人股骨头骨微血管内皮细胞的分离培养方法

发布时间：2018-12-05 20:01

【摘要】：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"鹅卵石"状。培养7~10 d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。
[Abstract]:Objective: to investigate the method of isolation and culture of human femoral head microvascular endothelial cells. Methods: from October 2013 to January 2014, 15 patients (2 males and 13 females) were treated with hip arthroplasty. The average age was 71.2 years (38 / 92). The cancellous bone in the femoral head was bitten into broken bone under aseptic condition and put into culture medium. The cells were isolated by enzyme digestion and density gradient centrifugation, and purified by differential adhesion method and selective medium method. The characteristics of cells were observed by inverted microscope and identified by vWF,CD31 immunofluorescence. Results: after 24 hours of primary culture, the number of cells observed under inverted microscope was positively correlated with the patient's age, and the older the cells were, the less the cells were. The cells were short fusiform, polygonal or cobblestone. After 7 days of culture, the cells grew dense, the cell fusion was whirlpool and the contact inhibition was obvious. The positive rate of vWF,CD31 immunofluorescence was 100, which indicated that the cells were bone microvascular endothelial cells. Conclusion: the method of isolation and culture of human femoral head microvascular endothelial cells is simple, stable, effective and reproducible.
【作者单位】：中日友好医院骨关节外科;北京协和医学院研究生院;中日友好医院临床医学研究所;
【基金】：国家自然科学基金资助项目(编号:81273972)~~
【分类号】：R329.2

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