Aβ_(1-42)诱导的小胶质细胞炎性反应对神经元细胞Mecp2蛋白表达的影响

Effect of Aβ_(1-42) induced microglial inflammatory response on the expression of Mecp2 protein in neuron cells

目的:体外研究Aβ_(1-42)诱导的小胶质细胞炎症反应对海马神经元细胞甲基化Cp G结合蛋白(Mecp2)表达的影响。方法:(1)用不同浓度的Aβ_(1-42)(终浓度分别为0,5,10,20,30μmol/L)处理小胶质细胞系(N9),24h后取上清液,ELISA检测每组N9细胞培养上清中炎症因子TNF-α、IL-1β的含量,选择合适的Aβ_(1-42)的终浓度。(2)用Aβ_(1-42)(终浓度为20μmol/L)及TLR4拮抗剂TAK-242(终浓度为1μg/ml)处理小胶质细胞系(N9),分为3组:正常对照组(con组)、单纯Aβ_(1-42)处理组(Aβ_(1-42)组)、Aβ_(1-42)处理后TAK-242干预组(TAK-242组)。24 h后取上清液,ELISA检测每组N9细胞培养上清中炎症因子TNF-α、IL-1β的含量。(3)实验分组同(2),24 h后收集培养液,分别干预生长良好的海马神经元细胞系(HT-22)。24 h后,通过Western Blot、免疫荧光染色方法检测每组HT-22细胞中Mecp2蛋白的表达。结果:(1)ELISA结果显示:与con组相比,Aβ_(1-42)处理后,N9细胞分泌的TNF-α、IL-1β的表达量随Aβ_(1-42)浓度的增加而增加(P<0.05)。(2)ELISA结果显示:与con组相比,Aβ_(1-42)组的TNF-α、IL-1β的表达水平明显提高(P<0.05);TAK-242组的细胞分泌TNF-α、IL-1β水平较Aβ_(1-42)组显著降低(P<0.05)。(3)Western Blot结果显示:与con组相比,Aβ_(1-42)组HT-22细胞中Mecp2蛋白表达明显增加(P<0.05);与Aβ_(1-42)组相比,TAK-242组HT-22细胞中Mecp2蛋白表达明显减少(P<0.05)。(4)免疫荧光染色结果显示:与con组相比,Aβ_(1-42)组HT-22细胞中Mecp2蛋白表达明显增强;而与Aβ_(1-42)组相比,TAK-242组HT-22细胞中Mecp2蛋白表达减弱。结论:Aβ诱导的小胶质细胞发生炎性反应,导致神经元的损伤,可能是由于这种炎性反应使得神经元细胞中Mecp2蛋白的表达量增加,从而损伤神经元。

Objective： The effect of Aβ（1-42） induced microglia inflammatory response on the expression of methylated Cp G binding protein（ Mecp2） in hippocampal neurons in vitro. Methods：（ 1） The treatment of microglia cell line（ N9） with different concentrations of Aβ（1-42）（ final concentration of 0,5,10,20,30 μmol/L）,24 h after the superna-tant,ELISA detection of each N9 cell culture supernatant in inflammatory factor TNF-α,IL-1β,the choice of Aβ（1-42） concentration.（ 2） The treatment of microglia cell line（ N9） with Aβ（1-42）（ final concentration of 20 μmol/L） and TLR4 antagonist TAK-242（ final concentration of 1 μg/ml）. It was divided into 3 groups： normal control group（ group con）,simple Aβ（1-42） treatment group（ group Aβ（1-42）） and TAK-242 intervention group after Aβ（1-42） treatment（ group TAK-242）.After 24 h,the supernatant was taken,and the content of the inflammatory factors TNF-α and IL-1β in the culture supernatant of N9 cells in each group was detected by ELISA.（ 3） The experimental grouping is the same as（ 2）,the culture fluid was collected after 24 h to interfere with the well grown hippocampal neuronal cell line（ HT-22）. After 24 h,the expression of Mecp2 protein in each HT-22 cell was detected by Western Blot and immunofluorescence staining.Results：（ 1） The results of ELISA showed that compared with the con group,after Aβ（1-42） treatment,the expression of TNF-α and IL-1β secreted by N9 cells increased with the increase of Aβ（1-42） concentration（ P〈0. 05）.（ 2） The results of ELISA showed that compared with the con group,the expression level of TNF-α and IL-1β in Aβ（1-42） group was significantly increased（ P〈0. 05）; The level of TNF-α and IL-1β secreted in the TAK-242 group were significantly lower than those in the Aβ（1-42） group（ P〈0. 05）.（ 3） The results of Western Blot showed that compared with the con group,the expression of Mecp2 protein in the Aβ（1-42） group HT-22 cells increased significantly（ P〈0. 05）. Compared with the Aβ（1-42） group,the Mecp2 protein expression in the HT-22 cells of the TAK-242 group decreased significantly（ P 0. 05）.（ 4） Immunofluorescence staining showed that compared with the con group,the expression of Mecp2 protein in the HT-22 cells increased significantly in the Aβ（1-42） group,while the expression of Mecp2 protein in the HT-22 cells of the TAK-242 group was weaker than that of the Aβ（1-42） group. Conclusion： Aβ induced inflammatory reaction in microglia can lead to neuronal damage,probably due to the increased expression of Mecp2 protein in neurons and the damage of neurons.