VEGF基因对兔超全厚皮片移植的影响及安全性研究

目的:探讨皮下局部注射质粒pcDNA3.1/Zeo( )-VEGF165对兔超全厚皮片存活的影响及安全性方面研究。方法:构建并体外扩增表达质粒pcDNA3.1/Zeo( )-VEGF165,以之直接注射转染兔背部超全厚皮片模型。通过与对照组比较,观察其对皮片活力的影响,并采集不同时期的组织进行免疫组化检查。结果:基因转染组在毛细血管周围及肌间隙短期可见VEGF沉积,与对照组相比皮片的平均成活面积显著增加。结论:皮下注射的基因在组织中能够短期表达VEGF,促进皮片血管重构,增加皮片存活能力,是一种简单有效的基因治疗方法。     

重组人VEGF基因治疗大鼠缺血TRAM皮瓣的观察

目的研究重组人VEGF基因对大鼠缺血TRAM皮瓣成活的影响,探讨基因治疗缺血皮瓣的可能性及疗效.方法动物实验分4组,实验组每只皮下注射PcDNA3.1VEGF 500μg;阳性对照组每只注射VEGF 100ng;空白对照组每只注射生理盐水1 ml;阴性对照组每只注射PcDNA3.1 500μg.用ELLSA法及免疫组化法测定VEGF基因表达水平,测定皮瓣成活面积及皮下组织中微血管密度,观察该基因对皮瓣成活的影响.结果实验组皮瓣成活面积及切片微血管密度显著高于阴性对照组及空白对照组(P＜0.05),与阳性对照组差异无显著性意义(P＞0.05).免疫组化显示实验组中有大量棕褐色抗原抗体复合物沉积在小血管内皮细胞胞浆内.结论直接皮下注射PcDNA3.1VEGF,可在注射部位表达具生物活性的VEGF,促进皮下微血管形成,提高缺血TRAM皮瓣的成活面积.     

电脉冲介导的血管内皮细胞生长因子基因治疗对大鼠随意型皮瓣成活的影响

目的探讨血管内皮细胞生长因子(vascularendothelialgrowthfactor,VEGF)质粒直接皮下注射,结合直流电脉冲刺激的基因治疗方案的血管生成效应及不同剂量质粒对皮瓣成活的影响。方法设计大鼠随意皮瓣模型,利用直流电脉冲将皮下注射的重组质粒PCMVMCSVEGFIRES2EGFP导入缺血皮瓣内。观察皮瓣成活的情况,利用苏木素伊红(HE)染色,观察VEGF基因的血管生成效应。结果皮下直接注射结合直流电脉冲成功地将重组质粒导入皮肤。基因转染后7d观察到明显的血管增生。基因治疗组皮瓣成活面积[80μg为(77.38±4.56)%,400μg为(82.57±5.21)%]显著大于对照组[(48.96±4.32)%]。但两基因治疗组皮瓣成活面积差异无统计学意义。结论VEGF质粒直接皮下注射结合直流电脉冲刺激能够高效转染大鼠皮肤,具有明显的血管生成效应,能够显著促进大鼠随意型皮瓣的成活。质粒的剂量对皮瓣成活的影响差异无统计学意义。     

血管内皮生长因子基因治疗与皮瓣延迟术对大鼠腹壁轴型皮瓣成活的影响

目的 探讨血管内皮生长因子基因治疗、皮瓣延迟术两种方法及二者联合应用对大鼠腹壁轴型皮瓣成活的影响.方法 制作大鼠以右侧腹壁浅动脉为血管蒂的超范围轴型皮瓣模型,分别采用皮下注射pcDNA4-VEGF165、皮瓣延迟术及二者联合应用,按处理方法的不同分为6组,①计算各组的皮瓣成活率;②取皮瓣组织标本行常规HE染色,检测平均微血管数目及内径;③取皮瓣组织标本行VEGF免疫组化染色检测VEGF的表达情况.各试验组与空白组相对比,各试验组之间两两对比.结果 各试验组的皮瓣成活率明显高于空白组,延迟同时基因治疗组的皮瓣成活率明显高于其他试验组;基因治疗组及联合应用组的平均微血管数目明壶高于延迟组及空白组;平均微血管内径:延迟组＞联合应用组＞基因治疗组＞空白组;免疫组化染色显示基因治疗组及联合应用组VEGF表达明显高于空白组及单纯延迟组.结论 皮下注射pcDNA4-VEGF165和皮瓣延迟均能有效地改善大鼠皮瓣的成活,但其作用机制不同,而二者的联合应用则能进一步地提高大鼠皮瓣的成活率.     

重组pcDNA3.1-hBMP-2转染人骨髓基质干细胞和对其增殖及血管内皮生长因子表达的影响

目的构建人骨形成蛋白-2(hBMP-2)真核表达载体pcDNA3.1-hBMP-2,转染人骨髓基质干细胞(MSCs),探讨基因转染对其增殖和血管内皮生长因子(VEGF)表达的影响. 方法利用重组DNA和基因克隆技术构建重组载体pcDNA3.1-hBMP-2;细胞培养和基因转染技术体外转染人MSCs;免疫细胞化学、原位杂交和蛋白印迹法检测细胞BMP-2的表达;通过流式细胞仪和VEGF探针原位杂交分析其对细胞增殖和VEGF表达的影响. 结果转染后细胞在mRNA水平和蛋白质水平均表达BMP-2;转染后S期细胞比例增多,提示细胞DNA的合成增加;BMP-2基因转染上调细胞VEGF的表达. 结论在脂质体介导下,pcDNA3.1-hBMP-2转染MSCs获得成功.基因转染后能促进细胞增殖并将通过使VEGF的表达增加促进血管再生,为进一步骨缺损的基因治疗及构建组织工程骨奠定了实验基础.     

脂质体介导血管内皮生长因子对不同时间断蒂大鼠随意型皮瓣的影响

目的 通过血管内皮生长因子基因对大鼠随意型皮瓣的转染,探讨基因治疗对不同时间断蒂的大鼠随意型皮瓣成活的影响.方法 以SD大鼠为实验模型制作背部随意型皮瓣,实验组注入脂质体包裹的PcDNAVEGF165(目的 基因组),对照组分别注入PcDNA(空白质粒组)和生理盐水(生理盐水组),于用药后1、3、5、7 d,每组每时相点分别随机选取10只断蒂,断蒂后7 d处死大鼠,观察下述指标:①皮瓣成活率.②皮瓣组织标本行常规HE染色检测平均微血管数目及内径.③行VEGF免疫组织化学染色检测VEGF表达情况.④取皮瓣组织标本在电镜下观察超微结构.结果 ①皮瓣成活率:1、3、5、7 d断蒂实验组皮瓣成活率分别为(45.45±12.24)%、(82.95±3.81)%、(85.00±3.38)%、(85.96±3.25)%.1 d断蒂实验组与对照组比较差异无统计学意义(P>0.05),3、5、7 d断蒂各实验组明显高于相应对照组(P<0.05),3、5、7 d断蒂各实验组则随着断蒂时间的延迟差异无统计学意义(P>0.05).②平均微血管数目及内径:各实验组与相应对照组比较差异有统计学意义(P<0.05).③各实验组VEGF染色深度明显高于对照组(P<0.05).④超微结构:实验组内有新生血管形成,内皮细胞内可见较多粗面内质网,线粒体等结构,组织内成纤维细胞增多,细胞合成代谢旺盛.结论 皮下注射脂质体介导VEGF基因可提高皮瓣成活率,促进早期断蒂,是一种简单,高效,经济,相对安全的基因治疗方法.     

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。     

血管内皮生长因子基因治疗大鼠缺血皮瓣的实验研究

目的　研究血管内皮生长因子 (VEGF)基因治疗对大鼠缺血皮瓣生存的影响。方法建立大鼠缺血皮瓣的动物模型 ,采用直接注射法转移 pcD2 /hVEGF12 1真核表达质粒于大鼠缺血皮瓣肉膜层 ,术后 7d ,应用苏木素 伊红 (HE)染色、单光子发射计算机断层摄影 (SPECT)及计算机图像分析软件等方法检测皮下血管密度、皮瓣血供和皮瓣成活率。结果　经VEGF基因治疗组的大鼠缺血皮瓣与对照组相比较具有皮下血管密度增加、血液供应增多和皮瓣成活率显著性增高 (P <0 .0 1)。结论　VEGF基因治疗能够诱导新血管形成、增加血流灌注 ,促进缺血皮瓣的生存。     

BMP-2重组质粒转染人骨髓基质干细胞及表达蛋白的诱导活性观察

目的 构建骨形态发生蛋白-2(BMP-2)真核表达质粒，使其在人骨髓基质干细胞(hBMSCs)中表达，并观察表达产物的诱导成骨活性。方法在脂质体介导下将BMP-2基因导入hBMSCs，流式细胞仪、ALP检测和VEGF探针原位杂交分析其对细胞增殖、ALP活性和VEGF表达的影响。并利用转染后的培养液上清诱导小鼠成纤维细胞(L929)，检测其骨钙素、Ⅱ型胶原表达。结果 转染后细胞稳定表达BMIP-2基因，S期细胞比例增多，ALP活性和VEGF的表达明显增加。且诱导L929细胞骨钙素、Ⅱ型胶原阳性表达。结论 BMP-2重组质粒转染hBMSCs后表达目的蛋白，促进自身增殖、分化和上调VEGF的表达，并诱导成纤维细胞向成骨细胞转化，为BMP-2基因治疗奠定了实验基础。     

血管内皮细胞生长因子(VEGF)基因转染骨髓间充质干细胞的表达

目的检测兔骨髓间充质干细胞(BMSc)经血管内皮细胞生长因子(VEGF165)基因转染后外源基因的表达，为进一步利用经基因转染的BMSc构建血管化组织工程骨组织打下基础。方法构建VEGF真核细胞表达载体，利用脂质体介导转染兔BMSc，使用原位杂交、免疫组织化学的方法检测VEGF165在BMSc中的表达。结果成功构建VEGF真核细胞表达载体，原位杂交、免疫组化方法显示经基因转染的BMSc中有阳性棕黄色颗粒出现，而未转染组呈现阴性结果。结论采用基因转移技术可以将VEGF转染至．BMSc中，并有外源性基因和蛋白的表达。     

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.     

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。     

血管内皮生长因子促进跨区供血反流轴型皮瓣成活的实验研究

目的研究血管内皮生长因子（vascular endothelial growth factor，VEGF）的局部皮下注射对大鼠背部跨区供血反流轴型皮瓣成活的影响及效果。方法取20只SD大鼠，制备8cm×2cm大鼠背部跨区供血反流轴型皮瓣模型，随机分成两组，每组10只。实验组：于皮瓣远端7．5cm及6．5cm处共选择4个对称位点，分别予100ng／100μlVEGF溶液50μl；对照组：每一位点予生理盐水50μl。术后1～7d行皮瓣大体观察，并于7d处死大鼠，切取皮瓣，行皮瓣成活率测定、组织学观察及血管密度检测。结果大体观察，实验组皮瓣成活面积明显大于对照组，实验组皮瓣成活面积15．55±0．27cm^2，对照组13．42±0．57cm^2，差异有统计学意义（P〈0.01）。组织学观察，实验组皮瓣血管密度34．40±3．75个／10倍光镜下视野，对照组21．00±3．16个／10倍光镜下视野，差异有统计学意义（P〈0.01）。镜下见实验组有大量新生肉芽组织形成，胶原纤维排列规则，成纤维细胞较多，炎性细胞浸润程度轻；对照组新生肉芽组织少，胶原纤维凝集成块，成纤维细胞少，炎性细胞浸润程度重。结论VEGF在皮瓣成活早期，通过促进缺血皮瓣新生血管形成，增加血管数量，改善缺血组织的血液供应，促进皮瓣成活；在皮瓣形成时局部、单次、足量应用VEGF是促进跨区供血反流轴型皮瓣远端成活的有效方法。     

脂肪基质血管成分移植促进随意皮瓣成活的实验研究

目的探讨脂肪基质血管成分(Stromal vascular fraction,SVF)移植是否可以促进随意皮瓣成活,及其作用的相关机制。方法分离4周龄Wistar大鼠脂肪中的SVF及骨髓中的单个核细胞(BM-MNCs),RT-PCR检测VEGF和bFGF在两种细胞中的表达。根据移植细胞的不同,将24只Wistar大鼠分成3组,分别为对照组、BM-MNCs组和SVF组。在大鼠背部设计一个10 cm×3 cm大小的矩形皮瓣,分别将含有5×107个SVF及BM-MNCs的混悬液各1 mL均匀注射至皮下组织层,对照组单纯注射1 mL PBS。2 d后,皮瓣掀开原位缝合,术后7 d统计皮瓣的成活率。取各组皮瓣相同部位的组织,实时定量PCR检测组织中VEGF和bFGF基因的表达。结果 SVF和BM-MNCs细胞中VEGF和bFGF的表达无明显差别(P>0.05)。皮瓣原位缝合后7 d,SVF组和BM-MNCs组皮瓣的成活率分别为(72.2±2.0)%和(76.4±3.1)%,均明显高于对照组的(56.8±4.6)%(P<0.05)。实时定量PCR检测发现SVF组和BM-MNCs组皮瓣组织中VEGF和bFGF基因的表达明显升高。结论 SVF移植入皮瓣后可以通过旁分泌生长因子如VEGF和bFGF等增加皮瓣的成活面积。     

Effect of transfection time on the survival of epigastric skin flaps pretreated with adenovirus encoding the VEGF gene

An experimental study was conducted to investigate the effect of time of adenovirus-mediated vascular endothelial growth factor (VEGF) gene therapy on the viability of epigastric skin flaps. Eighty-four male Sprague-Dawley rats were used. Skin flaps measuring 8 x 8 cm were marked on the ventral abdominal wall. The upper border of the flap was 1 cm above the costal margin, and the lower border was at the pubis and the inguinal fold. The lateral borders of the flap corresponded to the location of the distinct conversion of the thin ventral skin to the thick dorsal skin. Seven sites in the predicted area of necrosis on the outlined skin flaps were chosen for subdermal injections. All injections were administered by an individual who was blinded to the different treatment groups. The rats received either saline (control group I, N = 28) or adenovirus encoding green fluorescent protein (Ad-GFP; group II, N = 28) or Ad-VEGF (group III, N = 28). The epigastric island skin flaps based solely on the right inferior epigastric vessels were elevated either on the same day of injection (day 0 = 12 hours after transfection, N = 7) or on day 3 (N = 7), day 7 (N = 7), or day 14 (N = 7) after subdermal gene therapy. Flaps were sutured back to their native configuration. Flap viability was evaluated on day 7 after surgery. Sections of the flaps were examined histologically after undergoing hematoxylin-eosin staining. There was a significant reduction in mean percentage of necrotic flap area by 56%, 67%, 70%, and 54% in flaps transfected with Ad-VEGF, 12 hours, 3 days, 7 days, and 14 days before flap elevation, respectively ( < 0.05). There was no evidence that the mean percentage of skin necrosis in the Ad-GFP group was different than in the control group ( = 0.26). There was evidence of mild inflammation in flaps pretreated with Ad-GFP and Ad-VEGF compared with the control group. The authors demonstrated that adenovirus-mediated gene therapy of the abdominal skin after subdermal injections was technically feasible. This was demonstrated by the visualization of GFP expression in control experiments using a fluorescence microscope. In this study, adenovirus-mediated VEGF gene therapy promoted epigastric flap survival, which was not related to the time of transfection. These findings raise the possibility that pretreatment with VEGF gene therapy using an adenovirus vector may be applicable in patients at risk for plastic surgery.     

Effect of adenovirus-mediated gene transfection of vascular endothelial growth factor on survival of random flaps in rats

Objective: To evaluate the effect of local application of vascular endothelial growth factor ( VEGF ) via adenovirus-mediated gene transfer on survival of full thickness flaps selected randomly in rats.Methods: Thirty Sprague-Dawley rats weighing 480-520 g were used in this study. A dorsal flap (8 cm × 2 cm) in full thickness with the pedicle located at the level of the iliac crest was designed. Then the rats received 1 012 pfu replication-deficient recombinant adenovirus carrying VEGF ( AdCMV-VEGF group, n = 10 ), 1012 pfu recombinant β-galactosidase adenovirus ( AdCMV-Gal group, n = 10) and 1 ml saline (saline group, n = 10), respectively, in the distal two thirds of the proposed flap by means of subdermal injection at 8 different locations. Three days after treatment, the flaps were elevated as originally designed and sutured back in situ. The survival rate of the flaps was evaluated on day 7 after operation.Results: The survival rate of the flaps in the AdCMV-VEGF group increased significantly as     

Vascular endothelial growth factor gene therapy in improvement of skin paddle survival in a rat TRAM flap model

The use of growth factors in inducing angiogenesis and enhancing flap viability has provided promising results. Targeted gene therapy has evolved in hopes of increasing the longevity and effectiveness of these growth factor treatments. The purpose of this study was to examine the effect of preoperative treatment by vascular endothelial growth factor (VEGF) plasmid DNA on the survival of the skin paddle in a rat pedicled TRAM flap model. In part one of the study, VEGF plasmid DNA incorporated with lipofectamine was injected into the subcutaneous fascial layer of the upper abdominal walls of the rats. At 4 days postoperatively, biopsies were taken from the injected area for histology and VEGF protein quantification. In part two of the study, the rats were divided into three groups. In one experimental group, the VEGF plasmid DNA was injected into the subcutaneous fascial layer in the area where the TRAM flap would be elevated. In two control groups, the plasmid without VEGF DNA and saline were injected. The flaps were raised and replaced 4 days after injection. Flap survival was examined. Results showed that tissue receiving VEGF plasmid DNA injection revealed new vessel sprouting. The VEGF levels in these tissues were significantly higher than in the tissue not receiving VEGF plasmid DNA. In flap survival, the mean viable area of the skin paddles receiving preoperative VEGF plasmid DNA injection was significantly larger than that of flaps receiving no VEGF plasmid DNA and saline injection. This study demonstrated that preoperative subcutaneous injection of VEGF plasmid DNA could induce angiogenesis and improve TRAM skin paddle survival.     

Gene transfer of bFGF to recipient bed improves survival of ischemic skin flap.

BACKGROUND: The recipient bed is a promising target of angiogenic therapy to treat ischemic skin flaps. We delivered basic fibroblast growth factor (bFGF) gene to the recipient bed by a plasmid-based method with electroporation, and assessed the effects on flap viability in a rat dorsal skin flap model. METHODS: A 25 x 90 mm(2) axial skin flap was elevated on the back of male Sprague-Dawley rats. Two days before flap elevation, an expression plasmid vector containing the bFGF gene with the signal sequence was injected into the dorsal muscles beneath the skin flap, and then electroporation was delivered (FGF-E(+) group). As control, rats were injected with a plasmid vector containing LacZ gene (LacZ-E(+) group), instead of bFGF gene. Other groups of animals received plasmid vector containing bFGF (FGF-E(-) group) or LacZ (LacZ-E(-) group) gene without electroporation. Seven days later, the area of necrosis and neovascularisation of the skin flap were evaluated. RESULTS: The bFGF gene was successfully transferred to the dorsal muscles, and bFGF was expressed in muscle tissue. The area of flap necrosis (%) in the FGF-E(+) group (21.7+/-5.3%) was significantly smaller than that in the LacZ-E(+) (28.3+/-4.1%), FGF-E(-) (29.7+/-3.3%), and LacZ-E(-) (28.1+/-2.5%) groups. Postmortem angiograms and histological analyses showed that vascularisation in the distal part of the skin flap was significantly increased in the FGF-E(+) group compared with the other groups. CONCLUSION: These findings suggested that gene delivery of bFGF to the recipient bed muscles enhanced vascularity and viability of an ischemic skin flap, and that plasmid-based gene delivery with electroporation was a suitable delivery method.     

Adenovirus-mediated transforming growth factor-beta ameliorates ischemic necrosis of epigastric skin flaps in a rat model

BACKGROUND: Gene therapy has been recently introduced as a novel approach to treat ischemic tissues by using the angiogenic potential of certain growth factors. We investigated the effect of adenovirus-mediated gene therapy with transforming growth factor-beta (TGF-beta) delivered into the subdermal space to treat ischemically challenged epigastric skin flaps in a rat model. MATERIAL AND METHODS: A pilot study was conducted in a group of 5 animals pretreated with Ad-GFP and expression of green fluorescent protein in the skin flap sections was demonstrated under fluorescence microscopy at 2, 4, and 7 days after the treatment, indicating a successful transfection of the skin flaps following subdermal gene therapy. Next, 30 male Sprague Dawley rats were divided into 3 groups of 10 rats each. An epigastric skin flap model, based solely on the right inferior epigastric vessels, was used as the model in this study. Rats received subdermal injections of adenovirus encoding TGF-beta (Ad-TGF-beta) or green fluorescent protein (Ad-GFP) as treatment control. The third group (n = 10) received saline and served as a control group. A flap measuring 8 x 8 cm was outlined on the abdominal skin extending from the xiphoid process proximally and the pubic region distally, to the anterior axillary lines bilaterally. Just prior to flap elevation, the injections were given subdermally in the left upper corner of the flap. The flap was then sutured back to its bed. Flap viability was evaluated seven days after the initial operation. Digital images of the epigastric flaps were taken and areas of necrotic zones relative to total flap surface area were measured and expressed as percentages by using a software program. RESULTS: There was a significant increase in mean percent surviving area between the Ad-TGF-beta group and the two other control groups (P < 0.05). (Ad-TGF-beta: 90.3 +/- 4.0% versus Ad-GFP: 82.2 +/- 8.7% and saline group: 82.6 +/- 4.3%.) CONCLUSIONS: In this study, the authors were able to demonstrate that adenovirus-mediated gene therapy using TGF-beta ameliorated ischemic necrosis in an epigastric skin flap model, as confirmed by significant reduction in the necrotic zones of the flap. The results of this study raise the possibility of using adenovirus-mediated TGF-beta gene therapy to promote perfusion in random portion of skin flaps, especially in high-risk patients.