大鼠脂肪间充质干细胞的成骨分化

Osteogenic differentiation of rat adipose-derived mesenchymal stem cells

目的:观察大鼠脂肪间充质干细胞经成骨诱导向成骨细胞分化的生物学特性,探讨其作为骨组织工程种子细胞的可行性。方法:实验于2004-07/2006-03在中南大学湘雅医院中心实验室完成主要工作。①取健康SD大鼠双侧腹股沟区脂肪垫,消化法分离出脂肪间充质干细胞,接种入含有体积分数为0.1的新生牛血清的低糖DMEM培养基进行原代培养。②取第3代的脂肪间充质干细胞,用含有体积分数为0.1的新生牛血清、0.1μmol/L地塞米松、50μmol/L抗坏血酸、10mmol/Lβ-甘油磷酸钠的高糖DMEM培养基诱导其向成骨细胞分化。③于3,5,7,10,12,14,21d分别采用倒置显微镜观察细胞形态及增殖情况、Gomori改良钙钴法碱性磷酸酶染色、茜素红S钙结节染色和Ⅰ型胶原免疫细胞化学染色检测脂肪间充质干细胞成骨分化的情况。结果:①脂肪间充质干细胞原代细胞呈成纤维细胞样长梭形外观,传代稳定,细胞形态均一。②经成骨诱导,脂肪间充质干细胞体积增大,呈多角形;成骨诱导14d,Gomori改良钙钴法碱性磷酸酶染色,细胞胞浆内可见浅棕色至棕黑色的颗粒,平均染色阳性率为80%;碱性磷酸酶活性随时间的延长而逐渐增高[3,5,7,10,12,14d依次为(2.43±0.09),(3.60±0.08),(5.01±0.09),(7.75±0.07),(9.59±0.09),(10.94±0.10)μkat/L];成骨诱导21d,钙结节形成明显,茜素红S染色,呈红色结节;成骨诱导7d,Ⅰ型胶原免疫细胞化学染色,细胞胞浆呈棕黄色,胞核经苏木精复染为蓝色。结论:大鼠脂肪间充质干细胞经成骨诱导具有成骨细胞的生物学特性,可作为骨组织工程的种子细胞。

AIM： TQ investigate the biological features of rat adipose-derived mesenchymal stem cells （ADMSCs） to differentiate into osteoblast, and probe into the feasibility of ADMSCs as seed cells in bone tissue engineering. METHODS： The experiment was conducted in the Central Laboratory, Xiangya Hospital Affiliated to Central South University from July 2004 to March 2006. （1） ADMSCs were isolated from the inguinal fat pads excised from rats and digested with collagenase Ⅰ, and then incubated in low-glucose Dulbecco modified Eagle medium （DMEM） with 10% new-born bovine serum for primary culture. （2） The third-passage ADMSCs were cultured in high-glucose DMEM supplenmented with 10% new-born bovine serum, 0.1 μmol/L dexamethasone, 50 μmol/L ascorbic acid and 10 mmol/L β-glycerophosphat to differentiate into osteoblast. （3） At 3, 5, 7, 10, 12, 14 and 21 days later, the ADMSCs were observed under inverted microscope, and the differentiations to osteoblast were verified by Gomori staining, Alizarin red S staining and immunocytochemiscal staining. RESULTS： （1） ADMSCs were in fibroblast-shape and could stably passaged, during which ADMSCs kept the same shape in vitro. （2） After osteoblast-inducing culture, ADMSCs became larger and multi-angular. Fourteen days after culture, the positive expression of alkaline phosphatase activities （APA） was demonstrated by Gomori staining with a positive ratio of 80%, and APA was positively correlated with time [Which were （2.43±0.09）, （3.60±0.08）, （5.01±0.09）, （7.75±0.07）, （9.59±0.09）, （10.94±0.10）μkat/L respectively on the 3^rd, 5^th, 7^th, 10^th, 12^th and 14^th days]. The formation of mineralized nodules were verifed by Alizarin red S after 21 days culture. Cultured for 7 days, the type I collagen immunocytochemiscal staining appeared positive with brown cytoplasm and blue nucleus. CONCLUSION： After osteoblast-inducing culture, ADMSCs have the biological features of osteoblast and can be used as seeding cells for bone tissue engineering.