【作者】 韩德昌；

【导师】 韩新光；

【作者基本信息】 郑州大学 ， 口腔临床医学， 2009， 硕士

【摘要】 舌癌是口腔颌面部恶性肿瘤中最常见的口腔癌,其构成比在国内外均居第一。一般恶性程度较高,生长快,浸润性强。舌癌常早期发生颈淋巴结转移,且转移率较高,有文献报道高达60—80%,严重影响治疗效果,危害极大。近来的统计资料表明,舌癌的发病率有逐年上升的趋势,而且发病年龄趋向年轻化。但因其部位和功能的特殊,手术治疗容易造成毁伤和功能障碍,对人体的身心健康危害较大。因此,为提高舌癌的治愈率和生存率,提高患者的生活质量,分子靶向药物治疗作为生物治疗的重要组成部分也越来越受到重视,使得肿瘤的生物学治疗成为继手术、放疗和化疗之后的第四种治疗方法。故对舌癌侵袭机制和转移机制的研究越来越重视。尤其在细胞分子水平上,对舌癌细胞的侵袭和转移,从不同的方面取得了令人注目的进展。近年来发现Rho/ROCK信号转导通路是一个重要的信号通路,它的主要功能是调节肌动蛋白和细胞骨架重组,而细胞骨架的变化影响细胞的形态和结构,从而调节细胞的形态变化和运动。F—肌动蛋白细胞骨架不仅在细胞形态改变、细胞运动中有重要作用,还在细胞的各种生命活动如细胞增殖、细胞转录、细胞转化方面发挥重要作用。此外,Rho/ROCK通路还参与细胞周期的调控和抑制细胞凋亡等。侵袭和转移是肿瘤恶性程度的重要标志,也是患者死亡的主要原因。由于舌癌早期易发生颈部淋巴结转移,因而对于舌癌转移机制的研究成为肿瘤学家和口腔医学专家研究的热点。本实验研究旨在通过应用Rho/ROCK信号通路的特异性抑制剂Y—27632,用不同的浓度作用于体外培养的人舌癌Tca8113细胞,阻断Rho/ROCK信号转导通路,使用免疫荧光技术,通过激光扫描共聚焦显微镜,观察舌癌细胞内F—肌动蛋白数量和排列分布的变化、细胞骨架的重构和细胞形态的变化。同时,采用流式细胞术,对细胞内DNA含量进行检测,分析G₁、S和G₂/M期的细胞分布,观察Rho/ROCK通路对细胞周期的调控的影响,探讨其作用机制,为预防和治疗舌癌的侵袭、转移提供一种新的治疗思路和方法。目的应用Rho/ROCK信号通路的特异性抑制剂Y—27632,探讨Rho/ROCK信号转导通路对舌鳞癌F—肌动蛋白细胞骨架及对细胞周期的调控的影响。材料与方法用不同药物浓度的Rho/ROCK通路特异性抑制剂Y—27632（5μmol/L,10μmol/L,25μmol/L,50μmol/L）作用于体外培养的人舌鳞癌Tca8113细胞株24小时,用FITC标记的鬼笔环肽标记F—肌动蛋白,PI标记细胞内DNA,通过激光扫描共聚焦显微镜和免疫细胞化学法,观察舌癌细胞形态和细胞内F—肌动蛋白排列、分布和含量的变化。同时,采用流式细胞术,对细胞内DNA含量进行检测,分析细胞周期G₁、s和G₂/M各期的细胞分布。结果1.倒置显微镜下观察培养细胞为上皮样细胞形态,呈扁平、多角形、圆形、不规则性、梭性等多种形态,细胞异型性明显,多核、核分裂像少见;核浆比例增加,可见少量巨核细胞。2.激光扫描共聚焦显微镜下舌癌细胞的形态:Tca8113细胞为不规则形,细胞内微丝为粗大的较长的束状纤维,细胞膜伸出大量突起,其中许多微丝深入到细胞突起,即F-actin小体。细胞内F-actin微丝形成束状纤维,平行排列或纵横交错成网状贯穿整个细胞。Tea8113细胞actin聚集形成F-actin小体,大量的F-actin趋向于胞周分布,形成F-actin环,细胞核大,极性增强,偏向一侧,可见多个核仁。微丝在细胞的表面突起,排列形成许多微刺突、丝状伪足和片状伪足。经5μmol/L和10μmol/L处理后,细胞形态略有收缩,与对照组相比,变化不甚明显;经25μmol/L和50μmol/L处理后,细胞形态收缩,细胞生长稀疏,形状变规则,呈椭圆形或圆形,绿色荧光染色的F-actin显著减少,胞浆中所显示的微丝较短或消失,多呈直纤维状线型荧光,且数量明显减少,排列较稀疏,不规则,细胞边缘显示较强的荧光带或荧光环。3.SP免疫细胞化学法光镜下舌癌细胞的形态:见细胞呈圆形、椭圆形,细胞异型性不明显,核浆比例增加,胞核偏向一侧,可见少量巨核细胞,多核、核分裂像少见。4.平均荧光强度法和免疫细胞化学法测定F—肌动蛋白含量:实验组与对照组相比,均明显降低（P值均＜0.01）,差异具有显著性意义。5.流式细胞术检测细胞周期G₁、S和G₂/M各期的细胞分布结果:与对照组相比,除实验组（5μmol/L）P＞0.05外,其他各实验组,随Y-27632浓度的增大,使滞留在G₁期的细胞数量显著增多,经统计学分析,差异有显著性（P值均＜0.01）,说明G₁期的变化与抑制剂Y—27632浓度呈正相关系,与细胞内微丝结构发生改变相一致。结论1.Rho/ROCK信号通路参与人舌鳞癌Tca8113细胞F-肌动蛋白细胞骨架的重构和细胞形态的改变:2.F—肌动蛋白含量的变化与Rho/ROCK信号通路的特异性抑制剂Y—27632的浓度呈负相关系;3.Rho/ROCK信号通路也参与人舌鳞癌Tca8113细胞周期的调控。更多还原

【Abstract】 Carcinoma of tongue is common cancer of malignant tumor of oral and maxillofacial region,whose constituent ratio is the first at home and abroad.Tongue cancer usually has serious malignancy and invasion,high growth velocity.And it has high metastatic frequency of neck lymph node in early stage,which was reported to reach up to 60%-80%.Its harm to body is serious and therapeutic efficacy is seriously influenced.Recently,statistic data showed:the incidence rate of tongue cancer has gradually increasing tendency year by year.And age of onset tends to be younger.Because of special place and function,disfiguration and functional disturbance are easily caused by operation,and the health of body and mind is easily influenced.Therefore,for increasing healing rate and survival rate and raising quality of life of patients of tongue cancer,molecular targeted drug therapy is more and more important as the significant part of biotherapy.And biotherapy has gradually become the fourth choice for treatment of cancer after operation,radiotherapy,chemotherapy. So,studies on invasion and metastasis mechanisms of tongue cancer is more and more important,especially at cellular and molecular level,a noticeable advancement has been obtained in each aspect.In the past few years,Rho/ROCK signal transduction pathway was discovered as an important signal pathway,whose main function is to regulate actin and reconstruction of cytoskeleton,and the cellular shape and construction are influenced by the change of cytoskeleton,thus,Rho/ROCK signal pathway regulate the cellular morphologic change and motion.F-actin plays important role not only in the cellular morphologic change and motion,but also in the various kinds of cellular life activities,such as cell proliferation,cell transcription, cell transformation.In addition,Rho/ROCK signal transduction pathway mediates the regulation of cell cycle and apoptosis,et al.Invasion and metastasis are important marker of malignancy degree of tumor,and also are main reason of death of patients. Because of high metastatic frequency of neck lymph node of tongue cancer in early stage,as a result,studies on invasion and metastasis mechanisms of tongue cancer by oncologists and oral experts have become an investigative hot spot in medicine territory.The aim of this empirical study is to observe the changes of arrangement and distribution and content of F-actin,reorganization of F-actin cytoskeleton and the changes of morphology in human tongue squamous carcinoma Tca8113 cell lines with different concentration of Y-27632 to block Rho/ROCK signal pathway,by using the specific inhibitor Y-27632 of Rho/ROCK signal transduction pathway and immunofluorescent technique and laser scanning confocal microscopy.Meanwhile, the contents of DNA are detected by Flow cytometry and the distribution of G₁,S, G₂/M stage of cell cycle is analyzed and the effect of Rho/ROCK signal transduction pathway on regulation of the cell cycle is observed and analyzed in human tongue squamous carcinoma Tca8113 cell lines cultured in vitro.A kind of new therapeutic thinking and approach to prevent and cure invasion and metastasis of tongue squamous carcinoma may be put forward by investigating their mechanisms.ObjectiveTo investigate the effect of Rho/ROCK signal transduction pathway on F-actin cytoskeleton and regulation of the cell cycle in human tongue squamous carcinoma Tca8113 cell lines cultured in vitro by using the specific inhibitor Y-27632 of Rho/ROCK signal transduction pathway.Materials and methods The Tca8113 cells are treated with different concentration of Y-27632 （5μmol/L,10μmol/L,25μmol/L,50μmol/L） for 24h.Cellular F-actin stained with FITC-phalloidin and DNA stained with Propidium Iodide are examined by laser scanning confocal microscopy（LSCM） and immunocytochemical S-P method.Then the changes of arrangement and distribution and content of F-actin,and the changes of morphology are observed in Tca8113 cell lines.Meanwhile,the contents of DNA are detected by Flow cytometry and the distribution of G₁,S,G₂/M stage of cell cycle are analyzed in Tca8113 cell lines.Results1.The morphology of the Tca8113 cells cultured in vitro under inverted microscope is observed:the epithelial cellular shape,such as thin and flat,polygon, round and ellipse,irregularity and spindle,the significant heteromorphism,the seldom seen polynucleation and karyodieresis.And the morphology of the Tca8113 cells stained by HE method under common microscope shows:the cellular shape looks like round and ellipse,the nucleus is obviously manifested,the karyoplasmic ratio is raised,a few of the giant nucleus cell are seen.2.The cellular morphology and arrangement and distribution of the cellular F-actin of the Tca8113 cells under laser scanning confocal microscopy（LSCM） are observed:the irregular cellular shape,the bundle fibers of intra-cellular gross and long microfilament,protrusion of a great quantity cell processes on cellular membrane and a lot of microfilaments penetrated deeply in cell processes,that’s F-actin body.Of parallel arrangement and cross overlapping reticulated bundle fibers formed by F-actin micro filament appear in the whole cell.A great quantity of F-actin bodies assembled and formed by actin of the Tca8113 cells tend to distribute in cellular surrounding and form F-actin cycle with cell nucleus increasing and polarity strengthening,while the nucleus deviate to one-side,the Multiple nucleoli appear.A great quantity of microspikes,filopodium and lamellipodium are formed by a lot of surface projection of microfilaments on cellular membrane.In comparison with control group,the change of morphology in Tca8113 cell lines treated by Y-27632 with concentration of 5μmol/L and 10μmol/L is not obvious,but the others are obvious by Y-27632 with that of 25μmol/L and 50μmol/L,the shrinks of the cellular shape,the rarefaction of cell growth,the cellular shape appearing to be regular,such as round and ellipse,virent F-actin stained with FITC-phalloidin is obviously decreased,the microfilaments in kytoplasm becomes short or disappears or becomes straight and fibriform fluorescence,the F-actin microfilaments are rarefied and arranged irregularly;the brighter fluorescence band or fluorescence ring appear on the cellular margin.3.The morphology of human tongue squamous carcinoma Tca8113 cells stained by immunocytochemical S-P method under common microscope is observed:the cellular shape is round and oval,the heteromorphism is not manifest,the karyoplasmic ratio is raised,the nucleus deviate to one-side,there are a few of the giant nucleus cells and seldom polynucleation and karyodieresis.The shape of typical epithelial cell is not of significant difference.4.The results of the contents of F-actin detected by mean fluorescence intensity （MFI） and immunocytochemical S-P method in Tca8113 cells show:contrasted to control group,experiment groups is significantly induced（p<0.01）,the deviation of the contents of F-actin between control group and all experiment groups is significant.5.The results of the distribution of G₁,S,G₂/M stage of cell cycle are analyzed by Flow cytometry in Tca8113 cell lines showed:contrasted to control group,the number of Tca8113 cells detained at G₁ stage of the experiment groups is significantly increased with growing drug concentration,except the experiment group （5μmol/L）（P>0.05）,the deviation of the number of Tca8113 cells between control group and the others is significant.And the results suggest:the change of the number at G₁ stage of human tongue squamous carcinoma Tca8113 cell lines cultured in vitro is of positive correlation with the concentration of the specific inhibitor Y-27632,and is coincided with the changes of the construction of the cellular F-actin microfilament cytoskeleton in Tca8113 cells.Conclusions 1.Rho/ROCK signal transduction pathway participates in the reorganization of F-actin cytoskeleton and morphological changes in Tca8113 cell lines.2.The change of the content of F-actin in Tca8113 cells is of negative correlation with the concentration of the specific inhibitor Y-27632 of Rho/ROCK signal transduction pathway.3.Rho/ROCK signal transduction pathway participates in the regulation of cell cycle.更多还原