人脐带间充质干细胞衍生的外泌体对新生大鼠脑白质损伤小胶质细胞极化的影响

doi:10.3969/j.issn.1006-5725.2025.16.002

摘要/Abstract

摘要：

目的观察人脐带间充质干细胞衍生的外泌体（human umbilical cord mesenchymal stem cell-derived exosomes，HUC-MSC-Exo）对新生大鼠脑白质损伤（white matter injury，WMI）小胶质细胞极化的影响。方法将3日龄Sprague-Dawley大鼠随机分成假手术（Sham）组、缺氧缺血（hypoxia-ischemia，HI）组和HUC-MSC-Exo组纳入研究，每组12只大鼠。采用单侧颈总动脉结扎联合缺氧（8%氧气和92%氮气）构建WMI大鼠模型；超高速离心法提取外泌体（exosomes，Exo）并通过纳米流式、免疫印迹实验和透射电镜进行表征；脑立体定位仪辅助下侧脑室移植Exo（2×10⁸粒子/μL）并于HI后14 d进行脑组织样本获取。苏木素-伊红（hematoxylin-eosin，HE）染色观察脑组织形态学改变；尼氏染色观察脑组织尼氏小体形成；劳克坚劳蓝（luxol fast blue，LFB）染色观察脑组织髓鞘的形成；免疫荧光染色观察离子钙结合适配器分子1（ionic calcium binds adaptor molecule 1，Iba1）的定位表达；免疫印迹实验检测分化簇86（cluster of differentiation 86，CD86）、诱导型一氧化氮合酶（inducible nitric oxide synthase，iNOS）、肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）、白细胞介素-1β（interleukin-1β，IL-1β）和CD206、精氨酸酶1（arginase-1，Arg-1）、IL-10、转化生长因子-β（transforming growth factor-β，TGF-β）蛋白表达水平。结果纳米流式、免疫印迹实验和透射电镜结果显示，HUC-MSC-Exo粒子直径处于30~150 nm之间，呈现类椭圆形态并可见膜状结构，Exo标志物CD9、CD63、TSG101阳性而calnexin阴性表达。HE染色、尼氏染色和LFB染色显示，与Sham组相比，HI组出现脑组织结构破坏，表现为细胞形态改变、神经纤维排列紊乱及空泡化，尼氏小体溶解甚至消失，髓鞘形成受阻；HUC-MSC-Exo明显逆转了HI组病理学改变。免疫荧光染色和免疫印迹实验结果显示，小胶质细胞标志物Iba1主要表达于脑室下区（subventricular zone，SVZ），与Sham组相比，HI后SVZ区Iba1蛋白表达增加（t = 15.95、20.31，P < 0.01）；HUC-MSC-Exo明显降低了HI组Iba1蛋白表达（t = 10.35、11.01，P < 0.01）。免疫印迹实验结果显示，与Sham组相比，HI后M1小胶质细胞标志物（CD86和iNOS）和促炎细胞因子（TNF-α和IL-1β）的表达均明显增加（t = 10.98、7.68、15.13、13.13，均P < 0.01），M2小胶质细胞标志物（CD206和Arg-1）和抗炎细胞因子（IL-10和TGF-β）的表达亦增加（t = 14.26、9.38、8.82、7.42，均为P < 0.01）；与HI组相比，HUC-MSC-Exo降低了CD86、iNOS、TNF-α和IL-1β的蛋白表达（t = 9.79、5.81、8.06、7.03，均P < 0.01）而增加了CD206、Arg-1、IL-10和TGF-β的蛋白表达水平（t = 12.90、8.16、8.98、9.49，均P < 0.01）。结论 HUC-MSC-Exo通过调控小胶质细胞极化减轻新生大鼠WMI。

关键词: 人脐带间充质干细胞, 外泌体, 白质损伤, 小胶质细胞

Abstract:

Objective To observe the effect of human umbilical cord mesenchymal stem cell-derived exosomes （HUC-MSC-Exo） on microglial polarization in neonatal rats with white matter injury （WMI）. Methods Three-day-old Sprague-Dawley rats were randomly divided into sham group （Sham）， hypoxia-ischemia group （HI） and HUC-MSC-Exo group， with 12 rats in each group. Unilateral common carotid artery ligation combined with hypoxia （8% oxygen and 92% nitrogen） was used to construct a WMI rat model. Exosomes were extracted by ultra-high-speed centrifugation and characterized by nanoflow cytometry， western blot experiments and transmission electron microscopy. Brain stereotaxic-assisted inferior ventricular transplantation exo （2×10⁸ particles/μL） was performed and brain tissue samples were collected 14 days after HI. Hematoxylin-eosin （HE） staining was used to observe morphological changes of brain tissue. Nissl staining was used to observe Nissl body formation in brain tissue； Luxol fast blue （LFB） staining was used to observe the formation of myelin sheath in brain tissue. Immunofluorescence staining was used to observe the localized expression of ionic calcium binds adaptor molecule 1 （Iba1）. The protein expression levels of cluster of differentiation 86 （CD86）， inducible nitric oxide synthase （iNOS）， tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）， CD206， arginase-1 （Arg-1）， IL-10 and transforming growth factor-β （TGF-β） were detected by western blot. Results The results of nanoflow cytometry， western blot and transmission electron microscopy showed that the diameter of HUC-MSC-Exo particles was between 30 and 150 nm， and the oval-like shape and membrane-like structure were visible， and the exo markers CD9， CD63 and TSG101 were positive， while calnexin was negative. HE staining， Nissl staining and LFB staining showed that compared with the Sham group， the HI group had brain tissue structure destruction， which was manifested by cell morphological changes， nerve fiber arrangement disorder and vacuolation， Nissl body dissolution or even disappearance， and myelination was blocked. HUC-MSC-Exo significantly reversed the pathological changes in the HI group. The results of immunofluorescence staining and western blot showed that the microglial marker Iba1 was mainly expressed in the subventricular zone （SVZ）， and the expression of Iba1 protein in the SVZ region increased after HI compared with the Sham group （t = 15.95、20.31， P < 0.01）. HUC-MSC-Exo significantly reduced the expression of Iba1 protein in the HI group （t = 10.35、11.01， P < 0.01）. The results of western blot showed that the expressions of M1 microglia markers （CD86 and iNOS） and pro-inflammatory cytokines （TNF-α and IL-1β） were significantly increased after HI （t = 10.98、7.68、15.13、13.13， both P < 0.01）， and the expressions of M2 microglia markers （CD206 and Arg-1） and anti-inflammatory cytokines （IL-10 and TGF-β） were also increased after HI （t = 14.26、9.38、8.82、7.42， both P < 0.01）. HUC-MSC-Exo decreased the protein expression of CD86， iNOS， TNF-α and IL-1β （t = 9.79、5.81、8.06、7.03， all P < 0.01） and increased the protein expression levels of CD206， Arg-1， IL-10 and TGF-β compared to the HI group （t = 12.90、8.16、8.98、9.49， both P < 0.01）. Conclusion HUC-MSC-Exo attenuates WMI in neonatal rats by regulating microglial polarization.

Key words: human umbilical cord mesenchymal stem cells, exosomes, white matter injuy, microglia

中图分类号:

R741

王超,徐倩倩,张书绢,朱艳萍. 人脐带间充质干细胞衍生的外泌体对新生大鼠脑白质损伤小胶质细胞极化的影响[J]. 实用医学杂志, 2025, 41(16): 2447-2454.

Chao WANG,Qianqian XU,Shujuan ZHANG,Yanping ZHU. Effect of human umbilical cord mesenchymal stem cell-derived exosomes on microglial polarization in neonatal rats with white matter injury[J]. The Journal of Practical Medicine, 2025, 41(16): 2447-2454.

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参考文献 28

[1]	XIAO J. Role of the Gut Microbiota-Brain Axis in Brain Damage in Preterm Infants ［J］. ACS Pharmacol Transl Sci， 2024， 7（5）：1197-1204. doi:10.1021/acsptsci.3c00369 doi: 10.1021/acsptsci.3c00369
[2]	JANA A， GARG S， GHOSH S， et al. Generation of Functional Neurons from Mesenchymal Stem Cells Using Neural Differentiator and Engineered Peptide Hydrogel： Potential Therapeutic Lead for Traumatic Brain Injury ［J］. ACS Appl Mater Interfaces， 2024， 16（47）：64476-64493. doi:10.1021/acsami.4c12554 doi: 10.1021/acsami.4c12554
[3]	ZHANG X， ZHOU Y， YE Y， et al. Human umbilical cord mesenchymal stem cell-derived exosomal microRNA-148a-3p inhibits neointimal hyperplasia by targeting Serpine1［J］. Arch Biochem Biophys， 2022， 719：109155. doi:10.1016/j.abb.2022.109155 doi: 10.1016/j.abb.2022.109155
[4]	ABBASZADEH H， GHORBANI F， DERAKHSHANI M， et al. Human umbilical cord mesenchymal stem cell-derived extracellular vesicles： A novel therapeutic paradigm ［J］. J Cell Physiol， 2020， 235（2）：706-717. doi:10.1002/jcp.29004 doi: 10.1002/jcp.29004
[5]	陈珊，朱俊德，赵雪，等.神经干细胞来源外泌体对脑缺血再灌注损伤大鼠星形胶质细胞TGF⁃β1信号转导与炎症因子的影响［J］.实用医学杂志，2023， 39（13）：1600-1605.
[6]	LEE J， HAMANAKA G， LO E H， et al. Heterogeneity of microglia and their differential roles in white matter pathology ［J］. CNS Neurosci Ther， 2019， 25（12）：1290-1298. doi:10.1111/cns.13266 doi: 10.1111/cns.13266
[7]	ZHAO Y， GAN Y， XU G， et al. MSCs-Derived Exosomes Attenuate Acute Brain Injury and Inhibit Microglial Inflammation by Reversing CysLT2R-ERK1/2 Mediated Microglia M1 Polarization ［J］. Neurochem Res， 2020， 45（5）：1180-1190. doi:10.1007/s11064-020-02998-0 doi: 10.1007/s11064-020-02998-0
[8]	DONG C， CHEN M， CAI B， et al. Mesenchymal Stem Cell-Derived Exosomes Improved Cerebral Infarction via Transferring miR-23a-3p to Activate Microglia ［J］. Neuromolecular Med， 2022， 24（3）：290-298. doi:10.1007/s12017-021-08686-8 doi: 10.1007/s12017-021-08686-8
[9]	ZHANG Y， XIE Y， HAO Z， et al. Umbilical Mesenchymal Stem Cell-Derived Exosome-Encapsulated Hydrogels Accelerate Bone Repair by Enhancing Angiogenesis ［J］. ACS Appl Mater Interfaces， 2021， 13（16）：18472-18487. doi:10.1021/acsami.0c22671 doi: 10.1021/acsami.0c22671
[10]	HUANG Y， MA J， FAN Y， et al. Mechanisms of human umbilical cord mesenchymal stem cells-derived exosomal lncRNA GAS5 in alleviating EMT of HPMCs via Wnt/β-catenin signaling pathway ［J］. Aging （Albany NY）， 2023， 15（10）：4144-4158. doi:10.18632/aging.204719 doi: 10.18632/aging.204719
[11]	CARLONI S， CRINELLI R， PALMA L， et al. The Synthetic Cannabinoid URB447 Reduces Brain Injury and the Associated White Matter Demyelination after Hypoxia-Ischemia in Neonatal Rats ［J］. ACS Chem Neurosci， 2020， 11（9）：1291-1299. doi:10.1021/acschemneuro.0c00047 doi: 10.1021/acschemneuro.0c00047
[12]	VANNUCCI S J， BACK S A. The Vannucci Model of Hypoxic-Ischemic Injury in the Neonatal Rodent： 40 years Later ［J］. Dev Neurosci， 2022， 44（4/5）：186-193. doi:10.1159/000523990 doi: 10.1159/000523990
[13]	BOBIS-WOZOWICZ S， KMIOTEK K， KANIA K， et al. Diverse impact of xeno-free conditions on biological and regenerative properties of hUC-MSCs and their extracellular vesicles ［J］. J Mol Med （Berl）， 2017， 95（2）：205-220. doi:10.1007/s00109-016-1471-7 doi: 10.1007/s00109-016-1471-7
[14]	GAO X， GAO L F， ZHANG Y N， et al. Huc-MSCs-derived exosomes attenuate neuropathic pain by inhibiting activation of the TLR2/MyD88/NF-κB signaling pathway in the spinal microglia by targeting Rsad2 ［J］. Int Immunopharmacol， 2023， 114：109505. doi:10.1016/j.intimp.2022.109505 doi: 10.1016/j.intimp.2022.109505
[15]	ZHAI X， CHEN K， YANG H， et al. Extracellular vesicles derived from CD73 modified human umbilical cord mesenchymal stem cells ameliorate inflammation after spinal cord injury ［J］. J Nanobiotechnology， 2021， 19（1）：274. doi:10.1186/s12951-021-01022-z doi: 10.1186/s12951-021-01022-z
[16]	EBRAHIM N， SAIHATI H A AL， ALALI Z， et al. Exploring the molecular mechanisms of MSC-derived exosomes in Alzheimer's disease： Autophagy， insulin and the PI3K/Akt/mTOR signaling pathway ［J］. Biomed Pharmacother， 2024， 176：116836. doi:10.1016/j.biopha.2025.118042 doi: 10.1016/j.biopha.2025.118042
[17]	ZHANG J， WANG C， YANG G， et al. Olfactory mucosal mesenchymal stem cell-derived exosome Lnc A2M-AS1 ameliorates oxidative stress by regulating TP53INP1-mediated mitochondrial autophagy through interacting with IGF2BP1 in Parkinson's diseases ［J］. Cell Biol Toxicol， 2025， 41（1）：60. doi:10.1007/s10565-025-10009-7 doi: 10.1007/s10565-025-10009-7
[18]	ZHANG L， BAI W， PENG Y， et al. Human umbilical cord mesenchymal stem cell-derived exosomes provide neuroprotection in traumatic brain injury through the lncRNA TUBB6/Nrf2 pathway ［J］. Brain Res， 2024， 1824：148689. doi:10.1016/j.brainres.2023.148689 doi: 10.1016/j.brainres.2023.148689
[19]	ZHANG Z， ZOU X， ZHANG R， et al. Human umbilical cord mesenchymal stem cell-derived exosomal miR-146a-5p reduces microglial-mediated neuroinflammation via suppression of the IRAK1/TRAF6 signaling pathway after ischemic stroke ［J］. Aging （Albany NY）， 2021， 13（2）：3060-3079. doi:10.18632/aging.202466 doi: 10.18632/aging.202466
[20]	LI P， KASLAN M， LEE S H， et al. Progress in Exosome Isolation Techniques ［J］. Theranostics， 2017， 7（3）：789-804. doi:10.7150/thno.18133 doi: 10.7150/thno.18133
[21]	CHEN X， SAI Y， CUI W， et al. Human umbilical cord mesenchymal stem cell-derived exosomes combined with mouse nerve growth factor can more effectively ameliorate the motor disorder and brain pathological injury in mice with cerebral palsy ［J］. Adv Clin Exp Med， 2025. DOI： 10.17219/acem/192773 . doi: 10.17219/acem/192773
[22]	MARSTERS C M， NESAN D， FAR R， et al. Embryonic microglia influence developing hypothalamic glial populations ［J］. J Neuroinflammation， 2020，17（1）：146. doi:10.1186/s12974-020-01811-7 doi: 10.1186/s12974-020-01811-7
[23]	CUI L， LUO W， JIANG W， et al. Human umbilical cord mesenchymal stem cell-derived exosomes promote neurological function recovery in rat after traumatic brain injury by inhibiting the activation of microglia and astrocyte ［J］. Regen Ther， 2022， 21：282-287. doi:10.1016/j.reth.2022.07.005 doi: 10.1016/j.reth.2022.07.005
[24]	RONALDSON P T， DAVIS T P. Regulation of blood-brain barrier integrity by microglia in health and disease： A therapeutic opportunity ［J］. J Cereb Blood Flow Metab， 2020， 40（）：S6-S24. doi:10.1177/0271678x20951995 doi: 10.1177/0271678x20951995
[25]	YANG L， YU X， ZHANG Y， et al. Caffeine treatment started before injury reduces hypoxic-ischemic white-matter damage in neonatal rats by regulating phenotypic microglia polarization ［J］. Pediatr Res， 2022， 92（6）：1543-1554. doi:10.1038/s41390-021-01924-6 doi: 10.1038/s41390-021-01924-6
[26]	ZONG L， HUANG P， SONG Q， et al. Bone marrow mesenchymal stem cells-secreted exosomal H19 modulates lipopolysaccharides-stimulated microglial M1/M2 polarization and alleviates inflammation-mediated neurotoxicity ［J］. Am J Transl Res， 2021， 13（3）：935-951.
[27]	LIU W， RONG Y， WANG J， et al. Exosome-shuttled miR-216a-5p from hypoxic preconditioned mesenchymal stem cells repair traumatic spinal cord injury by shifting microglial M1/M2 polarization ［J］. J Neuroinflammation， 2020， 17（1）：47. doi:10.1186/s12974-020-1726-7 doi: 10.1186/s12974-020-1726-7
[28]	WANG J， WEI Q， YANG Y， et al. Small extracellular vesicles derived from four dimensional-culture of mesenchymal stem cells induce alternatively activated macrophages by upregulating IGFBP2/EGFR to attenuate inflammation in the spinal cord injury of rats ［J］. Front Bioeng Biotechnol， 2023， 11：1146981. doi:10.3389/fbioe.2023.1146981 doi: 10.3389/fbioe.2023.1146981

组别	Iba1相对阳性区域/%	Iba1蛋白表达
Sham组	4.03 ± 0.28	0.50 ± 0.03
HI组	10.69 ± 0.37^*	1.65 ± 0.05^*
HUC-MSC-Exo组	6.37 ± 0.22^△	1.03 ± 0.04^△
F值	130.99	207.07
P值	< 0.001	< 0.001

组别	CD86蛋白表达	iNOS蛋白表达	CD206蛋白表达	Arg-1蛋白表达
Sham组	0.54 ± 0.02	0.24 ± 0.03	0.31 ± 0.03	0.42 ± 0.02
HI组	0.87 ± 0.05^*	0.58 ± 0.05^*	0.55 ± 0.01^*	0.74 ± 0.05^*
HUC-MSC-Exo组	0.54 ± 0.06^△	0.21 ± 0.03^△	0.84 ± 0.04^△	1.12 ± 0.08^△
F值	15.96	27.25	84.72	36.77
P值	< 0.001	< 0.001	< 0.001	< 0.001

组别	TNF-α蛋白表达	IL-1β蛋白表达	IL-10蛋白表达	TGF-β蛋白表达
Sham组	0.45 ± 0.03	0.49 ± 0.04	0.41 ± 0.04	0.40 ± 0.02
HI组	0.89 ± 0.05^*	0.92 ± 0.03^*	0.75 ± 0.06^*	0.68 ± 0.03^*
HUC-MSC-Exo组	0.66 ± 0.02^△	0.46 ± 0.06^△	1.17 ± 0.09^△	1.03 ± 0.05^△
F值	34.43	29.96	31.84	66.83
P值	< 0.001	< 0.001	< 0.001	< 0.001