【作者】 安智广；

【导师】 许彦枝；

【作者基本信息】 河北医科大学 ， 口腔临床医学， 2010， 硕士

【摘要】 目的:口腔黏膜癌前病变是一种癌前状态,表现为口腔黏膜上皮细胞出现不同程度的不典型增生,但尚未发展成癌。新的WHO分类中指出癌前病变的最新定义为:“转变为鳞状细胞癌的可能性增加的发生改变的上皮。这种改变的上皮出现各种传统上称为上皮异常增生的细胞学和组织结构的变化”。目前公认的癌前病变和癌前状态包括白斑、红斑、扁平苔藓和口腔黏膜下纤维性变等。癌前病变如果不能及时治疗而任其发展,最终会发展成癌,严重影响治疗效果和预后,也给患者的生理及心理造成巨大影响。如何对口腔黏膜癌前病变进行治疗,阻断其向口腔癌发展,已经成为当前研究的热点。目前针对口腔黏膜癌前病变的治疗方法较多,主要有西药口服、全身及局部应用激素和免疫抑制剂、早期手术切除及中药治疗等。但应用西药疗效不稳定,激素及免疫抑制剂初期起效虽快但作用不持久且副作用大,而以往的中药治疗多结合局部激素应用,单纯应用中药治疗癌前病变的报道较少,缺乏足够的实验研究来证实中药的治疗效果。针对口腔黏膜癌前病变的特点,中医中药在治疗口腔黏膜癌前病变方面具有明显的优势:中药标本同治、疗效稳固;口服汤剂或中成药制剂,服用方法简便,患者的依从性好,易于接受;与西药相比副作用小等。鉴于以上的优势,中药在治疗癌前病变方面越来越受到人们的重视。本实验采用中药藓化饮对实验动物口腔黏膜癌前病变进行治疗干预,并通过流式细胞术和免疫组化方法观察治疗前后P53和COX-2表达情况,分析和探讨藓化饮对口腔黏膜癌前病变的逆转作用。为今后临床应用提供实验理论依据。方法:1.口腔黏膜癌前病变动物模型制备及分组取叙利亚金黄地鼠(SPF级)55只,6～8周龄,体长约10cm,体重90～110g,雌雄兼备,由北京维通利华实验动物技术有限公司提供,生产许可证编号:SCXK(京)2007-0001。按照完全随机化原则分为四组:空白对照组5只,癌前病变对照组10只,生理盐水治疗对照组10只,藓化饮治疗组30只。空白对照组每日正常饲养,不做处理,直至实验结束,与其余组一同处死并取材。癌前病变对照组、生理盐水治疗对照组、藓化饮治疗组均以0.5%DMBA涂抹双侧颊黏膜,每周三次,持续六周。2.中药藓化饮煎剂的制备先向组方中药中加入约8～10倍的水,浸泡40分钟～1小时,用武火加热20～30分钟至煮沸,保持约10分钟,再以文火煎约10分钟后,过滤,取汁;再加入相当于剩余滤渣量6倍的水,用武火加热20～30分钟至煮沸,用文火煎约10分钟,加入砂仁,再煎约6～8分钟,过滤,弃去滤渣。合并两次药液,浓缩直到需要的药液量(含生药1g/ml),分装,消毒,4℃储存备用。3.治疗方法生理盐水治疗对照组、藓化饮治疗组分别以生理盐水和中药藓化饮汤剂灌胃治疗,1.6ml/只,每天一次,持续八周,处死全部动物并取材,固定,镜检。4.免疫组化法检测P53及COX-2在各组中的表达取各组蜡块,常规石蜡切片,脱蜡至水,按照S-P试剂盒操作说明进行操作,镜下观察空白对照组,癌前病变对照组,生理盐水治疗对照组及藓化饮治疗组中的P53和COX-2的表达情况,并分析对比各组之间的表达情况是否存在差别,以评价中药藓化饮对口腔黏膜癌前病变的逆转作用。5.流式细胞术石蜡包埋组织在切片机上连续切取40-50μm厚的组织片5-8片,放入试管中;二甲苯脱蜡,梯度酒精水化;生理盐水冲洗,120目铜网轻搓组织,收集单细胞悬液;500目尼龙网过滤,离心,1000rpm,5分钟;加入一抗工作液,室温下静置40分钟;加入二抗工作液,室温下静置1小时;机检,读取结果。6.统计学方法所有采用SPSS 13.0统计软件,等级资料应用秩和检验方法分析,计量资料以均数±标准差(±S)表示,应用单因素方差分析对P53及COX-2检测结果进行统计学分析,α=0.05,P<0.05具有统计学意义。结果:1.在癌前病变对照组、生理盐水治疗对照组及藓化饮治疗组50例标本中,随机抽取10例标本在光镜下观察,均发现上皮分层不规则,基底细胞极性丧失,滴状钉突,细胞核多形性,核质比例增加,细胞核增大,不典型分裂像,核仁增大、数量增加及核深染等不典型增生表现,可说明癌前病变模型制备成功。2.免疫组化2.1 P53在空白对照组中阴性表达;在癌前病变对照组及生理盐水治疗对照组中均为强阳性表达;在藓化饮治疗组中为弱阳性表达。各组间差异有统计学意义,癌前病变对照组和生理盐水治疗对照组间差异无统计学意义。2.2 COX-2在空白对照组中阴性表达;在癌前病变对照组及生理盐水治疗对照组中均为强阳性表达;在藓化饮治疗组中为弱阳性表达。各组间差异有统计学意义,癌前病变对照组和生理盐水治疗对照组间差异无统计学意义。3.流式细胞术3.1 P53在各组中均有表达,空白对照组和藓化饮治疗组均为弱阳性表达,组间差异无统计学意义;癌前病变对照组及生理盐水治疗对照组中均为强阳性表达,组间差异无统计学意义;藓化饮治疗组与癌前病变对照组相比,组间差异有统计学意义。3.2 COX-2在各组中均有表达,空白对照组和藓化饮治疗组均为弱阳性表达,组间差异无统计学意义;癌前病变对照组及生理盐水治疗对照组中均为强阳性表达,组间差异无统计学意义;藓化饮治疗组与癌前病变对照组相比,组间差异有统计学意义。4.相关性研究表明:P53与COX-2在口腔癌前病变的发生发展过程中,存在明显正相关性。结论:1.应用诱癌剂DMBA,在一定的时间和频次下,可有效诱导金黄地鼠颊黏膜癌前病变。2.P53与COX-2在口腔黏膜癌前病变的发生和发展中有着重要的意义,P53与COX-2在口腔癌前病变的发生发展过程中,存在明显正相关性。3.中药藓化饮对口腔癌前病变具有明显治疗效果。更多还原

【Abstract】 Objective: The lesion precancerous of oral mucosa is a kind of precancerous state,showing off atypical hyperplasia of epithelium of oral mucosa in different extent,but it’s not be the cancer.The new categorization of WHO give the definition of lesion precancerous as that:Precursor lesions are defined as altered epithelium with an increased likelihood for progression to squamous cell carcinoma(SCC).The altered epithelium shows a variety of cytological and archi-tectural changes that have traditionally been grouped under the term dysplasia. Currently accepted precancerous lesions and precancerous conditions, includingleukoplakia, erythema, lichen planus and oral submucous fibrosis, etc. Without timely treatment of precancerous lesions and left unchecked, will ultimately develop into cancer, seriously affect the treatment and prognosis, but also to patients a tremendous impact on physical and psychological. How to treat oral precancerous lesions, blocking its development to oral cancer, has become a hotspot of current research. At present for the oral treatment of precancerous lesions of large, mainly Western oral, systemic and local use of steroids and immunosuppressive agents, early surgical excision and traditional Chinese medicine treatment. But the efficacy of Western medicine instability,and hormones and immune inhibitors, although the early onset of fast but its role is not sustainable and side effects are large; the most previous tradition of Chinese medicine connect with application of local hormones,so there’s little news about Chinese medicine treatment of precancerous lesions onlyand little study to prove the treatment effect of the Chinese medicine. Chinese medicine in the treatment of precancerous lesions of oral mucosa has obvious advantages for the characteristics of precancerous lesions of oral mucosa: Chinese medicine samples wen, effective solid; oral decoction or proprietary Chinese medicine preparation, taking the method is simple, patient compliance is good, easy to accept; with fewer side effects than western medicine and so on. In view of the above advantages, traditional Chinese medicine in the treatment of precancerous lesions of the increasing attention has been paid. In this study, drinking herbal Xianhuayin of experimental animals treated with oral precancerous lesion intervention, and by flow cytometry and immunohistochemistry to observe the before and after treatment P53 and COX-2 expression, analysis, and explore the Xianhuayin of drinking on the oral mucosa reversal of the role of precancerous lesions. For future clinical applications, experimental theory.Methods:1. Animal model of oral precancerous lesions and group preparationSyrian golden hamster get (SPF grade) 55, 6 to 8 weeks of age, body length of about 10cm, weight 90 ~ 110g, both male and female, from Beijing Tong Lihua Experimental Animal Technology Victoria Limited, the production license number: SCXK (Beijing) 2007-0001. In accordance with the principle of completely randomized into four groups: untreat control group, 5, precancerous lesions control group, 10, and saline-treat control group, 10, Xianhuayin-treat group, 30 to drink.The untreat control group, the normal daily feeding, untouched, until the end of the experiment, together with the other groups killed and drawn.Precancerous lesion control group, saline-treat control group, Xianhuayin-treat group are painted by 0.5% DMBA at the cheek pouch mucosa three times a week for six weeks.2. The brew of traditional Chinese medicine decoction of XianhuayinTraditional Chinese medicine in the first set of square by adding about 8 to 10 times as much water, soak 40 minutes to 1 hour, Wu Huo heat with 20 to 30 minutes to boil and maintain for about 10 minutes, and then about Huo Jian-Wen After 10 minutes, filtering, juice; then add the equivalent of six times the amount of the remaining residues in water, heated by Wu Huo 20 to 30 minutes to boil, with the text Huojian about 10 minutes, adding Amomum villosum, boil about 6 ~ 8 minutes, filtering, abandoned to the residues. Merging the two liquid, concentrated liquid until the required amount (including crude drug 1g/ml), packaging and sterilization, 4℃stored until required.3. Xianhuayin of traditional Chinese medicine treatment of drinking and saline gavageSaline-treat control group and Xianhuayin-treat group were treated with saline and drink decoction of Chinese medicine Xianhuayin gavage treatment, 1.6ml / only once a day for eight weeks , killed all the animals and subjects, fixed, microscopic examination.4. Immunohistochemical detection of P53 and COX-2 expression in all groupsTake wax blocks of each group, routine paraffin sections, dewaxing to water, in accordance with S-P kit instructions to operate, microscopic observation of the untreat control group, precancerous lesions control group,saline-treat control group and Xianhuayin-treat group, in the P53 and COX-2 expression, and analysis and comparison between the expression of each group whether there are differences, to evaluate the traditional Chinese medicine Xianhuayin precancerous lesions of oral mucosa reverse effect.5. Flow cytometryParaffin-embedded tissues on a continuous cut in the slicing machine to take the organization of 40-50μm thick film 5-8 tablets, placed in a test tube; xylene dewaxing, gradient alcohol hydration; saline wash, 120 mesh copper mesh Qingcuo organizations to collect a single cell suspension; 500 mesh nylon mesh filtration, centrifugation, 1000rpm, 5 minutes; to join an anti-working solution at room temperature, put it aside for 40 minutes; to join two anti-working fluid, put it aside for 1 hour at room temperature; machine inspection, read the results.6. Statistical MethodsUsing SPSS 13.0 statistical software, test results are the mean±standard deviation (±S) said that the application of analytical methods rank sum test P53 and COX-2 test results were statistically analyzed,α= 0.05, P <0.05 statistically significant.Results:1. Probabilistic selected specimens of 10 cases observed in the optical microscope, are found in irregular epithelial stratification, basal cell polarity loss, drop-shaped nail protrusion, pleomorphic nuclei, nuclear-cytoplasmic ratio increased, the nucleus increases, not like a typical split , nucleolus increases, increase in the number and nuclear deep-dyed so the performance of atypical hyperplasia, may indicate precancerous lesion models were successfully constructed.2. Immunohistochemistry2.1P53 in the untreat control group, negative expression; in the precancerous lesions control group and saline-treat control group were strongly positive expression; in the Xianhuayin-treat group for the weak positive expression. Differences between the groups was statistically significant, precancerous lesions control group and saline-treat control group, no significant difference.2.2COX-2 in the untreat control group, negative expression; in the precancerous lesions control group and saline-treat control group were strongly positive expression; in the Xianhuayin-treat group for the weak positive expression. Differences between the groups was statistically significant, precancerous lesions control group and saline-treat control group, no significant difference.3. FCM3.1P53 were expressed in all groups, the untreat control group and the Xianhuayin-treat group were weak expression, no statistically significant difference between the groups; the precancerous lesions control group and saline-treat control group were strongly positive expression, no statistically significant difference between the groups; untreat control group and precancerous lesions compared with the control group, statistically significant difference between the groups.3.2COX-2 were expressed in all groups, the untreat control group and the Xianhuayin-treat group were weak expression, no statistically significant difference between the groups; the precancerous lesions control group and saline-treat control group were strongly positive expression, no statistically significant difference between the groups; untreat control group and precancerous lesions compared with the control group, statistically significant difference between the groups.4. Correlation studies have shown that: P53 and COX-2 in oral precancerous lesions of the occurrence and development process, there is significant correlation.Conclusion:1.Applications lure cancer agent DMBA, in a certain time and frequency, it can be effectively induced hamster buccal mucosa precancerous lesions.2.P53 and COX-2 in oral precancerous lesions in the occurrence and development of great significance, P53 and COX-2 in oral precancerous lesions in the occurrence and development process, there is significant correlation.3.Medicine Xianhuayin in the treatment of precancerous lesions of drink has a noticeable effect.更多还原