SEMA3B基因真核表达载体的构建及对肺癌细胞恶性生物学行为的影响  

Construction of eukaryotic expression vector of SMEA3B and the functional research on malignant biological characteristics of lung cancer cells

作　　者：赵俊[1] 何爽[1] 郑云[1] 陈敏[1] 明凯华[1] 

机构地区：[1]广州医科大学附属广州市第一人民医院,广州510180

出　　处：《广州医药》2015年第6期4-8,共5页Guangzhou Medical Journal

基　　金：广州市医药卫生科技项目(20131A011023)

摘　　要：目的构建抑癌基因SEMA3B真核表达载体pc DNA3.1-SEMA3B,并检测其对肺癌A549细胞恶性生物学行为的影响。方法应用PCR扩增SEMA3B全长c DNA片段,构建真核表达载体pc DNA3.1-SEMA3B。克隆PCR、双酶切法、基因测序验证过表达载体构建成功。将pc DNA3.1-SEMA3B真核表达载体和空载体pc DNA3.1分别转染入A549细胞中,应用qRT-PCR、Western blot检测SEMA3B mRNA、蛋白表达水平的变化;MTS法检测细胞增殖;流式细胞仪检测细胞凋亡、细胞周期;克隆形成实验检测细胞集落形成能力。结果 SEMA3B基因扩增片段与预测片段一致,克隆成功,且测序鉴定证实真核表达载体构建成功。转染pc DNA3.1-SEMA3B真核表达载体可上调SEMA3B mRNA、蛋白表达水平,且可抑制A549细胞的增殖,诱导凋细胞亡,细胞被阻滞在G1期,抑制细胞集落形成能力。结论成功构建了SEMA3B基因真核表达载体,抑癌基因SEMA3B在肺癌恶性生物学进程中可能发挥重要作用。Objective To construct the eukaryotic expression vector of the cancer suppressor gene,SEMA3 B,and research the effects on malignant biological behavior of lung cancer A549 cells. Methods By reverse transcriptase-polymerase chain reaction（ RT-PCR）,the full length SEMA3 B gene was amplified and then was inserted into pc DNA3. 1. The recombinant plasmid pc DNA3. 1-SEMA3 B was confirmed correctly through double enzyme digestion and PCR identification,which was transfected into lung cancer A549 cells by lipid media transfection. The untransfected A549 and A549 transfected with pc DNA3. 1were used as controls. SMEA3 B gene was detected by qRT-PCR and western blot. MTS assay,flow cytometry,and colony formation test were performed to evaluate the effect of overexpression of SEMA3 B gene on A549 cell proliferation,apoptosis,cell cycle,and colony forming ability. Results The amplied fragment of SEMA3 B gene by PCR was consistent with the anticipated result,the SEMA3 B gene was cloned successfully. And the recombinant plasmid pc DNA3. 1-SMEA3 B was constructed successfully through gene sequence identification. After transfection of pc DNA3. 1-SEMA3 B,SEMA3B mRNA and protein expression levels were raised,and overexpression of SEMA3 B gene in A549 cells significantly inhibited the proliferation of A549 cells,induced apoptotic cell death,blocked cell cycle in the G1 phase,and suppressed cell colony-forming ability. Conclusion The recombinant pc DNA3. 1-SEMA3 B is constructed successfully. SEMA3 B gene can significantly inhibit the malignant biological behavior of lung cancer A549 cells.

关 键 词：SEMA3B 克隆 基因转染 增殖 肺癌 

分 类 号：R734.2[医药卫生—肿瘤]