摘要
目的研究蛋白酶体抑制对体外培养的星形胶质细胞(AS)IL-6分泌的影响。方法SD乳鼠皮层AS原代培养,并纯化鉴定;不同浓度(0.1,1,2.5,5μM)的蛋白酶体抑制剂(lactacystin)对第二代AS进行短期(24h)急性处理,另一批同期的AS应用较低浓度(0.5μM)的lactacystin长期(4周)慢性处理,应用RT-PCR及ELISA方法检测ASIL-6mRNA的表达和培养基中IL-6蛋白的分泌水平。结果纯化传代的皮层AS经GFAP免疫荧光鉴定,其阳性率可达99.45%;短期抑制组中,1,2.5,5μM蛋白酶体抑制剂可诱导ASIL-6mRNA表达和蛋白分泌增加,与对照组比较差异有显著性(P<0.01);长期抑制组中IL-6mRNA表达及蛋白分泌也较同期对照组增加,差异有统计学意义。结论一定程度蛋白酶体活性抑制可诱导培养的ASIL-6mRNA表达及蛋白分泌增加,提示蛋白酶体功能障碍后可能通过诱导AS分泌IL-6增加来参与阿尔茨海默病的病理改变。
Objective To investigate the effect of proteasome inhibition on the expression and secretion of IL-6 in cultured astrocytes in vitro. Methods The astrocytes were isolated from cortex of 24-hour-old SD rats for in vitro primary cultrue, then they were purified and identified by immunofluorescence technology. The second generation of astrocytes were treated with proteasome inhibitor （lactacystin） at different concentrations （0. 1. 1, 2. 5, 5 μM） for 24 hours and another concentration （0. 5 μM） for 4 weeks. The level of IL-6 mRNA in cultured astrocytes and protein secretion in the media were detected by RT-PCR and ELISA. Results The percentage of GFAP positive cells was about 99. 45% after astrocytes were purified by shaking. Groups of 24 h inhibition by lactacystin at 1, 2. 5 and 5 μM which induced astrocytes to express IL-6 mRNA and increase their secretion showd significant differences compared with the control group （P〈0.01）. The levels of IL-6 mRNA and protein were also increased in the group of 4 week inhibition by lactacystin, and there was statistical difference between these two groups. Conclusions Proteasome inhibition can induce increases of IL-6 mRNA and protein expression in cultured astrocytes. It suggests that the dysfunctional proteasome might involved in the pathology of Alzheimer＇s disease by inducing an increased expression of IL-6 from astrocytes in vitro.
出处
《卒中与神经疾病》
2008年第4期195-198,203,共5页
Stroke and Nervous Diseases
基金
国家杰出青年基金(30725019)
