Wnt5a对HaCaT细胞的增殖、周期及血管内皮生长因子的影响

Impact of Wnt5a on HaCaT Cell Proliferation,Cycle and Vascular Endothelial Growth Factor

目的:以HaCaT细胞为细胞模型,探讨Wnt5a能否激活非经典Wnt5a/Ca^2+信号传导途径,并检测其对HaCaT细胞增殖、周期和对血管内皮生长因子(VEGF)的影响,探讨Wnt5a在银屑病发病中的作用机制。方法:采用CCK-8检测不同浓度Wnt5a重组蛋白对HaCaT细胞增殖的影响;利用流式细胞仪技术测定Wnt5a对HaCaT细胞周期和Ca^2+浓度的影响;不同浓度Wnt5a和Frizzled5分别刺激HaCaT细胞后,分别采用RT-PCR和Western blot技术检测HaCaT细胞内VEGF在mRNA和蛋白水平的表达变化。结果:5μg/mL、15μg/mL和25μg/mL的Wnt5a能够不同程度的提高HaCaT细胞的增殖能力,促进细胞周期从G1期向S期分化,其中15μg/mL和25μg/mL组与空白组相比具有明显差异(P<0.01)。同时,15μg/mL和25μg/mL的Wnt5a可增加HaCaT细胞内Ca^2+浓度(P<0.05)。RTPCR和Western blot结果显示,Wnt5a和Frizzled5能不同程度的促进VEGF的表达,其中Wnt5a的效果更显著(P<0.05)。结论:外源性Wnt5a能促进HaCaT细胞增殖和周期,并增加细胞内Ca^2+浓度,进而激活Wnt5a/Ca^2+非经典信号传导途径,从而促进VEGF的表达。

Objective： To study whether exogenous Wnt5 a activation nonclassical Wnt5 a/Ca2＋ signaling pathway, and adopting HaCaT cell as cell model to determine the HaCaT cell prolifera- tion, cell cycle and impact of vascular endothelial growth factor （VEGF）. Methods： CCK-8 was used to detect the effect of different concentrations of recombinant protein Wnt5a on HaCaT cell proliferation, flow cytometry assay was used to detect the effect of exogenous Wnt5a on HaCaT cell cycle and Ca2＋concentrations. Then HaCaT cells were stimulated by Wnt5a and Frizzled5 in dif- ferent concentrations, RT-PCR and Western blot were used to detect the expression of VEGF mR- NA and protein levels in HaCaT cells. Results： Wnt5a can improve the proliferation of HaCaT cells and promote cell cycle progression from G1 to S phase. Meanwhile, the concentration of 15 μg/mL and 25 μg/mL Wnt5a can increase the Ca2＋ concentration in the HaCaT cell （P 〈 0. 05 ）. RT-PCR and Western blot showed that, Wnt5a and Frizzled5 can promote different levelsof expression of VEGF, and WntSa showed more significant effect （P 〈 0. 05 ）. Conclusion： WntSa can promote HaCaT cell proliferation and cell cycle, and increase intracellular Ca2＋ concentration, then activate WntSa/Ca2＋ non-classical signaling pathway, and promote the expression of VEGF.