肝癌细胞中血管内皮生长因子的自分泌及其意义

Autocrinism of vascular endothelial growth factor in human hepatocellular carcinoma cell and its significance

目的:研究人肝癌细胞中血管内皮生长因子(VEGF)的自分泌及其意义。方法:反转录PCR(RT-PCR)检测体外培养的人肝细胞肝癌细胞系SMMC7721、HHCC和HepG2中VEGF受体的表达。采用人工合成的反义寡核苷酸(ODN)抑制肝癌细胞VEGF的表达后,分别检测肝癌细胞增殖活性和超微结构的变化,流式细胞仪分析肝癌细胞周期以及VEGF受体蛋白表达的变化。结果:VEGf受体1(Flt-1)在SMMC7721细胞中有表达,而HHCC和HepG2细胞则表达VEGF受体2(KDR)。反义ODN对HHCC细胞的增殖有抑制作用,且与ODN的作用浓度呈线性关系,浓度为2.5μmol/L、5μmol/L和10μmol/L时细胞增殖抑制率分别为26%、41%和50%。反义ODN对SMMC7721细胞则无此作用,而正义ODN对上述2种细胞均无作用。反义ODN作用后,HHCC细胞出现S期比率下降,并且在细胞周期曲线中出现凋亡前期峰,而SMMC7721细胞无上述变化。反义ODN能降低HHCC细胞膜表面KDR的表达(阳性率分别为:反义组19.2%,正义组29.8%,对照组31.2%),对于SMMC7721细胞Flt-1的膜表面表达则无明显影响。结论:人肝癌细胞中存在VEGF的自分泌途径,其机制可能为通过诱导肝癌细胞膜表面KDR的表达及其信号转导作用,从而调节肝癌细胞的分裂增殖。

Purpose: To explore the autocrinism of vascular endothelial growth factor ( VEGF) in human hepatocellular carcinoma cell and its significance. Methods: The expression of VEGF receptors was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) in human hepatocellular carcinoma cell lines SMMC7721, HHCC and HepG2 in vitro. The expression of VEGF in human hepatocellular carcinoma cells was inhibited by synthetic antisense oligodeoxynucleotide (ODN). Phenotypic alterations in human hepatocellular carcinoma cells by antisense reduction of VEGF expression were observed. And the alterations for expression of VEGF receptors in human hepatocellular carcinoma cell membranes were analyzed by flow cytometry. Results: Flt-1 was expressed faintly in SMMC-7721 cell whereas KDR was expressed in HHCC cell and HepG2 cell in vitro. Compared to control, antisense ODN against VEGF inhibited HHCC cell proliferation in vitro in a dose-dependent manner and had no effects on SMMC-7721 cell and L929 cell. When the concentration of antisense ODN was 2.5 μmol/L,5 μmol/L and 10 μmol/L, the proliferation of HHCC cell was inhibited by 26%, 41 % , and 50% respectively. Compared with control, flow cytometry analysis showed a decrease in cell number in S phase and a pre-apoptosis peak in HHCC cell treated by antisense ODN. There was no alteration of cell cycle in SMMC-7721 cell. The positive rate of KDR expression in HHCC cell membrane was 19. 2%, 29. 8% and 31. 2% in the antisense ODN, sense ODN and control groups, respectively. The expression of KDR in HHCC cell membrane was markedly inhibited by antisense ODN against VEGF compared with the control group, as measured by flow cytometry. There was no alteration for expression of Flt-1 in SMMC-7721 cell membrane. Conclusions: VEGF produced by human hepatocellular carcinoma cells acts directly upon human hepatocellular carcinoma in an autocrinal manner and may promote the proliferation and differentiation of human hepatocellular carcinoma cells via the activation of the KDR-dependent signaling pathway.