WANG Hui1, WANG Ling1, LI Kaiª²Zong2, LING Rui1, SUN Baoª²Hua1, LI Xiaoª²Jun1,YI Jun1

    1Department of Vascular and Endocrine Surgery, 2Department of Hepatobiliary Surgery, Xijing Hospital, Fourth Military Medical University, Xi¬ðan 710033, China

    ¡¾Abstract¡¿ AIM: To estimate the effect of human peripheral blood derivedª²endothelial 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 6ª²day adherence cells marked by CMª²DiI were multipoint injected into the left local muscles of ischemic limbs(EPC group) and M199 was multipoint injected into the right local muscles of ischemic limbs (M199 group) in order to evaluate the therapeutic effect. Seven days after operation, 60 ¦Ìg FITCª²UEA 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 ¢ø 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¡À9.3)%, (33.8¡À11.7)%,(73.5¡À6.3)%and (38.9¡À8.2)%, 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¡À7.6)%,(88.9¡À5.4)% and (78.3¡À13.6)%, respectively; the positive rate of CD133 dropped to (3.8¡À8.7)%. The necrosis and density of capillary of the ischemic 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 ischemic limbs of nude mice in the EPC group. CONCLUSION: We can culture and obtain EPC from mononuclear cells of human peripheral blood in vitro under a certain condition. The transplanted EPC can incorporate into the local capillary networks and repair the ischemia of posterior limbs of nude mice. ......