【作者】 蔡扬；

【导师】 李秉琦；

【作者基本信息】 四川大学 ， 口腔临床医学， 2004， 博士

【摘要】 基因不稳定是人类肿瘤发生发展的中心环节,染色体不稳定(chromosome instability, CIN)是见于人类大多数恶性肿瘤中的一种基因不稳定形式,包括口腔鳞状细胞癌(Oral squamous cell carcinomas, OSCC)。非整倍体作为CIN的特征性表现形式可以出现在相当比例的OSCC及癌前病损,并与细胞的恶性转化进程有关,已经被成功地应用于预测口腔癌前病损的癌变进程,但其形成机制仍不清楚。近年来研究显示:细胞有丝分裂检查点(mitotic checkpoint)功能缺陷和中心体异常可能是肿瘤细胞CIN(非整倍体)形成的重要原因,但这一推测还没有在包括OSCC在内的人类大量实体瘤中得到全面证实。 因此,本研究旨在探讨口腔鳞状细胞癌细胞染色体不稳定的潜在机制及其相关调控,了解其与口腔黏膜癌变的关系及其在口腔癌前损害预后判断中的作用和意义。 研究材料与方法如下:(1)以7株体外培养的非整倍体口腔鳞状细胞癌(OSCC)细胞为研究对象,采用免疫荧光染色(IF)、激光共聚焦显微镜观察及荧光活化细胞分类(FACS)等方法,观察非整倍体OSCC细更多还原

【Abstract】 Genetic instability is currently considered to be central to the development of human cancer. Chromosome instability (CIN) exemplified by aneuploidy was seen in the early stage of many tumors, including oral squamous cell carcinomas (OSCC). It is currently considered that the functional defects of mitotic checkpoint and centrosome abnormalities may play important roles in the development of aneuploidy in tumor cells. This study is an attempt to elucidate the possible underlying mechanisms of CIN in oral squamous cell carcinoma and their possible roles in oral carcinogenesis.In this study, (1) Seven aneuploidy OSCC cell lines were investigated for the functional status of mitotic checkpoint by using FACA to investigate the percentage of cells arrested at G2/M after incubated in nocodazole, and the status of centrosomes in these cell lines were investigated by using double Immunofluorescence staining with antibodies against gamma-tubulin and pericentrin. (2) Formalin-fixed, paraffin-embedded tissues of 12 cases of normal oral epithelium, 22 case of dysplasia with different degree epithelium dysplasia and 32 cases of OSCC with different differentiation were investigated for centrosome status by using double immunofluorescence staining with antibodies against gamma-tubulin and cytokeratin. The differences and the change trend of centrosome status in these groups were statistically analyzed bySPSS 10.0. (3) The same OSCC cell lines were investigated for the expression of Cycling Cdk2, p27/KipK p21/Wafl by western blot and the correlation between the expression of the proteins and centrosome amplification were analyzed.The results showed: (1) All aneuploidy OSCC cell lines exhibited high percentages of cells arrested at G2/M phase of the cell cycle after 18 hours incubation in nocodazole indicating an intact mitotic checkpoint, while centrosome abnormalities, numerical amplification such as more than two centrosomes per cell as well as morphological defects such as clusters and/or huge in size of centrosomes, were observed in a fraction of OSCC cells (0.4%-18.8%) in all OSCC cell lines. (2) Normal oral epithelium showed normal centrosomes in epithelium cells, while 16 out of 22 cases (72.73%) of dysplasia (DYS) and 27 out of 32 cases (84.38%) of OSCC showed the evidence of centrosome amplification and morphological abnormalities characterized by huge size, clump or supernumerary centrosomes in a small fraction of epithelium or tumor cells. The percentage of cells with abnormal centrosomes increased gradually from mild-dysplasia epithelium to poorly differentiated OSCC, which positively correlated with the histological\cytologic grade of oral precancerous lesions and OSCC (r=0.955, P<0.01) . The percentage of cells with abnormal centrosomes in OSCC is higher than that in dysplasia (t= 5.26, PO.01), while there were no significantly differences between the dysplasia with OSCC outcome and that without OSCC outcomes. (3) The degree of centrosome amplification positively correlated with the ratios of protein expression of CyclinE/p21wafl (r =0.710, p<0.05) and CyclinE/p27kipI (r=0.848, pO.Ol )in all OSCC cell linesinvestigated.In all, it is suggested that (1) The CIN phenotype (aneuploidy) in OSCC cells may not attribute to the defects of mitotic checkpoint, while the abnormalities of centrosome is likely a contributing factor towards CIN in OSCC cells. (2) Centrosome amplification was an early event and that might play a role in the establishment and perhaps the progression of OSCC. There might be some direct mechanistic relationship between centrosome defects and the cellular morphological phenotype characteristic of dysplasia and OSCC, Centrosome amplification could be served as an alternative diagnostic indicator of dysplasia but not a effective predictive marker for the progression of oral precancerous lesions. (3) Unbalance of the expression of CyclinEN Cdl^ p27/Kipl, p21/Wafl might be responsible for centrosome amplification in OSCC cells and the increase of the ratios of CyclinE/p27kipl and/or CyclinE/p21wafl was likely to be one of the reasons for centrosome amplification in OSCC cells.更多还原