内皮祖细胞在膀胱细胞外基质上的生长及分化

背景：传统的组织工程膀胱支架材料本身无血管结构,植入体内后面临血管化不足的问题。 目的：观察内皮祖细胞与膀胱细胞外基质的生物相容性。 方法：分离培养兔内皮祖细胞,种植于兔膀胱细胞外基质上与其复合培养。将复合生长材料植入兔背部皮下检测其组织相容性。 结果与结论：兔内皮祖细胞能够在膀胱细胞外基质表面正常黏附、生长、增殖,细胞形态良好。内皮祖细胞-膀胱细胞外基质植入兔体内后1周时,可见材料周围炎症反应明显,粘连严重,出血较多;苏木精-伊红染色可见组织中较多炎性细胞浸润,胶原及弹性纤维排列松散。植入后8周时可见材料已降解成碎细丝状,与周围组织融合生长在一起,但质地略脆,易出血;苏木精-伊红染色可见组织中已无明显炎症细胞浸润反应,胶原及弹性纤维排列紧密并且有新生血管长入其中。结果表明内皮祖细胞与膀胱细胞外基质具有良好的相容性,复合培养物与体内组织具有良好的相容性。     

Chemoattraction of progenitor cells by remodeling extracellular matrix scaffolds

The chemotactic properties of a biologic scaffold composed of extracellular matrix (ECM) and subjected to in vivo degradation and remodeling were evaluated in a mouse model of Achilles tendon reconstruction. Following a segmental resection of the Achilles tendon in both C57BL/6 and MRL/MpJ mice, the defect was repaired with either an ECM scaffold composed of urinary bladder matrix (UBM) or resected autologous tendon. The surgically repaired and the contralateral tendons were harvested at 3, 7, and 14 days following surgery from each animal. Chemotaxis of multipotential progenitor cells toward the harvested tissue was quantified using a fluorescent-based cell migration assay. Results showed greater migration of progenitor cells toward tendons repaired with UBM-ECM scaffold compared to both the tendons repaired with autologous tissue and the normal contralateral tendon in both the MRL/MpJ and C57BL/6 mice. The magnitude and temporal pattern of the chemotactic response differed between the two mouse strains.     

Distinct tissue formation by heterogeneous printing of osteo- and endothelial progenitor cells

The organ- or tissue-printing approach, based on layered deposition of cell-laden hydrogels, is a new technique in regenerative medicine suitable to investigate whether mimicking the anatomical organization of cells, matrix, and bioactive molecules is necessary for obtaining or improving functional engineered tissues. Currently, data on performance of multicellular printed constructs in vivo are limited. In this study we illustrate the ability of the system to print intricate porous constructs containing two different cell types--endothelial progenitors and multipotent stromal cells--and show that these grafts retain heterogeneous cell organization after subcutaneous implantation in immunodeficient mice. We demonstrate that cell differentiation leading to the expected tissue formation occurs at the site of the deposited progenitor cell type. While perfused blood vessels are formed in the endothelial progenitor cell-laden part of the constructs, bone formation is taking place in the multipotent stromal cell-laden part of the printed grafts.     

Distinct patterns of microvascular endothelial cell morphology are determined by extracellular matrix composition.

Endothelial interactions with the extracellular matrix (ECM) play important roles in angiogenesis but whether specific ECM signals can determine specific cellular morphologies is unclear. The authors compared in vitro ECM-induced morphological responses of the phenotypically distinct human placental microvascular endothelial cells (HPMECs) with large vessel endothelial cells (HUVECs). HPMECs showed distinct patterns of reorganization in response to collagen-I or collagen-IV (monolayer disruption, sprouting, migration) and Matrigel or laminin-A (intussusception, cord formation, tubulogenesis), and an intermediate response to fibrin; whereas HUVECs responded similarly to collagen-1 and Matrigel (elongation, lattice formation, vacuolation) and showed little response to fibrin. Although the extent of collagen and Matrigel responses of HPMECs were increased by serum, acidic or basic fibroblast growth factor (aFGF, bFGF), or vascular endothelial growth factor (VEGF), and varied with matrix protein concentration, the basic patterns were matrix specific, and were independent of fibronectin. The collagen responses correlated with disruption of adherens and tight junctions and the formation of filopodial protrusions. Matrigel responses were associated with up-regulated junctional localization of VE-cadherin, and tubulogenesis developed mainly through paracellular remodeling rather than intracellular vacuolation. Overall, these findings suggest that distinct ECM interactions stimulate specific morphological responses. These signals may regulate morphological behaviour in the angiogenesis cycle, switching endothelial cells between migratory and vasculogenic phenotypes.     

内皮祖细胞的研究进展

骨髓含有内皮祖细胞, 能够迁移至外周血并分化为成熟内皮细胞,参与胚胎时期的血管生成、出生后的微血管新生以及肿瘤组织的发生。体内内皮祖细胞数量和活性受多种生理性及病理性因素的的影响。体外扩增后回输体内可以修复受损组织、器官的血管,促进器官功能恢复;抑制其活性,在一定程度上可以抑制肿瘤组织的生长。内皮祖细胞为缺血性疾病以及肿瘤的治疗提供了另一新的靶点。     

内皮祖细胞的体外培养

 目的 建立一个体外培养脐血来源内皮祖细胞（EPC）的培养体系。方法 脐带血经密度梯度离心获得单个核细胞,按本室已建立的培养体系细胞培养,免疫细胞化学和流式细胞术对培养7d后的细胞进行CD34、CD133、vWF、CD146及CD144鉴定。结果 接种后前5d为生长的潜伏期,细胞开始贴壁,无明显扩增。第6天平均每个视野下细胞数目为287+45;第9天细胞数为282+46;第12天开始,细胞进入对数生长期,细胞数为805+67（P<0.05）;第19天细胞继续增殖,细胞数为1115+182（P<0.05）;第23天时,细胞进入凋亡期,数量明显减少,为265+61（P<0.05）。vWF,CD146,CD144表达阳性。流式细胞术结果表明,梭形样细胞群体中,CD34阳性率为88.98%+5.15% (P<0.05),CD133阳性率为1.20%+1.44% (P<0.05)。结论 利用本实验室的培养体系成功培养出内皮祖细胞。     

Construction of multi-functional extracellular matrix proteins that promote tube formation of endothelial cells

We developed artificial extracellular matrix proteins designed to have collagen-binding activity and active functional units that promote network formation of vascular endothelial cells. We engineered a laminin-derived IKVAV sequence, which stimulates capillary network formation of vascular endothelial cells, to incorporate into an elastin-derived structural unit. The designed fusion protein also had a cell-adhesive RGD sequence and a collagen-binding domain derived from fibronectin. The resultant fusion protein could bind to collagen type I and promote angiogenic activity of collagen gel. The collagen-binding domain also had slight angiogenic activity; however, the designed fusion protein also enhanced cellular migration activity. The engineering strategy of designing multi-functional ECM proteins has a possibility for supporting current tissue engineering techniques.     

In vitro manipulation of endothelial progenitor cell adhesion to vascular endothelium and extracellular matrix by the phorbol ester PMA

Injection of endothelial progenitor cells (EPCs) into arteries for cell therapy is a promising field in regenerative medicine. However, adhesion of EPCs during capillary passage is restricted, and non-adhering cells are lost into circulation. Here we demonstrate that it is possible to achieve a three- to sevenfold higher rate of EPC adhesion to endothelium and extracellular matrix molecules after short-term activation with phorbol myristate acetate (PMA). In addition, differentiation and toxicity analyses of PMA activated EPCs showed no impact on cell differentiation and negligible impact on cell survival.     

Cyclic strain effects on human monocyte interactions with endothelial cells and extracellular matrix proteins

Human vascular endothelial cells (ECs) are exposed to various levels of hemodynamic forces, cyclic strain, and shear stress in vivo. Here, we examined the in vitro effects of the various levels (0-6%, 7-16%, and 17-25%) of strain at 60, 30, and 15 cycles per minute (cpm) on human monocyte adherence to endothelial cells and extracellular matrix protein preabsorbed surfaces. Monocyte adhesion to endothelial cells under cyclic strain significantly increased. At both 30 and 60 cpm, ECs under strains of 7-16% and 17-25% showed >52% and >117% higher monocyte adhesion than endothelial cells under static condition when monocytes were added for 0.5 h. This increase in monocyte adhesion to ECs under cyclic strain remained significantly higher even after 24 h of incubation. Human monocyte adhesion to extracellular matrix protein preabsorbed surfaces differed depending on the specific extracellular matrix protein. Monocytes adhered to collagen type I and fibronectin preabsorbed surfaces >50% under 0-6% strain, >23% under 7-16% strain, and >52% under 17-25% strain at 15 and 30 cpm compared to the collagen type V preabsorbed surface. However, when extracellular protein preabsorbed surfaces under cyclic strain were compared to the control static condition, monocyte adhesion did not significantly change on most of other surfaces. These results suggest that cyclic strain may play a role in the regulation of monocyte-endothelial cells/extracellular matrix interactions in vivo.     

Low-molecular-weight peptides derived from extracellular matrix as chemoattractants for primary endothelial cells.

The development of synthetic and naturally occurring scaffolds for tissue engineering applications has included strategies to promote attachment of specific cell types, control the rate of scaffold degradation, encourage angiogenesis, or otherwise modulate the host response. We have reported that bioscaffolds developed from porcine small intestinal submucosa (SIS) facilitate the constructive remodeling of tissues and recruit marrow-derived cells that persist long after the acute inflammatory stages have resolved. We have not yet determined which cells are recruited, the eventual fate of these cells, or via what mechanisms the events occur. We now have analyzed various molecular weight fractions of acid-hydrolyzed SIS by both functional and morphologic methods and have determined that fraction 4 (5 to 16 kDa) possesses chemoattractant activity for primary murine adult liver, heart, and kidney endothelial cells in vitro. Addition of fraction 4 to Matrigel plugs promoted in vivo vascularization when the plugs were implanted subcutaneously in mice. These results indicate that small-molecular-weight peptides derived from the degradation of porcine SIS are biologically active in the recruitment of murine endothelial cells in vitro and in vivo.     

他汀类药物对内皮祖细胞的相关作用

背景：他汀类药物作为临床上广泛应用的降脂类药物,其不同剂量与疗程对于内皮祖细胞有着不同的近期及远期作用。 目的：综述国内外他汀类药物对内皮祖细胞作用的现状和进展。 方法：应用计算机检索CNKI和Pubmed数据库中1995-01/2009-11关于内皮祖细胞和他汀类药物的文章,在标题中以“内皮祖细胞,他汀类药物”或“endothelial progenitor cells,statins”为检索词进行检索。选择文章内容与内皮祖细胞及他汀类药物有关者,同一领域文献则选择近期发表或发表在权威杂志文章。初检得到843篇文献,根据纳入标准选择关于内皮祖细胞及他汀类药物的19篇文献进行综述。 结果与结论：他汀类药物对于内皮祖细胞有多重的作用。在短期治疗和低剂量的情况下,对心血管疾病有保护作用,增强内皮祖细胞各方面功能;在长期、大剂量应用他汀类药物后,造成其数量减少,抑制了血管生成,这对于抑制肿瘤血管发生有一定的研究价值。     

兔外周血内皮祖细胞的分离培养、鉴定和体外标记

目的 研究新西兰大白兔外周血内皮祖细胞的分离、培养方法,对其进行功能鉴定,同时用增强型绿色荧光蛋白对细胞进行标记示踪,为后续实验研究做好准备.方法 ①以健康雄性新西兰大白兔为研究对象,密度梯度法分离兔外周血单个核细胞,采用专用的EGM-2 MV完全培养基对单个核细胞进行诱导分化培养,将其接种在人纤维连接蛋白包被培养板,动态观察细胞生长过程.②通过DiL标记的乙酰化低密度脂蛋白和FITC标记的凝集素UEA-1双染法鉴定血管内皮祖细胞,显示红色荧光的为吞噬了乙酰化低密度脂蛋白的细胞,绿色荧光为结合UEA-1的细胞,双染色为橙色荧光.③用携带绿色荧光蛋白基因的腺病毒液对培养7 d细胞进行感染,用荧光显微镜观察细胞绿色荧光蛋白表达情况.结果 细胞形态观察：新分离的骨髓单个核细胞呈圆形,第3～4天可观察到贴壁梭型细胞,第5～8天出现多个细胞团.乙酰化低密度脂蛋白和凝集素UEA-1双染法鉴定血管内皮祖细胞结果：在血管内皮祖细胞的胞质中,出现与乙酰化低密度脂蛋白结合的红色荧光聚集,阳性率达95%以上,与凝集素UEA-1结合率几乎达100%,2者双染色率达90%以上;腺病毒感染细胞后24 h即可表达EGFP,5 d表达最强,1～2周表达稳定,此后逐渐减弱;腺病毒感染MSCs效率约为95%.结论 从兔外周血中能成功地分离培养出具有血管内皮祖细胞特征的细胞群体;经Ad-EGFP感染后的EPCs可有效表达EGFP,且转染效率较高,是一种较好的EPCs标记方法.     

Mobilization of endothelial progenitor cells in sepsis

Inflammation Research - Sepsis, an intractable clinical syndrome, is often accompanied by severe vascular endothelial injury and barrier dysfunction. Previous evidence has shown that the endogenous...     

Recruitment of progenitor cells by an extracellular matrix cryptic peptide in a mouse model of digit amputation

Biologic scaffolds composed of extracellular matrix (ECM) have been used successfully in preclinical models and humans for constructive remodeling of functional, site-appropriate tissue after injury. The mechanisms underlying ECM-mediated constructive remodeling are not completely understood, but scaffold degradation and site-directed recruitment of both differentiated and progenitor cells are thought to play critical roles. Previous studies have shown that degradation products of ECM scaffolds can recruit a population of progenitor cells both in vitro and in vivo. The present study identified a single cryptic peptide derived from the α subunit of the collagen III molecule that is chemotactic for a well-characterized perivascular stem cell in vitro and causes the site-directed accumulation of progenitor cells in vivo. The oligopeptide was additionally chemotactic for human cortical neural stem cells, rat adipocyte stem cells, C2C12 myoblast cells, and rat Schwann cells in vitro. In an adult murine model of digit amputation, treatment with this peptide after mid-second phalanx amputation resulted in a greater number of Sox2+ and Sca1+,Lin- cells at the site of injury compared to controls. Since progenitor cell activation and recruitment are key prerequisites for epimorphic regeneration in adult mammalian tissues, endogenous site-directed recruitment of such cells has the potential to alter the default wound healing response from scar tissue toward regeneration.     

The interplay between extracellular matrix and progenitor/stem cells during wound healing: Opportunities and future directions

Skin wound healing, a dynamic physiological process, progresses through coordinated overlapping phases to restore skin integrity. In some pathological conditions such as diabetes, wounds become chronic and hard-to-heal resulting in substantial morbidity and healthcare costs. Despite much advancement in understanding mechanisms of wound healing, chronic and intractable wounds are still a considerable challenge to nations’ health care systems. Extracellular matrix (ECM) components play pivotal roles in all phases of wound healing. Therefore, a better understanding of their roles during wound healing can help improve wound care approaches. The ECM provides a 3D structure and forms the stem cell niche to support stem cell adhesion and survival and to regulate stem cell behavior and fate. Also, this dynamic structure reserves growth factors, regulates their bioavailability and provides biological signals. In various diseases, the composition and stiffness of the ECM is altered, which as a result, disrupts bidirectional cell-ECM interactions and tissue regeneration. Hence, due to the impact of ECM changes on stem cell fate during wound healing and the possibility of exploring new strategies to treat chronic wounds through manipulation of these interactions, in this review, we will discuss the importance/impact of ECM in the regulation of stem cell function and behavior to find ideal wound repair and regeneration strategies. We will also shed light on the necessity of using ECM in future wound therapy and highlight the potential roles of various biomimetic and ECM-based scaffolds as functional ECM preparations to mimic the native stem cell niche.     

MS-818 accelerates mobilization of endothelial progenitor cells and differentiation to endothelial cells.

MS-818 that is a synthetic pyrimidine compound and shown to have neurotrophic actions, enhanced basic fibroblast growth factor (bFGF)-induced angiogenesis in vivo. However, the mechanism and whether MS-818 affects endothelial cells (ECs) directly is not known. Here, the authors investigated whether MS-818 alone could induce angiogenesis and tried to clarify the mechanism of neovascularization by MS-818 in terms of angiogenesis and vasculogenesis. The authors show that MS-818 affects ECs directly and induces migration of and tube formation by ECs in vitro (angiogenesis). Furthermore, the authors demonstrate that MS-818 mobilizes endothelial progenitor cells (EPCs) from the bone marrow and potentiates their differentiation to ECs (vasculogenesis). The effect of MS-818 on the endothelial differentiation was further confirmed with an in vitro differentiation system using mouse embryonic stem cells. MS-818 activates the mitogen-activated protein kinase (MAPK) pathway but not the phosphoinositol 3-kinase (PI3K)-Akt pathway in ECs. These results indicate that MS-818, a synthetic compound, promotes both angiogenesis and vasculogenesis.     

组织工程肺种子细胞及其细胞外基质的研究与进展

背景：至今为止,构建组织工程肺并运用于临床尚并未取得较大进展,原因在于种子细胞来源及鉴定,移植时限选择,支撑架安全性,细胞分化程序的标准化,细胞外基质分泌的控制等多个方面都存在难点。 目的：总结组织工程肺的种子细胞来源及鉴定、细胞外基质替代物的研究与进展。 方法：检索中国期刊全文数据库(CNKI：1989/2011)和PubMed(1989/2011)数据库,检索词分别为“组织工程肺,细胞源,支架,细胞外基质,体内植入”和“Tissue-engineered Lung,Cell Source,Support Scaffolding,lung matrices,Vivo Implantation”,语言分别设定为中文和英文。阅读文题和摘要进行筛选,选择具有原创性,论点论据可靠且分析全面、密切相关的文章,排除重复性研究以及质量较差文章。共检索到72篇文章,按纳入及排除标准筛选后,共纳入36篇文章。 结果与结论：利用合适的细胞源与支撑架进行组织工程肺研究可培育出功能健全的可用来替换的肺组织,但目前组织工程肺的研究尚无重大进展。最近研究成功运用于临床的脱细胞气管为组织工程肺治疗肺部疾病带来新的希望。