【作者】 楼晓莉；

【导师】 宋建星；

【作者基本信息】 第二军医大学 ， 外科学， 2009， 博士

【摘要】 随意皮瓣是修复创面、重建功能、改善外形常用的皮瓣。但临床应用时往往受到严格的长宽比例的限制。临床上超过一定长宽比例的皮瓣远端常发生缺血坏死,目前尚无公认的最佳改善皮瓣存活的方法。皮瓣移植成功与两个因素有关:一是蒂部及血管网的血供营养,二是皮瓣基底及周边组织床新生微血管的及时长入。因此在蒂部血流一定的条件下,如何促进血管新生成为提高皮瓣存活面积一个关键因素。新生血管的及时形成对皮瓣的成活起着很重要的作用,如果阻断皮瓣与受床之间血运的建立,皮瓣的成活面积将减少。皮瓣远端缺血部分的成活过程与皮片相同,主要通过与受床间建立血运再血管化而成活。在目前众多的研究表明,血管内皮生长因子(VEGF)是一种有效的促血管新生因子,它能刺激血管内皮细胞有丝分裂和增加血管内皮细胞渗透性促进血管生成,诱导NO的生成[1],进而起到促进组织愈合的作用。VEGF有良好的生物学作用已经得到认可,很多学者也尝试了用各种不同的方法和途径通过外源性的VEGF作为补充来促进皮瓣的成活。组织在缺血后发生一系列的生物反应,组织的缺血引起VEGF浓度的升高,改变皮瓣的灌注,从而引起血管的舒张和新生血管的形成[2-3]。然而,由于皮瓣远端组织长时间严重的缺氧、缺血会使微血管及内皮细胞会受到直接损伤,使得内源性的VEGF来不及分泌,或在外源性的VEGF补充后远端组织的新生血管不能及时长入而导致坏死。在缺血后3h,我们就可以检测到VEGF受体VEGFR-2的表达,而在18h后就会发生衰减[4]。因此,我们在本实验中在使用VEGF的同时,联合引进了具有抗氧化,抗炎症,抗凋亡,可以提高机体抗缺氧能力的血红素氧化酶(HO-1)[5]。此酶能够延长随意皮瓣远端组织的抗缺氧时间,为VEGF促进新生血管的长入赢得了更加充足的时间,从而提高超长随意皮瓣远端组织的存活面积。HO-1是血红素代谢的起始限速酶,主要存在于细胞微粒体中。它能够将血红素分解为胆绿素、一氧化碳和铁[6-7],胆绿素在胆绿素还原酶的作用下进一步生成胆红素。胆红素和胆绿素能有效的清除氧自由基,有较强的抗氧化功能。在许多因素作用下能诱导产生HO-1表达,是细胞在缺血再灌注[8]、低氧损伤、内毒素血症[9]、组织排异[10]等应激过程中的一种自我保护机制。Bussolati等[11]提出VEGF诱导HO-1的表达,提高了HO-1在人内皮细胞中的活性。HO-1抑制剂能够消除VEGF在体内非炎症的促血管新生反应。先前的体外实验证实了HO-1能诱导VEGF的分泌,HO-1的大量表达会引起毛细血管样结构的增殖和形成[12-13]。Soares等[14]研究发现,VEGF和HO-1的表达会激发一个正反馈环的形成,即VEGF可以上调HO-1的表达,反之亦然。基因表达载体有病毒表达载体和非病毒表达载体。病毒表达载体有RNA逆转录病毒、DNA腺病毒和DNA腺相关病毒(AAV)等;而非病毒表达载体有质粒、脂质体、裸DNA和基因枪等。目前被研究应用最多也最方便的是腺病毒载体。腺病毒载体的优点有①宿主范围广,对人致病性低;②在增殖和非增殖细胞中感染和表达基因;③能有效进行增殖,滴度高;④不整合到染色体中,无插入致突变性;⑤能同时表达多个基因等。正是由于具有以上一些优点,腺病毒被极其广泛地应用于体外基因转导、体内接种疫苗和基因治疗等各个领域。在本实验中,我们将VEGF和HO-1有机的结合在一起,使其两者发挥更大的生物学效应。并以腺病毒为载体转染靶细胞,使其能持续表达目的蛋白,有助于克服传统的生物制剂半衰期短,活性低,成本高等缺点。以腺病毒在载体联合应用VEGF和HO-1能否提高随意皮瓣远端组织的成活面积,目前国内外未见相关报道。为此,我们设计了本实验课题,进行了以下三个部分的实验研究。1. Ad5-VEGF和Ad5-HO-1非增殖腺病毒的构建与制备目的:构建含人血管内皮细胞生长因子(VEGF165)和血红素氧合酶(HO-1)的重组腺病毒载体(Ad-VEGF165和Ad-HO-1),为后续的基因转染、体内实验提供实验基础。方法:引物合成后获取目的基因,将质粒PDC315-VEGF和PSUCMV-HO-1与含有5型腺病毒右臂的质粒pBHGE3通过Lipofectamine2000共转染至293细胞。共转染后9-14天出现病毒空斑,经过三次病毒空斑纯化,提取腺病毒DNA并应用PCR进行鉴定。将重组出的病毒在293细胞中反复大量扩增,通过氯化铯密度梯度离心法进行纯化,并用TCID50法计算病毒滴度。结果:经鉴定正确的腺病毒命名为Ad-VEGF165和Ad-HO-1。测得重组腺病毒Ad-VEGF滴度为1×1010 pfu/ml,病毒Ad-HO-1滴度为7.5×109 pfu/ml。结论:成功构建了复制缺陷型重组腺病毒Ad-VEGF165和Ad-HO-1,其滴度高,毒性低,可用于体外转染。2.腺病毒Ad-VEGF和Ad-HO的体外实验研究目的:探讨重组腺病毒Ad-VEGF165和Ad-HO-1转染成纤维细胞BJ后目的蛋白的表达情况和对BJ细胞生长、增殖分化的影响。方法:用报告基因Ad-EGFP转染靶细胞,观察其在荧光显微镜下的荧光强度和流式细胞仪来检测腺病毒的转染效率和强度。用ELISA的方法检测Ad-VEGF165在BJ细胞中定量表达,用免疫组化和WESTERN BLOT检测Ad-HO-1在BJ细胞中的蛋白表达情况。MTT检测腺病毒对BJ细胞增殖活性的影响。结果:腺病毒介导的目的基因对BJ细胞有较高的转染效率,随着MOI的增强效率增高,具有量效关系。ELISA可以检测到VEGF的有效表达,同时免疫组化和WESTERN BLOT也检测到了Ad-HO-1在BJ细胞中的蛋白表达。转染一周后MTT检测OD值,转染组细胞与未转染组细胞的OD值无显著性差异。结论:腺病毒介导的VEGF165和HO-1能在BJ细胞中有效表达,并且不会对BJ细胞的增殖活性产生影响。3. Ad-VEGF和Ad-HO联合应用提高大鼠超长皮瓣存活率的体内实验研究目的:探讨腺病毒介导的VEGF165和HO-1联合应用能否提高大鼠超长随意皮瓣远端组织的成活面积。方法:以SD大鼠背部两侧超长随意皮瓣为模型[15]。将动物随机分为5组:Ad-VEGF165+Ad-HO-1(A组)、Ad-VEGF165(B组)、Ad-HO-1(C组)、Ad-GFP(D组)、病毒保存液Ad-buffer(E组)。分别在掀起皮瓣深筋膜层分10小部分注射。术后一周采用颈椎脱臼法处死。观察各个皮瓣的大体情况,存活面积,常规病理切片HE染色,皮瓣组织的VEGF和HO-1免疫组化检测,CD31免疫组化和皮瓣新生微血管密度(MVD)计数,进行统计学分析。结果:大体解剖及病理切片显示:Ad-VEGF165+Ad-HO-1联合应用组皮瓣的成活面积及血管化程度明显高于其他四组,B组稍高于C、D、E三组,C、D、E三组之间无明显差异(P>0.05)。结论:腺病毒介导的VEGF165和HO-1联合应用能够提高大鼠超长随意皮瓣远端组织的成活面积,降低皮瓣覆盖创面的风险。综上所述,我们构建了携带VEGF165和HO-1目的基因的非增殖腺病毒Ad-VEGF165和Ad-HO-1,其对提高大鼠超长随意皮瓣远端组织的成活面积的疗效明显强于传统治疗。该方案充分发挥了HO-1的抗缺氧、抗凋亡、抗炎症的能力和VEGF165的协同作用,为提高皮瓣的长宽比和成活率提供了新的临床治疗策率和新的思路。更多还原

【Abstract】 Random flap is commenly used to wound covering, function reconstruction and appearance improvement. But the distant end of the flap which is beyond a certain length/width ratio can easily get necrosis.There are two critical success factors for flap grafting.One is the blood supply of the root flap.The other is neovascularization.If neovascularization is interrupted, survival area of the flap will be less.The surviving process of the distant end is similar with skin flap.As we all know, Vascular endothelial growth factor (VEGF), a mitogen that promotes vascular endothelial cells’proliferation and angiogenesis[16], is a 45-kDa glycoprotein secreted in the vascular wall by endothelial and smooth muscle cells. VEGF promotes angiogenesis in normal physiologic conditions such as wound repair and in pathologic conditions including atherosclerosis, diabetic retinopathy, tumor growth and metastasis formation.But in fact,vegf had disadvantages.Remote organizations of the flap suffered serious hypoxia and ischemic for a long time resulting in the injury of the vescular endothelial cells. There was not enough time for the endogenous secretion of VEGF. In order to compensate for disadvantage of VEGF, we adopted HO-1 which has the function of anti-oxidation, anti-inflammatory, anti-apoptotic for joint application.It can obviously enhance the ability of anti-hypoxia gaining time for secretion of vegf of the body, hoping increase the survival area of the remote ultra-long random flap.Several lines of evidence suggest that heme oxygenase-1 (HO-1) is a component of cellular defence mechanisms against oxidative stress-mediated injury.Heme oxygenase (HO) is the rate-limiting enzyme for heme degradation in mammalian cells. HO catabolizes cellular heme,which is a prooxidant, to carbon monoxide (CO), bilirubin and iron. Three HO isoforms have been characterized; each encoded by a different gene. An inducible form HO-1, is induced by a variety of stresses such as oxidized lipoproteins,cytokines, hemodynamic changes, angiotensin II and nitric oxide (NO) in vascular wall. HO-1 induction seems to function as an adaptive response against these injurious stimuli. Soares and colleagues have reported that has found that the expression of vegf and ho-1 can stimulate the formation of a positive feedback loop,that is the expression of vegf can upregulate the secreation of ho-1, and vice versa.Gene expresson includes virus expression vector which consists of retroviral RNA, adenovirus DNA and adeno-associated virus and non-virus expression which involves plasmid, liposomes, naked DNA and gene gun.Currently, adenovirus vector is the most convenient and most widely used.There are plenty of advantages of adenovirus.①It has wide host range and low pathogenicity for people.②It can infect the proliferation and non-proliferating cells and have expression of genes.③Have high proliferation and titer.④Not integrated into the chromosome and no insert Mutagenicity.⑤Expression of multiple genes at the same time. Precisely because of the advantage above, adenovirus is widely used in gene transduction in vitro, vaccination in vivo and gene therapy.This study was conducted to investigate the united biological activity of VEGF and HO-1 expression. Target cells were transfected by adenovirus which can sustainly express the target protein. It can overcome the disadvantages of traditional biological agents such as short half-life, low activity and high cost. There were no relevant reports at home and abroad about increasing survival area of remote random flap through adenovirus VEGF and adenovirus HO-1. We designed this experiment subjects and carried out three-part of this experimental study.1. Construction of Ad5 repilication-deficient adenovirusAd-VEGF165 and Ad-HO-1To construct the new recombinant adenoviruses Ad-VEGF165 and Ad-HO-1 by homologous recombination, the plasmid PDC315-VEGF and PSUCMV-HO-1 were co-transfected with pBHGE3 into 293 cells through Lipofectamine2000 respectively. The recombinant adenoviruses were confirmed by PCR analysis using specific primers, amplified at a large scale and purified by cesium chloride gradient centrifugation. The viral titer of Ad-VEGF165 achieved1×1010 pfu/ml while Ad-HO-1 7.5×109 pfu/ml with TCID50 method.2. Biological activity of Ad-VEGF165 and Ad-HO-1 in vitroFor the purpose of comparing the infection capability of adenoviruse, the normal cells BJ were cultured and transfected with Ad-EGFP. The results were observed under the fluorescence micro- scope. The transfection and protein expression of VEGF gene was determined by ELISA. Because the expression of HO-1 gene in recombinant adenoviruse Ad-HO-1 infected host cells was demonstrated to locate to the mitochondrion .So the protein expression of HO-1 gene was determined by western blot and imunohistochemisty. The results of ELISA, western blot and imunohistochemisty all show that Ad-VEGF165 and Ad-HO-1 expresse large numbers of taget proteins in BJ cells. MTT assay was performed to determine the safty of Ad-VEGF165 and Ad-HO-1 at various viral MOIs. The transfection was demonstrated by flow cytometry. The results showed that the growth and proliferation of BJ cells transfected by Ad-VEGF165 and Ad-HO-1 were the same as the normal BJ cells.So Ad-VEGF165 and Ad-HO-1 were safe to use.3. Antitumor efficacy of SG235-TRAIL in vivo25 female Sprague-Dawley rats weighing between 250 and 350g were used for this experiment. One randon flap was made on each dorm of the Sprague-Dawley mice(2 flaps each mouse).Then mice were allotted randomly into five groups equally: Ad-VEGF165+Ad-HO-1(A group)、Ad-VEGF165(B group)、Ad-HO-1(C group)、Ad-11B-GFP(D group)、Ad-buffer(E group) (n=5 mice/10 flap/group). Each flap underwent 10 dots injections in the deep fascia, with total dosage of 109 pfu per flap. After 10 days,all animals were killed and 3 small tissues of flap were removed and fixed in 10% neutral formaldehyde for 6h and paraffin-embedded, and 5-μm-thick consecutive sections were cut for H&E staining, immunohistochemistry and MVD counting. VEGF165 and HO-1 protein were detected by immunohistochemistry.The expression of CD31 was much more in A group than other 4 groups.Compared with the control group and other treatment groups, survival rate of combination use of Ad-VEGF165 and Ad-HO-1 was much higher.Obvious necrosis was observed by routine pathologic examination in the end of the long flap in the certain groups.Collectively, we have constructed 5-type repilication-deficient adenovirus Ad-VEGF165 and Ad-HO-1,whose joint application has obviously increased the survival area of remote random flap.The project has given full play to HO-1 for its ablity of anti-oxidation, anti-inflammatory, anti-apoptotic and synergy with VEGF(165). It provides a new clinical treatment strategy and new ideas to improve the aspect ratio of flap and its survival rate.更多还原