摘要
目的研究低分子肝素（LMWH）对不同肿瘤细胞系分泌血管内皮生长因子（VEGF）的影响，以及其对VEGF所诱导的肿瘤血管内皮细胞增殖的作用。方法采用酶联免疫吸附（ELISA）法，检测LMWH和肝素对肺腺癌A549细胞、肝癌HepG2细胞、结肠腺癌HCT116和HCT8细胞分泌VEGF和肿瘤坏死因子α（TNF-α）蛋白的影响。采用逆转录聚合酶链反应（RT-PCR）和实时定量PCR法，检测LMWH和肝素对HepG2细胞表达VEGF mRNA的影响。采用非放射性细胞增殖检测试剂盒和流式细胞仪，检测肿瘤条件培养基在有无LMWH作用时对脐静脉血管内皮细胞（HUVEC）增殖和细胞周期的影响。结果对照组、LMWH组和肝素组A549细胞中，VEGF蛋白的表达量分别为（1045．89±165．30）pg／ml、（782．45±67．17）pg／ml和（916．54±71．25）pg／ml；对照组、LMWH组和肝素组HCT116细胞中，VEGF蛋白的表达量分别为（955．76±51．14）pg／ml、（822．89±142．39）pg／ml和（951．77±188．22）ps／ml；对照组、LMWH组和肝素组HCT8细胞中，VEGF蛋白的表达量分别为（1290．62±41．23）pg／ml、（1063．34±63．82）ps／ml和（1257．14±11．40）pg／ml；对照组、LMWH组和肝素组HepG2细胞中，VEGF蛋白的表达量分别为（1083．00±134．35）pg／ml、（758．00±84．85）pg／ml和（874．00±22．62）pg／ml。LMWH可抑制不同肿瘤细胞系中VEGF蛋白的表达（均P〈0．05），但对TNF-α蛋白的表达没有影响。LMWH可抑制HepG2细胞中VEGF mRNA的表达。肿瘤条件培养基可以诱导HUVEC增殖，加入LMWH的肿瘤条件培养基中，HUVEC增殖减少，G0±G1期细胞比例增加。结论LMWH可抑制不同的肿瘤细胞系分泌VEGF，并具有抑制VEGF诱导的血管内皮细胞增殖的作用。
Objective To investigate whether low molecular weight heparin （LMWH） may suppress the expression and secretion of vascular endothelial growth factor （VEGF） from tumor cells in vitro and inhibit the VEGF-induced proliferation of human tumor vascular endothelial cells. Methods Human lung cancer cell line A549, human liver cancer cell line HepG2, human colon carcinoma cell lines HCT116 and HCT8 were used in this study. The expression levels of VEGF and TNF-α （ tumor necrosis factor-α） in the tumor ceils with or without pretreatment of LMWH/heparin were measured by standard sandwich ELISA technique. The VEGF mRNA level of HepG2 cells cultured with or without LMWH/heparin was determined by RT-PCR and real time PCR. Human umbilical vein endothelial cells （HUVEC） were cultured in tissue culture medium （TCM） with or without LMWH/heparin for 3 days. Then non-radioactive cell proliferation assay （MTS） kit and cell cycle assay by flow cytometry were performed to measure the proliferation of HUVEC. Results The VEGF levels in the control, LMWH, and heparin groups of the pulmonary adenocareinoma cell line A549 were （1045.89 ± 165.30） pg/ml, （782.45 ± 67.17） pg/ml and （916. 54 ± 71.25） pg/ml, respectively. The VEGF levels in the control, LMWH, and heparin groups of the colon adenocarcinoma cell line HCT116 were （955.76 ±51.14 ）pg/ml, （822.89 ± 142.39） pg/ml and （951.77 ±188.22） pg/ml, respectively. The VEGF levels in the control, LMWH, and heparin groups in the colon adenocarcinoma cell line HCT8 were （1290. 62 ± 41.23） pg/ml, （1063.34±63.82） pg/ml and （1257.14± 11. 40）pg/ml, respectively. The VEGF levels in the control, LMWH, and heparin groups in the liver cancer cell line HepG2 were （1083.00 ±134.35） pg/ml, （758.00±84. 85） pg/ml and （874. 00 ± 22.62） pg/ml, respectively. The VEGF expression levels in the above mentioned cell lines cultured in TCM were significantly reduced in the LMWH-treated groups compared with that of the control group （ P 〈 0.05）. But the level of TNF-α in TCM-cultured cells was unaffected by LMWH. The VEGF mRNA was reduced in the LMWH-treated HepG2 cell line. Moreover, TCM exhibited stimulating effect on proliferation of HUVEC and the effect was significantly impaired by LMWH treatment. Flow cytometric analysis revealed that LMWH treatment arrested HUVECs at the G1 phase of cell cycle. Conclusion LMWH can suppress the expression and secretion of VEGF by tumor cell lines and therefore have a potential inhibiting effect on angiogenesis induced by VEGF.
出处
《中华肿瘤杂志》
CAS
CSCD
北大核心
2009年第11期826-830,共5页
Chinese Journal of Oncology
关键词
低分子肝素
肿瘤细胞
血管内皮生长因子
Low molecular weight heparin （LMWH）
Tumor cells
Vascular endothelial growth factor （VEGF）
