【作者】 胡斌；

【导师】 陈子华；

【作者基本信息】 中南大学 ， 外科学， 2010， 博士

【摘要】 背景：大肠癌是世界上第三大常见肿瘤。尽管近年来在外科技术、化疗和放疗方面有很大改进,但是术后复发和转移仍然是大肠癌治疗的主要难题。因此需要有新的治疗策略来进一步提高大肠癌患者的预后。Id-1是HLH转录因子家族的一员,它在许多肿瘤中表达水平增高,而且它的高表达和治疗效果差呈正相关。目的：探讨Id-1基因在人大肠癌组织中的表达及临床意义；研究Id-1siRNA对人大肠癌SW480细胞药物敏感性的影响。方法：1.采用Real time PCR和Western blot方法检测56例手术切除大肠癌及远癌肠粘膜组织中Id-1 mRNA和蛋白的表达。采用免疫组织化学方法检测107例手术切除大肠癌组织及23例肠镜下活检非癌大肠粘膜上皮组织中Id-1的表达。结合大肠癌患者临床病理特征和术后随访资料分析临床意义。2.采用Id-1 siRNA干扰人大肠癌SW480细胞中Id-1基因的表达。实验分为空白对照组(未转染的大肠癌细胞)、阴性对照组(转染非特异性序列的大肠癌细胞)和Id-1 siRNA组(转染Id-1 siRNA的大肠癌细胞)。用Real time PCR检测Id-1 mRNA表达变化,Western blot检测Id-1蛋白表达变化。用MTT法观察单用Id-1 siRNA、单用5-FU,以及Id-1 siRNA联合5-FU时SW480细胞增殖的变化。进一步的实验中,用一种浓度的5-FU处理各实验组细胞,用流式细胞术观察细胞凋亡的变化。用小剂量的5-FU处理各实验组细胞,用划痕愈合实验观察各实验组细胞体外迁徙运动能力的变化。结果：1.在56例新鲜标本中,Real time PCR和Western blot结果显示：大肠癌组织中Id-1 mRNA和Id-1蛋白的平均表达水平明显高于远癌肠粘膜组织(P<0.01)。Id-1 mRNA和蛋白的表达水平与大肠癌的Dukes分期、肿瘤组织分化程度以及淋巴结转移有关(P<0.05)。免疫组化结果显示：大肠癌组织中Id-1蛋白的表达明显高于非癌大肠粘膜上皮组织(P<0.01)；Id-1蛋白的表达水平与大肠癌的Dukes分期及淋巴结转移有关(P<0.05)。生存分析显示,在107例大肠癌患者中,Id-1蛋白高表达组5年累积生存率明显低于低表达组(P<0.05)。2. Id-1 siRNA成功高效转入大肠癌SW480细胞内。Real time PCR检测Id-1 mRNA表达水平在Id-1 siRNA组减低,而在空白对照组,阴性对照组Id-1 mRNA的表达无明显变化；Western blot检测Id-1siRNA组Id-1蛋白表达较空白对照组和阴性对照组明显减低(P<0.01)。MTT实验结果显示：单用Id-1 siRNA对SW480细胞生长有一定抑制作用,但抑制作用较弱；单用化疗药物5-FU对细胞生长有一定抑制作用,而且这种抑制作用具有明显的浓度依赖性；Id-1 siRNA和5-FU联用时对SW480细胞生长的抑制作用显著增强(P<0.01),Id-1 siRNA组细胞对5-FU的易感浓度降低,半数抑制浓度(IC50)明显下调(P<0.01)。用一种浓度的5-FU处理各实验组细胞,流式细胞术结果表明细胞凋亡率在Id-1 siRNA组明显高于空白对照组和阴性对照组(P＜0.01)。用小剂量的5-FU处理各实验组细胞,划痕愈合实验结果表明细胞迁徙运动能力在Id-1 siRNA组明显低于空白对照组和阴性对照组(P＜0.01)。结论：1.Id-1的高表达不仅与大肠癌的发生、发展密切相关,而且可作为判断大肠癌患者预后的指标之一2. Id-1 siRNA能有效地沉默大肠癌SW480细胞Id-1 mRNA及蛋白的表达。下调Id-1能促进SW480细胞对化疗药物5-FU的敏感性。更多还原

【Abstract】 BACKGROUND:Colorectal cancer (CRC) is the third most common cancer worldwide. Despite advances in surgical technique, chemotherapy and radiotherapy, recurrence and metastasis are the main problem in the management of CRC. Therefore, new therapeutic strategies are needed to improve the survival of CRC patients. Id-1(Inhibitor of differentiation/DNA binding), a member of the helix-loop-helix (HLH) transcription factor family, is up-regulated in many types of human cancer and its expression levels are correlated with poor treatment outcome and shorter survival.OBJECTIVE:To detect the expression of Id-1 in colorectal carcinoma (CRC), and to evaluate its clinical significance. To investigate the changes of the drug sensitivity in Id-1 siRNA transfected SW480 cells.METHODS:1. Id-1 expression in 56 fresh CRC tissues as well as in 107 CRC patients who had accepted resection were detected by Real time PCR, Western blot and Immunohistochemistry. The relationship between Id-1 expression and clinicopathological features, prognosis was analyzed.2. The specific Id-1 small interference RNA (siRNA) was transfected into colorectal carcinoma SW480 cells using Iipofectamine2000. Three groups of cells were used in the vitro studies, including black control group (untransfected cells), negative control group (cells transfected with the non-silencing control siRNA) and Id-1 siRNA group (cells transfected with the Id-1 siRNA). The expression of Id-1 mRNA was detected by real time reverse transcription-polymerase chain reaction. The expression of Id-1 protein was measured by western blot. Drug sensitivity of the cells to 5-fluorouracil (5-FU) was analyzed with methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and flow cytometry. Cell migration and movement capacity was assessed using scratch healing experiment.RESULTS:1. Real time PCR and Western blot showed the expression level of Id-1 were significantly higher in CRC than intestinal mucosa distant from the tumor (P<0.01). Id-1 expression was associated with Dukes stages, grade of differentiation and lymph node metastasis (P<0.05). Similarly, immunohistochemistry showed the expression level of Id-1 was significantly higher in CRC than normal intestinal mucosa (P<0.01). The overexpression of Id-1 in CRC was correlated with Dukes stages and lymph node metastasis (P<0.05). Furthermore overexpression of Id-1 was associated with poor prognosis.2. Id-1 siRNA was transfected into the SW480 cells efficiently. The mRNA and protein of Id-1 in Id-1 siRNA group were significantly decreased compared with either negative control group or black control group (P<0.01). MTT results showed that Id-1 siRNA group had higher cell inhibitory after treated with 5-FU (P<0.01). Add 5-FU (102.4mmol/L), FCM results demonstrated that the apoptosis of Id-1 siRNA group had been increased compared with negative control group and black control group (P<0.01). Add 5-FU (1.6 mmol/L), scratch healing experiment results demonstrated that the migration and movement capacity of Id-1 siRNA group had been decreased compared with negative control group and black control group (P<0.01).CONCLUSION:1. Overexpression of Id-1 is closely associated with the progression of CRC, and might be regarded as a factor of poor prognosis in CRC.2. Id-1 siRNA led to the efficient and specific inhibition of Id-1 expression in CRC SW480 cells. Silencing of Id-1 expression can increase SW480 cells sensitization to 5-FU.更多还原