脂肪间充质干细胞的基本生物学特性及向成骨细胞诱导分化的实验研究

目的　研究脂肪间充质干细胞的基本生物学特性以及在特定培养条件下向成骨细胞分化 ,探讨其作为骨组织工程的种子细胞的可行性。方法　取 3周龄Lewis大鼠的腹股沟脂肪垫 ,消化法获得脂肪间充质干细胞 ,分别用脂肪诱导培养基和成骨诱导培养基诱导其向脂肪细胞与成骨细胞分化 ,组织化学染色、免疫细胞化学染色和Westernblot检测细胞分化的情况。结果　从成体大鼠脂肪组织中培养出脂肪间充质干细胞 ,原代脂肪间充质干细胞能自发分化为脂肪细胞 ,传代细胞在胰岛素和呋塞米的作用下生成脂滴 ,过氧化物酶体增殖物激活受体 (PPAR)γ表达增强 ,向脂肪细胞分化 ;在呋塞米、抗坏血酸、β 甘油磷酸钠的诱导下 ,脂肪间充质干细胞的碱性磷酸酶(ALP)活性检测显示诱导组与对照组差异有显著性 (P <0 .0 1) ,vonKossa染色出现钙结节 ,骨桥蛋白 (OPN)、骨形态发生蛋白 (BMP) 2免疫细胞化学染色阳性 ,Westernblot检测到诱导后细胞OPN、BMP2的表达。结论　从脂肪组织中可获得具有多分化潜能的间充质干细胞 ,经诱导后可分化为脂肪细胞和成骨细胞 ,有可能成为骨组织工程较理想的种子细胞之一     

膝关节滑膜间质干细胞分离、培养及其多向分化潜能特性的研究

目的 探讨晚期骨关节炎患者膝关节滑膜间质干细胞(synovium-derived mesenchymalstem cells,SMSCs)体外分离、培养的可行性及其在体外向脂肪细胞、成骨细胞和软骨细胞定向分化的特性.方法 取膝关节滑膜组织,胶原酶消化获得有核细胞.挑选单细胞克隆,筛选获得SMSCs.流式细胞技术检测细胞表面特异性抗原标志.培养至第三代,分别向脂肪细胞、成骨细胞和软骨细胞诱导分化.油红O染色鉴定向脂肪细胞分化;碱性磷酸酶染色、茜素红染色鉴定向成骨细胞分化;甲苯胺蓝染色鉴定向软骨细胞分化.RT-PCR检测脂肪细胞、成骨细胞标志基因.Ⅱ型胶原免疫组化染色检测软骨细胞Ⅱ型胶原的表达.结果 原代SMSCs体外培养呈葵花样细胞集落,传代后可见圆形巨噬样细胞和纺锤形成纤维样细胞,融合后呈成纤维细胞样生长.CD44、CD90呈阳性,CD34、CD71和CD45呈阴性.向脂肪细胞诱导21d,油红O染色阳性;RT-PCR检测有脂蛋白酶、乙二腈及PPARγ2表达;向成骨细胞诱导7、28 d,ALP,茜素红染色阳性,有ALP、Osteopontin及Osteocalcin表达;向软骨细胞诱导21d,甲苯胺蓝染色阳性,Ⅱ型胶原免疫组化染色阳性.结论 晚期骨关节炎患者膝关节滑膜组织可以分离、培养获得SMSCs. SMSCs具有向脂肪细胞、成骨细胞和软骨细胞发生定向分化的潜能.     

骨髓基质干细胞成骨-成脂分化及补肾中药对其影响的研究进展

骨髓基质干细胞在不同条件的诱导下，可以分化为成骨细胞、软骨细胞、成纤维细胞、脂肪细胞、神经细胞等多种细胞系，在骨髓微环境中骨和脂肪形成之间的关系是复杂的，成骨细胞与脂肪细胞均来源于骨髓基质干细胞 (MSCs)，并且在其向成骨细胞和脂肪细胞分化之间存在相互逆转的关系和很大程度的可塑性。骨质疏松患者骨量减少与骨髓腔中脂肪组织增加有关，这可能与骨髓中MSCs分化失衡，过多向脂肪细胞分化有关。中医从“肾主骨”、“髓生骨”理论出发，认为肾精不足所致的骨髓空虚是骨质疏松症发病的关键，补肾中药通过影响骨髓基质干细胞成骨-成脂分化，从而防止骨质疏松症的发生。     

脂肪基质干细胞培养及其定向分化为成骨细胞、软骨细胞特性的研究

目的研究脂肪基质干细胞（adipose—derived stem cells，ADSCs）分离培养的方法，探讨大鼠ADSCs在体外向成骨细胞、软骨细胞分化的能力。方法从成年SD大鼠腹股沟处无菌获取脂肪组织，胶原酶消化分离，培养出ADSCs。基础培养基传至第二代时改换诱导培养基，分别诱导向成骨、软骨细胞分化，培养2～4周，碱性磷酸酶（alkaline phosphatase，ALP）、Vonkossa染色鉴定向成骨细胞分化能力；阿新蓝染色鉴定向软骨细胞分化能力。RT—PCR检测成骨细胞、软骨细胞标志基因。结果大鼠脂肪能够分离培养出生长旺盛的ADSCs；向成骨细胞诱导，ALP、Vonkossa染色阳性，RT—PCR检测有ALP、Osteocalcin及Osteopontin表达；向软骨细胞诱导，阿新蓝染色阳性，RT—PCR检测有Ⅱ型胶原、X型胶原和Aggreean表达。结论大鼠脂肪组织可以分离培养出ADSCs，生物学特性与骨髓基质干细胞（mesenchymal stemcells，MSCs）相似，能够向成骨细胞、软骨细胞分化，有希望成为组织工程理想的种子细胞来源。     

大鼠脂肪基质干细胞的培养及其向成骨细胞分化的研究

目的：研究大鼠脂肪基质干细胞（adipose—derived stem cells，ADSCs）分离培养的方法，探讨大鼠脂肪基质干细胞在体外向成骨细胞分化的能力。方法：取成年Sprague—Dawley大鼠腹股沟处脂肪组织，胶原酶消化分离．接种于自制基础培养基巾分离传代。于基础培养基中传至第二代时改换诱导培养基，诱导向成骨细胞分化，培养2-4周．用碱性磷酸酶染色和Von Kossa染色鉴定成骨细胞分化能力。结果：大鼠脂肪中能够分离培养出生长旺盛的脂肪基质下细胞：经向成骨细胞诱导培养后，碱性磷酸酶染色和Von Kossa染色阳性，证实细胞能够分化为成骨细胞。结论：大鼠脂肪组织可分离培养出脂肪基质干细胞，生物学特性与骨髓基质干细胞（mesenchymal stem cells，MSCs）相似，能够向成骨细胞分化，有望成为组织工程的种子细胞来源。     

脂肪间充质干细胞的成骨诱导分化

目的　研究脂肪间充质干细胞 (MSCs)在特定培养条件下向成骨细胞分化 ,探讨其作为骨组织工程的种子细胞的可行性。方法　取 3周龄Lewis大鼠的腹股沟脂肪垫 ,消化法获得脂肪MSCs,用成骨诱导培养基诱导其向成骨细胞分化 ,组织化学染色、免疫细胞化学染色和Westernblotting检测细胞分化的情况。结果　从成体大鼠脂肪组织中培养出脂肪MSCs,能大量稳定增殖传代。在地塞米松、抗坏血酸、β-甘油磷酸钠的诱导下 ,脂肪MSCs的ALP活性增高 ,VonKossa染色出现钙结节 ,OPN、BMP - 2免疫细胞化学染色阳性 ,Westernblotting检测到诱导后细胞OPN、BMP - 2的表达 ,且随诱导时间延长表达增强。结论　从脂肪组织中可获得具有多分化潜能的MSCs,并能在体外稳定增殖传代 ,经诱导后可分化为脂肪细胞和成骨细胞 ,有可能成为骨组织工程较理想的种子细胞之一     

骨髓脂肪细胞与成骨细胞分化的信号转导调控机制

骨髓基质干细胞具有多向分化潜能和自我增殖能力.在不同调节因子的作用下分化为多种细胞,如成骨细胞、软骨细胞、脂肪细胞、成肌细胞、神经细胞和血管内皮细胞等.近年研究发现,骨质疏松的发生可能与骨髓内环境中骨髓基质干细胞向成骨细胞分化能力的减弱,使得骨髓脂肪细胞的数量增多,脂肪细胞与成骨细胞的比例失调有关.骨髓基质干细胞纵向分化为成熟骨髓脂肪细胞及成骨细胞的过程受到多种通路的调控和影响,而分化成熟的成骨细胞和脂肪细胞在一定条件下也可以相互转化,这提示我们以后是否可以通过药物或其他手段来抑制BMS向脂肪细胞的分化、增殖能力进而使更多成骨细胞生成,甚至通过脂肪细胞去分化再分化为成骨细胞,从而有效地刺激骨形成,以达到治疗骨丢失和骨代谢异常的目的.基于此,本文结合最新研究进展,重点叙述骨髓基质干细胞向成熟脂肪细胞与成骨细胞分化过程中的信号转导调节机制以及两条信号转导间信号转导因子的相互影响.同时叙述了成熟骨髓脂肪细胞和成骨细胞在一定条件下相互转化可能的机制,希望可以为以后研究开发骨组织合成代谢药物提供新思路.     

脂肪间充质干细胞的成骨诱导分化

目的研究脂肪间充质干细胞(MSCs）在特定培养条件下向成骨细胞分化，探讨其作为骨组织工程的种子细胞的可行性。方法取3周龄Lewis大鼠的腹股沟脂肪垫，消化法获得脂肪MSCs，用成骨诱导培养基诱导其向成骨细胞分化，组织化学染色、免疫细胞化学染色和Western blotting检测细胞分化的情况。结果从成体大鼠脂肪组织中培养出脂肪MSCs，能大量稳定增殖传代。在地塞米松、抗坏血酸、β-甘油磷酸钠的诱导下，脂肪MSCs的ALP活性增高，Von Kossa染色出现钙结节，OPN、BMP-2免疫细胞化学染色阳性．Western blotting检测到诱导后细胞OPN、BMP-2的表达，且随诱导时间延长表达增强。结论从脂肪组织中可获得具有多分化潜能的MSCs，并能在体外稳定增殖传代，经诱导后可分化为脂肪细胞和成骨细胞，有可能成为骨组织工程较理想的种子细胞之一。     

胰岛素样生长因子-1基因转染脂肪间充质干细胞向软骨细胞分化的研究

目的 观察胰岛素样生长因子(IGF)-1基因转染的脂肪间充质干细胞(ADSCs)向软骨细胞分化的效果.方法 原代培养兔ADSCs,免疫荧光法检测细胞表面抗原CD44、CIM9;脂质体介导人IGF-1基因转染兔ADSCs联合低浓度血清培养基向软骨细胞分化诱导,RT-PCR及Western blot方法检测IGF-1的表达,MMT法绘制细胞增殖曲线、甲苯胺蓝染色软骨结节、免疫组织化学检测Ⅱ型胶原的表达.结果 脂肪间充质干细胞CD44、CD109表达阳性,基因转染后细胞IGF-1表达阳性,细胞增殖速度增快,出现软骨结节,Ⅱ型胶原表达增高.结论 从脂肪组织中能够分离出增殖旺盛的ADSCs,IGF-1在ADSCs内获得稳定表达,细胞增殖能力增强,促进其向软骨细胞分化.     

骨髓腔内脂质代谢异常对原发性骨质疏松症的影响

骨髓腔内脂质代谢异常在骨质疏松症的发病中起重要作用。骨髓基质细胞是具有多向分化潜能的干细胞,在成脂诱导剂作用下可加速向脂肪细胞分化。过氧化物酶体增殖物活化受体γ和核心结合因子α1是骨髓基质细胞向脂肪细胞分化过程的关键,两者呈"此消彼长"关系。脂肪细胞不仅可抑制成骨细胞活动,还可促进破骨细胞形成和功能活化,从而引起成骨-破骨偶联失调,最终导致骨质疏松症。抑制骨髓基质细胞向脂肪细胞分化成为一种新的治疗骨质疏松症的途径。     

成人间充质干细胞体外成骨的初步研究

目的 建立一种分离和培养成人骨髓来源的间充质干细胞（MSCs）的方法,观察成人MSCs体外成骨潜能。方法 用Percoll分离液分离出骨髓中的单个核细胞,并在含10％胎牛血清的低糖DMEM培养液中培养。通过传代培养扩增MSCs。为促进成人MSCs体外成骨性分化,第5传代培养时加入成骨性添加剂,培养第4、12天分别用流式细胞仪分析成人MSCs表面分子的表达,并用碱性磷酸酶组化染色和Von Kossa染色。结果 成人MSCs是骨髓黏附细胞中有相应细胞表面蛋白表达和形态均的一细胞群。成骨性添加剂可作用于传代培养的成人MSCs,表现为培养皿表面有相互连接的结节状聚合体、碱酶染色阳性细胞数量增多、Von Kossa染色可见钙化的基质沉积。结论 所建立的成人MSCs的分离和培养条件可分选出骨髓黏附细胞中一组独特的细胞群,成人MSCs具有体外成骨潜能。     

Biological alchemy: engineering bone and fat from fat-derived stem cells

Adipose tissue contains a population of pluripotent stem cells capable of differentiating along multiple mesenchymal cell lineages. In this study the authors isolated these fat-derived stem cells successfully from Lewis rats and induced differentiation along adipogenic and osteogenic lineages in vitro and in vivo. Induction was stimulated by exposing stem cells to lineage-specific induction factors. Adipocyte-inducing media contained dexamethasone, insulin, and isobutyl-methylxanthine. Osteoblast inducing media contained dexamethasone, beta-glycerophosphate, and ascorbic acid. Undifferentiated stem cells were maintained in minimal essential media alpha and fetal bovine serum. At 10 days, cells cultured in adipogenic media differentiated into adipocytes in vitro, as evidenced by positive Oil red O staining of lipid vacuoles. At 21 days, cells cultured in osteogenic media differentiated into osteoblasts in vitro as demonstrated by Alizarin red staining of a calcified extracellular matrix and immunohistochemical staining for osteocalcin. Differentiated cells were seeded at a density of 5 x 106 cells onto 15 x 15-mm polyglycolic acid grafts and implanted subcutaneously into three groups of Lewis rats: Group I contained undifferentiated stem cell grafts, group II contained adipocyte grafts, and group III contained osteoblast grafts. At weeks 4 and 8, in vivo fat formation was demonstrated in group II rats, as confirmed by Oil red O staining. At 8 weeks, group III rats demonstrated in vivo bone formation, as confirmed by the presence of osteocalcin on immunohistochemistry and the characteristic morphology of bone on hematoxylin-eosin staining. Group I rats demonstrated no in vivo bone or fat formation at either time interval. These results demonstrate the ability to isolate pluripotent stem cells from adipose tissue, to induce their differentiation into osteoblasts and adipocytes in vitro, and to form bone and fat subsequently in vivo. This is the first published report of in vivo bone formation from fat-derived stem cells. These cells may eventually serve as a readily available source of autologous stem cells for the engineering of bone and fat.     

Revitalization of cortical bone allograft by application of vascularized scaffolds seeded with osteogenic induced adipose tissue derived stem cells in a rabbit model

Background  

Adipogenous tissue derived stem cells (ASC) are available in abundance in the human body and can differentiate in the presence of lineage-specific induction factors, for example, in myogenic, adipogenic, chondrogenic and osteogenic cells. The aim of this study was to evaluate the impact of osteogenic induced ASC’s (O-ASC) on revascularization and cellular repopulation of avital cortical bone employing a vascularized bovine scaffold.     

PEI/SPIO对骨髓间充质干细胞生物学特性的影响

目的探讨应用新型聚乙烯酰胺（PEI）包覆的超顺磁性氧化铁（SPIO）标记骨髓间充质干细胞（bMSCs）对其生物学特性的影响。方法分离、培养贵州小香猪bMSCs,选取第3代bMSCs,用含铁浓度为4、6、8、10、12μg/ml的DMEM/F12培养液孵育24h,未标记组为对照。通过普鲁士蓝染色、透射电镜检查、胎盼蓝染色、MTT检测及成骨、成软骨、成脂诱导分化实验,探讨不同PEI/SPIO浓度对bMSCs标记效率、细胞活性、增殖及分化的影响。结果铁浓度为8μg/ml以上的SPIO标记bMSCs后,普鲁士蓝染色标记效率均接近100%。与对照组相比,Fe浓度为4、6、8μg/ml的PEI/SPIO标记的bMSCs活细胞比率均在95%以上,差异无统计学意义（P〉0.05）;而Fe浓度为10μg/ml时,活细胞比率约为（80.24±1.34）%,Fe浓度为12μg/ml时,活细胞比率约为（75.44±2.33）%,两者对细胞的活性有明显的抑制作用（P〈0.05）。4、6、8g/ml的PEI/SPIO标记的bMSCs成骨、成软骨及成脂分化与正常对照组相比无明显差异,而10、12ug/ml组在诱导培养过程中大部分细胞死亡。结论含铁浓度8μg/ml是PEI/SPIO标记干细胞的适宜浓度,既能高效地标记bMSCs,又不影响bMSCs的细胞活性及增殖分化能力等生物学特性。     

Cell Surface Expression of Stem Cell Antigen-1 (Sca-1) Distinguishes Osteo-, Chondro-, and Adipoprogenitors in Fetal Mouse Calvaria

The flat bones of the skull (calvaria) develop by balanced cell proliferation and differentiation in the calvarial sutures and the bone tips. As the brain grows and the calvaria expand, cells within the sutures must remain undifferentiated to maintain suture patency, but osteoprogenitors also need to be recruited into the osteogenic fronts. The exact identity of calvarial osteoprogenitors is currently not known. We used immunomagnetic cell sorting to isolate Sca-1⁺ and Sca-1⁻ cells from fetal mouse calvaria and determined their differentiation potential in in vitro differentiation asssays and in vivo subcutaneous transplantations. Cells within the Sca-1⁺ cell fraction have a higher adipogenic potential, whereas cells within the Sca-1⁻ cell fraction have a higher osteogenic and chondrogenic potential. The Sca-1⁻ fraction retains its chondrogenic potential after in vitro expansion but not its osteogenic potential. The Sca-1⁺ fraction does not retain its adipogenic potential after in vitro expansion. Subcutaneous transplantation resulted in islands of bone and cartilage in implants that had been seeded with Sca-1⁻ cells. In conclusion, immunomagnetic cell sorting with Sca-1 antibodies can be used to separate a Sca-1⁺ cell fraction with adipogenic potential from a Sca-1⁻ cell fraction with osteogenic and chondrogenic potential. Isolation of pure populations of calvarial adipoprogenitors, osteoprogenitors, and chondroprogenitors will be beneficial for cellular studies of calvarial development, adipogenesis, osteogenesis, and chondrogenesis. Calvaria-derived osteogenic cell populations may be useful in craniofacial tissue regeneration and repair.     

人脂肪细胞的可塑性

目的 探索体外培养环境下人成熟脂肪细胞的去分化现象,旨在挖掘其作为种子细胞的潜能,为组织工程研究开辟新思路.方法 自成人吸脂术后抽吸物提取成熟脂肪细胞及脂肪组织来源干细胞(adipose-derived stromal cells,ASCs),天花板贴壁培养法诱导成熟脂肪细胞去分化,观察细胞形态变化,获得去分化脂肪细胞(dedifferentiated adipocytes,DA).相同的条件下,MTT比色法比较DA、ASCs活性并绘制细胞生长曲线;流式细胞仪鉴定DA、ASCs表面分子的表达;油红O染色、茜素红染色、阿尔辛蓝染色分别鉴定DA、ASCs成脂分化、成骨、成软骨分化能力.结果 人成熟脂肪细胞在体外培养环境下能去分化为成纤维细胞状DA;MTT比色法测细胞活性:DA、ASCs均有很强的增殖能力,两者差异无统计学意义;流式细胞仪测定:DA、ASCs中HLA-ABC、CD29、CD44均为阳性,CD45、CD34、CD106均为阴性;成脂分化2周,油红O染色可见DA、ASCs内出现红色脂滴;成骨分化2周,茜素红染色可见DA、ASCs内红色钙盐沉积;成软骨分化2周,阿尔辛蓝染色可见DA、ASCs内软骨基质沉积.结论 成熟的脂肪细胞在体外培养条件下可成为DA,DA具有很强的增殖活性,表达部分干细胞特征性表面蛋白,有成骨、成软骨及强大的成脂分化能力,有望成为组织工程优秀的种子细胞.

Abstract：

Objective To explore the dedifferentiation phenomenon of human mature adipocytes cultured in vitro and to discuss the possibility of using dedifferentiation adipocytes ( DA ) as seed cells.Methods Mature adipocytes and ASCs were harvested from human fat aspirates. Mature adipocytes were cultured and induced to DA by ceiling adherent culture method. Cell morphology were observed during the whole process. Viabilities of DA and ASCs were compared by MTT chromatometry and cell growth curves were drawn based on it. Cell surface markers of DA and ASCs were detected by flow cytometry. The adipogenic,osteogenic and chondrogenic ability of DA and ASCs were assessed by oil red O staining,alizarin bordeaux staining and alcian blue staining, respectively. Regults Human mature adipocytes can dedifferentiate into fibroblast-shaped DA. MTT chromatometry assay demonstrated that DA and ASCs both had strong reproductive activity, with no significant difference between them. Flow cytometry assay demonstrated that both DA and ASCs expressed HLA-ABC, CD29 and CD44, while didn't express CD45,CD34 and CD106. After two weeks of adipogenic differentiation, lipid droplets could be displayed by oil red O staining in both DA and ASCs. After two weeks of osteogenic differentiation, calcium salts mineralization in DA and ASCs could be detected by alizarin bordeaux staining. After two weeks of chondrogenic differentiation, matrix of cartilage cells in DA and ASCs could be detected by alcian blue staining. Conclusions Mature adipocytes can be dedifferentiated into DA in vitro. DA has strong reproductive activity, as well as osteogenic, chondrogenic ability and strong adipogenic ability. It expresses some of the stem cell-related cell surface proteins and is a promising seed cell for adipose tissue engineering.     

体内外人脂肪间充质干细胞向肝样细胞分化的实验研究

目的 分离、培养人脂肪间充质干细胞(hADSCs),研究其在体内外向类肝样细胞转化的能力.方法 采用Ⅰ型胶原酶消化皮下脂肪,贴壁分选去除造血细胞的方法获得较纯的干细胞.通过对形态观察、表面分子鉴定、透射电镜内部结构扫描以及成脂、成骨定向诱导对脂肪间充质干细胞进行鉴定.用EGF、FGF、OSM、HGF、TSA细胞因子体外诱导hADSCs向肝样细胞分化.尾静脉输注hADSCs至急性肝损伤裸鼠,观察hADSCs在裸鼠肝脏内定植、向肝样细胞分化情况,以及hADSCs对裸鼠肝损伤的保护作用.结果 分离获得的细胞呈长梭形贴壁生长,低表达CD34、CD45,高表达CD73、CD90、CD105,具有多向分化潜能.诱导细胞表现出肝样细胞形态,表达AFP、alb,并且和肝细胞一样具有吸脂性.体内诱导:移植细胞在裸鼠体内生存、分布,并且细胞表达人alb,细胞体积较裸鼠肝脏细胞大,形态不均一,与人肝脏细胞形态有差异.同时研究发现hADSCs可改善急性肝损伤裸鼠肝功能,降低转氨酶.结论 人脂肪间充质干细胞可在体内外向肝样细胞分化,分化的细胞具有肝细胞样生物学性状.hADSCs对裸鼠急性肝损伤具有一定的治疗作用.

Abstract：

Objective To isolate and culture human adipose derived mesenchynal stem cells (hADSCs) and study the potential of hADSCs to differentiate into hepatocyte-like cells. Methods To ensure the removal of contaminating hematopoietic cells, hADSCs were selected based on plastic adherence. Cell surface antigen was confirmed by flow cytometry; ultramicrostructure was detected by transmission electron microscopy; adipogenic and osteogenic differentiation of hADSCs was analyzed by oil Red O staining and yon Kossa staining. hADSCs were exposed to differentiation medium containing EGF,FGF,OSM, HGF,TSA in vitro. 10% CCl4 (100 μ1/20 g body weight )was injected into immune-deficient BALB/c-nu mice and hADSCs (5 × 105 cells) were simultaneously administrated from the tail vein. Blood samples were collected and concentration of aminotransferase and direct bilirubin were detected. Administration without hADSCs was used as a control. One month later, we sacrificed the mice and liver sections were examined by histochemical immunofluorescence with human ALB specific antibodies. Results hADSCs exhibited fibroblast-like morphology, and expressed CD73, CD90,CD105, and were lacking of CD34 and CD45. Adipogenic and osteogenic differentiation showed that hADSCs have the capacity of multidifferentiation. The differentiated cells showed hepatocyte-like cell morphologies and hepatocyte-specific markers including albumin (alb) and α-fetoprotein (AFP). The bioactivity assays revealed that these hepatocyte-like cells could uptake low-density lipoprotein (LDL).Histochemical immunofluorescence showed that hADSCs were incorporated into injured livers. Some human alb-positive cells were found in liver sections after implantation of undifferentiated hADSCs. Transaminase activity in the experimental group was lower than in the control group. Conclution hADSCs can differentiated into functional hepatocyte-like cells and can relieve CCl4 induced BALB/c-nu acute liver injury.     

大鼠MSCs体外分离培养及骨向分化的实验研究

目的:建立骨髓MSCs体外分离培养体系,并进行骨向分化诱导,以证实其多向分化潜能,为骨髓MSCs进一步的临床应用研究提供实验依据。方法:用密度梯度离心法分离大鼠骨髓MSCs,并对其形态学特征进行观察。用诱导剂对骨髓MSCs向成骨细胞进行诱导分化,并进行形态学观察和免疫细胞化学检测。结果:用密度梯度离心法成功分离获得了高纯度的骨髓MSCs。经骨向诱导后,ALP和矿化结节染色阳性。结论:采用密度梯度离心法成功建立了大鼠骨髓MSCs体外分离和培养体系,并能够向成骨细胞分化。     

Comparison of Multipotent Differentiation Potentials of Murine Primary Bone Marrow Stromal Cells and Mesenchymal Stem Cell Line C3H10T1/2

Murine C3H10T1/2 cells have many features of mesenchymal stem cells (MSCs). Whether or not the multipotent differentiation capability of C3H10T1/2 cells is comparable to that of primary bone marrow–derived MSCs (BM-MSCs) was investigated in this study. For in vitro osteogenic differentiation, both BM-MSCs and C3H10T1/2 cells differentiated to osteoblastic cell lineage and showed positive staining for alkaline phosphatase (ALP) and increased mRNA expression of Runx2, Col1αI, and osteocalcin. C3H10T1/2 cells and BM-MSCs induced similar amounts of bone formation in the biomaterials. Under chondrogenic induction in the presence of TGF-β1, cell pellets of both BM-MSCs and C3H10T1/2 cells formed cartilage-like tissues with cartilage matrix components including proteoglycan, type II collagen, and aggrecan. However, C3H10T1/2 cells presented lower adipogenic differentiation potential, with only about 10% C3H10T1/2 cells (but about 70% of BM-MSCs) being committed to adipogenesis. In this study we confirmed that C3H10T1/2 cells coimplanted with osteoconductive scaffolds can form bone spontaneously in vivo and that C3H10T1/2 cells have a basal level of osteocalcin expression, suggesting that they may be a good alternative source of primary BM-MSCs for investigating osteogenic and chondrogenic differentiation in bone or cartilage tissue engineering studies. Caution is needed when using C3H10T1/2 cells for adipogenic studies as they appear to have lower adipogenic potential than BM-MSCs.