本文選題：尼克酰胺N-甲基轉(zhuǎn)移酶　切入點：1-MNA　出處：《浙江大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：背景大腸癌是人類最常見的惡性腫瘤之一,在美國,2014年的報告顯示預(yù)計有136,830例新發(fā)病例和50,310例死亡病例,在中國,大腸癌的發(fā)病率已躍居第四位。尼克酰胺N-甲基轉(zhuǎn)移酶是參與體內(nèi)許多藥物及異源性物質(zhì)生物轉(zhuǎn)化和解毒的重要酶。研究發(fā)現(xiàn)其在多種腫瘤中過表達(dá),如大腸癌、腎癌、胃癌、乳頭狀甲狀腺癌、乳腺癌等,高表達(dá)的NNMT與腫瘤細(xì)胞的遷移和腫瘤分級正相關(guān),與細(xì)胞分化差和預(yù)后差有關(guān),本課題組之前的研究發(fā)現(xiàn)在大腸癌中NNMT可抑制細(xì)胞凋亡、加速細(xì)胞周期進(jìn)程而促進(jìn)細(xì)胞增殖,然而有關(guān)NNMT作為一種代謝酶是如何抑制細(xì)胞凋亡和加速細(xì)胞周期進(jìn)程而促進(jìn)細(xì)胞增殖的機制還不是很清楚,為此對這一機制做了研究。目的研究NNMT促進(jìn)大腸癌細(xì)胞增殖的機制。方法1.在SW480上構(gòu)建NNMT高表達(dá)細(xì)胞模型(SW480細(xì)胞內(nèi)源性低表達(dá)NNMT基因)和在HT29上構(gòu)建NNMT表達(dá)下調(diào)細(xì)胞模型(HT29細(xì)胞高表達(dá)NNMT基因)2.采用Real-Time PCR和Western-Blot檢測目的分子的表達(dá)量。3.采用MTT法、平板克隆形成及軟瓊脂集落形成研究細(xì)胞增殖情況。4.采用高效液相色譜法檢測1-MNA的水平及NNMT酶活性。5.采用流式細(xì)胞術(shù)檢測細(xì)胞周期、細(xì)胞凋亡、細(xì)胞內(nèi)ROS水平。6.分別采用ATP、ADP/ATP、NAD+/NADH檢測試劑盒檢測其水平。結(jié)果1.NNMT高表達(dá)的SW480/NNMT-1和SW480/NNMT-2細(xì)胞內(nèi)1-MNA的水平升高；NNMT表達(dá)下調(diào)的HT29/NNMTshRNA1#和HT29/NNMTshRNA2#細(xì)胞內(nèi)1-MNA的水平下降。2.1-MNA刺激SW480細(xì)胞后細(xì)胞生長曲線右移、平板克隆形成率和軟瓊脂集落形成率增加。3.1-MNA刺激SW480細(xì)胞后早期凋亡比例增加,S期比例增加。4.1-MNA刺激SW480細(xì)胞后,細(xì)胞內(nèi)ROS的水平降低。5. NNMT過表達(dá)的SW480/NNMT-1和SW480/NNMT-2細(xì)胞內(nèi)ATP水平升高,NNMT表達(dá)下調(diào)的HT29/NNMTshRNA1#和HT29/NNMTshRNA2##細(xì)胞內(nèi)ATP水平下降。6.1-MNA刺激SW480細(xì)胞后,細(xì)胞內(nèi)ATP的水平升高。結(jié)論1.NNMT通過增加大腸癌細(xì)胞內(nèi)1-MNA的水平導(dǎo)致細(xì)胞內(nèi)ROS的水平下降,從而抑制細(xì)胞凋亡和加速細(xì)胞周期進(jìn)程。2. NNMT經(jīng)1-MNA增加了大腸癌細(xì)胞內(nèi)ATP的水平,在能量代謝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.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R735.34

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