本文關(guān)鍵詞： 甲狀腺乳頭狀癌 高糖 Nrf2 小檗堿 PI3K/Akt信號(hào)通路　出處：《湖北中醫(yī)藥大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：第一章高糖對(duì)甲狀腺乳頭狀癌K1細(xì)胞增殖及PI3K/Akt/Nrf2信號(hào)通路的影響研究目的觀察不同濃度葡萄糖對(duì)甲狀腺乳頭狀癌K1細(xì)胞增殖的促進(jìn)作用;初步探討高糖對(duì)甲狀腺乳頭狀癌K1細(xì)胞核因子-E2相關(guān)因子2(nuclear factor erythroid 2-related factor 2,Nrf2)蛋白表達(dá)及核轉(zhuǎn)位的影響并研究其可能的上游信號(hào)通路。研究方法1.以不同濃度葡萄糖(5.5、10、25、50)mmol/L干預(yù)K1細(xì)胞,分別于12、24、36、48h采用MTT比色分析法檢測(cè)各組K1細(xì)胞增殖率,同時(shí)設(shè)相同滲透壓組作為對(duì)照。2.根據(jù)MTT結(jié)果,將甲狀腺乳頭狀癌K1細(xì)胞分為正糖組(5.5mmol/L葡萄糖)、高糖組(25mmol/L葡萄糖)和高滲組(25mmol/L甘露醇),各組均分別于0、12、24、48h提取細(xì)胞,采用Western Blot法檢測(cè)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)表達(dá)水平;采用細(xì)胞免疫熒光法分析各組Nrf2在細(xì)胞內(nèi)的分布情況;使用Spearman相關(guān)性分析法檢測(cè)高糖作用下K1細(xì)胞增殖分別與Nrf2、PI3K蛋白和p-Akt/Akt的相關(guān)性。研究結(jié)果1.隨葡萄糖濃度升高,K1細(xì)胞增殖率改變,25mmol/L葡萄糖濃度下K1細(xì)胞增殖達(dá)到峰值(148.76±5.19)%,50mmol/l葡萄糖濃度時(shí)K1細(xì)胞增殖率下降(84.72±3.26)%,與5.5mmol/l葡萄糖濃度時(shí)K1細(xì)胞增殖率比較均有統(tǒng)計(jì)學(xué)差異(p0.01)。2.同一葡萄糖濃度作用下隨時(shí)間延長(zhǎng)K1細(xì)胞增殖率發(fā)生改變,12h明顯升高,24h達(dá)峰,隨后下降。3.25mmol/L葡萄糖干預(yù)K1細(xì)胞24h時(shí),K1細(xì)胞內(nèi)Nrf2、PI3K和p-Akt/Akt表達(dá)及Nrf2在細(xì)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細(xì)胞核比例:91.2%,260/285)均達(dá)峰值,并且它們隨時(shí)間的變化均與K1細(xì)胞增殖率變化同步。4.Spearman相關(guān)性分析表明高糖組Nrf2、PI3K及p-Akt/Akt表達(dá)均與K1細(xì)胞增殖率呈正相關(guān),相關(guān)系數(shù)r分別為0.908、0.910、0.916,p0.01。5.滲透壓升高對(duì)K1細(xì)胞增殖及Nrf2、PI3K蛋白表達(dá)、p-Akt/Akt等均無(wú)明顯影響(p0.05)。研究結(jié)論1.甲狀腺乳頭狀癌K1細(xì)胞增殖率隨葡萄糖濃度和作用時(shí)間的變化而改變,25mmo/l葡萄糖濃度作用24h時(shí),K1細(xì)胞增殖率達(dá)高峰。2.高糖可能通過(guò)促進(jìn)Nrf2表達(dá)及核轉(zhuǎn)位,從而促進(jìn)甲狀腺乳頭狀癌K1細(xì)胞增殖,這可能與高糖上調(diào)K1細(xì)胞內(nèi)PI3K/Akt信號(hào)通路有關(guān)。第二章小檗堿抑制高糖誘導(dǎo)的甲狀腺乳頭狀癌K1細(xì)胞增殖及可能的機(jī)制研究目的觀察小檗堿對(duì)高糖誘導(dǎo)的甲狀腺乳頭狀癌K1細(xì)胞增殖的抑制作用;探討小檗堿對(duì)高糖誘導(dǎo)的甲狀腺乳頭狀癌K1細(xì)胞Nrf2蛋白表達(dá)及核轉(zhuǎn)位的影響,并初步探究其發(fā)揮作用的可能信號(hào)通路,以期為早期防治糖尿病合并甲狀腺乳頭狀癌治療提供新的思路和方法。研究方法1.高糖(25mmol/L)加入不同濃度的小檗堿(0、10、40、80)μmol/L共同干預(yù)K1細(xì)胞24h時(shí),采用MTT比色分析法檢測(cè)各組K1細(xì)胞增殖情況,設(shè)正糖(5.5mmol/L)作為對(duì)照。2.采用Western blot檢測(cè)各組Nrf2、PI3K、Akt、p-Akt表達(dá)水平,細(xì)胞免疫熒光法分析各組Nrf2在細(xì)胞內(nèi)的分布,設(shè)正糖(5.5mmol/L)作為對(duì)照。研究結(jié)果1.與正糖組比較,高糖組K1細(xì)胞增殖率、PI3K、p-Akt/Akt、Nrf2表達(dá)及Nrf2在細(xì)胞核分布的比例均顯著升高(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細(xì)胞核比例:(93.0%,265/285)vs(23.1%,45/195),p0.01)。2.與高糖組比較,高糖加入10、40、80μmol/L小檗堿后各組K1細(xì)胞增殖率((111.76±4.10)%、(70.03±2.18)%、(32.41±3.76)%)均下降(p0.05),80μmol/L小檗堿組增殖抑制作用最強(qiáng),同時(shí)40μmol/L小檗堿組增殖抑制作用高于10μmol/L小檗堿組。3.高糖加入不同濃度小檗堿后各組PI3K、p-Akt/Akt、Nrf2表達(dá)及Nrf2在細(xì)胞核分布的比例,與高糖組比較均下調(diào)(p0.05),且其變化均隨小檗堿的濃度升高而更加顯著。研究結(jié)論小檗堿呈濃度依賴性抑制高糖誘導(dǎo)的K1細(xì)胞增殖,這可能與小檗堿抑制高糖誘導(dǎo)的PI3K/Akt信號(hào)通路過(guò)度激活從而下調(diào)Nrf2蛋白表達(dá)及核轉(zhuǎn)位有關(guān)。
[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媯，

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