低渗结合自然沉降法分离兔骨髓间充质干细胞及其鉴定

背景：骨髓间充质干细胞数量极少,10万个骨髓有核细胞中才有1个骨髓间充质干细胞,如何成功分离并鉴定骨髓间充质干细胞是研究中的关键工作。 目的：建立完善的骨髓间充质干细胞体外分离培养体系,从细胞的形态、表型和功能方面鉴定骨髓间充质干细胞。 方法：低渗原理去除杂细胞,结合自然沉降分离骨髓间充质细胞,并对其形态学特征进行光镜观察。对骨髓间充质细胞的表型进行流式细胞仪检测。并将骨髓间充质细胞向成骨细胞和脂肪细胞进行诱导分化,行形态学观察和碱性磷酸酶、Von kossa、Ⅰ型胶原、油红O染色检测。 结果与结论：实验分离获取的骨髓间充质细胞经表型鉴定为：CD29(+),CD45(-);向成骨细胞诱导后,碱性磷酸酶、矿化结节染色及Ⅰ型胶原检测均为阳性;向脂肪细胞诱导后,油红O染色阳性。结果表明实验分离的骨髓间充质细胞为类成纤维细胞样非造血类干细胞,此类细胞具有向成骨细胞和脂肪细胞分化的功能。

Isolation and identification of rabbit bone marrow mesenchymal stem cells by low permeability combined with natural sedimentation

BACKGROUND:The number of bone marrow mesenchymal stem cells (BMSCs) was few. 100 000 bone marrow karyocytes contain 1 BMSC. How to successfully isolate and identify BMSCs is a key in the study. OBJECTIVE: To establish a perfect culture system in vitro of BMSCs; BMSCs were identified from cell morphology, phenotype and functional aspects. METHODS:Low permeability principle was used to remove mixed cell, and combined with the natural sedimentation to isolate BMSCs. Their morphological characteristics were observed by light microscopy. The cell phenotype of BMSCs was determined by flow cytometry. BMSCs were induced into osteoblasts and adipocytes. Cell morphology was observed and alkaline phosphatase (AKP), Von Kossa, collagen typeⅠ, oil red O detection were performed. RESULTS AND CONCLUSION: BMSCs were identified as phenotype: CD29(+), CD45(-). Following being induced into osteoblasts, AKP, mineralized nodules and collagen type Ⅰ staining were detected positively; to adipocytes, the oil red O staining was detected positively. Results suggest that isolated BMSCs are non-hematopoietic stem cells with fibroblast-like cell morphology. These cells had the functions of differentiating into osteoblasts and adipocytes.