脂肪间充质干细胞相关分化与炎症相关

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,*,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,*,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,*,脂肪间充质干细胞相关分化与炎症相关,威斯腾生物技术中心,威斯腾生物十二周年庆技术培训之,目录,脂肪间充质干细胞分离、培养和鉴定,脂肪间充质干细胞,骨关节炎和肥胖,脂肪间充质干细胞分化与炎症,脂肪间充质干细胞(ASCs).,Adipose tissue,is recognized as a complex,endocrine organ,that plays central roles in energy homeostasis,feeding,insulin sensitivity,and,inflammation,.,Adipose tissue contains different cell types besides adipocytes,including,adipose-derived stromal cells(ASCs).,ASCs were first reported by Frohlich in 1972,as,adherent,proliferating adipocyte precursors,.Different names have been used to describe the adherent cell population that can be expanded from lipoaspirates,e.g.lipoblast,pericytes,preadipocytes,processed lipoaspirates(PLA)cells,and many others.,Several works demonstrated the stem cell-like plasticity of ASCs and their capability of differentiating into cells of,mesodermal origin,such as,adipocyte,osteocyte,chondrocyte and myocyte,lineages.,Zuk PA,etc.Human adipose tissue is a source of multipotent stem cells.Mol Biol Cell 2002;13:4279-4295,PLA,or,SVF(stromal vascular fraction),To clarify nomenclature,the,International Fat Applied Technology Society,(IFATS)proposed to name,ASCs the isolated,plastic-adherent,multipotent cell population obtained from adipose tissue through different methods,Subcutaneous fat is an abundant and accessible source of both,heterogeneous stromal vascular fraction(SVF)cells,and a small number of relatively,homogeneous ASCs,.SVF cells include vascular endothelial cells and their progenitors,smooth muscle cells,immune regulatory monocytes/macrophages and T regulatory cells.,Caspar-Bauguil,S.,et al.,Adipose tissue lymphocytes:types and roles.J Physiol Biochem,2009.65(4):p.423-36.,Similarly to bone marrow mesenchymal stromal cells(BMSCs),ASCs were identifiedin vivo as cells with perivascular localization,in close contact with the other cell types.,Like other MSC types,ASCs cannot be,identified by a single specific marker,but multiple marker combinations are needed for the identification of the cell population.ASCs express the surface markers used for BMSC characterization,with some,peculiarities,.,BMSCs express,CD106,that is not or slightly expressed by ASCs;,by contrast,BM-MSCs lack the expression of,CD49d,which is expressed by ASCs.Following ex vivo expansion,some markers are normally expressed by ASCs,such as,CD13,CD29,CD34,CD54,CD73,CD90,CD105 and MHC I.,By contrast,markers of the angiogenic and haematopoietic lineages,such as,CD14,CD31,CD133,MHC II and CD45,are not or poorly expressed by ASCs.,Strem BM,Hicok KC,Zhu M,Wulur I,Alfonso Z,Schreiber RE,et al.,Multipotential differentiation of adipose tissue-derived stem cells.,Keio J Med 2005;54:13241,ASCs,的分离培养,1,）准备实验器材和试剂，置于超净工作台内紫外消毒等。,2,）取,3,周龄,SD,大鼠断颈处死，,750ml/L,乙醇浸泡,5-10min,，之后再更换酒精再浸泡,5,分钟，最后移入超净工作台内。,3,）在超净工作台内严格遵循无菌操作要求。用组织剪剪开腹部皮肤直达到,大腿内侧腹股沟处,。更换组织剪和组织镊，分别取出腹股沟处的,白色脂肪垫,，置入含有双抗,(,含,100U/mL,青霉素和,100g/mL,链霉素,),的,PBS,中。,4,）组织镊,剔除白色脂肪块上的血管、淋巴结等,其他组织。再用含双抗的,PBS,清洗,3,遍，置入青霉素瓶内剪碎为约,1mm,3,大小的组织块。,5,）将剪碎的组织块移入,75mL,超大培养瓶底部并涂布均匀，每瓶约放入,0.4g,脂肪组织块，将培养瓶翻转底部朝上，置入,37,、,5%CO2,孵箱内贴壁,1,小时左右。,6,）肉眼观察培养瓶底部没有明显水滴的时候，贴壁小心加入,510ml,的培养基（含改良型,-MEM,、,10%FBS,、,100U/mL,青霉素和,100g/mL,链霉素），勿将组织块吹下。,37,、,5%CO2,孵箱内培养，每,2-3,天更换培养基。,7,）待组织块周围生长出来的细胞充分融合时，用胰蛋白酶消化传代，细胞此时为,P1,代细胞，继续培养。之后的细胞在培养瓶中长满达到,70%-90%,融合时，继续后进行传代培养。,脂肪组织,组织块贴壁培养法培养,24,小时后，在组织块周围爬出少量的长梭形细胞,P1,代细胞在,3,天时融合率就能达到,80%,左右,ASCs,鉴定,脂肪干细胞的检测 图中为,P3,大鼠脂肪干细胞的流式分析检测结果，细胞的表面标记为,CD29+CD31,-,CD34+CD45,-,CD90+CD146,-,A,：,P3,大鼠脂肪干细胞；,B,为,ASCs,成脂诱导分化，油红,O,染色检测呈红色；,C,为,ASCs,成骨诱导分化，图中所示红色块状为钙结节；,D,为成软骨诱导分化，阿尔新蓝染色检测呈蓝色；,E,、,F,为脂肪干细胞的神经诱导分化，,F,免疫荧光神经细胞特异蛋白,-tubulin,与胚胎干细胞和骨髓干细胞相比，脂肪干细胞具有明显的优势。虽然胚胎干细胞具有全能性，能分化形成全部组织来源的细胞，但是其临床应用往往受到医学伦理的限制。骨髓干细胞是来自骨髓组织的多潜能间充质干细胞，其取材较为困难，不仅会给患者带来巨大的痛苦，而且获得的干细胞数量也很非常少；从人骨髓中只能,取到大约,0.001%,0.002%,的间充质干细胞，而从人白色脂肪组织中却能够,取到大约,1%,的脂肪干细胞。脂肪干细胞获取容易，还表现出较强的多向分化能力、免疫耐受性和分泌能力，这些优势都极大地促进了脂肪干细胞在组织工程和再生医学领域的应用，具有广阔的应用前景。,一氧化氮与骨关节炎,NO,是由一组已知的被称为一氧化氮合成酶,(NOSs),催化,L-,亮氨酸的一个胍基氮生产,L-,腺嘌呤基瓜氨酸和,NO,。,有三种亚型的,NOS,包括,NOS-1,、,NOS-2,和,NOS-3,。它们能利用一系列复杂的辅助因子和酶辅被作用物合成,NO,。,NOS-1(nNOS),和,NOS-3,（,eNOS,）是由钙,/,钙调蛋白调节的组成型,NOS,酶。,NOS-2,（,iNOS,）主要是由炎症刺激物刺激机体部位诱导产生。,高浓度的,NO,或它的衍生物与骨关节炎的发展有密切关系,，它通过抑制软骨细胞外基质（,ECM,）的合成和促进降解。同时,NO,还能抑制软骨细胞与合成代谢因子,IGF-1,的相互作用。,TGF-1,对于关节软骨的正常功能的维持具有重要的作用。,它能通过,Smad,信号通路调控细胞的增殖和胞外基质的合成或降解。细胞表面,TGF-1,配体的结合能导致受体活化的,Smads,磷酸化并且向细胞核内进行转移，参与这个过程的主要是,Smad2/3,，它能调节很多基因的转录表达。,一氧化氮（,NO,）是在急性和慢性炎症部位产生的一种自由基。,当,NO,浓度高于,115mM,时，细胞因子激活的巨噬细胞开始凋亡。同时，在成骨细胞中，高浓度的,NO,能降低,ALP,活性和细胞活力。,因此，当,NO,浓度高于正常细胞水平时，对细胞是有毒性的。,NO,能调节,TGF-,信号。,Prediction of nitric oxide concentrations in melanomas.Nitric Oxide,2010.,Mesenchymal stem cell-mediated immunosuppression occurs via concerted action of chemokines and nitric oxide.Cell Stem Cell,2008,.,Nitric oxide inhibits glomerular TGF-beta signaling via SMOC-1,J Am Soc Nephrol.2009.,Nitric oxide regulates vascular calcification by interfering with TGF-b signaling,Cardiovasc Res.2008.,表明,NO,在间充质前体细胞的增殖和分化中扮演重要角色。,The nitric oxide p