本文選題：食管鱗癌細(xì)胞 + 二甲雙胍(Metformin、Met)��； 參考：《新鄉(xiāng)醫(yī)學(xué)院》2015年碩士論文

【摘要】：背景二甲雙胍(Metformin、Met)作為臨床常用的一線降血糖藥物,不良反應(yīng)小,安全性好,對正常人無明顯降血糖作用。最近大量研究發(fā)現(xiàn)二甲雙胍可以抑制多種腫瘤細(xì)胞的生長,也有研究發(fā)現(xiàn)二甲雙胍可以增加一些放化療藥物的敏感性,其抗腫瘤機(jī)制大多是通過激活與腺苷酸活化蛋白激酶(adenosine monophosphate activated protein kinase、AMPK)相關(guān)的信號(hào)通路,從而抑制蛋白質(zhì)的翻譯和誘導(dǎo)細(xì)胞的凋亡。而二甲雙胍在抗食管癌方面鮮少研究。目的探討二甲雙胍對食管鱗癌EC-109細(xì)胞的增殖抑制作用及對聯(lián)合順鉑(DDP、 cisplatin)化療的增敏作用,并初步探討其分子機(jī)制,為二甲雙胍聯(lián)合順鉑治療食管癌提供理論依據(jù)。方法1.食管鱗癌EC-109細(xì)胞分為四組：空白對照組(A)、Met組(B)、DDP組(C)、Met+DDP組(D),其中B組設(shè)有5個(gè)濃度組(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組設(shè)有2個(gè)濃度組(C-a 4.17 μmol/L、C-b 8.33μmol/L),D組Met濃度均為5 mmol/L(聯(lián)合a組、聯(lián)合b組)。2.四甲基偶氮唑藍(lán)法(MTT)測定不同時(shí)間段(24 h、48 h、72 h)的細(xì)胞增殖抑制率,并計(jì)算二甲雙胍作用的IC50值,選用5 mmol/L的二甲雙胍濃度作為后續(xù)實(shí)驗(yàn)的濃度。3.流式細(xì)胞儀檢測各實(shí)驗(yàn)組作用48 h后對細(xì)胞周期的影響。4. Real Time-PCR法測定各組細(xì)胞作用48 h后的p21和JNK1的RNA表達(dá)情況；Western Blot法檢測各買驗(yàn)組細(xì)胞作用48 h后的p21和JNK1的蛋白表達(dá)情況。(其中第3、4步實(shí)驗(yàn)中的分組為：A組、B-a組、B-c組、C-a組、聯(lián)合a組,分別另命名為Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ組)結(jié)果1.MTT結(jié)果顯示：B組,在同一時(shí)間點(diǎn),不同濃度的二甲雙胍對細(xì)胞的增殖抑制率均顯著高于空白對照組(P0.05),且呈濃度依賴性(P0.05)；在二甲雙胍各濃度組內(nèi),作用48 h、72 h后的細(xì)胞增殖抑制率均高于作用24 h后(P0.05),作用72 h后的細(xì)胞增殖抑制率均高于作用24 h、48 h后(P0.05)。聯(lián)合a組和聯(lián)合b組作用24 h、48 h、72 h后對細(xì)胞的增殖抑制率均高于同時(shí)間段5 mmol/L·Met組和單藥順鉑組(P0.05)；同一時(shí)間點(diǎn)聯(lián)合a組與聯(lián)合b組比較有統(tǒng)計(jì)學(xué)差異(P0.05)。2.流式細(xì)胞儀檢測細(xì)胞周期,結(jié)果顯示二甲雙胍聯(lián)合或不聯(lián)合順鉑可誘導(dǎo)EC-109細(xì)胞周期阻滯在G1、S期,與對照組相比差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。3. Real Time-PCR結(jié)果顯示：以GAPDH為內(nèi)參,與Ⅰ組比較,Ⅱ、Ⅲ、Ⅳ、V組的p21基因表達(dá)明顯上調(diào),且Ⅴ組的p21基因表達(dá)上調(diào)較Ⅱ、Ⅲ、Ⅳ組明顯(P0.05)；與Ⅰ組比較,Ⅱ、Ⅲ、Ⅳ組JNK1基因表達(dá)上調(diào),但Ⅴ組的JNK1蛋白表達(dá)明顯下調(diào)(P0.05)。4.Western Blot結(jié)果顯示：與Ⅰ組比較,Ⅱ、Ⅲ、Ⅳ、Ⅴ組的p21蛋白表達(dá)明顯上調(diào),且V組的p21蛋白表達(dá)上調(diào)較Ⅱ、Ⅲ、Ⅳ組明顯(P0.05)；與Ⅰ組比較,Ⅱ、Ⅳ組JNK1蛋白表達(dá)上調(diào),但Ⅲ、Ⅴ組的JNK1蛋白表達(dá)下調(diào)(P0.05)。結(jié)論二甲雙胍對食管鱗癌EC-109細(xì)胞的增殖具有抑制作用,且呈時(shí)間濃度依賴性；二甲雙胍可以增加EC-109細(xì)胞對順鉑化療的敏感性；二甲雙胍聯(lián)合或不聯(lián)合順鉑作用于EC-109細(xì)胞后,細(xì)胞周期大部分阻滯在G1、S期；二甲雙胍對EC-109細(xì)胞發(fā)揮順鉑化療增敏作用,其機(jī)制是通過p21/JNK1信號(hào)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.

【學(xué)位授予單位】：新鄉(xiāng)醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R735.1

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本文編號(hào)：1851459