人股骨头骨微血管内皮细胞的分离培养方法

A method for isolated culture of bone microvascular endothelial cells of human femoral head

目的：探讨培养人股骨头骨微血管内皮细胞分离培养方法.方法：2013年10月至2014年1月15例行髋关节置换患者切除的内部无病变的股骨头,男2例,女13例;年龄38～92岁,平均71.2岁.无菌条件下将股骨头内松质骨咬成碎骨粒,放入培养基.采用酶消化法,密度梯度离心法分离细胞;差速贴壁法,选择性培养基法纯化细胞.倒置显微镜观察细胞特点,并采用vWF、CD31免疫荧光进行细胞鉴定.结果：原代培养24 h后倒置显微镜下观察细胞数量与患者年龄呈正相关,年龄越大细胞越少.培养4～5 d细胞呈短梭形、多角形或“鹩卵石”状.培养7～10d细胞生长密集,细胞融合呈漩涡状,接触抑制明显.vWF、CD31免疫荧光检测阳性率100％,表明细胞为骨微血管内皮细胞.结论：人股骨头骨微血管内皮细胞分离培养方法简单、稳定、有效、可重复性好,可以获得纯度较高的股骨头骨微血管内皮细胞.

Objcetive：To investigate the method of separation of culture of bone microvascular endothelial cells （BMECs） of human femoral head in vitro.Methods：From October 2013 to January 201 4,15 femoral heads without pathologic change from patients resected during hip replacement were selected involving 2 males and 13 females with a mean age of 71.2 years old ranging from 38 to 92.Cancellous bone in femoral head was bited into broken bone grain and transfered into medium in aseptic contidion.Cells were isolated by the methods of enzymic digestion and density gradient centrifugation,purified by differiential attachment.The characteristics of cells was observed by inverted microscope,vWF and CD31 immunofluorescence analysis was applied for identification of cells.Results：The number of cells was positively correlated with patients＇ age after 24 hours in primary culture.The older patients had the less cells numbered.After 4 to 5 days＇ culture,primary cells appeared short spindle,polygon shaped and cobblestone-like morphology.After 7 to 10 days＇ culture,primary cells proliferated densely,became fusion,arranged in swirl,and contact inhibition appeared significantly.Immunofluorescence staining revealed the cells were 100％ positive for vWF and CD31,and it showed that the cultured cells were BMECs.Conclusion：It was a simple,steady,effective method with good reproducibility,by which highly purified human BMECs can be obtained.