NNMT经1-MNA促进大肠癌细胞增殖及其机制研究

发布时间：2018-03-15 04:36

本文选题：尼克酰胺N-甲基转移酶　切入点：1-MNA　出处：《浙江大学》2016年硕士论文　论文类型：学位论文

【摘要】：背景大肠癌是人类最常见的恶性肿瘤之一,在美国,2014年的报告显示预计有136,830例新发病例和50,310例死亡病例,在中国,大肠癌的发病率已跃居第四位。尼克酰胺N-甲基转移酶是参与体内许多药物及异源性物质生物转化和解毒的重要酶。研究发现其在多种肿瘤中过表达,如大肠癌、肾癌、胃癌、乳头状甲状腺癌、乳腺癌等,高表达的NNMT与肿瘤细胞的迁移和肿瘤分级正相关,与细胞分化差和预后差有关,本课题组之前的研究发现在大肠癌中NNMT可抑制细胞凋亡、加速细胞周期进程而促进细胞增殖,然而有关NNMT作为一种代谢酶是如何抑制细胞凋亡和加速细胞周期进程而促进细胞增殖的机制还不是很清楚,为此对这一机制做了研究。目的研究NNMT促进大肠癌细胞增殖的机制。方法1.在SW480上构建NNMT高表达细胞模型(SW480细胞内源性低表达NNMT基因)和在HT29上构建NNMT表达下调细胞模型(HT29细胞高表达NNMT基因)2.采用Real-Time PCR和Western-Blot检测目的分子的表达量。3.采用MTT法、平板克隆形成及软琼脂集落形成研究细胞增殖情况。4.采用高效液相色谱法检测1-MNA的水平及NNMT酶活性。5.采用流式细胞术检测细胞周期、细胞凋亡、细胞内ROS水平。6.分别采用ATP、ADP/ATP、NAD+/NADH检测试剂盒检测其水平。结果1.NNMT高表达的SW480/NNMT-1和SW480/NNMT-2细胞内1-MNA的水平升高；NNMT表达下调的HT29/NNMTshRNA1#和HT29/NNMTshRNA2#细胞内1-MNA的水平下降。2.1-MNA刺激SW480细胞后细胞生长曲线右移、平板克隆形成率和软琼脂集落形成率增加。3.1-MNA刺激SW480细胞后早期凋亡比例增加,S期比例增加。4.1-MNA刺激SW480细胞后,细胞内ROS的水平降低。5. NNMT过表达的SW480/NNMT-1和SW480/NNMT-2细胞内ATP水平升高,NNMT表达下调的HT29/NNMTshRNA1#和HT29/NNMTshRNA2##细胞内ATP水平下降。6.1-MNA刺激SW480细胞后,细胞内ATP的水平升高。结论1.NNMT通过增加大肠癌细胞内1-MNA的水平导致细胞内ROS的水平下降,从而抑制细胞凋亡和加速细胞周期进程。2. NNMT经1-MNA增加了大肠癌细胞内ATP的水平,在能量代谢平衡方面有重要作用。
[Abstract]:Background colorectal cancer is one of the most common human malignant tumors in the United States, according to a 2014 report estimated 136830 new cases and 50310 death cases, in Chinese, the incidence of colorectal cancer has been ranked fourth. Nicotinamide N- methyltransferase is an important enzyme in the body of many drugs and biotransformation and heterologous materials detoxification. Study found that it is overexpressed in many tumors, such as colorectal cancer, renal cell carcinoma, gastric carcinoma, papillary thyroid carcinoma, breast cancer, migration and tumor grade high expression of NNMT with tumor cells is related with cell differentiation and poor prognosis, the research group previously found in colorectal cancer NNMT can inhibit apoptosis, accelerate cell cycle and promotes cell proliferation, but the NNMT as a metabolic enzyme is the mechanism of inhibiting apoptosis and accelerating cell cycle progression and promotes cell proliferation. Is not very clear, for this mechanism to do the research. Objective to study the mechanism of NNMT promoting proliferation of colorectal cancer cells. Methods SW480 1. on the construction of NNMT high expression cell model (SW480 cell endogenous NNMT gene low expression) and construct the down-regulation of NNMT expression cell model in HT29 (high expression of NNMT gene in HT29 cells) 2. the expression of.3. Real-Time and Western-Blot PCR molecular detection using MTT method, plate clone formation and soft agar colony formation assay and cell cycle by flow cytometry, and NNMT enzyme activity of.5. on the proliferation of.4. cells was determined by high performance liquid chromatography 1-MNA. Apoptosis, ROS level in.6. cells respectively. Using ATP, ADP/ATP, NAD+/NADH detection kit. The results increase the high expression of 1.NNMT SW480/NNMT-1 and SW480/NNMT-2 1-MNA in cell level; down regulated expression of NNMT HT29/ NNMTshRNA1# The cell growth curve..2.1-MNA decreased after the stimulation of SW480 cells and HT29/NNMTshRNA2# cells in the 1-MNA level, colony formation rate and colony formation rate increased after.3.1-MNA stimulation of SW480 cells early apoptosis ratio increased, the percentage of S phase increased.4.1-MNA stimulated SW480 cells, decreased the intracellular ROS level of.5. NNMT expression of ATP SW480/NNMT-1 and SW480/NNMT-2 cells increased, NNMT expression decreased levels of ATP HT29/NNMTshRNA1# and HT29/NNMTshRNA2##.6.1-MNA cells decreased after SW480 cells were stimulated and increased intracellular ATP levels. Conclusion 1.NNMT can increase the level of 1-MNA in colorectal cancer cells resulted in intracellular ROS levels decreased, thus inhibiting apoptosis and accelerating cell cycle progression of.2. NNMT increased by 1-MNA the level of ATP in colorectal carcinoma cells, plays an important role in energy metabolism.

【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.34

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