大鼠肌源性干细胞的分离、纯化和培养☆

背景：要获得满足临床需要的肌源性干细胞，体外筛选和扩增已成为关键环节。 目的：拟建立稳定高效的成年大鼠肌源性干细胞的分离培养、纯化方法。 方法：成年SD大鼠麻醉后，无菌条件下取骨骼肌，采用XI型胶原酶、Dispase和胰酶消化法获得肌源性干细胞，以密度梯度离心法和差速贴壁法进行纯化。记录细胞生长曲线，MTT比色法观察不同接种密度对细胞生长的影响，采用免疫细胞化学染色对细胞进行鉴定。 结果与结论：原代肌源性干细胞体积较小，贴壁缓慢，折光性较好，多呈球形、梭形或纺锤形，增殖缓慢。传代培养后，加入含体积分数为20%血清浓度的完全培养基，以1×109 L-1密度接种时活细胞数量最多，为适宜接种密度，传1~4代细胞生长状态良好。免疫细胞化学染色结果为desmin(+)，CD34(+)，CD45(-)，Sca-1(+)，证实通过体外原代培养获得了高纯度的肌源性干细胞，并成功扩增。

1101/Isolation, purification and cultivation of muscle derived stem cells from adult rats

BACKGROUND: In vitro screening and amplification are important links to harvest muscle-derived stem cells that are satisfactory to clinical requirement. OBJECTIVE: To probe into the method of isolation, culture and purification of skeletal muscle-derived stem cells from adult rats in vitro. METHODS: The skeletal muscle was obtained sterilely following adult Sprague Dawley rats were anesthetized. Muscle-derived stem cells were harvested using enzyme digestion with XI collagenase, Dispase and trypsogen, and then purified by Percoll density gradient centrifugation and differential adhesion method. Growth curves were recorded and MTT colorimetric technique was used to describe the effects of various kinds of inoculum density on cell growth. Cells were identified by immunocytochemistry. RESULTS AND CONCLUSION: Primary muscle-derived stem cells were less in volume, lower adherence and well refraction, appearing as globular or fusiform or spindle and slowly multiplication. Following subculture, complete medium containing 20% serum was added. Cell number was greatest when cell density was 1×109/L, which was the optimal density. Cells at passages 1-4 grew well. Cells showed desmin(+), CD34(+), CD45(-) and Sca-1(+) by immunocytochemistry. Results verified that high-purity muscle-derived stem cells can be obtained in vitro and amplified successfully following primary culture.