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目的:观察含mIL-12基因的质粒转染至SKOV3卵巢癌细胞,效应细胞存在情况下对卵巢癌细胞株血管内皮生长因子(VEGF)表达的影响。方法:用脂质体转染技术将基因质粒及空质粒转染至SKOV3卵巢癌细胞(SKOV3/IL-12)及SKOV3/neo;用ELISA法检测IL-12及INF-γ的表达;用逆转录聚合酶联反应(RT-PCR)半定量法测定SKOV3/IL-12+S(脾细胞)0,12,24,36,48,60hVEGFmRNA含量;用ELISA法测定细胞培养上清液中VEGF蛋白含量及其抑制率。结果:基因转染至SKOV3可检测到IL-12蛋白表达;分泌的IL-12蛋白具有活性,使脾细胞产生INF-γ,12h时迅速出现,作用后24h表达量最高;SKOV3/IL-12细胞与脾细胞作用12h后即出现VEGFmRNA表达抑制,24h达到高峰,与对照组有显著差异(P<0.05);培养24h后上清中VEGF蛋白含量减少,与对照组有显著差异(P<0.05)。结论:将IL-12基因转染至SKOV3卵巢癌细胞并表达IL-12;分泌的IL-12蛋白具有活性,使脾细胞产生INF-γ;SKOV3/IL-12与效应细胞脾细胞作用后产生INF-γ能在核酸水平下调VEGF并能抑制VEGF蛋白的表达。
Objective: To investigate the effect of plasmid containing mIL-12 gene on the expression of vascular endothelial growth factor (VEGF) in ovarian cancer cell line SKOV3 ovarian cancer cells in the presence of effector cells. METHODS: Gene and empty plasmids were transfected into SKOV3 ovarian cancer cells (SKOV3 / IL-12) and SKOV3 / neo by lipofection. The expression of IL-12 and INF-γ was detected by ELISA. The content of VEGF in SKOV3 / IL-12 + S (spleen cells) 0, 12, 24, 36, 48 and 60 h was measured by semi-quantitative RT-PCR. The VEGF protein in cell culture supernatant Content and inhibition rate. Results: The expression of IL-12 protein was detected in the transfected SKOV3 cells. The secreted IL-12 protein was active and produced INF-γ in spleen cells. After 12 hours, the expression of VEGFmRNA reached a peak at 24h, which was significantly different from that of the control group (P <0.05). The content of VEGF in the supernatant decreased after 24h incubation, which was significantly different from that of the control group (P <0.05 ). CONCLUSIONS: The IL-12 gene is transfected into SKOV3 ovarian cancer cells and express IL-12. The secreted IL-12 protein is active and produces IFN-γ in spleen cells. The effect of SKOV3 / IL-12 and effector cell spleen cells INF-γ can downregulate VEGF at the nucleic acid level and inhibit the expression of VEGF protein.