【作者】 仇永乐；

【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:口腔黏膜癌前病变与口腔癌的发生密切相关,近些年来,为了降低口腔癌的发病率,人们越来越重视对口腔黏膜癌前病变的预防和治疗。目前,口腔黏膜癌前病变的治疗手段较多,主要分为药物治疗和非药物治疗。非药物治疗如手术治疗、激光治疗、冷冻治疗等等,有着适应证的局限性。而西药治疗如:增生平、维甲酸等等,长期服用可能会出现一些毒副作用。近年来研究表明,中医中药治疗口腔黏膜癌前病变显示出明显的临床效果,也成为目前口腔黏膜癌前病变研究的热点。本课题通过建立口腔黏膜癌前病变动物模型,应用电镜和免疫组化的方法观察中药藓化饮对口腔黏膜癌前病变的逆转作用。方法:选取SPF级LVG叙利亚金黄地鼠55只,鼠龄六周,体质量80g。按完全随机设计方法将动物分为:空白对照组5只和实验组50只。实验组用于建立口腔黏膜癌前病变动物模型,其中又分为癌前病变对照组10只、藓化饮治疗组30只、生理盐水治疗对照组10只。将实验组50只动物,颊囊黏膜涂以0.5%DMBA丙酮溶液,每周3次,连续涂药6周后,藓化饮治疗组、生理盐水治疗对照组分别灌胃治疗。治疗8周时,各组同步取材。采用电镜及免疫组化方法,观察中药藓化饮对金黄地鼠颊黏膜癌前病变的逆转作用。结果:1癌前病变动物模型的建立实验组50只动物颊黏膜致癌六周后,双侧颊黏膜肉眼观全部为充血、糜烂、条索状皱折等表现。随机切取10例不同肉眼表现的部分病变部位,HE染色后观察,上皮黏膜表现为不同程度的异常增生。由此说明,经DMBA丙酮溶液致癌六周后,已成功建立金黄地鼠颊黏膜癌前病变的动物模型。2藓化饮对叙利亚金黄地鼠口腔黏膜癌前病变的逆转作用2.1肉眼观察空白对照组金黄地鼠颊黏膜表面光滑,呈淡粉色,透亮具有弹性,血管清晰。实验组50只动物涂DMBA丙酮溶液六周后,颊黏膜变得粘连、粗糙、无弹性、充血糜烂,甚至可见条索状皱折。中药藓化饮灌胃八周后,可见颊黏膜皱折减轻,黏膜表面逐渐光滑,充血糜烂面基本消失,但弹性较正常稍差。生理盐水灌胃治疗八周后与治疗前无明显改变。2.2光镜观察空白对照组金黄地鼠颊黏膜为角化的复层鳞状上皮,上皮和肌层之间为薄层结缔组织及少量散在的纤维细胞和血管。停涂DMBA丙酮溶液八周后,镜下观察金黄地鼠颊黏膜可见不同程度的异常增生。中药藓化饮灌胃治疗八周后,黏膜上皮层次逐渐清晰,上皮层数逐渐恢复正常,基底层细胞排列比较有规则,异型性的细胞减少。生理盐水灌胃治疗八周后与治疗前无明显改变。2.3扫描电镜观察空白对照组金黄地鼠颊黏膜上皮表现为多边形细胞,细胞间桥很明显,个体细胞表面显示很多网状排列的微嵴,微嵴围成均匀一致的小窝,整个细胞表面呈现一种蜂窝状外观。停涂DMBA丙酮溶液八周,细胞外形更不规则,细胞重叠,松解现象更为突出,微嵴肿胀变宽,不规则排列,呈卵石状外观,蜂窝状结构完全消失。中药藓化饮灌胃治疗八周后,细胞外形逐渐清晰,呈现出多边形。细胞表面大部分区域呈现蜂窝状结构,可见连续的细胞界限。生理盐水灌胃治疗八周与治疗前无明显改变。2.4透射电镜观察空白对照组金黄地鼠颊黏膜上皮细胞外形规则,细胞紧密相邻,桥粒丰富,张力纤维发育正常,基底膜完整。停涂DMBA丙酮溶液八周后,可见细胞间隙增宽,桥粒大量减少,张力纤维减少,细胞外形不规则,核仁边集,核外形不规则,核膜内陷,可见基底膜断裂。中药藓化饮灌胃治疗八周后,增宽的细胞间隙逐渐减小,桥粒和张力纤维逐渐丰富,细胞外形逐渐规则。生理盐水灌胃治疗八周与治疗前无明显改变。3藓化饮治疗后EZH2、PCNA在各组免疫组化染色结果EZH2、PCNA在空白对照组和藓化饮治疗组低表达,而在癌前病变对照组和生理盐水治疗对照组高表达。癌前病变对照组EZH2、PCNA高表达率与空白对照组相比,有显著性差异(P<0.05);藓化饮治疗组EZH2、PCNA高表达率与癌前病变对照组相比,有显著性差异(P<0.05);而藓化饮治疗组EZH2、PCNA高表达率与空白对照组相比,无显著性差异(P>0.05);生理盐水治疗对照组EZH2、PCNA高表达率与藓化饮治疗组相比,有显著性差异(P<0.05)。统计学分析,两指标表达具有相关性,表达吻合度具有统计学意义。结论:1使用0.5%DMBA丙酮溶液涂抹叙利亚金黄地鼠颊黏膜六周后,能成功建立金黄地鼠颊黏膜癌前病变动物模型。2藓化饮治疗八周后,叙利亚金黄地鼠颊黏膜充血糜烂面基本消失,细胞外形逐渐清晰,上皮层异型性的细胞减少,EZH2、PCNA高表达率明显下降。说明藓化饮对金黄地鼠颊黏膜癌前病变有明显逆转作用。更多还原

【Abstract】 Objective:There was a correlation between oral mucosal premalignant lesion and carcinoma of oral mucosa. These years, people had been paid more and more attention on the carcinoma of oral mucosa preservation and treatment to cut down the disease incidence of carcinoma of oral mucosa. Now, there were lots of therapeutic tool for oral mucosal premalignant lesion. And mainly divide into drug treatment and un-drug treatment. The un-drug treatment usually included operation treatment、laser therapy、freezing therapy and so on. But these ways had indication confine and higher recurrence of post-treatment, especially could not prevent the new lesion appearance in other part of oral cavity. So these defects greatly cut down the un-drug treatment possibility. Medical doctor treat such as ratinoic acid would had adverse reaction if long-term using. So, the emphasis of oral mucosal premalignant lesion drug treatment was to prevent the new lesion appearance in other part of oral cavity and deeply developed. And Chinese herbal medicine properly had these ideal condition. These years research showed that Chinese herbal medicine had played markedly therapeutic effect during the oral mucosal premalignant lesion treatment process. And it had become to oral mucosal premalignant lesion research hot spot. The topic investigated the role of Xianhuayin on the reversal of the premalignant lesion in the mucosa of cheek pouch in golden hamster by using immunohistochemical methodand electron microscope by premalignant lesion animal model generation.Methods:A total of 55 SPF LVG Syrian golden hamsters (6 weeks old, weight: 80 g) were randomly divided into untreated control group (n=5) and experimental group (n=50). Animals in the experimental group were used to generate the oral mucosal premalignant lesion models. Of these 50 animals with oral mucosal premalignant lesions, 10 hamsters were treated with normal saline (NS-treated group); 10 hamsters were untreated (premalignant lesion group); 30 hamsters were treated with Xianhuayin (Xianhuayin-treated group). The buccal mucosa of 50 hamsters in the experimental group was painted with DMBA (0.5% in acetone) as described previously. Application with DMBA was performed three times a week for a continuous of 6 weeks. Then intragastic administration was performed once a day for a continuous 8 weeks in Xianhuayin-treated group and NS-treated group. All the animals were sacrificed and the tissue samples were collected at the same time. To investigate the role of Xianhuayin on the reversal of the premalignant lesion in the mucosa of cheek pouch in golden hamster by using immunohistochemical method、electron microscope.Results:1 Premalignant lesion animal model generationSix weeks after treatment with DMBA acetone solution, in the experimental group, both sides of the cheek pouch mucosa of hamsters became adhered, lacking of elasticity, congested, anabrotic and streak-like ruffle. Histological analysis was performed by randomly collecting ten part of lesion tissues, which had different of visual manifestation. Variable degree of epithelial dysplasia was observed under light microscopic . So six weeks after treatment with DMBA acetone solution, it had successfully generated the premalignant lesion animal model.2 Role of Xianhuayin on the reversal of premalignant lesion in the oral buccal mucosa of golden hamsters2.1 Visual observationsThe cheek pouch mucosa of hamsters in the untreated-group exhibited smooth surface, light pink color, transparency, elasticity and clear blood vessels. Six weeks after treatment with DMBA acetone solution, the cheek pouch mucosa of hamsters in the experimental group became adhered, rough, lacking of elasticity, congestion, anabrosis and streak-like ruffle was evident . Eight weeks after intragastric administration with Xianhuayin, the adherence of cheek pouch mucosa became mitigated, smooth surface appeared gradually, and congestion as well as anabrosis disappeared, but the elasticity was still relatively poorer than the normal tissues. No significant changes were observed after the hamsters in the premalignant lesion group were intragastrically administered with normal saline .2.2 Light microscopic observationsBuccal mucosa of the normal hamsters in the untreated group was keratinized and stratified squamous epithelium under light microscope. Lamellar connective tissues, small amount of sporadic fibrocytes and blood vessels were present between epithelium and muscular layer . Variable degree of epithelial dysplasia were observed in the hamster of premalignant group 8 weeks after painting with DMBA was stopped. Eight weeks after treatment with Xianhuayin, the irregular epithelial mucosa gradually became distinct, the layer of epithelium was recovered to normality, basilar membrane cells were regularly organized and the number of atypical cells was reduced . No significant changes were observed after hamsters were intragastrically administered with normal saline .2.3 SEM analysisScanning Electronic Microscopic (SEM) analysis showed that in the hamster of untreated-group, the buccal mucosa contained polygonal cells and clear cell bridge. The surface of the individual cells exhibited honeycomb structures . Eight weeks after painting with DMBA was stopped, the epithelial cells were morphologically irregular, overlapped and loosened. Swelling and widening micro-crests were irregularly arranged and appeared as gravel shape. Eight weeks after intragastric administration with Xianhuayin, most of the cell surface exhibited honeycomb structures and continuous cellular border was observed in the hamsters of Xianhuayin-treated group . No significant changes were observed for the hamsters in NS-treated group compared with those in the premalignant group.2.4 TEM analysis Transmission Electronic Microscopic (TEM) analysis showed that in the hamsters of untreated group, buccal mucosal epithelial cells were morphologically regular, closely connected and contained abundant desmosomes. The tonofibrils were developed normally. Eight weeks after painting with DMBA was stopped, desmosomes and tonofibrils were significantly reduced, intercellular gap was increased, the shape of epithelial cells were irregular, the morphologically irregular nucleolus was marginally gathered and the caryotheca was invaginated. Eight weeks after intragastric administration with Xianhuayin, the widening intercellular gap became reduced gradually, desmosomes and tonofibrils were gradually becoming abundant and the cells were becoming morphologically regular. No significant difference was observed between the hamsters in NS-treated group and those in the premalignant group.3 Expression of EZH2、PCNA in different group after Xianhuayin treatmentEZH2、PCNA were rarely expressed or lower expressed in the untreated group and Xianhuayin-treated group, and they were higher expressed in the premalignant group and NS-treated group.The rate of EZH2、PCNA high expression of the premalignant lesion group compared with the untreated control group’s, it had statistical difference (P<0.05); The rate of EZH2、PCNA high expression of the Xianhuayin-treated group compared with the premalignant lesion group’s, it had statistical difference (P<0.05); The rate of EZH2、PCNA high expression of the Xianhuayin-treated group compared with the untreated control group’s, it had no statistical difference (P>0.05); The rate of EZH2、PCNA high expression of the NS-treated group compared with the Xianhuayin-treated group’s, it had statistical difference (P<0.05)Statistical analysis, there was a correlation between EZH2 and PCNA, and the expression goodness of fit had statistical difference.Conclusion:1 Six weeks after treatment with DMBA acetone solution, it had successfully generated the premalignant lesion animal model.2 Eight weeks after intragastric administration with Xianhuayin, congestion as well as anabrosis disappeared; the cells were becoming morphologically regular; the number of atypical cells was reduced; the rate of EZH2、PCNA high expression obviously decreased. It’s concluded that Xianhuayin had the obvious role on the reversal of premalignant mucosal lesions in the golden hamster buccal pouch.更多还原