血浆外泌体微 RNA-126-5p在儿童注意缺陷多动障碍中的表达及临床意义

目的探究血浆外泌体微 RNA-126-5p(miR-126-5p)在儿童注意缺陷多动障碍( ADHD)中的表达及临床意义。方法选取 2021年 7月至 2022年 7月在江苏省人民医院确诊的 ADHD病儿 70例为 ADHD组,同时纳入同期江苏省人民医院健康体检的志愿者 70例为对照组,采用韦氏幼儿智力量表第 4版( WISC-Ⅳ)和家长评定行为量表( SNAP-Ⅳ)评估研究对象的临床症状,采用 miRNA测序绘制研究对象的差异 miRNA表达谱后寻找关键的 miRNA,并利用受试者操作特征曲线( ROC曲线)评价 miRNA诊断 ADHD的价值,分析 miRNA与临床症状的相关性,同时分析 miRNA的生物学功能。结果与对照组比较, ADHD病儿 WISC-Ⅳ的计算言语理解指数( VCI)［(76.99±6.93)分比( 95.30±5.94)分］、知觉推理指数( PRI)［( 75.38±4.49)分比( 94.69±6.33)分］、工作记忆指数(WMI)［(76.35±7.01)分比(94.11±5.87)分］、加工速度指数( PSI)［(81.36±6.90)分比( 89.49±6.20)分］以及总智商( FSIQ)［(79.18±9.30)分比( 90.19±5.22)分］的分值显著降低,而 SNAP-Ⅳ中注意缺陷和多动 /冲动评分明显增高。同时 miRNA测序筛选出 11个差异 miRNAs,其中 miR-126-5p在 ADHD病儿的血浆外泌体表达升高,并与病儿临床症状存在相关性,且 ROC曲线显示 miR-126-5p的 AUC为 0.817,灵敏度为 78.6%,特异度为 91.4%。此外 miR-126-5p主要涉及突触囊泡循环、轴突导向、炎症介质以及长时程增强等生物学功能有关。结论 ADHD病儿血浆外泌体 miR-126-5p升高,具有诊断 ADHD的临床应用价值,同时具有调控突触可塑性和炎症介质参与 ADHD的病理机制。     

人尿源干细胞外泌体对退变髓核细胞生物学功能的影响

目的:探讨人尿源干细胞外泌体(USC-Exos)对退变髓核细胞(NPCs)生物学功能的影响。方法 :从健康成人尿液中获得人尿源干细胞(USCs)并进行体外培养,通过成骨、成软骨、成脂诱导分化及蛋白质免疫印迹(Western Blot,WB)技术对所提取细胞进行鉴定。使用USCs完全培养基培养USCs 48h后采用差速离心法从培养基中提取外泌体,应用电子显微镜、粒径分析及WB技术对USC-Exos进行形态、直径及表面标志物检测。从腰椎间盘突出症患者术中摘除的髓核中提取NPCs,并培养至P6。以加入50μg/ml USC-Exos干预的P6NPCs为实验组,以50μg/ml未培养过细胞的USCs完全培养基离心沉淀物干预的P6 NPCs为对照组,分别在干预后1～6d使用CCK-8法检测NPCs增殖情况。使用PKH26荧光染料标记USC-Exos,并用标记后的USCExos干预P6 NPCs,12h后在激光共聚焦显微镜下观察NPCs对USC-Exos的摄取情况。两组在干预后48h进行β-半乳糖苷酶衰老染色检测NPCs衰老比例,免疫荧光染色观察蛋白聚糖(ACAN)、Ⅱ型胶原(COL2)的表达。干...     

MicroRNAs in the regulation of autophagy and their possible use in age-related macular degeneration therapy

Age-related macular degeneration (AMD) is a progressive sight-impairing disease of the elderly. The pathogenic mechanisms of AMD are not well understood although both genetic and many environmental factors have been associated with the development of AMD. One clinical hallmark of AMD is the detrimental aggregation of damaged proteins. Recently, it has been suggested that the weakening of autophagy clearance is an important mechanism in the pathogenesis of AMD. Autophagy is important in the removal of damaged or no longer needed cellular material and its recycling. A considerable number of autophagy-targeting microRNAs (miRNAs), small RNA molecules and epigenetic regulators have been found to be either up- or down-regulated in AMD patients and experimental models. The important role of autophagy-targeting miRNAs is supported by several studies and can open the prospect of the use of these miRNAs in the therapy for AMD.     

胰岛保护和功能维护的研究进展

胰岛移植在糖尿病及其并发症的治疗中有很广阔的前景,但移植后功能不良、排斥反应和供体不足等是胰岛移植领域的瓶颈。优化胰腺保存方法对获取有效胰岛的数量及功能的维护有积极意义;在培养期间对胰岛进行抗排斥反应和抗凋亡处理,包括间充质干细胞（MSC）、MSC来源的外泌体、抗凋亡药物和基因修饰等可能成为临床胰岛移植胰岛保护与功能维护的重要方法;胰岛移植后抗立即经血液介导的炎症反应（IBMIR）药物的使用对于胰岛功能的保护也具有重要作用。本文围绕从胰岛制备到胰岛移植的全过程,探讨胰岛保护和功能维护相关策略,旨在为提升供体质量以弥补供体绝对数量的不足,提升胰岛移植效率提供参考。     

外泌体在肺移植排斥反应中的作用研究进展

肺移植术后排斥反应包括急性排斥反应(AR)和以慢性移植肺功能障碍(CLAD)为主要表现的慢性排斥反应,是影响同种异体移植物长期存活的主要因素。外泌体是真核生物细胞间通讯的一种细胞外纳米囊泡,可以携带复杂生物学信息,参与各种生理、病理过程,已成为排斥反应中的重要免疫介质,通过多种途径调控排斥反应的发生发展,在排斥反应的监测和治疗中亦发挥着关键作用。本文就肺移植术后排斥反应的类型、外泌体调控排斥反应的机制、外泌体作为生物标志物及其在排斥反应治疗中的应用做一综述,旨在为肺移植术后排斥反应的综合诊治提供新的方向。     

Recent advances of exosomes in immune modulation and autoimmune diseases

Exosomes are small membrane-bound vesicles (30-100?nm) that are secreted by different types of cells and they have been well documented to resemble saucers or flattened spheres under the electron microscope. Recently, evidence indicates that exosomes play important roles in the immune modulation and are associated with the immune pathogenesis of autoimmune diseases, including rheumatoid arthritis (RA), Sjogren’s syndrome (SS) and systemic lupus erythematosus (SLE). In this review, we will summarize current research advances of exosomes in immunoregulation, pathogenesis, diagnosis and therapeutics of autoimmune diseases.     

与缝隙连接43蛋白相关的星形胶质细胞通过外泌体对神经元保护作用的机制研究

目的:观察缝隙连接43(Cx43)蛋白参与星形胶质细胞来源的外泌体对神经元保护作用的机制.方法:采用C57BL/6胎鼠以原代培养法获取星形胶质细胞及神经元,建立共培养(Co-Culture)模型,给予神经元过氧化氢(H2O2)损伤,星形胶质细胞给予Cx43特异性阻断剂Gap26,分别建立(Co-Culture+H2O2)组及(Co-Culture+H2O2+Gap26)组,同时设单纯神经元损伤(Neuron+H2O2)组,通过蛋白免疫印记(WB)法,测定神经元骨架相关的紧密连接蛋白(ZO-1)、α-肌动蛋白(α-actin)、谷氨酸转运蛋白-1(GLT-1)、外泌体标记蛋白CD63和凋亡相关的Bax/Bcl-2比例的变化,采用高效液相色谱仪(HPLC)观察神经元谷氨酸(Glu),采用ELFA法检测氧化应激因子腺嘌呤二核苷酸磷酸(NAPDH)氧化酶活性.结果:与(Neuron+H2O2)组相比,(Co-Culture+H2O2)组的神经元ZO-1、α-actin、GLT-1和CD63的表达明显上调,而Bax/Bcl-2比例、NAPDH氧化酶活性则明显降低(P<0.05).在给予Gap26后,可明显抑制以上星形胶质细胞对神经元的此种作用(P<0.05).结论:Cx43蛋白可促进星形胶质细胞的外泌体被神经元吸收,可产生稳定细胞形态结构、加强兴奋性氨基酸转运及降低神经元凋亡和氧化应激损伤等神经保护功能.     

Exosome的生物学特性及其在免疫调节中的作用

Exosome最早由Trams在正常细胞和肿瘤细胞培养上清液中被发现,后来在研究网织红细胞最后成熟为红细胞阶段发现也释放同样的小囊泡状结构,并命名为Exosome.但这一发现并没有引起人们的重视.     

Detection of circulating exosomal miR-17-5p serves as a novel non-invasive diagnostic marker for non-small cell lung cancer patients

Exosome-shuttled bioactive miRNAs act as novel non-invasive biomarkers for cancer diagnosis have received increasing attention. In this study, we aimed to investigate the expression signatures of exosomal miRNAs and develop a serum exosome-derived miRNA panel for diagnosis of non-small cell lung cancer (NSCLC). The miR-17-92 cluster including 6 miRNAs (miR-17-5p, miR-18a-5p, miR-19a-3p, miR-19b-1-5p, miR-20a-5p and miR-92a-1-5p) was selected as potential diagnostic candidate molecule. Then, expression profiles of the candidate miRNAs were firstly analyzed in 43 pairs of serum samples from the training set by quantitative real-time PCR, and the dysregulated miRNA along with three tumor markers (carcinoembryonic antigen, CEA; cytokeratin 19 fragment, CYFRA21-1; squamous cell carcinoma antigen, SCCA) were further validated in two independent cohorts, which consisted of training set (including 100 NSCLC patients and 90 healthy controls) and validation set (including 72 NSCLC patients and 47 healthy controls). The expression of miR-17-5p was significantly up-regulated in NSCLC patients compared with the healthy controls (P < 0.001), suggesting that miR-17-5p might have considerable clinical value in the diagnosis of NSCLC. Based on the data from the training set, we next used a logistic regression model to construct a 4-molecule panel consisting of miR-17-5p and three tumor markers for NSCLC diagnosis. The performance of such 4-molecule panel was verified with an area under the ROC curve of 0.860 (95% CI = 0.802 to 0.906, sensitivity = 63.0% and specificity = 93.3%) and 0.844 (95% CI = 0.766 to 0.904, sensitivity = 76.4% and specificity = 76.6%) in the training set and validation set, respectively. In conclusion, the newly developed diagnostic panel consisting of exosomal miR-17-5p, CEA, CYFRA21-1 and SCCA may have considerable clinical value in the diagnosis of NSCLC.     

脐带间充质干细胞源外泌小体减轻糖尿病模型小鼠心肌纤维化

目的:探讨脐带间充质干细胞外泌小体对糖尿病模型小鼠心肌纤维化的保护作用。方法:6~8周C57BL/6雄性小鼠随机分为3组:对照组、糖尿病模型组和糖尿病+外泌小体组。采用高脂饮食连续喂养5周后,联合小剂量多次(45 mg/kg,每周1次,连续5周)腹腔注射链脲佐菌素的方法诱导糖尿病(血糖≥16.7 mmol/L)模型。糖尿病+外泌小体组在糖尿病造模后,经尾静脉每周注射1次外泌小体,连续注射4周;对照组和糖尿病组予等体积的生理盐水连续注射4周。利用小动物彩色多普勒超声诊断仪检测小鼠心功能指标;然后取腹主动脉血,用生化比色法检测葡萄糖和游离脂肪酸的含量;同时取心脏组织,HE染色观察心肌纤维的结构变化;Masson染色观察心肌纤维化水平。结果:超声心动图结果显示,与对照组相比,糖尿病组较对照组的左心室舒张末期内径和左心室收缩末期内径增大(分别P0.05和P0.01),射血分数和室壁缩短率下降(P0.01);糖尿病+外泌小体组较糖尿病组的左心室收缩末期内径减小,心室扩张减小,射血分数和室壁缩短率提高(P0.01)。生化比色法检测血糖及血浆游离脂肪酸结果显示,与对照组相比,糖尿病组血糖及血浆游离脂肪酸水平显著增高,差异有统计学意义(P0.01);糖尿病+外泌小体组可显著降低血糖及游离脂肪酸水平,差异有统计学意义(P0.01)。HE染色结果显示,对照组心肌细胞完整,排列整齐;糖尿病组心肌细胞出现肥大、断裂;糖尿病+外泌小体组心肌细胞较完整,排列较整齐。Masson染色结果显示,与对照组相比,糖尿病组纤维化面积显著增大,差异有统计学意义(P0.01);与糖尿病组比较,糖尿病+外泌小体组纤维化面积减小,差异有统计学意义(P0.01)。结论:局部注射人脐带间充质干细胞外泌小体可以减轻糖尿病小鼠心肌纤维化,并改善心脏功能。