血小板活化因子诱导大鼠肺微血管内皮细胞Src抑制的蛋白激酶C底物基因表达

目的 研究血小板活化因子(PAF)对大鼠肺微血管内皮细胞(RPMVEC)中Src抑制的蛋白激酶C底物(SSeCKS)mRNA表达的影响及其信号转导途径.方法 体外培养RPMVEC,采用细胞原位杂交技术检测不同刺激条件下RPMVEC中SSeCKS mRNA的表达变化.结果 正常RPMVEC中有少量SSeCKSmRNA表达,为棕黄色细颗粒状杂交信号,分布于胞质;用10~(-10)、10~(-9)、10~(-8)、10~(-7) mol/L的PAF分别孵育RPMVEC 1.5 h,SSeCKS mRNA表达随其浓度增加而升高(分别为0.054 9±0.000 7、0.059 9±0.001 8、0.069 0±0.001 8、0.075 4±0.001 9),与正常对照组(0.036 8±0.003 7)比较差异均有统计学意义(均P<0.05);10~(-7) mol/L PAF刺激RPMVEC 0.5、1.5、3、6、12、24 h,SSeCKS mRNA表达水平于0.5 h即显著升高(0.071 0±0.001 5),1.5 h达峰值(0.075 6±0.001 7),之后逐渐下降,24 h时(0.043 9±0.001 0)仍高于正常对照组(0.038 2±0.004 1,均P<0.05);10 μmol/L核转录因子-κB(NF-κB)抑制剂吡咯烷二巯基氨甲酸(PDTC)预处理RPMVEC 1 h可显著下调Par对SSeCKS mRNA的诱导效应(0.049 7±0.003 2比0.071 9±0.001 9,P<0.05),而蛋白激酶C(PKC)抑制剂双吲哚基顺丁烯二酰亚胺(BIM)预处理RPMVEC0.5 h则不影响此效应(0.069 7±0.002 1比0.071 9±0.001 9,P>0.05).结论 PAF呈浓度和时间依赖的方式上调RPMVEC中SSeCKS基因转录;NF-κB信号通路而非PKC参与了PAF的诱导效应.

Induction effect of platelet-activating factor on Src-suppressed C kinase substrate gene expression in rat pulmonary microvascular endothelial cells

Objective To investigate the effect of platelet-activating factor(PAF)on the production of Src-suppressed C kinase substrate(SSeCKS)mRNA in rat pulmonary microvascular endothelial cell (RPMVEC)and its signal transduction pathways.Methods Cellular in situ hybridization was performed to reveal changes in SSeCKS mRNA expression in the cultured RPMVECs after giving PAF stimulation.Results Normal RPMVECs expressed SSeCKS mRNA at a low level,which appeared throughout the cytoplasm with specific hybridization signals.1.5 hours of 10~(-10),10~(-9),10~(-8),10~(-7) mol/L PAF incubation induced a progressive increase in SSeCKS mRNA expression.When compared to the normal control group each difference had statistical significance(0.054 9±0.000 7,0.059 9±0.001 8,0.069 0±0.001 8,0.075 4±0.001 9 vs.0.036 8±0.003 7,respectively,all P＜0.05).Within 0.5,1.5,3,6,12,24 hours of 10~(-7) mol/L PAF challenge,the level of SSeCKS mRNA expression raised at 0.5 hour markedly (0.071 0±0.001 5),peaked at 1.5 hours(0.075 6±0.001 7),then began to decline gradually,and still persisted at a higher level than the normal control group until 24 hours(0.043 9±0.001 0 vs.0.038 2±0.004 1,all P ＜ 0.05).Pre-incubation of 10 μmol/L pyrrolidine dithiocarbamate(PDTC)that inhibits activity of nuclear factor-κB(NF-κB)in RPMVECs caused a conspicuous attenuation of PAF-induced SSeCKS mRNA expression(0.049 7±0.003 2 vs.0.071 9±0.001 9,P＜0.05),whereas no change of PAF-induced effect was found by pretreatment of protein kinase C(PKC)inhibitor bis-indolylmaleimide (BIM,0.069 7±0.002 1 vs.0.071 9±0.001 9,P＞0.05).Conclusion PAF can up regulate the expression of SSeCKS mRNA in a dose-and time-dependent manner in RPMVECs.It is NF-κB rather than PKC signal pathway that is involved in modulation of the intracellular signaling process.