摘要
目的:研究人外周血来源内皮祖细胞(EPC)移植对改善肢体缺血的作用.方法:人外周血单个核细胞在体外诱导扩增6d和14d后,分别检测其EPC特异性标志的表达,并将荧光染料标记后的培养第6日的贴壁细胞通过缺血局部多点注射移植到后肢缺血的裸鼠动物模型体内,以评价其治疗效果.结果:人外周血单个核细胞经体外诱导分化,培养第6日的贴壁细胞表达KDR,CD133,CD34和vWF,流式细胞仪检测其阳性率分别为(46.4±9.3)%,(33.8±11.7)%,(73.5±6.3)%和(38.9±8.2)%;培养第14日的贴壁细胞KDR,CD34和vWF的表达阳性率均增高,而CD133表达阳性率明显降低,流式细胞仪检测其阳性率分别为(81.5±7.6)%,(88.9±5.4)%,(78.3±13.6)%和(3.8±8.7)%;移植EPC后裸鼠缺血后肢的坏死情况和毛细血管密度均较对照组明显改善(P<0.05);移植EPC后缺血后肢肌肉石蜡切片中可见分散不均的红色和黄绿色双色荧光的细胞掺入.结论:从人外周血单个核细胞中可诱导出EPC,而且移植的EPC可以定向整合到后肢缺血局部,明显改善裸鼠后肢缺血.
AIM： To estimate the effect of human peripheral blood derived-endothelial progenitor cell（EPC） transplantation on the repairment of limb ischemia. METHODS： The mononuclear cells from human peripheral blood were cultured in M199 medium supplemented with 10% fetal bovine serum, 20 ng/mL VEGF and 4ng/mL bFGF in vitro. The specific markers of EPC were analyzed by FCS at day 6 and 14 during culture respectively. The bilateral femoral artery, great saphenous artery, iliac circumflex artery, and muscular branch of 15 Balb/c nude mice were ligated to cause limb ischemia. The 6-day adherence cells marked by CM-DiI were multipoint injected into the left local muscles of ischemic limbs（ EPC group） and M199 was multipeint injected into the right local muscles of ischemic limbs （ M199 group） in order to evaluate the therapeutic effect. Seven days after operation, 60μg FITC-UEA was injected via tail vein. One hour later, the mice were killed. The heart,liver,lung, spleen, kidney and limb gastrocnemius were taken and examined with fluorescence microscopy. The number of capillaries of ischemic limbs was measured by factor V1 related antigen immunohistochemical staining. RESULTS： The cultured cells at day 6 expressed KDR, CD133, CD34 and vWF,and the positive rates of KDR, CD133, CD34 and vWF were （46.4±9.3）%, （33.8±11.7）%,（73.5±6.3）%and （38.9± 8.2 ） %, respectively. At the 14th day during culture, the cells expressed KDR, CD34 and vWF and the positive rates of KDR,CD34 and vWF were （81.5 ±7.6）%, （88.9 ± 5.4）% and （78.3± 13.6）%, respeetively; the positive rate of CD133 dropped to （3.8±8.7 ） %. The necrosis and density of capillary of the isehemie limbs of nude mice were improved more obviously in the EPC group than that in the M199 group （ P 〈 0.05 ）. Red and green fluorescence were observed in the paraffin section of isehemie limbs of nude mice in the EPC group. CONCLUSION： We can culture and obtain EPC from mononuelear cells of human peripheral blood in vitro under a certain condition. The transplanted EPC can incorporate into the loeal capillary networks and repair the isehemia of posterior limbs of nude mice.
出处
《第四军医大学学报》
北大核心
2006年第14期1276-1278,共3页
Journal of the Fourth Military Medical University
基金
第四军医大学西京医院2004年临床高新技术资助项目(XJGX04031M26)
关键词
内皮
干细胞
移植
外周血
endothelium
stem cells
transplantation
peripheral blood
