摘要目的 探讨src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),10 mg/L LPS刺激1h、3h、6h、12h、24 h或0.1 mg/L、1 mg/L、10 mg/L LPS刺激24 h；蛋白激酶C抑制剂(BIM)预处理0.5h或50 nmol/L SSeCKS-siRNA转染PMVEC 48 h后再加入10 mg/L LPS孵育24 h,酶联免疫吸附法检测细胞培养液上清TNF-α量(ng/L).采用SPSS10.0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、10 mg/L LPS刺激PMVEC 24 h后的,TNF-α分泌量分别为(253.70±23.55)、(327.88±37.25)、(403.20±36.22) ng/L,均高于未刺激组(82.28±22.56) ng/L(均P =0.000)；10 mg/L LPS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24 h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.

abstractsObjective 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-way analysis 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-αsecreted 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-αin 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-αdecreased 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-αin PMVEC induced by LPS,relieving the inflammatory response of PMVEC.