碱性成纤维细胞生长因子在低强度脉冲超声波治疗骨折过程中的表达

目的:探索低强度脉冲超声波(LIPUS)在兔桡骨骨折愈合过程中碱性成纤维细胞生长因子(bFGF)的表达及分布情况,进而探讨LIPUS的作用机制.方法:将20只成年新西兰兔制作成双侧桡骨骨折模型.一侧桡骨接受每天20min的LIPUS治疗,另一侧作为对照.术后2、3、4、5周分别处死动物.应用兔疫组化方法观察bFGF在骨痂标本中的分布.结果:在骨折愈合早期的骨痂中,新生基质、未分化间充质细胞和骨细胞免疫组化染色阳性;而在成熟骨化的骨基质和分化末期的肥大的软骨细胞,染色阴性.同实验组相比,对照组阳性着色的细胞数及染色程度明显减弱.结论:LIPUS可以通过增加bFGF的合成来促进骨折愈合.     

低强度脉冲超声波促进骨折愈合的研究进展

超声波是一种超过人类听阈范围的高频声波,这种声波以压力波的形式存在,可对骨和周围组织产生微型压力作用.Wolff定律表明:骨的形成与结构随外力的大小与方向而重建,骨骼的生理反应和愈合过程可受机械力学影响.因此,研究超声的机械基础与骨组织对机械刺激之间的关系可能有一定的意义.超声波在理论上,可以代替一定的外力刺激,从而提供了一种促进骨折愈合的非侵袭性手段.研究者依照生物技术和生物工程原理,开展了大量基础和临床实验,积极探索超声波促进骨折愈合的机理.美国食品和药物管理局(FDA)分别于1994年10月与2000年2月对低强度超声波在加速新鲜骨折愈合及治疗骨折不愈合中的作用予以认可.作者就基础和临床研究对低强度脉冲超声波应用的评价进行综述.     

低强度脉冲超声治疗骨折

早在1952年就有动物实验研究表明,持续超声波可以刺激骨痂的形成。但直到30年后,才被较为系统地应用于临床治疗骨折,并取得了显著的疗效,从而引起众多学者的重视。近年来,一系列实验研究和临床应用都证实低强度脉冲超声(low-intensity ultrasound,LIUS)不仅可以有效地促进新鲜骨折的愈合,在骨不连的治疗上也有明显的作用。美国FDA分别于1994年10月和2000年2月相继批准在临床上应用低强度超声波治疗仪治疗新鲜骨折和骨不连。     

P物质对大鼠成纤维细胞碱性成纤维细胞生长因子及其受体表达的影响

目的　探讨P物质 (SP)对大鼠肉芽组织成纤维细胞碱性成纤维细胞生长因子 (bF GF)及其受体 (FGFR 1)表达的影响。方法　采用成纤维细胞体外培养和逆转录 多聚酶链反应(RT PCR)技术 ,观察SP在不同浓度 ( 1× 10 - 9～ 1× 10 - 5mol L)及孵育时间 ( 0、3、6、12、2 4h)情况下刺激成纤维细胞后 ,其bFGF、FGFR 1mRNA表达情况。结果　SP可上调大鼠成纤维细胞bF GF、FGFR 1mRNA表达。SP对bFGF的量 效曲线呈双相分布 ,最大效应浓度为 1× 10 - 7mol L。但SP仅在高浓度 ( 1× 10 - 6 ～ 1× 10 - 5mol L)时促进FGFR 1表达。在最大效应浓度 ( 1× 10 - 7～ 1× 10 - 5mol L)时 ,SP对bFGF、FGFR 1表达的上调作用分别于刺激细胞后 3、12h达高峰。结论　SP对大鼠肉芽组织成纤维细胞bFGF、FGFR 1基因表达存在直接影响 ,其表现形式与SP的刺激浓度及孵育时间有关 ,这可能是SP在创伤修复中发挥作用的机制之一。     

碱性成纤维细胞生长因子真核表达载体的构建及表达

目的构建人碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)真核表达质粒,研究其在体内外的表达.方法通过基因克隆技术,构建并大量制备pcDNA3.1/myc-His(-)-bFGF真核表达体系,RT-PCR和细胞免疫组织化学方法检测体外瞬时表达情况;直流电脉冲介导重组质粒pcDNA3.1/myc-His(-)-bFGF和pCD2-血管内皮细胞生长因子121(vascular endothelial growth factor,VEGF121)在兔颈部肌肉瓣内转移并表达,测定其促血管生成的生物学效应.结果构建的pcDNA3.1/myc-His(-)-bFGF真核表达体系成功转染体外培养HeLa细胞,目的基因在mRNA水平和蛋白水平均有表达.pcDNA3.1/myc-His(-)-bFGF和pCD2-VEGF121重组质粒分别转染在体肌瓣,获得外源基因高水平表达.转基因肌肉发生血管增生、血流增强的生物学效应.结论构建了人bFGF真核表达质粒,并可在体内外顺利表达,为进一步组织移植或组织工程基因治疗研究奠定了基础.     

碱性成纤维细胞生长因子对成骨细胞中转化生长因子-β1和碱性成纤维细胞生长因子mRNA表达的影响

目的　探讨外源性碱性成纤维细胞生长因子 (bFGF)对体外成骨细胞中的生长因子 :转化生长因子 β1 (TGF β1 )和bFGFmRNA表达的影响。 方法　将体外培养的新生SD大鼠颅骨成骨细胞 ,用不同浓度的bFGF(5～ 50 μg/L)进行处理 ,利用核酸原位分子杂交 ,检测两种生长因子在细胞中mRNA的表达。结果　依bFGF浓度的增加成骨细胞内TGF β1mRNA表达分别是对照组的 1 .5、1 .6、1 .9和 2 .0倍 ;bFGFmRNA表达则分别是对照组的 1 .2 8、1 .37、1 .40和 1 .51倍。bFGF组中 ,TGF β1和bFGFmRNA的表达量均明显高于对照组 (P <0 .0 1 )。结论　外源性bFGF可以对成骨细胞自分泌bGFG产生影响 ,还能够促进TGF β1的合成     

低强度超声波促进桡骨远端骨折愈合

目的　观察低强度脉冲超声对桡骨远端骨折愈合的促进作用。方法　 4 1例桡骨远端骨折患者 ,随机分为超声治疗组 (2 1例 )和对照组 (2 0例 )。超声组在骨折复位后以短臂石膏固定 ,每日以低强度脉冲超声治疗 15min ;对照组仅作复位和短臂石膏固定。记录两组患者骨折临床愈合时间和X线片上骨折线处灰度值的变化。结果　超声治疗组的临床愈合时间较对照组缩短 (32 0 4d± 2 5 8dvs 4 0 75d± 5 12d ,P <0 0 1)。超声治疗组骨折愈合前后X线片上骨折线处灰度值变化较对照组大。结论　低强度脉冲超声可明显加快桡骨远端骨折的愈合 ,促进骨折处局部骨的形成。     

碱性成纤维细胞生长因子在肾癌中的表达及意义

目的：探讨碱性成纤维细胞生长因子（b-FGF)在肾癌中的表达及意义。方法：采用斑点杂交技术测定20例肾癌患者癌组织,癌旁和周围正常肾组织中b-FGF mRNA的表达情况。结果：肾癌组织中b-FGF mRNA明显高于癌旁和周围正常肾组织（P＜0．01）,b-FGF mRNA的升高与肿瘤分期和分级密切相关,与肿瘤大小无明显关系。结论：b-FGF在肾癌组织中自身分泌,与肾癌的发生和发展密切相关。     

冲击波对骨折愈合过程中骨痂生成及血管内皮生长因子和碱性成纤维细胞生长因子蛋白表达的影响

目的研究冲击波对骨折愈合过程中骨痂生成及血管内皮生长因子(VEGF)和碱性成纤维细胞生长因子(bFGF)蛋白表达的影响。方法选用新西兰大白兔60只,随机分为6组:3 d、1周、2周、3周、4周和6周组,每组10只。制备双侧桡骨横断骨折模型,左侧为对照侧,右侧为冲击波治疗侧(14 kV,2000次)。分别于术后1、2、4和6周摄X线片,并评价骨痂生成量;于术后3 d、1周、2周、3周、4周和6周取材行病理检查,观察骨折愈合情况,并做免疫组化染色检测VEGF和bFGF蛋白的表达水平。对检测结果进行统计学分析。结果X线片示术后1、2、4、6周冲击波治疗侧骨痂生成量多于对照侧,差异均有统计学意义(P＜0．01)。病理检查显示术后4周治疗侧皮质骨生成量较对照侧多且致密,差异有统计学意义(P＜0．01),术后6周治疗侧皮质骨生成量较对照侧多,差异有统计学意义(P＜0．01)。免疫组化检测显示术后1、2和3周治疗侧VEGF阳性指数均较对照侧高,差异均有统计学意义(P＜0．05);术后3 d、1周、2周和3周bFGF阳性指数治疗侧均高于对照侧,差异均有统计学意义(P＜0．05)。结论冲击波能促进骨折愈合过程中VEGF、bFGF蛋白的表达,并能促进骨痂生成,加速骨折愈合。     

碱性成纤维细胞生长因子在骨愈合过程中的表达与合成

目的 通过对骨愈合过程中内源性碱性成纤维细胞生长因子（ｂａｓｉｃ ｆｉｂｒｏｂｌａｓｔ ｇｒｏｗｔｈ ｆａｃｔｏｒ,ｂＦＧＦ）合成变化的观察,探讨其成此过程的作用,以此加深对骨生理学的认识,同时为干预骨愈合提供理论依据。方法 选用ＳＤ大鼠并将其胫骨造成骨缺损,用免疫组织化学及原位杂交技术检测ｂＦＧＦ基因和蛋白质在骨闸组织成熟过程不同时期的变化情况。结果 免疫组织化学染色显示,从骨折后４天～３人,ｂ     

bFGF对体外关节软骨细胞创伤愈合与增殖的影响

目的：探讨碱性成纤维细胞生长因子（ｂａｓｉｃ ｆｉｂｒｏｂｌａｓｔ ｇｒｏｗｔｈ ｆａｃｔｏｒ,ｂＦＧＦ）对关节软骨细胞创伤愈合与增殖行为的影响。方法;采用体外创伤愈合模型及ＭＴＴ比色方法,对软骨细胞创伤愈合与增殖行为进行分析。结果：ｂＦＧＦ浓度为５ｎｇ／ｍｌ时,即可显著促进软骨细胞创伤愈合,浓度为５０ｎｇ／ｍｌ时,其促进作用达到最大值。     

骨折愈合过程中血管内皮细胞生长因子表达的实验研究

目的 观察骨折愈合过程中骨痂组织内血管内皮细胞生长因子（ｖａｓｃｕｌａｒ ｅｎｄｏｔｈｅｌｉａｌ ｇｒｏｗｔｈ ｆａｃｔｏｒ,ＶＥＧＦ）的表达,进一步探讨ＶＥＧＦ对骨折愈合的影响。方法 采用免疫组织化学方法愈合的不同阶段,骨痂组织内表达ＶＥＧＦ的细胞来源不同,同时表达程度也存在差异。结论 骨折愈合的不同时期,骨痂组织内间充质细胞、软骨细胞、成骨细胞等出现程度不同的ＶＥＧＦ表达,表明ＶＥＧＦ参与调节     

Effects of a Single Percutaneous Injection of Basic Fibroblast Growth Factor on the Healing of a Closed Femoral Shaft Fracture in the Rat

Recently, bioactive agents to stimulate bone formation have been available in the orthopedic field. We have shown previously that a single, local injection of basic fibroblast growth factor (bFGF) contributes to the formation of a larger cartilage (soft callus) but does not promote replacement of the cartilage by osseous tissue during experimental closed femoral fracture healing. Aiming at a clinical application, the present study was undertaken to clarify the effects of locally injected bFGF on bone (hard callus) formation and the mechanical properties of the callus in closed fracture healing in rats. Immediately after fracture, a carrier (200 μL of fibrin gel) containing 100 μg of bFGF or carrier alone was applied to the fracture site. At days 42 and 56 postfracture, the bone union rate, bone mineral density (BMD), and mechanical properties (strength and stiffness) of the callus were evaluated. Unexpectedly, with the exception of reduced stiffness in the FGF-injected callus at day 56, none of these parameters showed a significant difference between the control and the FGF-injected groups. Furthermore, the temporal expression pattern of OPN mRNA during healing was very similar between groups. We conclude that, in the healing of closed fractures of long bones, administration of bFGF forms a larger callus but does not necessarily accelerate the healing process.     

Local Application of Basic Fibroblast Growth Factor Minipellet Induces the Healing of Segmental Bony Defects in Rabbits

Fibroblast growth factor (FGF) has been reported to increase the volume of callus in a fracture model of rats. There are, however, no reports of successful repair of segmental bony defects by application of an FGF solution. In this study, the effects of basic FGF on the repair of segmental bony defects in the rabbit femur were examined. Minipellet, a new drug delivery system using atelocollagen, was employed to ensure effective delivery of FGF. Segmental bony defects (10 mm in length) were created in the right femurs of 19 rabbits. In pilot studies, no defects of this size healed spontaneously within 6 weeks. Bones were stabilized with miniexternal fixators. Minipellets containing basic FGF were implanted between fragments so as to bridge the two fragments. The healing processes were monitored radiographically and studied histologically. In rabbits in which FGF was added to the defect site at doses of 1.4 μg or higher, approximately 90% of the defects were filled with new bone and cartilage within 6 weeks after minipellet implantation. In rabbits receiving placebo minipellets, however, approximately 15% of the defects were filled by callus within 6 weeks. Furthermore, this callus did not change into mature bone. An injection of 2 μg of FGF solution to bony defects had no effect on the repair of segmental bony defects. These findings suggest that FGF plays a role in the production of adequate volumes of callus particularly in the initial stages of fracture healing and that sustained local release enables FGF to be effective at a low dose. In summary, large segmental bony defects healed after insertion of low-dose FGF minipellets. An adequate dose of FGF and an appropriate delivery system are required for successful healing of large bony defects. These findings imply the potential value of FGF minipellets in clinical practice. Received: 26 June 1997 / Accepted: 11 May 1998     

Do Vascular Endothelial Growth Factor and Basic Fibroblast Growth Factor Promote Phenytoin''s Wound Healing Effect in Rat? An Immunohistochemical and Histopathologic Study

BACKGROUND: This study was designed to determine the possible correlation between phenytoin and vascular endothelial growth factor and basic fibroblast growth factor in the wound healing process. METHODS: Sixty Wistar albino rats were divided into four groups, each containing 15 animals. The experimental groups received a daily phenytoin treatment (10 mg) for 3 or 7 days following 3-cm dorsal skin incisions on the midline; the control group incisions were treated with 0.5 mL of saline solution during the same time periods. After completion of treatments, all animals were killed, and skin tissue samples were obtained. RESULTS: Histopathologic examination of all groups revealed that there were significantly increased (p<0.05) amounts of fibroblasts, collagen deposition, and blood vessels in the phenytoin-treated groups when compared to the control groups. Although immunolocalization of vascular endothelial growth factor and basic fibroblast growth factor was weak in the 3-day phenytoin treatment groups, they were strongly expressed in the 7-day treatment group when compared to the control groups. CONCLUSION: The findings of this study demonstrate that the tissue alterations of the wound healing process could be accelerated by phenytoin and the potential local pathways of vascular endothelial growth factor and basic fibroblast growth factor.     

中西医结合治疗骨折微循环研究

本项研究采用大耳白兔为实验动物，采用外骨膜广泛剥离、部分肌肉切除的桡骨骨折的动物模型，采用自身对照法，分成对照组、实验组。对照组不包括软组织损伤及骨膜广泛剥离的骨折模型。利用组织学、电镜及血管灌注等方法对骨折愈合微循环变化进行了观察。并将软组织损伤对骨折愈合过程中的微循环及超微结构的变化进行了分析。术后１～８周随机分批处死动物，标本观察。实验结果显示：１．１至４周毛细血管代偿性扩张较对照组差，新生毛细血管管腔狭窄、闭塞。５周后骨折处微循环开始接近正常。２．骨皮质外侧四分之一的骨细胞变性坏死比较明显。３．形成骨痂的成骨细胞活性明显降低、胶原合成不良、骨折愈合相对迟缓。此项研究显示软组织损伤及骨外膜剥离与骨折愈合过程中的微循环改变具有直接关系。软组织损伤及骨外膜剥离可加重骨折断端的缺血，导致骨折愈合过程相对迟缓。     

Therapeutic Angiogenesis With Basic Fibroblast Growth Factor: Technique and Early Results

Background. Patients not amenable to complete myocardial revascularization by conventional methods present a difficult clinical problem. Here we present the early results and technical considerations of the administration of basic fibroblast growth factor for the induction of collateral growth using heparin-alginate slow-release devices in patients undergoing coronary artery bypass grafting.

Methods. Eight patients were enrolled. Patients were candidates if they had at least one graftable obstructed coronary artery and at least one major arterial distribution not amenable to revascularization, a serum creatinine level less than 3 mg/dL, ejection fraction greater than 0.20, and estimated operative mortality of less than 25%. During conventional coronary artery bypass grafting, 10 heparin-alginate devices, each containing either 1 μg or 10 μg of basic fibroblast growth factor, were implanted in the epicardial fat in multiple regions of the unrevascularizable territory and also in the distal distribution of a grafted or patent artery.

Results. There was no mortality and no evidence of renal, hematologic, or hepatic toxicity during follow-up. Three months after the operation, all patients remain free of angina. Seven patients were examined with stress perfusion scans. Three patients had clear enhancement of perfusion to the unrevascularized myocardium, 1 patient had a new fixed defect, and 3 had minimal overall change but had evidence of new small, fixed perfusion defects. Seven patients had improved or similar myocardial contractile function (ejection fraction at 3-month follow-up = 0.53 ± 0.22 versus 0.47 ± 0.14 preoperatively). One patient suffered a perioperative myocardial infarction in the area of basic fibroblast growth factor administration.

Conclusions. This preliminary study demonstrates the safety and technical feasibility of therapeutic angiogenesis with basic fibroblast growth factor delivered by heparin-alginate slow-release devices. Further studies examining the safety, clinical efficacy, and long-term results are ongoing.     

Elevated Serum Vascular Endothelial Growth Factor and Basic Fibroblast Growth Factor Levels in Patients with Thymic Epithelial Neoplasms

Neovascularization, an essential event for the growth of solid tumors, is regulated by a number of angiogenic factors, among which vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), are considered to exert potent angiogenic activity. In this study, we investigated whether serum VEGF and bFGF levels could be predictors of the development and extension of thymic epithelial neoplasms. The subjects of this study were 37 patients with thymoma, 6 with thymic carcinoma, and 23 healthy volunteers. Serum samples were collected before clinical treatment. Serum VEGF levels were significantly (P < 0.05) elevated in the patients with thymic carcinoma (1 080 ± 1 185 pg/ml) compared with those in the healthy volunteers (407 ± 589 pg/ml). Serum bFGF levels were also significantly (P < 0.05) elevated in the patients with thymic carcinoma (2 740 ± 631 pg/ml) compared with those in the healthy volunteers (1 728 ± 1 192 pg/ml). However, the serum VEGF and bFGF levels did not significantly differ between the patients with thymoma and the healthy volunteers. Serum VEGF and bFGF levels did not significantly differ according to the stage and pathological subtype of thymoma. Moreover, there was no correlation between the serum levels of VEGF and those of bFGF. Thus, while serum VEGF and bFGF levels may serve as markers for thymic epithelial tumors, it is unlikely that circulating VEGF and bFGF could be used as markers for assessing the progression of thymoma tumors. Received: November 10, 2000 / Accepted: May 15, 2001