【作者】 苗俊峰；

【导师】 宋代辉；

【作者基本信息】 山东大学 ， 口腔临床医学（专业学位）， 2013， 硕士

【摘要】 无限制的快速增殖是恶性肿瘤的主要特征,也是恶性肿瘤侵润、转移的基础。在人类肿瘤中已经发现和鉴定出许多种肿瘤抑制物,如Rb蛋白、P53蛋白等。近几年的研究发现,细胞命运决定因子Dachshundl(DACH1)细胞命运决定因子DACH1作为细胞/肿瘤内源性生长抑制物,能够抑制肿瘤的增殖。DACH1是黑腹果蝇Dachshund(Dac)基因在脊椎动物中的类似物。Dac基因是一种对细胞命运起决定作用的螺旋—转角—螺旋性核蛋白,它在果蝇属的眼睛以及肢体发育中起主要作用。DACH1作为一种内源性生长抑制物可在哺乳动物的正常组织中表达,作为内源性生长抑制物,与激活或转录的多种复合物有关；它并通过抑制对细胞迁移、粘附和生长起决定作用的基因的表达,从而抑制细胞的增殖。已有研究表明,在乳腺癌、卵巢癌中,DACH1在调节乳腺癌、卵巢癌肿瘤的增殖、侵润和转移中起重要作用，其中在分化较差的低分化肿瘤中DACH1其表达丧失,而在侵袭性肿瘤中,DACH1的表达较正常组织以及非侵袭性肿瘤均降低。DACH1在口腔癌中的表达及其作用尚未见报道,基于此我们设计实验,研究DACH1基因在口腔癌中的表达及作用。目的：检测细胞命运决定因子DACH1蛋白在口腔鳞癌和腺样囊性癌组织中的表达,并分析其与临床、病理特征及患者预后之间的关系。另外通过检测体外培养的鳞癌细胞系Tca8113，人腺样囊性癌细胞中DACH1的RNA表达水平,探讨DACH1在口腔鳞癌和腺样囊性癌发生发展中的作用,以期为口腔鳞癌和腺样囊性癌的诊断、治疗及预后判断提供新的依据。材料与方法：1.选取山东大学齐鲁医院2009年5月～2012年5月间收治的经病理检查证实的口腔鳞癌患者80例,腺样囊性癌18例,患者年龄在45-74岁之间,平均年龄为56.3岁。按照病理科诊断其中高分化鳞癌43例,中分化鳞癌27例,低分化鳞癌10例；通过DACH1蛋白在上述不同病理组织中阳性率的比较,及其表达与各临床病理指标之间关系的比较采用x2检验,DACH1蛋白在组织中表达之间的相关性分析采用Spearman秩检验相关分析。2.体外培养TCA8113舌鳞癌细胞系,人腺样囊性癌细胞,稳定传代后并用QIAGEN试剂盒提取总RNA,将总RNA逆转录为cDNA,然后用DACH1引物进行(Real-time fluorescence quantitative PCR, RFQ-PCR)实验,以β-act in作为内参通过相对定量法检测其DACH1转录mRNA的水平,从细胞分子层面检验DACH1的表达。结果：1.免疫组织化学结果显示,免疫组织化学结果示DACH1蛋白在高分化鳞癌、中分化鳞癌、低分化鳞癌中表达的阳性率依次减低；DACH1蛋白在腺样囊性癌组织中的表达较鳞癌高。2.RT-PCR显示体外培养的TCA8113舌鳞癌细胞系,人腺样囊性癌细胞的DACH1的mRNA表达水平与参考基因接近,之间没有差异(p>0.05),且两种细胞之间也未见明显差异(p>0.05)结论：DACH1蛋白在口腔鳞癌中的表达整体阳性率较低,鳞癌的分化程度越低,DACH1的表达水平越低；其在腺样囊性癌组织中的表达高于口腔鳞癌中的表达,与腺样囊性癌细胞增殖性比鳞癌差有关。DACH1的表达与肿瘤细胞的增殖相关。更多还原

【Abstract】 The main features of unrestricted rapidly proliferating malignant tumor, also malignant tumor invasion and metastasis basis. Have been found in human tumors and identified many types of tumor suppressor Rb protein, P53protein. In recent years, the study founds that cell fate decisions factor DACH1as endogenous growth inhibitor, can inhibit the proliferation of tumor. DACH1Dachshund (Dac), D. melanogaster gene analogues in vertebrates. Dac gene is a cell fate play a decisive role spiral-corner-spiral nucleoprotein, it plays a major role in the Drosophila eye and limb development. DACH1expression in the normal tissue of a mammal, as the growth of the endogenous inhibitor, and related to the activation or transcription of a variety of composite, by inhibition of cell migration, adhesion and growth play a decisive role in gene expression, thereby inhibiting cell proliferation. Studies have shown that in breast cancer, ovarian cancer, DACH1in the regulation of tumor growth, invasion and metastasis plays an important role, which is expressed in poorly differentiated tumors DACH1loss, invasive tumors, DACH1expression normal tissue as well as non-invasive tumors were reduced. DACH1expression in oral cancer and its role has not been reported. So we design the experimental study DACH1gene in the meaning of the expression and the role of oral cancer. Objective:This study was designed to evaluate the expression of DACHlin the oral squamous cell carcinoma and adenoid cystic carcinoma and to investigate the relationships between their expressions and the clinicopathological parameters and prognosis respectively. In addition by detecting the level of RNA expression in squamous cell carcinoma cell lines Tca8113and the adenoid cystic carcinoma cell. Thus we will find promising targets for diagnosis and therapy and potentially useful factors for detection the prognosis in the oral squamous cell carcinoma and adenoid cystic carcinoma.Methods:1.Oral squamous cell carcinoma patients with pathologically proven of Qilu Hospital were treated between May2009to May2012,80cases,18cases of adenoid cystic carcinoma in patients aged45to74years, with an averageage was56.3years old. Well differentiated according to the pathology of which43cases of squamous cell carcinoma, moderately differentiated squamous cell carcinoma27cases,10cases of poorly differentiated squamous cell carcinoma; DACH1protein in the different histopathological positive rate compared their expression with clinicopathological parameters relations X2test was used to compare the correlation analysis between DACH1protein in tissues correlation analysis using Spearman rank test.2.Cultured in vitro TCA8113tongue squamous cell carcinoma cell lines, human adenoid cystic carcinoma passaged and stable extraction of total RNA using Q.IAGEN kit, total RNA was reverse transcribed into cDNA, and then DACH1primers (Real-time fluorescence quantitative PCR, RFQ-PCR) experiments, β-actin as internal reference detection of its DACH1transcriptional mRNA levels by relative quantification inspection DACH1of expression, from the cellular and molecular level. Results:1. Immunohistochemistry results showed that immunohistochemistry results are shown DACH1protein in well-differentiated squamous cell carcinoma, in differentiated squamous cell carcinoma, followed by a reduction of poorly differentiated squamous cell carcinoma, the positive expression rate; DACH1protein in adenoid cystic carcinoma expression of high compared with squamous cell carcinoma.2.RT-PCR showed the TCA8113tongue squamous cell carcinoma cell lines in vitro adenoid cystic carcinoma DACH1mRNA level close to the reference gene, there was no difference (p>0.05) between the two cells did notsee a significant difference (p>0.05).Conclusion:DACH1protein in oral squamous cell differentiation, the lower the level of expression; expression in adenoid cystic carcinoma is lower than the expression in oral squamous cell carcinoma, that it is associated with the proliferation of cancer cells.更多还原