【摘要】 目的建立两种检测外周血平滑肌祖细胞(SMPCs)的绝对计数方法,评估了方法的观察者内和观察者间差异及初步应用。方法小鼠眼眶取血样本经抗体孵育后使用"裂解/一次洗涤法"处理,流式细胞仪检测。SMPCs定义为c-kit阴性/CD140b阳性/lineage阴性/sca-1阳性。SMPCs计算方法包括"微球法"和"流式直接体积法"。分别在采血后0、24和48 h后进行检测。SCI模型使用Allen’s打击装置造模,检测伤后24 h的SMPCs。结果微球法和直接体积法的观察者内可靠系数分别是0.947和0.839;观察者间可靠系数分别是0.911和0.768。SMPCs在血液样本中不稳定,静置24和48 h后丢失较多。SCI后24 h,SMPCs的数量较正常组和假手术组明显升高。结论建立了两种SMPCs绝对计数法,有较好的观察者内一致性和观察者间一致性。应用两种方法能够有效的检测到小鼠SCI后SMPCs的升高,为探讨其变化的意义提供了方法和依据。更多还原

【Abstract】 Objective To check absolute SMPCs count in mice with acute spinal cord injury. Methods The blood samples were obtained from Institute of Cancer Research( ICR). The samples were processed through now lyse /one-wash procedure and c-kit-/ CD140 b + / lineage-/ sca-1 + SMPCs were analyzed by exclusion of dead cells and by fluorescence-minus-one control. SMPCs were enumerated by two methods: 1) bead-based 123 count e Beads ( e Bioscience) Count; 2) ‘direct volume’-based Accuri C6 Flow Cytometer( BD) Count. The cells were immediately measured and after storage of blood samples for 24 h and 48 h,respectively. The SCI in mice were induced by Allen’s device and the number of SMPCs was detected 24 h after SCI. Results There were good intra-observer and inter-observer agreement in both methods. SMPCs were unstable in blood samples. The number of SMPCs increased after 24 h in spinal cord injury( SCI) mice. Conclusions Two reproducible protocols for SMPCs quantification are established. Acute spinal cord injury increases the smooth muscle progenitor cells in mice. These protocols should facilitate future studies to further define the role of SMPCs as cellular biomarkers in SCI.更多还原