猪脂肪间充质干细胞的分离培养及其成脂分化

脂肪间充质干细胞(Adipose mesenchymal stemcell,AMSCs)是一类来源于脂肪组织并具有多向分化潜能的干细胞。近年来的研究证明,脂肪组织具有取材方便和干细胞含量高的优势,有望在研究与应用领域成为骨髓干细胞的替代物。猪是一种比啮齿类更接近人类的模式动物,具有较强的脂肪沉积能力。本研究探讨了猪脂肪间充质干细胞的体外分离纯化、培养扩增和向脂肪细胞诱导分化的条件。采用Ⅰ型胶原酶消化分离脂肪微管基质成分,传代培养扩增,流式细胞仪检测细胞表面标记。取第3-7代AMSCs,采用不同方法诱导AMSCs向脂肪细胞分化,光学显微镜下可观察到诱导后的细胞内有高折光性的小脂滴出现,油红O染色成阳性,不同诱导方法诱导率不同。被诱导细胞用RT-PCR可检测到脂肪细胞分化标志基因LPL和PPARγ的表达。结果表明可以从脂肪组织中分离培养出AMSCs,经传代后可提高其纯度。CD44、CD105表达呈阳性,CD14、CD34、S-100、HLA-DR呈阴性,在合适的诱导条件下,可向脂肪细胞分化。     

应用原子力显微镜分析猪脂肪前体细胞的分化

脂肪形成过程中发生的异常变化与许多疾病的产生有着密切的关系。为深入了解脂肪形成的机制,利用原子力显微镜研究脂肪前体细胞向成熟脂肪细胞分化前后细胞形貌、超微结构和机械性能的变化。结果表明,脂肪前体细胞与成熟脂肪细胞在形貌上存在明显的差异。在超微结构的探测中成熟脂肪细胞表面粗糙度低于脂肪前体细胞。通过力曲线的分析得出,分化前后两种细胞的机械性质均发生改变。脂肪前体细胞在粘弹力、硬度和杨氏模量的研究中比成熟脂肪细胞都高出约20％。原子力显微镜在纳米尺度上分析脂肪前体细胞向成熟脂肪细胞分化过程中细胞膜的改变,其研究结果为进一步探讨脂肪形成机制提供可视化定量数据。     

干细胞的研究进展及临床应用前景

干细胞是一类具有自我复制和分化潜能的影响,它包括胚胎干细胞和成体干细胞。干细胞的发育受多种内在机制和微环境因素的影响。目前人类胚胎干细胞已可在体外培养。最新研究发现,成体干细胞可以横向分化为其他类型的细胞,为干细胞的广泛应用提供了基础。     

全反式维甲酸抑制猪脂肪间充质干细胞的成脂分化

分离培养猪脂肪间充质干细胞（adipose mesenchymal stem cells, AMSCs）,流式细胞仪鉴定其表面标记.利用MTT比色检测不同浓度的全反式维甲酸（all trans retinoic acid, ATRA）对猪AMSCs增殖的影响;光学显微镜下观察猪AMSCs向脂肪细胞分化的形态学变化;油红O染色提取法分析不同浓度ATRA对猪AMSCs成脂分化的影响;RT PCR检测脂肪细胞分化标志基因LPL和aP2 mRNA的变化.MTT比色结果显示,生理浓度（1×10^－9～1×10^－8 mol/L）和药理浓度（1×10^－7～1×10^－5 mol/L）ATRA对猪AMSCs增殖均没有影响.油红O染色提取法结果表明,除1×10^－7　mol/L ATRA对猪AMSCs成脂分化没有影响外,生理浓度（1×10^－9～1×10^－8 mol/L）和其它药理浓度（1×10^－6～1×10^－5 mol/L）ATRA均显著抑制猪AMSCs成脂分化（P<0.05）.RT-PCR检测显示,ATRA显著抑制脂肪细胞分化标志基因LPL和aP2 mRNA表达（P<0.05）.     

间充质干细胞特性与应用前景

间充质干细胞是中胚层发育的早期细胞,具备干细胞的基本特性。在发育的不同阶段和特定环境条件下,间充质干细胞可向骨、软骨、肌肉、神经、血管及血液细胞等多种方向分化。在成体的很多器官和组织中也存在着间充质干细胞,以备修复和再生所用。间充质干细胞易于体外培养,扩增迅速,可以分化为多种细胞,为干细胞生物工程提供了一个很好的种子细胞。在明确间充质干细胞生物学特性和分化的机制后,可在体外和体内将其定向诱导分化为多种细胞。间充质干细胞具有巨大的临床应用价值和科学研究价值。     

成体干细胞的生物学特点及应用前景

成体干细胞存在于人和哺乳动物组织中,具有自我更新和一定的分化潜能,现已从骨髓,软骨,血液,神经,肌肉,脂肪,皮肤,角膜缘,肝脏,胰腺等许多组织中获得成体干细胞,发现部分组织成体干细胞具有多向分化潜能,成体干细胞的研究在再生医学中有十分广阔的应用前景。     

脊髓神经干细胞的研究进展

近十多年来,人们对神经干细胞的研究进展迅速。本文从生长因子EGF和bFGF的反应性、分化特点、临床应用前景三个方面来综述脊髓神经干细胞的研究进展。为深入开展脊髓神经干细胞的基础研究及临床应用研究提供科学依据。     

人胚胎干细胞培养建系及其应用

简要概述了自1998年首次建立hES细胞系以来近6－7年国内外的现况、分离培养建系、鉴定标准和冻存技术发展、定向诱导分化及其应用等方面的研究进展。     

胚胎干细胞的研究进展

胚胎干细胞是来源于早期胚胎内细胞团的、具有多向分化潜能的细胞,已经广泛用于生命科学的许多领域,它在医学方面的应用成为医学领域的研究热点.本文对胚胎干细胞在诱导分化、基础研究和临床应用上的研究进展进行了综述,对今后的研究方向进行了展望.     

干细胞研究及其在临床医学上的应用

干细胞是一类具有自我更新和分化潜能的细胞,可以分为胚胎干细胞和成体干细胞两类。近来干细胞研究的二项重大技术突破：胚胎干细胞的体外培养和成体干细胞的横向分化,为干细胞在临床医学上的应用开辟了广阔的前景。     

Efficient generation of functional Schwann cells from adipose-derived stem cells in defined conditions

Schwann cells (SCs) are hitherto regarded as the most promising candidates for viable cell-based therapy to peripheral nervous system (PNS) injuries or degenerative diseases. However, the extreme drawbacks of transplanting autologous SCs for clinical applications still represent a significant bottleneck in neural regenerative medicine, mainly owing to the need of sacrificing a functional nerve to generate autologous SCs and the nature of slow expansion of the SCs. Thus, it is of great importance to establish an alternative cell system for the generation of sufficient SCs. Here, we demonstrated that adipose-derived stem cells (ADSCs) of rat robustly give rise to morphological, phenotypic and functional SCs using an optimized protocol. After undergoing a 3-week in vitro differentiation, almost all of treated ADSCs exhibited spindle shaped morphology similar to genuine SCs and expressed SC markers GFAP and S100. Most importantly, apart from acquisition of SC antigenic and biochemical features, the ADSC-derived SCs were functionally identical to native SCs as they possess a potential ability to form myelin, and secret nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and glia-derived neurotrophic factor (GDNF). The current study may provide an ideal strategy for harvesting sufficient SCs for cell-based treatment of various peripheral nerve injuries or disorders.     

牛脂肪间充质干细胞的分离、培养与鉴定

为了给组织工程提供种子细胞,对牛间充质干细胞(Adipose-derived stem cells,ADSCs)进行体外分离培养。首先应用胶原酶消化法分离牛ADSCs,进行体外培养、连续传代,并观察细胞的形态变化,通过细胞计数绘制生长曲线,细胞压片进行染色体分析,采用细胞免疫荧光化学方法检测细胞表面标记,利用成骨分化和成脂分化检测其分化能力。结果显示牛ADSCs体外培养时细胞形态呈成纤维细胞样,增殖稳定;Vimentin、CD49d、CD13表达呈阳性,CD34表达呈阴性;成骨诱导条件下的细胞碱性磷酸酶活性高,茜素红染色呈阳性;成脂诱导条件下细胞周围脂滴明显,油红-O染色呈阳性。结果证明牛ADSCs体外生长稳定、增殖速度快、定向分化能力强,简易的体外分离培养及诱导方法为其在组织工程中的应用奠定了基础。     

The use of adipose-derived stem cells as sheets for wound healing

Cellular therapies have shown immense promise in the treatment of nonhealing wounds. Cell sheets are an emerging strategy in tissue engineering, and these cell sheets are promising as a delivery method of mesenchymal stem cells to the wound bed. Cell sheet technology utilizes temperature dependent polymers to allow for lifting of cultured cells and extracellular matrix without the use of digestive enzymes. While mesenchymal stem cells (MSCs) have shown success in cell sheets for myocardial repair, examination of cell sheets in the field of wound healing has been limited. We previously developed a novel cell sheet composed of human adipose-derived stem cells (ASCs). Both single and triple layer cell sheets were examined in a full-thickness murine wound model. The treatment cell sheets were compared with untreated controls and analyzed at timepoints of 7, 14, 18 and 21 d. The ASC cell sheets showed increased healing at 7, 14 and 18 d, and this effect was increased in the triple layer cell sheet group. Future development of these cell sheets will focus on increasing angiogenesis in the wound bed, utilizing multiple cell types, and examining allogeneic cell sheets. Here we review our experiment, expand upon our future directions and discuss the potential of an off-the-shelf cell sheet. In the field of wound healing, such a cell sheet is both clinically and scientifically exciting.     

Human adipose-derived mesenchymal stem cells: serial passaging,doubling time and cell senescence

Adult adipose-derived mesenchymal stem cells (AD-MSC) are very interesting to our research group because they are easy to harvest, they are abundant in humans, and they have potential clinical applications in autologous cell therapy for disc degeneration. We examined these cells through sequential serial passages to assess osteogenic and chondrogenic capabilities, mean doubling time and cell senescence. Osteogenic and chondrogenic potencies were maintained through 13 passages. Mean passage doubling time increased significantly with increasing passage number. When donor age was evaluated, passages 1-4 from older donors had significantly longer doubling times compared to cells from younger donors. Passages 5-11 showed similar findings when analyzed by donor age. The mean percent senescence increased significantly with cell passaging, rising from 0% at passage 1 to 3.4% at passage 13. These novel data suggest that caution should be exercised when using AD-MSC with long passage times.     

支架材料对棕色脂肪源性干细胞向起搏细胞诱导分化的影响

目的观察不同三维支架材料对棕色脂肪来源干细胞（BADSCs）诱导分化成起搏细胞的效果,为构建生物起搏器提供实验依据。 方法将培养7 d的原代BADSCs分别种植到胶原海绵、明胶海绵和透明质酸水凝胶3种不同的材料中,在不同时间用光镜和扫描电镜观察细胞-支架复合体中细胞形态学的变化,免疫荧光染色检测心肌细胞、起搏细胞相关蛋白的表达。采用单因素方差分析。 结果细胞在3种支架上均能存活、增殖,LIVE/DEAD检测显示,培养3 d的胶原海绵、明胶海绵和透明质酸水凝胶3种细胞-支架复合物死细胞率分别为（46.35±1.50）%、（47.00±1.60）%和（1.76±1.08）%,其中细胞在透明质酸水凝胶中死亡率最低,并且细胞-透明质酸水凝胶复合物可自发性地搏动,三组比较差异具有统计学意义（F = 37.56,P < 0.05）。培养至2周时,胶原海绵、明胶海绵和透明质酸水凝胶中Connexin45细胞阳性率分别为（10.67±1.25）%、（13.67±1.25）%和（21.00±1.60）%,差异有统计学意义（F = 9.435,P < 0.01）,HCN2细胞阳性率分别为（11.00±1.60）%、（14.00±2.16）%和（34.33±3.68）%,差异有统计学意义（F = 17.52,P < 0.01）,HCN4细胞阳性率分别为（18.67±2.05）%、（13.00±1.60）%和（66.00±2.94）%,差异有统计学意义（F = 27.96,P < 0.01）,Sr细胞阳性率分别为（13.00±1.63）%、（14.33±1.24）%和（75.33±3.30）%,差异有统计学意义（F = 36.40,P < 0.01）,水凝胶中Connexin45、HCN2、HCN4和Sr的细胞阳性率均高于胶原海绵和明胶海绵,差异均具有统计学意义（P < 0.05）。 结论BADSCs在胶原海绵、明胶海绵和透明质酸水凝胶中均能很好地生长和分化,但透明质酸水凝胶更适用于组织工程化起搏器的构建。     

Syngeneic adipose-derived stem cells with short-term immunosuppression induce vascularized composite allotransplantation tolerance in rats

Background aimsA clinically applicable tolerance induction regimen that removes the requirement for lifelong immunosuppression would benefit recipients of vascularized composite allotransplantation (VCA). We characterized the immunomodulatory properties of syngeneic (derived from the recipient strain) adipocyte-derived stem cells (ADSCs) and investigated their potential to induce VCA tolerance in rats.MethodsADSCs were isolated from Lewis (LEW, RT1A^l) rats; their immunomodulatory properties were evaluated by means of mixed lymphocyte reactions in vitro and VCAs in vivo across a full major histocompatibility complex mismatch with the use of Brown-Norway (BN, RT1Aⁿ) donor rats. Two control and four experimental groups were designed to evaluate treatment effects of ADSCs and transient immunosuppressants (anti-lymphocyte globulin, cyclosporine) with or without low-dose (200 cGy) total body irradiation. Flow cytometry was performed to quantify levels of circulating CD4⁺CD25⁺FoxP3⁺ regulatory T cells (Tregs).ResultsCultured syngeneic ADSCs exhibited CD90.1⁺CD29⁺CD73⁺CD45⁻CD79a⁻CD11b/c⁻ phenotype and the plasticity to differentiate to adipocytes and osteocytes. ADSCs dramatically suppressed proliferation of LEW splenocytes against BN antigen and mitogen, respectively, in a dose-dependent fashion, culminating in abrogation of allo- and mitogen-stimulated proliferation at the highest concentration tested. Accordingly, one infusion of syngeneic ADSCs markedly prolonged VCA survival in LEW recipients treated with transient immunosuppression; of these, 66% developed tolerance. Total body irradiation provided no additional VCA survival benefit. An important role for Tregs in tolerance induction/maintenance was suggested in vivo and in vitro.ConclusionsTreatment comprising syngeneic ADSCs and transient immunosuppression (i) increased levels of circulating Tregs and (ii) induced tolerance in 66% of recipients of major histocompatibility complex–mismatched VCAs.