本文選題：臍血　切入點：間充質(zhì)干細胞　出處：《四川大學》2007年博士論文　論文類型：學位論文

【摘要】： 目的探討新生兒臍血間充質(zhì)干細胞(mesenchymal stem cells，MSCs)體外分離、純化、擴增，以及向成骨及脂肪細胞定向誘導分化的方法與條件。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淋巴細胞分離液密度梯度法、沉降紅細胞后密度梯度法及CD34+免疫磁珠負選法分離單個核細胞(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％胎牛血清進行MSCs培養(yǎng)傳代，獲得第3代集落生長細胞作流式細胞儀表面抗原測定并向成骨、脂肪細胞定向誘導分化，成骨細胞鈣沉積經(jīng)茜素紅染色，脂肪細胞胞漿油滴經(jīng)油紅染色證實。結果經(jīng)沉降紅細胞后分離的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)的細胞則難以形成集落：集落細胞表面抗原測定表達CD29、CD59、CD71而不表達CD34、CD45及HLA-DR等分子。集落細胞進行成骨、成脂肪細胞定向誘導分化，成骨定向誘導分化的細胞經(jīng)茜素紅染色胞漿中出現(xiàn)有大量的鈣沉積；成脂肪定向誘導分化的細胞油紅染色示胞漿充滿油滴空泡。結論新生兒臍血中可分離出MSCs，并可在體外進行培養(yǎng)擴增。以甲基纖維素沉降紅細胞后密度梯度離心分離的MNCs培養(yǎng)較為有效，集落細胞表達基質(zhì)細胞表面抗原，能夠向成骨細胞、成脂肪細胞定向誘導分化。 目的探討人臍帶靜脈內(nèi)皮下間充質(zhì)干細胞體外分離、擴增與成骨、成脂肪細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，獲取細胞培養(yǎng)、擴增，細胞呈集落生長后傳代，取傳代細胞進一步行免疫表型測定和誘導向成骨、成脂肪細胞分化。結果這種臍帶靜脈內(nèi)皮下細胞分離法，可獲得貼壁生長的細胞，呈短棒狀或梭形樣，易擴增和形成集落，有基質(zhì)細胞免疫表型表達，成骨誘導分化的細胞經(jīng)茜素紅染色胞漿中有大量的鈣沉積，成脂肪誘導分化的細胞經(jīng)油紅染色示胞漿充滿了油滴空泡。結論臍帶靜脈內(nèi)皮下存在有具間葉細胞分化能力的間充質(zhì)干細胞，并可在體外進行培養(yǎng)擴增形成集落細胞傳代，傳代細胞表達基質(zhì)細胞表面抗原，能夠向成骨細胞、成脂肪細胞分化。
[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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