血管内皮细胞生长因子及有关肢体缺血基因治疗的研究进展

目的 探讨血管内皮细胞生长因子（vascular endothelial growth.VEGF）基因治疗严重慢性肢体缺血性疾病的可能性。方法 综述近年来对有关ＶＥＧＦ的分子生物学、ＶＥＧＦ的促血管生成机理及通过基因转移表达ＶＥＧＦ改善缺血肢体血供所进行的研究。结果 临床前的研究显示,ＶＥＧＦ能够刺激动物血肢体侧支动脉的形成,即所谓的“治疗性的血管生成”;临床结果证实,能过基因转移表达的ＶＥＧＦ能够促进新生血管形成,使有严重肢体     

大鼠全脑缺血再灌注细胞凋亡的观察

本实验采用大鼠全脑缺血再灌注模型，观察缺血后不同时间点的神经元凋亡，以研究大鼠全脑缺血再灌注后脑组织的形态学改变。材料与方法一、动物分组健康、雄性Ｗｉｓｔａｒ大鼠４３只，体重２００～２５０ｇ，随机分成七组；假手术组（Ａ组，ｎ＝５），缺血组：分成缺血１０分钟组Ｂ组、缺血６小时组Ｃ组（各组ｎ＝５），缺血再灌注组：缺血１０分钟再灌注１天组Ｄ组、２天组Ｅ组、４天组Ｆ组、７天组Ｇ组（各组ｎ＝７）。二、脑缺血模型的制备采用Ｐｕｌｓｉｎｅｌｌｉ法［１］，制备大鼠全脑缺血模型。假手术组只分离出翼小孔、双侧颈总动…     

膀胱移行细胞癌VEGF表达及与血管形成定量的关系

为了探讨膀胱移行细胞癌中血管内皮生长因子（ＶＥＧＦ）表达及其与血管形成定量关系，应用免疫组织化学方法，对６２例原发性膀胱移行细胞癌及８例正常膀胱组织中ＶＥＧＦ进行检测，并对其在３０例浸润性膀胱癌组织中表达与血管形成定量关系进行了研究。结果发现正常膀胱组织均为阴性反应，膀胱癌组织中ＶＥＧＦ蛋白阳性表达率为５６％。低分化和浸润性癌中ＶＥＧＦ蛋白阳性表达率明显高于高分化和表浅性癌组（Ｐ＜００５），浸润性膀胱癌组织中血管形成定量与ＶＥＧＦ表达密切相关（Ｐ＜００１）。结果提示：ＶＥＧＦ表达对膀胱癌生物学行为有重要影响。ＶＥＧＦ是膀胱癌发生发展过程中一个主要血管生成因子，ＶＥＧＦ蛋白表达和血管形成定量有可能成为预测膀胱癌转移和预后的指标     

血管内皮生长因子在肾细胞癌中的表达及意义

研究肾细胞癌血管内皮生长因子（ＶＥＧＦ）的表达及其与肿瘤转移、分期、病理类型及预后的关系。采用抗ＶＥＧＦ的多克隆抗体免疫组织化学技术染色（ＬｓＡＢ法）研究６１例肾癌组织切片。结果显示：４５９％（２８／６１）的肾癌ＶＥＧＦ表达阳性，淋巴结和（或）血行转移的ＶＥＧＦ表达率（７７８％）明显高于非转移者（３２６％，Ｐ＜００１）；阳性表达者五年生存率（２９１％）明显低于阴性表达者（８４６％，Ｐ＜００１）；Ⅰ、Ⅱ期阳性表达低于Ⅲ、Ⅳ期（Ｐ＜００５）；但与性别、年龄及肿瘤的病理类型无关。ＶＥＧＦ除在癌细胞胞浆和胞膜表达外，尚表达于肿瘤基质血管和邻近肿瘤的正常肾小管胞浆、肾小球和血管内皮及血管平滑肌胞膜。认为ＶＥＧＦ除由肿瘤细胞合成外，可能尚表达于邻近肿瘤的正常肾小管胞浆，ＶＥＧＦ表达有助于肾癌预后判断及指导治疗，ＶＥＧＦ可能是肿瘤血管的良好标记物，设法抑制ＶＥＧＦ可望成为肾癌治疗的有效方法。     

凋亡抑制基因Bcl-2蛋白产物在沙土鼠海马缺血耐受中的表达

目的 研究沙土鼠短暂的脑缺血后海马ＣＡ１区Ｂｃｌ－２蛋白的表达,探讨Ｂｃｌ－２蛋白表达与脑缺血耐受之间的关系,方法 采用夹闭沙土鼠双侧颈总动脉造成脑缺血模型。６３只健康沙土鼠随机分成四组。非缺血对照组（Ａ组,ｎ＝５）;预处理对照组（Ｂ组,ｎ＝６）,单次脑缺血２ｍｉｎ;缺血预处理组（ＰＣ组,ｎ＝２６）与缺血对照组（ＩＲ组,ｎ＝２６）,有或无预处理缺血２ｍｉｎ,间隔３ｄ后缺血５ｍｉｎ,分别再灌４ｈ（Ｐ     

利多卡因对短暂脑缺血后海马区细胞凋亡的影响

目的 探讨利多卡因对短暂脑缺血后海马区迟发性神经元降解的影响。方法 脑缺血模型为兔脑四血管夹闭模型,２５只家兔随机分成三组：假手术组（Ｓｈ,ｎ＝５）;缺血组（Ｉｓ,ｎ＝１０）,夹闭双侧颈总动脉和椎动脉５ｍｉｎ后恢复脑灌注;利多卡因组（Ｌｉ,ｎ＝１０）,夹闭颈总和椎动脉前５ｍｉｎ给予利多卡因１０ｍｇ／ｋｇ。３组均于３ｄ后取脑行病理ＨＥ染色和ＴＵＮＥＬ染色,对民区阳性细胞进行计数。结果 ＨＥ染色发生缺     

胃癌组织中P15蛋白和血管内皮生长因子表达及其相关性研究

目的研究胃癌组织中Ｐ１５蛋白和血管内皮生长因子（ＶＦＧＦ）的表达及相关性。方法应用ＳＰ免疫组织化学方法检测了１６０例胃癌和５２例胃良性病变中Ｐ１５蛋白和ＶＥＧＦ的表达情况。结果Ｐ１５蛋白在胃癌中阳性率为４３．８％（７０／１６０例），在胃良性病变中的阳性率为６９．２％（３６／５２例）（Ｐ〈０．０５）。ＶＥＧＦ在胃癌中的阳性表达率为７５．０％（１２０／１６０例），在胃良性病变中的阳性表达率为７．７％（     

肝癌的微血管密度与血管内皮生长因子表达

目的 研究肝癌组织连续切片中微血管密度（ＭＶＤ）及血管内皮生长因子（ＶＥＧＦ）两者之间的关系。方法 肝癌组织标本５０例,采用抗人Ⅷ因子相关抗原、抗人ＶＥＧＦ进行免疫组织化学染色显示组织中微血管及ＶＥＧＦ阳性表达细胞。结果 ５０例肝癌组织中,ＶＥＧＦ表达阳性者２８例,阳性率为５６％。肝癌及癌旁正常肝组织ＭＶＤ测定结果显示,ＶＥＧＦ阳性表达组明显谪于阴性表达组,其差异有显著性（Ｐ〈０．０５）。结论 肝     

腹部手术中静脉持续滴注前列腺素E1对胃癌患者肝功能的影响

为观察前列腺素Ｅ１（ＰＧＥ１）对腹部手术患肝功能的影响，将５５例术前肝功能正常、准备行择期胃癌根治术的成年患，随机分成ＰＧＥ１组（ｎ＝２７）和对照组（ｎ＝２８），在静脉全麻下进行手术，开腹后，ＰＧＥ１组，以２０￣６０ｎｇ·ｋｇ＾－１·ｍｉｎ＾－１速率持续滴注ＰＧＥ１，至止手术结束，而对照组则以相同的速率持续滴注生理盐水，分别于术后第１、３、７、１０天测定谷丙转氨酶（ＧＰＴ）及谷草转氨酶（ＧＯＴ     

七氟醚及其它挥发性麻醉药对肝药酶的影响

目的：观察七氟醚及其它挥发性麻醉药对肝药酶的影响。方法：将４０只月龄２～３月的ＳＤ大鼠随机分：对照组（Ｃ组，ｎ＝１０，０ＭＡＣ）、氟烷组（Ｆ组，ｎ＝１０，０．５ＭＡＣ）、异氟醚组（Ｉ组，ｎ＝１０，０．５ＭＡＣ）和七氟醚组（Ｓ组，ｎ＝１０，０．５ＭＡＣ）。各组动物分别吸入相应浓度的麻醉药，３小时／天，共７天。结果：表现为Ｆ组及Ｓ组肝脏代谢戊巴比妥钠的能力明显高于对照组（Ｐ＜０．０１）；Ｉ组有高于对照组的倾向（Ｐ＞０．０５）。结论：七氟醚、氟烷和异氟醚有酶诱导作用或倾向。     

Vascular growth factor expression in a rat model of severe limb ischemia

BACKGROUND: In ischemic tissue hypoxia induces production of vascular growth factors, especially VEGF, which initiate local angiogenesis. Collateralization-or arteriogenesis-occurs at a distance from the ischemic tissue and depends on different growth factors such as FGF-2. A spatial discrepancy in endogenous growth factor production in limb ischemia may have implications for therapeutic angiogenesis. The present study elucidates if such spatial and temporal variation occurs. MATERIALS AND METHODS: A two-staged procedure was performed to generate severe long-lasting limb ischemia in 60 rats. At 1, 7, 28, and 56 days, subgroups were subjected to perfusion assessment with laser Doppler imaging and angiography. Muscle samples and foot skin were gathered to measure growth factor expression and signs of angiogenesis using immunohistochemistry. RESULTS: There was an early twofold increase (P < 0.05) in both VEGF and FGF-2 levels in distal muscle from the ischemic leg, but no significant rise in the thigh. The concentrations decreased over time with an exception for VEGF in soleus and FGF-2 in anterior tibial muscle, which remained high. An increased capillarity was noted (P < 0.05) in soleus after 28 days, and the number of BrdU-positive ECs was elevated in all ischemic samples at 56 days. Collateral arteries were observed on the angiograms after 7 days. CONCLUSIONS: The results suggest that in limb ischemia any major increase in vascular growth factor production is limited to ischemic tissue. The spatial and temporal distribution patterns of growth factor production are complex and to a great extent influenced by inflammation.     

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     

骨髓间充质干细胞联合血管内皮生长因子基因治疗肢体缺血的实验研究

目的 观察骨髓间充质干细胞(bone mesenchymal stem cell,BMSC)联合血管内皮生长因子(vascular endothelial growth factor,VEGF)基因治疗对家兔肢体缺血模型的疗效.方法 切除新西兰兔右后肢全长股浅动脉并结扎股深动脉以建立兔后肢缺血模型,随机分为空质粒对照组(EP组)、骨髓间充质干细胞组(BMSC组)、VEGF基因治疗组(VEGF组)及联合治疗组(BV组),每组各8只.分别于治疗后28 d及30 d进行动脉造影及VEGF免疫组化染色.结果 EP组、BMSC组及VEGF组的新生血管计数组间比较差异无统计学意义(P＞0.05).BV组的新生血管计数较其余3组明显增加,差异有统计学意义(F=35.47,P＜O.01).BMSC组及VEGF组的VEGF免疫组化染色呈阳性表达,与EP组比较差异有统计学意义(F=764.32,P＜0.01).BV组的VEGF免疫组化染色呈强阳性表达,与其余3组比较差异有统计学意义(F =764.32,P＜0.01).结论 BMSC联合VEGF基因治疗兔肢体缺血可使VEGF获得稳定而有效的表达,从而改善肢体缺血.     

Transmyocardial laser revascularization combined with vascular endothelial growth factor 121 (VEGF121) gene therapy for chronic myocardial ischemia--do the effects really add up?

OBJECTIVE: Different therapy strategies for coronary disease in conventionally untreatable patients have been developed, among them transmyocardial laser revascularization (TMLR) and the application of growth factors. The objective of our study was to determine whether a combined therapy of TMLR with a vascular endothelial growth factor(121) (VEGF(121)) plasmid is able to stimulate the development of sufficient collateral circulation and hereby to preserve cardiac function. MATERIALS AND METHODS: A severe stenosis of the left anterior descending artery was created in healthy pigs. After 1 week, perfusion and regional contractility were assessed at baseline. Afterwards, the ischemic area was treated with TMLR (n=8), intramyocardial injection of naked plasmid DNA encoding VEGF(121) (n=7), or both (n=7). Control animals were left untreated (n=8). After 3 months, the animals were re-examined and underwent immunohistological analysis. RESULTS: The number of capillaries increased only after injection of VEGF(121) plasmid alone compared to untreated ischemia and to the other therapy groups, whereas the number of arterioles was higher following TMLR treatment alone or in combination with VEGF(121) than it was in the case in untreated ischemic animals. However, only combined VEGF(121)+TLMR therapy resulted in an improvement in regional myocardial blood flow in comparison with 1 week ischemia, indicating the efficient development of collateral circulation. In contrast, better regional contractility compared to the 1-week baseline, as well as restoration of the pre-ischemic values, were achieved by both VEGF(121) and combined VEGF(121)+TLMR therapies. CONCLUSIONS: This study of chronic myocardial ischemia with a porcine model indicates a synergistic action of TMLR and VEGF(121) gene therapy. Combined treatment alone achieved an increase of regional myocardial perfusion, which accompanied arteriogenesis and corresponded with the restoration of regional function.     

血管内皮生长因子定向转染诱导缺血心肌血管生成的研究

目的观察血管内皮生长因子（VEGF）质粒直接心肌注射对兔急性心肌缺血后侧支循环形成的影响。方法建立兔左冠状动脉结扎的急性心肌缺血模型，取缺血边缘区以先期构建的真核表达质粒pcDNA3／VEGF165和真核载体pcDNA3的DNA分别多点心肌注射，给药后2、4周取材分别行逆转录-聚合酶链反应（RT-PCR）和蛋白浓度分析、苏木素-伊红（HE）染色、VEGF染色。结果治疗组VEGF165 mRNA含量较对照组明显增多；蛋白浓度分析转染后VEGF165显著增多，高峰出现于实验后2周；治疗组缺血心肌新生血管数目明显多于对照组。结论VEGF165外源性基因能在转染心肌细胞后成功表达，促进了缺血心肌血管新生和侧支循环形成。     

Appropriate control of ex vivo gene therapy delivering basic fibroblast growth factor promotes successful and safe development of collateral vessels in rabbit model of hind limb ischemia

PURPOSE: In our previous study, adenovirus-mediated ex vivo gene transfer of basic fibroblast growth factor promoted significant collateral vessel development in a rabbit model of hind limb ischemia. The present study examined how to control the efficacy and safety of this gene therapy, and also evaluated the feasibility of repeat application of this procedure. METHODS: Modified hFGF gene with the secretory signal sequence was adenovirally transferred to cultured autologous fibroblasts, and various numbers of the cells (2 x 10(5), 1 x 10(6), 5 x 10(6), or 2.5 x 10(7)) or vehicle was injected through the left internal iliac artery in rabbits in whom the left femoral artery had been excised 21 days previously. Twenty-eight days after cell administration, calf blood pressure ratio, angiographic score, blood flow in the internal iliac artery, and capillary density of muscle tissue were measured to analyze collateral vessel development and tissue perfusion in the ischemic limb. To assess delivery efficiency and viral contamination, the distribution of injected cells and the time course of blood anti-adenovirus antibody titer were examined in rabbits treated with various numbers of gene-transduced cells. In addition, animals received two injections, 21 days apart, of fibroblasts infected with adenovirus vector containing the luciferase gene, and luciferase expression was measured to evaluate whether the present therapy is repeatable. RESULTS: At 28 days after cell administration, significant collateral vessel development without detectable side effects was observed in rabbits who received 5 x 10(6) or 2.5 x 10(7) cells, compared with those who received vehicle, and no significant development was detected in animals with fewer than 5 x 10(6) cells (P <.01 for calf blood pressure ratio and capillary density, P <.05 for angiographic score and maximum blood flow). There was no difference in collateral augmentation between rabbits with 5 x 10(6) and 2.5 x 10(7) cells. However, in animals with 2.5 x 10(7) cells a large number of injected cells accumulated in the lungs, anti-adenovirus antibody titer increased significantly, and calf blood pressure in the left hind limb of two rabbits decreased immediately after injection. Luciferase analysis showed very low gene expression after repeated administration. CONCLUSION: These findings suggest that 5 x 10(6) is a suitable number of cells to induce appropriate collateral vessel development and minimize potential side effects of this procedure. Despite use of ex vivo gene transfer, repeat administration of the cells was not feasible.Clinical relevance Since the present study determined the appropriate conditions for effective and safe stimulation of collateral vessels, the clinical relevance of the ex vivo therapy might be carried forward. However, the findings raised another issue that should be resolved before clinical application; that is, the number of gene-transduced cells able to be injected was strictly limited. To estimate the therapeutic range of cell number in humans, additional experiments using large animals are desirable.     

血管内皮生长因子转染内皮祖细胞治疗大鼠缺血后肢的研究

目的 使用血管内皮生长因子(VEGF)转染内皮祖细胞(EPC)治疗大鼠缺血后肢,观察EPC、VEGF转染EPC对大鼠缺血后肢的新生血管和肢体成活的影响.方法 制作SD大鼠后肢缺血模型,将动物随机分为3组,每组6只.将构建的VEGF基因真核表达载体转染入骨髓来源的EPCs后通过尾静脉注射人大鼠体内,并与使用磷酸盐缓冲液(PBS)或EPC的动物进行比较,观察转染VEGF的EPCs在缺血部位的聚集和形成新生血管的情况.结果 (1)动物总残肢率比较,CELL组、VEGF组较PBS组明显增加的肢体恢复率(P<0.05),CELL组肢体恢复率较VEGF组差(P<0.05).(2)毛细血管密度与PBS组比较,各时间点中CELL、VEGF组MVD均明显增多(P<0.05).(3)缺血肢体VEGFa的表达:VEGF组的VEGF蛋白表达较PBS组、CELL组、明显增多(P<0.05);(4)手术后7、14、28 d,与PBS对照组比较,CELL、VEGF组细胞的血流灌注有较大程度的恢复(P<0.01).结论 VEGFa基因转染EPCs对缺血部位的血管新生有重要影响,联合应用VEGFa基因和EPCs治疗缺血后肢有较好的协同作用.     

Induction of angiogenesis by cationic lipid-mediated VEGF165 gene transfer in the rabbit ischemic hindlimb model

PURPOSE: The purpose of this study was to test the efficacy of a new cationic lipid formulation coupled with the cDNA encoding for the 165-residue form of vascular endothelial growth factor (VEGF(165)) to induce neovascularization and enhance blood flow in the rabbit ischemic hindlimb model. METHODS: Two days after removal of their right femoral arteries, rabbits received intramuscular injections of different concentrations of VEGF(165) or saline solution in the ischemic thigh. Tissue perfusion and increased neovascularization of the ischemic limb were assessed weekly on the basis of the calf blood pressure ratio for the ischemic/nonischemic limbs, regional blood flow to the skeletal muscles as measured with radioactive microspheres, postmortem angiography, and histology. RESULTS: At weeks 1 and 2 after surgery, animals treated with 1000 microgram of VEGF(165) had a 1.5-fold increase and a 2.5-fold increase, respectively, in the regional blood flow to both the adductor and gastrocnemius muscles of the ischemic limb. The blood pressure ratio was also greater in the treated animals than in the controls at weeks 2 and 3 after surgery. Early neovascularization in the VEGF(165) group was further documented at week 1 after surgery by more angiographically recognizable collateral vessels (angioscores were 64.13 +/- 2.51 and 38.28 +/- 3.82 for VEGF(165) and saline solution, respectively; P <.001) and by a threefold increase in the number of capillaries (vascular density) relative to the controls (P <.005). CONCLUSIONS: Intramuscular administration of a single dose of plasmid-liposomes encoding for VEGF(165) accelerates angiogenesis and increases blood flow in the rabbit hindlimb ischemic model. Therefore, this nonviral vector could be recommended for further testing for use in therapeutic angiogenesis.     

Determinants of ischemic injury to skeletal muscle

Purpose: Ischemic injuries to the lower extremity are often graded in severity according to their duration. Other determinants may also influence the extent of an injury, however, and may be equally significant contributors to the final outcome. The purpose of this study was to compare the relative influences of ischemic time, temperature, residual blood flow, muscle location, and fiber type on postischemic necrosis in a rabbit model of skeletal muscle ischemia-reperfusion injury.Methods: Animals' hindlimbs were rendered ischemic under differing conditions of each determinant and then reperfused for 48 hours. Necrosis in the rectus femoris, semimembranosus, anterior tibial, and soleus muscles was determined by nitroblue tetrazolium staining and computerized planimetry. The severity of each animal's injury was quantified by calculating the cumulative percentage of necrosis by weight of all muscles excised from the ischemic limb.Results: Four hours of ischemia at room temperature resulted in an average of 21% ± 7% necrosis. Lengthening the ischemic interval to 5 hours increased necrosis to 61% ± 4% (p < 0.01 vs 4 hours); however injuries were equally or more significantly influenced by changes in ischemic temperature or small changes in ischemic limb residual (collateral) blood flow. The most severe injuries of any encountered were observed when limbs were maintained at body temperature during ischemia (92% ± 9% necrosis after 5 hours of ischemia, p < 0.01 vs room temperature ischemia), whereas extremely small improvements in ischemic period residual flow (by allowing pelvic collateral cross-flow during ischemia) resulted in significant salvage in all muscles studied. Muscles predominating in fast-twitch fibers had significantly greater necrosis than did those richer in slow-twitch fibers; this difference was apparent only after longer periods (5 hours) of ischemia. Thigh muscles sustained significantly greater injuries than did distal hindlimb muscles, except in animals subjected to body temperature ischemia, where the distribution of necrosis was uniform.Conclusions: The results of this study indicate that muscle necrosis accompanying an ischemic event can be significantly influenced by numerous determinants in addition to ischemic time, each of which warrants careful clinical scrutiny when appraising the extent of an injury. (J VASC SURG 1994;19:623-31.)