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黑色素瘤细胞及细胞培养液促进小鼠缺血后肢血管新生

周涛, 胡朝晖, 周波, 符伟国, 王玉琦

复旦大学中山医院1 血管外科； 心内科，上海 200032； 浙江大学附属第二医院神经外科，杭州 310009；美国新墨西哥州大学药学院及生物医学和神经影像联合研究中心，阿尔伯克基 87131

摘要

本文旨在探讨小鼠黑色素瘤细胞及培养提取液对小鼠后肢缺血组织血管新生的影响，以及细胞趋化因子基质细胞衍 生因子1 (stromal cell derived factor 1, SDF-1)及其特异性趋化因子受体(CXC chemokine receptor 4, CXCR4)是否在这种影响中发挥 作用。制作小鼠后肢缺血模型，在腹部皮下注射小鼠黑色素瘤细胞B16-F10 或在缺血后肢肌肉注射细胞培养上清液，分别于 7，14 和21 天行激光多普勒扫描(laser Doppler perfusion imaging, LDPI)评估后肢缺血组织血流灌注情况，21 天后流式细胞术 分析小鼠骨髓细胞(bone marrow cells, BMCs)中的CXCR4 表达，碱性磷酸酶染色后组织学分析缺血后肢血管新生情况。结果显示，与对照组相比，肿瘤细胞和细胞培养液均可使BMCs 中CXCR4 阳性表达细胞数增加(P<0.05)，小鼠缺血后肢血流灌 注情况明显改善(P<0.05)，小鼠缺血后肢肌肉毛细血管密度显著增加(P<0.05)。以上结果提示，黑色素瘤细胞及细胞培养液 可以促进BMCs 中CXCR4 的表达，通过SDF-1/CXCR4 途径促进缺血组织的血管新生。

关键词： 局部缺血; 血管新生; 基质细胞衍生因子1 ; 特异性趋化因子受体; 激光多普勒血流扫描

分类号：R543

B16-F10 melanoma cells and cell culture supernatant enhance angiogenesis in mouse ischemic limb

ZHOU Tao, HU Zhao-Hui, ZHOU Bo, FU Wei-Guo, WANG Yu-Qi

Department of Vascular Surgery； Department of Cardiology, Zhongshan Hospital Affiliated to Fudan University, Shanghai 200032, China； Department of Neurosurgery, Second Affiliated Hospital, Zhejiang University, Hangzhou 310009, China； College of Pharmacy and Biomedical Research and Integrative Neuro-Imaging Center, University of New Mexico, Albuquerque, New Mexico 87131, USA

Abstract

Generation of therapeutic angiogenesis to enhance vascularization in the ischemic tissues is a method for treating ischemic tissues in atherosclerotic cardiovascular artery disease. The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor (CXC chemokine receptor 4, CXCR4) play a critical role in the process of post-natal neovascularization. The SDF-1-CXCR4 axis is a potential mechanism for the treatment of ischemic limb. Here, we investigated the role of CXCR4 in bone marrow cells (BMCs) in neovascularization induced by tumor cells and the supernatant of culture media in a murine hind-limb ischemia model which was made by resecting femoral artery and vein. After the injection of mouse melanoma cells B16-F10 (1×106 cells in 0.1 mL at the operation day, s.c.) into the abdomen or the cell culture supernatant (0.1 mL/d for 21 d after operation, i.m.) into the ischemic abductor muscle, the CXCR4 positive BMCs were analyzed by flow cytometry. The perfusion of the ischemic limb was evaluated by laser Doppler perfusion imaging (LDPI) on 7, 14 and 21 d after vascular injury operation. Capillary endothelial alkaline phosphatase (AP) was stained to quantify the presence of capillaries, and histological method was used to evaluate the capillary density as a measure of neovascularization in ischemic tissues. The proportions of CXCR4 positive BMCs were notably higher in ischemic limb injected with tumor cells or the supernatant compared to those in the control group (P<0.05). Injection of tumor cells or the supernatant resulted in significantly improved perfusion as measured by LDPI perfusion ratios on 7, 14 and 21 d after femoral artery and vein resection in mice, compared to the controls (P<0.05). Tissue samples harvested from the lower calf muscle at day 21 demonstrated increased capillary densities in mice receiving tumor cells (0.81±0.13) or the supernatant (0.63±0.05), compared with those in control group (0.44±0.09, P< 0.05). In conclusion, the injection of B16-F10 tumor cells or the supernatant induces the increase of CXCR4 positive cells in BMCs and the improvement of in vivo neovasculogenesis in mouse ischemic limb.

Key words: ischemia; neovascularization; chemokine stromal cell-derived factor-1; CXC chemokine receptor 4; laser Doppler perfusion imaging

收稿日期：2008-11-21　　录用日期：2009-02-19

通讯作者：符伟国　　E-mail: drfuweiguo@gmail.com

引用本文：

周涛, 胡朝晖, 周波, 符伟国, 王玉琦. 黑色素瘤细胞及细胞培养液促进小鼠缺血后肢血管新生[J]. 生理学报 2009; 61 (2): 139-145.

ZHOU Tao, HU Zhao-Hui, ZHOU Bo, FU Wei-Guo, WANG Yu-Qi. B16-F10 melanoma cells and cell culture supernatant enhance angiogenesis in mouse ischemic limb. Acta Physiol Sin 2009; 61 (2): 139-145 (in Chinese with English abstract).