血红素加氧酶-1重组载体的构建及在胃癌细胞中的初步研究  

Constraction of Heme Oxygenase-1 Recombinant Plasmid and It＇ s Research in Gastric Adenocarcinoma Cell

作　　者：王波[1] 刘庆军[1] 于景翠[2] 尹玉静[1] 魏旭冉[1] 周虹[1] 

机构地区：[1]军事医学科学院野战输血研究所,北京市100850 [2]哈尔滨医科大学第二临床医学院,哈尔滨市150081

出　　处：《医学分子生物学杂志》2010年第4期300-304,共5页Journal of Medical Molecular Biology

基　　金：资助项目：国家重点基础研究发展规划项目（973计划）（No.2009CB521702）

摘　　要：目的构建人血红素加氧酶-1（hemeoxygenase-1，HO-1）及其突变体的真核表达载体，观察其在胃腺癌细胞中HO-1表达和活性变化，研究HO-1活性变化的胃腺癌细胞对顺铂抗药能力的变化，为进一步研究HO-1对肿瘤细胞影响机制奠定基础。方法根据GenBank中HO-1cDNA序列设计引物，调取基因并克隆入pcDNA3．1（＋）质粒中，构建表达人野生型HO-1与突变型HO-1（HO-1G143H）的重组质粒。脂质体介导重组质粒转染胃腺癌细胞BGC823，用RT—PCR和Western印迹法分别检测细胞中HO-1mRNA的表达和蛋白表达水平，体外测定HO—1活性变化，应用顺铂进行体外抗药性实验。结果酶切鉴定和测序证实，HO-1真核表达载体构建成功；转染质粒后的BGC823，HO-1的mRNA和蛋白的表达水平明显上升；转入野生型质粒的细胞HO-1活性上升，转入突变型质粒的细胞HO-1活性下降；HO-1活性下降BGC823细胞抗顺铂杀伤能力增强。结论构建了HO-1野生型与突变型真核表达载体；将其转入胃腺癌细胞，引起了HO-1的mRNA和蛋白表达的增加和活性变化；体外实验表明，HO-1活性下降的BGC823细胞抗顺铂能力增强。Objective To construct eukaryotic expression plasmids expressing human heme oxygenase-1 （ HO-1 ） and its mutant for studying the influence of changes of HO-1 expression and en- zymatic activity in gastric adenoearcinoma cell, for study of the foundation of HO-1 and tumor cell. Methods According to HO-1 cDNA sequence in GenBank, The gene tained by PCR and cloned into expression vector pcDNA3.1 （ ＋ ） to fragment of HO-1 was ob- construct wild and mutant HO-1 recombinant plasmids. The plasmids were transfected into gastric adenocarcinoma cell BGC823 and were confirmed by PCR, western blot analysis. The expression and enzymatic activity of HO-1 were examined and the Cisplatin resistance of gastric adenocarcinoma cells with changed HO-1 was observed. Results The recombinant plasmids of HHO-1 and its mutant were constructed and identified through sequencing. BGC823 cells transfected with the plasmids showed elevated expression of HO-1 mRNA and protein. While the increased activity of HO-1 were showed in cells transfected with wild HO-1 recombinant plasmid, and the decrease were showed in cells transfected with mutant HO-1 recombinant plasmid. The cell resistance to Cisplatin was improved with the decreased activity of HO-1. Conclusion The recombinant plasmids of HHO-1 and its mutant were constructed successfully. Transfected plasmids into gastric adenocareinoma cell BGC823 lead to in-crease of HO-1 expression in RNA, protein, and changes of HO-1 activity. The experiments in vitro show the resistance of the cells with the decreased HO-1 activity to Cisplatin was improved.

关 键 词：血红素加氧酶-1 肿瘤 基因重组 

分 类 号：R722.17[医药卫生—儿科]