重组人VEGF基因治疗提高游离颗粒脂肪移植存活率的实验研究

目的:研究重组人血管内皮生长因子(VEGF)基因治疗对大鼠游离颗粒脂肪移植存活的影响。方法:SD雄性大鼠36只,分三组,于游离颗粒脂肪移植后分别注入重组的VEGF质粒(目的基因组),空白质粒(空白质粒组)和生理盐水(生理盐水组)。于术后7、15、30天分别采集移植的脂肪组织,计算植入物校正重量比并行HE染色观察一般组织病理改变,同时使用免疫组化法测定组织的VEGF表达水平及微血管密度。结果:目的基因组在移植物重量改变方面显著小于空白质粒组及生理盐水组(P<0.05),其VEGF表达及微血管密度均显著高于其他两组(P<0.01),空白质粒组及生理盐水组差异无显著性意义。此外,在空白质粒组及生理盐水组镜下存在大量囊样脂池、坏死区。结论:皮下注射的转VEGF基因的质粒在组织中能够表达VEGF,并诱导新血管的形成,增加血流灌注,减少移植组织的吸收,促进移植脂肪存活。     

重组人VEGF基因在游离组织移植中作用的实验研究

目的：研究重细人血管内皮生长因子（VEGF）基因治疗皮瓣、皮片及游离颗粒脂肪移植存活的影响。方法：将带有PCD-VEGF165的大肠杆菌接种到LB培养基中，通过碱分裂法制备PCD-VEGF165质粒，再用反蒸发法将质粒包埋于脂质体中。将108只雄性SD大鼠均分为三组，每组36只，每组再按注射的成分不同分三小组，分别为PCD-VEGF165目的基因组、PCD空白质粒组和生理盐水组，各组分别注入目的基因、空白质粒和生理盐水后在不同时间点计算组织成活率，同时取标本做成石蜡块行常规染色及免疫组化染色，检测VEGF165的表达及血管生长情况。结果：目的基因组的皮瓣及皮片成活面积较空白质粒和生理盐水组明显大（P〈0．01），游离脂肪重量减少的程度亦显著小于空白质粒组和生理盐水组（P〈0．05），三种移植组织中目的基因组VEGF的表达及微血管密度均显著高于其他两对照组（P〈0．01），目的基因组的血管直径明显小于对照组（P〈0．05），空白质粒组及生理盐水组差异无显著性意义。结论：注射转VEGF基因的质粒后可在组织中表达丰富的VEGF，诱导新血管的形成，增加血流灌注，并减少移植组织的吸收，最终促进移植皮瓣、皮片及脂肪的存活。     

重组人VEGF基因对游离组织移植物血管形成的作用

目的:研究重组人血管内皮生长因子(VEGF)基因对游离皮瓣、皮片及游离颗粒脂肪等移植物血管形成的作用。方法:将带有PCD-VEGF165的大肠杆菌接种到LB培养基中,通过碱分裂法制备PCD-VEGF165质粒,再用反蒸发法将质粒包埋于脂质体中。将108只雄性SD大鼠均分为三组,每组36只,每组再分三小组,分别为PCD-VEGF165目的基因组、PCD空白质粒组和生理盐水组,注入后在不同时间点取标本做成石蜡块行常规染色以观察血管生长情况。结果:三种移植组织中目的基因组微血管密度均显著高于其他两对照组(P<0.01),血管直径明显小于对照组(P<0.05),空白质粒组及生理盐水组差异无显著性意义(P>0.05)。结论:VEGF基因生物活性稳定,特异性强,持续时间长。注射转VEGF基因的质粒后可诱导移植组织周围新血管的形成,增加血流灌注。     

血管内皮生长因子基因改善大鼠腹壁下动脉皮瓣成活的实验研究

目的　通过血管内皮生长因子 (vascularendothelialgrowthfactor,VEGF)基因对腹壁下动脉皮瓣的转染 ,探讨应用VEGF基因对皮瓣成活的影响。方法　以SD大鼠为实验模型制作腹壁下动脉皮瓣 ,并分别注入脂质体包裹的PCD VEGF1 6 5(目的基因组 ) ,PCD(空白质粒组 )和生理盐水 (生理盐水组 )。术后 ,①通过腹腔注射荧光素钠估计皮瓣的血流量 ;②计算断蒂后各组大鼠皮瓣的成活面积 ;③取大鼠皮肤标本行常规染色检测平均血管数目及内径 ;④皮肤标本行VEGF免疫组化染色检测VEGF表达情况。结果　目的基因组、空白质粒组和生理盐水组的平均荧光染色面积分别为6 0 6 4%、30 15 %、2 9 89%(P <0 0 5 ) ,大鼠断蒂后的平均成活面积分别为 92 3%、30 5 %、31 8%(P<0 0 5 ) ,平均血管数目分别为 10 1 72、91 35、89 85 (P <0 0 5 ) ,平均血管内径分别为 2 6、31 0 9、32 5 1μm(P <0 0 5 ) ,免疫组化染色示目的基因组染色深度明显高于空白质粒组和生理盐水组 (P <0 0 5 )。结论　PCD VEGF1 6 5转染能够改善皮瓣的成活 ,且通过转染表达了丰富的VEGF1 6 5。     

内皮抑素基因对ACHN RCC中VEGF和MVD表达的影响

目的：利用已构建的带有内皮抑素基因真核表达载体质粒（pSecES）导入裸鼠人肾癌细胞系肾细胞癌（ACHN RCC）中,研究内皮抑素基因对血管内皮生长因子（VEGF）和微血管密度（MVD）表达的影响.方法：将荷瘤裸鼠随机分为3组,每组8只.干预组每只裸鼠瘤内3点注射复合物100μl,内含30μl梭华-Sofasttm和30μg pSecES质粒;对照组每只裸鼠瘤内3点注射复合物100μl,内含30μl梭华-Sofasttm和30μg pcDNA3.1质粒;空白组每只裸鼠瘤内3点注射生理盐水100μl.各组每只裸鼠间隔3天注射1次,连续注射3次.接种后第34天处死裸鼠,取肿瘤组织按SP法进行免疫组织化学染色,检测MVD和VEGF的表达情况.结果：干预组ACHN RCC的生长较对照组和空白组明显受到抑制,干预组CD34标记的MVD的表达和VEGF的表达较对照组和空白组低.结论：内皮抑素基因导入可以抑制荷瘤裸鼠ACHN RCC的生长,而且MVD和VEGF在ACHNRCC的表达减少.     

人血管内皮细胞生长因子基因转染大鼠骨髓间充质干细胞对脂肪颗粒移植存活率的影响

目的 检测大鼠骨髓间充质干细胞(BMSCs)经人血管内皮细胞生长因子(VEGF165)基因转染后,对脂肪颗粒移植存活率的影响.方法　利用FuGENE HD介导VEGF165基因体外转染SD大鼠BMSCs,与取自SD大鼠的脂肪组织混合后移植于大鼠背部,类似方法设立未转染组及DMEM液空白对照组.检测脂肪组织存活率及再生血管密度.结果　转染组存在外源性基因和蛋白的表达,脂肪组织存活率及再生血管密度高于未转染组,且两者均高于对照组.结论　转染VEGF165基因的BMSCs具有更强的促血管再生作用,可提高游离移植脂肪的存活率.     

自体移植脾组织VEGF、KDR表达与血管再生的实验研究

目的 研究自体移植脾组织血管再生及VEGF、KDR表达规律，阐明VEGF、KDR对移植脾组织血管再生的调控作用，为脾脏外科临床及实验研究提供理论依据。方法 健康Wistar大鼠70只，体重100—120g，随机分为7组，每组10只中又设脾切除自体脾移植组5只，假手术组5只，分别于术后7，14，30，60，90，120，180d进行：(1)自体移植脾组织病理学检测；(2)大鼠行主动脉插管灌注墨汁，光镜观测再生血管并采用图像分析测定其密度；(3)免疫组化抗VEGF、KDR抗体染色，图像分析定量，阐明其表达规律及与血管再生的关系。结果 (1)自体脾组织移植术后7d即有血管从大网膜向脾组织内伸展，移植脾组织内血管密度逐渐增大，至术后180d血管再生接近正常；(2)自体脾组织移植术后7d、14d，VEGF、KDR阳性染色细胞密度迅速升高，术后60d达高峰，以后逐渐降低，至术后180d VEGF、KDR阳性染色细胞密度趋向正常。结论 自体脾组织大网膜内移植术是简便有效的脾移植方法；移植脾组织新生血管由大网膜再生而来；术后移植脾组织内VEGF、KDR表达量升高，促进血管形成，血管再生完成后恢复正常水平。     

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.     

纳米血管内皮生长因子在治疗下肢缺血促进血管再生成中的作用

目的　利用家兔后肢缺血模型 ,观察纳米材料聚乳酸聚乙醇酸共聚物 (poly dl lactic co glycolicacid ,PLGA) ,包载血管内皮生长因子 (VEGF16 5 )基因 ,经局部肌肉注射后 ,外源基因在局部缺血组织中的转染及强度 ,以及缺血部位的血管新生状况。　方法　制备VEGF16 5 真核表达质粒 ,制备包载VEGF16 5 基因的纳米粒子 ,并检测其理化性质和体外释放曲线。建立家兔后肢缺血模型 2 4只 ,其中4只为对照组 ,只行股动脉及其分支结扎切断术 ;2只为空白纳米粒子组 ,局部缺血肌肉注射空白纳米粒子 ;10只应用裸质粒VEGF16 5 转染 ,8只采用纳米技术包载VEGF16 5 转染。直接缺血部位肌肉内多点注射 ,进行局部定位基因转染。术后 14d行血管造影 ,了解缺血部位侧枝形成情况。处死兔子 ,取股二头肌 ,内收大肌 ,做病理切片 ,免疫组化染色 ,观察VEGF16 5 的表达 ,测定毛细血管密度。应用逆转录 聚合酶链反应了解VEGF16 5 在骨骼肌中的表达 ,并对不同的转染技术进行半定量分析。　结果　转基因治疗 14d后 ,转染VEGF基因组血管造影可见明显新生血管和侧枝循环形成 ,免疫组化染色可见VEGF16 5 蛋白表达水平增高 ,缺血肌肉血管数增多 ,纳米VEGF16 5 治疗组与裸质粒VEGF16 5 治疗组的毛细血管密度明显高于对照组 ,有显著性差异 (P     

碱性成纤维细胞生长因子联合血管内皮细胞生长因子促进自体脂肪移植成活的动物实验研究

目的探讨碱性成纤维细胞生长因子(1bFGF)联合血管内皮细胞生长因子(VEGF)对大鼠自体脂肪移植血管新生及成活的影响。方法建立大鼠自体脂肪移植模型,将腹股沟处脂肪组织与生长因子蛋白溶液或生理盐水混合移植于大鼠背部。大鼠背部随机注射脂肪组织与生理盐水或0.5μg bFGF、1.0μg bFGF、2.0μgbFGF、0.5μg bFGF+0.5μg VEGF、1.0μg bFGF+1.0μg VEGF。12周后,对移植物进行成活率分析、组织学观察以及CD31免疫组织化学染色。结果向0.3 ml脂肪组织中添加2.0 Pg bFGF可显著提高移植物成活率及微血管密度。0.5μg bFGF组及1.0μg bFGF组与生理盐水组相比,差异无统计学意义(P0.05);但分别联合应用0.5μgVEGF和1.0μgVEGF以后,可显著升高移植物成活率,差异具有统计学意义(P0.05)。1.0μg bFGF+1.0μg VEGF联合应用组的移植物成活率和血管密度亦高于2.0μg bFGF单添加组。结论相比单独应用bFGF,VEGF与bFGF联合应用可刺激更多的血管生成,从而提高移植脂肪的成活率。     

Effect of rhVEGF gene transfection on survival of grafts after autologous free granular fat transplantation in rats

Objective： To investigate the effect of recombinant human vascular endothelial growth factor （rhVEGF） on autologous free granular fat grafts in rats.
Methods： Forty-eight Sprague Dawley （ SD ） rats, weighing 190-280 g and regardless sex, were randomly divided into three groups, sixteen in each. After fat transplantation, the rats were treated with plasmid DNA encoding rhVEGF protein （the experimental group ）, plasmid DNA （ the negative group） and normal saline （ the blank control group ）, respectively. At 3, 7, 15 and 30 days after transplantation, the rats were killed and the grafts were weighed, respectively. Histopathological changes were evaluated. Microvessel density and the expression of VEGF were examined by immunohistochemical staining and Western blotting.
Results： The weights of the negative and blank control groups were significantly reduced on the 7th, 15th and 30th days compared with those of the experimental group. The expression of VEGF and the microvessel density in the experimental group were significantly higher than the other two groups during the latter periods.
Conclusion： The plasmid encoding VEGF can induce expression of VEGF and angiogenesis in fat grafts and reduce the absorption of free fat grafts.     

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.     

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.     

Nerve graft prefabrication: preliminary study

This study aimed to produce prefabricated nerve graft as effective as autologous nerve graft without donor site morbidity for repairing segmental nerve defects. Thirty rats were used and were separated into three groups. In the first group, vein graft excised from jugular vein was sutured to make a bridge between epineural gaps of tibial and peroneal nerve. In the second group, one-quarter of the nerve diameter was incised after excision of the epineurial sheath, and the vein graft was sutured between epineurial gaps. In the third group, the vein graft was sutured between epineurial gaps, and plasmid including vascular endothelial growth factor (VEGF) gene were injected into muscle next to the nerve. Functional and morphological assessments were performed at the end of the 8 weeks. We prefabricated nerve graft by using autologous vein as a conduit material between two intact nerves and by gene therapy, which increases the VEGF level in the medium.     

人VEGF165基因在狗骨髓基质细胞中的表达

目的 :检测转染 pcDNA3 hVEGF165狗骨髓基质细胞的VEGF165mRNA表达。方法 :用脂质体介导转染狗骨髓基质细胞 ,用免疫组化及RT PCR检测VEGFmRNA的表达。结果 :重组质粒 pcDNA3 hVEGF165转染骨髓基质细胞后 ,免疫组化及RT PCR检测有VEGFmRNA的表达。结论 :采用脂质体介导的方法可将hVEGF165基因转染至狗骨髓基质细胞并表达外源性基因     

整合素连接激酶对瘢痕成纤维细胞VEGF的调控作用

目的 探讨整合素连接激酶(intergrin-linked kinase,ILK)在人瘢痕成纤维细胞中的表达及对成纤维细胞VEGF的调控作用.方法 8例人增生性瘢痕标本采用组织块法分离培养瘢痕成纤维细胞,选取第5～6代细胞备用.实验分为3组①对照组:不做任何处理,只用含10%FCS的DMEM培养液同步培养;②空质粒组:用空质粒转染人瘢痕成纤维细胞;③ILK cDNA表达质粒转染组:用ILK cDNA表达质粒转染人瘢痕成纤维细胞.首先通过免疫细胞化学染色法检测ILKcDNA转染前后成纤维细胞ILK、VEGF的表达变化;应用实时荧光定量PCR(RT-PCR)及Western blot检测成纤维细胞ILK、VEGF的mRNA和蛋白水平变化;最后采用酶联免疫吸附试验(ELISA法)检测3组成纤维细胞上清液中的VEGF蛋白含量.结果 免疫细胞化学染色结果显示瘢痕成纤维细胞胞浆中ILK表达阳性,VEGF表达不明显,经ILK cDNA转染细胞后ILK与VEGF表达均增强;RT-PCR显示ILK cDNA转染组VEGF mRNA表达(0.338±0.060)均高于对照组(0.022±0.001)和空质粒组(0.028±0.005,P＜0.05);Western blot结果显示ILK cDNA转染组VEGF蛋白表达(0.819±0.019)显著高于对照组(0.607±0.033)和空质粒组(0.591±0.024,P＜0.05);ELISA法检测ILK cDNA转染组的VEGF蛋白含量高于其他两组(P＜0.05).结论 ILK可以上调VEGF mRNA及蛋白水平,并且促进成纤维细胞分泌VEGF,ILK可能通过提高瘢痕成纤维细胞合成分泌VEGF而促进增生性瘢痕血管生成.

Abstract：

Objective To explore the expression of intergrin-linked kinase (ILK) and its effect on VEGF expression in fibroblasts from human hypertrophic scar. Methods Fibroblasts were isolated from hypertrophic scar of 8 patients and cultured in vitro. Then the cells were divided into three groups: ① Cells were cultured only in DMEM containing 10% FCS in the control group; ② Cells were transfected with empty plasmid in the empty plasmid group; ③ Cells were transfected with plasmid experessing ILKcDNA in the ILK cDNA plasmid transfection group. First, the expression of ILK and VECF was observed by immunocytochemistry before and after ILK cDNA transfection. Second, ILK and VEGF mRNA expression was investigated by real-time PCR ( RT-PCR). Third, the protein expression of ILK and VEGF was detected by Western blot. Finally, the protein level of VEGF in supernatant of fibroblasts was measured by ELISA. Results Before ILK cDNA transfection, the expression of ILK was positive and the VEGF expression was weak in cytoplasm of fibroblasts . After ILK cDNA transfection, both the expression of ILK and VEGF was enhanced. The level of VEGF mRNA was significantly higher in ILK cDNA transfection group (0.338 ±0.060) than that in control group (0.022 ±0.001) and empty plasmid group (0.028 ± 0. 005 , P ±0. 05 ). The level of VEGF protein was significantly higher in ILK cDNA transfection group (0. 819 ±0. 019) than that in control group (0. 607 ±0. 033) and empty plasmid group (0. 591 ±0. 024, P ＜0. 05). Secretion of VEGF increased remarkably in ILK cDNA transfection group comparing with the other two groups (P＜0. 05). Conclusions ILK could up-regulate the VEGF mRNA and protein level in human scar fibroblasts. It may play an important role in the angiogenesis in hypertrophic scar.