敲低圆柱瘤病(CYLD)基因抑制人脐静脉内皮细胞的增殖及迁移

Inhibition of proliferation and migration of human umbilical vein endothelial cells by knockdown of cylindromatosis( CYLD) gene

目的通过短发夹RNA(shRNA)敲低圆柱瘤病(CYLD)基因,观察抑制CYLD表达后对人脐静脉内皮细胞(HUVEC)生物学行为的影响并研究其参与的信号通路。方法将HUVEC分别感染CYLD shRNA腺病毒(实验组)及绿色荧光蛋白(GFP)空载腺病毒(对照组)。分别用CCK-8法检测细胞增殖,细胞划痕实验检测细胞迁移能力,Western blot法检测相关信号通路分子的水平。结果 CCK-8法结果显示,CYLD shRNA组在第1、2、3、4、5天5个时间点,细胞增殖降低;划痕实验结果显示划痕48 h,CYLD shRNA组细胞迁移速率明显降低;与GFP组相比,Western blot结果显示CYLD shRNA组的蛋白激酶B(AKT)、糖原合成激酶3α/β(GSK3α/β)磷酸化水平均降低。结论抑制CYLD基因表达,可抑制HUVEC的增殖能力及细胞迁移能力,是通过抑制AKT/GSK3α/β信号通路完成的。

Objective To down-regulate the expression of cylindromatosis（CYLD） gene by shRNA and investigate its effect on proliferation,migration and cell signal pathways of human umbilical vein endothelial cells（ HUVECs）. Methods HUVECs were infected by adenoviruses of CYLD shRNA（ experiment group） and GFP（ control group）,respectively. The proliferation of HUVECs was detected by CCK-8 assay; the cell migration was assessed by a wound healing assay; the related cell signal pathways were analyzed by Western blotting. Results CCK-8 showed that the proliferation of the CYLD shRNA-infected HUVECs were significantly lower than that of the control group at 1,2,3,4,5 days after infection. The wound healing assay revealed that the migration rate of the CYLD shRNA-infected HUVECs was significantly reduced compared with the control HUVECs at 48 hours. Western blotting indicated that the phosphorylation of protein kinase B（AKT） and glycogen synthase kinase 3α/β（ GSK3α/β） significantly decreased in the CYLD shRNA-infected HUVECs compared with the control HUVECs. Conclusion Inhibiting the expression of CYLD gene can suppress the proliferation and migration of HUVECs,which may be attributed to the inhibition of the AKT / GSK3α / β pathway.