大鼠脂肪干细胞体外定向诱导分化为成骨细胞的研究

Study on adipose tissue-derived stem cells of rat differentiating into the osteoblast in vitro

目的研究脂肪组织来源干细胞(adiposetissue-derivedstemcells,ADSCs)在体外分离培养、诱导分化为成骨细胞的能力,探讨其作为骨组织工程种子细胞的可行性。方法从SD大鼠腹股沟脂肪垫分离出脂肪组织,通过Ⅰ型胶原酶消化法获取原代ADSCs,适时传代。逐日倒置显微镜下观察细胞形态及增殖特征。第3代细胞用成骨诱导培养基向成骨细胞诱导分化,并进行碱性磷酸酶染色、VonKossa染色及免疫细胞化学染色鉴定成骨细胞特性。结果从大鼠脂肪组织中分离培养出ADSCs,体外形态类似成纤维细胞,增殖迅速。在地塞米松、抗坏血酸、β-甘油磷酸钠及1,25-(OH)2VitD3诱导下,ADSCs表现出成骨细胞特性ALP活性显著增高,VonKossa染色出现钙化结节,OC、Ⅰ型胶原免疫细胞化学染色阳性。结论ADSCs易于分离培养,体外扩增迅速,经矿化诱导后可分化为成骨细胞,可作为骨组织工程的种子细胞。

AIM ：To explore the capability of adipose tissue -derived steme cells differentiating into osteoblast in vitro, to evaluate the feasibility of ADSCs as seeding cells in bone tissue engineering. METHODS：The adipose tissue were isolated from the inguinal fat pads of the SD rats. Primary ADSCs were obtained by the methods of collagenase Ⅰ digestion. Subsequently, the ADSCs were cultured and subcuhured. Cell morphology and proliferation characteristics were observed under inverted phase contrast microscope every day. The third passage of ADSCs were induced into osteoblasts by the osteogenic inducing fluid. The osteogenic phenotype were examined by means of alkaline phosphatase （ALP） , Von Kossa stainings and immunocytochemistry. RESUILTS：ADSCs were successfully obtained and cultured from rat adipose tissue. They appeared fibroblast - like and could proliferate rapidly in vitro. After induced with dexamethasone, ascorbic acid, beta -sodium glycerophosphate and 1,25 - （OH）2 Vit D3, ADSCs epressed the characteristic of osteoblasts ： increasing of alkaline phosphatase （ALP） actiuity significantly, appearance of calcium node showed under Kossa staining and positive OC,collagen Ⅰ through immunocytochemistry. CONCLUSION：Rat ADSCs could be obtained easily. They have a strong capability of proliferation and osteogenic properties. This study showed that the rat ADSCs could be used as seed cells in the bone tissue engineering.