人胎骨髓MSCs的分离鉴定及其向肝细胞样细胞的分化

目的: 分离鉴定人胎骨髓中的间质干细胞(MSCs),探索其体外培养的生物学特性，并在化学因子作用下诱导其向肝细胞分化。方法: 利用细胞差速贴壁生长特性分离纯化人胎骨髓MSCs；利用流式细胞仪检测其细胞周期和表面标志；添加常规诱导液诱导其向脂肪、成骨方向分化;采用DMSO、β-Me和5-aza联合预诱导24 h,换用H-DMEM和 rh-HGF正式诱导人胎骨髓MSCs向肝细胞分化，并从形态学和特异性细胞化学染色等方面加以鉴定。结果: 从人胎骨髓中成功分离、纯化得到MSCs，P4代MSCs有92.3%的细胞处于G₀/G₁期；P5代MSCs有96.1%的细胞处于G₀/G₁期。流式细胞仪检测P3代MSCs结果显示：人胎骨髓MSCs表达CD29、CD44、CD105和CD106 ,不表达造血细胞标志CD34、CD45,不表达与GVHD相关的HLA-DR、CD80、CD86、CD40、CD40L。在经典的诱导条件下，人胎骨髓MSCs可快速向脂肪及成骨细胞分化；在上述诱导条件下，人胎肝MSCs可分化为类肝细胞,表达特异性抗原甲胎蛋白（AFP）和白蛋白（ALB）。结论: 人胎骨髓中含有丰富的MSCs,人胎骨髓来源的MSCs具有较强的多向分化潜能，经DMSO、β-Me和5-aza联合预诱导及rh-HGF、nictinion等化学因子的作用，易向肝细胞样细胞分化，且免疫原性弱,是组织工程（生物型人工肝）的较为理想的种子细胞。

Isolation and identification of human fetal bone-derived mesenchymal stem cells and their differentiation to hepatocyte like cells

AIM: To separate and identify the mesenchymal stem cells (MSCs) from human fetal bone and to study their differentiation to hepatocyte like cells under the action of chemical induction.METHODS: The MSCs from human fetal bone were isolated and purified according to the different growth characteristic of attaching to the wall of cell culture flask.The cell cycle and surface markers of MSCs were identified using flow cytometry.The MSCs were pre-induced by adding DMSO,β-Me and 5-aza for 24 h,then adding the inductive medium of H-DMEM and rh-HGF to induce their differentiation to hepatocyte like cells (HLCs).HLCs were identified by the typical morphological change and the expression of special protein with the method of immunocytochemistry.RESULTS: The MSCs derived from human fetal bone expressed adhesion molecules CD29+,CD44+,but not antigens of hematopoietic CD34,CD45,and not antigens related to GVHD,such as HLA-DR,CD80 and CD86.Exposure of these cells to above-mentioned inductive agents resulted in obvious morphological change and an increase in expression of AFP and ALB.CONCLUSION: The results suggest the existence of plentiful MSCs in human fetal bone.MSCs derived from human fetal bone can easily differentiate to HLCs,and they have a lower immunogenic nature,which may provide the ideal source for tissue engineering (bioartificial liver) for cellular therapeutics.