人绒毛膜来源的间充质干细胞成骨成脂分化潜能

目的:探讨人绒毛膜来源的间充质干细胞(hCDMSC)体外生长特性和成骨成脂分化潜能,证实人绒毛膜来源的间充质干细胞作为组织工程种子细胞的可行性.方法:取胎盘组织基蜕膜面用胶原酶和胰蛋白酶法分离培养,通过传代扩增观察细胞形态,MTT法检测细胞增殖曲线,体外向成骨、成脂诱导分化,茜素红和油红O染色鉴定细胞分化能力,RT-P...     

大鼠骨髓间充质干细胞的体外分离与培养

目的 建立体外分离纯化及培养扩增大鼠骨髓间充质干细胞的方法.方法 应用全骨髓贴壁培养法分离培养大鼠骨髓间充质干细胞,并进行细胞传代培养.倒置显微镜下观察细胞形态及生长特征,测定细胞生长曲线,通过流式细胞仪检测细胞表面标志物,取第3代细胞分别加入成骨、成脂诱导剂并采用碱性磷酸酶染色及Von Kossa染色鉴定成骨能力,以油红O染色鉴定成脂能力.结果 获取的大鼠骨髓间充质干细胞形态呈均一成纤维细胞样,并呈集落样生长.传至第3代细胞纯度可达97%以上,其细胞表型CD29、CD44、CD105、CD166呈阳性表达,CD34、CD80、CD86呈阴性表达.经成骨诱导后细胞碱性磷酸酶染色及Von Kossa染色呈阳性,成脂诱导后细胞油红O染色呈现阳性.结论 应用全骨髓贴壁法可以分离培养出高纯度的大鼠bMSCs,培养的细胞扩增迅速、生物学特性稳定,是一种较为理想的体外分离扩增bMSCs的培养体系.     

人骨髓来源间充质干细胞的体外分离方法及鉴定

目的探讨人骨髓来源间充质干细胞的体外分离方法及鉴定。方法密度梯度离心从人骨髓分离单个核细胞,接种培养3h后频繁换液,培养至第14d时用0.25%胰酶室温作用2min移种后继续培养至第21d。采用流式细胞术检测收获细胞的表型分子。收获细胞在特定条件下诱导培养至第14d,分别采用茜素红染色、甲苯胺蓝染色和油红染色进行鉴定。结果第21d时均一的长梭状成纤维样细胞铺满瓶底,它们高表达CD105、CD73、CD90并低表达CD45、CD34和CD14,能够被诱导分化为骨细胞、软骨细胞和脂肪细胞。结论密度梯度离心法结合贴壁筛选可以成功地从人骨髓分离获得间充质干细胞,接种初期频繁换液体及后期控制胰酶作用时间可以有效减少造血系细胞混杂。     

大鼠脂肪干细胞的体外分离培养及对脂肪颗粒移植的影响

目的分析体外大鼠脂肪干细胞(ADSCs)混合脂肪移植后的存活情况。方法无菌条件下切取大鼠腹股沟脂肪组织,消化、分离培养得到第3代ADSCs,行成骨诱导(茜素红染色)和成脂诱导(油红O染色)。用CM-Dil荧光标记第3代ADSCs,24只大鼠每只背部皮下3处分别植入1.5ml脂肪颗粒、1.5ml荧光标记的ADSCs(密度为5×107个细胞/ml)和0.9ml荧光标记的ADSCs+0.6ml脂肪颗粒。术后2周、1个月和3个月每次取出8只大鼠的移植物,石蜡切片HE染色观察病理变化,冰冻切片荧光显微镜下观察ADSCs定位。结果 ADSCs成骨诱导2周后茜素红染色阳性,成脂诱导3周后油红O染色阳性。ADSCs与脂肪颗粒混合移植能明显改善脂肪组织的病理学变化。结论体外分离培养的大鼠ADSCs具有成骨、成脂分化的潜能,能改善脂肪颗粒移植时的脂肪组织的液化吸收现象。     

大鼠脂肪间充质干细胞体外诱导成骨分化的实验性研究

目的探讨大鼠脂肪间充质干细胞体外分离培养和成骨诱导分化的可行性。方法取6周龄大鼠双侧腹股沟处脂肪，酶鹪法分离培养原代脂肪间充质干细胞，成骨诱导培养18d，检测细胞成骨表型转化：碱性磷酸酶、骨钙素的表达以及细胞矿化细胞外基质的能力。结果大鼠脂肪中能分离培养出一定量的脂肪间充质干细胞，在成骨诱导培养液作用下，可特异性表达成骨分化的标志：碱性磷酸酶、骨钙素，同时也具有矿化细胞外基质的能力。结论大鼠脂肪间充质干细胞可能是一种理想的骨组织工程种子细胞的来源。     

人脐带问充质干细胞体外培养、鉴定及其诱导分化的研究

目的通过体外分离、培养及鉴定人脐带间充质干细胞（humanumbilicalcordmesenchymalstemcells,hUCMSCs）,探讨其多向分化潜能。方法取正常足月新生儿脐带,采用组织贴壁培养法分离原代hUCMSCs,观察细胞生长形态。采用流式细胞仪技术检测hUCMSCs细胞表型及细胞周期。通过成神经细胞诱导和成脂肪细胞诱导,鉴定hUCMSCs的多向诱导分化能力。结果成功分离和培养hUCMSCs原代细胞,经流式细胞仪鉴定高表达间质细胞标志CD44和CDl05,阳性率为96．73％和96．10％,整合素受体CD29阳性率为99．53％;低表达造血系标志CD34和CD45阳性率为0．80％和1．91％,人白细胞抗原HLA-DR阳性率为1．41％。细胞周期检测hUCMSCs主要处于G0／G1期。hUCMSCs成神经细胞诱导,出现神经元样细胞;免疫组化检测神经巢蛋白（Nestin）呈阳性表达。hUCMSCs成脂肪细胞诱导,出现空泡样脂肪滴,油红O染色可见脂质沉积。结论hUCMSCs具有多向分化潜能,可跨胚层诱导分化为多种组织类型的细胞。     

脐静脉源MSC分离及生物学特性

目的探讨脐静脉源间充质干细胞（MSC）的分离及培养,观察其进一步分化的潜能。方法用胶原酶常规消化分离脐静脉内皮细胞,传代3周后,用免疫组化法和流式细胞术检测细胞免疫表型;观察该类细胞向脂肪和成骨细胞分化的潜能,油红O染色和von Kossa染色鉴定细胞性质。结果脐静脉内皮／内皮下细胞接种3～4周后以长梭形的成纤维样细胞为主。传代至F2代时细胞形态均一为长梭形。表型检测示该类细胞高表达CD29、CD44、CD54、CD49e、HLA-ABC和CD40分子,不表达造血细胞相关分子如CD34、CD45、HLA-DR,免疫组化分析α平滑肌肌动蛋白（α-SMA）阳性、血管性血友病因子（vWF）阴性;细胞经成脂诱导后镜下可见细胞内脂滴形成,3周后油红O染色阳性;经成骨诱导3周后细胞von Kossa染色为阳性。结论脐静脉内皮和内皮下含有MSC样细胞,在适当的条件下可向脂肪细胞和成骨细胞分化。脐带可成为实验和临床研究所用MSC的又一来源。     

体外诱导人牙龈成纤维细胞多向分化潜能的实验研究

目的 探索人牙龈成纤维细胞（hGFs）是否具有多向分化潜能, 为组织工程学提供新的细胞来源。方法 经志愿者知情同意后收集健康牙龈组织, 组织块法培养 hGFs。取第 3 代 hGFs 进行成骨、 成软骨和成脂诱导, 未分化诱导的细胞为对照组。分别用碱性磷酸酶 （ALP） 染色和茜素红染色、 阿利新蓝染色、 油红 O 染色检测细胞成骨、 成软骨和成脂能力。实时定量聚合酶链反应 （PCR） 检测细胞中成骨分化标志基因 ALP、 runt 相关转录因子 2 （Runx2）、成软骨分化标志基因聚集蛋白聚糖（AGR）和成脂分化标志基因过氧化物酶体增殖物激活受体γ2（PPARγ2）的表达。结果 成骨诱导组培养至 7 d 时 ALP 染色可见大量的蓝紫色沉淀, 28 d 时细胞周围有大量红染的钙结节沉积,而对照组无钙结节, 培养至 14 d 细胞中 ALP 和 Runx2 表达明显高于对照组（P < 0.01）。14 d 时成软骨诱导组阿利新蓝阳性, 成脂诱导组可见红色的脂肪滴, 而对照组 2 种染色为阴性, AGR、 PPARγ2 表达均明显高于对照组（P <0.01）。结论 hGFs 具有成骨、 成软骨和成脂分化的多向分化潜能。     

大鼠骨髓间充质干细胞体外培养以及成骨诱导

目的:分离培养大鼠骨髓间充质干细胞,研究其体外培养的生物特性。方法:获取骨髓间充质干细胞进行培养,倒置相差显微镜观察细胞生长情况,绘制生长曲线,流式细胞仪鉴定细胞表面抗原。在地塞米松、β-甘油磷酸钠、抗坏血酸的作用下,进行成骨诱导分化。结果:原代和传代培养的体外培养细胞呈现梭形、多角形外观,具有较强的生长增殖能力;细胞CD29抗原呈阳性表达,CD34呈阴性表达。分化细胞表现成骨细胞特性,茜素红染色有橘红色磷酸盐胞外基质沉积。结论:建立稳定可靠的分离培养大鼠骨髓BM-MSCs方法,分离出的BM-MSCs具有干细胞的生物特性,可用于组织工程中的种子细胞。     

成人骨髓间充质干细胞体外向视网膜细胞的诱导分化

目的研究取材于成人骨髓的间充质干细胞在一定诱导条件下向视网膜神经细胞的分化。方法成人骨髓经密度梯度离心得到的细胞,根据其高黏附特性体外培养获得间充质干细胞。利用流式细胞仪分析其细胞表型,在体外诱导使其向视网膜神经细胞分化并用免疫荧光法进行鉴定。结果从骨髓中分离培养的细胞具有成纤维细胞样形态,贴壁生长,表型相对均一,表面标志为CD90、CD44、CD147阳性;而CD34、CD38、CD45、CD14、HLA-DR阴性。体外诱导后可以得到表达nestin(神经干细胞标志物)、GFAP(神经胶质细胞标志物)和Rhodopsin(视网膜光感受器细胞标志物)阳性的细胞。结论从人骨髓中分离培养得到的间充质干细胞具有向视网膜神经细胞分化的潜能。     

骨质疏松症的诊治及应用间充质干细胞的治疗现状

骨质疏松症是一种系统性的代谢性骨骼疾病,其特征是骨量减少和骨微结构退化,导致骨骼脆性和骨折风险的增加。在成人中,骨髓间充质干细胞在骨骼中主要分化为成骨细胞和脂肪细胞,当其分化失衡,分化的脂肪细胞增多,成骨细胞减少,最终会演变成骨质疏松症。骨质疏松症越来越被认为是一个主要的公共健康问题,对全世界超过2亿人有影响,每年造成...     

Feridex标记胎盘来源的间充质干细胞分化为神经干细胞的实验研究

目的分离胎盘组织来源的间充质干细胞并诱导分化为神经干细胞,以期找到能成功标记神经干细胞的方法。方法将胎盘组织剪碎后消化、培养,加入神经干细胞诱导因子10ng/mL表皮生长因子（EGF）＋10ng/mL重组人碱性成纤维生长因子（bFGF）＋DMEM/F12,并分别添加相应浓度的血清,预诱导3d后加入DMEM/F12＋0.1μmol/L全反式维甲酸（RA）,10ng/mL胶质细胞系源性神经营养因子（GDNF）＋10ng/mL脑源性神经营养因子（BDNF）进行正式诱导7d。结果诱导10d后用Feridex标记诱导后的细胞,普鲁士蓝染色显示90%以上的干细胞胞质内出现细小的蓝色铁颗粒,Nestin染色显示阳性。结论胎盘来源的间充质干细胞能成功诱导为神经干细胞,且Feridex可成功标记诱导后的细胞。     

黄芪甲苷对脂肪源性干细胞体外生物学行为的影响

目的探讨黄芪甲苷(Astragaloside IV,Ast)对体外培养人脂肪源性间充质干细胞(human adipose-derived mesen-chymal stem cells,hADSCs)生物学行为的影响。方法应用CCK8和PCNA法检测不同浓度和时间Ast孵育对hADSCs增殖情况的影响,选取出Ast最适干预条件并对hADSCs进行培养,观察药物干预后细胞形态、表面标志物、成骨及成脂分化潜能。结果 CCK8和PCNA结果均显示,Ast 20 mg.L-1干预48 h后细胞增殖最为明显,进一步选取Ast20 mg.L-1干预hADSCs 48 h,与对照组相比,两组细胞CD29、CD44、CD105的阳性率均为96%-99%;两组细胞成骨诱导与成脂诱导结果也无差异。结论 Ast可促进ADSCs增殖,以20 mg.L-1、孵育48 h为最适干预条件,且该条件干预后并不影响hADSCs细胞形态,表面标志物水平及多向分化能力。     

间充质干细胞的免疫原性及其在移植中的应用

目的：探讨研究间充质干细胞的免疫原性以及rMSCs在临床移植中的应用。方法：使用免疫组化方法鉴定rMSCs表面ratLA-ABC、ratLA-DR、CD80、CD86分子,并使用流式细胞术检测其含量;免疫组化法rMSC表面MHCI、Ⅱ类分子的鉴定。结果：ratLA-ABC分子在大鼠间充质干细胞的表面的表达率为13.08%,同时ratLA-DR以及CD80、CD86分子在大鼠间充质干细胞的表面均未有不表达,而外周血淋巴细胞PBLs的表面对ratLA-ABC、ratLA-DR、CD80、CD86这些分子的表达率分别为92.71%、22.39%、14.08%、9.03%。光学显微镜下观察细胞染色情况,细胞核中有棕黄染色者为阳性细胞。结论：MSC具有多向分化潜能,特性稳定;MSC不表达MHCⅡ类分子,不表达或极低表达MHCI类分子,能逃脱宿主免疫系统的排斥,异基因MSC可以进入宿主细胞,并能长期存在。MSC具有低免疫原性,临床上大大降低移植排斥及移植物抗宿主病,提高临床移植的存活率。     

From bone to brain: human skeletal stem cell therapy for stroke

Human adult skeletal stem cells, a.k.a. mesenchymal stem cells or marrow stromal cells (MSCs), have been identified as precursors of several different mesenchymal cellular lineages, including osteoblasts, chondrocytes, myoblasts, adipocytes, and fibroblasts, as well as non-mesenchymal lineages including neurons and glial cells. Adult stem cell transplantation is a promising strategy for the treatment of stroke. MSCs are also used as a platform for gene therapies and therapeutic agents. In this review, we discuss recent progress of human skeletal stem cell biology, in vitro differentiation of MSCs into neural stem cells and neurons, MSC therapy for stroke, MSC aging and the challenge of autologous cell therapy for stroke in elderly patients.     

Mesenchymal stem cells: cell biology and potential use in therapy

Mesenchymal stem cells are clonogenic, non-haematopoietic stem cells present in the bone marrow and are able to differentiate into multiple mesoderm-type cell lineages e.g. osteoblasts, chondrocytes, endothelial-cells and also non-mesoderm-type lineages e.g. neuronal-like cells. Several methods are currently available for isolation of the mesenchymal stem cells based on their physical and immunological characteristics. Because of the ease of their isolation and their extensive differentiation potential, mesenchymal stem cells are among the first stem cell types to be introduced in the clinic. Recent studies have demonstrated that the life span of mesenchymal stem cells in vitro can be extended by increasing the levels of telomerase expression in the cells and thus allowing culture of large number of cells needed for therapy. In addition, it has been shown that it is possible to culture the cells in xeno-free environment without affecting their growth or differentiation potential. Finally, the mesenchymal stem cells seems to be hypoimmunogenic and thus allogenic mesenchymal stem cells transplantation is possible. It is envisaged that mesenchymal stem cells can be used in systemic transplantation for generalized diseases, local implantation for local tissue defects, as a vehicle for genes in gene therapy protocols or to generate transplantable tissues and organs in tissue engineering protocols. The results of these initial trials are very encouraging and several clinical trials are under way to study the efficacy and long-term safety of therapeutics based on mesenchymal stem cells.     

When stem cells meet immunoregulation

The clinical use of stem cells to prevent tissue injury or reconstruct damaged organs is constrained by different ethical and biological issues. Whereas the use of adult stem cells isolated from differentiated tissues is advantageous from the ethical point of view, the immune response of a host to implants of either embryonic or adult stem cells remains a critical problem. Embryonic stem cells can be rejected by an immunocompetent recipient as well as some types of adult stem cells. There is, however, a population of adult stem cells able to differentiate into the three mesenchymal lineages, osteocytes, chondrocytes, adipocytes that have the additional capacity of modulating the immune response by the activation of disparate mechanisms, among which the generation of antigen-specific CD4+CD25+FoxP3+ regulatory T lymphocytes.This short review will focus on the immunological properties of embryonic and adult stem cells are, with particular emphasis on the immunomodulatory function of mesenchymal stem cells and their interactions with regulatory T lymphocytes.     

Interleukin-17A inhibits adipocyte differentiation in human mesenchymal stem cells and regulates pro-inflammatory responses in adipocytes

The immune system is closely linked to human metabolic diseases. Serum levels of IL-6 increase with obesity and insulin resistance. Not only does IL-6 decrease the insulin sensitivity of human cells such as adipocytes, but it also regulates the lineage commitment of naïve T cells into interleukin (IL)-17A-producing CD4(+) T (Th17) cells. Although IL-17A exerts a variety of effects on somatic tissues, its functional role in human adipocytes has not been identified. In this work, we show that IL-17A inhibits adipocyte differentiation in human bone marrow mesenchymal stem cells (hBM-MSCs), while promoting lipolysis of differentiated adipocytes. We find that IL-17A increases both mRNA and protein secretion of IL-6 and IL-8 during adipocyte differentiation in hBM-MSCs. IL-17A up-regulates cyclooxygenase (COX)-2 gene expression and thereby increases the level of prostaglandin (PG) E₂ in differentiated adipocyes. The suppression of anti-adipogenic PGE₂ by COX inhibitors such as aspirin and NS-398 partially blocked the effect of IL-17A on adipocyte differentiation in hBM-MSCs. Therefore, IL-17A exhibits its inhibitory effect in part via the COX-2 induction in differentiated adipocytes. In addition, treatment with anti-IL-17A antibody neutralizes IL-17A-mediated effects on adipocyte differentiation and function. These results suggest that IL-17A plays a regulatory role in both the metabolic and inflammatory processes of human adipocytes, similar to other pro-inflammatory cytokines such as IL-1, IFNγ, and TNFα.     

Comparison of ganglioside expression between human adipose- and dental pulp-derived stem cell differentiation into osteoblasts

Human adipose-derived stem cells (hADSCs) and dental pulp-derived stem cells (hDPSCs) have been considered alternative sources of adult stem cells because of their potential to trans-differentiate into multiple cell lineages. This study investigated the possible role of gangliosides in the osteoblast differentiation of hADSCs and hDPSCs. First, we investigated characterization of hADSCs and hDPSCs using FACS analysis. Mesenchymal stem cell specific markers, CD44 and CD105, were expressed but not hematopoetic markers, CD45 and CD117 in both of hADSCs and hDPSCs. High-performance thin-layer chromatography analysis showed that increased gangliosides were associated with differentiation of hADSCs and hDPSCs into osteoblasts. RT-PCR analysis confirmed that osteoblast specific genes, ALP, BMP-2, collagen were expressed in differentiated osteoblasts, however, the another osteoblast specific gene, osteocalcin, was not expressed. When hADSCs and hDPSCs were cultured under osteoblast-differentiation conditions, alkaline phosphatase (ALP) activity was increased in comparison to hADSCs and hDPSCs. Furthermore, specifically both ALP activity and ganglioside expression increased more in hDPSCs-derived osteoblasts than hADSCs-derived osteoblasts. These results suggest that gangliosides play a more important role in regulating the osteoblast-differentiation of hDPSCs compared to hADSCs.