摘要
目的 建立犬骨髓间叶干细胞的体外分离、培养、扩增与鉴定方法 ,了解其生物学特性 ,为心肌梗死、心力衰竭及缓慢性心律失常的干细胞移植提供细胞材料。方法 抽取犬骨髓液 10ml,以DMEM 1∶1稀释 ,用 1 0 6 3g/mlpercoll密度分离液 ,以 6 0 0 g离心力、30min分离骨髓单个核细胞 ;以LG DMEM及 10 %FBS添加 10 0U/ml青霉素 ,10 0 μg/ml链霉素 ,2 5 μg/ml两性霉素B培养骨髓干细胞 ;利用细胞贴壁筛选法分离、培养、扩增骨髓间叶干细胞 ;应用干细胞表面标志蛋白检测 ,诱导剂诱导分化间叶组织细胞等方法进行犬骨髓间叶干细胞的鉴定。结果 分离出的犬骨髓单个核细胞约占细胞总数的 10 -4～ 10 -6,在LG DMEM与选择性血清培养基上生长良好。犬骨髓间叶干细胞孵育 2 4h即可见细胞贴壁生长 ,贴壁细胞约占接种单个核细胞总数的 10 -6。犬骨髓间叶干细胞增殖分裂迅速 ,38～ 4 8h内增殖多个细胞 ,约 72h即可增殖形成大的细胞克隆 ,7～ 10d即可布满瓶底。细胞融合时类似成纤维细胞 ,呈纺锤状 ,细胞小而密集 ,螺旋梳状排列。连续培育 10代以上 ,未见细胞形态、增殖特性发生改变。未见骨髓间叶干细胞自发分化其它类型细胞 ,仍维持原代培养细胞的增殖特性。犬骨髓间叶干细胞的表面标志SH2阳性 ,CD4 5则阴性。
Objective To study and establish the method for isolation,culture,proliferation and identification of canine bone marrow mesenchymal stem cells (MSCs).MethodsTen ml canine bone marrow aspirate,taken from the iliac crest of normal canines,were diluted 1∶1 with Dullbecco's Modified Eagle Medium (DMEM).The washed cells were resuspended in DMEM to a final volume of 10 ml and layered over an equal volume of 1.063 g/ml Percoll gradient solution.After centrifugation at 600 g for 30 minutes,the mononuclear cells(MNCs)were recovered from the gradient interface and washed with PBS.Percoll fractionated MNCs were suspended in DMEM containing 1 g/L of glucose supplemented with 10% fetal bovine serum,100 U/ml penicillin,100 μg/ml streptomycin,and 25 μg/ml amphotericin B.All cells were plated in 10 ml of medium in a culture dishes.The cultures were maintained at 37℃ in air of 5% CO 2,with an initial medium change at 24 hours after initial plating and then medium change every 3 or 4 days.The MSCs were identified by special cellular surface antigens and pluripotent committing differentiation potential.Results The methodology for isolation and expansion of MSCs was established.The mononuclear cells from canine bone marrow were separated on Percoll gradient sedimentation solution at 600 g for 30 minutes in small percentage (about 0.01～0.001%).The MSCs were set very good in culture of LG-DMEM supplemented with 10% selected fetal bovine serum.MSCs attached and grew as fibroblastic cells at 24 hours after initial plating,about 10 -6 out of mononuclear cells.The hematopoietic stem cells that did not attach to the dish were washed from the culturewith each medium change.MSCs grew rapidly,developed into visible symmetric colonies at about 3 days and reached confluence at 7 to 10 days.While the cells were permitted to proliferate to confluence,MSCs were spindle-shaped morphology,small and arranged like pectinate.As many as(1.1～1.5)×10 7 cells were generated by passage 3 from a 10 ml marrow aspirate.The MSCs did not diffe
出处
《中华心律失常学杂志》
2004年第1期51-57,共7页
Chinese Journal of Cardiac Arrhythmias
关键词
骨髓
间叶干细胞
分离培养
生物学特性
Mesenchymal stem cells
Bone marrow
Culture
Cardiomyocytes
Adipocytes
Osteocytes
Canine
