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目的观察糖尿病动物缺血诱导的骨髓内皮祖细胞(EPC)动员是否存在障碍,以及这种障碍是否和缺血诱导的血管内皮生长因子(VEGF)释放降低有关。方法链脲霉素40 mg/kg 诱导C57BI/6雄鼠糖尿病,非糖尿病组给予等量缓冲液。饲养2个月后,进行左侧股动脉高位结扎离断术造成后肢缺血模型,通过红四氮唑染色法与后肢血管造影确定造模成功。于术前及术后不同时间点采血(1天,3天,n=8;5天,7天及14天,n=5),三色流式细胞术检测两组动物外周血单个核细胞中c-Kit~+/Sca-1~+/flk-1~+早期 EPC 比例。ELISA 法测定相应时间点血浆 VEGF 水平。结果基础状态下,糖尿病组循环 EPC 数量较非糖尿病组明显减少[(0.60±0.03)%比(0.95±0.09)%,P<0.001],血浆 VEGF 水平低于试剂盒检测灵敏度。两组动物缺血诱导的骨髓早期 EPC 释放曲线相似,即术后1天显著增加,术后3天达峰,动员持续至2周以上。但是在 EPC 早期快速动员阶段(术后前3天),糖尿病组外周血早期 EPC 数量较非糖尿病组明显减少[1天,(1.16±0.29)%比(1.80±0.32)%,P<0.05;3天,(1.38±0.34)%比(2.37±0.52)%,P<0.05]。同时组织缺血也伴随着血浆 VEGF 浓度的显著增高:非糖尿病组血浆 VEGF 水平在术后一天快速增加并达到峰值,此后渐降至相对较低水平持续两周以上;而糖尿病组术后1天血浆 VEGF 快速释放明显降低[(73.1±18.6)pg/ml 比(128.5±44.2)pg/ml,P<0.05]。结论糖尿病动物基础状态下外周血早期 EPC 数量减少,组织缺血诱导的骨髓 EPC 动员障碍,这种障碍可能与缺血诱导的 VEGF 释放减少有关。
OBJECTIVE: To investigate whether ischemia-induced mobilization of bone marrow endothelial progenitor cells (EPCs) is associated with ischemia and whether this disorder is associated with decreased ischemia-induced vascular endothelial growth factor (VEGF) release. Methods Streptozotocin (40 mg / kg) induced diabetes in C57BI / 6 male rats, while non-diabetic rats received equal volume of buffer. After feeding for 2 months, the model of hindlimb ischemia was induced by high ligation and interruption of the left femoral artery. The model was successfully established by red tetrazolium staining and hindlimb angiography. Blood samples were collected before and at different time points after operation (1 day, 3 days, n = 8; 5 days, 7 days and 14 days, n = 5). Flow cytometry was used to detect the levels of peripheral blood mononuclear cells In the proportion of c-Kit ~ + / Sca-1 ~ + / flk-1 ~ + early EPC. Plasma VEGF levels were measured by ELISA at the corresponding time points. Results Under the basal conditions, the number of circulating EPCs in diabetic patients was significantly lower than that in non-diabetic patients (0.60 ± 0.03% vs 0.95 ± 0.09%, P <0.001). The plasma VEGF level was lower than the sensitivity of the kit. The two groups of animal ischemia-induced early bone marrow EPC release curve was similar, that was significantly increased after 1 day, 3 days after the peak, mobilization continued for more than 2 weeks. However, in the early stage of rapid EPC mobilization (3 days after surgery), the number of early EPCs in peripheral blood of diabetic patients was significantly lower than that of non - diabetic patients (1.16 ± 0.29% vs 1.80 ± 0.32%, P <0.05) 3 days, (1.38 ± 0.34)% vs (2.37 ± 0.52)%, P <0.05]. Tissue ischemia also accompanied by a significant increase in plasma VEGF concentrations: the level of plasma VEGF in non-diabetic patients increased rapidly after 1 day and peaked, then decreased to a relatively low level for more than two weeks; and diabetic patients after 1 day Rapid plasma VEGF release was significantly reduced (73.1 ± 18.6 pg / ml pg / ml (128.5 ± 44.2 pg / ml, P 0.05]. Conclusion The number of EPC in the peripheral blood of diabetic animals is reduced at the initial stage, and the ischemia-induced bone marrow EPC mobilization disorder may be related to the decrease of ischemia-induced VEGF release.