一种改良人骨髓间充质干细胞分离培养的方法

背景：分离培养鼠、兔骨髓间充质干细胞的科研实践较多，而组织工程临床实践人多以同种属种子细胞的科研为基础。目的：建立一种稳定、高效，满足临床和实验审需要的人骨髓问充质干细胞分离培养方法。方法：使用骨穿针于人髂前上棘处抽取骨髓和松质侣’，用15mL培养体系冲洗松质骨并收集冲洗液，采用直接培养法，利用细胞贴壁性能初筛细胞；流式细胞仪枪测细胞表面抗原（CD29、CD34、CD44、CD45、CDl05），分别进行成骨、成脂肪诱导分化，在诱导过程中进行细胞形态学观察对干细胞进行初步豁定。结果与结论：松质骨冲洗液直接培养法在培养第5天的时候出现细胞集落，细胞稳定表达CD29、CD44、CDl05，不表达CD34、CD45。成骨诱导20d后出现明显钙结节，茜素红染色呈红色结节。成脂诱导7d后脂滴明显形成，油红素O染色见大量脂质沉淀。结果可见采用松质骨冲洗液直接培养法可以从人松质骨中短时间内获得大量间充质干细胞，其具有良好的细胞形态，稳定表达的细胞抗原及成骨、成脂分化能力。

A modified method of isolating and culturing human bone marrow mesenchymal stem cells

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 lilac spine, and then the bone marrow, the spongy bone was flushed by 15 mL culture medium and then the rinse solution was collected. The ce~ls 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.