人BMP-2体外定向诱导犬BMSCs向成骨方向分化的实验研究

Experimental study of human BMP-2 on osteogenic induction in BMSCs of dogs in vitro

目的通过将犬骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)建立体外培养体系,运用人骨形态发生蛋白-2(bone morphogenetic protein-2,BMP-2)体外定向诱导分化为成骨细胞,为后期建立骨组织工程提供种子细胞。方法提取比格犬BMSCs,全骨髓贴壁法结合密度梯度离心法行体外分离培养,每日观察细胞生长变化。将生长形态良好的第3代BMSCs分成两组,实验组加入200 ng/ml人BMP-2的含血清培养基对其进行成骨诱导培养,对照组只用含血清的完全培养基培养,于诱导3周后行碱性磷酸酶染色、诱导4周后行茜素红染色与Von-Kossa染色以鉴定分化的成骨细胞。结果诱导3周后实验组碱性磷酸酶染色示细胞胞质内黑色颗粒阳性表达,对照组为阴性;诱导4周后实验组茜素红染色以及Von-Kossa染色均呈钙结节阳性表达,对照组均为阴性。实验组染色结果都表达成骨细胞的特性。结论体外分离培养的比格犬BMSCs,在诱导剂人BMP-2的作用下可以向成骨细胞定向分化。

Objective：To provide seed cells for bone tissue engineering in the late establishment by establishing the cul-ture system of bone marrow mesenchymal stem cells（ BMSCs）of dogs in vitro,and using human BMP-2 to make them in-duced to differentiate into osteoblasts. Methods：The extraction of BMSCs of adult beagle dogs was made,then the whole marrow adherence method and density gradient centrifugation were used to isolate and culture BMSCs in vitro,and observe the cell growth morphology everyday. The third generation BMSCs with good growth form was divided into two groups. The experimental group were cultured with adding 200ng/ml human BMP-2 containing fetal bovine serum（FBS）while the control group were cultured only with complete medium containing FBS. Then we used the detection of alkaline phosphatase staining after 3 weeks′induction,alizarin red staining and Von-Kossa staining after 4 weeks′induction to identify the differentiation of osteoblasts. Results：After 3 weeks of induction of experimental group with alkaline phosphatase,staining showed the cyto-plasm of positive expression of black particles,and it was negative in the control group;After 4 weeks of induction of experi-mental group with alizarin red staining and Von-Kossa staining showed positive expression of calcium nodules,and it was negative in the control group. All the staining results in the experimental group showed the characteristics of osteoblasts. Conclusion：BMSCs of dogs,which are extracted and cultivated in vitro,can directionally differentiate into osteoblasts under the action of human BMP-2.