GRIM-19基因的克隆及其对小鼠前列腺癌细胞株的促凋亡作用

目的构建GRIM-19基因真核表达质粒,探讨GRIM-19基因转染对小鼠前列腺癌rm-1细胞的促凋亡作用。方法采用RT-PCR及重组DNA技术构建pcDNA-GRIM-19真核表达质粒,体外进行基因转染。形态学观察,DNAladder检测转染后rm-1细胞凋亡,RT-PCR方法检测rm-1细胞caspase-3活性。结果扩增出GRIM-19cDNA全长,测序结果与GenBank记载基本一致。转染rm-1细胞48h后证明其能在真核细胞表达,形态学及共聚焦显微镜观察到pcDNA3.1-GRIM-19重组质粒组出现明显的细胞凋亡,并出现典型的DNAladder。转染pcDNA3.1-GRIM-19重组质粒组caspase-3表达强度较对照组及空质粒组明显上调(P<0.05)。结论成功克隆并构建鼠GRIM-19基因真核表达载体pcDNA3.1-GRIM-19,该载体可诱导鼠rm-1细胞凋亡。其凋亡机制与caspase-3酶活性有关。

Cloning of mouse GRIM-19 gene and its apoptotic effect on prostate cancer rm-1 cells

Objective To construct pcDNA3.1-GRIM-19 eukaryotic expression vector and to investigate its function in mouse prostate cancer rm-1 cells.Methods RT-PCR was performed on total RNA extracted from mouse spleen tissue to obtain the cDNA of GRIM-19,which was inserted into pMD-18T vector.DNA sequencing was tested before the amplified products were cloned into pcDNA3.1.The recombinant vector was transfected into rm-1 cells and its expression was examined by RT-PCR.The apoptotic effects of cells were observed by laser scanning confocal microscope(LSCM)and DNA ladder assay.Then,using the RT-PCR detected caspase-3 activity in rm-1 cells after transfection.Results The amplified products were confirmed as the cDNA of rm-1 by DNA sequencing,being capable of expression in rm-1 cells.Typical apoptosis was observed by LSCM and DNA ladder in recombinant plasmid group after 48h of transfection in rm-1 cells.Overexpression of GRIM-19 increased caspase-3 expression.Conclusion The eukaryotic expression vector for GRIM-19 was successfully constructed,which can be expressed in rm-1 cells and can induce the cell apoptosis.Its apoptosis mechanism may related to caspase-3 activity.