碱性成纤维细胞生长因子对预构皮瓣血管化及皮瓣成活的影响

预构皮瓣的研究越来越受到临床整形医师的重视 ,它为面颈部软组织缺损的修复和器官再造提供了新的手段 ,其基本概念是将知名血管束植入到皮瓣下方 ,待血管发芽与皮瓣原有血管血运沟通后再行移植。预构皮瓣血运重建受到一些因素的影响。碱性成纤维细胞生长因子 (b FGF)具有广泛的生物活性 ,可调节血管内皮细胞的有丝分裂 ,是有效的血管生成因子之一[1 ] 。内源性 b FGF的降低还可导致组织损伤的加重和修复的延迟 [2 ]。将 b FGF应用于皮瓣的预构 ,有可能促进皮瓣与植入血管束之间血运的早期沟通 ,从而有利于皮瓣的成活和缩短皮瓣预构的时…     

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.     

碱性成纤维细胞生长因子对预构皮瓣血管化及成活的影响

目的：探讨碱性成纤维细胞生长因子（basic fibroblast growth factor，bFGF）对预构皮拉血管化的作用及成活的影响。方法：将新西兰大白兔耳中央动、静脉通过皮下隧道植入颈部皮瓣下方，实验组滴入9цg bFGF，对照组滴入0.2ml生理盐水。于术后1，2，3周观察血管芽与皮瓣血管沟通及皮瓣成活范围。结果：预构皮瓣术后1，2周，实验组血管芽及皮瓣成活率明显高于对照组。结论：局部应用bFGF对预构皮瓣的血管芽发生有促进作用，有利于预构皮瓣的成活。     

血管内皮生长因子联合碱性成纤维细胞生长因子对大鼠后肢动脉硬化闭塞血管再生机制的研究

目的探讨血管内皮生长因子(VEGF)联合碱性成纤维细胞生长因子(b FGF)对大鼠后肢动脉硬化闭塞血管再生的机制。方法雄性SD大鼠60只,建立后肢动脉硬化闭塞模型,按随机数字表法将其分为4组:生理盐水组(NS组)、VEGF组、b FGF组及VEGF+b FGF组。NS组、VEGF组隔天于腹腔分别注射生理盐水1 m L、100μg/L rh VEGF 1 m L;b FGF组隔天于后肢股内侧多点肌肉注射100μg/L rhb FGF 1 m L;VEGF+b FGF组隔天于腹腔注射100μg/L rh VEGF 1 m L及于后肢股内侧多点肌肉注射100μg/L rhb FGF 1 m L,共治疗21 d。第30 d时行血管造影检查观察大鼠后肢动脉硬化闭塞血管再生情况;第3个月时,取后肢股内侧肌肉组织分别应用Western blot和RT-PCR法检测其组织中VEGF和b FGF蛋白和m RNA表达水平。结果 1血管造影结果显示,VEGF+b FGF组侧支血管新生条数明显多于b FGF组(P0.05)、VEGF组(P0.05)及NS组(P0.001),b FGF组和VEGF组也明显多于NS组(P0.05),b FGF组和VEGF组间比较差异无统计学意义(P0.05)。2 Western blot和RT-PCR检测结果均显示,VEGF和b FGF蛋白和m RNA表达在VEGF+b FGF组均明显高于NS组(P0.001)、VEGF组(P0.001)和b FGF组(P0.001);VEGF组和b FGF组组间比较差异无统计学意义(P0.05)。结论 VEGF和b FGF联合应用能够上调大鼠后肢缺血区组织中VEGF、b FGF的含量,促进血管内皮细胞增殖和分化、毛细血管出芽生长,使缺血区血管新生,为外周动脉疾病的临床治疗提供新方法。     

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.     

局部缓释血管内皮生长因子促进肠吻合口愈合的研究

目的探讨应用生物蛋白胶缓释血管内皮生长因子(VEGF)促进肠吻合口愈合的有效性。方法通过在兔盲肠穿孔建立腹腔感染的动物模型,12h后行肠切除及肠吻合。动物分成4组(每组32只),各组吻合口表面所涂试剂不同:生理盐水组、生物蛋白胶组、VEGF组及VEGF+生物蛋白胶组。于手术后第3、5、7及14天统计各组吻合口漏发生情况,测量未发生吻合口漏的动物的吻合口破裂压,检测吻合口组织羟脯氨酸含量及VEGF表达情况。结果生物蛋白胶组及VEGF组的吻合口漏总发生率低于对照组,但差异无统计学意义(P0.05),而VEGF+生物蛋白胶组则明显低于对照组(P0.05)。至术后第14天,VEGF+生物蛋白胶组的吻合口破裂压、吻合口组织羟脯氨酸含量及VEGF蛋白表达阳性细胞率(除VEGF组)均较其余3组明显升高(P0.05,P0.01)。结论应用生物蛋白胶局部缓释VEGF能够促进肠吻合口愈合,降低吻合口漏的发生率。     

兔股动脉和静脉预构轴型扩张皮瓣的实验观察

目的 通过兔股动脉、静脉预构轴型扩张皮瓣的微循环血流晕动态变化、光镜下结构的改变及其成活面积,为预构轴型扩张皮瓣的临床应用提供依据.方法 选择新西兰白兔40只,随机分为4组:预构轴型扩张皮瓣组、预构轴型不扩张皮瓣组、单纯预构轴型皮瓣组及无蒂游离皮瓣组,每组4只,前2组股动脉、静脉移位后,预构轴型扩张皮瓣组、预构轴型不扩张皮瓣组分别在肉膜深面置入容量为50 ml长方形皮肤软组织扩张器,预构轴型扩张皮瓣组7 d后开始注水;无蒂游离皮瓣组为对照组,未采取预构及扩张处理.定期对4组皮肤进行微循环血流量检测,并取样进行光镜观察.预构术后52 d,前3组形成以预构股动静脉血管束为蒂的岛状皮瓣,游离皮瓣组则形成无蒂游离皮瓣后均原位缝合,观察其成活面积.结果 预构轴型扩张皮瓣组较其他组微循环血流量增加,成活面积大[(97.54±2.73)%],光镜下改变显著(P<0.05).结论 扩张术能促进预构轴型皮瓣的血管化进程,明显增大预构轴型皮瓣成活面积,增加其移植的安全性.     

骨髓间充质干细胞促进兔预构皮瓣成活的实验研究

目的探讨新西兰大白兔骨髓间充质干细胞(BMSC)同种异体移植促进预构皮瓣的血管再生,从而提高预构皮瓣存活率的可能性。方法体外分离、培养、鉴定并标记新西兰大白兔的BMSC。在实验兔腹部两侧构建预构皮瓣,两侧皮瓣随机分为实验组和对照组,实验组在股血管周围经皮注射已标记的BMSC悬液,对照组注射等量生理盐水。注射后第7天追踪观察BMSC移植后的成活情况,术后第14天掀起以股动静脉为轴心血管的岛状皮瓣,分别对两组的岛状皮瓣进行BrdU/vWF的免疫荧光双标检测;对岛状皮瓣中的VEGF进行Western Blot半定量分析;岛状皮瓣形成后第7天,观察两组皮瓣的成活情况。结果 BMSC异体移植后成活良好;实验组皮瓣内VEGF蛋白表达量明显高于对照组;实验组皮瓣内BrdU标记阳性细胞胞浆内有vWF信号;将预构皮瓣制成岛状皮瓣后第7天,实验组和对照组皮瓣存活率分别为(93.1±2.6)%、(51.5±7.5)%,P<0.05。结论异体移植BMSC可促进预构皮瓣的血管再生,提高预构皮瓣存活率。     

VEGF促进预构皮瓣血管新生的实验研究

目的探讨VEGF重组蛋白促进大鼠预构皮瓣血管新生、提高皮瓣存活面积的可行性。方法建立大鼠腹部预构皮瓣模型;30只Wistar大鼠随机分为两组;局部应用VEGF165（组Ⅰ）、PBS（组Ⅱ）;4周后形成以植入血管为蒂的岛状皮瓣,原位缝合;术后7d对皮瓣存活、血管新生情况进行检测。结果组Ⅰ、组Ⅱ的皮瓣存活率分别为66．13％±9．9％,55．59％±13．06％（P〈0．05）;组Ⅰ与组Ⅱ比较,微血管显影血管网更丰富,范围更广,分支更粗,内含墨汁的血管在皮瓣的表皮真皮、皮下层均有分布;微血管计数组Ⅰ、组Ⅱ分别为25．83±6．33条／mm^2,26．5±5．61条／mm^2（p〉0．05）。结论VEGF可以促进预构皮瓣的血管新生,提高存活率。     

血管内皮生长因子促进预构皮瓣成活的实验研究

目的　研究使用重组人类血管内皮生长因子 (Vascularendothelialgrowthfactor,VEGF)对预构皮瓣存活的作用 ,探讨VEGF能否促进正常血供组织的血管化。方法　取大鼠自体尾动脉 8cm移植 ,两端分别与股动、静脉吻合 ,成环状植入下腹部皮下组织 ,对照组局部应用 0 9%NaCl和16 %聚乙烯乙醇 ,实验组将VEGF分别溶于 0 9%NaCl和 16 %聚乙烯乙醇局部应用。分别于术后 3,4 ,5周以植入的尾动脉为血管蒂于下腹部形成 3cm× 4cm大小皮瓣 ,游离掀起皮瓣后缝回原处 ,7d后运用面积仪测出存活皮瓣面积的百分比。结果　第 5周后的皮瓣存活率VEGF组 (75 0 0 % ,5 8 4 1% )明显优于实验组 (10 % ,2 5 % )。结论　VEGF有利于预构皮瓣的存活。     

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。     

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。     

Gene therapy with adenovirus-mediated VEGF enhances skin flap prefabrication

We investigated the feasibility in rats of enhancing skin-flap prefabrication with subdermal injections of adenovirus-encoding vascular endothelial growth factor (Ad-VEGF). The left saphenous vascular pedicle was used as a source for vascular induction. A peninsular abdominal flap (8 x 8 cm) was elevated as distally based, keeping the epigastric vessels intact on both sides. After the vascular pedicle was tacked underneath the abdominal flap, 34 rats were randomly divided into three groups according to treatment protocol. The implantation site around the pedicle was injected with Ad-VEGF in group I (n = 10), with adenovirus-encoding green fluorescent protein (Ad-GFP) in control group I (n = 14), and with saline in control group II (n = 10). All injections were given subdermally at four points around the implanted vessel by an individual blinded to the treatment protocol. The peninsular flap was sutured in its place, and 4 weeks later, an abdominal island flap based solely on the implanted vessels was elevated. The prefabricated island flap was sutured back, and flap viability was evaluated on day 7. Skin specimens were stained with hematoxylin and eosin for histological evaluation. In two rats from each group, microangiography was performed to visualize the vascularity of the prefabricated flaps. There was a significant increase in survival of prefabricated flaps in the Ad-VEGF group compared to the control groups: Ad-VEGF, 88.9 +/- 6.1% vs. Ad-GFP, 65.6 +/- 9.4% (P < 0.05) and saline, 56.0 +/- 3.4% (P < 0.05). Sections from four prefabricated flaps treated with Ad-GFP revealed multiple sites of shiny deposits of green fluorescent protein around the area of local administration 1 day and 3 weeks after gene therapy. Histological examination done under high-power magnification (x400) with a light microscope revealed increased vascularity and mild inflammation surrounding the implanted vessel in all groups. However, we were unable to demonstrate any significant quantitative difference with respect to vascularity and inflammatory infiltrates in prefabricated flaps treated with Ad-VEGF compared with controls. Microangiographic studies showed increased vascularity around the implanted pedicle, which was similar in all groups. However, vascularization was distributed in a larger area in the prefabricated flaps treated with Ad-VEGF. In this study, the authors demonstrated that adenovirus-mediated VEGF gene therapy increased the survival of prefabricated flaps, suggesting that it may allow prefabrication of larger flaps and have the potential to reduce the time required for flap maturation.     

Neovascularization in prefabricated flaps using a tissue expander and an implanted arteriovenous pedicle

Creating prefabricated flaps using tissue expanders in combination with the implantation of maximal blood flow vascular pedicles into suitable tissue areas represents a new tendency in the reconstruction of large skin defects. In 42 Chinchilla Bastard female rabbits weighing 3,700-4,600 g, skeletonized arteriovenous pedicles with maximal blood flow, dissected from the femoral and saphena magna bundles, were implanted underneath abdominal fasciocutaneous flaps. Oval tissue expanders of 250 ml were placed and fixed on the abdominal wall to expand these prefabricated flaps. The evaluation parameters were macroscopic observation, blood analysis, selective microangiography, histology, and scintigraphy. The study results showed that neovascularization in expanded prefabricated flaps was established from newly formed vessels generated from the implanted pedicles and their vascular connections with the originally available vasculature in the flap. After 20 days of prefabrication, the entirety of the expanded prefabricated flaps was perfused by blood flow supplied from newly implanted arteriovenous pedicles. The study indicated that an expanded prefabricated flap can be successfully created by the simultaneous implantation of a maximal blood flow pedicle in combination with flap expansion.     

Prefabrication of large fasciocutaneous flaps using an isolated arterialised vein as implanted vascular pedicle.

Flap prefabrication represents a new trend in microsurgical tissue transfer. Based on the concept of neovascularisation, in Chinchilla Bastard rabbits (n=40), an isolated venous pedicle dissected from the femoral and saphena magna vein was arterialised by end-to-end anastomosis to the femoral artery at the inguinal ligament. This arterialised venous loop was implanted beneath a random-pattern vascularised abdominal fasciocutaneous flap as large as 8 x 15 cm(2) to investigate the development of neovascularisation at various evaluating times of 4, 8, 12, 16 and 20 days. To prevent neoangiogenesis from occurring between the underlying vascular bed and abdominal flap, a silicone sheet with the corresponding dimension of 8 cm x 15 cm x 0.25 mm was placed and fixed on the abdominal wall. The flap viability and the neovascularisation process in the prefabricated abdominal skin flaps were evaluated by macroscopic observation, blood analysis, selective microangiography and histology. The experimental results showed that newly formed vessels originating from the implanted isolated venous pedicle were evident on the angiograms 4 days after pedicle implantation. In the 8- and 12-day groups, newly formed vessels became larger and some were connected to the originally available vasculature in the abdominal fasciocutaneous flaps. In the 20-day group, entire flaps were perfused by the blood flow supplied from the newly implanted venous pedicles through newly formed vessels and their vascular connections. This study indicated that large flap prefabrication can be created by implantation of an isolated arterialised venous pedicle into a random-pattern vascularised fasciocutaneous flap. Twenty days appears to be the minimal length of time required after arterialised venous pedicle implantation for the maturation of neovascularisation in the prefabricated flap.     

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.