二甲双胍增加食管鳞癌对顺铂化疗敏感性的机制研究

发布时间：2018-05-06 08:00

本文选题：食管鳞癌细胞 + 二甲双胍(Metformin、Met)　；参考：《新乡医学院》2015年硕士论文

【摘要】：背景二甲双胍(Metformin、Met)作为临床常用的一线降血糖药物,不良反应小,安全性好,对正常人无明显降血糖作用。最近大量研究发现二甲双胍可以抑制多种肿瘤细胞的生长,也有研究发现二甲双胍可以增加一些放化疗药物的敏感性,其抗肿瘤机制大多是通过激活与腺苷酸活化蛋白激酶(adenosine monophosphate activated protein kinase、AMPK)相关的信号通路,从而抑制蛋白质的翻译和诱导细胞的凋亡。而二甲双胍在抗食管癌方面鲜少研究。目的探讨二甲双胍对食管鳞癌EC-109细胞的增殖抑制作用及对联合顺铂(DDP、 cisplatin)化疗的增敏作用,并初步探讨其分子机制,为二甲双胍联合顺铂治疗食管癌提供理论依据。方法1.食管鳞癌EC-109细胞分为四组：空白对照组(A)、Met组(B)、DDP组(C)、Met+DDP组(D),其中B组设有5个浓度组(B-a 5 mmol/L、B-b 10 mmol/L、B-c 20 mmol/L、B-d 40 mmol/L、B-e 80 mmol/L),C组设有2个浓度组(C-a 4.17 μmol/L、C-b 8.33μmol/L),D组Met浓度均为5 mmol/L(联合a组、联合b组)。2.四甲基偶氮唑蓝法(MTT)测定不同时间段(24 h、48 h、72 h)的细胞增殖抑制率,并计算二甲双胍作用的IC50值,选用5 mmol/L的二甲双胍浓度作为后续实验的浓度。3.流式细胞仪检测各实验组作用48 h后对细胞周期的影响。4. Real Time-PCR法测定各组细胞作用48 h后的p21和JNK1的RNA表达情况；Western Blot法检测各买验组细胞作用48 h后的p21和JNK1的蛋白表达情况。(其中第3、4步实验中的分组为：A组、B-a组、B-c组、C-a组、联合a组,分别另命名为Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ组)结果1.MTT结果显示：B组,在同一时间点,不同浓度的二甲双胍对细胞的增殖抑制率均显著高于空白对照组(P0.05),且呈浓度依赖性(P0.05)；在二甲双胍各浓度组内,作用48 h、72 h后的细胞增殖抑制率均高于作用24 h后(P0.05),作用72 h后的细胞增殖抑制率均高于作用24 h、48 h后(P0.05)。联合a组和联合b组作用24 h、48 h、72 h后对细胞的增殖抑制率均高于同时间段5 mmol/L·Met组和单药顺铂组(P0.05)；同一时间点联合a组与联合b组比较有统计学差异(P0.05)。2.流式细胞仪检测细胞周期,结果显示二甲双胍联合或不联合顺铂可诱导EC-109细胞周期阻滞在G1、S期,与对照组相比差异具有统计学意义(P0.05)。3. Real Time-PCR结果显示：以GAPDH为内参,与Ⅰ组比较,Ⅱ、Ⅲ、Ⅳ、V组的p21基因表达明显上调,且Ⅴ组的p21基因表达上调较Ⅱ、Ⅲ、Ⅳ组明显(P0.05)；与Ⅰ组比较,Ⅱ、Ⅲ、Ⅳ组JNK1基因表达上调,但Ⅴ组的JNK1蛋白表达明显下调(P0.05)。4.Western Blot结果显示：与Ⅰ组比较,Ⅱ、Ⅲ、Ⅳ、Ⅴ组的p21蛋白表达明显上调,且V组的p21蛋白表达上调较Ⅱ、Ⅲ、Ⅳ组明显(P0.05)；与Ⅰ组比较,Ⅱ、Ⅳ组JNK1蛋白表达上调,但Ⅲ、Ⅴ组的JNK1蛋白表达下调(P0.05)。结论二甲双胍对食管鳞癌EC-109细胞的增殖具有抑制作用,且呈时间浓度依赖性；二甲双胍可以增加EC-109细胞对顺铂化疗的敏感性；二甲双胍联合或不联合顺铂作用于EC-109细胞后,细胞周期大部分阻滞在G1、S期；二甲双胍对EC-109细胞发挥顺铂化疗增敏作用,其机制是通过p21/JNK1信号通路起作用。
[Abstract]:Background Metformin (Met), as a clinically common front-line hypoglycemic drug, has small side effects and good safety. There is no obvious hypoglycemic effect on normal people. Recently, a large number of studies have found that metformin can inhibit the growth of various tumor cells. It is also found that two metformin can increase the sensitivity of some chemoradiotherapy drugs. Most of the antitumor mechanisms are by activating the signaling pathway associated with adenosine monophosphate activated protein kinase (AMPK), which inhibits the translation of protein and induces cell apoptosis. Metformin is rarely studied in the anti esophageal cancer. Objective to explore the effect of metformin on the EC-109 cells of esophageal squamous cell carcinoma. The inhibitory effect of proliferation and the sensitizing effect on DDP (cisplatin) chemotherapy and its molecular mechanism were preliminarily discussed. Methods 1. EC-109 cells were divided into four groups: blank control group (A), Met group (B), DDP group (C) and Met+DDP group (D), of which 5 concentrations were set in B group. B-a 5 mmol/L, B-b 10 mmol/L, B-c 20 mmol/L, B-d 40 mmol/L, B-e 80 mmol/L), C group with 2 concentration groups (C-a 4.17 mu, 8.33 micron). The IC50 value of 5 mmol/L was selected as the concentration of metformin as a follow-up test. The effect of the concentration of.3. flow cytometry on the cell cycle of each experimental group was 48 h, and the.4. Real Time-PCR method was used to determine the RNA expression of p21 and JNK1 after the action of 48 h in each group. Protein expression in 3,4 step experiment: group A, group B-a, B-c group, C-a group, combined a group, and respectively named as I, II, III, IV, V group) results, 1.MTT results showed: B group, at the same time point, the proliferation inhibition rate of different concentrations of metformin on cells was significantly higher than that of the blank control group (P0.05), and showed a concentration dependence. In each concentration group of metformin, the inhibitory rate of cell proliferation after 48 h and 72 h was higher than that of 24 h (P0.05). The inhibitory rate of cell proliferation after the action of 72 h was higher than that of 24 h and 48 h (P0.05). The combined a group and the combined B group were 24 h, 48, 72, and the proliferation inhibition rate of the cells was higher than that of 5 intervals. And single drug cisplatin group (P0.05), the same time point combined with a group and combined B group had statistical difference (P0.05).2. flow cytometry to detect cell cycle. The results showed that metformin combined with or not combined with cisplatin could induce EC-109 cell cycle arrest in G1, S phase, and the difference was statistically significant (P0.05).3. Real Time-PCR knot than the control group. The results showed that the expression of p21 gene in group II, III, IV, V was obviously up regulated with GAPDH as internal reference, and the up regulation of p21 gene expression in group V was significantly higher than that in group I, III and IV (P0.05). The expression of JNK1 gene in group II, III and IV was up regulated compared with group I, but the expression of JNK1 protein expression in group V (P0.05).4.Western Blot showed: and group I showed that: and group I The expression of p21 protein in group II, III, IV and V was obviously up-regulated, and the up regulation of p21 protein expression in group V was significantly higher than that in group II, III and IV (P0.05). Compared with group I, the expression of JNK1 protein in group II and IV was up regulated, but the expression of JNK1 protein in group III and V was down regulated (P0.05). Conclusion two metformin has a inhibitory effect on the proliferation of EC-109 cells in esophageal squamous cell carcinoma and is present. Concentration dependence; metformin can increase the sensitivity of EC-109 cells to cisplatin chemotherapy; metformin combined or not combined with cisplatin acting on EC-109 cells, the cell cycle is mostly blocked at G1, S, and metformin exerts cisplatin chemosensitization to EC-109 cells, and its mechanism is mediated by the p21/JNK1 signaling pathway.

【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.1

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