摘要
目的探讨8re抑制的蛋白激酶c底物（SSeCKS）对脂多糖（LPS）诱导大鼠肺微血管内皮细胞（PMVEC）分泌肿瘤坏死因子（TNF）-α的影响。方法体外培养Wistar大鼠PMVEC，按随机数字表法分组（n=4），10mg／LLPS刺激1h、3h、6h、12h、24h或0．1mg／L、1mg／L、10mg／LLPS刺激24h；蛋白激酶C抑制剂（BIM）预处理0．5h或50nmol／LSSeCKS-siRNA转染PMVEC48h后再加入10mg／LLPS孵育24h，酶联免疫吸附法检测细胞培养液上清TNF-α
量（ng／L）。采用SPSSl0．0软件进行分析，组间比较采用单因素方差分析，以P〈0．05为差异具有统计学意义。结果0．1、1、10mg／LLPS刺激PMVEC24h后的，TNF-α分泌量分别为（253．70±23．55）、（327．88±37．25）、（403．204-36．22）ng／L，均高于未刺激组（82．28±22．56）ng／L（均P=0．000）；10mg／LLPS刺激PMVEC后，TNF-α分泌量于1h升高（170．11±49．22）ng／L，6h达峰值（404．82±13．78）ng／L，刺激24h后仍维持高水平（395．67±36．23）ng／L，分别与未刺激组（84．60±23．61）ng／L比较：P=0．001，0．000，0．000；BIM预处理PMVEC后再给予LPS刺激，TNF-α分泌量（200．44±27．39）ng／L较LPS单独刺激（402．28±31．07）ng／L显著较少（P=0．000）；与LPS单独刺激比较（407．28±32．64）ng／L，SSeCKS-siRNA抑制SSeCKS表达后，LPS对PMVEC分泌TNF-α的诱导效应（195．20±13．28）ng／L也显著下降（P=0．000）。结论下调SSeCKS表达和蛋白激酶C活性可抑制LPS对PMVEC分泌TNF-α的诱导效应，从而减轻PMVEC的炎症反应。
Objective To study the role of src-suppressed C kinase substrate （SSeCKS） in the secretion of tumor necrosis factor （ TNF-α ） in rat pulmonary micro - vascular endothelial cells （ PMVEC ） induced by lipopolysaccharide （LPS）. Methods Wistar rat PMVEC cultured in vitro were randomly （random number） divided into several groups （n = 4） as per exposure to given dosage of LPS for different lengths of time and to different dosages of LPS for given length of time. After PMVEC exposed to 10 mg/L LPS for 1 hour （h）, 3 h, 6 h, 12 h and 24 h or 0. 1 mg./L, 1 mg/L and 10 mg/L LPS for 24 h, the levels of TNF-αin the supernatant of culture medium were examined by the method of enzyme linked immunosorbent assay （ELISA）. Another PMVEC was pre-treated by protein kinase C （PKC） inhibitor bis-indolylmaleimide （BIM） for 0. 5 h or had the transfection of SSeCKS-specific small interfering RNA （siRNA） for 48 h before 10 mg/L LPS challenge for 24 h, and subsequently the supernatant was also examined by ELISA. One-wayanalysis of variance （ANOVA） was employed for statistical analysis by SPSS version 10. 0 to compare values among all groups. A significant difference was presumed as a probability value 〈 0. 05. Results After PMVEC incubated with 0. 1 mg/L, 1 mg/L and 10 mg/L LPS for 24 hours, the levels of TNF-α secreted were （253.70 ± 23.55） , （327.88 ± 37.25） , （403.20± 36. 22） , respectively, which were higher than that in un-stimulated PMVEC （82. 28 ± 22. 56, all P = 0. 000 ）. After 10 mg/L LPS challenge for one hour, the level of TNF-α in the supernatant of PMVEC raised substantially （170. 11 ± 49.22）, peaked at the time of 6 h （404. 82 ± 13.78 ）, then persisted at a higher level until 24 h （ 395. 67± 36. 23） than that in un-stimulated PMVEC （84.60 ± 23.61, P = 0.001, 0.000, 0.000, respectively）. After PMVEC pre-incubated with BIM, the level of LPS-induced TNF-α decreased obviously （200.44 ± 27.39 vs. 402.28 ± 31.07, P = 0. 000）. Compared with LPS challenged PMVEC （407.28 ± 32. 64 ）, depletion of endogenous SSeCKS in PMVEC after inhibited by SSeCKS-siRNA significantly attenuated increase in the level of LPS-induced TNF-α （195.20 ± 13.28, P = 0. 000）. Conclusions Down-activation of SSeCKS and PKC can inhibit the secretion of TNF-α PMVEC induced by LPS, relieving the inflammatory response of PMVEC.
出处
《中华急诊医学杂志》
CAS
CSCD
北大核心
2012年第12期1349-1353,共5页
Chinese Journal of Emergency Medicine
基金
国家自然科学基金（81100053、81070054）
关键词
脂多糖
肺微血管内皮细胞
肿瘤坏死因子
src抑制的蛋白激酶C底物
蛋白激
酶C抑制剂
小分子干扰RNA
炎症
信号通路
Lipopolysaccharide
Pulmonary microvascular endothelial cells
Tumor necrosis factor
Src-suppressed C kinase substrate
Protein kinase C inhibitor
Small interfering ribonucleic acid
Inflammation
Signal pathway
