佛波酯联合脂多糖诱导THP-1炎症细胞模型的优化及评价

Optimization and evaluation of an inflammatory cell model in LPS-stimulated PMA-differentiated THP-1 cells

目的优化条件建立人急性单核白血病细胞THP-1炎症细胞模型,评价其对磷酸二酯酶抑制剂类抗炎剂咯利普兰的响应。方法 THP-1细胞经佛波酯(PMA)诱导分化、脂多糖(LPS)刺激,ELISA检测细胞上清肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)水平。考察PMA和LPS剂量及其作用时间获得较优模型;于LPS同时加入咯利普兰考察此THP-1炎症细胞模型对抗炎作用的响应。结果 THP-1单核细胞经PMA诱导分化24 h呈巨噬细胞形态,延长至48 h细胞形态不变,诱导的细胞经LPS刺激产生显著升高的TNF-α和IL-6。PMA在100 ng/mL较50 ng/mL诱导分化THP-1经LPS刺激所产生TNF-α水平显著升高,且随LPS剂量增加而增加并在LPS达0.2μg/mL后趋于恒定;IL-6在PMA 100 ng/mL及LPS≥1μg/mL时释放明显。100 ng/mL PMA联合0.5μg/mL LPS相较于0.1μg/mL LPS刺激THP-1产生TNF-α被咯利普兰抑制更显著。结论 100 ng/mL PMA诱导分化的THP-1细胞在LPS刺激下炎性因子水平显著升高;100 ng/mL PMA联合不低于0.2μg/mL LPS刺激THP-1细胞可获较优炎症细胞模型,此模型对抗炎剂作用更敏感。

Objective To develop an optimal inflammatory cell model from lipopolysaccharide（LPS）-stimulated phorbol ester（ PMA）-differentiated THP-1 cells,and investigate its response to anti-inflammatory agent phosphodiesterase inhibitor rolipram. Methods THP-1 cells were differentiated by PMA and stimulated by LPS to release inflammatory factors in cell supernatants,like tumor necrosis factor α（ TNF-α） and interleukin 6（ IL-6）,which were detected by ELISA. The doses and durations of both PMA and LPS treatment were optimized to develop the inflammatory cell model. Rolipram was added along with LPS after PMA differentiation to assess the response of cells to the anti-inflammatory agent. Results THP-1 cells showed no significant differences in cell morphology between PMA treatment for 24 hours and for 48 hours,but significantly high levels of TNF-α and IL-6 were released under LPS treatment. TNF-α level increased significantly after the differentiation by PMA at 100 ng/mL in comparison with that at 50 ng/mL,and it increased in a LPS dose-depended manner untill a plateau at 0. 2 μg/mL LPS; the secretion level of IL-6 increased remarkably when THP-1 cells were induced by PMA at 100 ng/mL and stimulated by LPS≥1 μg/mL. The inflammatory cell model made using PMA at 100 ng/mL and LPS at 0. 5 μg/mL was more sensitive to the anti-inflammatory agent rolipram,compared with that by 0. 1 μg/mL LPS. Conclusion PMA at 100 ng/mL was selected for the differentiation of THP-1 cells with the enhanced responsiveness to LPS stimulation; THP-1 cells by the induction of PMA at 100 ng/mL coupled with the stimulation of LPS at no less than 0. 2 μg/mL was an optimal inflammatory cell model for significant secretion of TNF-α and IL-6,which was sensitive to the action of anti-inflammatory agents.