神经干细胞条件培养液对帕金森病模型神经元损伤的保护作用  

Protective effect of neural stem cell-conditioned medium on neuronal injury in Parkinson′s disease model

作　　者：倪玮 周继勤 凌亚亭 吕孝瑞 牛冬冬 曾宇 仇昀 司雨 王子瑜 胡嘉波[1] NI Wei;ZHOU Jiqin;LING Yating;LYU Xiaorui;NIU Dongdong;ZENG Yu;QIU Yun;SI Yu;WANG Ziyu;HU Jiabo(School of Medicine,Jiangsu University,Zhenjiang Jiangsu 212013;Health Clinical Laboratories,Health BioMed Co.Ltd.,Ningbo Zhejiang 315042,China)

机构地区：[1]江苏大学医学院,江苏镇江212013 [2]海尔施生物医药股份有限公司医学检验所,浙江宁波315042

出　　处：《江苏大学学报（医学版）》2023年第4期284-291,共8页Journal of Jiangsu University：Medicine Edition

基　　金：校企协同创新项目(20200605)。

摘　　要：目的:探究神经干细胞条件培养液(neural stem cell-conditioned medium,NSC-CM)对6-羟基多巴胺(6-hydroxydopamine,6-OHDA)诱导的帕金森病(Parkinson′s disease,PD)体内外模型中神经元损伤的影响及可能机制。方法:采用6-OHDA脑立体定位注射SD大鼠的内侧前脑束建立体内PD模型,并在黑质和纹状体中注射NSC-CM进行治疗;免疫组织化学染色检测黑质和纹状体中多巴胺能神经元(dopaminergic neuron,DAN)丢失情况,免疫印迹实验检测黑质中线粒体融合蛋白1(mitofusin1,Mfn1)的表达水平。采用6-OHDA处理大鼠肾上腺髓质嗜铬细胞瘤PC12细胞建立体外PD细胞模型,并用NSC-CM进行预处理;检测PD细胞模型的乳酸脱氢酶(LDH)活性、细胞活性氧水平,免疫印迹实验检测Mfn1表达水平。液相色谱串联质谱(liquid chromatography-tandem mass spectrometry,LC-MS/MS)检测NSC-CM成分并进行生物信息学分析;流式细胞术和激光共聚焦显微镜观察PC12细胞对神经干细胞(neural stem cells,NSCs)线粒体的内化。比较去除线粒体前后NSC-CM对PC12细胞存活率及线粒体荧光强度的影响。结果:注射NSC-CM后,PD大鼠DAN丢失减少,Mfn1蛋白表达水平明显升高(P<0.05)。NSC-CM预处理后,PD细胞模型LDH活性明显降低(P<0.05),活性氧水平明显下降(P<0.05),Mfn1蛋白表达水平明显升高(P<0.05)。LC-MS/MS检测到NSC-CM中存在线粒体成分,并观察到PC12细胞对NSC-CM中线粒体的内化。去除线粒体NSC-CM引起PC12细胞存活率显著下降(P<0.05)和线粒体荧光强度显著减弱(P<0.05)。结论:NSC-CM可改善6-OHDA造成的神经元死亡、氧化应激水平升高和线粒体融合水平下降,而该保护作用可能与PC12细胞内化NSCs线粒体有关。Objective:To investigate the protective effect of neural stem cell-conditioned medium(NSC-CM)on 6-hydroxydopamine(6-OHDA)-induced neuronal injury in vitro and in vivo model of Parkinson′s disease(PD)and its potential mechanisms.Methods:The in vivo PD model was established by stereotaxic injection of 6-OHDA into the medial forebrain bundle of SD rats,and NSC-CM was injected into the substantia nigra and striatum for treatment.The loss of dopaminergic neuron(DAN)in the substantia nigra and striatum was detected by immunohistochemical staining.The expression level of mitofusin 1(Mfn1)in substantia nigra was detected by Western blotting;The PD cell model was established in vitro by treating rat adrenal medulla pheochromocytoma(PC12 cells)with 6-OHDA,and pretreated with NSC-CM.Lactate dehydrogenase(LDH)release and cellular reactive oxygen species(ROS)levels in PD cell model were detected,and the expression level of Mfn1 was detected by Western blotting.The components of NSC-CM were detected by liquid chromatography-tandem mass spectrometry(LC-MS/MS)and analyzed by bioinformatics analysis.Flow cytometry and laser confocal microscopy were used to observe the internalization of neural stem cells(NSCs)mitochondria by PC12 cells.The effects of NSC-CM on the cell viability and mitochondrial fluorescence intensity of PC12 cells were compared before and after removal of mitochondria.Results:After injection of NSC-CM,the loss of DAN was significantly decreased and the expression level of Mfn1 protein was greatly increased in PD rats(P<0.05).After NSC-CM pretreatment,the release of LDH and the level of ROS in the PD cell model were significantly decreased(P<0.05),and the expression level of Mfn1 protein was remarkably increased(P<0.05).LC-MS/MS detected the presence of mitochondrial components in NSC-CM,and observed the internalization of mitochondria in NSC-CM by PC12 cells.Removal of mitochondria from NSC-CM significantly decreased the survival rate of PC12 cells(P<0.05)and the fluorescence intensity of mitochondria(P<0.05)

关 键 词：神经干细胞 条件培养液 6-羟基多巴胺 帕金森病 线粒体融合蛋白1 

分 类 号：R741.02[医药卫生—神经病学与精神病学]