【摘要】 目的:探讨脂质体介导VEGF-C基因转染对人胃癌OCUM-2MD3细胞VEGF-C表达的影响。方法:双酶切pCRⅡ-VEGF-C重组质粒获得人VEGF-CcDNA片段,与真核表达载体pcDNA3.1连接构建pcDNA3.1-VEGF-C重组质粒,脂质体介导转染人胃癌OCUM-2MD3细胞并筛选,RT-PCR检测转染后VEGF-CmRNA表达水平,免疫组化法、流式细胞术检测VEGF-C蛋白表达变化。结果:经酶切鉴定,pcDNA3.1-VEGF-C重组质粒含人VEGF-CcDNA。转染组VEGF-CmRNA相对吸光度值为(0.73±0.16)%,较空载组(0.54±0.13)%表达显著上调(P<0.01)。免疫组化和流式细胞术检测转染组VEGF-C蛋白阳性表达率分别为(52.16±6.72)%、(41.83±3.65)%,较空载组(30.33±5.04)%、(30.16±3.23)%显著增加(P<0.01或0.05)。结论:脂质体介导VEGF-C基因转染人胃癌OCUM-2MD3细胞可显著增加VEGF-C表达水平,对探索胃癌淋巴转移机制及其治疗具有重要意义。更多还原

【Abstract】 Objective: To explore the effect of VEGF-C gene transfection on the expression level of VEGF-C protein in human gastric cancer cell OCUM-2MD3. Methods: The VEGF-C cDNA sequence, which was obtained from clone vector pCRⅡ-VEGF-C doubly digested with EcoRΙ and XhoΙ, was ligated into an eukaryotic expression vector pcDNA3.1 to construction the recombinant plasmid pcDNA3.1-VEGF-C. The pcDNA3.1-VEGF-C was transformed into human gastric cancer cell OCUM-2MD3 induced by liposome. The changes in the expression level of VEGF-C mRNA and protein were determined by RT-PCR, immunohistochemistry and flow cytometry (FCM). Results: After the digestion, the recombinant plasmid pcDNA3.1-VEGF-C was confirmed to contain the human VEGF-C cDNA sequence. After transfection, the VEGF-C mRNA level (0.38±0.11)%, the positive rate of VEGF-C protein expression in transformed group (52.16±6.72)% by immunohistochemistry, and (41.83±3.65)% by FCM were all dramatically higher than those of the control group(P<0.05 or 0.01), respectively. Conclusion: After VEGF-C gene transfection by liposome, the expression level of VEGF-C in human gastric cancer cell OCUM-2MD3 is greatly heightened. This may be helpful for exploring the mechanism of lymphatic metastasis and its gene therapy in gastric carcinoma.更多还原