摘要
目的:将人caspase-1基因真核表达载体转染肝癌细胞后观察其表达水平及对IL-1β水平的影响,为研究cas-pase-1与IL-1β的关系及其在慢性炎症与肿瘤中的作用提供依据。方法:利用阳离子聚合物(jetPEI)将pIRES2-EGFP-caspase-1重组表达载体和pIRES2-EGFP对照载体转染人肝癌细胞HepG2(HepG2/caspase-1、HepG2/mock),48小时后倒置相差荧光显微镜下观察表达情况,利用RT-PCR、Western blot方法检测两组细胞caspase-1表达水平,采用caspase-1底物显色分析研究的方法检测两组细胞中caspase-1的活性。夹心ELISA法检测细胞培养上清以及细胞裂解液中IL-1β的表达水平。结果:转染48小时后倒置相差显微镜下可见,pIRES2-EGFP-caspase-1和pIRES2-EGFP重组表达载体均能在HepG2细胞中高效表达;HepG2/caspase-1细胞中caspase-1 mRNA表达水平显著提高;经LPS刺激后,HepG2/caspase-1细胞中caspase-1总蛋白的水平明显升高;caspase-1的生物活性水平与对照组相比具有显著性差异(t=25.679,P<0.05);HepG2/caspase-1细胞培养上清中IL-1β的水平明显高于HepG2/mock组细胞(t=3.417,P<0.05),胞浆裂解液中的变化相对不显著(t=2.396,P>0.05)。结论:人caspase-1基因真核表达载体pIRES2-EGFP-caspase-1能够在肝癌细胞中高效表达caspase-1,且该重组表达载体在HepG2细胞中能提高活性IL-1β的表达。
Objective：To develop the eukaryotic expressing vector harboring human caspase-1 and to explore its effects on the expression of human IL-1β of the tumor cells.Methods：The verified vector pIRES2-EGFP-caspase-1 containing full length of human caspase-1 gene was transfected into HepG2 hepatoma cells（HepG2/caspase-1） in a transient transfection system mediated by jetPEI and controlled by pIRES2-EGFP transfectants（HepG2/mock）.The expression of the recombinant vector was observed under fluorescent microscope 48 h after transfection.Expression level of caspase-1 mRNA was detected by RT-PCR,protein level of caspase-1 was assessed by Western blot.Enzymatic activity of caspase-1 was analyzed by adding the caspase-1specific substrate Ac-YVAD-pNA into total cell lysates.Sandwich ELISA was employed to assay the expression level of IL-1β both in suspernatant and cytoplasm.Results：The vector was transfected into HepG2 hepatoma cells（HepG2/caspase-1） and controlled with pIRES2-EGFP transfectants（HepG2/mock）.48 hours later when the cells were observed under fluorescent microscope,bringt green fluorescence was seen within the HepG2 cells.By RT-PCR using total RNA isolated from HepG2/caspase-1,it was shown that mRNA level of caspase-1 was markedly increased.As further indicated by Western blot,the protein level of caspase-1 was significantly enhanced especially when cells were triggered with LPS,and active caspase-1 production was also markedly upregulated.Moreover,the expression level of IL-1β in the supernatant of HepG2/caspase-1 cells was significantly elevated in a sandwich ELISA assay.Conclusion：The recombinant vector could enhance the level of both total and active caspase-1 in HepG2 cells.Moreover,expression of trcombinant caspase-1 the level of active IL-1β is also increased.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2011年第1期29-33,共5页
Chinese Journal of Immunology
基金
国家自然科学基金(30772497)
山东省科技攻关计划(2008GG10002007)资助项目
