兔BMSCs体外培养及其向成骨细胞分化的实验研究

目的　探讨培养兔骨髓基质干细胞向成骨细胞的分化,为骨组织工程研究提供种子细胞。方法　取 2月龄新西兰大耳白兔,麻醉后取骨髓,直接进行原代培养,传代后观察其生长特性,绘制生长曲线并加诱导液使其 向成骨细胞方向分化,并分别用钙钴法检测碱性磷酸酶,茜素红染色检测钙结节,免疫组化染色检测Ⅰ型胶原,透 射电镜观察胞质中钙质成分。结果　原代培养中出现大量细胞克隆,传代后细胞呈旋涡状密集生长,加入诱导液 后细胞形态发生改变并向成骨细胞分化,胞质内见有呈黑色的碱性磷酸酶颗粒和Ⅰ型胶原反应产物,并见有多个 细胞形成的钙化结节,电镜下观察到胞质中含有许多基质小泡,几天内成骨细胞数可达1×106/L。结论　培养兔 骨髓基质干可向成骨细胞方向分化,作为骨组织工程的种子细胞。

Experimental Study on the Differentiation of Cultured Bone Marrow Stromal Stem Cells (BMSCs) of Rabbit into the Osteoblast in Vitro

Objective To investigate the differentiation of cultured bone marrow stromal stem cells (BMSCs) of rabbit into osteoblast to provide seed cells for bone tissue engineering. Method BMSCs were isolated from rabbit of two month and cultured to observe the live speciability, and the osteogenic inducing fluid was added to accelerate the differentiation of BMSCs into osteoblast. The osteogenic potential of bone marrow stromal stem cells was investigated by means of alkaline phosphatase (ALP) and alizarin red S (ARS) stainings, immunohistochemical staining and transmisson electron microscope (TEM). Results Mass clones of BMSCs were observed in primary culture and arranged in spite shape in passage culture. BMSCs were seen to be differentiated into osteoblast after the conditonal fluid was added, ALP positive dark granules and collagen Ⅰ positive product were found in the cytoplasm of the cultured cells, and ARS positive calcium nodule was seen formed by several cultured cells, and a lot of matrix vesicles was observed by TEM, and the number of osteoblast was 1×10 6/L. Conclusion BMSCs could be mass cultured to be differentiated into osteoblast as seed cells for bone tissue engineering.