脂肪間充質(zhì)干細(xì)胞相關(guān)分化與炎癥相關(guān)

1、脂肪間充質(zhì)干細(xì)胞相關(guān)分化與炎癥相關(guān)威斯騰生物技術(shù)中心威斯騰生物十二周年慶技術(shù)培訓(xùn)之威斯騰生物十二周年慶技術(shù)培訓(xùn)之目錄脂肪間充質(zhì)干細(xì)胞分離、培養(yǎng)和鑒定脂肪間充質(zhì)干細(xì)胞骨關(guān)節(jié)炎和肥胖脂肪間充質(zhì)干細(xì)胞分化與炎癥脂肪間充質(zhì)干細(xì)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 diff

2、erent 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. li

3、poblast, 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

4、. human adipose tissue is a source of multipotent stem cells. mol biol cell 2002; 13: 4279-4295plaorsvf(stromal vascular fraction)to clarify nomenclature, the international fat applied technology society (ifats) proposed to name ascs the isolated, plastic-adherent, multipotent cell population obtain

5、ed from adipose tissue through different methodssubcutaneous 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

6、, 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 local

7、ization, 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 pecul

8、iarities. 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, m

9、arkers 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;54:13241ascs的分

10、離培養(yǎng)1）準(zhǔn)備實驗器材和試劑，置于超凈工作臺內(nèi)紫外消毒等。2）取3周齡sd大鼠斷頸處死，750ml/l乙醇浸泡5-10min，之后再更換酒精再浸泡5分鐘，最后移入超凈工作臺內(nèi)。3）在超凈工作臺內(nèi)嚴(yán)格遵循無菌操作要求。用組織剪剪開腹部皮膚直達到大腿內(nèi)側(cè)腹股溝處。更換組織剪和組織鑷，分別取出腹股溝處的白色脂肪墊，置入含有雙抗(含100u/ml青霉素和100g/ml鏈霉素)的pbs中。4）組織鑷剔除白色脂肪塊上的血管、淋巴結(jié)等其他組織。再用含雙抗的pbs清洗3遍，置入青霉素瓶內(nèi)剪碎為約1mm3大小的組織塊。 5）將剪碎的組織塊移入75ml超大培養(yǎng)瓶底部并涂布均勻，每瓶約放入0.4g脂肪組織塊，將培養(yǎng)

11、瓶翻轉(zhuǎn)底部朝上，置入37、5% co2 孵箱內(nèi)貼壁1小時左右。6）肉眼觀察培養(yǎng)瓶底部沒有明顯水滴的時候，貼壁小心加入510ml 的培養(yǎng)基（含改良型-mem、10% fbs、100u/ml青霉素和100g/ml鏈霉素），勿將組織塊吹下。37、5% co2 孵箱內(nèi)培養(yǎng)，每2-3天更換培養(yǎng)基。7）待組織塊周圍生長出來的細(xì)胞充分融合時，用胰蛋白酶消化傳代，細(xì)胞此時為p1代細(xì)胞，繼續(xù)培養(yǎng)。之后的細(xì)胞在培養(yǎng)瓶中長滿達到70%-90%融合時，繼續(xù)后進行傳代培養(yǎng)。脂肪組織組織塊貼壁培養(yǎng)法培養(yǎng)24小時后，在組織塊周圍爬出少量的長梭形細(xì)胞p1代細(xì)胞在3天時融合率就能達到80%左右ascs鑒定脂肪干細(xì)胞的檢測 圖中

12、為p3大鼠脂肪干細(xì)胞的流式分析檢測結(jié)果，細(xì)胞的表面標(biāo)記為cd29+cd31-cd34+cd45-cd90+cd146-a：p3大鼠脂肪干細(xì)胞；b為ascs成脂誘導(dǎo)分化，油紅o染色檢測呈紅色；c為ascs成骨誘導(dǎo)分化，圖中所示紅色塊狀為鈣結(jié)節(jié)；d為成軟骨誘導(dǎo)分化，阿爾新藍染色檢測呈藍色；e、f為脂肪干細(xì)胞的神經(jīng)誘導(dǎo)分化，f免疫熒光神經(jīng)細(xì)胞特異蛋白-tubulin 與胚胎干細(xì)胞和骨髓干細(xì)胞相比，脂肪干細(xì)胞具有明顯的優(yōu)勢。雖然胚胎干細(xì)胞具有全能性，能分化形成全部組織來源的細(xì)胞，但是其臨床應(yīng)用往往受到醫(yī)學(xué)倫理的限制。骨髓干細(xì)胞是來自骨髓組織的多潛能間充質(zhì)干細(xì)胞，其取材較為困難，不僅會給患者帶來巨大的痛

13、苦，而且獲得的干細(xì)胞數(shù)量也很非常少；從人骨髓中只能 取到大約0.001%0.002%的間充質(zhì)干細(xì)胞，而從人白色脂肪組織中卻能夠取到大約1%的脂肪干細(xì)胞。脂肪干細(xì)胞獲取容易，還表現(xiàn)出較強的多向分化能力、免疫耐受性和分泌能力，這些優(yōu)勢都極大地促進了脂肪干細(xì)胞在組織工程和再生醫(yī)學(xué)領(lǐng)域的應(yīng)用，具有廣闊的應(yīng)用前景。一氧化氮與骨關(guān)節(jié)炎no是由一組已知的被稱為一氧化氮合成酶(noss) 催化l-亮氨酸的一個胍基氮生產(chǎn)l-腺嘌呤基瓜氨酸和no。有三種亞型的nos包括nos-1、nos-2和 nos-3。它們能利用一系列復(fù)雜的輔助因子和酶輔被作用物合成no。nos-1 (nnos) 和nos-3 （enos）是

14、由鈣/鈣調(diào)蛋白調(diào)節(jié)的組成型nos酶。nos-2（inos）主要是由炎癥刺激物刺激機體部位誘導(dǎo)產(chǎn)生。高濃度的no或它的衍生物與骨關(guān)節(jié)炎的發(fā)展有密切關(guān)系，它通過抑制軟骨細(xì)胞外基質(zhì)（ecm）的合成和促進降解。同時no還能抑制軟骨細(xì)胞與合成代謝因子igf-1的相互作用。tgf-1對于關(guān)節(jié)軟骨的正常功能的維持具有重要的作用。它能通過smad信號通路調(diào)控細(xì)胞的增殖和胞外基質(zhì)的合成或降解。細(xì)胞表面tgf-1配體的結(jié)合能導(dǎo)致受體活化的smads 磷酸化并且向細(xì)胞核內(nèi)進行轉(zhuǎn)移，參與這個過程的主要是smad2/3，它能調(diào)節(jié)很多基因的轉(zhuǎn)錄表達。一氧化氮（no）是在急性和慢性炎癥部位產(chǎn)生的一種自由基。當(dāng)no濃度高于1

15、15mm時，細(xì)胞因子激活的巨噬細(xì)胞開始凋亡。同時，在成骨細(xì)胞中，高濃度的no能降低alp活性和細(xì)胞活力。因此，當(dāng)no濃度高于正常細(xì)胞水平時，對細(xì)胞是有毒性的。no能調(diào)節(jié)tgf-信號。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. nit

16、ric 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在間充質(zhì)前體細(xì)胞的增殖和分化中扮演重要角色。the nitric oxide pathway modulates hemangioblast activity of adult hematopoietic ste

17、m cells. blood, 2005.p38 map kinase mediates nitric oxide-induced apoptosis of neural progenitor cells. j biol chem, 2001.no相關(guān)檢測在一定的snp濃度范圍內(nèi)，可以發(fā)現(xiàn)培養(yǎng)基中no濃度依賴于snp的濃度，隨snp濃度的增加培養(yǎng)基中no濃度也是增加的。低濃度的外源性no處理adscs，與對照組相比其增殖能力并沒有明顯的改變。隨著snp濃度（4mmol/l）的逐漸增加可以發(fā)現(xiàn)，snp能抑制adscs的增殖。adscs成軟骨分化過程中，no的產(chǎn)生趨勢與正常細(xì)胞無顯著區(qū)別成軟骨分化

18、中的adscs分泌的no高于正常培養(yǎng)細(xì)胞外源性no對adsc成軟骨分化的col ii1基因表達有抑制作用，并且抑制程度非濃度依賴性相關(guān)tgf-通路參與adscs成軟骨誘導(dǎo)smad-dependent and smad-independent pathways in tgf-b family signalling，nature,2003.受體調(diào)節(jié) smads(r- smads:smad1、2、3、5、8和9)。外源性no抑制adscs成軟骨分化過程中的tgf-1、smad3。外源性no對adscs成軟骨分化的作用，首先表現(xiàn)在對其自身分泌tgf-1的抑制上。低濃度外源性no對adsc增殖能力并沒有明顯的改變。隨著濃度（4mmol/l）的逐漸增加就可抑制adscs的增殖。no抑制adscs成軟骨分化，抑制了成軟骨細(xì)胞相關(guān)基因的表達。外源性no供體能抑制adscs本身產(chǎn)生tgf-1，并且對tgf-/smad3下游信號通路有抑制作用。我們的結(jié)果支持這樣一個假說，no可以作為一個分子約束來抑制tgf-的過度反應(yīng)。t lymphocytes play a central role in the initiation and maintenance of inflammatory processes.