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[p38丝裂原活化蛋白激酶基因沉默对细颗粒物(PM2.5)诱导的肝细胞癌基因及凋亡基因表达的影响]

[Effect of p38MAPK gene silencing on expression of oncogenes and apoptotic genes induced by PM(2.5) in hepatocytes].

作者信息

Wang B Y, Qin S J, Li R B, Li B R, Cai Y, Zheng K, Xu X Y, Xie H W

机构信息

Institute of Environment and Health, Shenzhen Center for Disease Control and Prevention, Shenzhen 518055, China; School of Public Health, University of South China, Hengyang 421001, China.

Institute of Environment and Health, Shenzhen Center for Disease Control and Prevention, Shenzhen 518055, China; Xiangya School of Public Health, Central South University, Changsha 410078, China.

出版信息

Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi. 2020 Oct 20;38(10):721-725. doi: 10.3760/cma.j.cn121094-20191129-00548.

Abstract

To study the effect of p38 mitogen-activated protein kinase (MAPK) gene silencing on expression of apoptotic genes and oncogenes in hepatocytes treated with PM(2.5). From June to September 2019, according to the p38MAPK gene mRNA sequence provided by GenBank, three interfering sequences were designed and synthesized, ligated into PLVX-shRNA2-puro after annealing, and the recombinant lentiviral vector was transfected into L02 hepatocytes. The p38MAPK silencing cells were identified by real-time fluorescent quantitative PCR and western blotting. The normal L02 cells and p38MAPK silencing cells were treated with 50 μg/mL PM(2.5) water soluble solution, 10 μmol/L positive control Cr(6+), and a blank control group was set up, the treatment time was 24 h. The mRNA levels of oncogenes (c-fos, c-myc, k-ras) , tumor suppressor gene (p53) and apoptotic genes (Caspase-3, Caspase-8, Caspase-9) were detected by real-time PCR. The protein levels of oncogenes and apoptotic genes were detected by Western blotting. The expression levels of p38MAPK mRNA and protein in p38MAPK gene silencing cells were significantly lower than those in L02 hepatocytes (<0.05) , and the p38MAPK gene silencing cell line was successfully constructed. Compared with the blank control group, the expression levels of the oncogenes c-fos, c-myc, k-ras and the apoptosis genes Caspase-3, Caspase-8 and Caspase-9 increased, the expression level of tumor suppressor gene p53 decreased in the L02 hepatocyte group treated with PM(2.5) water soluble matter, and the differences were statistically significant (<0.05) . Compared with the L02 hepatocytes group treated with PM(2.5) water soluble matter, the expression levels of the oncogenes c-fos, c-myc, k-ras and apoptosis genes Caspase-3, Caspase-8 and Caspase-9 decreased, the expression level of tumor suppressor gene p53 increased in the p38MAPK gene silencing cells group treated with PM(2.5) water soluble matter, and the differences were statistically significant (<0.05) . PM(2.5) has effects on the expression of oncogenes, tumor suppressor genes and apoptotic genes in L02 hepatocytes, while p38MAPK gene silencing can inhibit the effects of PM(2.5) on L02 hepatocytes.

摘要

研究p38丝裂原活化蛋白激酶(MAPK)基因沉默对经PM(2.5)处理的肝细胞中凋亡基因和癌基因表达的影响。2019年6月至9月,根据GenBank提供的p38MAPK基因mRNA序列,设计并合成了三条干扰序列,退火后连接到PLVX-shRNA2-puro中,将重组慢病毒载体转染至L02肝细胞。通过实时荧光定量PCR和蛋白质免疫印迹法鉴定p38MAPK沉默细胞。将正常L02细胞和p38MAPK沉默细胞用50μg/mL PM(2.5)水溶性溶液、10μmol/L阳性对照Cr(6+)处理,并设空白对照组,处理时间为24小时。通过实时PCR检测癌基因(c-fos、c-myc、k-ras)、抑癌基因(p53)和凋亡基因(Caspase-3、Caspase-8、Caspase-9)的mRNA水平。通过蛋白质免疫印迹法检测癌基因和凋亡基因的蛋白质水平。p38MAPK基因沉默细胞中p38MAPK mRNA和蛋白质的表达水平明显低于L02肝细胞(<0.05),成功构建了p38MAPK基因沉默细胞系。与空白对照组相比,经PM(2.5)水溶性物质处理的L02肝细胞组中癌基因c-fos、c-myc、k-ras以及凋亡基因Caspase-3、Caspase-8和Caspase-9的表达水平升高,抑癌基因p53的表达水平降低,差异具有统计学意义(<0.05)。与经PM(2.5)水溶性物质处理的L02肝细胞组相比,经PM(2.5)水溶性物质处理的p38MAPK基因沉默细胞组中癌基因c-fos、c-myc、k-ras以及凋亡基因Caspase-3、Caspase-8和Caspase-9的表达水平降低,抑癌基因p53的表达水平升高,差异具有统计学意义(<0.05)。PM(2.5)对L02肝细胞中癌基因、抑癌基因和凋亡基因的表达有影响,而p38MAPK基因沉默可抑制PM(2.5)对L02肝细胞的影响。

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