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目的以脂多糖(lipopolysaccride,LPS)刺激,模拟革兰阴性菌感染,观察体外培养大脑皮质星形胶质细胞分泌某些促炎性细胞因子及负性调控分子的变化,为进一步了解脑部炎症中星形胶质细胞的调节机制提供更丰富的实验依据。方法采用改良McCarthy法体外对大脑皮质星形胶质细胞进行培养,抗GFAP抗体荧光鉴定纯度后,用10ng/ml LPS刺激24h后收集细胞和培养上清,Western blotting检测SOCS-3(Suppressor of Cytokine Signaling-3,SOCS-3)蛋白水平,ELISA法检测培养上清中TNF-α和IL-6水平。结果 LPS刺激24h后,星形胶质细胞中SOCS-3有升高趋势,但与正常对照比较差异无统计学意义(P>0.05);星形胶质细胞培养上清中IL-6和TNF-α的水平明显升高,与正常对照比较差异均有统计学意义(P<0.01,P<0.05)。结论 LPS刺激能有效促进星形胶质细胞分泌促炎性细胞因子;SOCS-3的表达与星形胶质细胞分泌细胞因子的变化密切相关,是调控星形胶质细胞适度产生促炎性细胞因子的重要负反馈因子。
Objective To investigate the changes of some proinflammatory cytokines and negative regulatory molecules secreted by cultured cortical astrocytes in vitro by stimulating with lipopolysaccride (LPS). To further understand the effects of lipopolysaccharide (LPS) on brain inflammation The regulatory mechanism of astrocytes provides a richer experimental basis. Methods The cultured cortical astrocytes were cultured by modified McCarthy method. The purity of anti-GFAP antibody was identified by fluorescence. After stimulated with 10ng / ml LPS for 24 hours, the cells and the supernatant were harvested. The expression of SOCS-3 (Suppressor of Cytokine Signaling-3, SOCS-3) were detected by enzyme linked immunosorbent assay (ELISA). The levels of TNF-α and IL-6 in culture supernatants were detected by ELISA. Results After stimulated by LPS for 24 h, SOCS-3 in astrocytes increased, but there was no significant difference compared with normal control (P> 0.05). IL-6 and TNF in astrocyte culture supernatants -α increased significantly (P <0.01, P <0.05) compared with the normal control. Conclusion LPS stimulation can effectively promote the secretion of proinflammatory cytokines by astrocytes. The expression of SOCS-3 is closely related to the changes of cytokines secreted by astrocytes, Important negative feedback factor of the factor.