文章摘要
毛诗慧,李琳,孙逸梅,等.川芎嗪预处理增强骨髓间充质干细胞外泌体对脑缺血大鼠的神经保护和抗神经元凋亡作用[J].浙江中医药大学学报,2023,47(5):455-462.
川芎嗪预处理增强骨髓间充质干细胞外泌体对脑缺血大鼠的神经保护和抗神经元凋亡作用
Tetramethylpyrazine Pretreatment Enhances the Neuroprotective and Anti-neuronal Apoptosis Effects of Bone Marrow Mesenchymal Stem Cell-Derived Exosomes in Rats with Cerebral Ischemia
DOI:10.16466/j.issn1005-5509.2023.05.001
中文关键词: 脑缺血  川芎嗪  骨髓间充质干细胞  外泌体  凋亡  Akt
英文关键词: cerebral ischemia  tetramethylpyrazine  bone marrow mesenchymal stem cells  exosomes  apoptosis  Akt
基金项目:国家自然科学基金项目(81274113、81873028);浙江省公益技术应用研究计划(2016C33185)
作者单位
毛诗慧 浙江中医药大学基础医学院 杭州 310053 
李琳 浙江中医药大学基础医学院 杭州 310053 
孙逸梅 浙江中医药大学基础医学院 杭州 310053 
梁慧琦 浙江中医药大学基础医学院 杭州 310053 
江伟锋 浙江中医药大学基础医学院 杭州 310053 
许家 浙江中医药大学基础医学院 杭州 310053 
杨琰 浙江中医药大学基础医学院 杭州 310053 
储利胜 浙江中医药大学基础医学院 杭州 310053 
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中文摘要:
      [目的] 探讨川芎嗪(tetramethylpyrazine,TMP)预处理的骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)源性外泌体(exosomes, Exos)对脑缺血大鼠的神经保护和抗神经元凋亡作用。[方法] TMP预处理BMSCs 48 h,采用超高速离心法分离提取外泌体并鉴定。采用线栓法制备大鼠大脑中动脉阻塞(middle cerebral artery occlusion,MCAO)模型,实验分为假手术组、模型组、BMSCs源性Exos(BMSC-Exos)组和TMP预处理的BMSCs源性Exos(exosomes from TMP-pretreated BMSCs,TMP-BMSC-Exos)组。脑缺血90 min后,给药组分别经尾静脉注射BMSC-Exos(100 μg/500 μL)和TMP-BMSC-Exos(100 μg/500 μL)。采用Longa评分和角实验评价大鼠神经功能,2,3,5-氯化三苯基四氮唑(2,3,5-triphenyltetrazole chloride,TTC)染色检测大鼠脑梗死体积,神经元特异性核蛋白(neuronal nuclei,NeuN)和原位末端转移酶标记(terminal-deoxynucleoitidyl transferase mediated nick end labeling,TUNEL)免疫荧光双标检测神经元凋亡,免疫印迹法检测B细胞淋巴瘤-2(B cell lymphoma-2,Bcl-2)、Bcl-2相关X(Bcl-2 associated X,Bax)、磷酸化蛋白激酶B(phosphophorylated-protein kinase B,p-Akt)及蛋白激酶B(protein kinase B,Akt)的表达水平。[结果] BMSC-Exos和TMP-BMSC-Exos的形态、表面标记蛋白和粒径大小无显著差异。与BMSC-Exos比较,TMP-BMSC-Exos显著促进神经功能恢复(P<0.05),减小脑梗死体积(P<0.01),增加缺血周边区NeuN阳性细胞数量(P<0.05),减少凋亡神经元数量(P<0.05),提高Bcl-2和p-Akt蛋白表达水平,降低Bax蛋白表达水平(P<0.05,P<0.01)。[结论] TMP预处理可以提高BMSC-Exos对脑缺血的神经保护作用,其机制可能与TMP-BMSC-Exos抑制神经元凋亡有关。
英文摘要:
      [Objective] To investigate the neuroprotective and anti-neuronal apoptosis effects of tetramethylpyrazine(TMP) pretreated bone marrow mesenchymal stem cell-derived exosomes(BMSC-Exos) in rats with cerebral ischemia. [Methods] BMSCs were pretreated with TMP for 48 h, and exosomes were extracted by ultracentrifuge and identified. The rats were subjected to right middle cerebral artery occlusion(MCAO) using an intraluminal filament method. The rats were divided into sham operation group, model group, exosomes from BMSCs(BMSC-Exos) group, exosomes from TMP-pretreated BMSCs(TMP-BMSC-Exos) group. After 90 min of cerebral ischemia, BMSC-Exos(100 μg/500 μL) and TMP-BMSC-Exos(100 μg/500 μL) were injected through the tail vein, respectively. Neurological functions were evaluated by Longa score and the corner test, and the cerebral infarct volume was tested by 2,3,5-triphenyltetrazole chloride(TTC) staining, neuronal apoptosis was observed by neuronal nuclei(NeuN) and terminal-deoxynucleoitidyl transferase mediated nick end labeling(TUNEL) double immunofluorescence staining, and the protein expression levels of B-cell lymphoma-2(Bcl-2), Bcl-2 associated X(Bax), phosphophorylated-protein kinase B(p-Akt) and protein kinase B(Akt) were detected by Western blot. [Results] There were no significant differences in morphology, surface markers and particle size between BMSC-Exos and TMP-BMSC-Exos. Compared with BMSC-Exos group, TMP-BMSC-Exos significantly enhanced the neurological function recovery(P<0.05), reduced infarct volume(P<0.01), increased the number of NeuN positive cells(P<0.05) and reduced the number of apoptotic neurons(P<0.05). Furthermore, the protein expression levels of Bcl-2 and p-Akt were increased and the protein expression level of Bax was decreased(P<0.05, P<0.01). [Conclusion] TMP pretreatment could enhance the neuroprotective effect of BMSC-Exos on cerebral ischemia, and the mechanism may be related to inhibition of neuronal apoptosis.
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