兔脂肪基质细胞的分离培养及其成骨活性的研究

Isolating Culture and Osteogenic Potential of Human Adipose Derived Stromal Cells

目的探讨兔脂肪组织来源脂肪基质细胞的分离培养方法及其成骨活性.方法取成年新西兰大白兔腹股沟皮下脂肪组织1～2mL,采用机械切割及Ⅰ型胶原酶消化法从脂肪组织中分离获得脂肪基质细胞,原代培养及传代以后,以诱导培养基(内含10mmol/Lβ-甘油磷酸钠、10～8mol/L地塞米松、50mg/L维生素C)进行诱导培养.实验评估:应用形态学观察、碱性磷酸酶钙-钴法染色检测碱性磷酸酶,VonKossa钙化结节染色检测钙化结节的形成等方法来鉴定诱导分化所得细胞的成骨活性.结果行诱导培养的脂肪基质细胞呈多层成长,形态多为梭形,细胞外基质有白色钙化结节形成;细胞增殖速度减慢,生长周期变长.碱性磷酸酶钙钴法染色及VonKossa钙化结节染色后均表现为阳性,未诱导培养的脂肪基质细胞则表现为阴性.结论初步建立了一套由脂肪组织分离培养脂肪基质细胞的方法,并证明该细胞在体外诱导培养条件下具备向成骨细胞分化的能力.

Objective To explore the isolation method and ossify activity of human adipose tissue-derived stromal cells. Methods Collection of ADSCs：1 - 2 mL subcutaneous adipose tissues were harvested from inguinal groove of rabbits. The obtained ADSCs were isolated by mechanical cutting and type Ⅰ collagenase digestion. The cells were harvested and plated in inductive culture supplemented with 10 mmol/L Na β-glyeerophosphate and 10 -8 mol/L dexamethasone, 50 mg/L vitamin C. The activity of the ossify was determined by observation of morphology and alkali phosphatase after Gomori straining, and the formation of calcium nodus after Von Kossa staining. Results The induced ADSCs were muhiplayer, mostly fusiform, metuliform or cube, and then these cells were observed under inverted phase contrast microscope. White mineralized nods formed in extraecllular matrix of central cell masses. Gomori and Von Kossa staining demonstrated that these cells were expressed positively, whereas the non-induced cells were negative. Conclusions A method of isolating ADSCs is primarily established. These cells can differentiate into osteoblasts in vitro.