摘要
目的 探讨蛋白激酶C(PKC) 核因子κB(NF κB)信号转导通道对人肺动脉平滑肌细胞 (HPASMCs)增殖和血管内皮生长因子 (VEGF)表达的影响。方法 体外培养HPASMCs ,用工具药PKC激活剂 12 肉豆蔻酰 13 乙酸佛波酯 (PMA)和NF κB抑制剂二硫代氨基甲酸吡咯烷 (PDTC) ,将HPASMCs分为对照组、PMA组和PMA +PDTC组在常氧和缺氧条件下培养。逆转录 聚合酶链反应 (RT PCR)检测VEGFmRNA表达 ,Westernblot法检测VEGF和NF κB的抑制蛋白IκBα蛋白表达 ,免疫细胞化学法检测NF κBp6 5的表达和定位 ,流式细胞术检测细胞周期时相分布。结果 ( 1)NF κBp6 5胞核染色阳性率、IκBα蛋白相对表达量及细胞周期G2 /M % :常氧或缺氧PMA组与相应对照组、PMA +PDTC组比较差异均有显著性 (P均 <0 0 5 ) ;缺氧PMA组与常氧PMA组比较差异有显著性 (P <0 0 5 )。 ( 2 )VEGFmRNA和蛋白表达 :常氧对照组、PMA组、PMA +PDTC组组间差异均无显著性 (P均 >0 0 5 ) ;缺氧PMA组均高于缺氧对照组、缺氧PMA +PDTC组、常氧PMA组 ,差异均有显著性 (P均 <0 0 5 )。 ( 3)缺氧PMA组NF κB胞核染色阳性率、VEGF蛋白相对表达量、G2 /M %之间均呈正相关 (r =0 5 87～ 0 710 ,P均 <0 0 5 )。结论 常氧培养HPASMCs存在PKC NF κB信号转导通道 ;
Objective To investigate the effect of protein kinase C(PKC) nuclear factor kappa B(NF κB) signal transduction pathway on proliferation and expression of vascular endothelial growth factor(VEGF) in human pulmonary artery smooth muscle cells(HPASMCs) Methods Cultured HPASMCs in normoxia or hypoxia conditions were divided into three groups and stimulated with or without phorbol 12 myristate 13 acetate(PMA) and pyrrolidine dithiocarbamate(PDTC) in vitro The three groups were the control group,the PMA group and the PMA+PDTC group Reverse transcriptase polymerase chain reaction(RT PCR) was used to detect VEGF mRNA expression,and the expression of VEGF protein and the inhibitor protein IκBα were observed by Western blot,while the location and expression of NF κB p65 were observed by immunocytochemical staining,and cell cycle phases were analyzed by flow cytometry Results (1)As for the positive rate of nucleolar staining for NF κB p65,the relative expression of IκBα protein,and the percentage of G 2/M phases of cell cycle,there were significant differences between the PMA group andthe control group or PMA+PDTC group, both in normoxia and hypoxia conditions( P <0 05,respectively),and there was also a significant difference between the normoxia and hypoxia PMA groups( P <0 05) (2)There were no significant differences in VEGF mRNA and protein expression among the three groups( P >0 05,respectively)in normoxia,but the expression was higher in hypoxia PMA group than in hypoxia control and hypoxia PMA+PDTC or normoxia PMA group( P <0 05,respectively) (3)There was a positive correlation between the positive rate of nucleolar staining for NF κB p65,the relative expression of VEGF protein and the percentage of G 2/M phases of cell cycle in hypoxia PMA group( r =0 587-0 710, P <0 05,respectively) Conclusions There is a signal transduction pathway of PKC NF κB in HPASMCs The activity of PKC can be enhanced in hypoxia,concomitant NF κB activation or VEGF overexpression to be involved in the proliferation These results suggest that the activation of NF κB can be considered as a downstream of PKC signal transduction pathway,and the activation of PKC NF κB signal transduction pathway and VEGF overexpression may contribute to the process of hypoxic pulmonary hypertension
出处
《中华结核和呼吸杂志》
CAS
CSCD
北大核心
2004年第4期218-223,共6页
Chinese Journal of Tuberculosis and Respiratory Diseases
基金
国家自然科学基金资助项目 (3 9770 3 41)
教育部高等学校博士学科点专项科研基金资助项目([2 0 0 2 ] 173 )
