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24h；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0.5 μg孵育24h.内毒素处理后0、3、6、24h后,倒置显微镜下观察细胞形态改变,并计算6h时长短轴比值.24h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3h,细胞成双极杆状细胞,6h时最为明显,24h细胞形态又恢复至椭圆或阿米巴样.仅6-Bnz-cAMP能明显抑制6h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24h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.%Aim To observe the effect of protein kinase A (PKA) and Epac ( exchange proteins directly activated by cAMP)-the downstream effectors of cAMP onthe expression of inflammatory cytokines by the primary cultured microglia stimulated with endotoxin. Methods The 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 (xg 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 mi-croglia over short axis at 6 h was calcuated. The super-nate was collected at 24 h after endotoxin chanllenge and the levels of TNF-a, IL-1 p and IL-10 were meas-ured 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-a and IL-1 (3 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-a, 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 with endotoxin.
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