蛋白激酶C-α信号通路在尼古丁诱导的脐静脉内皮细胞PAI-1表达中的作用

The Role of Protein Kinase C-αSignal Pathway in Nicotine-induced Plasminogen Activator Inhibitor-1 Expression in Human Umbilical Vein Endothelial Cells

目的探讨蛋白激酶C-α(PKC-α)信号通路在尼古丁诱导的人脐静脉内皮细胞(HUVECs)纤溶酶原激活物抑制物-1(PAI-1)表达中的作用。方法体外培养HUVECs,随机分为4组:对照组、尼古丁组、STS组及尼古丁+STS组。收集各组细胞及上清液,采用酶联免疫吸附法(ELISA)测定上清液PAI-1蛋白含量,细胞逆转录-聚合酶链反应(RT-PCR)检测细胞PAI-1 mRNA表达,免疫荧光染色观察PKC-α在细胞内的定位变化,蛋白免疫印迹(Western Blot)法测定细胞胞浆与胞膜PKC-α的表达水平。结果尼古丁组PAI-1蛋白及mRNA表达水平较对照组升高,差异均有统计学意义(P均<0.01);尼古丁+STS组PAI-1蛋白及mRNA表达水平较尼古丁组下降,但仍高于对照组,差异均有统计学意义(P均<0.01)。对照组HUVECs中PKC-α的绿色荧光均匀分布于细胞浆中;尼古丁孵育后胞内PKC-α发生移位,在细胞膜出现明显的绿色荧光;STS处理组胞浆的绿色荧光减弱;尼古丁+STS组细胞膜荧光强度低于尼古丁组。尼古丁组胞膜PKC-α蛋白增高,胞浆PKC-α蛋白降低,胞膜/胞浆PKC-α蛋白表达增高,与对照组比较差异均有统计学意义(P均<0.01)。尼古丁+STS组胞膜PKC-α蛋白较尼古丁组降低,胞浆PKC-α蛋白较尼古丁组升高,胞膜/胞浆PKC-α蛋白较尼古丁组降低,差异均有统计学意义(P均<0.01)。胞膜PKC-α表达与PAI-1 mRNA及蛋白表达呈正相关(r值分别为0.813、0.882,P均<0.05);胞浆PKC-α蛋白与PAI-1 mRNA及蛋白表达呈负相关(r值分别为-0.744、-0.797,P均<0.05)。结论在尼古丁诱导HUVECs上调PAI-1表达过程中,伴随着细胞内PKC-α蛋白膜转位及表达变化,PKC-α信号通路参与内皮细胞纤溶紊乱。

Objective To investigate the possible role of protein kinase C-α(PKC-α)signaling pathway in nicotine-induced plasminogen activator inhibitor-1(PAI-1)expression in human umbilical vein endothelial cells(HUVECs).Methods HUVECs were cultured in vitro and randomly divided into four groups:control group(A),nicotine group(B),STS group(C),and nicotine+STS group(D).The PAI-1 protein content in the supernatant was determined by enzyme-linked immuno sorbent assay method,and the expression of PAI-1 mRNA in the supernatant was detected by cell reverse transcription-polymerase chain reaction(RT-PCR),the localization of PKC-αin cells was observed by immunofluorescence staining,and the expression of PKC-αin cytoplasm and membrane was determined by western blot.Results PAI-1 protein and mRNA in nicotine group were significantly higher than those in control group(P<0.01),PAI-1 protein and mRNA in nicotine+STS group was lower than nicotine group,but still higher than that in control group,the difference was statistically significant(P<0.01).The green fluorescence of PKC-αprotein was distributed in cytoplasm in control group,shifted in nicotine group.The green fluorescence appeared around the cell membrane and decreased in nicotine+STS group,and the fluorescence intensity of cell membrane in nicotine+STS group was weaker than that in nicotine group.Compared with control group.the cell membrane PKC-αprotein increased,the cytoplasm PKC-αprotein decreased,the PKC-αprotein expression ratio in membrane/cytoplasm increased in nicotine group(P<0.01).In nicotine+STS group,PKC-αcell membrane protein was lower than that in nicotine group,cytoplasm PKC-αprotein was higher than that in nicotine group,and PKC-αprotein in membrane/cytoplasm was lower than that in nicotine group(P<0.01).The expression of cell membrane PKC-αwas positively correlated with the expression of PAI-1 mRNA and protein(r=0.813 and 0.882,P<0.05),while the expression of cytoplasm PKC-αprotein was negatively correlated with the expression of PAI-1 mRNA and protein(r=-0.744,-0.797,P<0.05).Conclusion During nicotine-induced up-regulation of PAI-1 expression in HUVECs,PKC-αsignal pathway is involved in endothelial fibrinolysis,along with PKC-αprotein membrane transposition and expression changes.