【作者】 张军；

【导师】 赵增仁； 田延锋；

【作者基本信息】 河北医科大学 ， 外科学， 2015， 硕士

【摘要】 目的:结直肠癌是一种严重威胁人类生命健康的消化系统恶性肿瘤之一,统计分析报告显示其在常见癌症中位居第三位,发病率及病死率呈现出日益增高的趋势。结直肠癌是多种因素、多个基因相互作用逐步发展的结果,但其具体的发生发展机制尚不完全明确。Notch-1包含多种表皮生长因子样重复序列组成的细胞外区和多种不同结构域组成的细胞内区,相对分子质量约为30k D,羧基端位于细胞质内,氨基端位于细胞外。它是一个广泛存在于生物进化过程中的高度保守分子,参与细胞信号的传导,调控细胞的增殖、分化、凋亡,并且与肿瘤发生发展有密切关系。本课题组前期研究发现Notch-1在结直肠癌组织过表达,与结直肠癌的发生、发展有着一定的关联。通过实时荧光定量聚合酶链反应(q RT-PCR)检测结直肠癌患者组织中Notch-1的表达,同时用CCK-8法研究不同浓度DAPT对结直肠癌细胞增殖活性凋亡的影响,为深入了解Notch-1在结直肠癌发病机制中发挥的作用提供理论依据。方法:1收集2002年9月~2012年9月40例来自河北医科大学第一医院、河北医科大学第四医院结直肠癌手术标本。每例标本分别取自结直肠原发肿瘤组织及上(下)切缘正常黏膜且术后病理证实无癌组织侵犯的癌旁组织。新鲜标本离体后迅速置于液氮中,于-80℃保存。40名患者术前未接受任何化疗和放疗。2选取结直肠癌细胞株HCT116进行细胞培养。3应用q RT-PCR方法检测40例结直肠癌组织及对应结直肠正常黏膜组织中Notch-1的表达水平,比较两组间表达差异,并分析Notch-1的表达与结直肠癌患者临床病理特征的关系。4对HCT116细胞进行不同浓度DAPT处理,将不同浓度DAPT处理后细胞组作为实验组,将未处理细胞组作为对照组。5 HCT116细胞进行不同浓度的DAPT处理后1、2、3、4d,分别应用CCK-8法检测4个时点的OD值,并绘制出细胞生长曲线,观察对照组和实验组间细胞增殖变化情况。6应用流式细胞技术检测不同浓度DAPT处理后HCT116细胞凋亡情况,比较对照组和实验组之间的差异。7采用SPSS19.0及Graph Pad Prism 5.0统计软件,对实验结果进行分析,计量资料以中位数(四分位数)表示,两组间配对样本采用非参数检验、多组间数据比较采用单因素方差分析,重复测量数据采用双因素方差分析。绘制各临床病理资料的Kaplan-Meier生存曲线,采用时序检验比较两条曲线的差异。生存时间为自手术之日起至其死亡或末次随访日所经历的时间,以月为单位计算。采用Cox回归分析对基因表达和临床参数进行多因素生存分析,分析影响生存的危险因素,以P<0.05表示差异存在有统计学意义。结果:1 Notch-1在40例结直肠癌组织中表达量0.0568(0.0302,0.0757),正常黏膜组织中表达量0.0107(0.0068,0.0231),可见Notch-1在癌组织中的表达明显高于正常黏膜组织中的表达(P<0.0001),差异有统计学意义。2 Notch-1在肿瘤大于等于5cm表达水平为0.0686(0.0508,0.1321)明显高于肿瘤小于5cm表达水平0.0500(0.0286,0.0660)(ss=0.0236);Notch-1表达与淋巴结转移、年龄、性别、浸润深度无明显关系,各指标组间无统计学差异(P>0.05)。3 HCT116细胞不同浓度DAPT处理后Notch-1表达量出现下降。4 HCT116细胞不同浓度DAPT处理后1、2、3、4d,通过CCK-8法检测各组细胞增殖情况,结果显示:10μM DAPT可以明显抑制结直肠癌细胞的增殖,差异具有统计学意义(P<0.0001)。5 HCT116细胞中,实验组10μM早期凋亡率(2.785±0.4070)%明显高于对照组(0.2950±0.1530)%,差异有统计学意义(P=0.0046);实验组5μM早期凋亡率(0.8250±0.1934)%高于对照组(0.2950±0.1530)%,但差异无统计学意义(P=0.0981);实验组1μM早期凋亡率(0.6150±0.1472)%高于对照组(0.2950±0.1530)%,但差异无统计学意义(P=0.2063)。6随访生存率,并行单因素发现结直肠癌患者生存率与淋巴结有无转移(P=0.0002)及肿瘤侵润深度(P=0.0137)有关,与性别、年龄、肿瘤大小、Notch-1表达水平无关(P>0.05)。7多因素Cox生存分析:Cox多因素生存分析结果显示影响结直肠癌患者生存率独立危险因素是淋巴结有无转移(P=0.003),未发现性别、年龄、肿瘤大小、Notch-1表达水平、浸润深度与结直肠癌患者生存率有明显相关性(P>0.05)。结论:1 Notch-1在结直肠癌中高表达,提示其可能对结直肠癌具有癌基因作用。2 Notch-1在大于等于5cm的肿瘤表达水平上调更明显,提示其可能对判定结直肠癌恶性程度和预后有一定的意义。3 Notch-1可能通过促进结直肠癌细胞的增殖,发挥癌基因的作用。更多还原

【Abstract】 Objective : Colorectal carcinoma is one of the digestive system carcinoma which threat the health of human seriously, statistic reports shows that its rank in 3rd in common carcinoma, the morbidity and fatality are raised day by day. Colorectal carcinoma is a result of the interaction between multi-factors and multi-genes. Notch-1 consist of extracellular domain which comprised by a variety of epidermal growth factor repeats and intracellular domain comprised by different structures, relative molecular mass is 30 k D, C-terminal located in cytoplasm and N-terminal outside the cell membrane. It is a highly conserved molecular that exist in biological evolution widely, and participate in cell signal transmission, regulate proliferation, differentiation and apoptosis, and have a close relationship with tumorigenesis. We found that Notch-1 was overexpression in colorectal carcinoma, and had a relationship with tumorigenesis of colorectal carcinoma. In the work, we examined the expression of Notch-1 in tissues by q RT-PCR, and study the influence of different concentration of DAPT to proliferation activity and apoptosis of cancer cells, provide a theoretical foundation for understanding the role of Notch-1 take part in the pathogenesis of colorectal carcinoma.Methods:1 40 cases fresh specimens were collected, included carcinoma tissues and normal mucous tissues. All the specimens were come from the First Hospital of Hebei Medical University and Fourth Hospital of Hebei Medical University between September, 2002 and September 2012. All the patients were not received chemotherapy and radiotherapy before operation.2 HCT116 was selected for cell culture.3 The expression of Notch-1 in 40 cases cancer tissues and correspond normal tissues were detected by q RT-PCR, and analyzed the relationship between the expression of Notch-1 and clinicopathologic feature.4 Cells that treated by different concentration of DAPT served as experimental group, and untreated cells as control group.5 OD values were detected by CCK-8 at different times after the treatment was performed, to observe the proliferation difference between control group and experimental group.6 Flow cytometry was performed in control group and experimental group to compare the difference.7 All the results were analyzed by SPSS19.0 and Graph Pad Prism 5.0, the results were showed by median(quartile), Wilcoxon test was used to analyzed paired samples, One-way ANOVA was used to analyzed multiple groups comparison, Two-way ANOVA was used to analyzed repeated measure data. Kaplan-Meier survival curve was drawn. Survival time was between the operation day and the last follow up, calculated on month. Multi-factor survival analysis was performed by Cox regression analysis. P<0.05 was regarded as statistical significance.Results:1 The expression of Notch-1 in colorectal cancer tissues and normal tissues were 0.0568(0.0302,0.0757) and 0.0107(0.0068,0.0231), respectively. The expression of Notch-1 in cancer tissues was higher than in normal tissues(P<0.0001), the difference was statistically significance.2 The expression of Notch-1 in large tumor group(equal or greater than 5cm) were higher than small group(less than 5cm), the difference was statistically significance(P=0.0236). Notch-1 was independent with lymph nodes metastasis, age, gender, depth of invasion.3 DAPT could down-regulate the expression of Notch-1.4 Cell proliferation was detected by CCK-8 after the treatment of DAPT. The results shown that 10μM DAPT could suppress the cell propagation obviously, the difference was statistically significance(P<0.0001).5 Compared with control group( 0.2950±0.1530) %, 10μM DAPT promoted the early apoptosis rate obviously(2.785±0.4070), the difference was statistically significance(P=0.0046). 5μM and 1μM DAPT could also promote the apoptosis rate, but the difference has no statistically significance.6 Survival rate analysis shown that the survival rate of patients was correlated with lymph nodes metastasis(P=0.0002)and depth of invasion(P=0.0137), the difference was statistically significance.7 Cox multiple factor survival analysis shown that the lymph nodes metastasis was the independent risk factor that impacted the survival rate of colorectal carcinoma patients(P=0.003). The gender, age, tumor volume, Notch-1 level and depth of invasion were no relevance with patient survival rate.Conclusions:1 The expression of Notch-1 is high level in colorectal carcinoma, suggesting that Notch-1 may probably have a potential oncogene activity.2 The expression of Notch-1 is up-regulated even more in the tumor which is equal or greater than 5cm, suggesting that it has significance in judging grade malignancy and prognosis of colorectal carcinoma.3 Notch-1 may play an oncogene activity by promoting the proliferation of cancer cells.4 The survival rate of colorectal carcinoma patients was related to lymph nodes metastasis and tumor invasion depth.更多还原