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。

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.