细胞移植结合激光肌肉血管重建增强鼠缺血肢体新生血管形成

目的: 研究激光肌肉血管重建（TMR）和血管内皮祖细胞（EPCs）移植相结合能否增强缺血肢体新生血管形成。方法： 密度梯度法分离脐带血单个核细胞并体外培养，免疫组织化学和流式细胞术定量和定性分析。应用DiI标记注射入激光孔道和缺血区的EPCs。结扎裸鼠右侧髂外动脉造成急性肢体缺血模型，随机分为：TMR＋EPCs组：EPCs移植入缺血区肌肉的激光孔道；TMR组： 缺血区肌肉激光打孔；EPCs组：注射EPCs至缺血区肌肉；对照组：缺血肢体模型。超声多谱勒测定缺血和非缺血后肢的股动脉血流获得比值（FAFI），组织化学和免疫组织化学检查缺血肢体肌肉标本。结果： 贴壁细胞表达KDR、CD34、AC133、CD31和vWF，并且吞噬Ac-LDL，有内皮细胞功能，培养7 d的细胞流式细胞术检查CD34（62%±7%）和AC133（57.2%±9.8%）阳性。术后28 d，TMR＋EPCs、EPCs和TMR组FAFI明显高于各自基线水平；TMR＋EPCs和EPCs组FAFI明显高于对照组。TMR+EPCs组新生血管形成明显多于对照组。结论： TMR和EPCs移植结合明显改善了缺血肢体的灌注并增强了缺血肢体新生血管形成。

Cell transplantation combined with transmuscle laser revascularization augments neovascularization in rat ischemia hindlimb

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, P<0.01) and EPCs group (0.59±0.09, P<0.05) compared to control group (0.47±0.05). It was significantly higher in TMR +EPCs-，EPCs- and TMR group compared to baseline (TMR+EPCs group: 0.66±0.09 vs 0.39±0.07, P<0.01; TMR group: 0.54±0.12 vs 0.40±0.09, P<0.05; EPCs group: 0.59±0.09 vs 0.38±0.08, P<0.01; control group: 0.47 ±0.05 vs 0.39±0.08, P>0.05). 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<0.01).CONCLUSION: Nd: YAG-laser revascularization combined with the application of EPCs transplantation significantly ameliorates perfusion and augments neovascularization in this ischemic hindlimb model.