人脂肪来源间充质干细胞成骨及成软骨的潜能

Osteogenic and chondrogenic differentiation potential of human adipose-derived mesenchymal stem cells

背景：人脂肪来源间充质干细胞是种具有较强的体外增殖和多系分化能力的成体干细胞,可以从美容吸脂手术中获得,取材方便,原料来源丰富,在生物治疗应用方面蕴藏着巨大的价值。目的：体外分离、培养人脂肪来源间充质干细胞,探讨其基本生物学特性及成骨成软骨的潜能。方法：取美容吸脂获得的脂肪组织,采用Ⅱ型胶原酶消化法分离人脂肪来源间充质干细胞并进行体外培养;观察细胞形态、测定细胞周期、流式细胞仪鉴定细胞表面标志;取第3代细胞,分别加入成骨诱导培养基及成软骨诱导培养基行体外成骨及成软骨诱导。结果与结论：体外培养人脂肪来源间充质干细胞呈纤维样形态,原代细胞24h内贴壁,培养5-7d后开始形成细胞集落;经细胞周期检测显示G0/G1,S和G2/M所占比例分别为（88±2）%,（12±2）%和0.03%。经流式细胞仪检测CD29和CD105呈阳性表达,CD34和CD45呈阴性表达。RT-PCR检测显示,人脂肪间充质干细胞经成骨诱导分化后细胞中骨桥蛋白mRNA呈阳性表达,经软骨诱导分化后细胞中Ⅱ型胶原mRNA呈阳性表达。结果证实,实验成功体外分离、培养人脂肪来源间充质干细胞,其具有向成骨细胞和软骨细胞分化的潜能。

BACKGROUND：Human adipose-derived mesenchymal stem cells are a kind of adult stem cells with the high ability of proliferation and multi-lineage differentiation. They can be acquired from the cosmetic liposuction operation with rich sources of raw material and the selections are very convenient. They have a potential value in the field of bioremediation. OBJECTIVE：To establish a method of isolating and culturing human adipose-derived mesenchymal stem cells and investigate the proficiency of basic biological characteristics and the potential ability of osteogenic and chondrogenic differentiation. METHODS：The adipose tissue was taken from the liposuction operation, human adipose-derived mesenchymal stem cells were isolated by type II collagen enzyme digestion and then cultured in vitro. Cell morphology was observed, cell cycle was determined, and cell surface markers were identified with flow cytometry. Passage 3 cells were assigned to two induction groups A, B and two control groups A, B. Then the cells were induced to differentiate into osteoblasts and chondrocytes with different culture media in vitro. The differentiated cells were identified by histochemical staining, immunocytochemical staining and RT-PCR. RESULTS AND CONCLUSION：After in vitro culture, human adipose-derived mesenchymal stem cells showed the desmoids shape. The primary cells adhered to the wall in 24 hours, and then formed the colony after 5-7 days. The passaged cells adhered to the wall in 4-6 hours and maintained the same shape. Cell cycle studies showed that cells at G0/G1, S and G2/M accounted for （88±2）% （88±2）% and 0.03% respectively. The flow cytometry examination showed that the cells were positive for CD29 and CD105 but they were negative for CD45. RT-PCR showed that after osteogenic induction, human adipose-derived mesenchymal stem cells had positive osteopontin mRNA expression;after chondrogenic induction, cells had positive type II collagen mRNA expression. Human adipose-derived mesenchymal stem cells were successfully isolated and cultured and the cells exhibit the potential to differentiate into osteoblasts and chondrocytes.