锌依赖的金属蛋白酶1促进巨噬细胞凋亡  

Zinc-dependent metalloprotease 1 promotes apoptosis of RAW264.7 macrophages

作　　者：李鹏[1] 何永林[1] 张继明[1] 方陈城 

机构地区：[1]重庆医科大学基础医学院病原生物学教研室,重庆医科大学分子医学与肿瘤研究中心,重庆400016

出　　处：《细胞与分子免疫学杂志》2015年第12期1585-1587,1592,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：重庆市自然科学基金(cstc2012jj A10023)

摘　　要：目的构建锌依赖的金属蛋白酶1(zmp1)基因的真核表达质粒,探讨其对巨噬细胞凋亡的影响。方法以卡介苗(BCG)基因组DNA为模板,采用PCR法扩增zmp1基因;克隆到真核表达质粒p EGFP-N1的多克隆位点中,构建真核表达质粒p EGFP-N1-zmp1并转染RAW264.7细胞,荧光显微镜观察绿色荧光蛋白表达,实时定量PCR(qRT-PCR)检测zmp1 mRNA的水平;藻红蛋白标记的膜联素Ⅴ/7-氨基放线菌素D(annexinⅤ-PE/7-AAD)双染色结合流式细胞术检测Zmp1蛋白对细胞凋亡影响。结果扩增出zmp1基因;真核表达质粒经过XhoⅠ和Bam HⅠ双酶切及基因测序鉴定构建成功;转染细胞24 h后,经荧光倒置显微镜观察到绿色荧光;qRT-PCR结果显示转染p EGFP-N1-zmp1的RAW264.7细胞zmp1 mRNA的表达显著升高;转染后48 h,细胞早期凋亡率有所增加。结论在RAW264.7细胞中成功表达了Zmp1并能促进巨噬细胞凋亡。Objective To construct the eukaryotic expression vector of zinc-dependent metalloprotease 1（ zmp1） gene from Bacillus Calmette-Guerin（ BCG） and investigate its impact on the apoptosis of RAW264. 7 macrophages. Methods Zmp1 gene was amplified from the genome of BCG by PCR. The zmp1 gene fragment was inserted into multiple cloning sites of p EGFP-N1 to construct the eukaryotic expression vector p EGFP-N1-zmp1. The constructed p EGFP-N1-zmp1 was transfected into RAW264. 7 cells by LipofectamineTM2000. The expression of green fluorescent protein（ GFP） was observed by fluorescence microscopy. The zmp1 mRNA was detected by quantitative real-time PCR（ qR-PCR）. The effect of Zmp1 protein on the apoptosis of RAW264. 7 macrophages was detected by flow cytometry（ FCM）. Results With zmp1 gene amplified by PCR,we successfully constructed the recombinant vector p EGFP-N1-zmp1 as demonstrated by restriction enzyme analysis and sequencing. GFP was seen in RAW264. 7 cells 24 hours after transfected with the recombinant plasmid.As qRT-PCR showed,the expression level of zmp1 mRNA was up-regulated. The early apoptotic rate increased 48 hours after transfection. Conclusion The increased expression of Zmp1 in RAW264. 7 cells promotes the apoptosis of RAW264. 7 cells.

关 键 词：卡介苗 锌依赖的金属蛋白酶1(Zmp1) 真核表达 RAW264.7细胞 细胞凋亡 

分 类 号：R392.12[医药卫生—免疫学] R378.911[医药卫生—基础医学]