碱性成纤维细胞生长因子促进兔缺血后肢血管新生的实验研究

目的:了解局部应用碱性成纤维细胞生长因子(bFGF)对促进兔缺血后肢血管新生的作用。方法:25只日本大耳白兔随机分2组,外科结扎切断各兔股动脉及其分支,制作兔后肢缺血模型。试验组各兔在缺血后肢肌肉内多次注射重组人bFGF蛋白(n=15);对照组给予等剂量生理盐水(n=10)。术后4周,各兔行腹主动脉造影观察侧支血管形成情况,取内收肌和腓肠肌肌肉行病理切片HE染色应用图像分析系统统计血管密度,并用免疫组织化学方法检测内收肌和腓肠肌中VEGF阳性表达的血管数。结果:试验组兔侧支循环血管条数、血管密度及VEGF阳性表达的血管数均大于对照组(P<0.01),缺血状态得到改善。结论:在兔缺血后肢肌肉中注射bFGF蛋白可促进血管新生,增加兔缺血后肢血液灌注,改善肢体缺血状态。     

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.     

基质细胞衍生因子联合骨髓单个核细胞移植治疗大鼠肢体缺血的实验研究

目的将基质细胞衍生因子(SDF-1)用于自体骨髓单个核细胞(BM-MNC)移植治疗肢体缺血,初步探讨SDF-1促进血管新生的机理和疗效。方法建立大鼠左后肢缺血模型,将动物随机分为非缺血对照组,缺血对照组,SDF-1局部应用组,自体BM-MNC移植组以及两者联合应用组。检测缺血后24 h左后肢腓肠肌SDF-1含量及CD133+细胞数量;于移植后4周动脉造影后处死动物,免疫组化法检测缺血区新生血管密度,评价血管新生情况。结果缺血对照组24 h肌组织SDF-1含量为(1．31±0．20)ng/50μg总蛋白,非缺血对照组为(0．93±0．29)ng／50μg总蛋白(P<0．05); CD133+细胞数(2．10±0．62)vs．(0．24±0．10)个／HP,P<0．01。SDF-1局部应用可增加缺血区CD133+细胞浸润数量(3．64±0．69)个／HP(P<0．01);并可提高四周后组织内新生血管数量;SDF-1与自体骨髓单个核细胞移植联合应用,可进一步提高新生血管数量。结论SDF-1可能在肢体缺血后自体干细胞的动员中起重要作用,其局部应用可促进缺血区血管新生或进一步提高干细胞移植治疗缺血的疗效。     

局部miR-126基因治疗促进小鼠缺血后肢血管新生的观察

目的:通过体外构建重组腺病毒mi R-126,应用于小鼠缺血后肢腓肠肌局部治疗,观察mi R-126对缺血局部具有促血管新生作用。方法:重组腺病毒mi R-126包装、纯化、滴度的鉴定;C57小鼠随机分为A组(C57左侧缺血后肢手术组)、B组(空病毒C57左侧缺血后肢手术组)、C组(重组腺病毒mi R-126 C57左侧缺血后肢手术组)三组,制作小鼠缺血后肢模型,术后即刻将重组腺病毒mi R-126和腺病毒各50μl局部注射于小鼠缺血左后肢腓肠肌。分别于3、7、14天各组(3只)取左后肢腓肠肌做HE染色、CD31免疫组化染色、west ern bl ot检测Akt、ERK1/2、pAkt、pERK1/2蛋白水平以及实时定量PCR检测等。结果:各种检测结果显示C组较A、B两组血管内皮细胞增生明显,新生血管数目计数明显增多,mi R-126表达水平明显增高,尤其在第7天升高最为明显,以及VEGF、bFGF等介导的I P3和MAPK信号通路中ERK1、pERK1、AKT和pAKT蛋白水平表达明显增高。结论:mi R-126局部应用于缺血后肢,通过激活Akt、ERK1/2的相关通路,促进血管新生,利于缺血后肢功能恢复。     

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.     

骨髓干细胞移植促进缺血肢体血管新生的研究

我们通过建立大鼠肢体缺血模型。采用骨髓内皮祖细胞(EPC)移植于缺血肢体，观察缺血组织血管新生及血管内皮生长因子(VEGF)的表达情况，探讨自体骨髓干细胞移植在治疗肢体缺血性疾病中的价值。     

pEGFP-C1/Akt体外转染骨髓间充质干细胞对后肢缺血大鼠血管生成的影响

目的 探讨经肌肉注射转染pEGFP-C1/Akt的鼠骨髓间充质干细胞(MSCs)对后肢缺血大鼠血管生成的影响.方法 Wistar大鼠30只,制成双后肢缺血模型,双盲法随机分为基因治疗组(肌注经pEGFP-C1/Akt转染的MSCs)、非基因治疗组(肌注MSCs)及对照组(肌注PBS液).造模前、造膜后即刻及MSCs移植后1～7 d内,每天用红外线皮温仪测定大鼠后肢皮温变化.28 d时经动脉造影观察后肢血管生成情况; 免疫组化染色检测后肢毛细血管密度; 逆转录-多聚酶链反应(RT-PCR)和Western blot法检测后肢肌肉组织中Akt及血管内皮细胞生长因子(VEGF)的 mRNA和蛋白的表达.结果 移植3 d后基因治疗组大鼠后肢皮温升高明显.28 d时经动脉造影观察基因治疗组后肢侧支血管生成明显; 荧光显微镜观察有绿色荧光细胞在基因治疗组的内收肌和半膜肌分布.毛细血管密度: 基因治疗组为(7.1±0.3)个/高倍镜,非基因治疗组为(4.2±0.4)个/高倍镜,对照组为(1.3±0.2)个/高倍镜,各组间差异均有统计学意义(P＜0.01).Akt及VEGF的 mRNA和蛋白的表达分析: 基因治疗组Akt mRNA(2.44±0.14)和蛋白(1.12±0.13)及VEGF mRNA(1.11±0.11)和蛋白(0.97±0.13)表达水平均明显高于非基因治疗组Akt mRNA(1.58±0.13)和蛋白(0.78±0.12)及VEGF mRNA(0.78±0.14)和蛋白(0.67±0.11)以及对照组Akt mRNA(0.64±0.11)和蛋白(0.36±0.12)及VEGF mRNA(0.56±0.11)和蛋白(0.33±0.13)的表达水平(P＜0.01),后2组间比较差异亦均有统计学意义(P＜0.01).结论 pEGFP-C1/Akt体外转染骨髓MSCs促进后肢缺血大鼠血管生成的效果优于单纯MSCs治疗,为基因转染MSCs治疗缺血性疾病提供可能.     

血管内皮生长因子-碱性成纤维细胞生长因子基因治疗家兔肢体缺血的研究

目的　了解血管内皮生长因子 (VEGF)和碱性成纤维细胞生长因子 (bFGF)联合克隆基因 (VEGF bFGF)治疗兔下肢动脉缺血模型后新生血管和侧支形成状况。方法　应用 40只家兔制成下肢缺血模型 ,其中VEGF bFGF组 10只 ,VEGF组 12只 ,空载体组 8只 ,生理盐水 (NS)组 10只。构建pcDNA3 /VEGF和pcDNA3 /VEGF bFGF真核表达载体。转染缺血部位肌组织 ,行下肢血管造影。结果　血管造影计数显示 ,VEGF bFGF组在转染后 14d(1.98± 0 .2 2 ) ,2 8d (1.81± 0 .5 2 ) ,5 6d(2 .2 1± 0 .44 )和 3个月 (2 .10± 0 .2 2 ) ;VEGF组在 2 8d(1.3 8± 0 .2 9) ,5 6d(1.94± 0 .2 5 )和 3个月 (2 .2 4± 0 .3 1) ,新生血管形成较对照组显著增加 (P <0 .0 5 )。结论　VEGF bFGF真核表达载体可以获得局部高效表达 ,刺激新生血管生成 ,建立侧枝循环 ,改善肢体缺血。     

BMSCs移植治疗非急性期深静脉血栓的实验研究

目的:探讨BMSCs移植治疗非急性期深静脉血栓的可能性。方法:取新西兰大白兔20只,其中4只作为干细胞供体;余16只建立血栓模型,随机均分为A组(BMSCs移植组)和B组(对照组)。观察碱性成纤维细胞生长因子(bFGF)、血管内皮细胞生长因子(VEGF)蛋白的表达以及新生毛细血管数。结果:移植后14、28 d新生毛细血管数实验兔A组多于B组(P<0.05),A组VEGF、bFGF蛋白表达水平高于B组(P<0.05)。结论:BMSCs移植可以促进非急性期深静脉血栓的血管新生及侧支血管形成,改善静脉回流。     

大鼠缺血后肢成纤维细胞生长因子的表达及意义

“血管新生疗法”是慢性肢体缺血性疾病极有前景的治疗方案．已证实多种血管生长因子及其基因,如血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)等可明显促进缺血部位的血管新生,改善组织供血,并已在实验和临床研究中取得进展,但对缺血状态下机体内源性促血管新生机制尚未完全阐明。为此,我们造模大鼠后肢缺血,观察其     

rhG-HGF联合成纤维细胞生长因子促进下肢血管新生的实验研究

目的 探讨重组人肝细胞生长因子(rhG-HGF)联合成纤维细胞生长因子(bFGF)对下肢缺血动物模型血管新生的影响.方法 制作80只小鼠左下肢缺血模型,术后随机分为4组,每组20只:(1)生理盐水对照组;(2)bFGF组;(3)rhG-HGF组;(4)rhG-HGF+bFGF组.应用多谱勒超声血流测定、肌肉毛细血管密度测定,比较4组缺血肢体血流/正常肢体血流比值及肌肉毛细血管密度.结果 术后4周,4组缺血肢体血流/正常肢体血流比值及肌肉毛细血管密度为:rhG-HGF+bFGF组>rhG-HGF组>bFGF组>生理盐水组(均P<0.05).结论 rhGvHGF促进血管新生作用强于bFGF,两者联合应用有协同作用,可以更明显改善下肢缺血状况.     

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     

Enhanced angiogenesis and growth of collaterals by in vivo administration of recombinant basic fibroblast growth factor in a rabbit model of acute lower limb ischemia: dose-response effect of basic fibroblast growth factor.

The purpose of this study was to evaluate the effects of exogenous recombinant basic fibroblast growth factor (bFGF) on angiogenesis in severely ischemic tissue beds. We used a two-stage procedure to produce severe ischemia of the hindlimb of 34 New Zealand rabbits. The ischemic hindlimb received intramuscular injection of saline (group A), 1 microgram bFGF (group B), or 3 micrograms bFGF (group C), daily for 2 weeks. Tissue perfusion, skeletal muscle infarction, angiogenesis, and collateral growth were assessed by angiography, transcutaneous oximetry (TcPO2), quantitative spectrophotometric assay of triphenyltetrazolium chloride reduction in muscle, capillary density (capillaries per square millimeter), and capillary per muscle fiber ratio. There were no significant differences in baseline TcPO2 among the three groups for both thigh and calf measurements. Angiography revealed extensive perfusion of the left hindlimb in all the assessed bFGF treated animals. Both thigh and calf TcPO2 values showed a significant increase in all groups over the 14 days ischemia was induced (p less than 0.0001), but the two treatment groups exhibited a much more rapid rise in TcPO2 than the control group (p less than 0.0001). The capillaries per square millimeter and capillaries per muscle fiber ratios were significantly increased in all posttreatment measurements for all animals that received bFGF. The treatment groups with bFGF had a significant (p = 0.025) increase in thigh muscle viability compared with controls based on triphenyltetrazolium chloride reduction. Whereas there was evidence of muscle infarction in both the thighs of groups A and B, there was none in group C.(ABSTRACT TRUNCATED AT 250 WORDS)     

重组人内皮抑素对尿毒症腹膜透析大鼠腹膜新生血管形成的影响

目的 研究重组人内皮抑素对尿毒症腹膜透析（PD）大鼠腹膜新生血管形成的影响。 方法 40只雄性SD大鼠,按随机数字表法分为正常对照组、肾衰竭非透析组、4.25%PD组、重组人内皮抑素10 mg/kg PD组、重组人内皮抑素40 mg/kg PD组,每组8只。对PD组规律PD 28 d。重组人内皮抑素干预组在行规律PD期间,隔天1次皮下注射重组人内皮抑素,至透析第28天结束。28 d后取各组大鼠新鲜腹膜组织,RT-PCR法检测腹膜组织血管内皮生长因子（VEGF）、碱性成纤维细胞生长因子（bFGF） mRNA表达;免疫组化染色检测VEGF、bFGF蛋白表达。CD34染色观察腹膜组织毛细血管密度（MVD）。 结果 各组大鼠腹膜组织均表达VEGF和bFGF,肾衰竭非透析组、4.25%PD组VEGF及bFGF mRNA、蛋白表达均显著高于正常对照组（均P < 0.05）;重组人内皮抑素10 mg/kg PD组、40 mg/kg PD组VEGF及bFGF mRNA、蛋白表达均显著低于4.25%PD组（均P < 0.05）。肾衰竭非透析组、4.25%PD组腹膜组织MVD均显著高于正常对照组（均P < 0.05）;重组人内皮抑素10 mg/kg、40 mg/kg PD组腹膜组织MVD均显著低于正常对照组（均P < 0.05）。 结论 重组人内皮抑素可以有效抑制PD大鼠腹膜新生血管的形成,下调VEGF、bFGF mRNA及蛋白表达可能是其抑制腹膜新生血管形成的机制之一。     

Controlled delivery of vascular endothelial growth factor promotes neovascularization and maintains limb function in a rabbit model of ischemia

Purpose: Vascular endothelial growth factor (VEGF) modulates new blood vessel development and growth and has been suggested as a potential therapeutic agent that could alleviate debilitating claudication in patients. The objective of this study was to determine whether controlled, local delivery of a low dose of VEGF from an osmotic pump could promote neovascularization, limb perfusion, and functional improvements in the hind limbs of rabbits rendered partially ischemic by surgery. The effects of VEGF were compared with those of the vasodilator nitroglycerin (NTG) and to saline administered similarly. Methods: Thirty rabbits were randomly assigned to either VEGF (n = 10), NTG (n = 10), or saline (n = 10) treatment groups. Partial ischemia was induced in each left hind limb by surgical ligation of the common and superficial femoral arteries, leaving the internal iliac artery intact. The right limb of each animal served as a nonischemic control. Immediately after vessel ligations, a 28-day osmotic pump was implanted to deliver VEGF (0.22 μg/kg/day), NTG (17.8 μg/kg/day), or saline solution into the common iliac artery just proximal to the ligation site. Comparative vascularity between ischemic and nonischemic limbs within treatment groups and between groups was evaluated by (1) capillary counts from representative fields of hematoxylin and eosin stained muscle tissue taken from hind limbs at day 40; (2) digitized arteriograms of ischemic legs at day 40, which were used to quantify the complexity of vascular branching (fractal dimension index) and the total extent of vascularization (vascular density index); (3) measuring capillary refill times in ischemic limbs; and (4) observations of functional and trophic changes in ischemic limbs. Statistical differences between treatment groups were evaluated by one-way ANOVA. Results: Complexity of vascular branching and vascular density were significantly greater (p < 0.001) in VEGF-treated ischemic limbs compared with NTG- and saline-treated ischemic limbs. By postoperation day 14, all VEGF-treated ischemic limbs had restored capillary refill (p < 0.001), new hair growth, and greatly improved limb function and appearance. Saline-treated limbs exhibited ischemic changes, with poor capillary refill and negligible limb function. Capillary refill in NTG-treated ischemic limbs did not differ significantly from saline-treated limbs. Ischemic VEGF-treated limbs had significantly more capillaries compared with both ischemic and nonischemic limbs in saline-treated animals (p < 0.05). Ischemic NTG-treated limbs also had significantly more capillaries compared with ischemic limbs in saline-treated animals (p < 0.05). Because of high variability, however, capillary counts in VEGF-treated ischemic limbs did not differ significantly from those of contralateral nonischemic limbs, or from capillary counts in either ischemic or nonischemic limbs of NTG-treated rabbits. Conclusions: Controlled release of microgram quantities of VEGF significantly enhanced neovascularization and vascular perfusion in ischemic limbs compared with controls in this rabbit model of partial ischemia. In addition, VEGF-treated ischemic limbs demonstrated near-normal function and appearance, whereas NTG- and saline-treated ischemic controls remained noticeably impaired. This novel approach of VEGF delivery may prove clinically useful either alone or combined with revascularization procedures. (J Vasc Surg 1998;27:886-95.)     

应用转血管内皮生长因子基因治疗肢体缺血的研究

目的研究肌肉内转血管内皮生长因子(VEGF)基因治疗肢体缺血的可行性,比较各种治疗方法的疗效. 方法雄性新西兰大白兔50只,完全切除股动脉后随机分为3组.实验组为明胶海绵携载法转基因组(n=18)和肌肉内注射转基因组(n=18);只注射pcDNA3为对照组(n=14).通过直接注射法及明胶海绵携载法将构建的质粒pcDNA3-VEGF121基因转入缺血肌肉内,立即测定各组肢体髂内动脉血流量,在术后2天,1、2、3和4周应用逆转录-聚合酶链反应(RT-PCR)技术测定基因表达,术后30天通过测定缺血肢体髂内动脉血流量、动脉血管造影及组织学观察测定血管密度,评价侧支循环变化. 结果术后2天,转VEGF基因治疗的两实验组均测到基因表达,并均维持2周.术后立即测定的髂内动脉血流量各组间无明显差异.术后30天,缺血肢体髂内动脉血流量、肢体血管造影血管数目和肌肉组织血管密度转VEGF基因的两实验组均比对照组明显增高,差异有统计学意义(P<0.01),明胶海绵携载组较直接注射组亦有明显增高,差异有统计学意义(P＜0.05). 结论肌肉内转VEGF基因治疗可促进急性缺血肢体侧支循环、改善血供,明胶海绵携载法较直接注射法有更好的疗效.     

Functional outcome of new blood vessel growth into ischemic skeletal muscle

PURPOSE: The administration of angiogenic growth factors and the transfer of well-vascularized tissues have been shown to induce development of new blood vessels in ischemic muscle. The functional significance of these new vessels is unknown. The hypothesis of this study is that the transfer of vascularized muscle and the local infusion of basic fibroblast growth factor (bFGF) synergistically improve contractile function of ischemic skeletal muscle. METHODS: Twenty-six rabbits were divided into four groups. An ischemic hindlimb was created in each by ligating the right common iliac artery. The flap + bFGF group (n = 6) had transposition of a contralateral rectus muscle flap onto the thigh. Additionally, bFGF (3 ng/h) was continuously infused at the flap-thigh interface. In the flap group (n = 6), a similar muscle flap was created, but carrier solution was infused at the interface. In the bFGF group (n = 6), no muscle flap was created; instead, bFGF (3 ng/h) was infused into the external iliac artery of the ischemic limb. In the control group (n = 8), carrier solution was infused into the external iliac artery (no flap, no bFGF). After 1 week, the soleus muscle was isolated and stimulated. Maximum twitch tension, the fatigue index (force of contraction after 2 minutes of continuous stimulation/initial force of contraction), maximum recovery, and the number of limbs recovered (ie, limbs that achieve a force of contraction during the recovery period of > 75% of the force of the initial contraction at the start of continuous stimulation) were recorded. Blood vessel density (number of vessels per ***) was determined by immunostaining the soleus muscle with anti-alpha-actin antibody. RESULTS: All values were indexed to the contralateral normal limb. The flap + bFGF group showed significant improvement versus the control group in maximum twitch tension (1.07 +/- 0.13 vs 0.63 +/- 0.12, P < .05), maximum recovery (0.94 +/- 0.05 vs 0.58 +/- 0.05, P < .05), and the number of limbs recovered (5/5 vs 0/6, P < .05). This improved function correlated with increased vessel density (flap + bFGF group, 1.44 +/- 0.11 vs control group, 0.72 +/- 0.01, P < .05). CONCLUSION: Reperfusion of an ischemic limb with a well-vascularized muscle flap and local bFGF infusion promoted increased blood vessel density in distal ischemic muscle. This increased vascularity was associated with restoration of otherwise impaired muscle function. Improved function occurred rapidly (1 week). A transposed muscle flap provided a functional blood supply to the site of maximum ischemia; this could be used to salvage otherwise nonreconstructible ischemic limbs.