解剖学报 ›› 2022, Vol. 53 ›› Issue (4): 447-452.doi: 10.16098/j.issn.0529-1356.2022.04.007

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骨髓间充质干细胞来源的外泌体对小鼠肝库普弗细胞极化的影响

秦阳阳 许龙飞 王琪 韩晶 洪艳*
  

  1. 贵州医科大学组织学与胚胎学教研室, 贵阳  550004
  • 收稿日期:2020-03-06 修回日期:2022-02-17 出版日期:2022-08-06 发布日期:2022-09-11
  • 通讯作者: 洪艳 E-mail:hongyanb@163.com
  • 基金资助:
    Ngn3基因修饰对脐源性胰岛素分泌细胞分化调控的影响

Effect of exosomes derived from bone marrow mesenchymal stem cells on the polarization of mouse liver Kupffer cells

QIN Yang-yang  XU Long-fei  WANG Qi  HAN Jing  HONG Yan*    

  1. Department of Histology and Embryology, Guizhou Medical University, Guiyang 550004,China
  • Received:2020-03-06 Revised:2022-02-17 Online:2022-08-06 Published:2022-09-11
  • Contact: HONG Yan E-mail:hongyanb@163.com

摘要:

目的  探讨骨髓间充质干细胞(BMSCs)来源的外泌体(exosomes)对肝库普弗(Kupffer)细胞极化的影响。   方法  体外分离培养BMSCs后,经流式细胞术鉴定其表面分子表达,通过成骨和成脂诱导培养基诱导鉴定其分化潜能,通过外泌体提取试剂盒从BMSCs培养上清提取外泌体,电子显微镜观察其形态,并用流式细胞术鉴定表面分子表达。体外培养Kupffer细胞随机分为正常培养组、脂多糖(LPS)刺激组、LPS共培养组,光学显微镜下观察体外培养3组Kupffer细胞形态的变化。60只小鼠腹腔注射CCl4复制急性肝损伤模型,并随机分为PBS对照组和外泌体治疗组,HE染色观察体内两组肝组织病理学变化、眼球取血检测肝功能谷丙转氨酶(ALT)和谷草转氨酶(AST)的表达。Western blotting分别检测体外培养的3组Kupffer细胞和体内两组肝脏组织诱导型一氧化氮合酶(iNOS)和精氨酸酶1(ARG1)的表达,Real-time PCR法检测其iNOS、ARG1、白细胞介素(IL)-1β、肿瘤坏死因子(TNF)-α和C-X-C基序趋化因子(CXCL)-10的表达。   结果  成功分离出BMSCs,具有成骨细胞和成脂细胞分化能力,并表达CD105、CD45;电子显微镜观察到分离的外泌体呈囊泡状,并表达CD63和CD81;光学显微镜观察显示,BMSCs的外泌体能减弱Kupffer细胞活化,BMSCs的外泌体注射后能减弱肝脏组织的病理性改变(P<0.05),降低肝功能ALT 和AST的表达(P<0.05);Western blotting显示,体外实验LPS和外泌体共培养组与体内实验外泌体治疗组的ARG1的表达均增加 (P<0.05),iNOS均降低(P<0.05);Real-time PCR显示,体外LPS和外泌体共培养组与体内外泌体治疗组的iNOS、 IL-1β、TNF-α和CXCL-10的表达下降(P<0.05),ARG1的表达增加(P<0.05)。   结论  BMSCs来源的外泌体抑制肝Kupffer细胞向M1型极化。

关键词: 骨髓间充质干细胞, 外泌体, 库普弗细胞, 极化, 免疫印迹法, 小鼠

Abstract:

Objective  To investigate the  the effect of exosomes derived from bone marrow mesenchymal stem cells (BMSCs) on the polarization of liver Kupffer cells.     Methods  After BMSCs were isolated and cultured in vitro, their surface molecular expression was identified by flow cytometry, and their differentiation potential was induced and identified by osteogenic and lipogenic induction media. Exosomes were extracted from the supernatant of BMSCs culture by exosomes extraction kit, and their morphology was observed by electron microscopy, and surface molecular expression was identified by flow cytometry. In vitro cultured Kupffer cells were randomly divided into normal culture, lipopolysaccharide(LPS) stimulation and LPS co-culture groups, and morphological changes in Kupffer cells were observed under light microscope.Sixty mice were injected intraperitoneally with CCl4 to replicate acute liver injury model and randomized into PBS control and exosomes treatment groups, and the liver histopathology and the expression of liver functional alanine transferase(ALT) and aspartate aminotransferase(AST) were detected by HE staining. Western blotting was used to detect the  expressions of inducible nitric oxide synthase(iNOS) and arginase-1(ARG1) in the three groups of Kupffer cells cultured in vitro and the two groups of liver tissues in vivo. The expression of iNOS, ARG1, interleukin(IL)-1β, tumor necrosis factor(TNF)-α and C-X-C motif chemokine(CXCL)-10 were detected by Real-time PCR.    Results  BMSCs were successfully isolated and had the ability of differentiation of osteoblasts and adipocytes, and expressed CD105 and CD45. The exosomes were bubble-like and expressed CD63 and CD81. Light microscopy showed that exosomes of BMSCs attenuated Kupffer cells activation, and exosomes of BMSCs attenuated pathological changes of liver tissue after injection (P<0.05), decreased the high expression of ALT and AST in liver function (P<0.05); Western blotting showed that ARG1 expression increased in LPS and exosomes co-cultured in vitro and exosomes treated in vivo (P<0.05), iNOS decreased (P<0.05); Real-time PCR showed decreased expression of iNOS, IL-1β, TNF-α and CXCL-10 in LPS and exosomes co-cultured group and exosomes treated group (P<0.05), ARG1 expression increased (P<0.05).    Conclusion  Exosomes derived from BMSCs inhibited M1 type polarization of mouse Kupffer cells.

Key words: Bone marrow mesenchymal stem cell, Exosome, Kupffer cell, Polarization, Western blotting, Mouse

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