蛋白激酶C激活的荧光共振能量转移分析

摘要

Objective To investigate the changes in the expressions of Fas-associated death domain protein (FADD) and cellular-FLICE inhibitory protein (c-FLIP) in the articular cartilage of patients with Kashin-Beck disease (KBD) and the role of these proteins in the pathogenesis of KBD. Methods The cartilage samples were collected from patients with established diagnosis of KBD and osteoarthritis and from healthy control subjects undergoing amputation due to traffic accidents. The expressions of Fas-associated death domain protein (FADD) and cellular-FLICE inhibitory protein (c-FLIP) in the cartilage were detected by immunohistochemistry, and the positive chondrocytes were counted in different layers of the articular cartilage under microscope. Results The positivity rates of FADD in the middle layer of articular cartilage from patients with KBD [(28.68±2.19)%] and osteoarthritis [(35.40±2.34)%] were significantly higher than that in normal cartilage [(10.51 ±5.02)%, F=16.245, P=0.000], but the rates in the upper and deeper layers were comparable among the 3 groups (P=0.206-0.761). In KBD cartilage, FADD expression was the highest in the middle layer [(28.68±5.38)%] followed by the deeper layer [(17.94±8.38)%]. Compared with the healthy controls, KBD and osteoarthritis patients showed significantly higher FLIP expression in the upper layer of the cartilage (F=5.929, P=0.018) but similar expressions in middle and deeper layers. Conclusions KBD patients have significant increased FADD expression in the middle layer but decreased FLIP expression in the upper layer of the cartilage, suggesting that the death receptor pathway and its regulators play important roles in the pathogenesis of KBD.%目的 当前,检测蛋白激酶C(PKC)激活的方法尚缺乏灵敏性或直接性,这里我们提供了一种新的直接而灵敏的方法——利用荧光共振能量转移(fluorescence resonance energy transfer,FRET)技术来构建检测PKC激活的方法.方法 将表达PKC激活的报告分子(CKAR)的质粒单独转染或与表达甲状旁腺素1型受体(PTHR1)的质粒共转染HEK293细胞,培养72 h后用激光共聚焦显微镜检测FRET的变化,并以此判断甲状旁腺素或佛波酯是否激活PKC.结果 在只转染CKAR质粒的HEK293细胞,佛波酯降低了CKAR分子的FRET效率,并使青色荧光与黄色荧光的比值(C/Y)增加,而PTH( 1-34)未能改变C/Y的值.在共转染了CKAR和PTHR1质粒的HEK293细胞,PTH(1-34)则使C/Y增加.结论 PKC激活报告分子可用于检测PKC的激活,该方法也可作为PKC相关信号转导的实验平台.