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目的 观察骨髓间充质干细胞 (Mesenchymalstemcells ,MSCs)在软骨细胞培养条件下传代培养的主要生物学特性。方法 梯度离心分离幼兔骨髓有核细胞 ,培养分离MSCs并进行传代培养 ,观测在体外软骨细胞培养条件下MSCs形态、生长特点及Ⅰ型和Ⅱ型胶原、蛋白多糖聚集体合成分泌以及碱性磷酸酶活性等方面的变化。结果 ①MSCs原代细胞呈可见的均匀分布的簇状生长 ,呈长梭形 ,在传代培养中形态特性无明显变化、均质性明显提高 ;②各代Ⅰ型胶原免疫组化均为阳性 ,Ⅱ型胶原免疫组化均为阴性。③在各代培养中碱性磷酸酶染色均为阴性 ,对甲苯胺蓝存在极弱的异染性反应 ;④细胞外基质中硫酸糖胺多糖含量亦很低。结论 传代培养中骨髓MSCs保持了常规低糖培养条件下的基本生物学特点 ,说明常用的软骨细胞基础培养条件同样适合骨髓MSCs的培养 .
Objective To observe the main biological characteristics of subcultured mesenchymal stem cells (MSCs) under the conditions of chondrocyte culture. Methods MSCs were separated by gradient centrifugation. MSCs were cultured and subcultured. The morphology and growth characteristics of MSCs and the synthesis and secretion of type I and type II collagen and proteoglycan aggregates were observed under chondrocyte culture in vitro. The effects of alkaline phosphatase Enzyme activity and other aspects of change. Results ① The primary cells of MSCs showed clustered growth in uniform distribution with a long fusiform shape. There was no obvious change in the morphological characteristics of subcultured MSCs, and the homogeneity was obviously improved. ② The immunohistochemical staining of type Ⅰ collagen in all generations was positive, Ⅱ Collagen immunohistochemistry were negative. ③ in all generations of culture were negative for alkaline phosphatase, p-toluidine blue there is a very weak allodynic reaction; ④ extracellular matrix sulfated glycosaminoglycans content is also very low. Conclusion MSCs in subculture maintain the basic biological characteristics of conventional low-glucose culture conditions, indicating that the basic culture conditions of chondrocytes are also suitable for the culture of bone marrow MSCs.