人胎儿肝脏间充质干细胞的体外培养及生物学特性研究

目的建立一种在体外分离、培养人胎儿肝脏来源的间充质干细胞(MSC)的方法,并研究其生物学特性。方法采用双酶消化法分离获取人胎儿肝脏MSC并进行体外培养,待细胞达80%以上融合时传代,各传代细胞依次记为P1～P10代细胞,并于倒置相差显微镜下观察细胞形态。取P3、P7、P10代细胞,连续培养7d,观察分析细胞生长曲线。取P5代细胞,采用流式细胞仪检测MSC表面CD34、CD44、CD105、CD13、HLA-ABC、HLA-DR等抗原标志的表达。取P4代细胞,进行体外成骨细胞诱导培养后,采用碱性磷酸酶染色鉴定。冻存传代细胞,分别于2、6个月后复苏细胞,台盼蓝染色计数复苏后细胞存活率。结果原代细胞3d贴壁,6d后开始快速增长并形成集落,11d左右达80%～90%融合,多呈纤维样;传代培养后细胞维持纤维样形态。传代细胞生长曲线具有共同特征:潜伏期约24～48h,对数增殖期约3～4d,对数增殖期后第5～6天进入平台期。流式细胞仪检测显示,MSC表面CD34、HLA-DR呈阴性表达,CD44、CD105、CD13呈阳性表达,HLA-ABC呈弱阳性表达。碱性磷酸酶染色显示,诱导后细胞的细胞核染成均一的淡蓝色。冻存复苏后细胞存活率达90%以上,且与未冻存传代细胞相比具有相同的生长特性。结论本研究成功地从人胎儿肝脏培养出MSC,为MSC应用于科学研究和临床治疗提供了新的细胞来源。

Study on culture and biological characterstics of human fetal liver mesenchymal stem cells in vitro

Objective To establish the isolation and culture method of human fetal liver mesenchymal stem cells （MSC） in vitro,and explore their biological properties. Methods Separation by double enzyme digestion to obtain MSC from human fetal liver and cultured in vitro until the cells reached 80% confluence and passaged,and the order of passage cells recorded as P1 -P10 generation cells,and the morphology was observed under inverted phase contrast microscope. The P3,P7,P10 generation cells were collected and continuous cultured for 7d,observed and analyzed the cell growth curve. Detecting the surface markers of CD34、CD44、CD105、CD13、HLA-ABC、HLA-DR of the P5 generation cells by using flow cytometry. Inducing the P4 cells into osteoblast cells in vitro and identifying by alkaline phosphatase staining. Recover the cryopreserved passage cells respectively in 2,6 months,and detected cells survival rate after resuscitation by trypan blue staining. Results Original generation of cells 3d pasted the wall,started the swift growth and formed the colony after 6d,about 11d reached 80% -90% fusions,and showing the desmoid shape. After the transfer of generation raise,cells maintained desmoid shape. The passage cells growth curves had the common characteristic：latent period was 24 -48 h,logarithm multiplication period was 3 -4 d,and 5 -6 d entered the platform period after the logarithm multiplication period. The flow cytometry examination showed that the expression of CD34,HLA-DR were negative,CD44,CD105,CD13 were positive,and HLA-ABC were positive on the surface of MSC. The alkalinity phosphatase staining demonstrated that the cell nucleus were dyed the homogeneous pale blue. After the recovery,the cells survival percentage reached above 90%,compared with the uncryopreserved passage cells,the cryopreserved passage cells had the same growth characteristic. Conclusion The study had successfully cultured MSC from human fetal liver,which provide a new source of cells for scientific research and clinical treatment.