四氧嘧啶诱导的糖尿病对兔骨髓内皮祖细胞及循环内皮祖细胞功能的影响

Effect of Alloxan-induced diabetes mellitus on the functions of bone marrow-derived and circulating endothelial progenitor cells

目的 探讨糖尿病对兔骨髓内皮祖细胞（EPC）与循环EPC功能的影响，并观察骨髓EPC和循环EPC自体移植对糖尿病兔急性心肌缺血的改善作用。方法 四氧嘧啶法制造家兔糖尿病模型，8周后糖尿病兔分别抽取骨髓或外周血后于体外培养骨髓EPC及循环EPC，进行细胞鉴定并分别测定两种细胞的集落数、增殖力、黏附功能、成血管能力。糖尿病兔随机分为骨髓EPC组（n＝6）、循环EPC组（n＝6）及糖尿病对照组（n＝6），并选取正常家兔作为正常对照组（n＝6）进行细胞培养实验。骨髓EPC组、循环EPC组及糖尿病对照组兔制造急性心肌缺血模型后，分别心肌内自体移植骨髓EPC、循环EPC及培养液。4周后激光共聚焦显微镜观察移植细胞，Masson染色测定心肌纤维化面积，免疫组化法测定血管密度，超声心动图分析心功能的变化，实时定量PCR法测定血管内皮生长因子（VEGF）及碱性成纤维细胞生长因子（bFGF）的mRNA表达水平。结果 与正常对照兔比较，糖尿病兔循环EPC的集落数、增殖力、黏附力、成血管能力下降（均P〈0.05），骨髓EPC的集落数、增殖力、黏附力下降，但与正常对照兔比较差异均无统计学意义（均P〉0.05），成血管能力较正常对照兔下降（P＝0.023）。与循环EPC组及糖尿病对照组比较，骨髓EPC组心肌纤维化面积较小（6.98%±0.94%比13.03%±2.97%、15.84%±4.74%，均P＝0.001），血管密度[（792±87）比（528±71）、（372±77）条/mm2，均P〈0.001]较大，VEGF（6.25±2.33比2.19±1.01、1.55±0.52，均P〈0.001）及bFGF（6.38±2.65比1.24±0.76、1.18±0.82，均P〈0.001）的mRNA表达水平较高，左心室射血分数（61%±4%比47%±5%、50%±10%，均P〈0.05）较高。与糖尿病对照组比较，循环EPC组血管密度增加，但心肌纤维化面积、VEGF和bFGF的mRNA表达水平、左心室射血分数差异无统计学意义。结论 糖尿病损害了兔循环EPC功能，也影响了骨髓EPC成血管功能；糖尿病兔骨髓EPC自体移植可通过促血管新生及旁分泌作用减少心肌纤维化并改善心功能。

Objective To explore whether diabetes mellitus （DM） impairs functions of bone marrow-derived endothelial progenitor cells （BM-EPC） and circulating EPC.Methods Diabetic model of rabbit was induced by Alloxan injection and the rabbits were then randomly divided into three groups： BM-EPC group, circulating EPC group, and DM group, with six rabbits in each group. Another 6 normal rabbits were enrolled as normal control group as well. 8 weeks later, BM-EPC and circulating EPC from diabetic and healthy rabbits were isolated and cultured. Colony number, proliferation, adhesion and tube formation function were detected. Exogenous diabetic BM-EPC and circulating EPC were analyzed for therapeutic efficacy in acute ischemia model of diabetic rabbits. Left ventricular （LV） function was assessed using Echocardiography. Capillary density and fibrosis area were evaluated by confocal laser scanning microscope （CLSM） and Masson-trichrome staining. The mRNA expression of vascular endothelial growth factor （VEGF） and basic fibroblast growth factor （bFGF） was analyzed using real-time quantitive PCR.Results Colony number, proliferation, adhesion and tube formation function of diabetic circulating EPC were significantly reduced compared with healthy rabbits. DM impaired tube-forming ability of BM-EPC, but did not influence colony number, proliferation and adhesion function. Compared with circulating EPC and control group, BM-EPC group had fewer fibrosis area （6.98%±0.94% vs 13.03%±2.97% and 15.84%±4.74%, both P=0.001）, higher capillary density [（792±87） vs （528±71） and （372±77） vessels/mm2, both P〈0.001], higher mRNA expression of VEGF （6.25±2.33 vs 2.19±1.01 and 1.55±0.52, both P〈0.001） and bFGF （6.38±2.65 vs 1.24±0.76 and 1.18±0.82, both P〈0.001）, higher left ventricular ejection fraction （LVEF） （61%±4% vs 47%±5% and 50%±10%, both P〈0.05）.Conclusions DM not only impaired functions of circulating EPC, but also influenced tube formation function of BM-EPC. Auto transplantation of BM-EPC may rescue the ischemic myocardium by neovascularization and paracrine effect in diabetic rabbits.