脐血内皮祖细胞治疗糖尿病大鼠下肢缺血的实验研究

摘要

目的 研究脐血内皮祖细胞(EPC)治疗糖尿病下肢缺血的有效性及机制,为临床治疗糖尿病足病(DF)提供理论依据. 方法 取产妇足月产脐血分离单核细胞,培养7d,流式细胞仪鉴定细胞,并计数活细胞数.将Wistar雄性大鼠分成3组:(1)糖尿病大鼠射线照射后结扎双后肢股动脉,尾静脉注射血管EPC(DE组).(2)糖尿病大鼠结扎双后肢股动脉,尾静脉注射缓冲液(DP组).(3)正常大鼠射线照射后结扎双后肢,尾静脉注射EPC(NC组).用绿色荧光(GFP)示踪EPC,Ⅷ因子免疫组化检测肌纤维间毛细血管数,RT-PCR检测双后肢肌肉血管内皮生长因子(VEGF) mRNA水平. 结果 DE、NC组右后肢腓肠肌溃疡及缺血明显好转,有明显荧光,肌纤维间毛细血管数多,VEGF表达高,DP组好转不明显,无荧光,毛细血管数少,VEGF表达少,其差异有统计学意义(P＜0.05). 结论 脐血EPC治疗糖尿病大鼠下肢缺血有效.%Objective To study the therapeutis efficacy and its mechanism of umbilical cord blood endothelial progenitor cells (EPC) treatment for lower limb ischemia of diabetic rats. Methods The umbilical cord blood mononuclear cells of parturients with full-term delivery were cultured for 7 days. The cells were identified by flow cytometry and the living cells were counted. Wistar male rats were divided into three groups: (1) DE group, in which the ligation of the femoral artery of the lower limbs of diabetic rats was performed after the γ-ray radiation for 7～10 days, then a tail intravenous EPC transplantation was done; (2) DP group, in which the ligation of the femoral artery of the lower limbs of diabetic rats was adopted after the γ-ray radiation for 7～10 days, then a tail intravenous PBS injection was given; and (3) Control group, in which the ligation of the femoral artery of the lower limbs of normal rats was adopted after the y-ray radiation for 7～10 days, then a tail intravenous EPC transplantation was realized. A green fluorescent photometer (GRP) was used to trace the EPC. The Ⅷ factor immunohistochemical stain was applied to detect the number of capillaries between the muscular fibers. The RT-PCR detection was employed to exam the VEGF mRNA level of the muscles of lower limbs. Results In the DE group and control group, the ulcers and ischemia of the gastrocnemius muscles of the right lower limbs were significantly improved, which was manifested as bright fluorescence, more capillaries between the muscular fibers, and increased VEGF expression. In the DP group, the improvement was not obvious, which was manifested as without fluorescence, less capillaries, and weak VEGF expression. The difference among the groups was statistically significant (all P<0. 05). Conclusion The treatment with EPC for the ischemia of lower limbs of diabetic rats is effective.