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该文探讨甜蜜素联合柠檬黄对人克隆结肠腺癌细胞(Caco-2细胞)损伤的机理,以高内涵细胞成像分析(high content analysis,HCA)技术为平台,析因设计实验方差分析为联合毒性的评价方法、实时荧光定量PCR(quantificational Real-time polymerase chain reaction,q RT-PCR)和Western blot为检测方法对此进行探究。结果表明,甜蜜素与柠檬黄均会显著地抑制Caco-2细胞的增殖,且呈现剂量–效应关系,IC50分别为11.37±0.15 g/L和1.21±0.12 g/L。与单独组相比,联合组显著地升高细胞内Ca2+浓度、升高活性氧(reactive oxygen species,ROS)水平、抑制细胞增殖、增大细胞膜通透性、降低线粒体膜电位。联合组在细胞内Ca2+浓度、ROS水平上表现为协同作用,在线粒体膜电位变化上表现为相加作用。q RT-PCR和Western blot显示,联合组显著提高Bax、细胞色素c(cytochrome c)、胱冬肽酶-3(caspase-3)等基因的表达,并上调Bax和下调Bcl-2的蛋白质水平,同时提高细胞色素c、剪切胱冬肽酶-3(cleaved-caspase-3)的蛋白质水平来抑制Caco-2细胞增殖。
This article explores the mechanism of sodium cyclamate on the human colon adenocarcinoma cells (Caco-2 cells) injury mechanism to high content of cell imaging analysis (high content analysis, HCA) technology as a platform, factorial design analysis of variance as a joint Toxicity evaluation methods, quantitative real-time polymerase chain reaction (q RT-PCR) and Western blot detection methods to explore this. The results showed that both cyclamate and lemon yellow could significantly inhibit the proliferation of Caco-2 cells, and showed a dose-effect relationship with IC50 of 11.37 ± 0.15 g / L and 1.21 ± 0.12 g / L, respectively. Compared with the single group, the combination group significantly increased intracellular Ca2 + concentration, increased reactive oxygen species (ROS) levels, inhibited cell proliferation, increased cell membrane permeability and decreased mitochondrial membrane potential. The combination group showed a synergistic effect on intracellular Ca2 + concentration and ROS level, and showed an additive effect on mitochondrial membrane potential changes. q RT-PCR and Western blot showed that the combination group significantly increased the expression of Bax, cytochrome c, caspase-3 and other genes, upregulated Bax and down-regulated the protein level of Bcl-2 , While increasing cytochrome c, cleaving the protein level of cleaved-caspase-3 to inhibit Caco-2 cell proliferation.