【摘要】 目的:观察硫酸化八肽胆囊收缩素(cholecystokinin octapeptide,CCK-8)对肿瘤坏死因子α诱导大鼠滑膜细胞株RSC-364核因子κB的影响,以及CCK-A/B受体是否参与这一过程。方法:实验于2003-02/2004-02在河北医科大学法医学教研室分子生物学实验室完成。取大鼠滑膜细胞株RSC-364经肿瘤坏死因子α(10 μg/L)、sCCK-8(10-8,10-7,10-6 mol/L)、CCK受体拮抗剂丙谷胺及溶剂单独或联合应用孵育:①孵育3h,用反转录-聚合酶链反应技术检测细胞CCK-A受体及CCK-B受体mRNA的表达。②孵育1h,用电泳迁移率检测核因子κB相对活性。③孵育30 min,用Western blot检测胞浆IκB蛋白表达的相对水平。结果:①细胞CCK-A受体及CCK-B受体mRNA的表达:RSC-364细胞固有表达CCK-A/B受体,肿瘤坏死因子α(10 μg/L)可使CCK-A受体和CCK-B受体mRNA的表达分别上调148%和173%(P<0.01)。肿瘤坏死因子α和CCK-8(10-8～10-6 mol/L)联合孵育细胞,CCK-A受体和CCK-B受体mRNA表达与肿瘤坏死因子α组相比分别增高47%,56%,30%和57%,13%,24%(P<0.05,0.01)。②核因子κB相对活性:肿瘤坏死因子α组明显高于对照组(294.45±36.48,0,P<0.01);肿瘤坏死因子α+CCK-810-8,10-7,10-6 mol/L组高于肿瘤坏死因子α组(470.69±56.76,489.37±64.95,558.90±74.15,P<0.05,0.01);CCK-8的作用可被丙谷胺减弱(400.79±39.06)。③IκB蛋白表达的相对水平:肿瘤坏死因子α组明显低于对照组(139.43±30.76,220.79±34.58,P<0.01),肿瘤坏死因子α+CCK-810-8,10-7,10-6 mol/L组低于肿瘤坏死因子α组(95.26±8.54,84.15±8.77,63.28±16.13,P<0.05),并可被丙谷胺所抑制(137.22±20.33,P<0.01)。结论:CCK-8对肿瘤坏死因子α诱导的RSC-364核因子κB活性具有正向调节作用,并能降低IκBα蛋白水平,提示CCK-8在类风湿性关节炎发病过程中可能具有调控作用,此作用可能通过滑膜细胞上的CCK受体实现。更多还原

【Abstract】 AIM:To investigate the effect of sulfated cholecystokinin octapeptide(sCCK-8)on tumor necrosis factor-α(TNF-α)induced nuclear factor-κB(NF-κB)activity in rat fibroblast-like synovial cell strain RSC-364,and its receptor mechanisms.METHODS:The experiment was conducted in the Laboratory of Molecular Biology,Department of Forensic Medicine of Hebei Medical University between February 2003 and February 2004.RSC-364 cells were stimulated by TNF-α in the presence or absence of sCCK-8(10-8,10-7,10-6 mol/L),CCK receptor antagonist proglumide and dissolvent:① The expressions of CCK-A receptor(CCK-AR)and CCK-B receptor(CCK-BR)mRNA were assayed by reverse transcription polymerase chain reaction(RT-PCR)after 3-hour incubation.② The NF-κB binding activity was analyzed by electrophoretic mobility shift assay(EMSA)after 1-hour incubation.③After 30-minute incubation,the IκB protein level in the cytoplasma was detected by Western blot.RESULTS:① mRNA expressions of CCK-AR and CCK-BR:Both CCK-AR and CCK-BR were constitutively expressed by RSC-364.TNF-α(10 μg /L)could up-regulate the mRNA expressions of both CCK-AR and CCK-BR by 148% and 173% respectively(P < 0.01).sCCK-8,at concentrations from 10-8 mol/L to 10-6 mol/L,significantly increased CCK-AR mRNA expression by 47%,56%,30%,and CCK-BR,57%,13% and 24%,after TNF-α exposure(P < 0.05,P < 0.01).② Relative activity of NF-κB:It was significantly higher in the TNF-α group than that in the control group(294.45±36.48,0,P < 0.01),and that in combination of TNF-α and sCCK-8 of all concentration group was remarkably higher than that in the TNF-α group(470.69±56.76,489.37±64.95,558.90±74.15,P < 0.05,0.01).The effect of sCCK-8 was abrogated by a CCK receptor antagonist proglumide(400.79±39.06).③ Western Blot results:The IκB protein level in the TNF-α group was obviously lower than that in the control group(139.43±30.76,220.79±34.58,P < 0.01),and that in all combination groups were lower than that in the TNF-α group(95.26±8.54,84.15±8.77,63.28±16.13,P < 0.05),which could be attenuated by proglumide(137.22±20.33,P < 0.01).CONCLUSION:CCK-8 can up-reguate the activity of NF-κB in RSC-364 induced by TNF-α,and down-regulate the IκBα protein level,which indicate that CCK-8 might be a possible regulator on the pathogenesis of rheumatoid arthritis through its receptors on synoviocytes.更多还原