摘要
目的 研究脐血来源血管内皮祖细胞 (endothelialprogenitorcells,EPC)的移植对局部缺血的恢复作用。方法 脐血CD133+ 细胞在体外诱导扩增 10～ 14d后 ,收集贴壁梭形细胞 ,检测其内皮细胞特异标志的表达。将荧光标记的贴壁细胞从尾静脉移植到后肢单侧缺血的裸鼠体内。结果从脐血CD133+ 细胞中可诱导出EPC ,表现为表达内皮细胞标志的贴壁细胞。移植后 ,可整合到缺血部位新生血管内。移植EPC后裸鼠缺血后肢的毛细血管密度、血流灌注及坏死程度均较对照组明显改善 :缺血手术后 2周EPC组的缺血肢 /正常肢的血流比由 19 1%± 3 1%恢复为 77 3%± 5 6 % ,而对照组仅恢复为 4 0 6 %± 3 4 % (P <0 0 0 1) ;缺血后肢的完全恢复率由对照组的 1/10上升至 7/12 (P <0 0 5 )。缺血局部VEGFmRNA上调 ,体外VEGF对EPC具有趋化作用。 4h内趋化到下腔的细胞数为817个± 33个 ,而对照组为 4 74个± 6 2个 (P <0 0 5 )。结论 脐血CD133+ 细胞能诱导EPC ,体内移植后能促进肢体缺血的恢复 ,而缺血局部上调的VEGF表达可能是EPC定向整合到缺血局部的关键因素。
Objective To investigate the feasibility of transplanting cord blood CD133 + cells derived endothelial progenitor cells (EPC) in therapeutic vasculogenesis. Methods CD133 + cells from the cord blood of 52 neonates were cultured in fibronectin-coated flask in M199 medium supplemented with 10% fetal bovine serum, 50 ng/ml vascular endothelial growth factor (VEGF), 20 ng/ml interleukin-3 (IL-3) and 50 ng/ml stem cell factor (SCF). The cell markers of spindle-shaped adherent cells were determined with flow cytometry. The left femoral artery, great saphenous artery, iliac circumflex artery, and vein, and muscular branch of 22 Balb/c nude mice were cut to cause limb ischemia. One day after the unilateral ischemic limb surgery half million adherent cells were transplanted into 12 nude mice via tail vein (EPC group) and M199 was injected into the tail veins of 10 nude mice (M199 group). Fluorescence Dil, laser Doppler perfasion imaginer (LDPI) and immunohistochermistry were Laser Doppler perfusion imaginer (LDPI) was used to trace the transplanted cells and monitor the blood perfusion and capillary density of ischemic limbs. The ratio between the blood perfusion of the operated limb and of the non-operated opposite limb was recorded. Two to four mice in each group were killed 4, 7, 14, and 21 days after the operation and the gastrocnemius muscles of bilateral hind limbs were taken to count the number of capillaries. The VEGF mRNA levels of the ischemic and nonischemic limbs were examined with semi-quantitative RT-PCR. Seven days after the operation, fluorescein isothiocyanate (FITC)-binded ulex europaeus agglutinin-1 (UEA-1) was injected via tail vein to 3 EPC group mice. Thirty minutes later, the mice were killed. The heart, lung, liver, spleen and limb muscles were taken and examined with fluorescence microscopy. EPC were added into the upper chamber of Coster Transwell and chemotactic fluids of M199 with or without VEGE were added into the lower chamber. Four hours later the number of EPC in the lower chamber was counted so as to examine t he chemotactic effect of VEGE. Results Numerous cell clustersc; spindle-shaped adherent cells and cord-like structures, developed from the culture of cord blood CD133 + cells. These adherent cells expressed vascular endothelial growth factor receptor 2 (VEGFR-2), VE-cadherin, CD31, von Willebrand factor (vWF) and combined with ulex europaeus agglutinin-1 (UEA-1). Transplanted EPC survived and were incorporated into the capillary networks in the ischemic limbs of nude mice. The ratio between the blood perfusion of the ischemic limb and non-ischenmic limbs was 19.1%±3.1%. Two weeks after the transplantation, the ratio between the blood perfusion of the ischemic limb and non-ischenmic limbs of the EPC group was 77.3%±5.6%, significantly higher than that of the M199 group (40.6%±3.4%, P <0.001). CD31 histochemical staining showed that the density of capillaries in the gastrocnemius muscles of ischemic hind limb was significantly higher 7, 14, and 21 days after operation in the EPC group than in the M199 group ( P <0.05) RT-PCR showed obvious VEGF bands in the ischemic hind limb muscles, but not in the non-ischemic muscles. The number of EPC immigrating into the lower chamber of the Costa transwell was 817±32.5, significantly higher than that of the control group (473.5±61.5, P <0.05). Conclusion Cord blood CD133 + cells derived EPC is a robust cell source for therapeutic neovascularization. Upregulated expression of VEGF may account for the homing of transplanted EPC to ischemic tissue.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2003年第16期1437-1441,共5页
National Medical Journal of China
基金
国家重点基础研究发展规划资助项目 (0 0 1CB5 10 1)
国家攀登计划资助项目 (95 专 10 )
