血管内皮祖细胞移植提高裸鼠缺血皮瓣存活率

目的探讨血管内皮祖细胞(endothelialprogenitorcell,EPC)移植促进早期断蒂的缺血皮瓣的血管新生,从而提高皮瓣存活率的可能性。方法体外分离、培养人脐血中EPCs,免疫组织化学进行鉴定,然后移植于裸鼠随意皮瓣,皮瓣早期断蒂,裸鼠分两组:实验组(EPCs移植)、对照组(M199注射),术后皮瓣4d断蒂。观察皮瓣的存活率、激光多普勒血液监测仪监测血流灌注、CD34免疫组织化学检测皮瓣毛细血管密度、激光共聚焦检测EPCs在皮瓣内的密度。结果脐血中分离培养的EPCs表达CD34、KDR及CD133,实验组EPCs移植裸鼠皮瓣后,整合到缺血部位新生血管中,与对照组的皮瓣存活率分别为(60.3±2.1)%、(34.2±1.8)%(P<0.05);而且两组毛细血管密度、血流灌注差异均有统计学意义(P<0.05);实验组术后第7、11天时二组皮瓣中的EPCs密度分别为(75.2±6.5)个/mm2、(305.4±26.5)个/mm2,而对照组均为0个/mm2(P<0.05)。结论脐血中的EPCs体外培养后移植体内可促进缺血皮瓣的血管新生,提高存活率。     

VEGF gene transfer mobilizes endothelial progenitor cells in patients with inoperable coronary disease

BACKGROUND: Direct transfection of ischemic myocardium with naked plasmid DNA encoding for vascular endothelial growth factor-165 (VEGF165) has been shown to mobilize endothelial progenitor cells (EPCs). This study examined the kinetics of circulating EPCs isolated from peripheral blood mononuclear cells after gene transfer, and their role in neovascularization of ischemic myocardium. METHODS: The mononuclear cell population was isolated from peripheral venous blood samples of patients with functional class III or IV angina receiving intramyocardial VEGF165 gene transfer. Peripheral blood mononuclear cells were examined by an in vitro EPC culture assay and fluorescent-activated cell sorting. The data were compared with a control group consisting of patients who had undergone off-pump coronary artery bypass grafting without receiving gene transfer. RESULTS: Coinciding with a rise in VEGF levels, mobilization of EPCs increased significantly over base line for 9 weeks after the treatment (121+/-14 cells/mm2 versus 36.8+/-8 cells/mm2, p < 0.0005), followed by a subsequent decrease. Fluorescent-activated cell sorting analysis confirmed culture assay data, with a statistically significant rise in cells expressing vascular endothelial-cadherin, CD51/61 [alphavbeta3], CD62E [E-selectin], CD34, and KDR. The control group failed to show significant mobilization of EPCs. CONCLUSIONS: Mobilization of EPCs with resultant postnatal vasculogenesis, may play a role in revascularizing ischemic myocardium following human gene transfer with VEGF165.     

血管内皮祖细胞和血管内皮生长因子促进预构皮瓣血管新生作用的比较

目的 比较血管内皮祖细胞(endothelial progenitor cells,EPCs)与血管内皮生长因子(vascular endothelial growth factor,VEGF)在促进预构皮瓣血管新生作用上的差异,探讨EPCs移植提高预构皮瓣存活面积的可行性.方法 分离雄性Wistar大鼠(45只)一侧股血管柬,转位植入腹部皮下,建立预构皮瓣实验模型.将体外诱导分化的EPCs(组Ⅰ,n=15)和VEGF(组Ⅱ,n=15)分别注射于皮瓣局部,对照组仅注射PBS溶液(组Ⅲ,n=15).4周后形成以植入血管为蒂的岛状皮瓣,原位缝合;术后7 d对皮瓣存活率、血管密度计数进行检测.结果 组Ⅰ、组Ⅱ、组Ⅲ的皮瓣存活率分别为(87.26±10.13)%、(66.13±9.9)%、(55.59±13.06)%,组Ⅰ分别与组Ⅱ和组Ⅲ比较,差异均有统计学意义(P<0.001);微血管密度分别为:(38.67±9.52)个/mm~2、(25.83±6.33)个/mm~2、(26.5±5.61)个/mm~2(P<0.05).结论 EPCs促进预构皮瓣血管新生的作用优于VEGF,局部应用骨髓来源的EPCs可以有效地提高预构皮瓣存活面积.     

骨髓血管内皮祖细胞移植提高缺血皮瓣存活率的研究

目的：探讨大鼠骨髓来源的血管内皮祖细胞（endothelial progenitor cells,EPCs）移植促进缺血皮瓣的血管新生,从而提高皮瓣存活率的可能性。方法：在Wistar大鼠背部形成2cm×8cm蒂部位于双侧髂棘连线上的随意型超比例皮瓣,建立缺血皮瓣模型。采用密度梯度离心法从Wistar大鼠骨髓中分离出骨髓单个核细胞,并在专有的诱导培养基（EG-M2MV）中培养,贴壁筛选法分离,诱导分化为EPCs;对细胞的形态、表型、功能加以鉴定。将EPCs注射移植于iWstar大鼠背部的缺血皮瓣,大体观察皮瓣的存活率,vWF免疫酶染色法检测皮瓣毛细血管密度。以缺血皮瓣单纯注射磷酸盐缓冲液（PBS）的Wistar大鼠作为对照组。结果：骨髓中分离培养的EPCs呈现典型的＂铺路石＂样外观,表达CD34、Flk-1及CDl 33表型,能够摄取DIL-ac-LDL和特异性结合FITC-UEA-1。注射EPCs组的缺血皮瓣存活率以及毛细血管密度显著高于对照组（P〈0.05）。结论：骨髓中的EPCs在体外培养后注射移植于皮瓣内,可以促进缺血性皮瓣的血管新生,提高缺血性皮瓣的存活率。     

Transplanted endothelial progenitor cells augment the survival areas of rat dorsal flaps

Endothelial progenitor cells (EPCs) have been identified in peripheral blood, and have been reported to be incorporated into ischemic regions such as the ischemic hindlimb. In this study, we examined whether or not transplantation of EPCs is useful for salvaging surgical flaps in vivo. At the same time, we quantitatively compared the neovascularization ability of transplanted EPCs and that of mature endothelial cells (ECs). ECs obtained from the aorta of rats by explantation and passaged several times were used in the present study. EPCs were obtained from the blood of rat hearts. The blood samples were separated by density gradient centrifugation. Light-density mononuclear cells (MNCs) were collected and cultured on plastic plates coated with rat plasma vitronectin. Cells attached at day 7 of culture were deemed to be EPCs. Then PBS (control), ECs, or EPCs (3.0 x 10(5) suspended in 1.0 ml PBS) were injected at the middle of a flap. Seven days after surgery, the survival lengths of the flaps were evaluated. EPC-transplanted groups revealed statistically significant augmentation of survival length compared with the other two groups (p < 0.003). EPC-transplanted groups had significantly more angiographically detectable blood vessels (p < 0.003) and significantly higher capillary density (p < 0.03) than the other two groups. Confocal microscopy revealed that EPCs were incorporated into enhanced neovascularization. These results suggest that transplantation of EPCs may be useful for salvaging surgical flaps, and EPCs are superior to ECs in neovascularization ability.     

转染血管内皮生长因子基因的小鼠NIH3T3细胞移植促进皮瓣早期血运重建的研究

目的探讨转染血管内皮生长因子（VEGF）基因的小鼠NIH3T3细胞移植对缺血皮瓣的血管新生和皮瓣存活率的影响。方法体外PcDNA3．1（-）／VEGF165质粒转染小鼠NIH3T3细胞,免疫组化方法检测小鼠NIH3T3细胞体外表达VEGF的情况,CM-DiI标记小鼠NIH3T3细胞。将小鼠随机分为3组：A组[PcDNA3．1（-）／VEGF165质粒转染的NIH3T3细胞移植]、B组（单纯NIH3T3细胞移植）、C组（单纯DMEM培养基注射）。每只小鼠背侧皮下按组分别注射细胞悬液和培养基,注射后酶联免疫吸附（ELISA）法连续检测大鼠血浆VEGF浓度,注射后第4天掀起一个蒂在尾侧的4．0cm×1．5cm的随意皮瓣。术后第7天分别观察皮瓣的存活率、血流灌注、皮瓣毛细血管密度、NIH3T3细胞在皮瓣内的分布和存活情况。结果转染VEGF165基因的小鼠NIH3T3细胞体外和体内检测均高表达VEGF165蛋白。A组的皮瓣存活率、毛细血管密度、血流灌注比值均显著高于另外两组（P〈0．05）。结论转染VEGF基因的小鼠NIH3T3细胞皮下移植可促进缺血皮瓣的血管新生,提高存活率。     

人脐血CD133+血管内皮祖细胞的培养、鉴定与分化研究

目的探讨从人脐血中分离、体外培养CD133^＋血管内皮祖细胞（EPCs）的方法及其生长特性和鉴定。方法采用密度梯度离心法结合MACS分选法提取人脐血中CD133^＋细胞,进行流式细胞仪检测纯度,予EBM-2培养液接种培养,观察其生长特性;利用Dil-LDL及FITC-Lectin摄取实验、细胞免疫组织化学等进行鉴定。结果经流式细胞仪检测CD133^＋细胞平均占单核细胞的（1．13±0．10）％,磁珠分选所得CD133^＋细胞平均纯度为（91．45±1．04）％;CD133^＋细胞贴壁生长,可分化为梭形血管内皮细胞及形成集落;CD133^＋细胞培养过程中Dil-LDL及FITC-Lectin摄取阳性,双染阳性率为（95．83±1．72）％;CD133^＋细胞培养1周后经免疫组织化学检测CD34、Ⅷ因子阳性率分别为（95．83±2．23）％和（95．92±1．43）％,与人脐静脉内皮细胞比较差异无统计学意义;CD133^＋细胞体外培养4d、1周可形成小血管结构。结论免疫磁珠分选法可获取较高纯度CD133^＋血管内皮祖细胞,在生长因子作用下诱导其分化为成熟血管内皮细胞。     

VEGF基因体外转染大鼠骨髓间充质干细胞的实验研究

目的:探讨脂质体介导血管内皮细胞生长因子(VEGF)基因转染大鼠骨髓间充质干细胞(MSCS)应用于基因治疗的可行性、安全性。方法:体外分离、培养、鉴定MSCs,PcDNA3.1(-)/VEGF165质粒转染MSCs,转染后用免疫荧光和ELISA检测MSCs表达VEGF蛋白的情况,MTT检测MSCs对VEGF质粒转染的敏感性。结果:骨髓中分离得到MSCs,流式细胞检测显示MSCs不表达CD34和CD45,但表达CD90。透射电镜观察可见细胞浆中含大量粗面内质网和分泌颗粒。VEGF基因转染MSCs后第5天抗VEGF免疫荧光染色约90%的MSCs呈阳性,ELISA检测结果显示PcDNA3.1(-)/VEGF165质粒转染组细胞培养上清液中VEGF含量明显高于对照组,并于转染后第5天达到峰值。MTT检测结果显示VEGF质粒转染对MSCs增殖无影响。结论:MSC可作为VEGF基因转染的靶细胞用于基因治疗。     

红细胞生成素对慢性肾衰竭大鼠外周血内皮祖细胞的影响

目的 探讨红细胞生成素（EPO）对慢性肾衰竭大鼠外周血内皮祖细胞（EPC）数量和功能的影响。 方法 采用分阶段5/6肾切除制备大鼠慢性肾衰竭模型。实验动物按数字随机表法分为4组（均n = 7）：假手术组、慢性肾衰竭组（模型组）、30 U/kg EPO干预组（小剂量组）和50 U/kg EPO干预组（大剂量组）。大鼠皮下注射EPO 6周后,取其外周血分离培养EPC,并检测EPC数量及其增殖、黏附和形成血管结构的能力。 结果 与假手术组比较,慢性肾衰竭大鼠外周血EPC数量及其增殖、黏附与形成血管结构的能力均显著下降（均P < 0.05）。应用EPO治疗能显著增加慢性肾衰竭大鼠外周血EPC数量（P < 0.05）,改善外周血EPC增殖、黏附及形成血管结构的能力（均P < 0.05）,并且呈剂量依赖性。 结论 EPO可改善慢性肾衰竭大鼠外周血EPC的数量和功能。     

Significance of circulating endothelial progenitor cells in patients with fracture healing process

Fracture healing is a complex bone formation process, and neovascularization may contribute to new bone regeneration. The circulating endothelial progenitor cell (EPC) mobilization and homing could involve in neovascularization and vasculogenesis. In this study, we investigate the changes of circulating EPC during bone fracture healing, and the possible contribution of EPCs to increased neovascularization and fracture healing. The number of circulating EPCs was monitored in twenty‐four patients with long bone traumatic fracture within the first 48 h and at 3, 5, 10, and 14 days post‐fracture. The mononuclear cells which isolated from peripheral blood were analyzed by flow cytometry. Peripheral blood counts of leukocytes and platelets were measured by hematology analyzer. The amount of peripheral EPCs significantly increased in patients with fracture compared to age‐matched healthy control subjects within the first 48 h after injury, and peaked at 3 days post‐fracture. There was no significant difference in the change trend of early EPCs between male and female, but the number of early EPCs was significantly greater in younger patients compared to older patients. A comparison of the EPCs levels between patients with severe injury (ISS > 16) and patients with mild injury (ISS ≤ 16) revealed no statistically significant difference. The level of early EPCs was inverse correlation with the level of plate after fracture, but no correlation with the level of peripheral leucocytes. These findings suggest traumatic fracture may induce the mobilization of EPCs into the peripheral circulation. The increased EPCs may contribute to neovascularization and involve in fracture healing. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1860–1866, 2012     

A novel and simplified method of culture of human blood-derived early endothelial progenitor cells for the treatment of ischemic vascular disease

Endothelial progenitor cells (EPCs) consist of two different subpopulations named early (eEPCs) and late EPCs (lEPCs) that are derived from CD14(+) and CD14(-) circulating cells, respectively. These cells are regularly cultured over fibronectin-coated surfaces in endothelial basal medium (EBM)-2 supplemented with insulin-like growth factor (IGF-1), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF). We have developed a new and simplified method for culturing human EPCs obtained from peripheral blood and tested their ability to preserve cardiac function following infarction. We first demonstrated that eEPCs derived from human peripheral blood mononuclear cells (PBMCs) and cultured in EBM-2 medium supplemented with autologous serum (10%) over fibronectin-coated surfaces (10 μg/ml) in the presence of IGF-1 (50 ng/ml) only, have a secretome similar to eEPCs cultured under regular conditions with IGF-1, VEGF, EGF, and FGF. Our data also indicate that IGF-1 modulates PBMC secretome in a dose-dependent manner. In another series of experiments, we showed that PBMCs cultured in suspension in bags (S-PBMCs) in basal medium supplemented with fibronectin and IGF-1 secrete significant amounts of stem cell factor (SCF, 31.3 ± 3.1 pg/ml)), hepatocyte growth factor (HGF, 438.6 ± 41.4 pg/ml), soluble tumor necrosis factor receptor 1 (sTNFR1, 127.1 ± 9.9 pg/ml), VEGF (139.3 ± 9.6 pg/ml), and IGF-1 (147.2 ± 46.1 pg/ml) but very low levels of TNF-α (13.4 ± 2.5 pg/ml). S-PBMCs injected intravenously into NOD SCID mice migrated to the injured myocardium, reduced cardiac fibrosis, enhanced angiogenesis, and preserved cardiac function after myocardial infarction (MI) in a manner similar to eEPCs cultured under standard conditions. In conclusion, we show in this study a refined and optimized method for culturing eEPCs. Our data indicate that S-PBMCs are composed of several cell populations including eEPCs and that they secrete high amounts of antiapoptotic, anti-inflammatory, and proangiogenic factors capable of preserving cardiac function following MI.     

Endothelial progenitor cells and rheumatic disorders

In human adults, new blood vessels may form via endothelial sprouting from pre-existing endothelial cells/angioblasts (angiogenesis) or via the recruitment of circulating endothelial progenitor cells (EPCs) (vasculogenesis). EPCs are a population of bone marrow-derived cells able to differentiate into mature endothelial cells and participating in the formation of new blood vessels. The molecular phenotype of EPCs and processes leading to their mobilization from bone marrow and homing to neovascularization sites remain unclear. There is still debate regarding methods for their quantification and isolation. In the field of rheumatology, EPCs have been studied in multiple myeloma and inflammatory rheumatic disorders. In myeloma, data suggest that EPCs could be reliable biomarkers of tumor angiogenesis, growth and antiangiogenic therapy efficacy. Recent studies suggest that EPCs are involved in synovial vascularization, and may contribute to the increased cardiovascular morbidity and mortality in rheumatoid arthritis, known features of this disease. In systemic lupus erythematosus, preliminary data suggest that EPCs are decreased. Results available in systemic sclerosis are consistent with the hypothesis that EPCs are recruited during active disease; however, their levels may be depleted as the disease progresses and under chronic ischemic conditions. EPCs are important in vasculogenesis, and may be involved in other systemic features of inflammatory rheumatic disorders.     

Surgical injury induces the mobilization of endothelial progenitor cells

BACKGROUND: Endothelial progenitor cells (EPCs) are derived from the bone marrow and incorporate into the foci of tumor neovascularization to increase tumor growth. We hypothesized that surgery induces the mobilization of EPCs. METHODS: C57BL/6 mice were assigned randomly to standardized laparotomy or anesthesia-only treatment groups (n=102 mice). Animals were killed at 6, 24, 48, and 72 hours. Bone marrow EPCs were detected by blood flow cytometric dual staining for stem cell antigen-1/cKit. Circulating EPCs were characterized in blood by vascular endothelial growth factor receptor 2(+)/macrophage activating complement-1(-) staining. EPCs were detected in splenic homogenates by dual staining for lectin and acetylated low-density lipoprotein uptake. Plasma vascular endothelial growth factor was determined by enzyme-linked immunosorbent assay. RESULTS: Surgery induced increases in bone marrow and splenic EPC levels (0.2% +/- 0.01% vs 2.9% +/- 0.3%) at 24 hours and in circulating EPC levels (2.5% +/- 0.01% vs 35.2% +/- 6%) at 48 hours compared with control subjects (P <.001). Surgical injury also caused an increase in vascular endothelial growth factor release (81 +/- 8 vs 14 +/- 2 pg; P>.02). CONCLUSIONS: EPCs were mobilized by surgical injury, which may have implications for residual and metastatic tumor growth during the perioperative period.     

缺氧诱导因子转染内皮祖细胞治疗大鼠缺血后肢

目的 使用缺氧诱导因子-1α(HIF-1α)转染内皮祖细胞(EPC)治疗大鼠后肢缺血,观察EPC、HIF-1α转染EPC对大鼠缺血后肢血管新生和肢体成活的影响.方法 制作SD大鼠后肢缺血模型,将动物随机分为3组,每组6只.将构建的HIF-1α基因真核表达载体转染入EPCs后通过尾静脉注射入大鼠体内,并与注射磷酸盐缓冲液(PBS)或EPC的大鼠进行比较,观察转染HIF-1α对新生血管形成的影响.结果 (1)EPC组、HIF组较PBS组肢体恢复率明显增加(P<0.05),EPC组肢体恢复率较HIF组差(P<0.05).(2)与PBS组比较,各时间点中EPC、HIF组微血管密度(MVD)均明显增多(P<0.05),HIF组较EPC组明显增高(P<0.05).(3)HIF组的HIF与血管内皮生长因子(VEGF)蛋白表达较PBS组、EPC组明显增多(P<0.05).PBS组Capase-3的表达较EPC组、HIF组明显增多(P<0.05).(4)术后7 d,各组的大鼠肢体灌注均明显降低,但EPC、HIF组细胞的血流灌注较PBS组多(P<0.01).术后14、21 d,与PBS对照组比较,HIF组的血流灌注恢复明显(P<0.01),EPC组血流灌注较HIF组少(P<0.05).结论 EPCs对大鼠缺血后肢的局部血管新生有明显促进作用,联合应用HIF-1α和EPCs有更优的治疗效果.     

Accelerated flap prefabrication with vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is a potent promoter of angiogenesis that has been shown to enhance revascularization of ischemic tissues, including skin flaps. This study was designed to investigate the value of a single topical application of recombinant human VEGF to accelerate flap viability in a rat model of a non-ischemic prefabricated flap. Prefabricated flaps were created in 48 Sprague-Dawley rats. An autologous tail artery loop was anastomosed to the femoral artery and vein, and implanted subcutaneously in the lower abdomen. Flaps were divided into two groups of 24 each. At the time of loop implantation the control group received 0.9 percent NaCl or a 16 percent vol/wet polyvinyl alcohol (PVA) solution: the treatment group received VEGF in 0.9 percent NaCl or VEGF in PVA. The PVA gel was used to facilitate topical application In each group, 3- x 4-cm flaps nurtured by the tail artery pedicle were elevated and resutured into place after 3, 4, and 5 weeks. The percentage of surviving skin of each flap was determined by planimetry 7 days after flap elevation. Mean skin survival areas at 3, 4, and 5 weeks were control group 0 percent. 8 percent and 17.5 percent; and VEGIF-treated group, 6 percent, 40 percent, and 66.7 percent respectively VEGF significantly improved flap survival by 5 weeks (p = 0.02). These results suggest that VEGF can accelerate maturation of prefabricated flaps. This approach could expand the application of flap prefabrication as a resource for reconstructive surgery.     

Enhancement of ischemic flap survival by prefabrication with transfer of exogenous PDGF gene

Treatment of skin flaps by means of gene therapy has been introduced recently as a novel approach to enhance viability of ischemic skin flaps. Transfer of the platelet-derived growth factor (PDGF) to enhance survival of the ischemic skin flap has not been explored. In this study, the authors investigated the effect of the transfer of the PDGF cDNA on survival and vascularity of the ischemic random flap in a rat model, and compared the effects of PDGF gene therapy to those of vascular endothelial growth factor (VEGF) gene therapy. A total of 45 adult Sprague-Dawley rats were randomly divided into four groups. The PDGF gene therapy group (n=10) received the plasmid containing the PDGF cDNA with liposome injected to the dermis of the flap. A saline control group (n=10) received physiologic saline only, and the vector control group (n=10) received liposome plus vector without the PDGF gene segment. In the fourth group (n=15), the VEGF gene was transferred to the flap. Seven days later, a dorsal random flap including the injection area was raised. One rat each from the saline and vector control groups died during the study period and were excluded. The viability of the flap and vascularity within the flaps were assessed 7 days after flap elevation. The PDGF plasmid-treated flaps had significantly greater survival area (60.8+/-7.8 percent) compared with the flaps treated with saline (52.3+/-5.0 percent) and those treated with liposome and vector (50.7+/-5.9 percent). PDGF gene therapy had effects on survival of the flap similar to VEGF gene therapy (57.6+/-5.2 percent, after transfer of VEGF cDNA). Neovascularization with the flap tissues was confirmed by immunohistochemical staining of von Willebrand factor, a marker specific for angiogenesis. The number of newly-formed blood vessels in the transgenic flaps was significantly greater than that of the vessels in the flaps receiving the saline. The findings of this study indicate that transfer of the PDGF cDNA effectively enhances neovascularization of the ischemic skin flap and increases the viability of the flap, and transfer of the PDGF gene is as efficient as transfer of the VEGF gene in improving viability of the skin flap. This study suggests that PDGF gene therapy may be a novel strategy for the treatment of ischemic skin flaps.     

Vascular endothelial growth factor gene therapy with intramuscular injections of plasmid DNA enhances the survival of random pattern flaps in a rat model.

The objective of this study was to determine the effects of the naked plasmid DNA encoding vascular endothelial growth factor (VEGF) on the survival of random flaps on rats. Thirty Sprague-Dawley rats whose random flaps were elevated on the back were randomised into three groups of 10 animals each. In the experimental group, the naked plasmid DNA encoding VEGF was injected directly into the panniculus carnosus of the flap. In the two control groups, either control plasmid DNA or physiologic saline was injected. After 7 days, the flaps were evaluated with the following devices: RT-PCR for the expression of VEGF gene, immunohistochemistry for the expression of VEGF protein, histology for vascular density, single photon emission computerised tomography for RBC in the flap, and image analysis for flap survival area. Notably increased expressions of VEGF mRNA and VEGF protein were found in the treatment group. Vascular density was markedly more increased in the treatment group than those in the two control groups (P < 0.01). Compared with the two control groups, the flap treated with VEGF plasmid DNA showed a more significantly enhanced tissue viability: 87 +/- 5 versus 47 +/- 6% for the control plasmid DNA group and 46 +/- 5% for the saline group (P < 0.01). Our results indicated that the VEGF gene therapy was able to enhance the survival of random pattern flaps by inducing angiogenesis.     

人脐血内皮祖细胞体内促进血管重建的实验研究

目的：通过观察人脐血内皮祖细胞（EPC）在裸鼠体内缺氧状态下分化成内皮细胞的现象，初步研究其促进血管重建的作用。方法：根据EPC表面标记CD133，采用免疫磁珠法分离培养EPC。设计EPC裸鼠成瘤实验，观察瘤体内血管增生情况；将荧光标记的EPC注射于裸鼠腹部皮瓣中，观察EPC参与皮瓣血管重建情况；将EPC接种于聚羟基乙酸（PGA）中，移植于裸鼠皮下，观察EPC参与血管重建。结果：原代EPC贴壁后呈梭形，从第7天开始数量明显增加，呈克隆状生长。透射电镜观察到成熟内皮细胞最具特征性的细胞器——weibel-Palade小体。裸鼠成瘤实验：抗人vWF免疫荧光显示大量EPC参与肿瘤血管生成；裸鼠皮瓣实验：荧光标记EPC参与裸鼠皮瓣血管生成；PGA移植实验：抗人vWF免疫酶组织化学染色显示EPC参与血管生成。结论：EPC参与血管重建，具有促进血管新生，加速缺血组织血管化的作用。     

Improvement of skin paddle survival by application of vascular endothelial growth factor in a rat TRAM flap model

The effect of vascular endothelial growth factor (VEGF) on skin flap survival and its ability to induce a pharmacological delay by promoting angiogenesis in a flap was studied in a rat transverse rectus abdominis musculocutaneous flap, using a 3 x 8-cm skin paddle with the inferior epigastric vessels as its main vascular supply. Forty-three Sprague-Dawley rats were divided into four groups. In group 1, VEGF was injected into the femoral vein after the flap was elevated. In group 2, VEGF was injected intra-arterially into the flap through the superior epigastric artery after the flap was elevated. In group 3, VEGF was injected into the subcutaneous fascial layer in the area where the flap would be dissected, and the flap was then raised 7 days after injection. In group 4, the flap was dissected and replaced, using saline injection as the control. On postoperative day 5, the survival area of each skin paddle was measured and the flap was harvested for histological analysis. The results showed that the mean survival area +/- standard deviation for the skin paddle was 6.82 +/- 4.89 cm2 (28.4 +/- 20.4% of the whole skin paddle) in the control group, and 4.2 +/- 3.0 cm2 (17.5 +/- 12.5%) and 6.02 +/- 5.97 cm2 (25.1 +/- 24.9%) in the groups with VEGF systemic and intra-arterial administration respectively. The skin survival area in the group with preoperative subcutaneous administration of VEGF was 17.85 +/- 2.88 cm2 (74.4 +/- 12%), which was significantly higher than the other three groups (p < 0.01). Histological semiquantitative analysis showed increased neovascularization in the flap treated with VEGF preoperatively. The data demonstrate that preoperative treatment with VEGF can induce angiogenesis and enhance skin paddle survival in a musculocutaneous flap.