脐血及脐带静脉内皮下间充质干细胞的分离扩增与分化

发布时间：2018-03-21 17:05

  本文选题：脐血　切入点：间充质干细胞　出处：《四川大学》2007年博士论文　论文类型：学位论文

【摘要】： 目的探讨新生儿脐血间充质干细胞(mesenchymal stem cells，MSCs)体外分离、纯化、扩增，以及向成骨及脂肪细胞定向诱导分化的方法与条件。方法无菌条件下收集新生儿脐血60～120ml，枸橼酸钠抗凝，以Ficoll-HyPaque淋巴细胞分离液密度梯度法、沉降红细胞后密度梯度法及CD34+免疫磁珠负选法分离单个核细胞(mononuclear cells，MNCs)。分离获得的MNCs采用L-DMEM培养基+10％胎牛血清或Mesencult~(TM)培养基+10％胎牛血清进行MSCs培养传代，获得第3代集落生长细胞作流式细胞仪表面抗原测定并向成骨、脂肪细胞定向诱导分化，成骨细胞钙沉积经茜素红染色，脂肪细胞胞浆油滴经油红染色证实。结果经沉降红细胞后分离的MNCs，，使用Mesencult~(TM)培养基+10％胎牛血清培养成功率高，第3代可出现明显的集落生长，而另两种方法分离培养的细胞则难以形成集落：集落细胞表面抗原测定表达CD29、CD59、CD71而不表达CD34、CD45及HLA-DR等分子。集落细胞进行成骨、成脂肪细胞定向诱导分化，成骨定向诱导分化的细胞经茜素红染色胞浆中出现有大量的钙沉积；成脂肪定向诱导分化的细胞油红染色示胞浆充满油滴空泡。结论新生儿脐血中可分离出MSCs，并可在体外进行培养扩增。以甲基纤维素沉降红细胞后密度梯度离心分离的MNCs培养较为有效，集落细胞表达基质细胞表面抗原，能够向成骨细胞、成脂肪细胞定向诱导分化。 目的探讨人脐带静脉内皮下间充质干细胞体外分离、扩增与成骨、成脂肪细胞分化的方法与条件。方法脐带先用无菌生理盐水冲洗，去除脐静脉中的瘀血，再沿静脉血管灌入12～15ml含1mg／mlⅡ型胶原酶的磷酸盐缓冲生理盐水(PBS)，6～8h后灌洗离心，获取细胞培养、扩增，细胞呈集落生长后传代，取传代细胞进一步行免疫表型测定和诱导向成骨、成脂肪细胞分化。结果这种脐带静脉内皮下细胞分离法，可获得贴壁生长的细胞，呈短棒状或梭形样，易扩增和形成集落，有基质细胞免疫表型表达，成骨诱导分化的细胞经茜素红染色胞浆中有大量的钙沉积，成脂肪诱导分化的细胞经油红染色示胞浆充满了油滴空泡。结论脐带静脉内皮下存在有具间叶细胞分化能力的间充质干细胞，并可在体外进行培养扩增形成集落细胞传代，传代细胞表达基质细胞表面抗原，能够向成骨细胞、成脂肪细胞分化。
[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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