【作者】 郭强中；

【导师】 李云英；

【作者基本信息】 广州中医药大学 ， 中医五官科学， 2011， 博士

【摘要】 背景喉癌是头颈部常见的恶性肿瘤之一,血瘀证是其临床常见的中医分型。金喉片是具有活血化瘀功效的中药复方制剂,既往的研究发现金喉片能够有效改善声带瘀血状态及重建声带局部微循环,并能够抑制喉癌内血管增生,对抗喉癌细胞引起的血小板聚集和癌栓形成,减少血栓对喉癌细胞的保护,达到延缓癌瘤生长的目的。通过研究金喉片对血瘀证模型大鼠的血液流变学和舌象的影响可以定位其改善血瘀证的作用途径,通过研究金喉片对喉癌裸鼠移植瘤中抑癌基因PTEN和致癌基因survivin的影响可以明确其抗癌的作用机理,整合这两方面的研究成果就可以为临床治疗血瘀型喉癌提供实验基础。声带息肉、成人型喉乳头状瘤和喉鳞状细胞癌分别属于中医“喉喑”、“喉瘤”和“喉菌”范畴,声带息肉属于喉部良性增生性病变,喉乳头状瘤属于喉部良性肿瘤,也属于喉癌前病变,而喉鳞状细胞癌则属于喉部恶性肿瘤,从喉部良性增生性病变逐渐过渡到喉部恶性肿瘤的机制至今未明。通过联合检测致癌基因Survivin和抑癌基因PTEN在上述三种疾病中的表达差异可以揭示喉鳞状细胞从增生到癌变的变化机制,再结合前面的动物实验研究就可以为金喉片治疗不同阶段的血瘀型喉喑病在剂量、疗程和时机的选择上提供参考依据。目的观察具有活血化瘀功效的金喉片对血瘀证模型大鼠的血液流变学和舌象的影响,以及对喉癌裸鼠移植瘤中致癌基因survivin和抑癌基因PTEN的影响,以此来了解金喉片的活血化瘀作用和治疗喉癌的功效；同时研究声带息肉、成人型喉乳头状瘤和喉鳞状细胞癌三种疾病中Survivin和PTEN的变化规律,以探讨喉鳞状细胞从增生到癌变的变化机制,为金喉片治疗喉喑疾病的合理应用建立参考依据。方法1.将随机分组的32只雄性SD大鼠进行14天的血瘀证造模,按折算后的金喉片常规有效剂量的1倍、3倍和6倍分别对造模后的大鼠进行14天的灌胃,并设立生理盐水对照组,观察在造模开始前、造模后及用药后大鼠血液流变学的变化。2.将培养好的喉癌细胞接种到随机分组的32只雌雄各半裸鼠背部皮下,按折算后的金喉片常规有效剂量的1倍、3倍和6倍分别对裸鼠进行灌胃,并设立生理盐水对照组,观察裸鼠背部瘤体变化。在第28天取瘤,并进行瘤体重量和体积检测,分析瘤体切片中致癌基因survivin和抑癌基因PTEN的表达差异。3.选取符合临床与病理诊断的声带息肉18例、成人型喉乳头状瘤10例和喉鳞状细胞癌18例,将每个患者的病理蜡块标本各连续切片三张,分别进行HE染色、Survivin和PTEN免疫组化检测。比较致癌基因Survivin和抑癌基因PTEN在声带息肉、喉乳头状瘤和喉鳞癌三种疾病鳞状上皮细胞中的表达差异。结果1.血瘀证模型大鼠按照378mg/Kg的剂量服用金喉片后血浆粘度出现了非常显著的下降(P<0.01),同时伴有高密度脂蛋白的显著升高(P<0.05)和低密度脂蛋白的显著降低(P<0.05),相应大鼠舌体的瘀暗程度减轻也最为明显(P<0.05),红细胞压积和全血粘度无显著变化(P>0.05)2.按照3300mg/Kg的剂量服用金喉片的裸鼠其背部皮下的喉癌移植瘤瘤体体积最小,重量最轻。全部裸鼠背部瘤体的平均密度为1.202g/cm3。Survivin和PTEN在瘤体中的阳性率均体现出高剂组<中剂组<低剂组<空白组的规律特点,且两者的阳性率呈正相关关系(P<0.01)3.声带息肉的Survivin和PTEN阳性率均显著低于喉乳头状瘤和喉鳞状细胞癌(P<0.05),而喉乳头状瘤的Survivin和PTEN阳性率均低于喉鳞状细胞癌,但均没有显著性差异(P>0.05),两种免疫组化形成声带息肉<成人型喉乳头状瘤≤喉鳞状细胞癌的表达规律。在声带息肉、喉乳头状瘤及喉鳞状细胞癌三种疾病中,PTEN的阳性率均显著低于Survivin (P<0.05)。Survivin和PTEN在高分化喉鳞癌中的阳性率要显著低于中分化喉鳞癌(P<0.05)。结论1.常规有效剂量的金喉片能够通过升高高密度脂蛋白和降低低密度脂蛋白来显著降低血瘀证模型大鼠的血浆粘度及改善其舌体血瘀程度,从而达到改善血瘀证的目的。2.6倍有效剂量的金喉片则能够有效抑制喉癌裸鼠移植瘤的生长,并且降低致癌基因Survivin在癌瘤中的表达,抑癌基因PTEN的阳性表达始终低于Survivin,表现出一种从属性的应激表达模式。3. Survivin和PTEN在喉良性增生性疾病的鳞状细胞中部分表达,而在良性和恶性肿瘤中过度表达,体现出随恶性程度增加而逐渐升高的表达态势,同时在声带息肉、喉乳头状瘤和喉鳞癌中前者的阳性率均高于后者。Survivin和PTEN虽然功能相反,但作用机制不同,非对立消长关系,而是主从表达关系,PTEN是出于竞争目的而从属于Survivin的表达,两者的统一决定了鳞状细胞癌变的速度和恶性程度。更多还原

【Abstract】 ObjectivesThrough exploring the changes on hemorheology and blood liqid of rats with blood stasis syndrome and weight or size of subcutaneous laryngocarcinoma on backside of nude mice aroused by Gold Throat Tablets (GTT), composed by Chinese herbs, to find the mechanism of GTT in improving circulation (IC) and removing haemostasis (RH) and restraining Laryngeal Cancer (LC) development.Through exploring the differentiation on positive expressing rates between oncogene Survivin and tumor-suppresser gene PTEN (Phosphatase and tensin homolog deleteted on chromosome Ten) on Vocal Cord Polyps (VCP), adult type Laryngeal Papilloma (LP), and Laryngeal Squamous Cell Carcinoma (LSCC), to reveal the mechanism in canceration of human laryngeal squamous cells, from benign proliferative lesion, benign tumor to malignant tumor in larynx.Methods1. Randomly allocated 32 SD rats into high, middle, low (according to 6:3:1 proportion)dose and blank control groups averagely and made them into blood stasis syndrome models through 14 days. To observe the changes took place on hemorheology and blood liqid between before and after feeding GTT 14 days in each group.2. To vaccinate cultivated laryngeal carcinoma cells into subcutaneous backs of 32 nude mice randomly allocated into high, middle, low (according to 6:3:1 proportion) dose and blank control groups. To observe the changes on weight and size of tumors originated from these cells for 28 days, then analyzing the density of tumors and pathological expression of oncogene Survivin and tumor-suppressor gene PTEN on immunohistochemical slices after separating tumors from back of nude mice.3.After picked out 18 cases of vocal cord polyps,10 cases of adult type laryngeal papilloma, and 18 cases of laryngeal squamous cell carcinoma for hematoxylin-eosin (HE) staining and immunohistochemical process of Survivin and PTEN with three continuous section preparations, the differentiations of positive expression rates of Survivin and PTEN in the same human laryngeal squamous cells areas among three diseases would be compared.Result1. The plasma viscosity and stasis dark degree in tongues of low dose group SD rats descended very obviously companied with remarkable ascending of high density lipoprotein (HDL) and dropping of low density lipoprotein (LDL) (P<0.01), no prominent changes took place in hematokrit (HCT) and whole blood viscosity (WBV) (P>0.05)2.The weight and size of subcutaneous laryngocarcinoma on backs of high dose group nude mice both were the smallest. The average density of all tumors was 1.202g/cm3. The positive expressing rates of Survivin and PTEN with positive correlation both revealed the following tendency:high dose group <middle dose group<low dose group<blank control group.3. The positive expressing rates of Survivin and PTEN in vocal cord polyps were obviously more lower than in adult type laryngeal papilloma and laryngeal squamous cell carcinoma (P<0.05), which showed no obvious difference between them (P>0.05). The positive expressing rates of Survivin were always higher than PTEN in VCP, adult type LP, well and moderate differentiated LSCC evidently (P<0.05). The positive rates of these two genes hid a positive correlation to each other (P<0.05)Conclusion1. Regular effective dose of GTT can obviously reduce the plasma viscosity of SD rats with blood stasis syndrome through lowering LDL and lifting HDL, accompanied with remarkable improvement of stasis dark degree in tongues.6 times regular doses of GTT can prevent overgrowth of transplanted laryngocarcinoma on nude mice and excessive expression of oncogene Survivin.2. Survivin and PTEN can express moderately in laryngeal squamous cells of benign proliferative lesion, but excessively in benign and malignant laryngeal tumors. The positive expressing rates can increase according to the grade of malignancy in LSCC.3. PTEN, expressing for competition purpose, shows a subordinative relationship with Survivin. Although they have opposite functions in determine whether the canceration of laryngeal squamous cells take place or not, Survivin is always playing leading role and making predominant impact on development of benign proliferative lesion, benign and malignant tumor in larynx.更多还原