脂多糖预处理活化星形胶质细胞来源细胞外囊泡可保护神经元  

Protective effect of lipopolysaccharide-preconditioned astrocyte extracellular vesicles on neurons

作　　者：孙海涛 黄永辉[1] 龚爱华[2] 曹兴兵[1] 李震 Sun Haitao;Huang Yonghui;Gong Aihua;Cao Xingbing;Li Zhen(Affiliated Hospital of Jiangsu University,Zhenjiang 212000,Jiangsu Province,China;Jiangsu University,Zhenjiang 212000,Jiangsu Province,China)

机构地区：[1]江苏大学附属医院,江苏省镇江市212000 [2]江苏大学,江苏省镇江市212000

出　　处：《中国组织工程研究》2022年第13期1985-1992,共8页Chinese Journal of Tissue Engineering Research

基　　金：镇江市科技项目(SH2020053),项目负责人:黄永辉。

摘　　要：背景:中枢神经系统疾病往往与神经元凋亡密切相关。星形胶质细胞与神经元联系紧密,来源于星形胶质细胞特别是活化星形胶质细胞的细胞外囊泡能否抑制谷氨酸所致损伤神经元的凋亡,发挥神经保护作用,尚未见报道。目的:探究星形胶质细胞胞外囊泡(astrocyte extracellular vesicles,AS-EVs)和脂多糖预处理的星形胶质细胞胞外囊泡(LPS-preconditioned astrocyte extracellular vesicles,LPAS-EVs)对谷氨酸诱导神经元损伤的作用及可能机制。方法:体外培养星形胶质细胞,并利用脂多糖预处理建立诱导活化模型,收集上清,超离法提取细胞外囊泡并进行蛋白定量和鉴定。利用PC12细胞进行初步实验,先探究AS-EVs及LPAS-EVs对PC12细胞增殖的影响,再建立谷氨酸诱导的PC12细胞和原代神经元损伤模型,实验分4组:PBS组(与EVs组同体积的PBS处理24 h)、谷氨酸组(10 mmol/L谷氨酸处理24 h)、AS-EVs组(200 mg/L AS-EVs预处理24 h+10 mmol/L谷氨酸处理24 h)、LPAS-EVs组(200 mg/L LPAS-EVs预处理24 h+10 mmol/L谷氨酸处理24 h)。CCK-8法检测PC12细胞存活率,Western blot检测细胞凋亡相关蛋白Bax和Bcl-2表达。结果与结论:①成功提取星形胶质细胞并建立脂多糖诱导的星形胶质细胞活化模型;②成功提取AS-EVs及LPAS-EVs,经鉴定符合细胞外囊泡形态学标准,相关标志物表达阳性;③AS-EVs及LPAS-EVs对PC12细胞增殖无明显影响;④与谷氨酸组相比,AS-EVs和LPAS-EVs能够提高PC12细胞存活率,且LPAS-EVs作用强于AS-EVs(P均<0.05,n=3);⑤AS-EVs和LPAS-EVs能够抑制促凋亡蛋白Bax的表达,促进抗凋亡蛋白Bcl-2的表达(P均<0.01,n=3),从而抑制PC12细胞和原代神经元凋亡,发挥神经保护作用,且LPAS-EVs的作用强于AS-EVs(P均<0.05);⑥结果表明,星形胶质细胞所释放的细胞外囊泡能够抑制谷氨酸所致损伤神经元的凋亡,具有神经保护作用,并且在受到脂多糖刺激后,这种作用得到增强。BACKGROUND:Central nervous system diseases are often closely related to neuronal apoptosis.Astrocytes are closely related to neurons.It remains poorly understood whether extracellular vesicles,which are derived from astrocytes,especially activated astrocytes,may inhibit glutamate-induced neuronal apoptosis,thereby exerting neuroprotective effect.OBJECTIVE:To investigate the role and possible mechanism of astrocyte extracellular vesicles(AS-EVs)and lipopolysaccharide-preconditioned astrocyte extracellular vesicles(LPAS-EVs)in glutamate-induced neuronal injury.METHODS:Astrocyte was cultured in vitro and induced activation model was established by lipopolysaccharide.The supernatant of cells of two groups was collected and extracellular vesicles were extracted by hypervelocity centrifuges,and then identified.PC12 cells were used for preliminary experiments.First,we investigated the effects of AS-EVs and LPAS-EVs on PC12 cell proliferation,and then established the PC12 cells and primary neurons injury model induced by glutamate.Experiment was divided into four groups:PBS group(PBS of the same volume as EVs groups,24 hours),glutamate group(10 mmol/L glutamate,24 hours),AS-EVs group(200 mg/L AS-EVs,24 hours+10 mmol/L glutamate,24 hours),and LPAS-EVs group(200 mg/L LPAS-EVs,24 hours+10 mmol/L glutamate,24 hours).The survival rate of PC12 cells was determined by CCK-8 assay.Western blot assay was used to detect the expression of apoptosis related proteins Bax and Bcl-2.RESULTS AND CONCLUSION:(1)Astrocyte was extracted and lipopolysaccharide-induced astrocyte activation model was established successfully.(2)AS-EVs and LPAS-EVs were successfully extracted,which met the morphological standards of extracellular vesicles and showed positive expression of related markers.(3)AS-EVs and LPAS-EVs had no significant effect on PC12 cells proliferation.(4)Compared with glutamate group,AS-EVs and LPAS-EVs could improve the survival rate of PC12 cells,and the effect of LPAS-EVs was stronger than that of AS-EVs(all P<0.05,n=3).(5)AS-EVs

关 键 词：星形胶质细胞 细胞外囊泡 脂多糖 PC12细胞 神经元 凋亡 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学]