【摘要】 目的:探讨白细胞介素1β(IL-1β)刺激不同时间对大鼠脑微血管内皮细胞(rCMEC)的环氧合酶(COX)活性及其mRNA表达和PGE2释放的影响。方法:建立rCMEC培养,进行Ⅷ因子相关抗原鉴定。细胞长至融合状态后加入IL-1β(30μg/L),分别刺激0.5、1、2、4、8、12、24 h,ELISA测定细胞内COX-1、COX-2的活性及细胞外液PGE2含量,荧光实时定量PCR检测COX-1、COX-2的mRNA表达量,扩增产物进行熔解曲线图及琼脂糖电泳分析,并与未刺激的rCMEC作比较。结果:①Ⅷ因子抗体免疫组化染色可见90%以上的培养细胞呈阳性,确认为rCMEC。②IL-1β刺激4 h时细胞培养液PGE2含量已明显高于未刺激组(P<0.05);12 h时PGE2含量达到最大值(P<0.01);24 h时PGE2含量有所回降,但与未刺激组比较仍有显著差异(P<0.05)。③IL-1β刺激不同时间rCMEC内COX-1活性与未刺激组相比无统计学差异(P>0.05);COX-2活性在第8 h时已明显高于未刺激组(P<0.05),12 h活性达峰值(P<0.01),24 h活性有所回降,但仍具显著差异(P<0.05)。④IL-1β刺激不同时间COX-1 mRNA表达与未刺激组比较无明显差异(P>0.05);未刺激组在本实验条件下未检测到COX-2 mRNA表达,IL-1β刺激1h时可见COX-2 mRNA表达,4 h时COX-2 mRNA表达至峰值,而后开始回降,第12 h时已未见表达。熔解曲线图显示无非特异性扩增;琼脂糖电泳可见扩增基因与目的基因长度相符,结果与荧光定量PCR一致。结论:IL-1β作用下,rCMEC释放的PGE2持续增加并于12h达峰值,这一过程主要与COX-2 mRNA表达激活及COX-2活性增加有关。更多还原

【Abstract】 AIM: To study the cyclooxygenase(COX) activity and its mRNA expression,and PGE2 release from rats cerebral microvascular endothelial cells(rCEMC) stimulated by IL-1β(30 μg/L) at different times.METHODS: rCMEC were cultured,and identified by immunohistochemistry for von Willebrand factor(Ⅷ factor,a marker for all endothelial cells) in cytoplasm of the cells.After rCEMC grew to confluency,they were stimulated with IL-1β for 0.5,1,2,4,8,12 and 24 h,respectively.Activity of COX-1 and COX-2 in rCEMC and production of PGE2 in the conditioned media were detected by ELISA.COX-1 and COX-2 mRNA expressions were measured by real-time quantity PCR.The amplification product was tested by melting curve and identified by electrophoretic gel.RESULTS: ① Positive immunostaining for Ⅷ factor was present diffusely in the cytoplasm in more than 90% rCMEC.② Compared to the cells without IL-1β stimulation,the production of PGE2 increased significantly(P<0.05) at 4 h after rCEMC were incubated with IL-1β and reached the top level at 12 h(P<0.01),then declined thereafter at 24 h(P<0.05).③ There was no significant difference on COX-1 activity between IL-1β group and non-IL-1β group.COX-2 activity increased significantly compared with those in non-IL-1β(P<0.05) at 8 h after rCEMC were incubated with IL-1β and reached the top level at 12 h(P<0.01),then declined thereafter at 24 h(P<0.05).④ There was no significant difference on COX-1 mRNA expression between IL-1β group and non-IL-1β group.COX-2 mRNA was induced and became detectable at 1 h,and reached the top level at 4 h,then declined thereafter at 8 h and became undetectable by 12 h and 24 h after incubation with IL-1β.The melting curve showed there was no nonspecific amplification and electrophoretic gel showed the lengths of amplification products accorded with the predicted lengths.CONCLUSION: While rCEMC are stimulated by IL-1β,the excretion of PGE2 increases and reaches the top level at 12 h,which is related with its induction on COX-2 mRNA expression and COX-2 activity.更多还原