趋化因子CC配基2对α?Synuclein诱导的小胶质细胞增殖及神经元凋亡的影响

Study on the effect of CCL2 on the proliferation of microglia and apoptosis of Neuron induced byα-Synuclein

目的探讨趋化因子CC配基2(CCL2)对α?突触核蛋白(α?Synuclein)介导的小胶质细胞增殖及神经元凋亡的影响。方法体外分离培养原代小胶质细胞和原代神经元。小胶质细胞分为4组,依次为对照组、CCL2组、α?Synuclein组、CCL2+α?Synuclein组。对照组中加入等量的PBS,CCL2组细胞中加入含有CCL2浓度为0.05ng/μL的培养液。α?Synuclein组细胞中加入含有α?Synuclein浓度为0.2ng/μL的培养液。CCL2+α?Synuclein组中加入含有0.05ng/μL CCL2、0.2ng/μLα?Synuclein的细胞培养液。培养24h后,检测小胶质细胞增殖情况及细胞中α?Synuclein蛋白水平,同时检测细胞培养液中TNF-α、IL-1β、NO含量。用各组小胶质细胞培养液培养原代神经元,观察神经元凋亡情况及神经元中Cleaved Caspase-3、Akt、p-Akt水平。结果 CCL2组、α?Synuclein组、CCL2+α?Synuclein组小胶质细胞增殖活性及分泌TNF-α、IL-1β、NO水平明显高于对照组(P<0.05)。CCL2组、CCL2+α?Synuclein组小胶质细胞中α?Synuclein水平明显高于对照组(P<0.01)。CCL2组、α?Synuclein组、CCL2+α?Synuclein组小胶质细胞培养液作用后的神经元凋亡率及Cleaved Caspase-3蛋白水平明显高于对照组,pAkt水平低于对照组(P<0.01)。结论 CCL2促进α?Synuclein引起的小胶质细胞增殖和分泌TNF-α、IL-1β、NO的能力,对α?Synuclein引起的神经元凋亡也具有促进作用,促凋亡作用机制可能与Akt信号通路有关。

Objective To investigate the effects of CCL2 on the proliferation of microglia and apoptosis of Neuron induced by α-Synuclein .Methods Primary microglia and primary neurons were isolated and cultured in vitro .The microglia were divided into 4 groups ,which were the control group ,the CCL2 group ,theα-Synuclein group ,the CCL2＋ α-Synuclein group .In the control group ,the amount of PBS was added ,and the cells in the CCL2 group were added with the culture solution containing 0.05ng/μL concentration of CCL2.α-Synuclein group cells were added to culture medium containing a 0.2ng/μL concentration of α-Synuclein . CCL2＋ α-Synuclein group were added with CCL20.05ng/μL ,0.2ng/μL α-Synuclein .After 24h culture ,the proliferation of micro-glia and the level of α-Synuclein ,the contents of TNF-α,IL-1βand NO in cell culture medium were detected .The primary neurons were cultured with cell culture medium in each group ,the neuron apoptosis was observed and the levels of Cleaved Caspase-3 Akt , p-Akt and were observed .Results The proliferation activity and secretion of TNF-α,IL-1β,NO in the CCL2 group ,the α-Synuclein group ,the CCL2＋ α-Synuclein group were significantly higher than that in control group （P〈0.05） .The level of α-Sy-nuclein in CCL2 group and CCL2＋ α-Synuclein group was significantly higher than that in control group （P〈0.01） .The apopto-sis rate and Cleaved Caspase-3 protein level of CCL2 group ,α-Synuclein group and CCL2＋ α-Synuclein group were significantly higher than those in control group ,the level of p-Akt was lower than that of control group （P〈0.01） .Conclusion CCL2 pro-motes the proliferation of the microglia and secretion of TNF-α,IL-1β,NO induced by α-Synuclein ,and can promotes the apoptosis of neuron ,the mechanism may be related to Akt signaling pathway