程序性细胞死亡因子4基因对川崎病血清诱导血管内皮细胞凋亡的影响

Effect of siRNA interference with PDCD4 gene on apoptosis of vascular endothelial cells induced by kawasaki disease serum

目的研究程序性细胞死亡因子4(PDCD4)对川崎病病人血清诱导的血管内皮细胞凋亡的作用。方法研究时间为2017年3月至2018年4月。采集邵阳学院附属第一医院川崎病病人血清,处理血管内皮细胞,实时荧光定量PCR(realtime PCR)和蛋白质印迹法检测细胞中PDCD4 mRNA和蛋白表达水平。在血管内皮细胞中转染PDCD4 siRNA,用川崎病血清培养,以Realtime PCR和蛋白质印迹法检测细胞中PDCD4表达水平。MTT方法检测细胞增殖,流式细胞术检测细胞凋亡,蛋白质印迹法检测细胞中活化型半胱氨酸天冬氨酸蛋白酶3(C-Caspase-3)、活化型Caspase-9(C-Caspase-9)蛋白水平。结果川崎病病人血清处理后的血管内皮细胞中PDCD4表达上调[mRNA:(1.00±0.01)比(3.51±0.36);蛋白:(0.26±0.08)比(0.57±0.06)]。PDCD4 siRNA可以下调川崎病病人血清诱导的血管内皮细胞中PDCD4表达水平[mRNA:(0.91±0.09)比(0.41±0.06);蛋白:(0.65±0.08)比(0.30±0.05)]。川崎病病人血清处理后的血管内皮细胞增殖能力降低[(100.00±0.00)%比(72.01±7.32)%],细胞凋亡率升高[(6.32±0.58)%比(21.26±2.31)%],细胞中的C-Caspase-3、C-Caspase-9蛋白水平升高。下调PDCD4可以提高川崎病血清条件下血管内皮细胞增殖能力[(71.57±9.05)%比(89.52±6.80)%],减少细胞凋亡[(22.58±1.79)%比(16.47±1.24)%]和细胞中C-Caspase-3、C-Caspase-9蛋白表达。结论下调PDCD4减少川崎病病人血清诱导的血管内皮细胞凋亡。

Objective To study the role of PDCD4 in the apoptosis of vascular endothelial cells induced by serum from patients with Kawasaki disease.MethodsThe research period was from March 2017 to April 2018.The serum of patients with Kawasaki diseasewas collected and used to culture vascular endothelial cells from The First Affiliated Hospital of Shaoyang University.Realtime PCR and Western blot were used to detect the expression of PDCD4 in cells.into Vascular endothelial cells was transfected with PDCD4 siRNA,and then cultured with Kawasaki disease serum.Realtime PCR and Western blot were used to detect the expression level of PDCD4 in cells.MTT assay was used to detect cell proliferation and apoptosis was detected by flow cytometry.Western blot was used to detect the level of C-Caspase-3 and C-Caspase-9 protein in the cells.ResultsThe expression of PDCD4 was increased in vascular endothelial cells treated by Kawasaki disease serum[mRNA:(1.00±0.01)vs.(3.51±0.36);protein:(0.26±0.08)vs.(0.57±0.06)].PDCD4 siRNA can reduce the expression level of PDCD4 in vascular endothelial cells[mRNA:(0.91±0.09)vs.(0.41±0.06);protein:(0.65±0.08)vs.(0.30±0.05)].Compared with those treated with vehicle,the proliferation of vascular endothelial cells after treatment with Kawasaki disease serum decreased[(100.00±0.00)%vs.(72.01±7.32)%],the rate of apoptosis increased[(6.32±0.58)%vs.(21.26±2.31)%],the level of C-Caspase-3 and C-Caspase-9 protein in cells increased.Down regulation of PDCD4 could enhance the proliferation of vascular endothelial cells in Kawasaki disease[(71.57±9.05)%vs.(89.52±6.80)%],reduce apoptosis[(22.58±1.79)%vs.(16.47±1.24)%]and expression of C-Caspase-3 and C-Caspase-9 protein in cells.ConclusionDownregulation of PDCD4 reduces apoptosis of vascular endothelial cells induced by serum from Kawasaki disease.