Cell transplantation combined with transmuscle laser revascularization augments neovascularization in rat ischemia hindlimb / 中国病理生理杂志

ABSTRACT

AIM: To determine the combined effect of transmuscle laser revascularization (TMR) and endothelial progenitor cells(EPCs) treatment on ischemic hindlimb of nude rats.METHODS: Mononuclear cells (MNCs) isolated from human umbilical cord-blood (HUCB) by density gradient centrifugation were expanded in vitro. Immunocytochemistry and flow cytometry studies were performed. EPCs were labeled with 1, 1'- dioctadecyl-1 to 3, 3, 3', 3'- tetramethyl-indocarbocyanine perchlorate (DiI) before injected into the laser induced channels or ischemic region. Acute ischemic limb was created in 4 groups of nude rats by ligating right external iliac artery. All animals were divided randomly into the following four groups TMR+EPCs group local transplantation of EPCs into laser channels; TMR group transmuscular channels were created without EPCs; EPCs group EPCs were injected into ischemic hindlimb; control group ischemic model without TMR or EPCs. All rats underwent femoral artery ultrasonic blood flow measurements of the ischemic and nonischemic limbs to obtained a flow ratio [femoral artery flow index (FAFI) right femoral artery flow /left femoral artery flow] at baseline (after ligating artery immediately) and 28 days postoperation, and then the samples of ischemic limb muscle underwent histochemical and immunohistologic analysis. RESULTS: The attached cells expressed endothelial cell (ECs) markers (KDR, CD34, CD31, AC133 and von Willebrand factor) and exhibited function similar to that of ECs judged by Ac-LDL incorporation. Flow cytometric analysis disclosed that AT cells were positive for CD34 (62%?7%) and AC133 (57.2%?9.8%) at day 7 of culture. 28 days after therapy, FAFI was significantly higher in the TMR +EPCs (0.66?0.09, P0.05). FAFI in the control group was unchanged and no difference was found between TMR group and control group. TMR+EPCs (5.66?0.77), TMR (4.96?0.31) as well as EPCs (4.68?0.44) treatment resulted in an increased number of capillaries in the treated regional area compared with control group (2.60?0.31, P