【摘要】 目的 构建人反义VEGF121 cDNA真核表达质粒,研究反义基因治疗对膀胱癌细胞增殖的影响。方法将VEGF121反向克隆入真核表达载体pcDNA3中,酶切鉴定。用此真核表达重组质粒转染人膀胱癌细胞株T24,G418筛选获得阳性克隆,RT-PCR法测定转染前后T24细胞中VEGF mRNA表达,ELISA法检测转染前后T24细胞中VEGF蛋白表达。利用MTT法、软琼脂克隆形成试验、流式细胞仪和电镜等检测转染前后T24细胞的生物学性状。结果 获得反义VEGF121 cDNA质粒表达重组质粒。VEGF121反义RNA部分阻断了T24细胞中VEGF表达,转染后肿瘤细胞VEGF mRNA和VEGF蛋白表达都明显减少;转染后单个细胞的克隆形成能力明显减弱;形态学超微结构显示细胞器扩张和肿胀,核染色质边集,凝集成块,可见明显的凋亡改变。结论成功构建了反义VEGF121 cDNA真核表达质粒,转染该质粒可明显抑制膀胱癌细胞增殖,为膀胱癌的基因治疗提供了一定的实验依据。更多还原

【Abstract】 Purpose To construct and identify eukaryotic expression recombinant plasmid bearing human VEGF121 cDNA and evaluate the effect of antisense gene therapy on bladder cancer. Methods We inserted VEGF121 into eukaryotic expression vector pcDNA3 and constructed pcDNA3-As- VEGF121. Restriction endonuclease analysis was used to confirm the reverse orientation of the VEGF cDNA. The recombinant plasmid was transfected into human bladder cancer cell line T24 and the positive done was screened by G418. VEGF mRNA and protein expression of T24 cells before and after transfection were detected by reverse transcription PCR and ELISA, respectively. The biological characteristics of T24 cells before and after transfection were also inspected. Results The pcDNA3-As-VEGF121 recombinant plasmid was obtained. VEGF expression was blocked by antisense VEGF RNA. The done formation ability of single cell was decreased after transfection. The obvious apoptosis and inhibition of proliferation of T24 cells after transfection were observed by electron microscope,flow cytometer and MTT methods. Conclusions The successful construction of antisense VEGF121 eukaryotic expression recombinant plasmid and the biological characteristics of the transfected cell may provide the basis for the development of antiangiogenic gene therapy to bladder cancer.更多还原