摘要
目的探究人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,HUCMSC)源外泌体对氧糖剥夺复氧人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)凋亡的抑制作用,阐明其可能的机制。方法培养HUCMSC,将收集的细胞上清液使用超速离心法提取HUCMSC分泌的外泌体(HUCMSC-exosomes,HUCMSC-EXO)并对其进行鉴定,将HUVEC随机分为正常对照组、模型组(氧糖剥夺/复氧组)和分别添加不同浓度的HUCMSC-EXO(20μg/ml、40μg/ml、60μg/ml)处理组(在模型组的基础上加入HUCMSC-EXO)。倒置相差显微镜形态学观察各组HUVEC细胞的表观变化,使用CCK8试剂测算各组HUVEC增殖抑制率,Western blot检测相关凋亡蛋白Caspase-3、Bax、Bcl-2的表达水平。另设模型组、HUCMSC-EXO+模型组、抑制剂+模型组和HUCMSC-EXO+抑制剂+模型组,Western blot观察HUCMSC-EXO、抑制剂及两者共用对HUVEC细胞HIF-1α及Bax的表达水平的影响。结果成功提取和鉴定HUCMSC-EXO,在倒置相差显微镜下,与正常对照组比较,模型组和不同浓度HUCMSC-EXO处理组的HUVEC体积缩小,变圆,连接疏散,生长状态较差;CCK8检测,HUCMSC-EXO处理组较模型组的细胞活力显著上升,差异有统计学意义(t=9.23,P<0.05),并与剂量呈正相关。Western blot检测,与对照组比较,模型组HUVEC的Caspase-3[(0.296±0.038),(0.879±0.088);t=14.92,P<0.05]、Bax[(0.234±0.034),(0.762±0.084);t=14.36,P<0.05]表达水平上调,Bcl-2的表达水平下调[(0.863±0.103),(0.387±0.059);t=9.85,P<0.05],均差异有统计学意义;与模型组比较,HUCMSC-EXO处理组HUVEC的Caspase-3(0.586±0.075)、Bax(0.311±0.055)水平下调(t=6.24,11.01,均P<0.05),Bcl-2(0.665±0.071)水平上调,均差异有统计学意义(t=7.45,P<0.05)。抑制剂干预实验显示,与HUCMSC-EXO+抑制剂+模型组比较,抑制剂+模型组HIF-1α蛋白[(0.348±0.055),(0.388±0.077);t=1.04,P>0.05]和Bax蛋白[(0.363±0.069),(0.370±0.064);t=0.18,P>0.05]表达差异无统计学意义(均P>0.05),但�
Objective To explore the inhibitory effect of exosomes secreted by human umbilical cord mesenchymal stem cells(HUCMSC)on apoptosis of human umbilical vein endothelial cells(HUVEC)after model group(oxygen-glucose deprivation reoxygenation),and to clarify its possible mechanism.Methods Human umbilical cord mesenchymal stem cells were cultured.The collected cell supernatant was stored in a centrifugal tube.The exosomes secreted by human umbilical cord mesenchymal stem cells were extracted by ultracentrifugation and identified.Human umbilical vein endothelial cells were randomly divided into control group,model group and different concentrations of HUCMSC-EXO(20μg/ml,40μg/ml,60μg/ml)treatment groups(adding HUCMSC-EXO into the model group).The morphological changes of HUVEC cells in each group were observed by inverted phase contrast microscope,and the proliferation inhibition rate of HUVEC in each group was measured by CCK-8 reagent.Western blot was used to detect the expression of apoptosis-related proteins Caspase-3,Bax,Bcl-2 and hypoxia-associated protein hypoxia inducible factor 1α(HIF-1α).Inhibitor(HIF-1αinhibitor)+model group and HUCMSC-EXO+inhibitor+model group were added on the basis of the above experiments.Western blot analysis was performed to observe the effects of HUCMSC-EXO,inhibitor and both of them on HIF-1αand Bax expressions in HUVEC.Results HUCMSC-EXO was successfully extracted and identified.Compared with the control group,the volume of HUVEC in the model group and the HUCMSC-EXO group with different concentrations decreased,became round,connected and evacuated,and the growth state was poor under the inverted phase contrast microscope.CCK-8 detection showed that the cell viability in the HUCMSC-EXO group was significantly higher than that in the model group,the difference was statistically significant(t=9.23,P<0.05).Western blot analysis showed that compared with the control group,the expression levels of Caspase-3((0.296±0.038),(0.879±0.088);t=14.92,P<0.05),Bax((0.234±0.034),(0.762±0.0
作者
叶益超
李晓红
史新宇
张震文
刘晓银
陈健
张哲
武卫周
王景景
周红霞
王毅
Ye Yichao;Li Xiaohong;Shi Xinyu;Zhang Zhenwen;Liu Xiaoyin;Chen Jian;Zhang Zhe;Wu Weizhou;Wang Jingjing;Zhou Hongxia;Wang Yi(Logistics University of People's Armed Police Forces,Tianjin 300309,China;Institution of Brain Trauma and Neurology Disease of People's Armed Police Forces,Tianjin Key Laboratory of Neurotrauma Repair,Tianjin 300162,China;Academy of Medical Engineering and Translational Medicine,Tianjin Uniersity,Tianjin 300072,China;Department of Neurology,Tianjin Hospital,Tianjin 300211,China)
出处
《中华行为医学与脑科学杂志》
CAS
CSCD
北大核心
2019年第12期1057-1063,共7页
Chinese Journal of Behavioral Medicine and Brain Science
基金
国家自然科学基金项目(81771352,81772018,81671222,81771350)
天津市杰出青年科学基金项目(18JCJQJC48500)
天津市天津医院院级课题(TJYY1810)。
关键词
人脐带间充质干细胞
外泌体
氧糖剥夺/复氧
人脐静脉内皮细胞
细胞凋亡
Human umbilical mesenchymal stem cells
Exosomes
Oxygen-glucose deprivation reoxygenation
Human umbilical venous endothelial cells
Apoptosis
