中文摘要：

目的观察环磷腺苷下游主要效应分子PKA和Epac对内毒素刺激的原代培养小胶质细胞分泌炎症细胞因子的影响。方法 24孔板接种原代培养的新生大鼠脑小胶质细胞,按不同药物处理分为6组：组1,先用DMSO及HBSS分别孵育30 min,然后HBSS孵育24 h;组2,先用DMSO及HBSS分别孵育30 min;组3,先用DMSO及6-Bnz-cAMP 100nmol分别孵育30 min;组4,先用DMSO及8-pCPT-2＇-O-Me-cAMP 50 nmol分别孵育30 min;组5,先用50nmol的H-89及6-Bnz-cAMP 100 nmol分别孵育30 min;组6,先用50 nmol的H-89及8-pCPT-2＇-O-Me-cAMP 50 nmol分别孵育30 min,最后组2～组6内毒素0.5μg孵育24 h。内毒素处理后0、3、6、24 h后,倒置显微镜下观察细胞形态改变,并计算6 h时长短轴比值。24 h时收集各组上清液,用ELISA法测定TNF-α、IL-1β及IL-10浓度。结果内毒素刺激后3 h,细胞成双极杆状细胞,6 h时最为明显,24 h细胞形态又恢复至椭圆或阿米巴样。仅6-Bnz-cAMP能明显抑制6 h时细胞形态的改变,预给予H-89,能完全逆转6-Bnz-cAMP对细胞形态学的影响。内毒素刺激24 h后,6-Bnz-cAMP能明显减少TNF-α、IL-1β的分泌,促进IL-10的释放,H-89能完全逆转该效应。给予50 nmol的8-pCPT-2＇-O-Me-cAMP仅减少TNF-α的分泌,H-89不影响8-pCPT-2＇-O-Me-cAMP的作用。结论较Epac,PKA对内毒素刺激的小胶质细胞释放炎症细胞因子发挥主要的作用。

英文摘要：

Aim To observe the effect of protein kinase A（PKA） and Epac（exchange proteins directly activated by cAMP）-the downstream effectors of cAMP on the expression of inflammatory cytokines by the primary cultured microglia stimulated with endotoxin.MethodsThe primary cultured microglia cells from brains of new-born rats were embedded in the 24 pore plate then treated with different drugs as the following： group 1 in which microglias were incubated with 10% DMSO for 30 min,then HBSS for 30 min,finally HBSS for 24 h;group 2 with 10% DMSO and HBSS for 30 min respectively,group 3 with 10% DMSO and 100 nmol 6-Bnz-cAMP for 30 min respectively,group 4 with 10% DMSO and 50 nmol 8-pCPT-2′-O-Me-cAMP for 30 min respectively,group 5 with 50 nmol H-89 and 100 nmol 6-Bnz-cAMP for 30 min respectively,group 6 with 50 nmol H-89 and 50 nmol 8-pCPT-2′-O-Me-cAMP for 30 min respectively,finally 0.5 μg endotoxin for 24 h for group 2 to 6.The morphologic changes were observed with an inverted microscope 0,3 h,6 h and 24 h after endotoxin stimluation and the ratio of long axis of microglia over short axis at 6 h was calcuated.The supernate was collected at 24 h after endotoxin chanllenge and the levels of TNF-α,IL-1β and IL-10 were measured by ELISA.Results The morphology of microglia changed to bipolar rod shapes 3 h after endotoxin challenge,reached peak at 6 h and then returned to oval or amoeboid at 24 h.Only treatment with 6-Bnz-cAMP could significantly inhibit the morphologic change at 6 h,but pretreatment with H-89 completely reversed the effect of 6-Bnz-cAMP.Treatment with 6-Bnz-cAMP could significantly inhibited the overexpression of TNF-α and IL-1β and enhance the secretion of IL-10,while these effects were completely reversed by H-89.Treatment with 8-pCPT-2′-O-Me-cAMP only slightly reduced the expression of TNF-α,meanwhile this effect was not influenced by H-89.Conclusion Compared with Epac,PKA shows more important effects on the expression of inflammatory cytokines by the primary cultured microglia stimulated