碱性成纤维细胞生长因子的研究进展

碱性成纤维细胞生长因子是一种多肽细胞生长因子,具有广泛的生理功能。碱性成纤维细胞生长因子的研究近年来取得了迅速发展,尤其是基因的表达及临床应用等方面。本文就该生长因子基因的克隆和表达,生物功能及临床应用研究进展作一概述。     

碱性成纤维细胞生长因子研究进展

碱性成纤维细胞生长因子是细胞生长和分化的重要调节因子,具有促血管生成、细胞增殖、细胞趋化、细胞迁移等活性,在细胞分化和机体发育过程中发挥重要作用。碱性成纤维细胞生长因子通过与细胞膜表面的特异性配体结合,进而引发细胞内的一系列级联反应,从而产生各种生物学效应。本文对碱性成纤维细胞生长因子的生物学基础、信号转导、生物学功能以及临床应用研究进展作一综述。     

生长因子在骨折愈合过程中作用的研究进展

骨折愈合是一个复杂的病理生理过程,涉及多种细胞和生长因子的协同作用。随着分子生物学和基因工程的发展,生长因子在骨折愈合过程中的基础研究和临床应用取得了较大的进展。本文就转化生长因子β．碱性成纤维细胞生长因子、血小板衍生生长因子、表皮生长因子、神经生长因子等五种生长因子在骨折愈合过程中的作用及近年来的研究进展作一综述,并分析五种生长因于在法医学中的应用前景。     

重组人碱性成纤维细胞生长因子在大肠杆菌中的高效表达*

利用基因改造的方法可以优化外源基因在大肠杆菌中的表达。利用逆转录PCR技术从原代培养的人成纤维细胞中克隆出人碱性成纤维细胞生长因子基因,在不改变氨基酸序列的前提下对该基因的上游部分序列进行改造,并将其插入表达载体pET-3c,转入大肠杆菌阳BL21（DE3）,IPTG诱导表达,表达蛋白占菌体总蛋白的30％以上,并具有良好的生物活性。     

抗菌肽与碱性成纤维细胞生长因子基因的融合及其在酵母中的表达

从重组质粒ｒＢＳ上切下柞蚕抗菌肽Ｄ基因片段,切去终止密码后连接到重组穿梭质粒ｐＶＴ－ＧＦ上碱性成纤维细胞生长因子ｃＤＮＡ的５′端,使密码框正确排列,构建成融合基因重组质粒ｐＶＴ－ＣＤＧＦ,转化到酵母中进行表达。转化子酵母蛋白粗提物用Ｅ．ｃｏｌｉＫ１２Ｄ３１作指示菌进行抑菌圈测试,初步检出具有抑菌活性,用ＥＬＩＳＡ检测证明其具有碱性成纤维细胞生长因子的抗原性。     

人碱性成纤维细胞生长因子基因克隆、cDNA5'端修饰及在大肠杆菌中的表达

采用cDNA PCR技术 ,从人胎盘cDNA文库DNA中克隆了人碱性成纤维细胞生长因子 (hbFGF)基因。用PCR突变法对其 5 端序列进行修饰 ,将天然和修饰后的hbFGF基因分别克隆至表达载体 pBV2 2 1,免疫印迹和SDS PAGE结果证明 ,经修饰后的基因在大肠杆菌DH5α中获得了表达 ,表达量占菌体总蛋白的 9%。     

成纤维细胞生长因子在骨修复中的作用和应用

成纤维细胞生长因子 ( FGF)是一类与肝素有高亲和性的多聚肽 ,最初根据它能刺激成纤维细胞再生而命名。现已发现有 1 8个成员 ,即 FGF1- FGF18。目前研究得最多的是 FGF1和 FGF2 。根据它们等电点 ( PI)的不同 ,FGF1又称为酸性成纤维细胞生长因子 ( a FGF,PI为 5 .6) ,FGF2 又称为碱性成纤维细胞生长因子 ( b FGF,PI为 9.6) ,它们均通过自分泌和 /或旁分泌途径在组织修复中发挥重要作用。成纤维细胞生长因子受体 ( FGFR)属于免疫球蛋白超家族成员 ,目前已确定了 4种由独立基因编码的人 FGFR。它们是一种跨膜蛋白质 ,分为细…     

大鼠卵巢颗粒细胞aFGF及bFGF基因表达的研究

成纤维细胞生长因子(FGFs)是一类多功能因子,具有广泛的生物学作用,它对卵泡发育的许多方面如颗粒细胞的生长及分化、纤维蛋白溶酶原活化剂的释放、血管生成等都有影响。FGFs按等电点的不同分为酸性成纤维细胞生长因子(aFGF)和碱性成纤维细胞生长因子(bFGF),两者有55％的同源性并作用于同一受体。从已有资料分析,aFGF和bFGF在进     

bFGF通过MAPK信号通路调节Myostatin表达

肌肉抑制素Myostatin是TGF-β(transforming growth factor-β)超家族成员之一,在哺乳动物及非哺乳动物中均能够抑制肌细胞的增殖和分化,是骨骼肌生长的负调控因子。但Myostatin基因自身是如何被调控的目前仍然不十分清楚。该实验发现,碱性成纤维细胞生长因子bFGF(basic fi broblast growth factor)能够上调Myostatin的表达。进一步分析表明,bFGF对于Myostatin的表达调控存在剂量和时间依赖性。同时实验还发现,MAPK信号通路部分地介导了bFGF对Myostatin基因的调节作用。     

牛碱性成纤维细胞生长因子在酵母系统中的表达

牛碱性成纤维细胞生长因子在酵母系统中的表达方向东陈淳谢志伟耿解萍王小宁戚正武（中国科学院上海生物化学研究所国家分子生物学实验室上海２０００３１）碱性成纤维细胞生长因子（ｂａｓｉｃｆｉｂｒｏｂｌａｓｔｇｒｏｗｔｈｆａｃｔｏｒ,ｂＦＧＦ）是ＦＧＦ家族中具有代表性的成员。分子中无糖基化位点,为一单链阳离子多肽,与肝素有很强的亲和能力。它广泛存在于机体的各种组织中,是重要的神经营养因子和血管生成因子,在胚胎发生、生长发育及血管形成等多种生理过程中起重要作用。     

bFGF与黑色素关系的研究进展

碱性成纤维细胞生长因子(bFGF)是一种具有多种生物学效应的细胞因子。目前对bFGF与黑色素关系的研究主要集中在bFGF对黑色素细胞的生长产生的影响和对黑色素生成的调控等方面,并进而探讨研究治疗黑色素相关疾病的机理,综述了在这些方面的研究进展。     

Structure-function relationship of basic fibroblast growth factor: site-directed mutagenesis of a putative heparin-binding and receptor-binding region.

Basic residues Arg-118, Lys-119, Lys-128, and Arg-129 within a putative heparin-binding and receptor-binding region of the 155-amino acid form of basic fibroblast growth factor (bFGF) have been changed to neutral glutamine residues by site-directed mutagenesis of the human bFGF cDNA. The bFGF mutant (M6B-bFGF) was expressed in E. coli and purified to homogeneity. When compared to wild type bFGF, M6B-bFGF showed in cultured endothelial cells a similar receptor-binding capacity and mitogenic activity, but a reduced affinity for heparin-like low affinity binding sites, a reduced chemotactic activity, and a reduced capacity to induce the production of urokinase-type plasminogen activator. In vivo, M6B-bFGF lacked a significant angiogenic activity. Modifications of both the primary and the tertiary structure of bFGF appear to be responsible for the modified biological properties of M6B-bFGF, thus confirming the possibility to dissociate at the structural level some of the biological activities exerted by bFGF on endothelial cells.     

Mutations in the heparin-binding domains of human basic fibroblast growth factor alter its biological activity

Eleven structural analogues of human basic fibroblast growth factor (bFGF) have been prepared by site-directed mutagenesis of a synthetic bFGF gene to examine the effect of amino acid substitutions in the three putative heparin-binding domains on FGF's biological activity. After expression in Escherichia coli, the mutant proteins were purified to homogeneity by use of heparin-Sepharose chromatography and analyzed for their ability to stimulate DNA synthesis in human foreskin fibroblasts. Recombinant human bFGF 1-146 and [Ala69,Ser87]bFGF, an analogue where two of the four cysteines had been replaced by alanine and serine, were equipotent to standard bovine basic fibroblast growth factor. Substitution of aspartic acid-19 by arginine in the first heparin-binding domain yielded a molecule that stimulated a higher total mitogenic response in fibroblasts as compared to bFGF. In addition, replacement of either arginine-107 in the second domain or glutamine-123 in the third domain with glutamic acid resulted in compounds that were 2 and 4 times more potent than bFGF. In contrast, substitution of arginine-107 with isoleucine reduced the activity of the molecule by 100-fold. Combination of domain substitutions to generate the [Glu107,123]bFGF and [Arg19,Lys123,126]bFGF mutants did not show any additivity of the mutations on biological activity. Alterations in the biological activity of the analogues was dependent on both the site of and the type of modification. Increased positive charge in the first domain and increased negative charge in the second and third domains enhanced biological potency. The altered activities of the derivatives appear to be due in part to changes in the affinity of the analogues for heparin. We conclude that changes in all three of the putative heparin-binding domains result in altered mitogenic activity and heparin interaction of basic fibroblast growth factor.     

Neutralizing antibodies inhibit the binding of basic fibroblast growth factor to its receptor but not to heparin

Polyclonal antibodies were prepared against recombinant basic fibroblast growth factor (bFGF) that reacted only with bFGF but not acidic FGF. These antibodies were able to inhibit various biological activities of bFGF such as the ability of bFGF to stimulate DNA synthesis in 3T3 cells, proliferation and migration of bovine capillary endothelial cells (BCEC), and neurite extension in pheochromocytoma (PC12) cells. The anti-bFGF antibodies also inhibited the mitogenic activity of subendothelial cell extracellular matrix for BCEC, demonstrating that the growth factor component in extracellular matrix required for supporting BCEC proliferation was bFGF. Anti-bFGF antibodies inhibited the cross-linking of bFGF to its high affinity receptor on BCEC cells. However, these antibodies did not inhibit the binding of bFGF to heparin-Sepharose or to the low affinity receptors of BCEC which have been demonstrated to be heparin-like molecules. These results suggest that bFGF has distinct domains for binding to high affinity cellular receptors and for binding to heparin.     

Basic fibroblast growth factor accumulates in the nuclei of various bFGF-producing cell types

The intracellular localization of basic fibroblast growth factor (bFGF) was studied in BHK-21 cells transfected with an expression vector containing the complementary DNA (cDNA) of the human bFGF gene (pbFGF). The intracellular location of bFGF was determined using indirect immunofluorescence. The antibodies used were polyclonal antibodies directed against either recombinant human bFGF or recombinant Xenopus bFGF. The nuclei of transfected cells that produce bFGF, but not the nuclei of untransfected cells, were labeled strongly by the antibodies. The nuclear staining was totally abolished when anti-bFGF antibodies preadsorbed with bFGF were used. Several types of endothelial cells known to produce bFGF were also stained in their nuclei by the antibodies. Nuclear extracts prepared from transfected cells were found to contain bFGF as determined using heparin-sepharose affinity chromatography, followed by Western blot analysis of fractions, which stimulated the proliferation BHK-21 cells. The mitogenic activity associated with the nuclei was not destroyed when isolated cell nuclei were digested by trypsin. It is therefore likely that the nucleus associated bFGF is intranuclear. These findings suggest that some biological activities of bFGF may be mediated by nuclear bFGF binding proteins or by the direct binding of bFGF to DNA.     

Heparin potentiates the action of acidic fibroblast growth factor by prolonging its biological half-life

The mechanism(s) by which heparin influences the biological activities of acidic and basic fibroblast growth factors (aFGF and bFGF) is not completely understood. One mechanism by which heparin could alter the biological activities of aFGF and bFGF is by altering their biological half-lives. We investigated the possibility that heparin potentiates aFGF-induced neurite outgrowth from PC12 cells by prolonging its biological half-life. Under conditions where heparin potentiated aFGF-induced neurite outgrowth, we observed that heparin increased the biological half-life of aFGF from 7 to 39 hr. We determined that greater than 25 hr of exposure to active aFGF was required for induction of neurite outgrowth. If aFGF activity was maintained for greater than 25 hr by periodic readdition of factor, heparin no longer potentiated aFGF-induced neurite outgrowth. These observations strongly suggest that heparin potentiates the activity of aFGF by prolonging its biological half-life. The protease inhibitors hirudin, leupeptin, and pepstatin A did not potentiate aFGF-induced neurite outgrowth, indicating that proteases inhibited by these inhibitors are not responsible for the loss of aFGF activity that we observed. However, aprotinin potentiated aFGF neurite-promoting activity approximately sevenfold, indicating that proteases that are inhibited by aprotinin are at least partially responsible for aFGF inactivation. These observations suggest that heparin regulates the activity of aFGF by regulating its proteolytic degradation, thereby regulating its biological half-life.     

Structural features in heparin which modulate specific biological activities mediated by basic fibroblast growth factor

The biological activity of basic fibroblast growth factor (bFGF)is influenced greatly by direct binding to heparin and heparansulphate (HS). Heparin-derived oligosaccharides have been utilizedto determine the structural requirements present in the polymerthat account for bind ing to bFGF. We had previously demonstratedthat fragments >6 mer can inhibit the interaction betweencell surface heparan sulphate proteoglycan (HSPG) and bFGF,and bFGF-induced proliferation of adrenocortical endothelial(ACE) cells. In contrast, oligosaccharides > 10 mer can enhancethe binding of bFGF to its high-affinity receptor or supportbFGF-induced mitogenesis in ACE cells (Ishihara et al., J. Biol.Chem., 268, 4675–4683, 1993). We have extended these studiesto size- and structure-defined oligosaccharides from heparin,2-O-desulphated (2-O-DS-) heparin, 6-O-desulphated (6-O-DS-)heparin, carboxyreduced (CR-) heparin and carboxy-amidomethylsulphonated(AMS-) heparin. Oligosaccharides from these polymers were fractionatedon a bFGF-affinity column and were assessed as inhibitors orenhancers of specific bFGF-derived biological activities. Theresults of these studies indicate that both 2-O-sulphate andthe negative charge of the carboxy group [L-iduronic acid (IdoA)residues] are required for specific interactions of heparin-derivedoligosaccharides with bFGF and for modulation of bFGF mitogenicactivity. In addition, the charge of the carboxy groups in uronicacids can be replaced by other functional groups with a negativecharge, such as the amidomethyl sulphonate moiety describedhere. basic fibroblast growth factor heparan sulphate heparin oligosaccharides     

The fibroblast growth factor family: Structural and biological properties

This article sumarizes the structural and biological properties of the family of fibroblast growth factors (FGF). Basic FGF (bFGF) and acidic FGF (aFGF) are the best characterized members of this family. bFGF and aFGF are potent modulators of cell proliferation, motility and differentiation. They are also potent angiogenesis factors in vivo. Some of the important biological characteristics of bFGF and aFGF discussed in the review include the affinity of bFGF and aFGF for heparin, their lack of secretion in culture and their association with extracellular matrix. Recently, several oncogenes, 40–50% homologous in sequence to bFGF and aFGF have been identified. These include int-2, hst, K-fgf and FGF-5. The structural and biological properties of these FGF-related oncogenes are also discussed.     

Correlation of cell migration, cell invasion, receptor number, proteinase production, and basic fibroblast growth factor levels in endothelial cells

The levels of endogenous basic fibroblast growth factor (bFGF) in seven clones of cultured bovine capillary endothelial (BCE) cells were assayed, and their relation to cell morphology, bFGF receptor number, cell migration, amniotic membrane invasivity, and proteinase levels were studied. Immunoblotting experiments with anti-bFGF IgG demonstrated that cells from these clones contained different amounts of bFGF. The cells containing high levels of bFGF had a spindle or elongated appearance at confluence and a low number of high affinity receptors for bFGF. The cells containing low levels of bFGF had a cobblestone-like appearance and a higher number of high affinity receptors. When exposed to 10 ng/ml bFGF, cells containing a low level of bFGF took on an elongated appearance with a crisscross pattern similar to that seen with the high producer bFGF cells. The endogenous bFGF levels of the BCE cell clones correlated with the extent of cell migration after wounding of a monolayer and the degree of invasion of the human amniotic membrane. Cells from the clone with the highest endogenous bFGF level migrated well, invaded the amnion membrane without the addition of exogenous bFGF, and were relatively unaffected by the addition of bFGF. Cells from the clone containing the lowest level of bFGF did not migrate or invade under normal conditions. However, the addition of bFGF to the culture medium strongly enhanced both of these processes. The inclusion of anti-bFGF IgG in the media suppressed cell migration and invasion. The plasminogen activator (PA) activities of cell lysates of the clones, assayed by the 125I-fibrin plate technique, indicated that the PA levels did not correlate with the bFGF levels. Metalloproteinase activities in the conditioned medium, assayed by gelatin zymography, correlated with the endogenous bFGF levels, suggesting that the degree of expression of metalloproteinases might be critical for cell migration and invasion. These data suggest that endogenous bFGF may have an important role for migration and invasion of BCE cells during neovascularization via the induction and/or activation of specific metalloproteinases.