【作者】 李抒；

【导师】 韩忠朝；

【作者基本信息】 中国协和医科大学 ， 内科学， 2006， 博士

【摘要】 用G-CSF动员的外周血单个核细胞(PBMNCs)自体移植能够改进下肢闭塞性动脉硬化和糖尿病足的肢体缺血状况。然而动员的外周血单个核细胞治疗肢体缺血的机制却不清楚。我们对后肢缺血模型裸鼠移植CD34~+的动员的外周血单个核细胞和CD34去除的动员的外周血单个核细胞，然后进行疗效的比较，以便进一步探讨PBMNCs的治疗机制。CM-DiI荧光标记后的人的动员的外周血单个核细胞[1×10~6]局部肌肉注射移植到单侧后肢缺血的裸鼠体内。在细胞移植后的3天、7天、14天、21天、28天进行了血流灌注、毛细血管密度、及缺血后肢坏死情况的动态观察。与PBS对照组比较，CD34~+和CD34去除的PBMNCs组的血流恢复程度，毛细血管密度以及缺血后肢的恢复情况都有明显改善：当进行CD34~+和CD34去除的PBMNCs两组比较时，结果发现CD34去除的PBMNCs组的治疗效应减弱。荧光示踪发现移植细胞聚集在小动脉周围或者散在的分布于毛细血管网，在CD34~+的PBMNCs移植组可以观察到移植细胞整合到缺血部位的新生毛细血管内，但在CD34去除的PBMNCs移植组没有观察到这样的现象。用Western blotting和ELISA方法检查表明缺血肌肉有明显升高的VEGF表达，同时发现红色荧光标记的移植细胞与表达VEGF的绿色免疫荧光重叠现象，提示VEGF是由移植的PBMNCs局部分泌的。上述研究结果表明移植G-CSF动员的PBMNCs不但可以通过整合到血管壁的机制促进血管生长，还可以通过提供细胞因子特别是VEGF的机制促进血管生长。CD34去除削弱了动员的PBMNCs移植治疗肢体缺血的血管新生效应。更多还原

【Abstract】 Objective Autolougous transplantation of granulocyte colony-stimulating factor (G-CSF)-mobilized human peripheral blood mononuclear cells (PBMNCs) improves limb ischemia in patients with arteriosclerosis obliterans of lower extremities and with diabetic foot. However, the mechanism of action of PBMNCs remains elusive. The present study was designed to investigate the role of CD34~+ stem/progeitor cells in neovascularization by comparing the therapeutic effects of G-CSF-mobilized PBMNCs and CD34-depleted G-CSF-mobilized PBMNCs transplantation in ischemic hindlimbs of nude mice. We also tried to reveal the mechanisms of action through which the transplanted cells effectively acted in vivo in improving critical limb ischemia Methods. Peripheral blood leukapheresis products were obtained from healthy volunteers who received subcutaneous administration of G-CSF. Mononuclear cells were separately isolated from the human products of leukapheresis by Ficoll-Paque density gradient centrifugation. The G-CSF-mobilized PBMNCs were divided into two parts. One part was the G-CSF-mobilized PBMNCs and the other part was CD34-depleted更多还原