Chinese Medical Sciences Journal ›› 2021, Vol. 36 ›› Issue (1): 35-42.doi: 10.24920/003753

• 论著 • 上一篇    下一篇

骨肉瘤MG63细胞中VEGF-C/VEGFR-3/iNOS信号转导对人脐静脉内皮细胞增殖的促进作用

吕杰,原杰,徐朝健,郝家齐,秦一川,王小强,王永峰()   

  1. 山西医科大学第二医院骨科,太原 030001,中国
  • 收稿日期:2020-04-11 接受日期:2020-05-14 出版日期:2021-02-07 发布日期:2021-02-07
  • 通讯作者: 王永峰 E-mail:wyfwf8@163.com

VEGF-C/VEGFR-3/iNOS Signaling in Osteosarcoma MG63 Cells Mediates Stimulatory Effects on Human Umbilical Vein Endothelial Cell Proliferation

Jie Lv,Jie Yuan,Chaojian Xu,Jiaqi Hao,Yichuan Qin,Xiaoqiang Wang,Yongfeng Wang()   

  1. Department of Orthopaedics, the Second Hospital of Shanxi Medical University, Taiyuan 030001, China
  • Received:2020-04-11 Accepted:2020-05-14 Online:2021-02-07 Published:2021-02-07
  • Contact: Yongfeng Wang E-mail:wyfwf8@163.com

摘要:

目的探讨血管内皮生长因子-C(vascular endothelial growth factor-C,VEGF-C)/血管内皮生长因子受体-3(vascular endothelial growth factor receptor-3,VEGFR-3)信号转导对人骨肉瘤MG63细胞中一氧化氮(nitric oxide,NO)的生成和诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)的表达以及对人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)增殖的影响。

方法MG63细胞分别以VEGF-C单独处理(VEGF-C组),VEGF-C联合iNOS抑制剂氨基胍AG(AG组)以及VEGF-C联合VEGFR-3抑制剂MAZ-51(MAZ-51组)处理,将未处理的MG63细胞作为对照。采用硝酸还原酶比色法测定NO生成量,逆转录-聚合酶链反应(reverse transcription polymerase chain reaction,RT-PCR)和Western blot分别检测iNOS的mRNA和蛋白水平。为研究MG63细胞中VEGF-C/VEGFR-3/iNOS信号通路对HUVECs增殖的影响,实验设立如下6组:HUVECs组、HUVECs+MG63组、HUVECs+VEGF-C组、HUVECs+MG63+VEGF-C组、HUVECs+MG63+VEGF-C+AG组及HUVECs+MG63+VEGF-C+MAZ51组。用细胞计数试剂盒(Cell Counting Kit-8,CCK-8)、5-乙炔基-2'-脱氧尿嘧啶核苷(5 ethynyl 2' deoxyuridine,EdU)掺入法和增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)定量表达评估各组HUVECs的增殖情况。

结果VEGF-C促进了MG63细胞中iNOS在基因(LSD-t=4.152,P<0.01)和蛋白水平的表达(LSD-t=3.486,P<0.01),增加了NO释放(LSD-t=3.774,P<0.01)。经AG或MAZ51处理均可降低上述作用(mRNA:LSD-t=9.183,P<0.001;LSD-t=8.639,P<0.001;protein:LSD-t=5.170,P<0.001;LSD-t=7.255,P<0.001;NO:LSD-t=10.326,P<0.001;LSD-t=10.540,P<0.001)。HUVECs与MG63细胞和/或VEGF-C共同培养可显著促进HUVECs的增殖(EdU:LSD-t=5.374,P<0.001;LSD-t=2.984,P<0.05;LSD-t=8.526,P<0.001;PCNA: LSD-t=9.267,P<0.001;LSD-t=5.515,P<0.001;LSD-t=14.873,P<0.001)。经AG(EdU:LSD-t=10.770,P<0.001;PCNA:LSD-t=19.940,P<0.001)或MAZ51(EdU:LSD-t=6.950,P<0.001;PCNA:LSD-t=14.001,P<0.001)处理后,MG63细胞和VEGF-C对HUVECs增殖的诱导作用减弱。

结论VEGF-C激活VEGFR-3可促进在人骨肉瘤MG63细胞iNOS的表达和NO的产生,进而诱导HUVECs的增殖。

关键词: 骨肉瘤, 血管内皮生长因子-C, 血管内皮生长因子受体-3, 诱导型一氧化氮合酶, 肿瘤血管生成

Abstract:

Objective To assess the effects of vascular endothelial growth factor-C (VEGF-C)/vascular endothelial growth factor receptor-3 (VEGFR-3) signaling on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in human osteosarcoma MG63 cells and the subsequent impact on the proliferation of human umbilical vein endothelial cells (HUVECs).

MethodsMG63 cells were treated with VEGF-C alone (VEGF-C group), VEGF-C + iNOS inhibitor aminoguanidine (AG; AG group), and VEGF-C + VEGFR-3 inhibitor MAZ51 (MAZ51 group); untreated MG63 cells were used as controls. NO production was evaluated by a colorimetric method involving nitrate reductase. Meanwhile, mRNA and protein levels of iNOS were examined by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. To explore the effect of VEGF-C/VEGFR-3/iNOS signaling of MG63 cells on proliferation of HUVECs, we set up six groups: HUVECs, HUVECs+MG63, HUVECs+VEGF-C, HUVECs+MG63+VEGF-C, HUVECs+MG63+VEGF-C+AG, and HUVECs+MG63+VEGF-C+MAZ51 groups. The proliferation of HUVEC cells was assessed by Cell Counting Kit-8 (CCK-8), 5-ethynyl-2’-deoxyuridine (EdU) incorporation assay, and proliferating cell nuclear antigen (PCNA) expression quantitation.

ResultsVEGF-C treatment enhanced iNOS expression at both gene and protein levels (mRNA: LSD-t=4.152, P<0.01; protein: LSD-t=3.486, P<0.01) and increased NO release of MG63 cells (LSD-t=3.774, P<0.01); treatment with either AG or MAZ51 decreased these effects (mRNA: LSD-t=9.183, P<0.001; LSD-t=8.639, P<0.001; protein: LSD-t=5.170, P<0.001; LSD-t=7.255, P<0.001; NO production:LSD-t=10.326, P<0.001; LSD-t=10.540, P<0.001). Interestingly, co-incubation of HUVECs with MG63 cells and/or VEGF-C significantly promoted HUVEC proliferation (EdU: LSD-t=5.374, P<0.001; LSD-t=2.984, P<0.05; LSD-t=8.526, P<0.001; PCNA: LSD-t=9.267, P<0.001; LSD-t=5.515, P<0.001; LSD-t=14.873, P<0.001).The proliferation effects of HUVEC induced by MG63 cells and VEGF-C attenuated by the treatment of AG (EdU: LSD-t=10.770, P<0.001; PCNA: LSD-t=19.940, P<0.001) or MAZ51 (EdU: LSD-t=6.950, P<0.001; PCNA: LSD-t=14.001, P<0.001).

ConclusionIn human osteosarcoma MG63 cells, activation of VEGFR-3 by VEGF-C promotes iNOS expression and NO production, which subsequently induces HUVEC proliferation.

Key words: osteosarcoma, VEGF-C, VEGFR-3, iNOS, tumor angiogenesis

基金资助: 山西省自然科学基金(201801D121220)

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