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

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

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.