马脂肪间充质干细胞的分离培养与生物学特性试验

为马的关节炎、肌腱和韧带损伤的临床治疗提供种子细胞,本试验通过采集马颈部脂肪组织,采用Ⅰ型胶原酶消化法分离脂肪间充质干细胞,并进行传代培养;绘制细胞生长曲线、测定细胞群体倍增时间;通过流式细胞术检测P3代细胞表面标记物,RT-PCR法扩增细胞表面标记物目的基因片段;油红O和茜素红染色法测定脂肪间充质干细胞的成脂和成骨诱导分化能力。结果发现:马脂肪间充质干细胞体外培养条件下呈长梭形和典型的旋涡状,生长状态良好、折光性强;经测定细胞生长曲线呈典型的"S"型,符合Logistic生长曲线规律;P3、P6和P9代细胞群体倍增时间分别为21.5 h、26 h、36 h;流式细胞术检测结果显示,P3代细胞高表达间充质干细胞表面标志物CD44、CD90和CD105,不表达造血系细胞表面标志物CD45;PCR扩增得到CD44、CD90、CD105和CD73特异性目的片段;油红O和茜素红染色证明,马脂肪间充质干细胞具有成脂和成骨诱导分化能力。     

绵羊骨髓间充质干细胞分离培养与鉴定

旨在分离绵羊骨髓间充质干细胞(sheep bone marrow mesenchymal stem cells, SBMSC),建立绵羊骨髓间充质干细胞系,为相关研究提供种子细胞。取新生健康绵羊肋骨为试验材料,利用贴壁法分离获得SBMSC;取生长良好的P3代细胞通过免疫荧光,进行CD73、CD90、CD105、CD106表型鉴定;取生长良好的P3代细胞分别进行成软骨、成脂、成骨方向的诱导,对诱导结果分别进行阿利新蓝、油红O和茜素红染色,鉴定分化结果。SBMSC呈成纤维样贴壁生长;体外培养P3代细胞表面标记物CD73、CD90、CD105和CD106完全符合标准;P3代细胞经软骨、脂肪、骨方向诱导3周后,经阿利新蓝染色,细胞呈亮蓝色;经油红O染色,细胞质内出现橙红色脂滴;茜素红染色表明聚集的细胞团中央能形成钙化结节。说明SBMSC经体外诱导培养后可向软骨细胞、脂肪细胞和成骨细胞分化,并具有明显的成软骨、成脂和成骨表型,表明SBMSC具有多向分化潜能。通过贴壁培养法建立了SBMSC完整的体外培养体系,为绵羊育种等方面提供种子细胞。     

骨碎补总黄酮对缺氧环境中犬骨髓间充质干细胞成骨分化潜能的影响

拟研究骨碎补总黄酮对缺氧环境中犬骨髓间充质干细胞(BMSCs)成骨分化潜能的影响.运用骨碎补总黄酮(TFDR)干预低氧浓度(10％)环境中犬BMSCs 4周后诱导成骨分化,倒置显微镜观察茜素红染色BMSCs钙结节形成,比色法检测碱性磷酸酶(ALP)活性水平,流式细胞术检测细胞线粒体膜电位,激光共聚焦显微镜和RT-PCR...     

犬脂肪间充质干细胞的分离培养及分化潜能研究

为获得犬脂肪间充质干细胞,本试验取犬腹股沟皮下脂肪组织,分别利用组织培养法和酶消化法分离犬脂肪来源间充质干细胞,对比观察不同来源细胞的形态和增殖特征,并通过诱导液促进细胞向成骨细胞和成脂细胞方向分化,检测其分化潜能。结果表明,通过组织培养法培养的青年犬脂肪组织,可获得大量脂肪间充质干细胞,该细胞生长旺盛,形态均一,可分化为碱性磷酸酶染色阳性的成骨细胞和油红O染色阳性成脂细胞。组织培养法分离培养犬脂肪间充质干细胞操作简单,可为细胞移植治疗等研究提供充足的细胞来源。     

崂山奶山羊脂肪间充质干细胞系的建立及鉴定

旨在建立崂山奶山羊脂肪间充质干细胞系并对其进行初步鉴定.采集崂山奶山羊脂肪组织,在Ⅰ型胶原酶的分解消化作用下,将脂肪组织中的单核细胞进行分离并扩增培养;测定其生长曲线,利用Real time RT-PCR技术对其表达的干细胞因子进行鉴定;将传至第3代的脂肪间充质干细胞进行成骨诱导分化,利用茜素红染色进行鉴定.结果显示,崂山奶山羊脂肪间充质干细胞形态为长梭形、星形等成纤维细胞样细胞形态,但比成纤维细胞饱满,第3代细胞生长至3 d左右时细胞进入指数生长期,生长至6~7 d时进入平台期;经成骨诱导分化后,经茜素红染色可见骨结节被染成深红色.综上提示,崂山奶山羊脂肪间充质干细胞具有诱导分化潜能.     

蒙古羊骨髓间充质干细胞的分离培养及多向诱导分化

本研究旨在建立蒙古羊骨髓间充质干细胞(bone marrow mesenchymal stem cell,BMSC)体外分离、纯化、增殖方法,诱导其向脂肪细胞、成骨细胞及软骨细胞分化。穿刺法抽取蒙古羊骨髓组织,采用密度梯度离心法结合贴壁培养法分离纯化BMSC,增殖,并测定其生长曲线及细胞倍增时间。对第3代蒙古羊BMSC进行成脂、成骨及成软骨诱导,观察诱导后的细胞形态。采用油红O、茜素红、HE等染色法在组织学水平对BMSC进行成脂、成骨和成软骨分化鉴定。结果显示,蒙古羊BMSC呈现均一梭形成纤维细胞样生长,生长曲线呈S型,生长增殖能力良好。在不同分化诱导之后,细胞呈现脂肪细胞、成骨细胞及软骨细胞的表型特征。表明获得的蒙古羊BMSC具有多向分化潜能,所分离细胞确为骨髓间充质干细胞。     

滩羊骨髓间充质干细胞分离与培养

利用全骨髓法与差速贴壁法分离和培养滩羊骨髓间充质干细胞（BMSCs），观察细胞形态及成脂诱导分化实验进行鉴定。结果显示原代培养的细胞呈圆形或三角形，24h后大部分细胞均贴壁，7d左右可达80％-90％融合，体外成脂诱导分化获得脂肪样细胞，油红染色为阳性。因此，全骨髓法与差速贴壁法可有效分离培养滩羊的骨髓间充质干细胞，是一种比较理想的分离培养方法。     

野猪骨髓间充质干细胞的分离培养与生物学特性

采用全骨髓培养法对野猪股骨中骨髓间充质干细胞（Bone mesenchymal stem cells,BMSC）进行分离、培养并传代,建立野猪骨髓间充质干细胞体外培养方法及对其生物学特性进行观察研究。结果显示,细胞形态呈梭形,漩涡状生长,生长曲线为典型S型,在诱导液作用下,可分化为成脂肪样细胞。结果表明,通过本方法能够分离到较纯的BMSC,为野猪资源保存和体细胞核移植提供技术支持。     

崂山奶山羊永生化骨髓间充质干细胞的诱导分化研究

为研究崂山奶山羊永生化骨髓间充质干细胞(BMSCs)的诱导分化潜能,采用P3代BMSCs和P30代TERT-BMSCs进行体外成脂和成神经诱导分化。结果显示,当细胞传至P30代时,TERT-BMSCs生长旺盛;在成脂诱导后,P3代BMSCs和P30代TERT-BMSCs均能观察到细胞中透明脂滴的形成,油红O染料染色后可见脂滴被染成红色;在成神经诱导后,P3代BMSCs和P30代TERT-BMSCs均能形成树突样或三角形的形态,甲苯胺蓝染色后可观察到细胞中尼氏体的存在。综上提示,永生化的崂山奶山羊BMSCs在多次传代后仍具有较强的多向诱导分化能力,这为永生化MSCs的广泛应用提供了理论依据。     

南阳牛骨髓间充质干细胞的分离培养及多向分化

本研究旨在建立南阳牛骨髓间充质干细胞(bone marrow-derived mesenchymal stem cells,BMSCs)体外分离培养方法,在此基础上研究其生物学特性和多向分化的能力。采用骨髓穿刺法取3月龄小牛的肋骨骨髓,分离培养BMSCs,传代培养并测定其生长曲线,RT-PCR检测Oct4、Nanog、Sox2基因的表达,然后取P3 BMSCs分别向神经和脂肪细胞进行诱导分化,并利用组织学染色技术和RT-PCR技术进行鉴定。结果表明,分离得到的BMSCs大小均匀,多呈梭形的成纤维细胞样生长;RT-PCR可检测到干细胞因子Oct4、Nanog、Sox2的表达;不同代次细胞生长曲线呈S型,一般在第3天时进入指数生长期,第7天后进入平台期;成神经诱导后,甲苯胺蓝染色可见明显的尼氏体结构,RT-PCR检测ENO2和GFAP基因表达呈阳性;成脂肪诱导后,油红O染色后可见大量的脂滴存在,RT-PCR检测Leptin和PPAR基因表达呈阳性。试验证明,成功分离得到了南阳牛BMSCs,且其具有多向诱导分化潜能。     

镉对体外培养鸡骨髓基质细胞成骨分化的毒性作用

骨是镉毒性作用的主要靶器官之一,但其对鸡骨髓基质细胞(bone marrow stromal cells,BMSCs)增殖和成骨分化的毒性作用仍不清楚.本研究利用差速贴壁纯化法获得鸡BMSCs,加入不同浓度镉处理不同时间,采用CCK-8法检测细胞增殖,碱性磷酸酶(alkaline phosphatase,ALP)和茜素...     

端粒逆转录酶基因修饰对绵羊骨髓间充质干细胞端粒酶活性影响的研究

骨髓间充质干细胞(BMSC)具有多向分化的特性,但其生命周期有限,为进一步探讨端粒逆转录酶(TERT)对端粒酶活性的影响,本试验利用将外源性TERT导入间充质干细胞技术,构建重组表达载体pcDNA3.1-EGFP-TERT,将其转染BMSC,通过筛选及鉴定,结果显示成功构建了重组表达载体pcDNA3.1-EGFP-TERT.将TERT-BMSCs在体外进行传代培养及生长曲线的测定,利用PCR技术对其原有干细胞因子的表达情况进行了鉴定.结果显示,TERT-BMSC具有快速扩增的能力,细胞形态良好,且仍具有干细胞特性.本试验结果揭示了外源性TERT基因的表达能激活绵羊BMSC的端粒酶活性.     

Impact of the source and serial passaging of goat mesenchymal stem cells on osteogenic differentiation potential:implications for bone tissue engineering

Background: Adult mesenchymal stem cells(MSCs) can be conveniently sampled from bone marrow, peripheral blood, muscle, adipose and connective tissue, harvested from various species, including, rodents, dogs, cats, horses,sheep, goats and human beings. The MSCs isolated from adult tissues vary in their morphological and functional properties. These variations are further complicated when cells are expanded by passaging in culture. These differences and changes in MSCs must be considered prior to their application in the clinic or in a basic research study. Goats are commonly used as animal models for bone tissue engineering to test the potential of stem cells for bone regeneration. As a result, goat MSCs isolated from bone marrow or adipose tissue should be evaluated using in vitro assays, prior to their application in a tissue engineering project.Results: In this study, we compared the stem cell properties of MSCs isolated from goat bone marrow and adipose tissue. We used quantitative and qualitative assays with a focus on osteogenesis, including, colony forming unit, rate of cell proliferation, tri-lineage differentiation and expression profiling of key signal transduction proteins to compare MSCs from low and high passages. Primary cultures generated from each source displayed the stem cell characteristics,with variations in their osteogenic potentials. Most importantly, low passaged bone marrow MSCs displayed a significantly higher and superior osteogenic potential, and hence, will be the preferred choice for bone tissue engineering in future in vivo experiments. In the bone marrow MSCs, this process is potentially mediated by the p38 MAPK pathway. On the other hand, osteogenic differentiation in the adipose tissue MSCs may involve the p44/42 MAPK pathway.Conclusions: Based on these data, we can conclude that bone marrow and fat-derived MSCs undergo osteogenesis via two distinct signaling pathways. Even though the bone marrow MSCs are the preferred source for bone tissue engineering, the adipose tissue MSCs are an attractive alternative source and undergo osteo-differentiation differently from the bone marrow MSCs and hence, might require a cell-based enhancer/inducer to improve their osteogenic regenerative capacity.     

Isolation and Differentiation of Mesenchymal Stem Cells From Bovine Umbilical Cord Blood

Currently, mesenchymal stem cells (MSCs) are used in veterinary clinical applications. Bone marrow and adipose tissue are the most common sources of stem cells derived from adult animals. However, cord blood which is collected non‐invasively is an alternative source of stem cells other than bone marrow and adipose tissue. Moreover, high availability and lower immunogenicity of umbilical cord blood (UCB) haematopoietic stem cells compared to other sources of stem cell therapy such as bone marrow have made them a considerable source for cell therapy, but MSCs is not highly available in cord blood and their immunogenicity is poorly understood. In this study, the cells with spindle morphology from 7 of 9 bovine UCB samples were isolated and cultured. These mesenchymal stromal cells were successfully differentiated to osteocytes, chondrocytes and adipocytes. In addition, Oct‐4 and SH3 were determined by RT‐PCR assay. It is the first report of isolation, culture, characterization and differentiation of bovine umbilical stem cells.     

CRISPR系统促进脂肪干细胞成骨分化与抑制成脂分化的研究

本研究旨在优化组织培养法分离小鼠脂肪间充质干细胞（adipose-derived stem cells,ASCs）,为研究成骨分化和成脂分化在间充质干细胞分化过程中的相互影响奠定基础。通过细胞形态学观察、细胞生长曲线和流式仪器检测所分离获得的间充质干细胞的特性,利用CRISPR-dCas9系统在快速促进间充质干细胞成骨分化的前提下观察其对成脂分化的影响,并通过生化染色、实时荧光定量PCR和免疫细胞学等手段进行分析。结果显示,接种3~5 d后可见细胞从组织块周围爬出,光镜下可见细胞形态多为成纤维细胞样的梭形细胞,且形态单一均匀,具有较高的爬出率,可以大大提高脂肪间充质干细胞的分离效率;通过CRISPR-dCas9系统激活Runx2和Osterix基因后可以促进间充质干细胞的成骨分化,实时荧光定量PCR及油红O染色结果显示,CRISPR-dCas9系统可以同时抑制间充质干细胞的成脂分化;通过CRISPR-dCas9-KRAB系统同时抑制成骨相关基因Runx2和Osterix后可以促进成脂分化。本研究利用组织贴壁法成功获得了高纯度的脂肪间充质干细胞,具有间充质干细胞的特性和分化能力;利用CRISPR系统可以同时过表达Runx2和Osterix两个基因,可以在进成骨分化的同时抑制成脂分化,表明成脂分化和成骨分化的相关性,为基因编辑在间充质干细胞诱导分化和临床应用方面提供了新的思路和方法。     

Characterization of Nucleated Cells From Equine Adipose Tissue and Bone Marrow Aspirate Processed for Point-of-Care Use

The objective of this study was to compare nucleated cell fractions and mesenchymal stromal cells (MSCs) from adipose tissue to bone marrow processed by a point-of-care device that are available for immediate implantation. A paired comparison using adipose and bone marrow from five horses was done. The number of nucleated cells, viability, total adherent cells on day 6 of culture and colony-forming unit fibroblasts (CFU-Fs) were determined. Gene expression for markers of stemness, adipogenic, chondrogenic, osteogenic lineage, and collagen formation was measured in total RNA isolated from adherent adipose and bone marrow cells. Day 6 adherent adipose-derived MSC was frozen briefly, whereas day 6 adherent bone marrow–derived MSC was passaged two additional times to obtain adequate cell numbers for chondrogenic, osteogenic, and adipogenic cell differentiation assays. The total cell count per gram was significantly greater for bone marrow, whereas total adherent cells per gram and the CFU-F per million nucleated cells on day 6 were significantly greater for the adipose. In undifferentiated adherent cells, relative gene expression for CD34, adipogenic, and chondrogenic markers and collagen II was significantly lower in the adipose-derived cells. Conversely, expression of collagen I was significantly higher in the undifferentiated adipose-derived cells. Cell density and total RNA were higher in differentiated adipogenic and osteogenic cultures of adipose cells and in chondrogenic cultures of bone marrow cells. This cell preparation method provides a stromal vascular fraction with a large proportion of multipotent MSCs. There are differences in the cells obtained from the two sources. This method can provide an adequate number of multipotent cells from adipose tissue for immediate implantation.