成年大鼠骨髓多能成体祖细胞的克隆化培养及其鉴定  

Clonal culture of rat hone marrow-derived multipotential adult progenitor cells and study of their biological properties

作　　者：冀凯宏[1] 熊俊[1] 向正华[2] 胡凯猛[1] 王英[1] 刘厚奇[1] 

机构地区：[1]第二军医大学发育生物学研究中心组织胚胎学教研室,上海200433 [2]第二军医大学发育生物学研究中心生物化学教研室,上海200433

出　　处：《中华血液学杂志》2006年第7期474-478,共5页Chinese Journal of Hematology

基　　金：国家青年海外合作基金(30428001);国家自然科学基金(90208026)

摘　　要：目的优化培养条件,获得体外克隆化培养的成年大鼠骨髓多能成体祖细胞(rMAPC),并对其进行表型分析及分化潜能鉴定。方法选择低血清条件培养基全骨髓直接贴壁法初步分离大鼠骨髓干细胞,用极限稀释法进行克隆化培养;用流式细胞术及免疫细胞化学法对克隆化培养的 rMAPC 进行表型分析,并进行体外成脂肪、成骨及成神经细胞诱导,RT-PCR 检测 Oct 4及三个胚层早期标志物,确定其表型及分化潜能。结果单个细胞来源 rMAPC 在体外扩增至20代仍保持良好的生长状态;流式细胞术及细胞免疫组化检测结果显示 CD71、α-SMA 及 Vimentin 表达阳性;CD34、CD44、CD45表达阴性;细胞周期分析显示 S+G_2+M 期细胞约占17%,而处于 G_0/G_1期的细胞占83%:rMAPC 体外可被诱导向脂肪细胞、骨细胞及神经细胞分化;RT-PCR 检测到 Oct 4及三个胚层早期标志物的表达。结论体外克隆化培养的 rMAPC 能维持良好的干细胞生物学特性。Objective To optimize the culture conditions for clonal isolation of rat bone marrow-derived multipotential adult progenitor cells （ rMAPC ） and identify their surface markers and differentiation potentials. Methods By using a low concentration of fetal bovine serum culture medium, rMAPCs were primarily isolated from bone marrow by attachment culture and clonal-like cells were selected by single cell limiting dilution. The surface antigens of the cloned rMAPC were analyzed by flow cytometry and immunocytochemistry. Multi-differentiation capacities were evaluated by lipoblasts and osteoblasts and neuroblasts differentiation induction. The expressions of Oct-4 and three embryonic germ layer markers were detected by RT-PCR. Restilts Single cell-derived rMAPC could be expanded to passage 20 in vitro which still maintained active proliferation ability. The expanded rMAPCs expressed CD71, α-SMA and vimentin, but not CD34, CIM4 and CD45. About 83% of the rMAPCs was in the resting phase（ G0 ＋ G1 ） of cell cycle and 17% in S ＋ G2 ＋ M phase. They could be induced to differentiate into adiopogenic cells, osteogenic cells and neural like cells. RT-PCR demonstrated that there were expressions of oct-4 gene and three embryonic germ layer markers on the rMAPCs. Conclusions Cloned rMAPC can maintain the phenotypes of stem cell during in vitro culturing. It might be an potential adult stem cell source for therapeutic stem cell transplanting and tissue engineering.

关 键 词：骨髓 多能成体祖细胞 单克隆培养 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]