血管内皮生长因子C靶向RNA干扰重组载体的构建和效应检测

Construction and identification of small interfering RNA expression vector targeting vascular endothelial cell growth factor-C

目的:构建针对人血管内皮生长因子C小干扰RNA表达载体,并观察其在胃癌细胞中的干扰效果。方法:实验于2006-02/2007-02在河南省分子医学重点学科开放实验室完成。①实验材料:小干扰RNA表达载体pSilencer3.1为美国Ambion公司产品;胃癌细胞SGC7901为河南省分子医学重点学科开放实验室保存;血管内皮生长因子C基因的干扰片断和聚合酶链反应引物(152bp)由上海生工生物公司合成。②实验过程:根据pSilencer3.1-H1载体要求设计靶向血管内皮生长因子C基因的两对小干扰RNA,退火后连接入载体相应位点,构建重组表达质粒。经酶切和测序鉴定后分别转染胃癌细胞株SGC-7901,经G418筛选,获得稳定表达的细胞株。③实验评估:采用反转录-聚合酶链反应和Westernblot法检测血管内皮生长因子CmRNA和蛋白水平的表达。结果:①重组质粒的酶切、核苷酸序列分析结果:经酶切和测序鉴定证实成功构建了血管内皮生长因子C小干扰RNA表达载体,建立了稳定转染细胞株。②稳定转染的细胞株中血管内皮生长因子CmRNA和蛋白表达:反转录-聚合酶链反应和Western blot显示转染后血管内皮生长因子C表达与对照组相比有不同程度的降低,以pSilencer3.1-neo-VEGF-C1尤为明显,抑制血管内皮生长因子C表达,不影响细胞的生长。结论:成功构建了血管内皮生长因子C小干扰RNA表达载体及稳定转染的胃癌细胞株。

AIM： To construct Vascular Endothelial Growth Factor-C （VEGF-C） small interfering RNA （siRNA） expression vector and evaluate its expression in gastric cancer cells. METHODS： The experiment was performed at the Henan Key Science Opening Laboratory of Molecular Medicine from February 2006 to February 2007. ①The siRNA expression vector pSilencer3.1 was purchased from Ambion Company. Gastric cancer cells SGC7901 was kept in Henan Key Science Opening Laboratory of Molecular Medicine. VEGF-C interfering fragment and polymerase chain reaction primer （152 bp） were bought from Shanghai Shenggong Biological Company. ②Two pairs of hairpin siRNA template oligonucleotides for VEGF-C based on pSilencer3.1-H1 vector were designed. These oligonucleoUdes were annealed and ligated into vector respectively. After confirmation by DNA sequencing, positive recombinant plasmids were transfected into gastric cancer cells SGC7901. Stable call lines of G418-resistance were acquired.③Expression of VEGF-C mRNA and protein was detected by Westem blot and reverse transcripUon-polymerase chain reaction （RT-PCR）. RESULTS： ①Enzymolysis and nucleotide sequence analysis of recombinant plasmids showed that two VEGF-C siRNA eukaryotic expression vectors were constructed successfully. Stable cell lines were established. ②Expression of VEGF-C mRNA and protein was obviously down-regulated compared with the control group, especially pSilencer3.1-neo-VEGF-C1, and VEGF-C expression did not affect the cell growth. CONCLUSION： The VEGF-C siRNA eukaryotic expression vectors are successfully constructed and stably expressed in gastric cancer calls.