脐带间充质干细胞外泌体的分离和鉴定

目的探讨脐带间充质干细胞的外泌体的获取与鉴定方法。方法通过组织块贴壁法从胎儿脐带分离和培养脐带间充质干细胞并通过RiboTMExosome Isolation Kit收集外泌体,采用电镜和流式细胞仪鉴定外泌体。结果第二代脐带间充质干细胞表面CD45、CD34和HLA-DR呈阴性表达,CD29、CD44和CD105呈阳性表达;在透射电镜下观察到脐带间充质干细胞外泌体呈圆形或椭圆形,大小不均匀,直径30~100 nm,有完整的膜结构,内含低密度物质;流式细胞检测外泌体CD9、CD63、CD81和CD83呈阳性表达。结论在培养脐带间充质干细胞的培养基中可以收集到外泌体,可以通过电镜和流式细胞仪对脐带间充质干细胞的外泌体进行鉴定。     

脂肪间充质干细胞来源外泌体的功能

外泌体是细胞外膜质纳米囊泡,将蛋白质、核酸(DNA和RNA)转运到靶细胞中,介导局部和系统的细胞间通信,从而改变受体细胞的行为.这些小泡在许多生物功能中发挥重要作用,如脂肪合成、免疫调节、神经再生和肿瘤调节等.脂肪间充质干细胞目前被认为是细胞治疗和再生医学领域中一种功能丰富的工具,可产生和分泌多种外泌体,继承细胞的多种...     

间充质干细胞外泌体治疗肺部疾病的研究进展

肺部疾病作为一种呼吸系统常见病,与重工业快速发展、环境污染等密切相关,死亡率极高。近来研究发现间充质干细胞(mesenchymal stem cells,MSCs)能够自主归巢至疾病损伤部位、修复受损组织并参与调节全身炎症和免疫反应,在临床上有较好的应用前景。外泌体是一种细胞外膜囊泡,可能通过调节细胞间通讯参与生物系统中的各种活动,分泌至细胞外环境的外泌体也会影响宿主免疫系统。而MSCs外泌体作为MSCs旁分泌过程中的一种介质,携带有蛋白质、脂质、m RNA和microRNA等生物活性物质,比基于细胞的治疗有更高的安全性,其潜在的医学价值受到了研究者的广泛关注。本文综述了近年来研究中MSCs外泌体在常见肺部疾病治疗中的作用机制,将为其在未来的临床应用方面提供依据。     

间充质干细胞源性外泌体miRNAs参与损伤心血管细胞修复的研究进展

外泌体是由各种组织和细胞所释放的细胞外囊泡,在细胞之间充当各种分子(包括蛋白质,脂质和RNA)的载体,从而调节或干预特定的生理过程.MiRNAs是一类小的非编码RNA,能够靶向多个mRNA并诱导其降解或翻译抑制以调控相关基因表达.目前对外泌体内微小RNA(miR-NAs)的研究最为广泛.间充质干细胞(MSCs)是一种多...     

人嗅黏膜间充质干细胞来源外泌体的分离鉴定及生物学特性研究

外泌体是指释放到细胞外微环境中的直径约50～130 nm的纳米级的膜性囊泡。嗅黏膜间充质干细胞（olfactory mucosa mesenchymal stem cells,OM-MSCs）作为一类新发现的间充质干细胞,在许多疾病中均具有治疗作用,且其内在机制与其旁分泌的外泌体密切相关,但OM-MSCs外泌体的分离、鉴定及生物学特性的研究尚未见报道。本研究采用超速离心法提取OM-MSCs培养液中的外泌体,应用流式细胞术及免疫荧光进行细胞鉴定后,分别用透射电子显微镜、纳米粒径分析及Western印迹对外泌体形态、颗粒大小和表面的特异性分子标志进行分析鉴定。采用CCK8增殖实验,Western印迹和划痕实验,分析其对人脑微血管内皮细胞增殖和迁移的影响。电镜、Western 印迹和纳米粒径分析的结果显示：OM-MSCs来源外泌体形态多为圆形,直径约为40～150 nm;表达外泌体标记物CD63,CD81;CCK-8法检测显示：不同浓度的OM-MSCs源外泌体可提高人脑微血管内皮细胞的增殖活性,且其增殖促进作用具有浓度依赖性（P<0.05）。Western 印迹检测结果显示：相比空白对照组,OM-MSCs源外泌体可显著提高内皮细胞的增殖细胞核抗原蛋白质水平表达（P<0.01）,细胞划痕实验结果显示,OM-MSCs源外泌体可增强内皮细胞的迁移能力,且高于对照组（P<0.01）。本研究表明：通过超速离心法可以分离纯化获得OM-MSCs源外泌体,且该外泌体具有促进人脑微血管内皮细胞迁移和增殖的作用。     

骨髓间充质干细胞外泌体中miRNA在视网膜新生血管形成中的作用及机制

视网膜血管疾病如早产儿视网膜病变、糖尿病视网膜病变和视网膜静脉阻塞等以异常增生的视网膜新生血管为主要病理表现。骨髓间充质干细胞来源外泌体通过旁分泌作用传递生物活性分子介导细胞间的物质与信息交换。其中,miRNA等内容物在传递信息中起关键作用,可调控缺血缺氧环境下内皮细胞的增殖、管腔形成和新生血管的形成。并且能够通过血视网膜屏障而不引起免疫、炎症反应,在眼科疾病治疗中极具潜力。本文总结骨髓间充质干细胞衍生外泌体中miRNA在视网膜新生血管形成中的作用和可能的作用机制,以期为外泌体在眼科疾病诊治中的应用拓宽新思路。     

间充质干细胞外泌体在糖尿病并发症中的作用

外泌体是一类由各种细胞分泌的直径40～100 nm的胞外囊泡,由细胞内复杂的产生机制生成后分泌到胞外,由于其内含蛋白质、脂质和核酸,所以在细胞间的通讯上发挥着巨大作用。近年来,各种细胞来源的外泌体与疾病治疗的相关性研究有了许多进展,特别是间充质干细胞外泌体,拥有强大的免疫调节能力和组织修复能力,因此其在糖尿病并发症的治疗上拥有巨大的潜力。本文将对外泌体的来源、间充质干细胞和外泌体的生物学特性、组织修复特性及其在糖尿病肾病、糖尿病中枢神经病变、糖尿病血管病变及糖尿病皮肤病变中的治疗潜力作一综述。     

脐带间充质干细胞——组织工程的新视角

目的 从脐带中分离培养脐带间充质干细胞(mesenchymal stem cell, MSC) 并进行鉴定,阐明其多向分化的潜在作用.方法 收集健康胎儿脐带,分离培养脐带中的间充质干细胞,以流式细胞仪对培养的间充质干细胞进行细胞表面标志检测,多种成分联合诱导其向脂肪、成骨方向分化,细胞化学染色检测诱导后的细胞变化.结果 脐带中分离培养的间充质干细胞不表达造血细胞系的标志CD34、CD45、HLA-DR,强表达CD105、CD44、CD90,在适当的诱导条件下可向脂肪及成骨方向分化.结论 脐带中存在具有多向分化潜能的间充质干细胞.     

Isolation and characterization of mesenchymal stem cells from caprine umbilical cord tissue matrix

Cord tissue fills the umbilical cord around the blood vessels and contains types of stem cells (mesenchymal stem cells or MSCs) that are not generally found in cord blood. MSCs are the stem cells that give rise to many of the “support tissues” in the body, including bone, cartilage, fat and muscle. Umbilical Cord Tissue cells (UCTs) possessing the capacity to differentiate into various cell types such as osteoblasts, chondrocytes and adipocytes have been previously isolated from different species including human, canine, murine, avian species etc. The present study documents the existence of similar multipotential stem cells in caprine UCTs having similar growth and morphological characteristics. The cells were isolated from caprine umbilical cord and cultivated in DMEM (low glucose) supplemented with 15% FBS, L-glutamine and antibiotics. Primary culture achieved confluence in 5–7 days having spindle shaped morphology. The cells were morphologically homogeneous, showed robust proliferation ability with a population doubled time of 92.07 h as well as normal karyotype. In vitro self-renewal capacity was demonstrated by colony-forming unit assay (CFU). The cells expressed MSC specific markers and showed multi-differentiation capability into adipogenic and osteogeneic. The results indicated that caprine UCTs (cUCTs) were isolated and characterized from umbilical cord tissue which can be used for tissue regeneration.     

Human mesenchymal stem cells isolated from the umbilical cord

Mesenchymal stem cells (MSCs) are known as a population of multi-potential cells able to proliferate and differentiate into multiple mesodermal tissues including bone, cartilage, muscle, ligament, tendon, fat and stroma. In this study human MSCs were successfully isolated from the umbilical cords. The research characteristics of these cells, e.g., morphologic appearance, surface antigens, growth curve, cytogenetic features, cell cycle, differentiation potential and gene expression were investigated. After 2weeks of incubation, fibroblast-like cells appeared to be dominant. During the second passage the cells presented a homogeneous population of spindle fibroblast-like cells. After more than 4months (approximately 26 passages), the cells continued to retain their characteristics. Flow cytometry analysis revealed that CD29, CD44, CD95, CD105 and HLA-I were expressed on the cell surface, but there was no expression of hematopoietic lineage markers, such as CD34, CD38, CD71 and HLA-DR. Chromosomal analysis showed the cells kept a normal karyotype. The cell cycle at the third passage showed the percentage of G(0)/G(1), G(2)/M and S phase were 88.86%, 5.69% and 5.45%, respectively. The assays in vitro demonstrated the cells exhibited multi-potential differentiation into osteogenic and adipogenic cells. Both BMI-1 and nucleostemin genes, expressed in adult MSCs from bone marrow, were also expressed in umbilical cord MSCs. Here we show that umbilical cords may be a novel alternative source of human MSCs for experimental and clinical applications.     

脐静脉和骨髓来源的间充质干细胞的比较研究

间充质干细胞(MSCs)的来源有限,成人骨髓是MSCs的主要来源,这极大地限制了其在实验和临床中的应用。为拓宽MSCs来源,从细胞形态、生长特性、免疫表型和多向分化能力等四个方面对人脐静脉来源和成人骨髓来源的间充质干细胞进行了比较研究。结果表明,人脐静脉来源和成人骨髓来源的 MSCs具有相似的生物学特征,成纤维细胞样形态生长,并具有强大的体外扩增和多向分化能力。人脐静脉来源的MSCs可替代成人骨髓MSCs,作为满足实验和临床需要的重要来源。     

脐带间充质干细胞对内毒素血症诱发的大鼠急性肝功能损伤的影响

目的评价脐带间充质干细胞（hUC-MSCs）对内毒素血症诱发的大鼠急性肝功能损伤的影响及其与凋亡机制的关系。 方法6周龄雄性SD大鼠18只,随机分为3组,分别是对照组（C组）、内毒素血症组（M组）和内毒素血症+hUC-MSCs治疗组（M+cells组）,每组6只。大鼠腹腔注射5 mg/kg脂多糖（LPS）诱导内毒素血症模型,并经尾静脉注射含20×10⁶个hUC- MSCs。4 h时检测血清谷草转氨酶（AST）和谷丙转氨酶（ALT）,ELISA方法检测肿瘤坏死因子（TNF-α）、白细胞介素6（IL-6）,HE常规染色鉴定肝脏组织病理,Western Blot法检测肝脏组织抗凋亡蛋白Bcl-2、促凋亡蛋白Bax、凋亡信号调节激酶1（ASK1）、应激活化蛋白激酶即JUN氨基末端激酶（JNK）蛋白的表达。多组间比较采用单因素方差分析,相关分析选用pearson。 结果（1）C组AST、ALT、TNF-α和IL-6浓度分别为（74.66±6.39）U/ L、（40.07±6.07）U/ L、（37.74±3.08）ng/L和（0.42±0.07）ng/L;与M组比较（310.75±9.13）U/L、（107.04±10.04）U/ L、（160.32±4.88）ng/L和（0.90±0.09）ng/L,差异具有统计学意义（P均 < 0.05）,M组AST、ALT、TNF-α、IL-6浓度分别为（310.75±9.13）U/L、（107.04±10.04）U/ L、（160.32±4.88）ng/ L和（0.90±0.09）ng/L,与M+cells组比较（204.49±15.36）U/L、（71.24± 7.34）U/ L、（117.61±9.37）ng/ L和（0.60±0.10）ng/L,差异具有统计学意义（P均 < 0.05）。（2）C组大鼠肝细胞形态正常,可见肝小叶结构清晰,肝汇管区无炎性细胞浸润,M组大鼠肝小叶散在点状坏死肝细胞伴炎性浸润,肝细胞间隙散布增生的Kuffer细胞,M+cells组大鼠肝小叶炎性细胞浸润及肝细胞间隙Kuffer细胞浸润改善。（3）与C组比较,M组大鼠肝脏组织JUN、ASK1和Bax蛋白表达均增高（P均 < 0.05）,Bcl-2蛋白表达降低（P < 0.05）;与M组比较,M+cells组大鼠肝脏组织JUN、ASK1和Bax蛋白表达降低（P均 < 0.05）,Bcl-2蛋白增加（P < 0.05）。（4）单因素相关分析显示大鼠血清ALT、AST与TNF-a指数呈正相关（r值分别为0.9580、0.9865,P均< 0.05）,大鼠血清ALT、AST与IL-6指数呈正相关（r值分别为0.9892、0.9630,P均 < 0.05）,大鼠血清ALT、AST分别与BAX、ASK1、JNK指数均呈正相关（r值分别为0.9993、0.9851、0.7901、0.9864、0.9557、0.7128,P均 < 0.05）,大鼠血清ALT、AST分别与BCL-2指数均呈负相关（r值分别为-0.8824、-0.9338,P均 < 0.05）,大鼠血清TNF-α分别与BAX、ASK1、JNK指数均呈正相关（r值分别为0.9466、0.8958、0.6025,P均< 0.05）,大鼠血清TNF-α与BCL-2指数呈负相关（r = -0.6025,P均 < 0.05）,大鼠血清IL-6分别与BAX、ASK1、JNK指数均呈正相关（r值分别为0.9941、0.9997、0.8679,P均< 0.05）,大鼠血清IL-6与BCL-2指数呈负相关（r = -0.8078,P均 < 0.05）。 结论hUC-MSCs具有减轻内毒素血症大鼠急性肝功能损伤的作用,其机制与抑制肝脏细胞凋亡相关。     

Conditioned medium derived from umbilical cord mesenchymal stem cells regenerates atrophied muscles

We investigated the regenerative effects and regulatory mechanisms of human umbilical cord mesenchymal stem cells (UC-MSCs)-derived conditioned medium (CM) in atrophied muscles using an in vivo model. To determine the appropriate harvest point of UC-CM, active factor content was analyzed in the secretome over time. A muscle atrophy model was induced in rats by hindlimb suspension (HS) for 2 weeks. Next, UC-CM was injected directly into the soleus muscle of both hind legs to assess its regenerative efficacy on atrophy-related factors after 1 week of HS. During HS, muscle mass and muscle fiber size were significantly reduced by over 2-fold relative to untreated controls. Lactate accumulation within the muscles was similarly increased. By contrast, all of the above analytical factors were significantly improved in HS-induced rats by UC-CM injection compared with saline injection. Furthermore, the expression levels of desmin and skeletal muscle actin were significantly elevated by UC-CM treatment. Importantly, UC-CM effectively suppressed expression of the atrophy-related ubiquitin E3-ligases, muscle ring finger 1 and muscle atrophy F-box by 2.3- and 2.1-fold, respectively. UC-CM exerted its actions by stimulating the phosphoinositol-3-kinase (PI3K)/Akt signaling cascade. These findings suggest that UC-CM provides an effective stimulus to recover muscle status and function in atrophied muscles.     

Comparative proteomic analysis of mesenchymal stem cells derived from human bone marrow,umbilical cord,and placenta: Implication in the migration

Umbilical cord (UC) and placenta (P) have been suggested as alternatives to bone marrow (BM) as sources of mesenchymal stem cells (MSC) for cell therapy, with both UC‐ and P‐MSC possess immunophenotypic and functional characteristics similar to BM‐MSC. However, their migration capacity, which is indispensable during tissue regeneration process, is unclear. Under defined conditions, the migration capacity of BM‐ and P‐MSC was found 5.9‐ and 3.2‐folds higher than that of UC‐MSC, respectively. By the use of 2‐DE and combined MS and MS/MS analysis, six differentially expressed proteins were identified among these MSC samples, with five of them known to be involved in cell migration as migration enhancing or inhibiting proteins. Consistent with their migration capacity, the levels of migration enhancing proteins including cathepsin B, cathepsin D and prohibitin,were significantly lower in UC‐MSC when compared with those in BM‐ and P‐MSC. For the migration inhibiting proteins such as plasminogen activator inhibitor‐1 (PAI‐1) and manganese superoxide dismutase, higher expression was found in the UC‐MSC. We also showed that the overexpression of the PAI‐1 impaired the migration capacity of BM‐ and P‐MSC while silencing of PAI‐1 enhanced the migration capacity of UC‐MSC. Our study indicates that PAI‐1 and other migration‐related proteins are pivotal in governing the migration capacity of MSC.     

Fetal mesenchymal stem cells derived from human umbilical cord sustain primitive characteristics during extensive expansion

Stem cells of fetal origin lie between embryonic and adult stem cells in terms of potentiality. Because of the ethical controversy surrounding embryonic stem cells and the relatively inferior quality of adult stem cells, the use of fetal stem cells would be an attractive option in future therapeutic applications. Here, we have investigated primitive characteristics of human umbilical-cord-derived fetal mesenchymal stem cells (UC fMSCs) during extensive expansion. We have successfully isolated and cultured UC fMSCs from all UC samples, but with two early fungal contaminations. UC fMSCs proliferated without significant evidence of morphological changes, and the average cumulative population-doubling level was over 25 for about 3 months. UC fMSCs showed the positive expression of several CD markers, known to be related to MSCs, including CD73 (SH-3, 4), CD90 (Thy-1), CD105 (SH-2), CD117 (c-kit), and CD166 (ALCAM). They demonstrated primitive properties throughout the expansion period: multilineage differentiation potentials examined by functional assays, a variety of pluripotent stem cell markers including Nanog, Oct-4, Sox-2, Rex-1, SSEA-3, SSEA-4, Tra-1–60, and Tra-1–81, minimal evidence of senescence as shown by β-galactosidase staining, and the consistent expression of telomerase activity. These results suggest that UC fMSCs have more primitive properties than adult MSCs, which might make them a useful source of MSCs for clinical applications. This work was supported by the Seoul R&BD Program (10548).