CAJ | 학술논문

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。
목적：통과장견골수간충질간세포（ bone marrow mesenchymal stem cells，BMSCs）건입체외배양체계，운용인골형태발생단백-2（bone morphogenetic protein-2，BMP-2）체외정향유도분화위성골세포，위후기건립골조직공정제공충자세포。방법제취비격견BMSCs，전골수첩벽법결합밀도제도리심법행체외분리배양，매일관찰세포생장변화。장생장형태량호적제3대BMSCs분성량조，실험조가입200 ng/ml인BMP-2적함혈청배양기대기진행성골유도배양，대조조지용함혈청적완전배양기배양，우유도3주후행감성린산매염색、유도4주후행천소홍염색여Von-Kossa염색이감정분화적성골세포。결과유도3주후실험조감성린산매염색시세포포질내흑색과립양성표체，대조조위음성；유도4주후실험조천소홍염색이급Von-Kossa염색균정개결절양성표체，대조조균위음성。실험조염색결과도표체성골세포적특성。결론체외분리배양적비격견BMSCs，재유도제인BMP-2적작용하가이향성골세포정향분화。
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.