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.     

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。     

pcDNA3.1(+)载体携带血管内皮生长因子治疗大鼠缺血皮瓣

目的 观察注射真核表达载体pcDNA3.1(+)携带血管内皮生长因子(VEGF)基因后对大鼠缺血皮瓣存活的影响.方法 构建重组质粒pcDNA3.1(+)-VEGF,将SD大鼠随机分成3组,每组10只.pcDNA3.1(+)-VEGF组:于背部皮肤皮下多点注射pcDNA3.1(+)-VEGF 30 μg/100μl;VEGF组:多点注射VEGF蛋白100 ng/100μl;生理盐水(NS)组:注射生理盐水100μl,注射2d后在其背部形成7 cm×3 cm的全厚随意型皮瓣,48 h后按原设计掀起皮瓣并原位缝合.术后10 d测量皮瓣的成活面积,计算成活面积百分比,并行免疫组织化学检测.结果 皮瓣成活面积百分比:pcDNA3.1(+)-VEGF组为(79.1±3.5)％,VEGF组为(62.2±2.7)％,NS组为(59.9±3.1)％,pcDNA3.1(+)-VEGF组皮瓣成活面积明显高于其他两组(P＜0.05).结论 皮下注射pcDNA3.1(+ )-VEGF可促进新生血管的形成,比单纯注射VEGF蛋白更能提高皮瓣的成活率.     

低能量体外震波扩大随意皮瓣成活面积的实验研究

目的 观察低能量体外震波(extracoral shock wave,ESW)扩大随意皮瓣成活面积的可能性.方法 40只SD大鼠分为对照组和治疗组,各20只.在鼠背部制作蒂位于头侧的随意皮瓣,长10 cm,蒂宽3 cm,分离后原位缝合.治疗组术后即用1500脉冲0.09 mJ/mm2的ESW对皮瓣进行干预,对照组不作处理.术后第1、3、5、7、10天用激光多普勒血流计测定皮瓣的血流量,大体观察皮瓣内血管分布情况,组织学检查微血管密度,计算皮瓣的存活面积和存活率.结果 治疗组皮瓣的中部、远部在各时间段的相对血流量值明显高于对照组:术后10 d,治疗组和对照组的微血管密度分别为31.5±4.9和10.3±2.8,皮瓣成活率分别为(78.0±8.0)%和(48.4±6.0)%,成活区的长宽比分别为2.52:1和1.51:1.两组差异均有统计学意义.结论 低能量ESW通过增加皮瓣的血流量、促进血管新生而扩大随意皮瓣的成活面积,为扩大随意皮瓣的临床用途提供可能.     

皮瓣延迟与血管内皮细胞生长因子对大鼠背部皮瓣成活影响的比较

目的 将皮瓣延迟与采用血管内皮细胞生长因子（vascular endothelial growth factor，VEGF）对皮瓣成活的影响进行对比研究。方法3月龄SD大鼠30只，随机分为生理盐水组、皮瓣延迟组及VEGF组，每组10只。应用背部超长、宽比随意皮瓣模型。皮瓣延迟组采用双蒂皮瓣延迟，延迟时间为7d，之后断头端蒂，形成蒂部位于尾端的单蒂皮瓣；VEGF组形成单蒂皮瓣，于皮瓣中、远段均匀分为4点，局部皮下注射含400ng VEGF溶液100μl；生理盐水组于局部皮下注射生理盐水100μl，余同VEGF组。单蒂皮瓣完全形成后5d，计算皮瓣成活率，切取皮瓣组织，进行微血管密度分析、微血管直径测量和微血管断面面积测量。结果VEGF组皮瓣成活率与皮瓣延迟组接近，差异无统计学意义（P〉0．05）。皮瓣延迟组内部微血管平均直径明显大于VEGF组和生理盐水组，VEGF组皮瓣内部微血管密度明显大于生理盐水组和皮瓣延迟组，差异均有统计学意义（P〈0．05）。皮瓣延迟组和VEGF组相比，其微血管断面面积接近，差异无统计学意义（P〉0．05）。结论皮瓣延迟后皮瓣内部主要表现为微血管扩张，应用VEGF后，皮瓣内部主要表现为微血管增生。二者均能有效增加皮瓣内部微血管断面面积，提高皮瓣成活率，但其作用途径不同。     

碱性成纤维细胞生长因子促进缺血皮瓣微循环重建的实验研究

目的探讨碱性成纤维细胞生长因子对缺血皮瓣微循环重建的影响。方法以 Wistar 大鼠为实验动物,在其背部形成7cm×1cm 随意皮瓣,通过测定皮瓣存活面积、病理切片、电镜观察、组织化学染色、图像分析技术等手段进行观察。结果给药组以毛细血管增生为特征,微血管断面面积及皮瓣存活率均显著高于对照组。结论碱性成纤维细胞生长因子可促进缺血皮瓣微循环重建。     

凝血酶肽促进缺血创面愈合与皮瓣存活的实验研究

目的探讨凝血酶受体激活肽(TP508)对促进缺血创面愈合与皮瓣存活的作用.方法SD大鼠66只,制作部分缺血创面(16只)、完全缺血创面(16只)、正常创面(18只)及皮瓣(16只)模型,每一模型又分为TP508治疗组和等渗盐水对照组.术后第3、7、10、14天,将创面或皮瓣坏死轮廓描记至醋酸纸七,输入计算机求出创面面积或坏死面积.结果术后7 d和14 d,TP508组正常创面面积仅为对照组的73.7%和45.4%.术后7 d,TP508组部分缺血创面面积为(99.8±30.7)mm2,而对照组为(128.0±43.4)mm2.术后第10天,TP508组完全缺血创面面积为(293.0±34.0)mm2,对照组为(352.4±41.2)mm2.术后第7天,TP508组皮瓣坏死面积为对照组皮瓣坏死面积的80.4%,第14天为56.8%.结论 TP508对促进大鼠缺血创面愈合和皮瓣存活均有显著作用.     

皮神经营养血管对扩大穿支蒂皮瓣存活面积的实验研究

目的 探讨皮神经营养血管对扩大穿支皮瓣存活面积的机制.方法 雄性SD大鼠30只,10只用于灌注解剖研究大鼠背部穿支血管、皮神经走行;20只随机分为两组,均设计3 cm×10 cm以旋髂深动脉皮穿支为蒂的背部长轴皮瓣,实验组皮瓣长轴与后正中线成0°、对照组呈30°.术后1 d、7 d尾静脉注射荧光素钠观察皮瓣血流情况;术后7d观察两组皮瓣存活率,并观察皮瓣的血管网分布.结果 大鼠旋髂深动脉皮穿支恒定,背部皮神经纵向分布,有丰富的血管网伴行.术后1 d实验组的血流灌注面积为42.85%,对照组皮瓣为37.94%,二者差异无统计学意义(P＞0.05);术后7 d两组皮瓣血流灌注面积分别为84.07%、58.55%,皮瓣存活面积分别为83.93%、59.95%,差异均有统计学意义(P＜0.01).术后7 d实验组皮瓣血管网密度高于对照组.结论 皮神经营养血管改善穿支皮瓣存活状况,沿皮神经走向切取穿支皮瓣可明显增大皮瓣存活面积.     

苦碟子注射液对大鼠随意皮瓣存活的影响

目的：观察苦碟子注射液研究苦碟子注射液对大鼠随意型皮瓣存活的影响对大鼠随意皮瓣成活的影响。方法：将2～3月龄重SD大鼠24只,采用随机抽签法分为2组（实验组和对照组,每组12只）。采用改良的大鼠随意型皮瓣制作方法造模,实验组每天腹腔注射苦碟子注射液5 ml/kg ,对照组每天腹腔注射生理盐水5 ml/kg。术后第7天颈椎脱臼处死大鼠分别进行皮瓣存活面积比的检测,同时取皮瓣近中远端组织经组织染色切片后光镜下观察,免疫组化法检测血管内皮生长因子（VEGF）的表达。结果：术后7 d,实验组皮瓣存活面积比为（70．432±3．867）％,显著高于对照组的（50．498±2．346）％（P＜0．01）;实验组皮瓣组织水肿、炎症细胞浸润情况比对照组明显减轻;切片观察出现较多新生血管,Ⅱ区新生血管密度（（30．11±5．53）/mm2,与对照组（20．13±4．11））/mm2比较,差异有统计学意义（P＜0．01）,Ⅲ区坏死严重。实验组VEGF表达量为（4867．31±452．36）,与对照组的VEGF表达量（2387．45±768．46）比较,差异有统计学意义（P＜0．01）。结论：苦碟子注射液可以提高VEGF的表达量、促进毛细血管新生,减轻皮瓣炎性浸润,还可以明显提高大鼠随意型皮瓣的成活率,为临床皮瓣移植研究提供了新的思路。     

腺病毒介导VEGF和HO-1联合治疗缺血随意皮瓣的实验研究

目的 探讨复制缺陷型腺病毒介导的血管内皮生长因子(VEGF165)和血红素氧化酶(HO-1)联合应用,对提高大鼠超长随意皮瓣成活面积的影响和作用机制.方法 将25只健康纯种成年雌性SD大鼠的背部两侧各设计一6cm×1cm大小随意皮瓣.皮瓣远端皮下分别注射目的基因联合应用组 (Ad-VEGF165+Ad-HO-1)、基因对照组(Ad-VEGF165)、基因对照组(Ad-HO-1) 、绿色荧光蛋白基因对照组(Ad-11B-GFP)、病毒保存液(Ad-buffer)为空白对照组,共5组.术后7d,分别测量皮瓣的成活面积、HE染色及目的蛋白和CD31免疫组化表达情况及皮瓣新生微血管密度(MVD)检测等.结果 目的基因联合应用组皮瓣成活面积、目的蛋白表达及MVD与其他4组比较,其差异有显著统计学意义(P ＜0.01).结论 以复制缺陷型腺病毒为载体,联合应用VEGF和HO-1基因治疗能显著增加大鼠随意皮瓣的成活面积,促进皮瓣存活.     

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.     

Microencapsulated Myoblasts Transduced by the Vascular Endothelial Growth Factor (VEGF) Gene for the Ischemic Skin Flap

Background  

This experimental study aimed to explore the influence of locally administered microencapsulated vascular endothelial growth factor (VEGF)-secreting myoblasts on the survival of the ischemic skin flap in rats and to elucidate the underlying molecular mechanism.     

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.     

Effects of microneedle length and duration of preconditioning on random pattern skin flaps in rats

To date, the surgical delay of skin flaps is the most common and reliable method that increases skin flap survival. In this study, we aimed to increase skin flap viability using preconditioning by microneedling. Seventy-two Sprague Dawley rats were randomly divided into control, surgical flap delay (SFD), and four microneedling groups (7 or 14 days of preconditioning with 0.5 mm or 1 mm needles). Modified McFarlane flaps were raised on the back of rats. In Group I, a caudal pedicled skin flap was raised and the flap survival rate was assessed on postoperative day 14. In the SFD group, a bipedicled flap was created and after 14 days of surgical delay, all skin flaps were raised. In the microneedling groups, 0.5 mm or 1 mm needles were used for 7 or 14 days. The flap survival rates of all microneedling and SFD groups were significantly higher than the control group. The plasma levels of vascular endothelial growth factor (VEGF) did not significantly differ between groups, but the VEGF level of skin samples in the SFD group was higher than the control group. The vessel counts of all microneedling and SFD groups were statistically higher than the control group in all skin samples taken before raising the flaps, but skin samples taken 14 days after raising the skin flap did not show any difference between groups. We showed that preconditioning by microneedling can be used to improve the viability of critical ischemic skin flaps at a level similar to surgical delay.     

Vascular endothelial growth factor (VEGF) expression and the effect of exogenous VEGF on survival of a random flap in the rat.

The induction of endogenous vascular endothelial growth factor (VEGF) production in the skin flap with ischemic injury and the effect of exogenous VEGF on survival of the ischemic skin flap were studied in rats. A dorsal flap model (3x10 cm(2)) was used in this study. In Part I, biopsies were taken from the flap at 2.5, 5.5, and 8.5 cm distances from the distal edge at 0, 6, 12, and 24 h after the flaps were sutured. Malonyldialdehyde (MDA) and VEGF(165) protein level were measured. In Part II, exogenous VEGF (1 microg/ml) was injected subdermally into the flaps in 14 rats before the flaps were replaced. Flaps that received a saline injection were used as the controls. The skin paddle survival was measured on postoperative day five. The results showed that the MDA level in the distal part of the flap significantly increased at 24 h postoperatively when compared to MDA in other parts of the flap. However, VEGF levels in the distal part of the flap significantly decreased when compared to the middle part of the flap. Subdermal injection of exogenous VEGF to the distal area of the flap could significantly improve survival of the distal flap (89% of total skin paddle) when compared to the control, which had a 64% mean percent survival. We conclude that production of endogenous VEGF protein is significantly increased in the skin flap with mild ischemia, but decreased in the flap with severe ischemia. Administration of exogenous VEGF could significantly enhance survival of ischemic flaps.     

碱性成纤维细胞生长因子促进缺血皮瓣微循环重建的实验研究

目的探讨碱性成纤维细胞生长因子对缺血皮瓣微循环重建的影响。方法以Ｗｉｓｔａｒ大鼠为实验动物，在其背部形成７ｃｍ×１ｃｍ随意皮瓣，通过测定皮瓣存活面积、病理切片、电镜观察、组织化学染色、图像分析技术等手段进行观察。结果给药组以毛细血管增生为特征，微血管断面面积及皮瓣存活率均显著高于对照组。结论碱性成纤维细胞生长因子可促进缺血皮瓣微循环重建。     

碱性成纤维细胞生长因子促进缺血皮瓣微循环重建的实验研究

OBJECTIVE: This study was to determine whether the basic fibroblast growth factor (bFGF) could promote reconstruction of microcirculation in the ischemic portion of a random skin flap. METHODS: Five methods were used in the experiment: measurement of the survival area of the skin flap; light and electron microscopic studies; immunohistochemical observation of the endothelial cell and microvessel image analysis technology (IAT). RESULT: The survival area and microvessels in the skin flap were significantly increased in the treated group. The results indicate that bFGF can stimulate the proliferation and differentiation of endothelial cells in vivo and promote the microcirculation of the ischemic flap. CONCLUSION: bFGF could promote reconstruction of microcirculation of the ischemic skin flap.     

Angiogenic effects of injected VEGF165 and sVEGFR-1 (sFLT-1) in a rat flap model

BACKGROUND: Injections of single-dose vascular endothelial growth factor (VEGF)(165) have been advocated as a therapeutic tool for angiogenesis in ischemic flaps. We challenged this thesis by employing both VEGF(165) and vascular endothelial growth factor receptor-1 (VEGFR-1) (for competitive inhibition of VEGF signal transduction) in different experimental settings of an ischemic rat flap model. MATERIAL AND METHODS: 80 isogenic rats were divided in two groups of 40 animals (groups 1A-1D and 2A-2D). The ischemic target was a 7 x 7-cm epigastric island flap, based on the right inferior epigastric pedicle. Group 1 received flap treatment 1 week prior to flap elevation by test substance injection into its flap panniculus carnosus: 1 ml NaCl 0.9% (1A), 1 ml Dulbecco's modified Eagle's medium (1B), 1.0 microg VEGF(165) (1C), and 10 microg sFLT-1 with 1.0 microg VEGF(165) (1D). sFLT-1 is a soluble receptor for VEGF and is able to prevent VEGF signaling through the cell surface receptor. Group 2 had the same flap treatment at the day of flap elevation. RESULTS: In group 1C we found the most vital flap tissue, without reaching significance. Compared with group 1D, however, significantly more flap tissue maintained vital. In groups 2A-2D, no significant results were found with respect to flap survival. CONCLUSIONS: Local application of single-dose VEGF(165) 1 week prior to ischemia dose not have significant clinical angiogenic effects. In this experimental setting, VEGF(165)-induced angiogenic effects can be significantly inhibited by adding sFLT1 in vivo. A single-dose of VEGF(165) under ischemic conditions causes no significantly better flap survival in this model.     

腺病毒载体介导人VEGF cDNA促进随意型皮瓣成活的实验研究

目的探讨以腺病毒载体局部应用血管内皮细胞生长因子基因对大鼠缺血随意型皮瓣成活的影响。方法30只SD大鼠随机分成3组,每组10只,于鼠背设计蒂在尾侧髂嵴水平的随意型皮瓣,面积为2cm×8cm,A组皮瓣远端23顺皮瓣长轴分10个点真皮下注入1012PFU的携带人血管内皮细胞生长因子基因的腺病毒(AdCMVVEGF),B组同法注入同剂量的携带β半乳糖苷酶基因的腺病毒(AdCMVGal),C组注入1ml的生理盐水,3d后掀起皮瓣并原位缝合,术后7d观察实验结果。结果每组皮瓣成活面积的百分比分别为A组(8591±254)%,B组为(5956±118)%,C组为(6148±109)%,A组成活率显著高于B组和C组,差异有非常显著性意义(P<001),B组与C组比较差异无显著性意义(P>005)。原位杂交、免疫组化显示,应用AdCMVVEGF治疗的成活皮瓣中,VEGF有表达,组织学切片显示,AdCMVVEGF治疗皮瓣中肉芽组织增生,新生血管大量形成。结论以腺病毒为载体局部应用VEGF基因能增加缺血皮瓣的成活面积,该法具有高效的特点。