| 赵佳伟,贾蓉蓉,王景辉,田 颖,袁一航,王玉刚,施 敏.间充质干细胞外泌体对小鼠急性肝衰竭的保护作用[J].,2020,(7):1246-1252 |
| 间充质干细胞外泌体对小鼠急性肝衰竭的保护作用 |
| Protective Effect of Exosomes Derived from Mesenchymal Stem Cells on Carbon Tetrachloride Induced Acute Liver Failure in Mice |
| 投稿时间：2019-09-02 修订日期：2019-09-26 |
| DOI：10.13241/j.cnki.pmb.2020.07.009 |
| 中文关键词: 小鼠骨髓间充质干细胞 外泌体 四氯化碳 急性肝衰竭 |
| 英文关键词: Mouse bone marrow mesenchymal stem cells Exosomes Carbon tetrachloride Acute liver failure |
| 基金项目:国家自然科学基金面上项目（81570549，81871912，81970530）；上海市医学重点专科项目（ZK2019C12） |
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| 中文摘要: |
| 摘要 目的：研究小鼠骨髓间充质干细胞（Mouse bone marrow mesenchymal stem cells, mBMMSC）来源的外泌体（Exosome, Exo）对四氯化碳（Carbon tetrachloride, CCl4）诱导的急性肝衰竭（Acute liver failure, ALF）的保护作用。方法：采用全骨髓培养法提取Balb/c小鼠原代骨髓间充质干细胞并利用流式细胞仪进行鉴定，用试剂盒混合细胞上清后提取干细胞分泌的外泌体，透射电镜（Transmission electron microscope, TEM）鉴定外泌体形态，纳米粒径追踪分析仪（Nanoparticle tracking analysis, NTA）鉴定外泌体的粒径，Western Blot鉴定外泌体表面的标志蛋白。激光共聚焦显微镜（Confocal laser scanning microscope, CLSM）下观察肝细胞内吞外泌体。小鼠肝脏的免疫荧光切片观察外泌体的肝脏内分布。Balb/c小鼠随机分为健康对照组（Healthy）、CCl4损伤组（CCl4）、CCl4损伤外泌体治疗组（Exo）。观察小鼠7 d内的生存时间。检测血清中谷丙转氨酶（ALT）、谷草转氨酶（AST）、乳酸脱氢酶（LDH）、尿酸（UA）的含量，H&E染色组织切片观察肝脏病理变化，流式细胞仪和磁珠细胞试剂盒（Cytometric bead array, CBA）检测血清中白介素6（IL-6）、白介素12（IL-12）和γ干扰素的含量（IFN-γ）。结果：成功提取并鉴定了小鼠骨髓间充质干细胞与其外泌体，NTA测得干细胞外泌体的粒径在100 nm左右，电镜下外泌体呈现囊泡状，Western Blot测得外泌体表达CD9和CD63蛋白。CLSM下观察到L02肝细胞内吞外泌体且随时间递增，免疫荧光切片观察到外泌体靶向肝脏且在肝脏组织中均匀分布。动物实验表明相比于CCl4组，Exo组小鼠的生存时间显著延长，血清中ALT、AST、LDH和UA的含量显著降低，肝脏组织切片显示肝脏损伤明显减少，血清中IL-6, IL-12和IFN-γ的含量显著降低。结论：小鼠骨髓间充质干细胞分泌的外泌体可能通过抑制炎症减轻四氯化碳诱导的急性肝衰竭。 |
| 英文摘要: |
| ABSTRACT Objective: To investigate the protective effect of exosomes(Exo) derived from mouse bone marrow mesenchymal stem cells(mBMMSC) on carbon tetrachloride（CCl4）-induced acute liver failure（ALF）. Methods: The primary bone marrow mesenchymal stem cells were extracted from Balb/c mice by a whole bone marrow culture method and were further confirmed by flow cytometry. Exosomes derived from mesenchymal stem cells were extracted through a Total Exosome Isolation Reagent. The morphology of exosomes was observed by TEM and the size distribution of obtained exosomes was measured by NTA. The surface markers of exosomes were detected by Western Blot. Endocytosis of exosomes by L02 cells was observed under CLSM and its distribution was observed by immunofluorescence sections. The survival rate and lifetime of Balb/c mice within 7 days were observed and recorded. The serum level of ALT, AST, LDH, UA were measured by a fully automatic biochemical analyzer. Hematoxylin-eosin of liver tissue was conducted to observe the histopathological changes. Cytokine level of IL-6, IL-12 and IFN-γ in mouse serum were detected with a Cytometric bead array by flow cytometry. Results: mBMMSCs and its exosomes were successfully extracted and identified. The size of exosomes was around 100 nm measured by NTA and the morphology was observed under TEM to be a shape of vesicle. Expression of CD9 and CD63 protein was proved by Western Blot. Besides, endocytosis of exosomes by L02 cells in a time-dependent way was observed under CLSM. And the distribution of targeted exosomes in liver was observed by immunofluorescence sections Compared with CCl4 group, the lifetime of Exo mice were dramatically prolonged. The serum level of ALT, AST, LDH and UA were significantly decreased and liver damage of histopathological section was substantially improved in Exo group. Cytokine level of IL-6, IL-12 and IFN-γ in Exo mouse serum were extensively decreased compared to CCl4 group. Conclusion: Exosomes derived from mBMMSC may alleviate carbon tetrachloride induced acute liver failure by inhibition of inflammation. |
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