兔骨髓间充质干细胞的分离培养及成骨诱导

Isolation,culture and osteogenic induction of rabbit bone marrow mesenchymal stem cells

背景:骨髓间充质干细胞具有多向分化潜能,且可大量体外扩增培养,是重要的组织工程种子细胞。但尚无统一的体外培养及定向诱导方法。目的:探讨体外定向诱导兔骨髓间充质干细胞分化为成骨细胞的可行性。方法:应用密度梯度离心法从兔四肢骨中分离纯化间充质干细胞,应用密度为1.073g/mL的Percoll分离液,3000r/min×30min离心,区别于相关报道的Ficoll分离液,2000-2500r/min×(20-30)min离心以及全骨髓培养法体外扩增至第3代,分别在普通培养基(对照组)和成骨诱导培养基(实验组)中培养。结果与结论:成功获得大量高纯度骨髓间充质干细胞。经成骨诱导后,实验组骨钙素含量明显高于对照组(P<0.05)。实验组碱性磷酸酶和钙结节染色阳性,对照组均阴性。结果表明使用密度梯度离心法可成功建立兔骨髓间充质干细胞的分离培养体系,骨髓间充质干细胞可定向诱导为成骨细胞。

BACKGROUND： Bone marrow mesenchymal stem cells are important seed cells for tissue engineering because of their multi-directional differentiation potential and possibility to be amplified and cultured in vitro But there have been no uniformed methods of in vitro culture and oriented differentiation. OBJECTIVE： To investigate the feasiblity of in vitro oriented differantation of rabbit bone marrowmesenchymal stem cells into osteoblasts METHODS： Rabbit bone marrow mesenchymal stem cells were isolated and purified using density gradient centrifugation （1.073 g/mL Percoll separation solution for centrifugation at 3 000 r/min for 30 minutes, which was different from Ficoll separation solution for centrifugation at 2 000-2 500 r/rain for 20-30 minutes as well as whole bone marrow culture method）. After in vitro amplification, passage 3 bone marrow mesenchymal stem cells were cultured with common culture medium （control group） and osteoblast induction culture medium （experimental group） RESULTS AND CONCLUSION： A large number of high purity bone marrow mesenchymal stem ceils were successfully obtained. After osteogenic induction, the content of osteocalcin in the experimental group was significantly higher than that in the control group （P 〈 0.05）. Alkaline phosphatase and calcium tubercle staining were positive in the experimental group, but they were negative in the control group. These findings suggest that density gradient centrifugation can be used to isolate and culture rabbit bone marrow mesenchymal stem cells, and using this method, bone marrow mesenchymal stern cells can be induce-differentiated into osteoblasts.