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中华普通外科学文献(电子版) ›› 2019, Vol. 13 ›› Issue (02) : 97 -102. doi: 10.3877/cma.j.issn.1674-0793.2019.02.003

所属专题: 文献

论著

血管内皮细胞生长因子促进肝内胆管细胞癌生长的机制研究
王伊菲1, 梁锐明2, 韦广滟1, 储鸿鹏1,()   
  1. 1. 510080 广州,中山大学附属第一医院肝外科
    2. 510080 广州,中山大学附属第一医院临床试验中心
  • 收稿日期:2018-12-02 出版日期:2019-04-01
  • 通信作者: 储鸿鹏
  • 基金资助:
    广州市科技计划项目(201704020099)

Functional mechanism of vascular endothelial growth factor in promoting the growth of intrahepatic cholangiocarcinoma

Yifei Wang1, Ruiming Liang2, Guangyan Wei1, Hongpeng Chu1,()   

  1. 1. Department of Liver Surgery, Guangzhou 510080, China
    2. Clinical Trials Unit, the First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, China
  • Received:2018-12-02 Published:2019-04-01
  • Corresponding author: Hongpeng Chu
  • About author:
    Corresponding author: Chu Hongpeng, Email:
引用本文:

王伊菲, 梁锐明, 韦广滟, 储鸿鹏. 血管内皮细胞生长因子促进肝内胆管细胞癌生长的机制研究[J]. 中华普通外科学文献(电子版), 2019, 13(02): 97-102.

Yifei Wang, Ruiming Liang, Guangyan Wei, Hongpeng Chu. Functional mechanism of vascular endothelial growth factor in promoting the growth of intrahepatic cholangiocarcinoma[J]. Chinese Archives of General Surgery(Electronic Edition), 2019, 13(02): 97-102.

目的

探究血管内皮细胞生长因子(VEGF)及其受体对肝内胆管细胞癌(ICC)细胞生长调控的作用机制。

方法

Western blotting检测VEGF在12例ICC患者肿瘤组织和癌旁正常组织中的表达水平。采用外源性重组人源VEGF(rhVEGF)处理ICC细胞株Huh28后,采用细胞计数检测细胞生长情况,5-溴脱氧尿嘧啶核苷(BrdU)实验检测细胞增殖,流式细胞术检测细胞凋亡。Western blotting检测rhVEGF处理后Huh28细胞中VEGF受体VEGFR1及VEGFR2的表达情况。使用特异性抗体分别阻断VEGFR1及VEGFR2,通过ELISA法检测细胞的凋亡水平。采用慢病毒shRNA构建稳定敲低VEGFR2的ICC细胞株Huh28-shVEGFR2(实验组)和对照细胞株Huh28-shNC(对照组)。采用Huh28-shVEGFR2及Huh28-shNC细胞在10只裸鼠中构建皮下瘤模型,观察肿瘤的生长情况。

结果

VEGF在ICC肿瘤组织中蛋白表达水平上调,显著高于癌旁正常组织(P<0.01)。外源性rhVEGF可以促进Huh28细胞生长,抑制细胞凋亡,而对细胞增殖能力无明显影响。rhVEGF处理后Huh28细胞中磷酸化的VEGFR1及VEGFR2表达水平升高(P<0.05)。VEGFR2抗体可以显著逆转rhVEGF介导的抗凋亡作用(P<0.05),而VEGFR1抗体则无明显效果。皮下瘤模型结果显示,与对照组相比,肿瘤生长在实验组中受到显著抑制,差异有统计学意义(P<0.05)。

结论

VEGF是通过VEGFR2依赖的信号通路抑制ICC细胞凋亡,从而促进ICC细胞生长。

Objective

To investigate the role of vascular endothelial growth factor (VEGF) and VEGF receptors on cell growth of intrahepatic cholangiocarcinoma (ICC).

Methods

Western blotting was performed to detect the expression of VEGF in tumour tissues and paired normal tissues from twelve patients with ICC. ICC cell line Huh28 was treated with exogenous recombinant human VEGF (rhVEGF). Cell growth was evaluated by cell counting, proliferation was detected by BrdU cell proliferation assay, and apoptosis was detected by flow cytometry. The expression of VEGF receptors VEGFR1/VEGFR2 in Huh28 cells after rhVEGF treatment were detected by Western blotting. VEGFR1 and VEGFR2 were blocked by specific antibodies, and cell apoptosis was examined by apoptosis-ELISA assay. The stably knockdown VEGFR2 cell line Huh28-shVEGFR2 (experimental group) and the control cell line Huh28-shNC (control group) were established using shRNA lentivirus. Subcutaneous tumour models in ten nude mice with Huh28-shVEGFR2 and Huh28-shNC were used to observe tumour growth.

Results

The protein expression of VEGF was up-regulated in ICC tumour tissues than in matched normal tissues (P<0.01). Exogenous rhVEFG could promote the growth of Huh28 cells, suppressed cell apoptosis, without significant effect on cell proliferation ability. The expression of phosphorylated VEGFR1 and VEGFR2 in Huh28 cells were up-regulated after rhVEGF treatment (P<0.05). VEGFR2 antibody could significantly reverse the anti-apoptosis effect of rhVEGF (P<0.05), while VEGFR1 antibody did not. Subcutaneous tumour models indicated that tumour growth in experimental group (Huh28-shVEGFR2) was significantly inhibited compared with the control group (Huh28-shNC), the difference was significant (P<0.05).

Conclusion

VEGF promotes ICC cells growth through inhibiting apoptosis of ICC cells in a VEGFR2-dependent signalling pathway.

图1 Western blotting检测VEGF在胆管细胞癌肿瘤组织(T)和癌旁正常组织(N)中的蛋白表达水平  **P<0.01
图2 外源性VEGF对Huh28细胞生长的影响 A为细胞计数实验检测rhVEGF处理后不同时间点的细胞数目;B为流式细胞术检测hVEGF处理后细胞的凋亡情况,SM为饥饿培养基,*P<0.05;C为BrdU实验检测hVEGF处理后细胞的增殖能力
图3 VEGFR2参与VEGF介导的抗凋亡作用 A为Western blotting检测rhVEGF处理后VEGF受体活化情况;B为ELISA检测细胞凋亡情况,SM为饥饿培养基,*P<0.05;C为Western blotting验证shRNA敲低效率的结果;D为动物模型皮下移植瘤生长曲线
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