二甲双胍对人食管癌细胞系KYSE450增殖和凋亡的影响及其机制

发布时间：2018-03-28 00:12

本文选题：二甲双胍　切入点：食管癌　出处：《郑州大学》2015年硕士论文

【摘要】：研究背景:食管癌是世界上常见的恶性肿瘤之一,在我国的发病率和死亡率高达世界第1位,发病世标率第6位,死亡世标率位于第12位。早期食管癌症状轻微,当出现临床表现而就医时,大部分已处于中晚期,失去了手术治疗的机会,而此时化疗成为治疗食管癌重要手段之一。但目前所用的化疗药物不良反应较大,病人耐受性差,且容易产生抗药性.治疗食管癌寻找新的有效的药物是当今研究的热点。研究显示,二甲双胍能抑制结肠癌、乳腺癌、肝癌以及神经胶质瘤等多种肿瘤细胞的生长,Kobayashi等研究显示,二甲双胍可以抑制TT、KYSE30及KYSE70 3种食管癌细胞系的增殖。本实验旨在通过观察不同浓度二甲双胍作用于食管癌细胞系KYSE450,在细胞水平观察药物对食管癌细胞增殖的抑制作用以及凋亡,并在分子基因水平初步探讨产生抑制作用可能的机制。目的:检测食管癌细胞系KYSE450,观察经不同浓度二甲双胍药物刺激,且在同一浓度不同时间作用下,m TOR通路下游蛋白4EBP1和S6K1含量的变化,探讨二甲双胍在食管癌恶性细胞中的过程及在其中的可能的作用及作用机制。方法:体外常规培养KYSE450细胞,分为对照组(不使用药物刺激),不同浓度实验组(5μmol/L、10μmol/L、20μmol/L、40μmol/L,采用MTT法检测不同浓度的二甲双胍作用不同时间对食管癌细胞增殖的抑制情况,并使用流式细胞仪器检测不同分组凋亡率的差异;同时在分子水平采用RT-PCR以及Western blot技术检测药物作用后KYSE450细胞中4EBP1和S6K1的m RNA和蛋白的表达的变化。结果:1.不同浓度二甲双胍对食管癌KYSE450细胞的抑制作用:MTT法实验结果显示,不同浓度二甲双胍对食管癌KYSE450细胞的增殖均有抑制作用,并且随着二甲双胍浓度的提高以及二甲双胍处理时间的延长,抑制率逐渐上升,抑制作用逐渐增强。这一结果表明在一定浓度和时间范围内,二甲双胍对细胞产生的抑制作用呈现时间-剂量依赖性,差异均存在统计学意义(P0.05)。2.不同浓度二甲双胍对食管癌KYSE450细胞的凋亡的影响流式细胞仪检测结果显示,5,10,20m M的二甲双胍作用于食管癌KYSE450细胞的48h后,细胞凋亡率分别为(20.8±1.35)%、(30.1±1.56)%、(39.7±1.44)%,而不加二甲双胍空白对照组细胞凋亡率为(5.8±1.92)%,结果表明,随着二甲双胍药物浓度的提高,食管癌KYSE450凋亡率也在逐渐上升,实验组与空白对照组比较,差异有统计学意义(P0.05)。3.不同浓度二甲双胍对食管癌KYSE450细胞4EBP1,S6K1 m RNA表达的影响。RT-PCR结果显示,与对照组比较,不同浓度二甲双胍作用后4EBP1和S6K1m RNA表达水平明显下降(P0.05),并且与二甲双胍的药物浓度相关。4.二甲双胍单药对食管癌KYSE450细胞作用后4ebp1,S6K1蛋白表达的变化Western-blot检测结果显示。与对照组比较,不同浓度二甲双胍作用48h后,4EBP1,S6K1蛋白表达水平明显下降(P0.05)。结论:1.二甲双胍能够抑制人食管癌KYSE450细胞增殖并诱导凋亡2.其机制可能与4EBP1和S6K1基因和蛋白表达下调有关。
[Abstract]:Background: esophageal cancer is one of the most common malignancies in the world, in our country, the incidence and mortality rate as high as first in the world, the incidence of the world standard rate of sixth, the standard rate is twelfth deaths. Early esophageal cancer with mild symptoms, when clinical manifestations and medical treatment, most have been lost in the late. The chance of surgery, while chemotherapy has become an important means of treatment of esophageal cancer. But the chemotherapy adverse drug reaction is currently used in the larger, patients of poor tolerance, and easy to produce drug resistance. For the treatment of esophageal cancer is the focus of current research and effective new drugs. Studies show that metformin can inhibit colon cancer, breast cancer, liver cancer and glioma tumor cell growth, Kobayashi research showed that metformin can inhibit TT, KYSE30 and KYSE70 in 3 esophageal carcinoma cell line proliferation. This study is to observe the different The concentration of metformin in human esophageal cancer cell line KYSE450, in the inhibition of cellular level effects on the proliferation of esophageal cancer cells and apoptosis, and the preliminary mechanism of inhibition may study at the molecular level. Objective: to detect the esophageal cancer cell line KYSE450, to observe the different concentration of metformin drug stimulation, and at the same concentration at different time under the effect of changes of M TOR pathway downstream protein 4EBP1 and S6K1 content, to explore the process of metformin in malignant cells in esophageal carcinoma and the possible effect and mechanism of them. Methods: conventional in vitro cultured KYSE450 cells were divided into control group (without the use of drugs, stimulation) groups with different concentrations (5 mol/L, 10 mol/L, 20 mol/L, 40 mol/L, MTT was used to detect different concentrations of metformin effects on esophageal cancer cell proliferation inhibition, and using flow cytometry instrument To detect the apoptosis rate of different groups; at the same time at the molecular level by RT-PCR and Western blot m RNA and the detection of drug action of protein 4EBP1 and S6K1 in KYSE450 cells after the expression changes. Results: 1. different concentrations of metformin on human esophageal cancer KYSE450 cells inhibited by MTT method: the experimental results showed that the inhibitory effect of different concentration metformin has on human esophageal cancer KYSE450 cells proliferation, and increased with the increase of the concentration of metformin and metformin treatment time, the inhibition rate increased gradually, the inhibition gradually increased. The results show that in a certain range of concentration and time, effect of metformin on the cells showed time dose dependent, the differences were statistically significant (P0.05) test results showed that the effects of different concentrations of metformin on.2. apoptosis of esophageal cancer cell line KYSE450 by flow cytometry, 5,10 20m M, metformin in KYSE450 esophageal carcinoma cell 48h, cell apoptosis rates were (20.8 + 1.35)% and (30.1 + 1.56)% and (39.7 + 1.44)%, without metformin in blank control group apoptosis rate was (5.8 + 1.92)%. The results show that with two a biguanide drug concentration increased, the apoptosis of esophageal cancer KYSE450 rate is also rising, the experimental group compared with control group, the difference was statistically significant (P0.05.3.) with different concentrations of metformin on esophageal carcinoma cell line KYSE450 4EBP1,.RT-PCR S6K1 m influence RNA expression results showed that compared with the control group, the expression of RNA 4EBP1 and S6K1m different concentration levels were significantly decreased after metformin (P0.05), and the concentration of.4. metformin single drug effect on esophageal carcinoma KYSE450 cells after 4ebp1, Western-blot to detect changes in the expression of S6K1 protein. Compared with control group, different concentration After metformin treatment, the expression level of 4EBP1 and S6K1 protein decreased significantly (P0.05). Conclusion: 1. metformin can inhibit the proliferation and induce apoptosis of human esophageal cancer KYSE450 cells 2., the mechanism may be related to the downregulation of 4EBP1 and S6K1 gene and protein expression, 48h.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.1

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