人脐带间充质干细胞的成脂诱导分化及其相关基因鉴定

目的:探讨人脐带间充质干细胞(hUC-MSCs)的分离提取、体外诱导分化为脂肪细胞的能力及其相关基因的表达情况。方法:取新生儿脐带经组织培养法提取后分离培养于αMEM完全培养基中,经大量纯化与扩增后,采用倒置显微镜观察其形态与细微结构;流式技术检测其细胞周期及表面标志;以含有成脂诱导剂的αMEM培养基对P3的hUC-MSCs进行培养,诱导其向脂肪细胞方向分化,对其对诱导后的细胞进行检测。应用油红"O"染色对其进行定性鉴定;应用实时定量RT-PCR对LPL、Leptin的基因含量进行测定。结果:经过组织培养法后,细胞呈贴壁生长,细胞呈梭性或旋涡状,形态不规则,多数有凸起,细胞核较大,核仁明显;第7代以前的细胞具有较强的生长活性;流式技术检测发现此类细胞高表达CD44、CD73和CD105等细胞表面标记,而几乎不表达CD34、CD45、CD31和HLA-DR。培养至第3代的细胞约72.724%的细胞处G1期、S期的细胞仅占18.069%,第7代时G1期细胞约为83.875%、S期为9.606%左右;经成脂诱导剂诱导分化后,细胞经油红"O"染色,分化为脂肪细胞的细胞着色并呈红色,实时定量RT-PCR结果显示该部分细胞表达脂肪细胞的标志性基因LPL和Leptin。结论:P7以前的hUC-MSCs具有较强的生长分化能力,可以向脂肪细胞进行分化并表达一定量的特定标志性基因。

Oriented Adipogenic Differentiation of Human Umbilical Cord Mesenchymal StemCells and Identification of the Relative Genes

Objective： To extract and isolate the human Umbilical Cord Mesenchymal Stem Cells （hUC-MSCs） from umbilical cord, induce the oriented adipogenic differentiation and identify the biological characteristics. Methods： The hUC-MSCs were isolated from umbilical cord tissue of newborn babies and cultured in αMEM complete medium. After large-scale purification and amplification, the morphology of cells was observed by using an inverted microscope. The cell cycle and surface markers were detected by flow cytometry, hUC-MSCs were cultured and induced into adipocyte in an adipogenic agent αMEM medium for P3 and the induced cells were detected and confirmed. The cells were stained by Oil Red ＂O＂ for its qualitative identification and the Leptin gene expression was determined by using real-time quantitative RT-PCR for LPL. Results： After tissue culture, the cells grown in an adherent way. The cells were spindle or spiral-shaped, irregular, and most of them have raised and large nuclei and prominent nucleoli. Cells Before the 7th generations exhibited a strong growth activity. There was the expression of markers of cell surface, such as CD44, CD73 and CD105, etc, but the CD34, CD45, CD31 and HLA-DR had not expression. Approximately, 72.72 percent of the cells in the 3rd generation were at the G1 stage while 18.07 percent were at the S stage. For the cells in the 7th generation, 83.88 percent of the cells were at the G1 stage while 9.61 percent were at the S stage. After staining the adipogenicly differented cells utilizing Oil Red ＂O＂, the cell were stained and appear red. The real-time detection of RT-PCR results indicated the existence of the characteristic genes of the adipogenicly differented cells, LPL and Leptin. Conclusions： The hUC-MSCs before P7 have a strong ability to differentiate toward adipocytes and to express certain amount of the characteristic genes.