【作者】 张凯；

【导师】 刘铜军；

【作者基本信息】 吉林大学 ， 外科学， 2010， 博士

【摘要】 目的:研究大肠癌肿瘤转导通路中蛋白表达水平,通过对我科室大肠癌患者术后切除的标本(癌组织及癌旁正常粘膜)中蛋白表达情况的研究,筛选出阳性表达蛋白,选择针对该蛋白的小分子抑制剂来处理不同的大肠癌肿瘤细胞株,研究其对肿瘤细胞的杀伤作用、对细胞周期的影响及机制,并与化学药物奥沙利铂联用观察是否具有协同效应。方法:收集26个大肠癌病人的组织标本,包括癌组织和癌旁正常组织,进行pathway array实验,研究其组织当中的蛋白质分子表达水平;根据pathway array结果,选取Her2作为切入点,在Sw480、Sw620、Ht29、Hct116、Hct15、Caco2细胞株中的基因表达水平,通过Western技术证实其表达情况;应用HER2特异性小分子抑制剂AG879对大肠癌癌细胞增殖方面的作用,进行pathway array研究;选取IC50处理肿瘤细胞株后进行细胞周期分析,研究Her2在大肠癌细胞周期方面的作用;分别用奥沙利铂与AG879对肿瘤细胞进行处理,然后联合应用这两种药物研究其协同效应。结果:26对标本当中,每对标本我们都杂交了117种磷酸化和非磷酸化蛋白,共检测到32种蛋白,其中存在两倍以上变化的蛋白有8种,p-PKCα、p-HER2、CyclinD1、P27在癌组织中高表达,ERK、XIAP、p-PKCα/βII、b-catenin在癌旁正常粘膜组织中高表达;AG879对在Sw480、Sw620、Ht29、Hct116、Hct15、Caco2细胞株有明显的抑制生长作用,故证明HER2有促进大肠癌癌细胞增殖的作用;与对照组相比较,应用AG879细胞株出现了G1捕获,证明HER2也参与大肠癌细胞周期的调节;进行pathway array分析,我们杂交了117种磷酸化和非磷酸化蛋白,共检测到32种蛋白,其中存在两倍以上变化的蛋白有8种。HER2可以上调ERK、P27、XIAP,CyclinD1的表达,抑制p-PKCα/βII、b-catenin的表达。结论:在大肠癌组织中,可以发现有多种蛋白质分子的异常表达。p-PKCα、p-HER2、CyclinD1、P27在癌组织中高表达,ERK、XIAP、p-PKCα/βII、b-catenin在癌旁正常粘膜组织中高表达。HER2参于大肠癌细胞的增殖、周期、凋亡的调节,可以上调ERK、P27、XIAP,CyclinD1的表达,抑制p-PKCα/βII、b-catenin的表达。AG879对6种大肠癌细胞株具有杀伤作用,并且和奥沙利铂联合用药具有协同效应,同时能够减少奥沙利铂的剂量,从而为大肠癌患者手术后化疗个体化进行初步的研究和探索。更多还原

【Abstract】 Purpose:1.By colorectal cancer tissue and adjacent normal tissues pathway array experiment to study the protein expression level differences molecules, screening positive for protein, as a research starting point; 2. Select for positive protein-specific inhibitor AG879 on the 6 kinds of colon cancer cell line treated to study the inhibitor on the tumor cells and tumor signal transduction pathways and cell cycle. 3. Study of conventional chemotherapy drugs and AG879 in combination whether the tumor cell lines with a synergistic effect.Methods:1.collecting 26 patients with colorectal cancer tissue samples, including cancer tissue and adjacent normal tissues to carry out pathway array experiment to study the organization of which the protein molecule expression level; 2, according to pathway array results, select the Her2 as a starting point, in the Sw480, Sw620, Ht29, Hct116, Hct15, Caco2 cell lines in gene expression level, by Western technology, confirmed that expression; 3, the application of small molecule inhibitors AG879 colorectal cancer cell proliferation and the role of conducting pathway array studies; 4, select the IC50 of different cell lines deal with these tumor cell lines after cell cycle analysis, study the role of Her2 in colorectal cancer cell cycle; 5, respectively oxaliplatin and AG879 on the tumor cells for processing, and then combined these two drugs to study the synergy effect.Results:1.26 pairs of samples among each pair of specimens we are all hybrids of the 117 kinds of phosphorylation and non-phosphorylated proteins were detected 32 kinds of proteins, of which there is more than twice the change in the protein are 8, including p-PKCα、p-HER2、CyclinD1、P27 in the colorectal carcinoma was significantly high expression; 2, AG879 in Sw480, Sw620, Ht29, Hct116, Hct15, Caco2 cell lines significantly inhibited the growth effect, it is proof of HER2 can promote colorectal cancer cell proliferation effect; 3, and compared with the control group, the application AG879 cell line appeared in G1 arrest, proof of HER2 are also involved in colorectal cancer cell cycle regulation; 4 to carry out pathway array analysis, we cross the 117 kinds of phosphorylation and non-phosphorylated proteins were detected in 32 species protein, in which there is more than twice the change, there are six kinds of proteins. HER2 can be reduced p-PKCα/βII and b-catenin expression, at the same time can up-regulate ERK、P27、XIAP and CyclinD1.Conclusion:1, in colorectal cancer, we found a variety of abnormal protein expression. p-PKCα, p-HER2, CyclinD1, P27 is highly expressed in cancer tissues, ERK, XIAP, p-PKCα/βII, b-catenin in adjacent normal mucosa epithelium. 2, HER2 Participation in colorectal cancer cell proliferation, cell cycle and apoptosis regulation, can up-regulate ERK, P27, XIAP, CyclinD1 expression, inhibition of p-PKCα/βII, b-catenin expression.3, AG879 on the six kinds of colon cancer cell line has a killing effect, and, and oxaliplatin in combination with synergistic effect, while reducing the dose of oxaliplatin.更多还原