小鼠胚胎干细胞定向诱导分化为软骨样细胞的初步研究

Preliminary study of chondrogenic differentiation of mouse embryonic stem cells

目的 全反式维生素A酸(RA)联合转化生长因子-β1(TGF-β1)及骨形态发生蛋白-2(BMP2)等多种因子定向诱导小鼠胚胎干细胞(ESC)分化,探索这几种细胞因子对ESC的成软骨定向诱导分化作用。方法 小鼠ESC在小鼠胚胎成纤维细胞(MEF)饲养层上培养扩增,在1×109mol/L全反式RA条件下制备拟胚体(EB),贴壁后联合应用TGF-β1及BMP2诱导分化,经形态学、免疫细胞化学和RT-PCR检测细胞性质。结果 ESC在悬浮培养下,经RA初步诱导后给予TGF-β1及BMP2等因子继续诱导,贴壁后EB周边可爬出多角形分化细胞,诱导分化细胞和EB表达Ⅱ型胶原和软骨发生早期特异性转录因子Scleraxis。结论 在RA预诱导EB分化后,TGF-β1和BMP2等因子可定向诱导小鼠ESC表达软骨特异性相关基因和蛋白,为胚胎干细胞成为软骨组织工程种子细胞提供了可能。

Objective To study the differentiation of mouse embryonic stem cells ( ESC ) induced by retinoic acid (RA) , transforming growth factor-β1 (TGF-β1 ) and bone morphogeneic protein-2 (BMP2) , and explore chondro-genic induction effect of these factors. Methods After being in vitro cultured and expanded, mouse ES cells were made into embryoid bodies (EBs) with the medium added 1×10-9mol/L all trans-RA. The EBs were induced by TGF-β1 , BMP2 and other factors. The induced cells derived form EBs were analyzed by morphology, immunocyto-chemistry and reverse transcription-polymerase chain reaction (RT-PCR). Results EBs were formed by ES cells cultured in suspension with RA for five days. When attached to the dish, EBs would give rise to a large number of polygonal cells given TGF-β1 , BMP2 and other induction factors. Induced EBs and differentiation cells expressed type Ⅱ collagen and transcription factor of Scleraxis, which usually expresses during early stage of embryo chondrogenesis. Conclusion After 1×10-9mol/L RA pre-stimulating the differentiation of EB, TGF-β1, and BMP2 can induce mouse ES cells to express specific gene and protein of chondrocytes, which provided the possibility for ES cells to become seed cells for cartilage tissue engineering.