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目的:研究VEGF-C对体外培养的宫颈癌HeLa细胞增殖和凋亡的影响;研究VEGF-C受体KDR、信号通路PI3K、MAPK在VEGF-C对宫颈癌增殖和凋亡调控中的作用。方法:应用重组人VEGF-C蛋白体外刺激宫颈癌HeLa细胞,MTT法检测细胞增殖,流式细胞仪检测细胞周期和凋亡,Western blot检测增殖与凋亡相关基因Bcl-2、CyclinD1蛋白表达;应用KDR-Ab、信号通路PI3K抑制剂LY294002、信号通路MAPK抑制剂PD98059预处理HeLa细胞,再进行VEGF-C刺激,观察上述指标的变化。结果:重组VEGF-C(50ng/μl)刺激HeLa细胞后增殖指数增加(2.13 vs 1),细胞周期S期比率增多[(64.26±0.20)%vs(30.91±0.09)%,P<0.05],细胞凋亡率降低(3.29±0.35 vs 7.44±0.55,P<0.05);Bcl-2、Cyclin D1表达增加(P<0.05)。KDR-Ab、LY294002预处理后与VEGF-C组相比增殖指数降低,细胞周期S期比率下降,凋亡指数升高,Bcl-2、Cyclin D1表达降低。PD98059预处理后,与VEGF-C组相比增殖指数降低、细胞周期S期比率下降、Bcl-2、Cyclin D1表达降低,但对VEGF-C诱导的凋亡无明显影响。结论:外源性VEGF-C作用于肿瘤细胞自身的KDR受体,激活细胞内信号传导通路MAPK途径和(或)PI3K途径诱导Cyclin D1表达,使肿瘤细胞S期加快,促进细胞周期的进程,进而促进He-La细胞增殖;通过PI3K途径诱导Bcl-2表达,抑制凋亡。
Objective: To study the effect of VEGF-C on the proliferation and apoptosis of cervical cancer HeLa cells cultured in vitro and to study the role of VEGF-C receptor KDR, PI3K and MAPK in the regulation of proliferation and apoptosis of cervical cancer. Methods: HeLa cells were stimulated with recombinant human VEGF-C protein in vitro. Cell proliferation was detected by MTT assay. Cell cycle and apoptosis were detected by flow cytometry. The expressions of Bcl-2 and CyclinD1 proteins were detected by Western blot. HeLa cells were pretreated with KDR-Ab, PI3K inhibitor LY294002, and MAP9 inhibitor PD98059, and then VEGF-C stimulation was performed to observe the changes of these indexes. Results: Proliferation index (2.13 vs 1) was increased after HepG2 cells were stimulated with recombinant VEGF-C (2.32 vs 1) (P <0.05) (3.29 ± 0.35 vs 7.44 ± 0.55, P <0.05). The expressions of Bcl-2 and Cyclin D1 increased (P <0.05). Compared with VEGF-C group, KDR-Ab and LY294002 pretreatment decreased proliferation index, S phase ratio of cell cycle, apoptosis index, Bcl-2 and Cyclin D1 expression decreased. PD98059 pretreatment, compared with VEGF-C group, proliferation index decreased, cell cycle S phase ratio decreased, Bcl-2, Cyclin D1 expression decreased, but no significant effect on VEGF-C induced apoptosis. CONCLUSION: Exogenous VEGF-C acts on tumor cell’s own KDR receptor, activates intracellular signal transduction pathway MAPK pathway and / or PI3K pathway to induce Cyclin D1 expression, accelerates S phase of tumor cells and promotes cell cycle progression, Thereby promoting the proliferation of He-La cells; inhibiting the apoptosis by inducing the expression of Bcl-2 through the PI3K pathway.