摘要
目的研究富含微小核糖核酸(miR)-132-3p的神经干细胞(NSC)释放的外泌体(EX)对缺氧且无糖(OGD)损伤的内皮细胞功能的保护作用。方法利用SPF级C57BL/6新生小鼠20只,原代培养NSC,采用装载miR-132-3p的慢病毒感染NSC获得NSC^(miR-132-3p),同时采用装载Scramble序列的慢病毒感染NSC获得NSC^(NC)。提取NSC^(NC)和NSC^(miR-132-3p)释放的EX,分别获得NSC-EX和NSC-EX^(miR-132-3p)。将EX与OGD受损小鼠脑微血管内皮细胞(mBMEC)共培养,采用血管形成检测试剂盒、CCK8试剂盒、划痕实验、流式细胞技术、实时荧光定量逆转录聚合酶链反应和Western blotting分别检测mBMEC血管形成能力、增殖能力、迁移能力、凋亡、miR-132-3p表达和磷酸化细胞外调节激酶(pERK1/2)水平;丝裂原活化的细胞外信号调节激酶1/2(MEK1/2)通路抑制剂(PD0325901)处理检测MEK1/2-ERK1/2通路对NSC-EX^(miR-132-3p)功能的调控作用。采用Student’st检验法比较上述各指标的组间差异。结果NSC-EX处理显著增加OGD诱导下mBMEC细胞的增殖能力(0.34±0.04vs0.23±0.04)、迁移能力[(238.45±18.72)μmvs(175.38±12.53)μm]、血管形成能力[(9±2)条vs(3±1)条]及ERK1/2磷酸化水平(0.27±0.02vs0.07±0.01),减少细胞凋亡[(22.5±3.1)%vs(35.7±4.7)%],差异均具有统计学意义(t=2.98,P=0.042;t=4.92,P=0.008;t=4.65,P=0.011;t=16.09,P=0.001;t=6.05,P=0.004)。与NSC-EX处理相比,NSC-EX^(miR-132-3p)处理更有效增加mBMEC细胞的增殖能力(0.45±0.06vs0.34±0.04)、迁移能力[(346.51±19.28)μmvs(238.45±18.72)μm]、血管形成能力[(15±3)条vs(9±2)条]、miR-132-3p表达(5.76±0.58vs2.85±0.39)和ERK1/2磷酸化水平(0.47±0.03vs0.27±0.02),减少细胞凋亡[(15.8±2.9)%vs(22.5±3.1)%],差异均具有统计学意义(t=2.97,P=0.041;t=6.27,P=0.003;t=3.05,P=0.038;t=7.16,P=0.002;t=9.69,P=0.001;t=3.06,P=0.04)。PD0325901处理降低NSC-EX^(miR-132-3p)对细胞增殖能力(0.28±0.03vs0.45±0.06)、迁移能力[(193.21±18.93)μmvs(346.51±19.28)μm]、血管形成能力[(6±2)条vs(15±3)条]、凋亡[(29.4±3.4)%vs(15.8±2.9)%]和ERK1/2磷酸化水平(0.16±0.01vs0.47±0.03)的作用效果,差异均具有统计学意义(t=5.05,P=0.007;t=9.23,P=0.005;t=4.67,P=0.009;t=5.82,P=0.004;t=17.14,P=0.001)。结论miR-132-p通过激活MEK1/2-ERK1/2信号通路显著增强NSC-EX对OGD损伤的mBMEC功能的保护作用。
ObjectiveTo investigate the effects of micro RNA(miR)-132-3p enriched neural stem cell-derived exosomes(NSC-EXs)on oxygen and glucose deprivation(OGD)injured mouse brain microvessel endothelial cells(mBMECs).MethodsPrimary neural stem cells(NSCs)were isolated and cultured.NSC^(NC)was obtained by transfecting NSCs with lentivirus loaded with scramble sequence,and NSC^(miR-132-3p)was obtained from NSCs transfected with lentivirus loaded with miR-132-3p vector.NSC-EXs and NSC-EXsmiR-132-3pwere extracted from NSCs and NSC^(miR-132-3p)culture medium,respectively;EXs were co-cultured with OGD treated mBMECs,and angiogenesis kit,CCK8 kit,scratch test,Annexin V-PE/7-AAD kit,quantitative Real-time polymerase chain reaction,and Western Blotting were used to detect the tube formation,proliferation,migration,apoptosis,miR-132-3p level,and phosphorylation of extracellular signal-regulated kinase 1/2(ERK1/2)in OGD treated mBMECs.Mitogen extracellular signal regulated kinase(MEK1/2)inhibitor(PD0325901)was used to measure the effect of NSC-EXsmiR-132-3pon regulating MEK1/2-ERK1/2 signaling pathway.Comparisons between two groups were analyzed byttest.ResultsCompared to OGD group,NSC-EXs-treated group were significantly with increased proliferation(0.34±0.04vs0.23±0.04,t=2.98,P=0.042),migration[(238.45±18.72)μmvs(175.38±12.53)μm,t=4.92,P=0.008],tube formation(9±2vs3±1,t=4.65,P=0.011),and ERK1/2 phosphorylation(0.27±0.02vs0.07±0.01,t=16.09,P=0.001),while decreased apoptosis of mBMECs[(22.5±3.1)%vs(35.7±4.7)%,t=6.05,P=0.004].Compared with NSC-EXs-treated group,NSC-EX^(miR-132-3p)-treated group was more effective on proliferation(0.45±0.06vs0.34±0.04,t=2.97,P=0.041),migration(346.51±19.28vs238.45±18.72,t=6.27,P=0.003),tube formation(15±3vs9±2,t=3.05,P=0.038),miR-132-3p expression(5.76±0.58vs2.85±0.39,t=7.16,P=0.002),and ERK1/2 phosphorylation(0.47±0.03vs0.27±0.02,t=9.69,P=0.001),while more decreased apoptosis of mBMECs[(15.8±2.9)%vs(22.5±3.1)%,t=3.06,P=0.04].MEK1/2 inhibitor(PD0325901)treatment could partially abolish the effects of NSC-EX^(miR-132-3p)in increasing the proliferation(0.28±0.03vs0.45±0.06,t=5.05,P=0.007),migration(193.21±18.93vs346.51±19.28,t=9.23,P=0.005),tube formation(6±2vs15±3,t=4.67,P=0.009),and ERK1/2 phosphorylation(0.16±0.01vs0.47±0.03,t=17.14,P=0.001),while more decreased apoptosis of mBMECs(29.4±3.4)%vs(15.8±2.9)%,t=5.82,P=0.004.ConclusionMiR-132-3p could enhance the effects of NSC-EXs on protecting multiple mBMECs physiological functions from OGD-induced injury through activating MEK1/2-ERK1/2 signaling pathway.
作者
马晓瑭
王艳
李素青
刘金花
石雨萌
潘群文
Ma Xiaotang;Wang Yan;Li Suqing;Liu Jinhua;Shi Yumeng;Pan Qunwen(Guangdong Key Laboratory of Age-Related Cardiac and Cerebral Diseases,Department of neurology,Affiliated Hospital of Guangdong Medical University,Zhanjiang 524001,China)
出处
《中华脑血管病杂志(电子版)》
2022年第3期172-181,共10页
Chinese Journal of Cerebrovascular Diseases(Electronic Edition)
基金
国家自然科学基金面上项目(81770500,81870580)
广东省自然科学基金(2019A1515011574)