小檗碱下调Nrf2活性抑制高糖诱导的甲状腺乳头状癌K1细胞增殖的研究

发布时间：2018-02-27 03:06

  本文关键词： 甲状腺乳头状癌 高糖 Nrf2 小檗碱 PI3K/Akt信号通路　出处：《湖北中医药大学》2017年硕士论文　论文类型：学位论文

【摘要】：第一章高糖对甲状腺乳头状癌K1细胞增殖及PI3K/Akt/Nrf2信号通路的影响研究目的观察不同浓度葡萄糖对甲状腺乳头状癌K1细胞增殖的促进作用;初步探讨高糖对甲状腺乳头状癌K1细胞核因子-E2相关因子2(nuclear factor erythroid 2-related factor 2,Nrf2)蛋白表达及核转位的影响并研究其可能的上游信号通路。研究方法1.以不同浓度葡萄糖(5.5、10、25、50)mmol/L干预K1细胞,分别于12、24、36、48h采用MTT比色分析法检测各组K1细胞增殖率,同时设相同渗透压组作为对照。2.根据MTT结果,将甲状腺乳头状癌K1细胞分为正糖组(5.5mmol/L葡萄糖)、高糖组(25mmol/L葡萄糖)和高渗组(25mmol/L甘露醇),各组均分别于0、12、24、48h提取细胞,采用Western Blot法检测Nrf2、磷脂酰肌醇-3激酶(phosphoinositide 3-kinase,PI3K)、蛋白激酶B(protein kinase B,Akt)和磷酸化Akt(phosphorylated-Akt,p-Akt)表达水平;采用细胞免疫荧光法分析各组Nrf2在细胞内的分布情况;使用Spearman相关性分析法检测高糖作用下K1细胞增殖分别与Nrf2、PI3K蛋白和p-Akt/Akt的相关性。研究结果1.随葡萄糖浓度升高,K1细胞增殖率改变,25mmol/L葡萄糖浓度下K1细胞增殖达到峰值(148.76±5.19)%,50mmol/l葡萄糖浓度时K1细胞增殖率下降(84.72±3.26)%,与5.5mmol/l葡萄糖浓度时K1细胞增殖率比较均有统计学差异(p0.01)。2.同一葡萄糖浓度作用下随时间延长K1细胞增殖率发生改变,12h明显升高,24h达峰,随后下降。3.25mmol/L葡萄糖干预K1细胞24h时,K1细胞内Nrf2、PI3K和p-Akt/Akt表达及Nrf2在细胞核分布的比例(Nrf2:0.869±0.075、PI3K:0.783±0.020、p-Akt/Akt:0.480±0.048、Nrf2细胞核比例:91.2%,260/285)均达峰值,并且它们随时间的变化均与K1细胞增殖率变化同步。4.Spearman相关性分析表明高糖组Nrf2、PI3K及p-Akt/Akt表达均与K1细胞增殖率呈正相关,相关系数r分别为0.908、0.910、0.916,p0.01。5.渗透压升高对K1细胞增殖及Nrf2、PI3K蛋白表达、p-Akt/Akt等均无明显影响(p0.05)。研究结论1.甲状腺乳头状癌K1细胞增殖率随葡萄糖浓度和作用时间的变化而改变,25mmo/l葡萄糖浓度作用24h时,K1细胞增殖率达高峰。2.高糖可能通过促进Nrf2表达及核转位,从而促进甲状腺乳头状癌K1细胞增殖,这可能与高糖上调K1细胞内PI3K/Akt信号通路有关。第二章小檗碱抑制高糖诱导的甲状腺乳头状癌K1细胞增殖及可能的机制研究目的观察小檗碱对高糖诱导的甲状腺乳头状癌K1细胞增殖的抑制作用;探讨小檗碱对高糖诱导的甲状腺乳头状癌K1细胞Nrf2蛋白表达及核转位的影响,并初步探究其发挥作用的可能信号通路,以期为早期防治糖尿病合并甲状腺乳头状癌治疗提供新的思路和方法。研究方法1.高糖(25mmol/L)加入不同浓度的小檗碱(0、10、40、80)μmol/L共同干预K1细胞24h时,采用MTT比色分析法检测各组K1细胞增殖情况,设正糖(5.5mmol/L)作为对照。2.采用Western blot检测各组Nrf2、PI3K、Akt、p-Akt表达水平,细胞免疫荧光法分析各组Nrf2在细胞内的分布,设正糖(5.5mmol/L)作为对照。研究结果1.与正糖组比较,高糖组K1细胞增殖率、PI3K、p-Akt/Akt、Nrf2表达及Nrf2在细胞核分布的比例均显著升高(增殖率:(126.64±5.41)%vs(87.31±3.67)%;PI3K:(0.425±0.019)vs(0.272±0.039);p-Akt/Akt:(0.446±0.021)vs(0.168±0.035);Nrf2:(0.597±0.014)vs(0.308±0.026);Nrf2细胞核比例:(93.0%,265/285)vs(23.1%,45/195),p0.01)。2.与高糖组比较,高糖加入10、40、80μmol/L小檗碱后各组K1细胞增殖率((111.76±4.10)%、(70.03±2.18)%、(32.41±3.76)%)均下降(p0.05),80μmol/L小檗碱组增殖抑制作用最强,同时40μmol/L小檗碱组增殖抑制作用高于10μmol/L小檗碱组。3.高糖加入不同浓度小檗碱后各组PI3K、p-Akt/Akt、Nrf2表达及Nrf2在细胞核分布的比例,与高糖组比较均下调(p0.05),且其变化均随小檗碱的浓度升高而更加显著。研究结论小檗碱呈浓度依赖性抑制高糖诱导的K1细胞增殖,这可能与小檗碱抑制高糖诱导的PI3K/Akt信号通路过度激活从而下调Nrf2蛋白表达及核转位有关。
[Abstract]:Chapter 1 Effects of high glucose on papillary thyroid carcinoma K1 cell proliferation and PI3K/Akt/Nrf2 signaling pathway Objective: To observe the effect of different concentrations of glucose on the proliferation of K1 cells of papillary thyroid carcinoma; preliminary study on high glucose related factors on the nuclear factor -E2 in papillary thyroid carcinoma (K1 2 nuclear factor erythroid 2-related factor 2, Nrf2) expression and nuclear translocation of protein and to study its possible upstream signaling pathways. 1. research methods with different concentrations of glucose (5.5,10,25,50) mmol/L on K1 cells, respectively in 12,24,36,48h using MTT colorimetric analysis method was used to detect the proliferation rate of K1 cells in each group, and the same osmotic pressure as control group.2. according to MTT results, the papillary thyroid carcinoma K1 cells divided into normal glucose group (5.5mmol/L glucose), high glucose group (25mmol/L glucose) and hypertonic group (25mmol/L mannitol), were equally different from 0,12,24,48h. Take a cell, using Western Blot method to detect Nrf2, phosphatidylinositol -3 kinase (phosphoinositide, 3-kinase, PI3K), protein kinase B (protein kinase B, Akt) and phosphorylated Akt (phosphorylated-Akt, p-Akt) expression level; analysis of distribution of each Nrf2 in the cell by cell immunofluorescence analysis; K1 cell proliferation method under the action of high glucose and Nrf2 were detected using Spearman correlation, the correlation between PI3K protein and p-Akt/Akt. Results: 1. when the glucose concentration increased, the rate of change of the proliferation of K1 cells, the proliferation of K1 cells in 25mmol/L glucose peak (148.76 + 5.19)%, 50mmol/l glucose concentration decreased the proliferation rate of K1 cells (84.72 + 3.26)%. And there were statistically significant differences in K1 cell proliferation rate of 5.5mmol/l glucose concentration (P0.01) of.2. with a glucose concentration with time under the action of the K1 cell proliferation rate changed, 12h increased significantly, 24h The peak, then decreased.3.25mmol/L glucose intervention K1 cells 24h, K1 cells in Nrf2, PI3K and p-Akt/Akt and Nrf2 expression in nucleus distribution ratio (Nrf2:0.869 + 0.075, PI3K:0.783 + 0.020, p-Akt/Akt:0.480 + 0.048, Nrf2: 91.2%, the proportion of nucleus 260/285) Jun Dafeng value, and they change with time and the proliferation of K1 cells the rate of change of synchronous.4.Spearman correlation analysis showed that high glucose group Nrf2, PI3K and p-Akt/Akt expression and K1 cell proliferation rate was positively correlated, the correlation coefficient r was 0.908,0.910,0.916 p0.01.5., the increase of osmotic pressure on the proliferation of K1 cells and the expression of PI3K protein, Nrf2, had no effect on p-Akt/Akt (P0.05). The conclusion of the study of papillary carcinoma K1 cell proliferation 1. thyroid rate changes with glucose concentration and time change, 25mmo/l glucose concentration 24h, peaked at.2. high glucose can promote Nrf2 cell proliferation rate of K1 The expression and nuclear translocation, thus promoting the proliferation of thyroid papillary carcinoma K1 cells, which may be related to PI3K/Akt signaling pathway in high glucose induced increase of K1 cells. The second chapter of berberine inhibits the proliferation of papillary thyroid carcinoma K1 cells induced by high glucose and the possible mechanism of inhibition Objective: To observe the effects of berberine on the proliferation of papillary thyroid carcinoma K1 cells induced by high glucose. To investigate the effect of berberine on glucose induced; the expression of Nrf2 in papillary thyroid carcinoma K1 cell protein and nuclear translocation, and to explore its possible signal pathway play, in order to provide new ideas and methods for the prevention of diabetes complicated with early carcinoma of papillary thyroid treatment. Methods 1. high glucose (25mmol/L) with different concentrations of berberine (0,10,40,80) mol/L 24h joint intervention K1 cells, using MTT colorimetric assay to detect the proliferation of K1 cells, with normal glucose (5.5mmol /L) as 瀵圭収.2.閲囩敤Western blot妫，

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