局部注射内皮祖细胞改善糖尿病大鼠后肢血管病变

Local injection of endothelial progenitor cells can improve the hind-limb ischemic vascular disorder of diabetic rats

目的探讨局部注射同种异体内皮祖细胞（EPCs）对糖尿病大鼠后肢血管病变的作用，并探讨其可能机制。方法腹腔注射链佐星制备SD大鼠糖尿病模型，然后将其两后肢股动脉结扎，制成血管病变模型。将模型分为两组，移植组大鼠左后肢为实验侧，自结扎部位以下每0．5cm注射分离白糖尿病大鼠外周血的以荧光染料标记的EPCs悬液0．5ml（含EPCs2．5×10^5个），右后肢为对照侧，注射等量的磷酸盐缓冲液；空白对照组不注射任何溶液。注射EPCs后1周，采用酶联免疫吸附试验检测后肢局部肌肉组织中血管内皮生长因子（VEGF）的表达；注射EPOs后28d，切取局部肌肉组织，荧光显微镜下观察组织切片中荧光染料的染色情况，免疫组织化学法检测von Willebrand因子（vWF）表达情况，并计算毛细血管密度。结果注射EPCs后1周，移植组实验侧组织中VEGF含量高于对照侧和空白对照组，差异有统计学意义（P〈0．05），而空白对照组和对照侧的差异无统计学意义（P〉0．05）。注射EPCs后28d，荧光显微镜下可见注射部位组织中蓝染的毛细血管内皮细胞；注射EPCs的左后肢局部毛细血管密度高于未注射的对照侧和空白对照组，差异有统计学意义（P〈0．05）。结论同种异体EPCs注射至糖尿病大鼠缺血后肢能分化为毛细血管内皮细胞，从而改善局部供血，局部VEGF分泌增加可能参与这一过程。

Objective To study the effect of allotype endothelial progenitor cells （EPCs） injection on the hind-limb ischemic vascular disorder of diabetic rats. Methods EPCs of diabetic rats were isolated and cultured. Immunofluorescent assays were used to detect the expression of CD34, CD133 and Flk-1. EPCs were injected locally on the ischemic hind-limb area after labeled by DAPI. ELISA assays were used to detect the expression of VEGF in the ischemic area. Immunohistocheraistry was performed to detect the expression of vWF and the number of positive stained capillary was counted. Results EPCs from diabetic rats were successfully isolated and cultured and their phenotypes were CD34^＋ CD133^＋ Flk-1^＋. The labeled EPCs were successfully implanted into the ischemic area and differentiated into endothelial cells. The secretion level of VEGF were significantly increased one week after transplantation. The density of capillary in the ischemic area was increased 4 weeks after transplantation, too. Conclusion Allotype EPCs can differentiate into endothelial cells after implanted into the hind-limb ischemic area of diabetic rats. The possible role is to increase the secretion level of VEGF.