本文選題：臍血　切入點(diǎn)：間充質(zhì)干細(xì)胞　出處：《四川大學(xué)》2007年博士論文　論文類型：學(xué)位論文

【摘要】： 目的探討新生兒臍血間充質(zhì)干細(xì)胞(mesenchymal stem cells，MSCs)體外分離、純化、擴(kuò)增，以及向成骨及脂肪細(xì)胞定向誘導(dǎo)分化的方法與條件。方法無菌條件下收集新生兒臍血60～120ml，枸櫞酸鈉抗凝，以Ficoll-HyPaque淋巴細(xì)胞分離液密度梯度法、沉降紅細(xì)胞后密度梯度法及CD34+免疫磁珠負(fù)選法分離單個核細(xì)胞(mononuclear cells，MNCs)。分離獲得的MNCs采用L-DMEM培養(yǎng)基+10％胎牛血清或Mesencult~(TM)培養(yǎng)基+10％胎牛血清進(jìn)行MSCs培養(yǎng)傳代，獲得第3代集落生長細(xì)胞作流式細(xì)胞儀表面抗原測定并向成骨、脂肪細(xì)胞定向誘導(dǎo)分化，成骨細(xì)胞鈣沉積經(jīng)茜素紅染色，脂肪細(xì)胞胞漿油滴經(jīng)油紅染色證實(shí)。結(jié)果經(jīng)沉降紅細(xì)胞后分離的MNCs，，使用Mesencult~(TM)培養(yǎng)基+10％胎牛血清培養(yǎng)成功率高，第3代可出現(xiàn)明顯的集落生長，而另兩種方法分離培養(yǎng)的細(xì)胞則難以形成集落：集落細(xì)胞表面抗原測定表達(dá)CD29、CD59、CD71而不表達(dá)CD34、CD45及HLA-DR等分子。集落細(xì)胞進(jìn)行成骨、成脂肪細(xì)胞定向誘導(dǎo)分化，成骨定向誘導(dǎo)分化的細(xì)胞經(jīng)茜素紅染色胞漿中出現(xiàn)有大量的鈣沉積；成脂肪定向誘導(dǎo)分化的細(xì)胞油紅染色示胞漿充滿油滴空泡。結(jié)論新生兒臍血中可分離出MSCs，并可在體外進(jìn)行培養(yǎng)擴(kuò)增。以甲基纖維素沉降紅細(xì)胞后密度梯度離心分離的MNCs培養(yǎng)較為有效，集落細(xì)胞表達(dá)基質(zhì)細(xì)胞表面抗原，能夠向成骨細(xì)胞、成脂肪細(xì)胞定向誘導(dǎo)分化。 目的探討人臍帶靜脈內(nèi)皮下間充質(zhì)干細(xì)胞體外分離、擴(kuò)增與成骨、成脂肪細(xì)胞分化的方法與條件。方法臍帶先用無菌生理鹽水沖洗，去除臍靜脈中的瘀血，再沿靜脈血管灌入12～15ml含1mg／mlⅡ型膠原酶的磷酸鹽緩沖生理鹽水(PBS)，6～8h后灌洗離心，獲取細(xì)胞培養(yǎng)、擴(kuò)增，細(xì)胞呈集落生長后傳代，取傳代細(xì)胞進(jìn)一步行免疫表型測定和誘導(dǎo)向成骨、成脂肪細(xì)胞分化。結(jié)果這種臍帶靜脈內(nèi)皮下細(xì)胞分離法，可獲得貼壁生長的細(xì)胞，呈短棒狀或梭形樣，易擴(kuò)增和形成集落，有基質(zhì)細(xì)胞免疫表型表達(dá)，成骨誘導(dǎo)分化的細(xì)胞經(jīng)茜素紅染色胞漿中有大量的鈣沉積，成脂肪誘導(dǎo)分化的細(xì)胞經(jīng)油紅染色示胞漿充滿了油滴空泡。結(jié)論臍帶靜脈內(nèi)皮下存在有具間葉細(xì)胞分化能力的間充質(zhì)干細(xì)胞，并可在體外進(jìn)行培養(yǎng)擴(kuò)增形成集落細(xì)胞傳代，傳代細(xì)胞表達(dá)基質(zhì)細(xì)胞表面抗原，能夠向成骨細(xì)胞、成脂肪細(xì)胞分化。
[Abstract]:Objective to investigate the methods and conditions of isolation, purification, amplification and differentiation into osteoblasts and adipocytes of mesenchymal stem cells of neonatal umbilical cord blood in vitro. The density gradient method of Ficoll-HyPaque lymphocyte isolate was used. Mononuclear cells of mononuclear cells (MNCs) were isolated by density gradient method after sedimentation and negative selection of CD34 immunomagnetic beads. The isolated MNCs was cultured in L-DMEM medium (10% fetal bovine serum) or Mesencultmann (TM) medium 10% fetal bovine serum for MSCs culture. The third generation of colony growth cells were determined by flow cytometry and differentiated into osteoblasts. Calcium deposition of osteoblasts was stained with alizarin red. Results MNCs isolated after sedimentation of red blood cells were cultured on Mesencultte TMM medium (10% fetal bovine serum) with high success rate, and colony growth could be observed in the third generation. However, the other two methods were difficult to form a colony: CD29, CD59, CD71, CD34, CD45, HLA-DR and other molecules were expressed in colony cell surface antigen assay. Colony cells were osteoblast and adipoblasts were induced to differentiate. There was a large amount of calcium deposition in the cytoplasm of osteogenic cells induced by alizarin red staining. Conclusion MSCs can be isolated from umbilical cord blood of newborn and can be cultured and amplified in vitro. Density gradient centrifugation with methylcellulose after sedimentation of red blood cells shows that the cytoplasm is filled with oil droplets. The culture of isolated MNCs was more effective. Colony cells express stromal cell surface antigens and differentiate into osteoblasts and adipoblasts. Objective to investigate the methods and conditions for the isolation, expansion and osteogenesis of human umbilical vein mesenchymal stem cells in vitro and the differentiation of adipoblasts. Then 12 ~ 15ml of phosphate buffer saline containing 1mg / ml type 鈪

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