远隔缺血预适应血清外泌体对神经细胞DNA甲基化水平及缺血耐受相关基因表达的影响  

Effects of serum exosomes of remote ischemic preconditioning on neuronal DNA methylation level and ischemic tolerance related gene expression

作　　者：邹明新[1] 崔钧贺 刘娜 张璞 邵国[2,3,4] Zou Mingxin;Cui Junhe;Liu Na;Zhang Pu;Shao Guo(Faculty of Biology Science and Technology,Baotou Teachers College,Science and Technology University of Inner Mongolia,Baotou 014030,China;不详)

机构地区：[1]内蒙古科技大学包头师范学院生物科学与技术学院,014030 [2]包头医学院内蒙古自治区低氧转化医学重点实验室 [3]首都医科大学宣武医院低氧适应转化医学北京重点实验室 [4]内蒙古科技大学包头医学院第二附属医院消化疾病研究所检验科

出　　处：《中国脑血管病杂志》2021年第2期107-114,共8页Chinese Journal of Cerebrovascular Diseases

基　　金：国家自然科学基金资助项目(82060337、81660307);内蒙古自治区高等学校科学研究项目(NJZY18384)。

摘　　要：目的研究人远隔缺血预适应(RIPC)后产生的血清外泌体对人神经母细胞瘤细胞系(SH-SY5Y神经细胞)DNA甲基化及氧糖剥夺(OGD)耐受的影响。方法6名男性在校大学生志愿者(身高:170~180 cm;体质量:60~80 kg;状态:上午、空腹)使用RIPC训练仪进行缺血预适应并采集肘中静脉血和血清,使用透射电镜方法对外泌体形态进行检测和鉴定。构建神经细胞OGD模型,当正常细胞培养至70%细胞密度时,撤去1640培养基改用无糖培养基,将OGD处理的SH-SY5Y细胞与外泌体孵育后进行处理,分为正常对照组(C组)、氧糖剥夺组(OGD组)、OGD+HuE-C组(SH-SY5Y细胞经过OGD处理后与正常人血清外泌体共孵育)、OGD+HuE-RIPC组(SH-SY5Y细胞经过OGD处理后和RIPC人血清外泌体共孵育)。3-(4,5-二甲基噻唑-2-基)-5-(3-羧基喹啉)-2-(4-磺基苯基)-2H-四唑(MTS)法检测细胞对OGD的耐受,亚硫酸氢钠限制性内切酶试验(COBRA)分析神经细胞基因组甲基化程度,实时聚合酶链反应(PCR)和Western Blot分析血管内皮生长因子(VEGF)和促红细胞生成素(EPO)的基因及蛋白表达水平。结果透射电镜结果显示,超速离心法可以成功分离血清外泌体。显微镜下观察正常SH-SY5Y细胞胞体丰满,经历OGD后,大量细胞呈皱缩高亮的凋亡状态;加入HuE-C外泌体后细胞形态恢复有限;加入HuE-RIPC外泌体后,SH-SY5Y细胞形态有了较为明显的恢复。形态学分析结果显示,RIPC外泌体对缺血状态下神经细胞有保护作用。MTS结果显示,在OGD模型中,将人RIPC血清外泌体与SH-SY5Y细胞共孵育后,SH-SY5Y细胞活力较OGD组明显提高(P<0.01)。COBRA实验结果显示,在SH-SY5Y细胞OGD模型中加入人RIPC血清外泌体后,SH-SY5Y细胞Alu甲基化程度下降(P=0.048),LINE-1非甲基化水平升高(P=0.040),LINE-1甲基化水平下降(P=0.004),细胞基因组甲基化呈显著下调趋势。实时PCR和Western Blot检测结果显示,加入RIPC血清外泌体后,OGD模型中,SH-SY5Y细�Objective To explore the effects of serum exosomes produced after human remote ischemic preconditioning(RIPC)on DNA methylation of SY5Y nerve cells regarding glucose and oxygen deprivation(OGD)tolerance.Methods 6 male college student volunteers(Height:170-180 cm;Body weight:60-80 kg;Status:Morning,fasting)used RIPC training device for ischemic preconditioning and their blood and serum samples from intermedian cubital veins were collected.Exosomes were detected and identified by transmission electron microscopy.An OGD model of nerve cells was constructed.When normal cells were cultured to 70%cell density,the 1640 medium was removed and replaced with a glucose-free medium.SH-SY5Y cells treated with OGD were incubated with exosomes,and were divided into control group(C group),OGD group,OGD treated SH-SY5Y cells incubating with normal human serum exosomes(OGD+HuE-C)group,OGD treated SH-SY5Y cells incubating with RIPC human serum exosomes(OGD+HuE-RIPC)group.3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxyquinoline)-2-(4-sulfophenyl)-2H-tetrazole(MTS)method was used for detecting cell tolerance of OGD,sodium bisulfite restriction endonuclease test(COBRA)for nerve cell genome methylation analysis,real-time polymerase chain reaction(PCR)and Western Blot for gene and protein expression analysis of vascular endothelial growth factor(VEGF)and erythropoietin(EPO).Results The results of transmission electron microscopy showed ultracentrifugation method can successfully separate serum exosomes.Normal SH-SY5Y cells were observed to be plump under the microscope.A large number of cells were shrunken and bright in an apoptotic state after OGD.After adding HuE-C exosomes,SH-SY5Y cell morphology was slightly restored;after adding HuE-RIPC exosomes,SH-SY5Y cell morphology has a more obvious recovery.Morphology analysis showed that RIPC exosomes had a protective effect on nerve cells under ischemia;MTS results showed that in OGD model,after co-incubation of SH-SY5Y cells with HuE-RIPC exosomes,nerve cell viability was significantly higher t

关 键 词：远隔缺血预适应 外泌体 缺血耐受 神经保护 

分 类 号：R743.3[医药卫生—神经病学与精神病学]