小鼠脂肪来源干细胞向成骨及软骨细胞的诱导分化

Adipose-derived stem cells differentiate into osteoblasts and chondrocytes

背景:脂肪干细胞常从脂肪组织中分离获取,脂肪组织在全身均有分布,极易大量获得,取材时对供区损伤小。目的:进一步验证小鼠脂肪来源干细胞的体外分离、培养方法,并在特定条件下诱导其向成骨细胞和软骨细胞分化,探讨其作为组织工程种子细胞的可行性。方法:取昆明种雄性小鼠附睾处脂肪,Ⅰ型胶原酶消化法和差速贴壁法获取、纯化脂肪干细胞并进行细胞表型鉴定。采用成骨诱导培养基诱导脂肪干细胞,进行钙钴法碱性磷酸酶染色和茜素红钙结节染色检测细胞分化情况;采用软骨诱导培养基诱导脂肪干细胞,进行甲苯胺蓝染色,番红O染色,Ⅱ型胶原免疫组织化学检测细胞分化情况。结果与结论:脂肪干细胞呈梭形贴壁生长,原代7-9d可达90%融合,传至第3代后细胞形态趋向一致,传代的脂肪干细胞生长曲线呈"S"形。细胞特异性抗原测定CD29、CD44表达阳性,而CD34、CD45呈阴性表达。成骨诱导分化后细胞碱性磷酸酶染色和茜素红染色呈阳性。软骨诱导分化后细胞番红O染色、Ⅱ型胶原免疫组织化学染色和甲苯胺蓝染色呈阳性。证实从脂肪组织中可分离到具有分化潜能的干细胞,可在体外稳定增殖传代,经诱导后可分化为成骨细胞、软骨细胞,其具有来源广泛,取材方便等优点,可成为组织工程理想的种子细胞。

BACKGROUND： Adipose-derived stem cells can be separated and obtained from fat tissue. Fat tissue distributes in the whole body, and can be easily obtained in large quantities and has less damage to the donor site when drawing. OBJECTIVE： To identify the methods of in vitro isolating and culturing of mice adipose-derived stem cells, to induce the adipose-derived stem cells to differentate into chondrocytes and osteoblasts and to investigate the feasibility of being seed cells in tissue engineering. METHODS： The adipose-derived stem cells were isolated from the epididymal fat tissue of Kunming mice. Primary adipose-derived stem cells were obtained and purified by collagenase [ digestion and differential adherence method. The adipose-derived stem cells were induced with osteogenic induction medium, and then gomori alkaline phosphatase staining and alizarin red calcium nodules staining were performed to detect the differentiation of adipose-derived stem cells; the adipose-derived stem cells were induced with cartilage induction medium, and the toluidine blue staining, safranin-O staining and type TI collagen immunohistochemistry testing were performed to detect the differentiation of adipose-derived stem cells. RESULTS AND CONCLUSION： The adipose-derived stem cells were spindle-shaped and in adherent growth. After primary cultured for 7-9 days, the cells could reach 90% confluence. After passaged to the third generation, the cell morphology was in consistency, and the growth curve of the passaged adipose-derived stem cells presented ＂S＂ shape. The expressions of CD29 and CD44 antigens were positive detected with cell-specific antigen test, but the expressions of CD34 and CD45 were negative. After osteoblast-inducing culture, the differentiation of adipose-derived stem cells towards osteoblasts was verified positively by alkaline phosphatase staining and alizarin red staining. After chondrocyte-inducing culture, the differentiation of adipose-derived stem cells into the chondrocyte was verified positively by oil red O staining, type ][[ collagen immunohistochemical staining and toluidine blue staining. The adipose-der＇1ved stem cells with differentiation potential can be isolated from the fat tissues and can be the cells can be stably passaged and differentiated in vitro. The adipose-derived stem cells can be differentiated into osteoblasts and chondrocytes after induction, and the adipose-derived stem cells have the advantages of rich sources and easily obtained which can be considered as the ideal seed cells of tissue engineering