大鼠骨髓间充质干细胞体外分离培养及成骨成脂诱导

目的建立大鼠骨髓间充质干细胞（BMSCs）体外分离、纯化、扩增的方法及诱导BMSCs成骨成脂分化,提供理想的组织工程种子细胞。方法采用贴壁筛选法分离BMSCs,通过不断传代进行纯化和扩增培养,并绘制细胞生长曲线。诱导后检测油红O染色和矿化结节。结果 BMSCs在体外分离培养扩增,细胞形态为长梭形,流式检测分析P3和P5细胞表面标记物CD44、CD90为阳性,CD45为阴性。细胞生长曲线呈S形。经成脂诱导培养,脂肪的特异性油红O染色为阳性;经成骨诱导培养,形成了矿化结节。结论本实验分离培养的细胞具有BMSCs的表面标记特征和诱导分化为脂肪细胞和成骨细胞的潜能,为BMSCs在组织工程中的应用提供基础理论和实验依据。     

大鼠骨髓间充质干细胞原代培养及成骨成脂分化能力染色鉴定

目的：实验旨在建立一套简便有效的骨髓间充质干细胞原代培养、诱导分化及染色方法,观测骨髓间充质干细胞生物学特性,及其成骨、成脂分化潜能,为后续进行骨髓间充质干细胞基因修饰实验做好准备。方法本实验通过全骨髓培养法分离骨髓间充质干细胞,并用诱导分化培养液做定向诱导培养,观察骨髓间充质干细胞向成骨及成脂肪细胞分化过程中的细胞形态学变化,进行成骨成脂细胞染色鉴定,以探讨骨髓间充质干细胞的分离培养方法、生长规律及向成骨成脂细胞分化的条件。结果(1)通过全贴壁法成功进行了骨髓间充质干细胞原代及传代培养并绘制出第4代细胞生长曲线;(2)通过茜素红染色验证了骨髓间充质干细胞的成骨分化能力;(3)通过油红O染色验证骨髓间充质干细胞的成脂分化能力。结论全贴壁法提取的大鼠骨髓间充质干细胞经传代4次左右可达到一定纯度,在一定诱导条件下,经特定染色方法鉴定,可分化为成骨细胞,成脂细胞。     

大鼠骨髓间充质干细胞体外培养和鉴定

目的探讨大鼠骨髓间充质干细胞（BMSCs）体外分离培养方法及其生物学特性。方法采用全骨髓贴壁培养法获得大鼠骨髓间充质干细胞,取第3代骨髓间充质干细胞,通过成骨、成脂肪诱导分化以及流式细胞仪分析其表面标记（CD29、CD34、CD45、CD90）等鉴定BMSCs特征。结果全骨髓贴壁培养法获得的BMSCs,原代和传代培养具有活跃的增殖能力;BMSCs经诱导后可分别向成骨、成脂转化;流式分析表面标志分子高表达CD90（96.5%）、CD29（92.3%）,低表达CD34（0.89%）、CD45（1.41%）。结论全骨髓贴壁培养法可有效地分离和扩增BMSCs,分离培养的BMSCs具有潜在的多向分化能力。     

人和大鼠骨髓间充质干细胞体外培养和生物学特性的对比研究

目的 对比体外分离培养的人和SD大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)的生长情况和生物学特性.方法 骨髓穿刺抽取健康成人的骨髓,SD大鼠处死后从股骨取骨髓,采用密度梯度法分离出BMSCs,MTT法测定两种细胞的生长曲线,用成骨、成脂培养基对细胞进行定向诱导分化.结果 两种原代培养的BMSCs生长到第3代后,细胞形态均一、呈梭形排列,生长曲线提示人的BMSCs第9、15代仍有较强的增殖能力,第20代后增殖速度明显减慢.大鼠的BMSCs第8、16代增殖能力也很强,第30代 仍有活力.2种干细胞都能成功诱导分化为骨细胞和脂肪细胞.结论 人和大鼠骨髓间充质干细胞均能在体外分离培养,并具有多向分化潜能,大鼠的骨髓间充质干细胞增殖能力更强于人的骨髓间充质干细胞.     

大鼠骨髓间充质干细胞向脂肪细胞诱导分化的研究

目的探讨大鼠骨髓间充质干细胞（BMSCs）的分离纯化和定向成脂诱导。方法采用密度梯度离心法分离纯化骨髓间充质干细胞,免疫组织细胞化学法检测BMSCs表面抗原。脂肪诱导剂诱导BMSCs分化为脂肪细胞,利用油红O染色进行检测。结果免疫组织细胞化学检测结果显示,BMSCs表达CD29、CD44、CD90、CD106,不表达CD34、CD45。BMSCs经脂肪诱导剂诱导后,油红O染色阳性。随着诱导时间的延长,油红O阳性细胞比例增加。结论体外培养的BMSCs在一定诱导条件下能够向脂肪细胞分化,并且随着诱导时间的延长,脂肪细胞比例不断增加。     

大鼠骨髓间充质干细胞分离培养及鉴定

目的 建立一套简单易行有效的骨髓间充质干细胞(BMSCs)的培养、纯化、鉴定的方案,初步了解BMSCs的基本性状和功能特点.方法 取4周龄雄性SD大鼠,全骨髓贴壁培养BMSCs,对细胞相关的性状进行观察及分析,流式鉴定表面抗原,诱导其成骨、成脂分化,并进行染色鉴定等.结果 流式检测结果CD29、CD90呈阳性反应,CD34、CD45呈阴性反应,成骨诱导分化后茜素红染色呈阳性反应,成脂诱导分化后油红染色呈阳性反应.结论 全骨髓培养法是一种相对简单易行的BMSCs分离纯化的方法,可获得纯度高,活性好,性状均一的BMSCs.     

大鼠骨髓间充质干细胞的体外分离培养与鉴定

目的建立大鼠骨髓间充质干细胞(bone marrow-derived mesenchymal stem cells,BMSCs)体外分离培养方法。方法采用全骨髓贴壁法分离培养BMSCs,进行形态学观察,绘制生长曲线,流式细胞仪分析细胞周期、检测细胞表面抗原标志物,并进行成脂、成骨分化诱导。结果获取的BMSCs形态呈均一成纤维细胞样,并呈集落样生长。生长曲线呈S形,细胞周期显示86.02%P3代细胞为G0/G1期,保持活跃的扩增能力。BMSCs高表达CD44、CD90、低表达CD34、CD45。成脂诱导21 d后,可见细胞的胞浆内出现大量红染脂滴。成骨诱导21 d后,可见大量橘红色矿化结节形成。结论全骨髓贴壁培养法可成功有效地分离培养BMSCs。     

诱导骨髓间充质干细胞向成骨细胞分化的实验研究

目的 明确大鼠骨髓间充质干细胞(bone mensenchymal stem cells,BMSCs)在体外全骨髓培养条件下的生物学特性及其向成骨细胞分化的能力,探索更为简便有效的BMSCs体外培养方法.方法 提取大鼠原代BMSCs,用酠EM完全培养基和成骨诱导条件培养基体外培养,倒置相差显微镜和HE染色观察细胞形态;台盼蓝活细胞计数绘制细胞生长曲线;ALP检测试剂盒检测ALP含量变化和von Kossa染色检测细胞矿化结节,以判断细胞是否向成骨细胞分化.结果 全骨髓培养法在体外培养的大鼠原代BMSCs贴壁生长,呈梭形或多角形;使用Dex-SaMEM向成骨诱导后的BMSCs具有与成骨细胞在体外培养时相似的特征,倍增时间延长;合成ALP能力显著增强(P<0.05),von Kossa染色出现阳性的棕褐色或棕黑色团块的钙化结节.结论 全骨髓培养法取材方便,经成骨诱导培养的BMSCs表现出成骨细胞的形态特征和生物学特性,该方法可作为骨组织工程种子细胞培养的常规方法.     

兔自体骨髓间充质干细胞移植修复关节软骨缺损的研究

目的:研究将体外培养的兔骨髓间充质干细胞(MSCs)定向诱导分化为成骨细胞的方法, 探讨其作为组织工程化骨的种子细胞的可行性.方法:采用Percoll分离液,从兔的骨髓组织中分离骨髓间充质干细胞,用低糖型的DMEM( Dulbecco's modified Eagle's medium)培养液中添加成骨诱导剂地塞米松、β-甘油磷酸钠及抗坏血酸进行培养.诱导2周后钙-钴法染色检测碱性磷酸酶(ALP)表达, 诱导4周后VonKos-sa染色检测钙结节形成.结果:第3代兔MSCs呈典型的成骨细胞形态,诱导2周ALP染色阳性率达80%,诱导4周后VonKossa染色可见钙结节形成.结论:经过分离纯化后的兔骨髓基质细胞在体外培养条件下可以分化为成骨细胞.     

不同氧浓度微环境对大鼠骨髓间充质干细胞成骨及成脂肪分化的影响

目的 观察不同氧浓度微环境对间充质干细胞分化的影响,对比分析间充质干细胞在体外常规培养与机体生理的微环境中分化情况的差异.方法 从大鼠骨髓中分离获取间充质干细胞;体外培养扩增后,取第3代骨髓间充质干细胞,以5×105/mL浓度接种到6孔培养板中,贴壁后分别加入成骨和成脂诱导分化因子;并分别给予高压氧(1.5kPa,60min)和低压氧(-1.5kPa,60min)处理, 1次/d×5d.诱导2周后进行钙结节茜素红染色和Von Kossa染色检测成骨分化情况;油红染色检测脂肪样细胞形成数量.结果 与常规分化组比较,高压氧组钙化结节数量明显增加约为常规组的(1.5±0.1)倍(P<0.01);脂肪样细胞数量也明显增加,约为常规组的(1.6±0.2)倍(P<0.01).低压氧组钙化结节数量明显减少约为常规组的(0.6±0.2)倍(P<0.01);脂肪样细胞数量明显减少,约为常规组的(0.5±0.1)倍(P<0.01).结论 高浓度氧微环境可以促进大鼠骨髓间充质干细胞成骨和成脂分化,低浓度氧则表现为抑制.     

肿瘤干细胞与肝癌干细胞相关研究进展

随着干细胞研究领域发展的日趋成熟,科学家们发现干细胞的这一特性和癌细胞之间有惊人的相似性.肿瘤可能起源于正常干细胞的转化,相似的信号通路可能既调节干细胞也调节癌细胞的自我更新,且癌细胞中可能含有"肿瘤干细胞".本文阐述了目前研究较少的肝癌干细胞的最新研究进展.     

整合素与表皮干细胞

皮肤是人体最大的器官 ,表皮位于皮肤最外层 ,是能够自我更新的组织 (约每个月更新 1次 ) [1 ] 。基底层的表皮干细胞(keratinocytestemcell ,KSC)作为组织特异性干细胞 ,具有强大的增殖能力 ,是表皮中各类细胞的起源。近年来 ,由于组织工程化皮肤、皮肤遗传性疾病病因及肿瘤的     

成体干细胞与肺

<正>干细胞是一类具有自我更新和高度增殖、分化潜能的细胞,能够产生高度分化的功能细胞。按分化潜能可分成三类细胞,即全能干细胞、多能干细胞和单能干细胞。按生存阶段的不同可分为胚胎干细胞和成体干细胞两大类。     

Mesenchymal stem cells

It has become clear that adult mammalian bone marrow contains not one but two ostensibly discrete populations of adult stem cells. The first and by far the most fully characterized are the hematopoietic stem cells responsible for maintaining lifelong production of blood cells. The biological characteristics and properties of the second marrow resident population of stem cells, variously termed bone marrow stromal cells or mesenchymal stem cells, are in contrast much less well understood. In vitro, cultures established from single-cell suspensions of bone marrow from a wide range of mammalian species generate colonies of adherent marrow stromal cells, each derived from a single precursor cell termed a colony-forming unit-fibroblast (CFU-F). Culture conditions have been developed to expand marrow stromal cells in vitro while maintaining the capacity of these cells to differentiate into bone, fat, and cartilage. A significant portion of our current knowledge of this population of cells is based on analysis of the properties of these culture expanded cells, not on the primary colony-initiating cells. In this article, we will focus on methodologies to prospectively isolate stromal progenitors from mouse and human bone marrow and will review current data that suggest stromal progenitors in the bone marrow in situ are associated with the outer surfaces of blood vessels and may share identity with vascular pericytes.     

间充质干细胞的培养特性及其相关细胞因子表达

目的:探讨体外培养间充质干细胞的方法及其生物学特性,并检测与其抑制T淋巴细胞增殖活性减轻GVHD机制的细胞因子的表达。方法:通过密度梯度离心及贴壁筛选法分离、纯化及培养间充质干细胞,观察其生物学特性,并进行免疫细胞化学染色观察间充质干细胞是否表达TGF-β1、HGF。结果:采用贴壁筛选法培养间充质干细胞原代经7天贴壁后变为梭形,经14~17天可长满瓶底,传代后3~4天即可长满,免疫分型示CD29+,CD44+,CD34-,CD45-,免疫细胞化学染色显示高度表达TGF-β1及HGF。结论:密度剃度离心结合贴壁筛选法培养间充质干细胞可获得纯度较高,生物学特性稳定的间充质干细胞;间充质干细胞可能通过TGF-β1与HGF两种细胞因子抑制T淋巴细胞增殖减轻移植后GVHD。     

Expansive effects of aorta-gonad-mesonephros-derived stromal cells on hematopoietic stem cells from embryonic stem cells

Background Hematopoietic stem cells (HSCs) give rise to all blood and immune cells and are used in clinical transplantation protocols to treat a wide variety of refractory diseases, but the amplification of HSCs has been difficult to achieve in vitro. In the present study, the expansive effects of aorta-gonad-mesonephros (AGM) region derived stromal cells on HSCs were explored, attempting to improve the efficiency of HSC transplantation in clinical practice.Methods The murine stromal cells were isolated from the AGM region of 12 days postcoitum (dpc) murine embryos and bone marrow（BM）of 6 weeks old mice, respectively. After identification with flow cytometry and immunocytochemistry, the stromal cells were co-cultured with ESCs-derived, cytokines-induced HSCs. The maintenance and expansion of ESCs-derived HSCs were evaluated by detecting the population of CD34+ and CD34+Sca-1+cells with flow cytometry and the blast colony-forming cells (BL-CFCs), high proliferative potential colony-forming cells (HPP-CFCs) by using semi-solid medium colonial culture. Finally, the homing and hematopoietic reconstruction abilities of HSCs were evaluated using a murine model of HSC transplantation in vivo.Results AGM and BM-derived stromal cells were morphologically and phenotypically similar, and had the features of stromal cells. When co-cultured with AGM or BM stromal cells, more primitive progenitor cells (HPP-CFCs ) could be detected in ESCs derived hematopoietic precursor cells, but BL-CFC’s expansion could be detected only when co-cultured with AGM-derived stromal cells. The population of CD34+ hematopoietic stem/progenitor cells were expanded 3 times,but no significant expansion in the population of CD34+Sca-1+ cells was noted when co-cultured with BM stromal cells. While both CD34+ hematopoietic stem/progenitor cells and CD34+Sca-1+ cells were expanded 4 to 5 times respectively when co-cultured with AGM stromal cells. AGM region-derived stromal cells, like BM-derived stromal cells, could promote hematopoietic reconstruction and HSCs’ homing to BM in vivo.Conclusions AGM-derived stromal cells in comparison with the BM-derived stromal cells could not only support the expansion of HSCs but also maintain the self-renewal and multi-lineage differentiation more effectively. They are promising in HSC transplantation. Chin Med J 2005; 118(23):1979-1986     

Development and application of stem cells

Stem cells are defined by two important characteristics, the ability to proliferate by a process of self-renewal and the potential to form at least one specialized cell type. Transient population of pluripotent or multipotent stem cells first appear during the development at the first days post coitum.     

Development and application of stem cells

Stem cells are defined by two important characteristics: the ability to proliferate by a process of self-renewal and the potential to form at least one specialized cell type. Transient population of pluripotent or multipotent stem cells first appear during the development at the first days post coitum. The cells of the inner cell mass (ICM) of the blastocyst, of which embryonic stem cells (ES) are the in vitro counterpart, can give rise to any differentiated cell type in the three primary germ layers of the embryo (endoderm, mesoderm and ectoderm).1-3 These cells gradually mature into committed, organ- and tissue-specific stem cells or adult stem cells, such as neural stem cells, mesenchymal stem cells, hematopoietic stem cells, etc. Over the past years, studies have focused on two aspects: molecular level and application, and some new methods and technology have been used.     

Therapeutic potential of motor neurons differentiated from embryonic stem cells and induced pluripotent stem cells

Degeneration of motor neurons (MN) caused by disease or injury leads to paralysis and is fatal in some conditions. To date, there are no effective treatments for MN disorders; therefore, cell therapy is a promising strategy to replace lost MN. Embryonic stem (ES) cells isolated from the inner cell mass of mammalian blastocysts self-renew and are pluripotent because they differentiate into cell types of the three germinal layers. Reprogramming of adult cells to a state similar to ES cells, termed induced pluripotent stem (iPS) cells, has been recently reported. It is well established that pluripotent cell types can give rise to specialized phenotypes, including neurons. Mouse, monkey and human MN can be differentiated from ES and iPS cells using procedures generally involving embryoid bodies formation and stimulation with retinoic acid and Sonic hedgehog. Differentiated MN express characteristic molecular markers such as Islet1, HB9 and Choline acetyltransferase, exhibit electrophysiological maturity and are able to form synaptic contacts similar to neuromuscular junctions in vitro. Furthermore, transplanted MN promote functional recovery in animal models of neurodegenerative diseases and MN injury. The potential clinical applications of stem cell-derived MN was enhanced after iPS cell derivation, which makes possible the generation of patient-specific pluripotent cells for autologous cell replacement therapies and may be used for drug development and disease modeling. This review summarizes MN differentiation protocols from ES and iPS cells in regard to neuronal differentiation efficiency, expression of MN markers and functional properties in vitro, as well as their therapeutic effects after grafting.     

Reprogramming of adult human neural stem cells into induced pluripotent stem cells

Background Since an effective method for generating induced pluripotent stem cells (iPSCs) from human neural stem cells (hNSCs) can offer us a promising tool for studying brain diseases,here we reporte...