脊柱手术出血中间充质干细胞的分离鉴定及数量变化

背景：临床骨科松质骨区手术中有较多红骨髓随术区出血而浪费丢失。 目的：分析从脊柱手术出血中分离骨髓间充质干细胞的可行性及骨髓间充质干细胞数与收集血液时间和年龄的关系。 方法：入选3 例腰椎退变不稳和16例腰椎爆裂骨折患者,20~30岁 4例、30~40岁 5例、40~50岁5例、50~60岁 2例,64岁1例、65岁1例、67岁1例,均行后路椎弓根钉棒系统内固定治疗。术中手锥钻开椎弓根拔出手锥后立即收集0~5 min时段的钉道出血标本4例各5 mL,用于细胞鉴定。另15例拔出手锥后0~5 min、5~10 min、10~15 min 3个时段,取椎骨出血标本各5 mL分离出其中的单个核细胞接种于6孔板,培养3 d后全量换液除去未贴壁细胞、苏木精原位染色并计数。 结果与结论：免疫细胞化学法鉴定显示贴壁细胞细胞膜抗原CD34、CD14表达阴性,CD44、CD29表达阳性。0~5 min时间段的椎骨出血标本中的贴壁细胞数为262.47±172.20,5 min以后出血标本中的贴壁细胞数极少甚至没有。0~5 min时间段组血液标本中的骨髓间充质干细胞数随年龄增长而减少。

A preliminary study of the number and identification of mesenchymal stem cells in spinal surgery bleeding

BACKGROUND:A great amount of red bone marrow in the cancellous bone losses during orthopedic surgery. OBJECTIVE:To explore the feasibility to isolate bone marrow mesenchymal stem cells (BMSCs) during spinal surgery bleeding and to investigate the relationship of the number of BMMSCs with age and time. METHODS:Three cases of degenerative lumbar instability and 16 cases of lumbar burst fractures were selected to undergo posterior pedicle screw fixation, including 4 cases of 20-30 years, 5 of 30-40 years, 5 of 40-50 years, 2 of 50-60 years, 1 of 64 years, 1 of 65 years, and 1 of 67 years. 5 mL of vertebra blood was collected from 4 cases within 0-5 minutes after the awl for pierced vertebra was pulled out, and 5 mL of vertebral blood from other 15 cases was collected within 0-5, 5-10 and 10-15 minutes after the awl was pulled out, respectively. Mononuclear cells were isolated and incubated into 6-well plates, and then cultured for 3 days. The medium was renewed completely to remove unadherent cells. Hematoxylin-eosin staining was applied for cell counting. RESULTS AND CONCLUSION:The membrane mark showed that CD44 and CD29 were positive while the CD34 and CD14 were negative. The number of BMSCs isolated within 0-5 minutes was 262.47±172.20, and fewer or no BMSCs were found within 5-10 or 10-15 minutes. The number of BMMSCs from vertebra bleeding of 0-5 minutes had a decreased tendency with aging.