摘要目的 探讨细菌脂蛋白(BLP)耐受所致核转录因子-κB(NF-κB)活化受抑制的分子机制.方法 以不同浓度BLP(10、100、1 000 ng/ml)预处理人急性单核细胞白血病细胞(THP-1)诱导BLP耐受,再以不同浓度BLP(0、10、100、1 000 ng/m1)刺激经BLP预处理(耐受组)或未经BLP预处理(对照组)的THP-1细胞,采用酶联免疫吸附法(ELISA)检测培养上清中肿瘤坏死因子-α(TNF-α)的释放,确定最适的BLP预处理和刺激浓度.然后按此条件处理细胞不同时间(0、0.5、1、2、6 h)并提取蛋白,用蛋白质免疫印迹法(Western blotting)检测NF-κB亚单位p50和p65的表达、核转位及磷酸化情况.结果 对照组中10、100、1 000 ng/mlBLP可剂量依赖性刺激THP-1活化并产生TNF-α(pg/ml:184.86±32.51、3 215.88±167.09、6 042.96±245.37),耐受组中100 ng/ml BLP预处理几乎完全抑制不同剂量BLP诱导的TNF-α释放.故最适BLP预处理浓度为100 ng/ml,刺激浓度为1 000 ng/ml.Western blotting检测表明,在BLP耐受的细胞质中p50蛋白表达明显高于对照组(0 h:542.9±15.6比272.8±13.2,0.5 h:558.0±16.9比236.4±11.8,1 h:524.7±17.5比211.6±9.8,2 h:584.9±15.6比222.4±12.3,均P＜0.01),而两组间p65蛋白无明显差异.BLP刺激还可诱导对照组细胞中p50和p65发生核转位,即细胞核中p50和p65蛋白增加(1 h p50:344.2±13.6比79.0±5.2,p65:78.4±4.5比0,均P＜0.05),而耐受组细胞核中p50和p65均无明显变化.另外,BLP刺激还可诱导对照组细胞中p65的536位丝氨酸发生快速磷酸化(0.5 h:0.67±0.08比0.04±0.01,1 h:0.71±0.11比0.04±0.01,均P＜0.05).但是在BLP刺激的耐受组细胞中磷酸化p65蛋白水平无明显变化.结论 BLP耐受的THP-1细胞中抑制性NF-κB亚单位p50表达上调,而具有转活化能力的亚单位p65的核转位及磷酸化均受到抑制,可能是BLP耐受中TNF-α等NF-κB依赖的基因表达减少的分子机制之一.

abstractsObjective To approach the nuclear factor-κB (NF-κB) nuclear translocation mechanism in bacterial lipoprotein (BLP) tolerance. Methods Human monocytic THP-1 cells were first pretreated with 10, 100, 1 000 ng/ml BLP for 20 hours to induce BLP tolerance. Then THP-1 cells without BLP pretreatment (control group) or with BLP pretreatment (tolerance group) were stimulated with 0, 10, 100,1 000 ng/ml BLP again for 6 hours. The tumor necrosis factor-α (TNF-α) content in culture medium was measured by enzyme linked immunosorbent assay (ELISA) in order to determine the most suitable BLP pretreatment and stimulation concentration. Western blotting was used to detect the protein level, nuclear translocation and phosphorylation of NF-ΚB p50 and p65 in the cells of control and tolerance groups treated with respective conditions for 0, 0.5, 1, 2 and 6 hours. Results In control group BLP stimulation (10,100, 1 000 ng/ml) could induce THP-1 activation and TNF-α production (pg/ml: 184.86 ± 32. 51,3 215. 88±167. 09, 6 042. 96±245. 37) in a dose-dependent manner. In tolerance group, 100 ng/ml BLP pretreatment resulted in almost complete inhibition of TNF-α production as induced by 10-1 000 ng/ml BLP stimulation. Therefore, 100 ng/ml BLP pretreatment and 1 000 ng/ml stimulation were selected for following cell treatment. Western blotting analysts showed that there was an increase of p50 protein level in BLP-tolerant cells comparing with control group (0 hour: 542. 9±15. 6 vs. 272. 8±13. 2, 0. 5 hour: 558. 0±16. 9 vs. 236. 4±11.8, 1 hour: 524. 7±17. 5 vs. 211. 6±9. 8, 2 hours: 584. 9±15. 6 vs. 222. 4±12. 3, all P＜0. 01), whereas the p65 protein level was similar between the two groups. BLP stimulation also induced the nuclear translocation of p50 and p65 in control group (1-hour p50: 344. 2±13. 6 vs. 79. 0±5. 2, p65:78. 4 ±4.5 vs. 0, both P＜0. 05), but not in tolerance group. In addition, the phosphorylation of p65 at serine 536 was induced after BLP stimulation in control THP-1 cells (0. 5 hour: 0. 67±0. 08 vs. 0. 04±0. 01,1 hour: 0.71±0.11 vs. 0.04±0.01, both P＜0.05), but this change was not detected in BLP-tolerant cells. Conclusion It was found that in BLP-tolerant cells, the expression of inhibitory subunit p50 was increased and the nuclear translocation and phosphorylation of p65 with trans-activation ability was inhibited.These changes are likely responsible for the reduced gene expression of NF-ΚB dependent genes in BLP-tolerant cells.