关键词: 松质骨, 骨髓间充质干细胞, 分离, 培养, 鉴定, 成骨分化, 成脂肪分化, 干细胞

Abstract:

BACKGROUND: Experiments for the isolation and culture of rat or rabbit bone marrow mesenchymal stem cells are more common, but the clinical experiments of tissue engineering are often on the basis of the seed cells of the same species.
OBJECTIVE: To establish a protocol for isolating and culturing human bone marrow mesenchymal stem cells which is stable and efficient and consistent with the needs of clinical and laboratory experiments.
METHODS: Bone marrow and spongy bone were obtained from the human anterior superior iliac spine, and then the bone marrow, the spongy bone was flushed by 15 mL culture medium and then the rinse solution was collected. The cells were primary screened on the basis of adhesion function by direct cultivation method; the surface antigens, including CD29, CD34, CD44, CD45 and CD105 were detected by flow cytometry. Bone marrow mesenchymal stem cells were differentiated into osteoblasts and adipocytes, and the differentiated mesenchymal stem cells were identified by morphological observation.
RESULTS AND CONCLUSION: Cell colony could be seen at 5 days after cultured with spongy bone washing liquid. These cells were uniformly negative for CD34, CD45 and positive for CD29, CD44 and CD105. Calcium nodules were observed after osteogenic induction for 20 days and positive for alizarin red staining. The lipid droplets could be seen after adipogenic induction for 7 days and oil red O staining showed a large number of lipid deposition. The study shows that a large amount of mesenchymal stem cells can be isolated and cultured from adult human spongy bone in short time by direct cultivation methods, and the mesenchymal stem cells are uniform in morphology, the cells can express the antigens stably and differentiate into osteoblasts and adipocytes.