CAJ | 학술논문

目的研究肿瘤坏死因子(TNF)α诱导人肝癌细胞系MHCC-97H分泌白细胞介素8(IL-8)及p38丝裂原活化蛋白激酶(MAPK)和核因子-κB (NF-κ B)信号通路对其的调节作用.方法酶联免疫吸附法检测不同浓度的TNF α(0、1、5 ng/ml)干预人肝癌细胞MHCC-97H 24 h和相同浓度的TNFα(1 ng/ml)干预不同时间后,细胞上清液中IL-8的浓度; Western blot和免疫荧光方法检测TNFα(1 ng/ml)刺激MHCC-97H细胞后,p38 MAPK磷酸化蛋白的表达及其分布;非放射性NF-κ B p50/p65转录因子活性试剂盒检测TNFα干预MHCC-97H细胞后核蛋白中活性NF-κB p65蛋白的含量.多组均数比较采用单因素方差分析,多个样本均数间两两比较用q检验.结果 TNFα明显促进肝癌细胞分泌IL-8,具有显著的时间和浓度依赖性:TNFα干预0、6、12、18、24h,IL-8的浓度分别为(47.45±9.78)pg/ml、(497.41±52.83)pg/ml、(694.14±44.43)pg/ml、(909.35±97.22) pg/ml、(1093.59±75.72) pg/ml (F=144.04,P＜0.01);TNFα0、1、5、10 ng/ml干预24h,IL-8浓度分别为(47.45±9.12) pg/ml、(1093.59±75.72)pg/ml、(1372.77±85.10) pg/ml和(1614.55±153.52) pg/ml(F=364.14,P＜0.01).TNFα刺激肝癌细胞后,p38 MAPK磷酸化蛋白表达明显上调,给予p38 MAPK通路特异性抑制剂(SB203580)可以显著抑制TNFα诱导的IL-8的分泌(P值均＜0.01)且呈浓度依赖性(F=165.92,P＜0.01).免疫荧光结果显示TNFα促进肝癌细胞NF-κ B p65的核转位,且呈浓度依赖性(TNF α0、1、5、10 ng/ml处理肝癌细胞1h后细胞核蛋白表达NF-κ B p65对应吸光度值分别为0.52±0.04、2.75±0.15、3.13±0.18、3.81±0.14 (F=1266.42,P＜0.05),而SB203580则可以部分抑制NF-κB p65的核转位(F=141.20,P＜0.05).结论 TNFα通过p38 MAPK-NF-κB信号途径调节人肝癌细胞系MHCC-97分泌IL-8.
목적 연구종류배사인자(TNF)α유도인간암세포계MHCC-97H분비백세포개소8(IL-8)급p38사렬원활화단백격매(MAPK)화핵인자-κB (NF-κ B)신호통로대기적조절작용.방법 매련면역흡부법검측불동농도적TNF α(0、1、5 ng/ml)간예인간암세포MHCC-97H 24 h화상동농도적TNFα(1 ng/ml)간예불동시간후,세포상청액중IL-8적농도; Western blot화면역형광방법검측TNFα(1 ng/ml)자격MHCC-97H세포후,p38 MAPK린산화단백적표체급기분포;비방사성NF-κ B p50/p65전록인자활성시제합검측TNFα간예MHCC-97H세포후핵단백중활성NF-κB p65단백적함량.다조균수비교채용단인소방차분석,다개양본균수간량량비교용q검험.결과 TNFα명현촉진간암세포분비IL-8,구유현저적시간화농도의뢰성:TNFα간예0、6、12、18、24h,IL-8적농도분별위(47.45±9.78)pg/ml、(497.41±52.83)pg/ml、(694.14±44.43)pg/ml、(909.35±97.22) pg/ml、(1093.59±75.72) pg/ml (F=144.04,P＜0.01);TNFα0、1、5、10 ng/ml간예24h,IL-8농도분별위(47.45±9.12) pg/ml、(1093.59±75.72)pg/ml、(1372.77±85.10) pg/ml화(1614.55±153.52) pg/ml(F=364.14,P＜0.01).TNFα자격간암세포후,p38 MAPK린산화단백표체명현상조,급여p38 MAPK통로특이성억제제(SB203580)가이현저억제TNFα유도적IL-8적분비(P치균＜0.01)차정농도의뢰성(F=165.92,P＜0.01).면역형광결과현시TNFα촉진간암세포NF-κ B p65적핵전위,차정농도의뢰성(TNF α0、1、5、10 ng/ml처리간암세포1h후세포핵단백표체NF-κ B p65대응흡광도치분별위0.52±0.04、2.75±0.15、3.13±0.18、3.81±0.14 (F=1266.42,P＜0.05),이SB203580칙가이부분억제NF-κB p65적핵전위(F=141.20,P＜0.05).결론 TNFα통과p38 MAPK-NF-κB신호도경조절인간암세포계MHCC-97분비IL-8.
Objective To identify the role of p38 MAPK- NF-κB signaling pathway in TNF-α induced IL-8 production in human hepatocellular carcinoma cells.Methods The concentrations of IL-8 from MHCC-97H cells were measured by an enzyme-linked immunosorbent assay (ELISA).The phosphorylation of p38 MAPK was analyzed by Western blot and immunofluorescence.NF-κB p65 protein nuclear translocation was determined by non-radioactive NF-κB pS0 / p65 transcription factor activity kit and immunofluorescence.Results The IL-8 production from MHCC-97H cells challenged with TNFα significantly increased in a timedependent (F =144.04,P ＜ 0.01) and dose-dependent (F =364.14,P ＜ 0.01) manners,as compared with those without TNFα challenge.TNFα up-regulated the phosphorylation levels of p38 MAPK and increased the translocation of NF-κB p65 protein into the nucleus,also proved by immunofluorescence staining.p38MAPK inhibitor (SB203580) could significantly inhibit IL-8 production in a dose-dependent manners (F=65.47,P ＜ 0.01),and partially inhibited NF-κB p65 nuclear translocation in a dose-dependent manner ( F =141.20,P ＜ 0.05).Conclusion TNF-α could increase the production of IL-8 in MHCC-97H cells and p38 MAPK- NF-κB pathways seem to play a central role in the regulation of IL-8 production.