凝血酶激活小胶质细胞的时间及浓度依赖性研究

Study on time and concentration dependence of thrombin activation on microglia

目的:用不同浓度的凝血酶在设定时间内刺激小胶质细胞,确定凝血酶激活小胶质细胞的最佳浓度。再以最佳浓度分别作用不同时间,以探讨该浓度凝血酶作用小胶质细胞激活的最佳时间。方法:体外培养小胶质细胞N9,分为正常对照组、凝血酶5U/mL组、10U/mL组、15U/mL组、20U/mL组、25U/mL组、30U/mL组,作用时间为6h,用ELISA法检测上清液中肿瘤坏死因子-α(TNF-α)含量、Griess法检测一氧化氮(NO)含量,以确定凝血酶激活小胶质细胞的最佳浓度。然后以最佳浓度分别作用于小胶质细胞3、6、12、24h,再次用上述方法检测细胞上清液中TNF-α、NO含量,确定凝血酶激活小胶质细胞的最佳时间。结果:凝血酶浓度为5U/mL时,TNF-α含量与正常对照组比较差异无统计学意义(P=0.632),凝血酶浓度在10-20U/mL时,TNF-α含量随凝血酶剂量增加而逐渐升高,20U/mL时TNF-α的含量达到最高(P<O.001)。不同浓度凝血酶刺激组,细胞上清液中NO含量比较,组间差异有统计学意义(f=365.819,P<0.001)。凝血酶浓度为5U/mL时,NO的含量为4.44(±0.12)μmol,与正常对照组比较差异有统计学意义(P=0.018),且NO含量随着凝血酶浓度增加而升高,各组与正常对照组比较差异均有显著意义(P<0.001)。结论:凝血酶诱导小胶质细胞激活的最佳浓度为20U/mL,最佳时间为12h。

Objective:To determine the optimum concentration of thrombin activated mieroglia (MG)by stimulating microglia with different concentration of thrombin (T)during the setting time and explore the optimum time for the activation of raicroglia at the concentration of thrombin.Method:Mieroglia N9 cells cultured in vitro were divided into the normal control group,T5U/mL group, T10 U/mL group,T15 U/mL group,T20U/mL group,T25U/mL group and T30U/mL group. The stimulating time lasted for 6 hours.TNF-α and the content of nitric oxide (NO)were detected by Griess method with ELISA in the supernatant,so as to determine the optimum concentration of thrombin activated mieroglia.Then the optimum concentration of TNF-α and NO in cell supernatant was detected with the best concentration of 3,6,12,24 hours respectively,and the optimum time for thrombin to activate mg was determined.Result..When thrombin concentration was 5U/mL,the TNF-α content was not significantly different from that of the control group (P =0.632).When thrombin concentration was 10U/mL-20U/mL,the content of TNF-α increased with the increase of thrombin dose,and the content of TNF-α reached the highest level at 20U/mL (P<0.001).The contents of NO in the stimulation groups with different T concentration in the supernatant were statistically significant (f=365.819,P<0.001).As T concentration was 5U/mL,the content of NO was 4.44+0.12(m),and there was significant difference compared with that in the control group (P= 0.018),and the content of No with T concentration increased.Each group had a significant difference from the normal control group (P<0.001)Conclusion :The optimal concentration of thromhin-induced microglia activation is 20u /ml and the optimal time is 12 hours.