信号转导及转录化因子5对肺癌诊断价值的实验研究.pdf

目 录 1 信号转导及转录活化因子 5 对肺癌诊断价值的实验研 究„„„„„„„„„„„„„„„„„„„„„„„„„„2 2 中文摘要„„„„„„„„„„„„„„„„„„„„„„„2 3 英文摘要„„„„„„„„„„„„„„„„„„„„„„„4 4 前言„„„„„„„„„„„„„„„„„„„„„„„„„6 5 材料与方法„„„„„„„„„„„„„„„„„„„„„„11 6 结果„„„„„„„„„„„„„„„„„„„„„„„„„29 7 讨论„„„„„„„„„„„„„„„„„„„„„„„„„46 8 结论„„„„„„„„„„„„„„„„„„„„„„„„„54 9 参考文献„„„„„„„„„„„„„„„„„„„„„„„55 10 英汉缩略词对照表„„„„„„„„„„„„„„„„„„„60 11 致谢„„„„„„„„„„„„„„„„„„„„„„„„„61 12 综述„„„„„„„„„„„„„„„„„„„„„„„„„63 1 信号转导及转录活化因子5对肺癌诊断价值 的实验研究 摘要 目的: 为了检测信号转导和转录激活因子5(signal transducer and activator of transcription 5,STAT5) 、癌胚抗原(carcinoembryonic antigen,CEA) 、细胞角蛋白19片段(Cytokeratin 19 fragment,Cyfra21-1) 三者分别在正常人胚肺MRC-5细胞和人肺腺癌A549细胞中的表达情 况，并使用受度者工作特征曲线（ROC 曲线），探索STAT5能否可以 对肺癌早期诊断具有重要价值。方法：1.采用免疫细胞化学SP法检测 传代培养的正常人胚肺MRC-5细胞和人肺腺癌A549细胞中STAT5、 CEA、Cyfra21-1蛋白表达的表达。用Olympus BX60带照相系统显微 镜采集图片，使用IPP(Image-Pro Plus)6.0 图像分析软件分析所采集的 图片，通过计算机所测图片MOD(mean optical density,平均光密度)值 判断图片染色强度，得到相应的量化值；2.利用荧光定量PCR方法检 测在正常人胚肺MRC-5细胞和人肺腺癌A549细胞中STAT5 mRNA 、 CEA mRNA、Cyfra21-1 mRNA 的基因表达量。通过利用ROC 曲线分 析在人肺腺癌A549细胞中STAT5 、CEA 、Cyfra21-1的蛋白及mRNA 表达情况，利用曲线下面积比较三者单检与联合检测的诊断价值。结 果： 1.免疫细胞化学染色图片分析结果： （l ）STAT5蛋白、CEA蛋 白和Cyfra21-1蛋白在正常人胚肺MRC-5细胞中的表达均明显低于它 2 们各自在人肺腺癌A549细胞中的表达，经统计学软件分析计算，它 们各自在两组细胞中的表达水平差异有显著统计学意义(P0.01) 。(2) 在A549细胞中分别计算STAT5、CEA 、Cyfra21-1蛋白的ROC 曲线下 面积（Az ）分别为0.766、0.771、0.749 。三者的曲线下面积(Az)分别 比较，三者之间差异均不具有统计学意义（P ﹥0.05 ）。2.荧光定量 PCR方法检测结果显示: （1）STAT5、CEA、Cyfra21-1和β-actin扩增 之后各自的产物特异都达到较高水平；扩增效率分别为97.96% 、 96.77%、95.19%、97.73% 。（2 ）STAT5 mRNA 、CEA mRNA、Cyfra21-1 mRNA在正常人胚肺MRC-5细胞中的基因表达水平均明显低于它们 各自在人肺腺癌A549细胞中的表达，经统计学软件分析之后，它们 各自分别在两组细胞株中的表达差异均具有统计学意义(P0.05) 。(3) 分别计算STAT5mRNA 、CEAmRNA 、Cyfra21-1 mRNA 的ROC 曲线下 面积（Az ）分别