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背景糖尿病小鼠呈现缺血诱导的骨髓内皮祖细胞(EPC)动员障碍,但其分子机制目前尚未明了。目的观察缺血诱导的EPC动员障碍是否与糖尿病状态下骨髓EPC动员相关的信号通路改变有关。方法链脲霉素40mg/kg诱导C57BL/6雄鼠糖尿病,左侧股动脉高位结扎离断术造成单侧后肢缺血模型。于术前及术后1、3、7d不同时间点采血(n=6～10),流式细胞术检测外周血单个核细胞中细胞因子受体(c-Kit)+/干细胞抗原1(Sca-1)+/胎肝激酶1(Flk-1)+早期EPC比例。酶联免疫吸附测定(ELISA)法测定相应时间点血浆及骨髓血管内皮生长因子(VEGF)及间充质衍生因子1α(SDF-1α)水平。免疫印迹法测定术后3d骨髓单个核细胞VEGF及其受体2(VEGFR2)、蛋白激酶B(PKB,Akt)与内皮型一氧化氮合酶(eNOS)及其磷酸化产物、基质金属蛋白酶9(MMP-9)及其组织抑制因子1(TIMP-1)的蛋白表达水平,明胶酶谱法测定骨髓上清MMP-9酶活性。结果与非糖尿病组相比,糖尿病小鼠呈现缺血诱导的骨髓EPC动员障碍,动员高峰期其外周血早期EPC数量显著减少,并伴随血浆及骨髓中SDF-1α及VEGF释放减少[VEGF(85.3±22.8)比(164.1±30.4)ng/L,P<0.01;SDF-1α:(731±52)比(1267±174)ng/L,P<0.01],骨髓组织细胞VEGF、VEGFR2蛋白表达受抑、Akt与eNOS磷酸化减弱、MMP-9蛋白表达减少及其酶活性受损(P<0.05),而TIMP-1蛋白表达水平增加(P<0.05),且糖尿病组骨髓MMP-9酶活性受抑与其循环中EPC水平减低相关(P<0.01)。结论糖尿病动物缺血诱导的骨髓EPC动员障碍可能与组织缺血后SDF-1α/VEGF/Akt/eNOS/MMP-9信号通路的活化受损有关。
BACKGROUND Diabetic mice show ischemia-induced mobilization of bone marrow endothelial progenitor cells (EPCs), but the molecular mechanisms are not yet clear. Objective To observe whether ischemic-induced EPC mobilization disorder is related to the alteration of the signal pathways involved in the mobilization of bone marrow EPCs under diabetic conditions. Methods Streptozotocin 40 mg / kg induced C57BL / 6 male diabetic rats, left femoral artery ligation and disconnection caused unilateral hindlimb ischemia model. Blood samples were collected before surgery and at different time points (n = 6 ~ 10) at 1, 3 and 7 days after operation. Flow cytometry was used to detect the levels of c-Kit / stem cell antigen 1 -1) + / fetal liver kinase 1 (Flk-1) + early EPC ratio. The levels of vascular endothelial growth factor (VEGF) and mesenchymal stem cell factor 1α (SDF-1α) in plasma and bone marrow at the corresponding time points were determined by enzyme linked immunosorbent assay (ELISA). The expression of VEGFR2, PKB, Akt, eNOS and their phosphorylated products in bone marrow mononuclear cells were detected by Western blotting three days after operation. The expressions of matrix metalloproteinase 9 (MMP-9) and its tissue inhibitor of metalloproteinase-1 (TIMP-1) protein levels were determined by enzyme-linked immunosorbent assay. MMP-9 activity was measured by gelatin zymography. Results Compared with non-diabetic group, diabetic mice exhibited ischemic-induced mobilization of bone marrow EPCs with significantly reduced number of early EPCs in the peripheral blood during mobilization and concomitant reduction of SDF-1α and VEGF release in plasma and bone marrow [VEGF (85.3 (22.1 ± 30.4) ng / L, P <0.01; SDF-1α: (731 ± 52) vs (1267 ± 174) ng / L, P <0.01). The expression of VEGF and VEGFR2 in bone marrow The phosphorylation of Akt and eNOS decreased, the expression of MMP-9 decreased, the activity of MMP-9 decreased (P <0.05) and the expression of TIMP-1 protein increased (P <0.05) Inhibition was associated with a decrease in circulating EPCs (P <0.01). Conclusion The imbalance of mobilization of bone marrow EPC induced by ischemia in diabetic animals may be related to the impaired activation of SDF-1α / VEGF / Akt / eNOS / MMP-9 signaling pathway after tissue ischemia.