激酶非催化的C-叶域蛋白质1对人脐静脉内皮细胞衰老作用的影响及机制

Molecular mechanism of overexpression of kinase non-catalytic C-lobe domin containing on senescence of human umbilical vein endothelial cells

目的:探讨激酶非催化的C-叶域蛋白质1(KNDC1)对人脐静脉内皮细胞(HUVEC)衰老作用的分子机制。方法:体外分离培养HUVEC,选择第6代HUVEC转染KNDC1腺病毒为实验组,另设空白对照组与阴性对照组。显微镜下观察细胞形态变化,通过q PCR检测内皮细胞的KNDC1的mRNA的表达,用β-半乳糖苷酶染色检测细胞衰老情况,Westem blot检测衰老相关蛋白,分光光度法检测内皮细胞抗氧化酶活性。结果:第6代的HUVEC呈现出有别于前期传代细胞的衰老特征,细胞变得扁平,体积增大,增殖能力降低;实验组KNDC1的mRNA水平较对照组增高(239%~344%,P<0.05)及蛋白质表达也显著增高(249%~441%,P<0.05),实验组HUVEC的SA-β-gal染色阳性率显著上升(P<0.01),与KNDC1腺病毒转染剂量呈剂量依赖性。实验组磷酸化p53较对照组增加[(244.3±19.5)vs.(113.5±9.0),P<0.05]及抗氧化酶SOD及GPX含量(0.84±0.079);(0.82±0.062)较对照组下降,差异有统计学意义(P<0.05)。结论:KNDC1过度表达可能通过启动p53信号通路增加ROS表达,进而加速细胞衰老的进程。

Objective： To investigate the non catalytic kinase domain protein 1 C-leaves（ KNDC1）on human umbilical vein endothelial cells（ HUVEC） molecular mechanism of senescence. Methods： HUVEC were isolated and cultured in vitro,the sixth generation of HUVEC transfected with KNDC1 adenovirus as the experimental group,blank control group and negative control group. Cell morphological changes were observed under microscope. The expression of endothelial cells for the detection of q PCR KNDC1 mRNA,the aging situation with beta galactosidase staining cells,Westem blot detected protein,antioxidant enzyme activity of endothelial cell were detected by spectrophotometry. Results： The sixth generation of HUVEC showing a characteristic different from the early senescence of cultured cells,cells became flat,the volume increases,the decrease of proliferation; KNDC1 in the experimental group the level of mRNA was higher than those in control group（ 239%-344%,P 〈0. 05） and protein expression was significantly increased（ 249%-441%,P〈 0. 05）,the positive rate of SA-was significantly increased in experimental group HUVEC beta-gal the staining（ P 〈0. 01）,in a dose-dependent manner with KNDC1 transfection dose. The experimental group of phosphorylated p53 increased compared to the control group [（ 244. 3 ± 19. 5） vs.（ 11 3. 5 ± 9,P〈 0. 05] and antioxidant enzyme SOD and GPx content（ 0. 84 ± 0. 079）;（ 0. 82 ± 0. 062） decreased compared with the control group,the difference was statistically significant（ P〈 0. 05）. Conclusion： Overexpression of KNDC1 may increasethe expression of ROS by initiating p53 signaling pathway,and then accelerate the process of cell senescence.