人c-myc反义真核表达载体的构建及其对BT325细胞c-Myc蛋白表达的抑制作用

Construction of a eukaryotic expression vector of human antisense c-myc gene and its inhibitory effect on c-Myc expression in BT325 cells

目的：构建人c-myc第三外显子及3’非翻译区反义真核表达载体，用脂质体介导法转染人胶质瘤BT325细胞，以期降低其c-Myc表达水平．方法：采用基因重组方法构建真核表达载体;用G418筛选抗性细胞克隆;用免疫细胞化学ABC法检测细胞蛋白质的表达;用MTT法测定顺铂作用下，未转染及转染组细胞的存活率．结果：c-myc反义真核表达载体在BT325细胞中稳定表达，转染细胞c-Myc表达水平显著降低;c-Myc表达减弱细胞对顺铂诱导凋亡的敏感性．结论：抑制c-mycDNA第三外显子及其3’端非翻译区反义表达载体能够显著抑制。c-myc基因的表达;c-Myc在化疗药物诱导肿瘤细胞凋亡的过程中有重要作用．

AIM: To construct a eukaryotic expressing vectorof antisense targeting human c--myc third exon as well as its3' non--translational region and to block the c Myc expressionin BT325 cells. METHODS: A eukaryotic expressing vectorwas constructed by molecular cloning, which was trans feetedinto BT325 cells. Then MTT was used to assay the survivalpercentage of gene transfected and normal cells. RESULTS:c--ablys antisense expressing vector expressed stably in BT325cells and blocked their c--Myc expression remarkably. Theblocking of c--Myc expression lowered BT325 cells sensitivityto cisplatin induced apoptosis. CONCLUSION: The antisensetargeting c--,nyc third exon and non--translational region significantly inhibit c myc expression.