骨质疏松大鼠脂肪基质细胞成骨能力的研究

目的:探讨骨质疏松大鼠的脂肪基质细胞有无成骨能力。方法:采用去势法构建SD大鼠骨质疏松模型,设置2组样本,分别为19月龄骨质疏松SD雌性大鼠,正常19月龄SD雌性大鼠。每组大鼠分别取腹股沟脂肪垫中脂肪,用胶原酶消化法培养获得脂肪间充质干细胞,绘制并分析细胞生长曲线;再用经典的骨向诱导液诱导2组ASCs,比较2组细胞骨向分化能力有无差别。结果:骨质疏松大鼠与正常老龄大鼠的ASCs骨向分化能力无明显差别。结论:可用去势法构建SD大鼠骨质疏松模型,并可取其细胞行进一步研究。老龄骨质疏松大鼠的ASCs也可以考虑作为种子细胞促进其自体骨愈合,从而为利用自体ASCs促进骨质疏松骨愈合打下一定的理论基础。     

脂肪干细胞对神经创伤修复的研究进展

在医学上,神经系统对神经损伤的自我修复能力往往有限。因此,探索有效修复损伤神经的方法已经成为近年来的研究热点。研究发现脂肪干细胞对各类损伤神经都有一定的修复作用,可作为修复神经损伤的种子细胞。脂肪干细胞不仅具有干细胞的特性而且还具有自身的优点;首先它属于成体细胞,来源于中胚层,具有多种分化的潜能;其次免疫原性较低,易于取材;另外脂肪干细胞移植后的风险较低,因此被认为是修复神经损伤的优秀种子细胞。就脂肪干细胞的特性及脂肪干细胞在神经创伤修复方面的研究进展和存在的问题进行综述。     

Collagen-Hydroxyapatite Scaffolds Induce Human Adipose Derived Stem Cells Osteogenic Differentiation In Vitro

Mesenchymal stem cells (MSCs) play a crucial role in regulating normal skeletal homeostasis and, in case of injury, in bone healing and reestablishment of skeletal integrity. Recent scientific literature is focused on the development of bone regeneration models where MSCs are combined with biomimetic three-dimensional scaffolds able to direct MSC osteogenesis. In this work the osteogenic potential of human MSCs isolated from adipose tissue (hADSCs) has been evaluated in vitro in combination with collagen/Mg doped hydroxyapatite scaffolds. Results demonstrate the high osteogenic potential of hADSCs when cultured in specific differentiation induction medium, as revealed by the Alizarin Red S staining and gene expression profile analysis. In combination with collagen/hydroxyapatite scaffold, hADSCs differentiate into mature osteoblasts even in the absence of specific inducing factors; nevertheless, the supplement of the factors markedly accelerates the osteogenic process, as confirmed by the expression of specific markers of pre-osteoblast and mature osteoblast stages, such as osterix, osteopontin (also known as bone sialoprotein I), osteocalcin and specific markers of extracellular matrix maturation and mineralization stages, such as ALPL and osteonectin. Hence, the present work demonstrates that the scaffold per se is able to induce hADSCs differentiation, while the addition of osteo-inductive factors produces a significant acceleration of the osteogenic process. This observation makes the use of our model potentially interesting in the field of regenerative medicine for the treatment of bone defects.     

Paracrine Effect of Mesenchymal Stem Cells Derived from Human Adipose Tissue in Bone Regeneration

Mesenchymal stem cell (MSC) transplantation has proved to be a promising strategy in cell therapy and regenerative medicine. Although their mechanism of action is not completely clear, it has been suggested that their therapeutic activity may be mediated by a paracrine effect. The main goal of this study was to evaluate by radiographic, morphometric and histological analysis the ability of mesenchymal stem cells derived from human adipose tissue (Ad-MSC) and their conditioned medium (CM), to repair surgical bone lesions using an in vivo model (rabbit mandibles). The results demonstrated that both, Ad-MSC and CM, induce bone regeneration in surgically created lesions in rabbit''s jaws, suggesting that Ad-MSC improve the process of bone regeneration mainly by releasing paracrine factors. The evidence of the paracrine effect of MSC on bone regeneration has a major impact on regenerative medicine, and the use of their CM can address some issues and difficulties related to cell transplants. In particular, CM can be easily stored and transported, and is easier to handle by medical personnel during clinical procedures.     

Efficient Generation of Neural Stem Cell-Like Cells from Rat Adipose Derived Stem Cells After Lentiviral Transduction with Green Fluorescent Protein

Neural stem cells (NSCs) can be isolated from nervous tissues or derived from embryonic stem cells. However, their procurement for clinical applications is limited, and there is a need for alternative types of cell that have NSCs properties. In the present study, the differentiation potential of rat adipose-derived stem cells (ADSCs) was evaluated by infecting these cells with a lentiviral vector-encoding green fluorescent protein (GFP). ADSCs transduced with lentivirus were able to generate NSC-like cells, without any effects on their growth, phenotype, and normal differentiation potential. NSC-like cells derived from ADSCs formed neurospheres and expressed high levels of the neural progenitor marker nestin. In the absence of selected growth factors, these neurospheres differentiated into neurons expressing NeuN and MAP2 and GFAP-expressing glia, as determined by immunocytochemistry, Western blotting, and quantitative real-time polymerase chain reaction. These results demonstrate that ADSCs can be induced to generate neurospheres that have NSC-like properties and may thus constitute a potential source of cells in stem cell therapy for neurological disorders.     

胚胎干细胞体外分化为造血干细胞

造血干细胞移植已成为治疗白血病、再生障碍性贫血、重症免疫缺陷征、地中海贫血、急性放射病、某些恶性实体瘤和淋巴瘤等造血及免疫系统功能障碍性疾病的成熟技术和重要手段,另外这一技术还被尝试用于治疗艾滋病,已取得积极的效果。但是由于移植需要配型相同的供体,并且过程复杂,使得造血干细胞移植因缺少配型相同的供体来源以及费用昂贵而不能被广泛应用。胚胎干细胞是一种能够在体外保持未分化状态并且能进行无限增殖的细胞,在适合条件下能够分化为体内各种类型的细胞,研究胚胎干细胞分化为造血干细胞,不仅可作为研究动物的早期造血发生的模型,而且可以增加造血干细胞的来源,还可以通过基因剔除、治疗性克隆等方法来解决移植排斥的问题,从而为造血干细胞移植的发展扫除了障碍,因此有着重要的研究价值和应用前景。现对胚胎干细胞体外分化为造血干细胞的诱导方法,诱导过程中的调控机制,并对胚胎干细胞分化为造血干细胞的存在问题和发展前景进行讨论。     

Induction of Osteogenic Differentiation of Adipose Derived Stem Cells by Microstructured Nitinol Actuator-Mediated Mechanical Stress

The development of large tissue engineered bone remains a challenge in vitro, therefore the use of hybrid-implants might offer a bridge between tissue engineering and dense metal or ceramic implants. Especially the combination of the pseudoelastic implant material Nitinol (NiTi) with adipose derived stem cells (ASCs) opens new opportunities, as ASCs are able to differentiate osteogenically and therefore enhance osseointegration of implants. Due to limited knowledge about the effects of NiTi-structures manufactured by selective laser melting (SLM) on ASCs the study started with an evaluation of cytocompatibility followed by the investigation of the use of SLM-generated 3-dimensional NiTi-structures preseeded with ASCs as osteoimplant model. In this study we could demonstrate for the first time that osteogenic differentiation of ASCs can be induced by implant-mediated mechanical stimulation without support of osteogenic cell culture media. By use of an innovative implant design and synthesis via SLM-technique we achieved high rates of vital cells, proper osteogenic differentiation and mechanically loadable NiTi-scaffolds could be achieved.     

Enhanced Osteogenesis of Adipose Derived Stem Cells with Noggin Suppression and Delivery of BMP-2

Bone morphogenetic proteins (BMPs) are believed to be the most potent osteoinductive factors. However, BMPs are highly pleiotropic molecules and their supra-physiological high dose requirement leads to adverse side effects and inefficient bone formation. Thus, there is a need to develop alternative osteoinductive growth factor strategies that can effectively complement BMP activity. In this study, we intrinsically stimulated BMP signaling in adipose derived stem cells (ASCs) by downregulating noggin, a potent BMP antagonist, using an RNAi strategy. ASCs transduced with noggin shRNA significantly enhanced osteogenic differentiation of cells. The potency of endogenous BMPs was subsequently enhanced by stimulating ASCs with exogenous BMPs at a significantly reduced dose. The level of mineralization in noggin shRNA treated ASCs when treated with BMP-2 was comparable to that of control shRNA treated cell treated with 10-fold more BMP-2. The complementary strategy of noggin suppression + BMP-2 to enhance osteogenesis was further confirmed in 3D in vitro environments using scaffolds consisting of chitosan (CH), chondroitin sulfate (CS), and apatite layer on their surfaces designed to slowly release BMP-2. This finding supports the novel therapeutic potential of this complementary strategy in bone regeneration.     

脂肪干细胞的诱导分化及其来源的研究

目的:通过组织块培养法得到脂肪干细胞(adipose-derived stem cells,ADSCs),探讨其诱导分化潜能,并初步研究ADSCs的来源。方法:用脂肪组织块培养法培养原代人ADSCs。第三代ADSCs进行成脂和成骨诱导分化,分别用油红O和茜素红S染色进行鉴定。脂肪组织块培养七天后取脂肪组织进行Hematoxylin-eosin Staining(HE)染色观察ADSCs组织分布。结果:用脂肪组织块培养法成功培养出原代人ADSCs。ADSCs传代到第8代,依然保持着良好的增殖能力和细胞形态。ADSCs能成功诱导成脂肪细胞和骨细胞。通过对培养七天后的脂肪组织块进行HE染色,发现ADSCs主要分布在脂肪组织的间质血管和结缔组织周围。结论:用脂肪组织块培养出来的ADSCs具有成脂和成骨分化的潜能。ADSCs主要定位于间质血管和结缔组织周围。     

MIP-3α 联合成骨诱导因子诱导大鼠脂肪干细胞向成牙本质样细胞分化*

目的:观察巨噬细胞炎性蛋白-3α(MIP-3α)对大鼠脂肪干细胞(Adipose derived stem cells,ASCs)向成牙本质样细胞体外分化作用的影响。方法:分离、培养并鉴定大鼠ASCs;以MIP-3α联合成骨诱导因子(地塞米松,β-甘油磷酸钠,以及抗坏血酸)诱导第3代大鼠ASCs向成牙本质样细胞定向分化。诱导培养1、4、7d后,分别测定碱性磷酸酶(alkaline phosphatese,ALP)活性,并用RT-PCR及Western Blot检测成牙本质细胞的标志基因dspp及标志物牙本质涎蛋白(DSP)。结果:与单独加入成骨诱导因子相比,MIP-3α与成骨诱导因子联合应用能使ALP活性、dspp的mRNA表达以及DSP升高。结论:本研究显示MIP-3α与成骨诱导因子联合应用可以增强成牙本质细胞相关基因以及蛋白的表达,为牙齿再生种子细胞的寻找开辟了一条新思路。     

脂肪来源间充质干细胞体外定向诱导分化血管细胞的研究进展

脂肪来源的间充质干细胞具有较强的体外增殖能力,因具有生物学特性稳定、来源充足、体外培养条件低等优势已引起各国学者的关注。脂肪间充质干细胞凭借其多向分化潜能,是人体干细胞库潜在的重要来源之一。目前,研究人员已成功地在体外将其诱导为内皮细胞,成骨细胞、成软骨细胞、脂肪前体细胞、平滑肌细胞,心肌细胞、神经样细胞等。就脂肪来源的间充质干细胞体外定向诱导分化血管细胞的研究进展作一综述。     

Extracts of Adipose Derived Stem Cells Slows Progression in the R6/2 Model of Huntington's Disease

Stem cell therapy is a promising treatment for incurable disorders including Huntington''s disease (HD). Adipose-derived stem cell (ASC) is an easily available source of stem cells. Since ASCs can be differentiated into nervous stem cells, it has clinically feasible potential for neurodegenerative disease. In addition, ASCs secrete various anti-apoptotic growth factors, which improve the symptoms of disease from transplanted ASCs. Thus, cell-free extracts of ASCs (ASCs-E) could be a potential candidate for treatment of HD. Here, we investigated effects of ASCs-E on R6/2 HD mouse model and neuronal cells. In R6/2 HD model, injection of ASCs-E improved the performance in Rotarod test. ASCs-E also ameliorated striatal atrophy and mutant huntingtin aggregation in the striatum. In Western blot increased expressions of p-Akt, p-CREB and PGC1α were noted by injection of ASCs-E, when comparing to the R6/2 HD model. Neuro2A neuroblastoma cells treated with ASCs-E showed increased expression of p-CREB and PGC1α. In conclusion, ASCs-E delayed disease progression in animal model of HD by restoring of CREB-PGC1α pathway and could be a potential resource for treatment of HD.     

脂肪干细胞在再生医学中的应用

再生医学是一门研究如何促进创伤与组织再生及功能重建的新兴学科,主要通过研究干细胞分化、机体等正常组织创伤修复与再生等机制来维持、修复、再生或改善损伤组织和器官功能。脂肪干细胞（adipose-derived stem cells,ASCs）是近年来从脂肪组织中分离得到的一种具有多向分化潜能的干细胞,是一种足量的、可用于实际的、有一定吸引力的自体细胞代替的供体资源,并能够广泛的用于组织修复、再生、发育的可塑性及细胞治疗等研究中。阐述了脂肪干细胞在旁分泌、软组织重建及损伤修复、骨骼肌重建、心血管重建、神经系统重建及癌症转移与入侵方面的作用模式,概括总结了目前利用脂肪干细胞参与的临床治疗方法,以期对脂肪干细胞在再生医学中应用研究提供参考。     

Human Adipose Tissue Derived Mesenchymal Stem Cells Aggravate Chronic Cyclosporin Nephrotoxicity by the Induction of Oxidative Stress

The aim of this study was to investigate whether hATMSCs protect against cyclosporine (CsA)-induced renal injury. CsA (7.5 mg/kg) and hATMSCs (3×10⁶/5 mL) were administered alone and together to rats for 4 weeks. The effect of hATMSCs on CsA-induced renal injury was evaluated by assessing renal function, interstitial fibrosis, infiltration of inflammatory cells, and apoptotic cell death. Four weeks of CsA-treatment produced typical chronic CsA-nephropathy. Combined treatment with CsA and hATMSCs did not prevent these effects and showed a trend toward further renal deterioration. To evaluate why hATMSCs aggravated CsA-induced renal injury, we measured oxidative stress, a major mechanism of CsA-induced renal injury. Both urine and serum 8-hydroxydeoxyguanosine(8-OHdG) levels were higher in the CsA+hATMSCs group than in the CsA group (P<0.05). An in vitro study showed similar results. Although the rate of apoptosis did not differ significantly between HK-2 cells cultured in hATMSCs-conditioned medium and those cultured in DMEM, addition of CsA resulted in greater apoptosis in HK-2 cells cultured in hATMSCs-conditioned medium. Addition of CsA increased oxidative stress in the hATMSCs-conditioned medium. The results of our study suggest that treatment with hATMSCs may aggravate CsA-induced renal injury because hATMSCs cause oxidative stress in the presence of CsA.