门图测试相关性分析计算机嗓音测试及电声门测试各参数中,计算机嗓音测试中谐噪比与电声门图中HNR、Jitter、Shimmer、NNE相关性最好,p<0.001,可见HNR是最稳定的测试指标；其次,计算机语音分析中,Jitter、Shimmer、NNE与电声门图相关性也比较好,大部分p<0.01。CQP与Jitter、Shimmer正相关,有统计学意义(p<0.05)；CQP与HNR负相关,有统计学意义(p<0.01)。(8)各组分别分析心理听觉评估与嗓音声学测试结果的相关性①息肉组R与Jitter、Shimmer、HNR相关性显著,有统计学意义(p<0.001),G与HNR相关性显著有统计学意义(p<0.001),考虑息肉组声带基频、振幅的异常主要引起了声音粗糙的改变。B与NNE,G与Jitter、Shimmer相关性也较明显,有统计学意义(p<0.01)。②轻度非典型增生组,Jitter、Shimmer与G、R均关系密切,HNR与G、R亦关系密切,经spearman等级相关检验分析,有显著统计学意义(p<0.001)。③中度非典型增生组,仅基频微扰与声音质量关系密切,尤其是总嘶哑度,有显著统计学意义(p<0.001)；其次是粗糙声,有明显统计学意义(p<0.01)；再次是气息声(p<0.01)。(9)各组分别分析心理听觉评估与电声门图测试结果的相关性①息肉组G与Jitter、Shimmer、NNE、CQ有相关性,且为正相关,经等级相关检验,有统计学意义(p<0.05)；G与HNR为负相关,有统计学意义(p<0.05)；R与Shimmer、CQ为正相关,经相关分析,有统计学意义(p<0.05)。②轻度非典型增生组G与Jitter、HNR相关性明显,与前者正相关,与后者负相关,均有显著统计学意义(p<0.001)；R与Jitter、NNE、CQP、HNR亦相关,与前3者正相关,与HNR负相关,均有统计学意义(p<0.01)。③中度非典型组中,在所有相关系数中,G与Jitter相关性较高,但无统计学意义。其他参数之间相关性亦无统计学意义。结论1.组织病理学研究方面(1)Ki67在预测病变恶变趋势方面敏感、且有特异性。病理活检时行Ki67表达检查可为明确诊断提供方向,有助于判断疾病的预后转归。(2)Ki67与TGFβ1在三组的表达关系：息肉组二者表现出正相关关系；轻度及中度不典型增生组均表现出负相关关系,尤其是在中度不典型增生组,二者负相关关系显著。(3)病理研究说明声带由良性过渡到轻、中度不典型增生的过程中,Ki67表达阳性率的逐渐增多提示在所有活动的细胞周期(G1、S、G2和有丝分裂期)中细胞增殖活性逐渐增强。喉癌前病变的发病机制可能是G1、S、G2和有丝分裂期中细胞增殖活性增强。(4)TGFβ1不仅参与了声带息肉组织细胞间质中纤维组织增生,还可能参与了轻、中度不典型增生组织细胞间质中纤维组织增生,提示轻度、中度不典型增生病变细胞中纤维组织的增生也是基本病理改变之一,且轻、中度不典型增生病变纤维组织增生较息肉组明显。(5)本研究中TGFβ1抑制恶变、抑制上皮细胞增殖作用不明显。2.临床研究方面(1)中医慢喉喑临床路径围手术期治疗方案治疗声带良性增生病变效果显著,在改善声带振动频率、幅度、音质、声门闭合情况诸方面疗效显著。(2)息肉组,Jitter、Shimmer、HNR、CQ、G、R均为敏感指标。嘶哑声越严重,声带振动频率不规则性越大,幅度波动不规则性越大。(3)轻度不典型增生组,Jitter、Shimmer、HNR、G、R更为敏感。总嘶哑度越严重,声带振动频率不规则性越大,谐音成分越少；粗糙度越严重,声带振动频率不规则性越大,声带闭合时间越长,谐音成分越少。(4)中度不典型增生组,仅Jitter、G稍敏感。(5)三个病理分组各自参数相关性分析中发现,听觉心理评估的G是最敏感、稳定的测试指标。声学测试的Jitter、Shimmer、HNR较敏感、稳定。尤其是轻度不典型增生组,G、Jitter、HNR在关系尤为密切。到中度不典型增生组时,除G与Jitter,余各嗓音参数均不敏感。(6) Jitter、Shimmer、HNR、G、R相结合多参数分析可作为判断病情、疗效、复查随访的指标；(7)嗓音指标敏感度高,但特异性不强,对疾病诊断、定性无法提供确切依据。但可作为判断临床疗效的客观依据。更多还原

【Abstract】 ObjectivesNearly 10 years, with the development of phoniatrics, there are more and more attentions of clinicians, and phoniatrics gradually had been expanded from a simple means of scientific research to quantitative analysis means of laryngeal vocal function and clinical research of voice diseases. As accelerating the pace of life and work, different physiological stages (Voice change time, menopause), endocrine, mental emotional factors, environmental factors such as dust in the air, we have been finding more and more patients with hoarseness, and finding that vocal cord polyps and vocal cord nodules are the most common cause of hoarseness. Laryngocarcinoma is one of the most common malignant tumorof head and neck carcinoma in recent years, its incidence is rising, and it is a serious threat to human life. The development from benign lesions of the vocal cords to throat cancer is a gradual evolution process, so the potential forecast about malignant transformation of laryngeal precancerous lesions is very important. The basic pathological features of laryngeal precancerous lesions is epithelial dysplasia, and therefore the study of laryngeal epithelium from normal to malignant cell proliferation during the transition process, may reflect the degree of laryngeal epithelial hyperplastic lesions. In recent years, the research about relation between transforming growth factor-β1(TGFβ1)and tumor occurrence is fairly active. Transforming growth factor and tumor occurrence, development and tumor cell proliferation, differentiation and apoptosis are closely related. TGFβ1 as a pleiotropic growth factor, is expressed as inhibition of laryngeal cancer cell cycle-associated antigen Ki67 (Ki-67 Antigen) is a non-protein nuclear antigen in proliferation nucleus, and it is currently the best detected indicator of cell proliferation activity. Study about TGFβ1, Ki67 in benign hyperplasia vocal fold lesions (including benign and precancerous lesions) aspects and their correlation is less. This study attempts to find indicators of voice and vocal cord pathology correlation, filtering and effective voice parameters and immunohistochemical indicators for contracting the disease, provide the basis for prognosis. This study attempts to find indicators of voice and vocal cord pathology correlation, filtering effective voice parameters and immunohistochemical indicators so as to provide the basis for determine the condition andr the prognosis.We mant toserve the following purpose:1. From the molecular level to discuss the pathogenesis of voice disease;2. To provide objective diagnostic evidence for quantifying voice disease and determing the extent of voice disorders-an objective, scientific, data based evaluation;3. Attempt to establish an objective voice, multi-parameter assessment model;4. To search the intrinsic link between voice disorders’pathology and laryngeal function tests;5. To provide the basis for determing whether vesting or prevention of the disease-investigating laryngeal precancerous lesions;6. To provide basis for observation and treatment evaluation to prevent recurrence after the operation.MethodsBy inclusion, exclusion criteria we have randomly selected these pathological paraffin from December 2007 to October 2010.According to pathology outcome these cases were divided into three groups:vocal cord polyp group with cases, mild dysplasia group with 38 cases, moderate dysplasia group with 11 cases. Age:18 years-65 years of age (including 18 and 65 years) with an average age of 50.28 years; Gender:male 36, female 40 cases; course:1-123 months,12 months or less 35 cases,29 cases in 12-60 months and 12 cases in 60 months or moreWe have done research on organization pathology and clinical research. On the one hand, we did organization pathologic research, prepared pathological slice by conventional method, the two-step EnVision detection system were performed to carry out TGFβ1, Ki67 immunohistochemical staining, image acquisition conditions were fixed, then we have combined the average percentage of positive cells and the level scores of staining intensity to develop one semi-quantitative scoring method- H integral, We have respectively recorded TGFβ1, Ki67 expression in the three H points. For statistical purposes, I have further divided H points into 4 H-level points: 0 (0, negative),1 (1-100 points, weakly positive),2 (101-200, medium positive),3 (201-200, strongly positive). We have orderly done TGFβ1, Ki67 H expression analysis in each pathological group. And in each pathology group we have done correlation analysis between TGFβ1 and Ki67.The other research is Clinical research, fixed check conditions, we have entried preoperative and postoperative voice acoustic parameters (fundamental frequency perturbation Jitter, amplitude perturbation Shimmer, normalized noise energy NNE, harmonic to noise ratio HNR, Maximum hponation time MPT, S/Z), electronic EGG parameters (Jitter, Shimmer, NNE, HNR, Contact quotient perturbation CQP, Contact quotient CQ), stroboscopic laryngoscopy subjective shape evaluation parameters (mucosal wave, phase closure), doctor of psychological auditory evaluation parameters (total hoarse voice G, rough sound R, breath sound B). The two parameters-mucosal wave,closure phase are divided into three levels:0 is full loss of function,1 is partly loss of function and 2 is functioning properly. The three parameters G, R, B are divided into four levels:0, normal,1 mild,2 moderate,3 severe. In each pathological group we contrasted parameters before and after the surgery, and within each pathological group analysed the correlation of each kind of parameters。Result1. Research on organization pathology(1) Expression of Ki67 in different pathology groups:The positive rate in each group were significantly different compared (p <0.001). Polyp group, the weakly positive rate is highest, morethan 18.5%; mild atypical hyperplasia, the weakly positive rate is also main, reaching 57.9%;moderate atypical hyperplasia, the strongly positive rate is highest in positive rate, reaching 36.4%.In the different pathology grouping, the Ki67 expression positive rate assumes along with the pathological change degree’s aggravation advances gradually the tendency. Especially polyp group and the mild non-typical group masculine gender rate difference is obvious. After the card side examination, the difference has statistics significance (p<0.05)(2)Expression of TGFβ1 in different pathology groups:The positive rate in each group were significantly different compared (p<0.001). Polyp group, the medium positive rate is highest, reaching 40.7%; mild atypical hyperplasia, the medium positive rate is main, reaching 63.2%; moderate atypical hyperplasia, the mainly positive rate is weakly positive rate, reaching 45.5%.In the different pathology groups, TGFβ1 the expression masculine gender rate also advances gradually along with the pathological change degree’s aggravation, and the difference is obvious. After the card side examination, the difference has statistics significance (p<0.001)(3)Each group separately analyzes the expression relevance between Ki67 and TGFβ1:①The polyp group:Ki67 expression was positively correlated with the expression of TGFβ1, the correlation coefficient was notstatistically significant, r=0.227, p=0.255;②Mild atypical proliferation group:The Ki67 expression assumes the inverse correlation relations with TGFβ1 the expression, but the correlation coefficient has non-statistical significance, r=-0.216, p=0.193;③Moderate atypical proliferation group:The Ki67 expression assumes the remarkable inverse correlation relations with TGFβ1 the expression.The correlation coefficient was statistically significant, r=-0.760, p=0.007。(4)The three groups’distribution characteristic at age, the sex, course It is not every obvious.2. Clinical research(1)In different pathology groups voice acoustics test result comparison has indicated that there is non-statistics significance (P>0.05) in three group with computer acoustics results before the surgery.(2)In different pathology groups voice acoustics test result comparison has indicated that by comparing the three groups after the technique, between mild non-typical groups and the polyp group, polyp group S/Z surpasses the mild non-typical proliferation group explicitly, and there is statistics significance (p<0.05) (3)Voice acoustics test result comparison between before and after technique, computer analysis of speech sound each parameter has the distinct improvement besides S/Z, the difference is remarkable, particularly at Jitter, Shimmer, the NNE aspect the difference is remarkable (p≤0.001).(4) In different pathology groups the electroacoustical gate chart test result has indicated Shimmer, CQP dropped after the surgery contrasting by before the surgery, the difference is very remarkable (respectively is p<0.003、p<0.002).Also about Jitter、HNR, the difference is very remarkable (respectively is p=0.001、p<0.05)(5)In erery pathology grouping aroud surgery the frequency dodges laryngoscope subjective shape appraisal result comparison after technique, the mucous membrane wave has been distinct improved, the glottis closed situation also has been changed for the better, Statistics significance remarkable (P<0.001)。(6) In erery pathology groups psychological sense of hearing appraisal comparison by before and after surgery has showed polyp group G and mild non-typical proliferation group B obtained distinct improvement, it is remarkable statistics significance (P≤0.001);(7) Voice acoustics test EGG test relevance analysis:In the various parameters of computer voice test the relevance among EGG, HNR, Jitter, Shimmer and NNE is better(p<0.01), especially with HNR best(p<0.001).(8) Each group analyze the psychological sense of hearing appraisal and voice acoustics test result relevance separately:①Polyp group,R and Jitter, Shimmer, the HNR relevance is remarkable, has statistics significance (p<0.001)②The mild non-typical proliferation group, Jitter, Shimmer and G, R relate closely(p<0.001).③the moderate non-typical proliferation group, only the Jitter and the sound quality relations are close, particularly always hoarse, has the remarkable statistics significance(p<0.001).(9) Each group analyze the psychological sense of hearing appraisal and the electroacoustical gate chart test result relevance separately:①Polyp group G and Jitter, Shimmer, NNE, CQ have the relevance, positive related, after the rank related examination, has statistics significance (p<0.05) ②Mild non-typical proliferation group relevance of G and Jitter, HNR is obvious, being positive related with the former, with the latter inverse correlation. All has the remarkable statistics significance (p<0.01)③Moderate non-typical group, in all correlation coefficients, G and the Jitter relevance is high, but non-statistics significance.Conclusion1. Research on organization pathology(1)Ki67 becomes sensitive at forecasting pathological malignant change, and has the specificity. Carry out fashionable Ki67 expression inspection when you do pathology biopsy may get clear diagnosis direction, may judge disease’s prognosis extension to turn over.(2)The expression relationship of Ki67 and TGFβ1 in the three groups: polyps group showed a positive correlation between the two; mild and moderate dysplasia showed a negative correlation, especially in moderate dysplasia, a negative correlation between the two relations significantly.(3)Pathological studies show that in the transition process from polyp to the mild and moderate dysplasia, Ki67 expression rate’s gradually increasing tips in all activities of the cell cycle (G1, S, G2 and mitosis) cell proliferation activities have been gradually increasing. The pathogenesis of laryngeal precancerous lesions might be G1, S, G2 and mitosis period enhanced cell proliferation.(4)Not only TGFβ1 has participated in the fiber structure proliferation in intercellular substance of the vocal cord polyp organization but also possibly participated the same activity in intercellular substance of the lightly and the moderate atypical proliferation organization. It is suggested that in the mild and moderate atypical proliferation pathological change cell fiber structure’s proliferation is also one of basic pathology changes. And for mild and moderate dysplastic lesions fibroplasia was significantly more than polyps lesions(5) In this study, the function of TGFβ1 at inhibition of malignant transformation and inhibition of epithelial cell proliferation is not obvious.2. Clinical Research(1)Obey the perioperative clinical pathway on troditional chinese medicine chronic hoarseness we treat the vocal cord benign proliferation diseases remarkable effectively, and in improveming vocal cord vibrational frequency, the scope, the acoustic fidelity, the glottis closed situation various aspect curative effect is remarkable.(2)In the polyp group, Jitter, Shimmer, HNR, CQ, G, R are the sensitive targets. G is more serious, the vocal cord vibrational frequency irregularity is bigger, the scope fluctuation irregularity is bigger.(3)In the mild atypical proliferation group, Jitter, Shimmer, HNR, G, R are more sensitive. G is always more serious, the vocal cord vibrational frequency irregularity is bigger, the harmonics ingredient is less; R is more serious, the vocal cord vibrational frequency irregularity is bigger, the vocal cord closing time is longer, the harmonics ingredient is less.(4)In the moderate atypical proliferation group, Jitter, G are only slightly sensitive.(5)Three pathology groups in the respective parameter relevance analysis we have discovered that sense of hearing psychology appraisal’s G is most sensitive, the stable test target. Acoustics test’s Jitter, Shimmer, HNR are sensitive, stable. Especially the mild atypical proliferation group, G, Jitter, HNR are especially close in the relations. While in moderate atypical proliferation group, eliminates G and Jitter, other various voices parameter is insensitive.(6) Unify Jitter, Shimmer, HNR, G, R -ulti parameter analysis may provide evidence for judging the condition, the curative effect, the reexamination along with the target.(7)The voice parameters are sensitive, but the specificity is not strong, so they are unable to provide the accurate basis for disease diagnosis. However, they can be used as an objective basis to judge the clinical effect更多还原