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目的探讨蛋白激酶C-ε(PKC-ε)家族在肺成纤维细胞向纤维粘连蛋白趋化运动中的作用。方法于2006年10月至2007年4月在北京世纪坛医院内科实验室和美国内布拉斯加大学呼吸科实验室进行。(1)人胚肺成纤维细胞体外培养。(2)以纤维粘连蛋白作为趋化因子,采用Boyden趋化小室技术测定人胚肺成纤维细胞的趋化运动。(3)将人胚肺成纤维细胞转染PKC-ε小干扰RNA(siRNA),24h后应用蛋白质印迹法测定PKC-ε的表达。(4)分别采用PKC-ε非选择性抑制剂(CalphostinC)和选择性抑制剂(Ro-31-8220)及转染PKC-εsiRNA的人胚肺成纤维细胞,研究PKC-ε在肺成纤维细胞趋化运动中的调节作用。结果CalphostinC和Ro-31-8220显著抑制人胚肺成纤维细胞向纤维粘连蛋白的趋化运动[细胞迁移数分别为对照组的(68.50±4.68)%和(59.94±5.20)%,P<0.01]。成纤维细胞转染PKC-εsiRNA,孵育24h后PKC-ε的表达显著减少,成纤维细胞在纤维粘连蛋白作用下的趋化运动显著减弱[迁移的细胞数是对照组的(82.56±2.80)%,P<0.05)]。结论PKC-ε参与肺成纤维细胞趋化运动的调节,进而在肺成纤维细胞介导的组织重构中发挥作用。
Objective To investigate the role of protein kinase C-ε (PKC-ε) family in the chemotaxis of lung fibroblasts to fibronectin. Methods From October 2006 to April 2007 at the Shijitan Hospital Internal Medicine Laboratory in Beijing and the Nebraska University Respiratory Laboratory. (1) Human embryonic lung fibroblasts cultured in vitro. (2) Using fibronectin as a chemokine, Boyden chemotactic chamber technique was used to determine the chemotactic activity of human embryonic lung fibroblasts. (3) Human embryonic lung fibroblasts were transfected with small interfering RNA (siRNA) of PKC-ε, and the expression of PKC-ε was determined by Western blot 24 hours later. (4) PKC-ε non-selective inhibitor (CalphostinC) and selective inhibitor (Ro-31-8220) and PKC-εsiRNA transfected human embryonic lung fibroblasts were used to study the PKC-ε in lung fibroblasts Modulation of cell chemotaxis. Results Calphostin C and Ro-31-8220 significantly inhibited the chemotaxis of human embryo lung fibroblasts to fibronectin [(68.50 ± 4.68)% and (59.94 ± 5.20)%, respectively, P <0.01 ]. PKC-εsiRNA expression was significantly decreased in fibroblasts transfected with PKC-εsiRNA, and the chemotactic activity of fibroblasts was significantly weakened after fibronectin treatment [the number of migrated cells was 82.56 ± 2.80% , P <0.05)]. Conclusions PKC-ε is involved in the regulation of lung fibroblast chemotaxis and thus plays a role in lung fibroblast-mediated tissue remodeling.