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1、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
2、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,p
3、readipocytes,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 s
4、ource 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 di
5、fferent 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/macroph
6、ages 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 t
7、ypes.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 slightl
8、y 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
9、,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
10、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1mm3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-M
11、EM、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；
12、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
13、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
14、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
15、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
16、.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 pathway modulates hemangioblast activity of adult hematopoietic stem cells.Blood,2005.,p38 MAP kinase mediates nitric oxide-induced apoptosis of ne
17、ural progenitor cells.J Biol Chem,2001.,NO相关检测,在一定的SNP浓度范围内，可以发现培养基中NO浓度依赖于SNP的浓度，随SNP浓度的增加培养基中NO浓度也是增加的。,低浓度的外源性NO处理ADSCs，与对照组相比其增殖能力并没有明显的改变。随着SNP浓度（4mmol/L）的逐渐增加可以发现，SNP能抑制ADSCs的增殖。,ADSCs成软骨分化过程中，NO的产生趋势与正常细胞无显著区别成软骨分化中的ADSCs分泌的NO高于正常培养细胞,外源性NO对ADSC成软骨分化的col II1基因表达有抑制作用，并且抑制程度非浓度依赖性相关,TGF-通路参与AD
18、SCs成软骨诱导,Smad-dependent and Smad-independent pathways in TGF-b family signalling，nature,2003.,受体调节 Smads(R-Smads:Smad1、2、3、5、8和9)。,外源性NO抑制ADSCs成软骨分化过程中的tgf-1、smad3。,外源性NO对ADSCs成软骨分化的作用，首先表现在对其自身分泌TGF-1的抑制上。,低浓度外源性NO对ADSC增殖能力并没有明显的改变。随着浓度（4mmol/L）的逐渐增加就可抑制ADSCs的增殖。NO抑制ADSCs成软骨分化，抑制了成软骨细胞相关基因的表达。外源性NO
19、供体能抑制ADSCs本身产生TGF-1，并且对TGF-/SMAD3下游信号通路有抑制作用。,我们的结果支持这样一个假说，NO可以作为一个分子约束来抑制TGF-的过度反应。,T lymphocytes play a central role in the initiation and maintenance of inflammatory processes.Accumulation of T cells at inflammatory sites is one of the characteristic features of chronic inflammatory diseases.B c
20、ells promotes VAT T cell activation and proinflammatory cytokine production,which potentiate M1 macrophage polarization and insulin resistance.,炎症与成脂分化,Wang,L.,Y.Zhao,and S.Shi,Interplay between mesenchymal stem cells and lymphocytes:implications for immunotherapy and tissue regeneration.J Dent Res,2012.91(11):p.1003-10.,PBLCs分离,After coculture 2 days,After coculture 4 days,PPT模板下载：行业PPT模板：节日PPT模板：PPT素材下载：PPT图表下载：优秀PPT下载：PPT教程：Word教程：Excel教程：资料下载：PPT课件下载：范文下载：试卷下载：教案下载：,谢谢关注！,
