CAJ | 학술논문

目的探讨LPS诱导的巨噬细胞杀瘤效应以及与PKC相关的分子机制.方法两种巨噬细胞系P388D1和RAW264.7用LPS刺激、或先用PMA长期处理下调PKC表达、或用L-NAME阻断iNOS后再以LPS刺激,检测其杀伤肿瘤细胞系P815(MTT法)及分泌IL-1,TNF-α(ELISA法)和NO(Griess试剂)的作用;并用Western blot法检测PMA长期作用后两株细胞系中PKC亚型的表达.结果LPS刺激的RAW264.7细胞有杀伤靶细胞的功能,而P388D1几乎没有杀伤作用.PMA预处理(1μg/mL,24h)后可明显抑制LPS诱导的杀瘤作用.因上述结果提示PKC在巨噬细胞杀瘤活性中可能的重要作用,比较了PMA处理后两株细胞PKC亚型的表达:Western blot结果显示,在所检测的PKCα,β1,β2,δ及ε5种亚型中,在P388D1细胞均有表达,而在RAW264.7细胞仅有PKCα,PKCβ1,PKCδ表达;1μg/mL PMA作用24h后明显下调了RAW264.7细胞PKCα,PKCβ1和PKCδ的表达,在P388D1则有PKCα、PKCδ和PKCε下调,而PKCβ1和PKCβ2不被下调.结合LPS诱导的与PKC活化有关的IL-1、TNF-α及NO的产生,发现在P388D1细胞几乎不产生NO,而在RAW264.7细胞,NO合成酶抑制剂L-NAME不仅能阻断LPS诱导的NO的产生,而且明显抑制LPS诱导的杀瘤活性.结论 LPS诱导的巨噬细胞杀瘤作用主要是NO的杀伤作用来完成而非巨噬细胞直接与瘤细胞作用所致;而该作用与PKCβ活性密切相关.
목적탐토LPS유도적거서세포살류효응이급여PKC상관적분자궤제.방법량충거서세포계P388D1화RAW264.7용LPS자격、혹선용PMA장기처리하조PKC표체、혹용L-NAME조단iNOS후재이LPS자격,검측기살상종류세포계P815(MTT법)급분비IL-1,TNF-α(ELISA법)화NO(Griess시제)적작용;병용Western blot법검측PMA장기작용후량주세포계중PKC아형적표체.결과LPS자격적RAW264.7세포유살상파세포적공능,이P388D1궤호몰유살상작용.PMA예처리(1μg/mL,24h)후가명현억제LPS유도적살류작용.인상술결과제시PKC재거서세포살류활성중가능적중요작용,비교료PMA처리후량주세포PKC아형적표체:Western blot결과현시,재소검측적PKCα,β1,β2,δ급ε5충아형중,재P388D1세포균유표체,이재RAW264.7세포부유PKCα,PKCβ1,PKCδ표체;1μg/mL PMA작용24h후명현하조료RAW264.7세포PKCα,PKCβ1화PKCδ적표체,재P388D1칙유PKCα、PKCδ화PKCε하조,이PKCβ1화PKCβ2불피하조.결합LPS유도적여PKC활화유관적IL-1、TNF-α급NO적산생,발현재P388D1세포궤호불산생NO,이재RAW264.7세포,NO합성매억제제L-NAME불부능조단LPS유도적NO적산생,이차명현억제LPS유도적살류활성.결론 LPS유도적거서세포살류작용주요시NO적살상작용래완성이비거서세포직접여류세포작용소치;이해작용여PKCβ활성밀절상관.
Objective: To investigate the role of PKC isoforms in the regulation of LPS-triggered tumoricidal activity in macrophages and further elucidate its signal nechanisms. Methods: Two macrophage cell lines (P388D1 and RAW264.7) were stimulated by LPS alone, or with long-term of PMA pretreatment. Then cytotoxicities to P815 cells (by MTT assay) and IL-l, TNF-α (by ELISA) and nitric oxide (NO) production (by Griess reagent) in supernatants were measured. Western blot for PKC isoforms after long-term PMA pretreatment was analyzed. Results: RAW264.7 cells were stimulated with LPS to kill target tumor cells P815, whereas P388D1 cells failed to develop such an ability. Down-regulation of PKC isoforms by chronic treatment with PMA significantly inhibited the LPS-induced cytotoxicity in RAW264.7 cells. In unstimulated state, Western blotting with rabbit antiserum specific for the PKCα,β1, β2, δ or ε showed all 5 isoforms were detected in P388D1 cells, while only PKCα, PKCβ1 and PKCδ were detected in RAW264.7 cells. Exposure of the cells to long-term of PMA treatment significantly down-regulated the expression of PKCα , PKCβi and PKCδ in RAW264.7 cells. But in P388D1 cells, although PKCα, PKCδ and PKCεwere down-regulated, the expression of PKCβ1 and PKCβ2 could not be regulated. Comparing with LPS-induced IL-1, TNF-α and NO production by the two macrophage cell lines, P388D1 failed to produce NO.In RAW264.7 cells, LPS-induced NO production and antitumor activity was attenuated by the addition of L-NAME, an iNOS inhibitor. Conclusion: The results indicated a critical role of PKCβ in LPS-induced antitumor activity and this cytotoxicity is mainly due to PKC-β mediated NO production by RAW264.7 cells, but not a direct cytotoxic activity.