PDGF-D对人肝癌细胞增殖及VEGF表达影响研究

The Influence of Platelet-derived Growth Factor D on the Proliferation of Human Hepatocarcinoma Cells and Expression of VEGF

目的:研究血小板源性生长因子D(PDGF-D)对人肝癌细胞株BEL-7402增殖及其血管内皮生长因子(VEGF)表达的影响。方法:体外培养肝癌细胞株BEL-7402和肝癌旁非瘤性细胞株QSG-7701,采用RT-PCR方法检测PDGF-D与PDGFRβmRNA在BEL-7402和QSG-7701的表达情况;将浓度分别为0(对照)、5、10、20、50、100、200μg/mL的人重组PDGF-DD蛋白加入BEL-7402中,采用四甲基偶氮唑蓝比色法检测肝癌细胞的生长曲线;流式细胞仪检测细胞周期变化;半定量RT-PCR检测VEGF及PDGFRβmRNA表达情况,ELISA检测PDGF-DD干预细胞后培养上清中VEGF蛋白的表达情况。结果:PDGF-D及PDGFRβmRNA在BEL-7402中高表达,与QSG-7701相比差异有统计学意义(P<0.05)。PDGF-DD干预细胞后,可促进人BEL-7402增殖,浓度为100μg/mL时达最高峰;细胞周期分布变化,G_0/G_1期细胞数减少,S期细胞数增加,与对照组相比差异有统计学意义;RT-PCR结果显示,VEGF及PDGFRβ RI值,实验组(除5μg/mL组外)与对照组相比差异有统计学意义;ELISA结果显示,加入PDGF-DD各浓度组VEGF蛋白浓度较对照组增高,差异有统计学意义,并呈量效依赖性关系。结论:PDGF-DD能促进BEL-7402的增殖,上调PDGFRβ及VEGF的表达。PDGF-D及其信号传导系统在肝癌的发生、发展中可能发挥重要的作用,可作为肝癌预后预测指标和治疗靶点。

Objective： To investigate the influence of platelet-derived growth factor D （PDGFD） on the proliferation of BEL-7402 human liver tumor cells and the expression of vascular endothelial growth factor （VEGF）. Methods： Human liver tumor cell strain BEL-7402 and paracarcinoma cell strain QSG-7701 were cultured in vitro. PDGF-D and PDGFRI3 mRNA expression were assessed by reverse transcription-polymerase chain reaction （RT-PCR）. BEL-7402 was treated with different concentrations （0, 5, 10, 20, 50, 100, and 200 pg/mL） of exogenous PDGF-D. Cell proliferation was measured using the MTT assay. The effect of exogenous PDGF-D on the cell cycle of BEL-7402 cells was assessed by flow cytometry. The expression of VEGF was detected by RT-PCR and ELISA in BEL-7402 cells that were exposed to different concentrations of PDGF-D. Results： PDGF-D and PDGFR~ mRNA expression in human liver tumor cell strain BEL-7402 was considered significantly higher than that in paracarcinoma cell strain QSG-7701 （P〈0.05）. Exogenous PDGF-D promoted proliferation of tumor cells in a dose-dependent manner from 10 pg/mL to 100 pg/mL （P〈0.05）. The effect of PDGF-D at 100 pg/mL was stronger than at other concentrations. Incubation of tumor cells with PDGF-D markedly decreased the percentage of cells in G0/G1-phase and increased the percentage of cells in S-phase. Compared with the control group, VEGF and PDGFRβ mRNA expression in tile experimental groups （PDGF-D from 10 μg/mL to 200 μg/mL） was increased （P〈0.05）. VEGF protein expression in the experimental groups was significantly enhanced by exogenous PDGF-D in a dose-dependent manner, except in the 5 pg/mL group. Similarly, VEGF protein expression in the experimental groups was significantly higher than that in the control group. Conclusion： PDGF-D can promote proliferation of hepatocaroinoma BEL-7402 cells and increase expression of VEGF in a dose-dependent manner. PDGF-D may play an important role in the development of HCC and can be used as an index for prognosis evaluation and a target of treatment for liver cancer.