VEGF-C基因高表达促进胃癌细胞增殖及克隆和小管形成的实验研究

摘要

目的:构建人血管内皮生长因子(VEGF)-C基因慢病毒表达载体,并研究其在胃癌细胞中的功能.方法:采用SYBR Green相对定量逆转录聚合酶链反应(qRT-PCR)及蛋白免疫印迹法(Western blot)检测6种不同类型的胃癌细胞系MKN-45、MKN-28、NCl-N87、BGC-823、AGS和SGC-7901中VEGF-C mRNA及其蛋白表达水平；通过构建VEGF-C基因慢病毒表达载体进行细胞增殖、克隆形成和内皮细胞管形成实验,按实验组(MKN-45-GV/C)、对照组(MKN-45-GV)和空白细胞组(MKN-45)加以比较.结果:qRT-PCR和Western blot实验表明,在6种胃癌细胞中均可检测到VEGF-C mRNA及其蛋白的表达,其中在胃癌细胞MKN-45中表达最低(0.45±1.44),SGC-7901中表达最高(31.13±0.75)；增殖实验结果显示,与对照组比较,实验组细胞增殖数量明显增加(P＜0.001);克隆形成结果显示,实验组和对照组细胞在每平均视野形成克隆数分别为6.23±0.32和1.40±1.67,克隆形成率分别为85％和31％(P＜0.05)；内皮细胞管形成实验结果显示,实验组、对照组和空白细胞组小管数目分别为8.14±1.25、12.76±0.79和18.46±0.72 (P＜0.01),小管长度分别为98.56±3.71、105.17±1.34和151.62±2.51 (P＜0.05).结论:VEGF-C基因真核表达载体构建成功,VEGF-C基因促进胃癌细胞增殖、克隆形成和小管形成.%Objective: To the construction and biological characteristics of recombinant lentvi-ral human vascular endothelial growth factor-C gene plasmid in gastric cancer cells. Methods: The VEGF-C gene and protein levels were tested by the SYBR Green transcriptase relative quantitative polymerase chain reaction (qRT-PCR) method and protein immune imprinting method (Western blot method). Differences of the VEGF-C mRNA and protein expression levels among MKN-45, MKN-28,NCI-N87,BGC-823,AGS and SGC-7901 gastric cancer cell lines were analysed. We assayed cell growth activity, colony formation, and endothelial tube formation by constructed recombinant lentiViral human vascular endothelial growth factor-C gene plasmid. The whole experiment was divided into the experimental group(MKN-45-GV/C), the control group(MKN-45-GV)and the blank group(MKN-45). Results: VEGF-C gene and protein expression in all of the six gastric cancer cell lines with different levels, in which the minimum expression was detected of VEGF-C was found in MKN-45 gastric cancer cell line as 0.45 ± 1.44, and the maximum expression in SGC-7901 cell line as 31.13 ± 0.75. The cell proliferation assay indicated: compared with control group, the experimental group showed a significant increase of cell proliferation volume (P＜0.001); Colony formation results demonstrated: experimental group and control group had the cloned number of 6.23 ±0.32 and 1.40 ± 1.67 respectively per average view. The efficiency of colony formation was 85% and 31 % respectively (P＜0.05). The endothelial tube formation experiment showed: the experimental group, control group and blank group had tubular number of 8.14 ± 1.25, 12.76 ±0.79 and 18.46 ±0.72 respectively, the length of tube was 98.56 ±3.71, 105.17 ±1.34 and 151.62 ±2.51 respectively. Conclusion: Recombinant lentiViral human vascular endothelial growth factor-C gene plasmid was successfully constructed. VEGF-C gene promoted the cell growth, colony formation, and endothelial tube formation.