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中华脑血管病杂志(电子版)

中华脑血管病杂志(电子版) ›› 2022, Vol. 16 ›› Issue (03) : 172 -181. doi: 10.11817/j.issn.1673-9248.2022.03.006

论著

富含miR-132-3p的神经干细胞释放的外泌体激活MEK1/2/-ERK1/2通路改善缺氧无糖诱导的脑微血管内皮细胞损伤
马晓瑭1, 王艳1, 李素青1, 刘金花1, 石雨萌1, 潘群文1,†()   
  1. 1. 524001　广东湛江，广东医科大学附属医院神经病学研究所　广东省衰老相关心脑疾病重点实验室
  • 收稿日期:2021-09-08 出版日期:2022-06-01
  • 通信作者: 潘群文
  • 基金资助:
    国家自然科学基金面上项目(81770500，81870580); 广东省自然科学基金(2019A1515011574)

MiR-132-3p-enriched NSC-derived exosomes meliorate mouse brain microvessel endothelial cells injury via MEK1/2/ERK1/2 pathway

Xiaotang Ma1, Yan Wang1, Suqing Li1, Jinhua Liu1, Yumeng Shi1, Qunwen Pan1,†()   

  1. 1. Guangdong Key Laboratory of Age-Related Cardiac and Cerebral Diseases, Department of neurology, Affiliated Hospital of Guangdong Medical University, Zhanjiang 524001, China
  • Received:2021-09-08 Published:2022-06-01
  • Corresponding author: Qunwen Pan
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马晓瑭, 王艳, 李素青, 刘金花, 石雨萌, 潘群文. 富含miR-132-3p的神经干细胞释放的外泌体激活MEK1/2/-ERK1/2通路改善缺氧无糖诱导的脑微血管内皮细胞损伤[J]. 中华脑血管病杂志(电子版), 2022, 16(03): 172-181.

Xiaotang Ma, Yan Wang, Suqing Li, Jinhua Liu, Yumeng Shi, Qunwen Pan. MiR-132-3p-enriched NSC-derived exosomes meliorate mouse brain microvessel endothelial cells injury via MEK1/2/ERK1/2 pathway[J]. Chinese Journal of Cerebrovascular Diseases(Electronic Edition), 2022, 16(03): 172-181.

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NSCmiR-132-3p，同时采用装载Scramble序列的慢病毒感染NSC获得NSCNC。提取NSCNC和NSCmiR-132-3p释放的EX，分别获得NSC-EX和NSC-EXmiR-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-EXmiR-132-3p功能的调控作用。采用Student’s t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±0.04 vs 0.23±0.04）、迁移能力[（238.45±18.72）μm vs（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±0.02 vs 0.07±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-EXmiR-132-3p处理更有效增加mBMEC细胞的增殖能力（0.45±0.06 vs 0.34±0.04）、迁移能力[（346.51±19.28）μm vs（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±0.58 vs 2.85±0.39）和ERK1/2磷酸化水平（0.47±0.03 vs 0.27±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-EXmiR-132-3p对细胞增殖能力（0.28±0.03 vs 0.45±0.06）、迁移能力[（193.21±18.93）μm vs（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±0.01 vs 0.47±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。

关键词: 神经干细胞, 外泌体, 微小核糖核酸, 脑微血管, 内皮细胞
Objective

To 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).

Methods

Primary neural stem cells (NSCs) were isolated and cultured. NSCNC was obtained by transfecting NSCs with lentivirus loaded with scramble sequence, and NSCmiR-132-3p was obtained from NSCs transfected with lentivirus loaded with miR-132-3p vector. NSC-EXs and NSC-EXsmiR-132-3p were extracted from NSCs and NSCmiR-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-132-3p on regulating MEK1/2-ERK1/2 signaling pathway. Comparisons between two groups were analyzed by t test.

Results

Compared to OGD group, NSC-EXs-treated group were significantly with increased proliferation (0.34±0.04 vs 0.23±0.04, t=2.98, P=0.042), migration [(238.45±18.72)μm vs (175.38±12.53)μm, t=4.92, P=0.008], tube formation(9±2 vs 3±1, t=4.65, P=0.011), and ERK1/2 phosphorylation (0.27±0.02 vs 0.07±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-EXmiR-132-3p-treated group was more effective on proliferation (0.45±0.06 vs 0.34±0.04, t=2.97, P=0.041), migration(346.51±19.28 vs 238.45±18.72, t=6.27, P=0.003), tube formation (15±3 vs 9±2, t=3.05, P=0.038), miR-132-3p expression (5.76±0.58 vs 2.85±0.39, t=7.16, P=0.002), and ERK1/2 phosphorylation(0.47±0.03 vs 0.27±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-EXmiR-132-3p in increasing the proliferation (0.28±0.03 vs 0.45±0.06, t=5.05, P=0.007), migration (193.21±18.93 vs 346.51±19.28, t=9.23, P=0.005), tube formation(6±2 vs 15±3, t=4.67, P=0.009), and ERK1/2 phosphorylation (0.16±0.01 vs 0.47±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).

Conclusion

MiR-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.

Key words: Neural stem cells, Exosomes, micro RNA, Brain microvascular, Endothelial cells
图1 倒置显微镜白光下神经干细胞呈神经球样结构（图a），免疫荧光染色发现细胞核（DAPI，蓝色荧光，图b）、神经干细胞标记蛋白（Nestin，绿色荧光，图c）和新生细胞标记蛋白（Brdu，红色荧光，图d），图e所示为三组荧光重合（DAPI，蓝色荧光；Nestin，绿色荧光；Brdu，红色；重合，青色）。
图2 纳米颗粒分析仪检测神经干细胞分泌的外泌体（NSC-EX）粒径和浓度。图a为NSC-EX数据，图b为富含miR-132-3p的NSC-EX
图3 电子透射显微镜检测神经干细胞分泌的外泌体（NSC-EX）形态和粒径。图a为NSC-EX，图b为富含miR-132-3p的NSC-EX
图4 Western blot检测外泌体标志性蛋白CD63和TSG101表达注：NSC为神经干细胞，EX为外泌体
图5 神经干细胞分泌的外泌体（NSC-EX）与小鼠脑微血管内皮细胞融合。图a为DPAI标记细胞核；图b为PKH26标记NSC-EX；图c为NSC-EX与细胞融合
图6 装载miR-132-3p的慢病毒（LV）感染获得miR-132-3p高表达神经干细胞（NSC）。图a为LV-Scramble control转染NSC获得NSCNC；图b为LV-miR-132-3p转染获得NSCmiR-132-3p
表1 各组细胞及NSC-EX中miR-132-3p表达比较（
xˉ
±s）
组别 只数 miR-132-3p表达（倍数）
NSCNC 3 1.03±0.14
NSCmiR-132-3p 3 5.23±0.42
NSC-EX 3 1.05±0.13
NSC-EXmiR-132-3p 3 8.41±0.75
PBS处理mBMEC 3 0.99±0.16
NSC-EX处理mBMEC 3 2.85±0.39
NSC-EXmiR-132-3p处理mBMEC 3 5.76±0.58
表2 各组mBMEC存活能力、迁移距离及血管形成数比较（
xˉ
±s）
组别 只数

增殖能力

（OD值）

 迁移能力（μm） 血管形成能力（条）
对照组 3 0.51±0.07 414.18±21.51 18±3
OGD处理组 3 0.23±0.04a 175.38±12.53a 3±1a
NSC-EX组 3 0.34±0.04b 238.45±18.72b 9±2b
NSC-EXmiR-132-3p组 3 0.45±0.06c 346.51±19.28c 15±3c
NSC-EXmiR-132-3p+PD0325901组 3 0.28±0.03d 193.21±18.93d 6±2d
图7 神经干细胞来源外泌体（NSC-EX）和NSC-EXmiR-132-3p对缺氧且无糖（OGD）条件下小鼠脑微血管内皮细胞（mBMEC）血管形成能力的影响。图a为对照组mBMEC血管形成数；图b为OGD处理后mBMEC血管形成数；图c为NSC-EX处理下mBMEC血管形成数；图d为NSC-EXmiR-132-3p处理下mBMEC血管形成数；图e为NSC-EXmiR-132-3p+PD0325901处理下mBMEC血管形成数
表3 各组mBMEC凋亡的比较（
xˉ
±s）
组别 只数 凋亡率（%）
对照组 3 5.8±1.2
OGD处理组 3 35.7±4.7
NSC-EX组 3 22.5±3.1
NSC-EXmiR-132-3p组 3 15.8±2.9
NSC-EXmiR-132-3p+PD0325901组 3 29.4±3.4
图8 神经干细胞来源外泌体（NSC-EX）和NSC-EXmiR-132-3p对缺氧且无糖（OGD）条件下小鼠脑微血管内皮细胞（mBMEC）凋亡的影响。图a为对照组mBMEC凋亡水平；图b为OGD处理后mBMEC凋亡水平；图c为NSC-EX处理下mBMEC凋亡水平；图d为NSC-EXmiR-132-3p处理下mBMEC凋亡水平；图e为NSC-EXmiR-132-3p+PD0325901处理下mBMEC凋亡水平注：PE和7-AAD为Annexin V-PE/7-AAD凋亡检测试剂盒的2种染料
表4 各组细胞中ERK1/2磷酸水平的比较（
xˉ
±s）
组别 只数 磷酸化ERK1/2（倍数）
对照组 3 0.54±0.03
OGD处理组 3 0.07±0.01
NSC-EX组 3 0.27±0.02
NSC-EXmiR-132-3p组 3 0.47±0.03
NSC-EXmiR-132-3p+PD0325901组 3 0.16±0.01
图9 ERK1/2磷酸化水平检测。a为正常处理组；b为缺氧且无糖处理组；c为神经干细胞来源外泌体处理组；d为富含miR-132-3p的神经干细胞外泌体（NSC-EXmiR-132-3p）处理组；e为NSC-EXmiR-132-3p+MEK1/2通路阻断剂（PD0325901）处理组注：ERK为细胞外调节激酶，p-ERK为磷酸化细胞外调节激酶
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