论文部分内容阅读
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 knock down the gene of CYLD by short hairpin RNA (shRNA) and to observe the effect of inhibiting the expression of CYLD on the biological behavior of human umbilical vein endothelial cells (HUVECs) and to study its signaling pathways. Methods HUVECs were infected with CYLD shRNA adenovirus (experimental group) and green fluorescent protein (GFP) negative control adenovirus (control group) respectively. Cell proliferation was detected by CCK-8 assay, cell migration assay was performed by cell scratch assay, and levels of related signaling molecules were detected by Western blot. Results The results of CCK-8 assay showed that the cell proliferation of CYLD shRNA group was decreased at 5, 1, 2, 3, 4 and 5 days. Scratch assay showed that the cell migration rate of CYLD shRNA group was significantly decreased Compared with GFP group, Western blot results showed that phosphorylation of protein kinase B (AKT) and glycogen synthase kinase 3α / β (GSK3α / β) in CYLD shRNA group decreased. Conclusion Inhibition of CYLD gene expression inhibits the proliferation and migration of HUVECs by inhibiting the AKT / GSK3α / β signaling pathway.