人碱性成纤维细胞生长因子cDNA的克隆及在毕赤酵母中的表达
|
摘要
碱性成纤维细胞生长因子(basic fibroblast growth factor , 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,是一种具有广泛生物活性的肽类物质,对酸和热敏感,在体内分布极为广泛,是一种重要的神经营养因子和血管生成因子,能促进创伤愈合与组织修复,也能促进某些细胞再生,在胚胎生长、发育及血管形成等多种生理过程中起重要作用。
本研究从人神经胶质瘤细胞总RNA中钓取hbFGF cDNA序列,利用基因重组技术构建人bFGF cDNA克隆载体pMD18-T-hbfgf及hbFGF表达载体pPICZα-hbfgf。将hbFGF表达载体转化X-33毕赤酵母,建立兼具原核系统良好操作性和真核系统后加工性的重组hbFGF毕赤酵母真核表达体系。通过PCR和SDS-PAGE的鉴定,筛选出高表达重组hbFGF的酵母菌株,为进一步实验奠定了基础。结果表明,本研究正确克隆了人bFGF cDNA序列,构建的重组hbFGF毕赤酵母真核表达体系经甲醇诱导能够分泌具有活性的重组hbFGF蛋白,相对分子量为18KD;重组hbFGF的表达具有累积效应,产量在一段时间内随表达时间延长而增加。本研究为选用毕赤酵母作为生物反应器生产hbFGF基因工程药物提供了实验依据。
bFGF was first obtainded from bovine pituitary and nerve tissue byGospodarowicz in 1974. It was denominated fibroblast growth factor because itcan stimulate 3T3 cells proliferation .bFGF locates 4q25 of being chromosomeand it was made up of two intron and three exon .
The distribution of basic fibroblast growth factor(bFGF) in vivo is verybroad., they can be found in brain, heart, bone, eyes, adrenal gland and placenta .Cells cultivated in vitro, such as vascular smooth muscle cells ,vascularendothelial cells, oligodendrocyte and some tumor cells includingchondrosarcoma cells, neurospongiom cells can also generate bFGF. It has beenproved that bFGF is a cytokine with multi-biological activities in vivo and invitro.
bFCF is a broad-spectrum mitogen and inducing factor of morphogenesiaand differentiation, having evident chemotaxis to cells derived frommesoblastic. Studies have proved that bFCF is a angiogenesis factor, with thefuction of promoting wound healing and tissue repair. It can promote cellregeneration too.
The praeparatum which are used in the market are proteins expressed fromEscherichia coli . Pichia Pastoris express system which widely used in gene
engineering has the properties of simple operation, distinct genetic background,lower cost price. The system has eukaryote's folding and secrete mechanism,can accomplish the post-translational modification process which is theproperties of eukaryote including proteolysis , folding, glycosylation, has rapidgrowth,simple nutritional ingredient,and can accommodate the needs oflarge-scale industrialization .So we build an expression system of recombinantbfgf and seek to produce recombinant protein with gene-tic engineeringtechnology.1、Cloning of hbfgf cDNA sequenceWe first obtained the cDNA sequence encoding hbFGF by RT-PCR, inwhich the template RNA derives from glioma cell U251. Then the purified PCRproduction was cloned to pMD18-T vector. The results of sequencing showedthat the recombinant cloning vector pMD18-T-hbfgf was constructedcorrectly,so we can continue to do the expression of recombinant hbFGF.2、Construction of recombinant hbFGF expression system(1)Construction of hbFGF expression vector pPICZα-hbfgfWith the recombinant plasmid of pMD18-T-hbfgf as template, the sequenceencoding hbFGF was obtained by PCR. The endonuclease sites of XhoI andEcoRI were added into the two ends of interested sequence. Then it was clonedto pPICZαvector after being digested with these two endonucleases. The resultsof sequencing demonstrated that the construct was correct.(2)Transformation pPICZα-hbfgf into Pichia pastoriswe transformed purified pPICZα-hbfgf vector into Pichia pastoris andscreened on YPD plates containing 100μg/ml Zeocin. After 72 hours, dozens oftransforming colonies appeared .
(3)Expression of recombinant hbFGF in Pichia pastorisSelected several clones from YPD/Zeocin plates, and induced them by0.5% methanol in BMMY media. Analyzed the expression product by activitydetermination and SDS-PAGE.Results:① Through being detected the time course expression of positiveclone, it was found that there was a cumulative effect of production during thefirst 3 days and reach the peak at the end;In the next two days, the expression ofrecombinant protein was steady;Then the production of recombinant proteinbegan reduce. ②Analyzed the purified recombinant protein by SDS-PAGE, aprotein of 18 kDa molecular weight appeared. These results indicated that thefunctional recombinant hbFGF could be induced in methylotropic yeastexpression system and the secreting of recombinant protein had cumulativeeffect, which increased depending on time change. ③When the positive clonewas incubated in BMMY media with different pH varing from 3.0 to 6.0, thesecretion of recombinant protein had a highest yield at about pH 5.0.The results indicated that the effective expression system of recombinanthbFGF had been constructed. The variant Pichia pastoris can secret recombinanthuman basic fibroblast growth factor , which can exhibits activity and has amolecular weight of 18kD.
本研究从人神经胶质瘤细胞总RNA中钓取hbFGF cDNA序列,利用基因重组技术构建人bFGF cDNA克隆载体pMD18-T-hbfgf及hbFGF表达载体pPICZα-hbfgf。将hbFGF表达载体转化X-33毕赤酵母,建立兼具原核系统良好操作性和真核系统后加工性的重组hbFGF毕赤酵母真核表达体系。通过PCR和SDS-PAGE的鉴定,筛选出高表达重组hbFGF的酵母菌株,为进一步实验奠定了基础。结果表明,本研究正确克隆了人bFGF cDNA序列,构建的重组hbFGF毕赤酵母真核表达体系经甲醇诱导能够分泌具有活性的重组hbFGF蛋白,相对分子量为18KD;重组hbFGF的表达具有累积效应,产量在一段时间内随表达时间延长而增加。本研究为选用毕赤酵母作为生物反应器生产hbFGF基因工程药物提供了实验依据。
bFGF was first obtainded from bovine pituitary and nerve tissue byGospodarowicz in 1974. It was denominated fibroblast growth factor because itcan stimulate 3T3 cells proliferation .bFGF locates 4q25 of being chromosomeand it was made up of two intron and three exon .
The distribution of basic fibroblast growth factor(bFGF) in vivo is verybroad., they can be found in brain, heart, bone, eyes, adrenal gland and placenta .Cells cultivated in vitro, such as vascular smooth muscle cells ,vascularendothelial cells, oligodendrocyte and some tumor cells includingchondrosarcoma cells, neurospongiom cells can also generate bFGF. It has beenproved that bFGF is a cytokine with multi-biological activities in vivo and invitro.
bFCF is a broad-spectrum mitogen and inducing factor of morphogenesiaand differentiation, having evident chemotaxis to cells derived frommesoblastic. Studies have proved that bFCF is a angiogenesis factor, with thefuction of promoting wound healing and tissue repair. It can promote cellregeneration too.
The praeparatum which are used in the market are proteins expressed fromEscherichia coli . Pichia Pastoris express system which widely used in gene
engineering has the properties of simple operation, distinct genetic background,lower cost price. The system has eukaryote's folding and secrete mechanism,can accomplish the post-translational modification process which is theproperties of eukaryote including proteolysis , folding, glycosylation, has rapidgrowth,simple nutritional ingredient,and can accommodate the needs oflarge-scale industrialization .So we build an expression system of recombinantbfgf and seek to produce recombinant protein with gene-tic engineeringtechnology.1、Cloning of hbfgf cDNA sequenceWe first obtained the cDNA sequence encoding hbFGF by RT-PCR, inwhich the template RNA derives from glioma cell U251. Then the purified PCRproduction was cloned to pMD18-T vector. The results of sequencing showedthat the recombinant cloning vector pMD18-T-hbfgf was constructedcorrectly,so we can continue to do the expression of recombinant hbFGF.2、Construction of recombinant hbFGF expression system(1)Construction of hbFGF expression vector pPICZα-hbfgfWith the recombinant plasmid of pMD18-T-hbfgf as template, the sequenceencoding hbFGF was obtained by PCR. The endonuclease sites of XhoI andEcoRI were added into the two ends of interested sequence. Then it was clonedto pPICZαvector after being digested with these two endonucleases. The resultsof sequencing demonstrated that the construct was correct.(2)Transformation pPICZα-hbfgf into Pichia pastoriswe transformed purified pPICZα-hbfgf vector into Pichia pastoris andscreened on YPD plates containing 100μg/ml Zeocin. After 72 hours, dozens oftransforming colonies appeared .
(3)Expression of recombinant hbFGF in Pichia pastorisSelected several clones from YPD/Zeocin plates, and induced them by0.5% methanol in BMMY media. Analyzed the expression product by activitydetermination and SDS-PAGE.Results:① Through being detected the time course expression of positiveclone, it was found that there was a cumulative effect of production during thefirst 3 days and reach the peak at the end;In the next two days, the expression ofrecombinant protein was steady;Then the production of recombinant proteinbegan reduce. ②Analyzed the purified recombinant protein by SDS-PAGE, aprotein of 18 kDa molecular weight appeared. These results indicated that thefunctional recombinant hbFGF could be induced in methylotropic yeastexpression system and the secreting of recombinant protein had cumulativeeffect, which increased depending on time change. ③When the positive clonewas incubated in BMMY media with different pH varing from 3.0 to 6.0, thesecretion of recombinant protein had a highest yield at about pH 5.0.The results indicated that the effective expression system of recombinanthbFGF had been constructed. The variant Pichia pastoris can secret recombinanthuman basic fibroblast growth factor , which can exhibits activity and has amolecular weight of 18kD.
引文
1. Gospodsrowicz D.Purification of a fibroblast growth factor from bovine pituitary「J」.J Bid Chem, 1975,250:2515-2520.
2.Trowell O A. Growth of tissues in vitro. VI. The effects of some tissue extracts on the growth of periosteal fibroblasts[J]. J Exp Biol, 1939,16:60-70.
3. Abraham JA,Whang JL.Tumolo A,et al Human basic fibroblast growth factor nucleotide sequence and genomic organization,EMBO J,1986,5;2523-2528
4. Muthukrishnan L,Warder E,Mcneil PL.Basic fibroblast growth factor is efficiently released from a eytolsolie storage site through plasma membrane disruption of endothelial cells.J Cell Physial ,1991,148:1-7.
5. Kurokawa T, Sasada R, Iwane M,et al . Cloning and expression of cDNA encoding human basic fibroblast growth factor . FEBSL ett, 1987, 213: 189 -194
6. Takayama H,Ray J,Raymon HK,et al. Basic fibroblast growth factor increase dopaminergic graft survival and function in a rat model of Parkinson's disease Nat Med,1995,1:53-58
7. Fawcett JW, Barker RA, Dunnett SB. Dopaminergic neuronal survival and the effects of bFGF in explant, three dimensional and monolayer cultures of embryonic rat ventral mesencephalon「J」.Exp Brain Res, 1995, 106(2):275-282
8.Abraham J A ,Mergia A Whang . Nucleotide sequence of a bovine clone encoding the angiogenic protein[J] . Basic Fibroblast Growth Factor Science , 1986 ,233 :545 -546.
9.Gospodarowicz D. Localisation of a fibroblast growth factor and its effect alone and with hydrocortisone on 3T3 cell growth [J]. Nature, 1974,249: 123-127.
10.Incue K,Sakai I,Hattori M.The cell-adhesive effect of basic fibroblast growth factor on piturtary cells in vitro. J Endocr,1991;130:381.
11. Battler A,Scheinowitz M,BorA.et al.Intoracoronary injection of bFGF enhances angiogenesis in infracted swine myocardium,J Coll Cardio (Am),1993:22(7):2001
12. Harada K,Grossman W,Friedman M,et al.bFGF improves myccardial function in chronically ischemic poreine hearts ,J Clin Invest,1994,94:623.
13. Gospodarowiez D,Ferrara N,Schwegerer L,et al.Structural characterization and Biological function of fibroblast growth factor .Endocrine Rev,1987;8(2):95-100.
14. Eriksson AE ,Cousens LS ,Weaver LH , et al . Three dimensional structure of human basic fibroblast growth factor [J]. Proc Natl Acad Sci USA,1991, 88 :34-41
15. Gospodarowicz D , Cheng J , Lui GM , et al . Isolation of brain fibroblast growth factor by heparinsepharose affinity chromat ography :identity with pituitary fibroblast growth factor [ J ] . Proc Natl Acad Sci USA, 1984, 81: 6963 -6967.
16. Kiefer MC , Stephans JC , Crawford K , et al . Ligand affinity cloning and structure of a cell surface heparan sulfate proteoglycan that binds basic fibroblast growth factor [J ] . Proc Natl Acad Sci USA 1990 ,87 :69-85
17. Hoffman R S. The growth -activating effect of extracs of adult and embryonic tissues of the rat on fibroblast coloies in culture[ J ] . Growth , 1940, 4:361 -371.
18. Luo Y, Lu W, Mohamedali KA , et al.The glycine box : a determine inant of specificity for fibroblast growth factor [J].Biochemistry, 1998,37:16-25.
19. Paulo M. et al. New observations on the intracellular localization and release of bFGF. J Cell Biol 1991,47:201-207.
20.Sommer A , Brewer M T, Thompson R C , et al. A form of human basic fibroblast growth factor with an extended amino terminus[J]. Biochem Biophys Res Commun, 1987 , 144 (2) : 543-550.
21. Heath W F , Cantrell A S , Mayne N G , et al . Mutation in the heparin domains of human basic fibroblast growth factor alter its biological activity[J ] .Biochemistry , 1991 , 30(22):5608-5615.
22.Estape D, Heuvel J , Rinas U. Susceptibility towards intramolecular disulphide-bond formation affects conformational stability and folding of human basic fibroblast growth factor[J ]. BiochemJ , 1998 , 335(Pt2) : 343 -349.
23. 向军俭 陈彤, 邹海燕等. 重组碱性成纤维细胞生长因子生物活性检测方法的研究[J] . 暨南大学学报(自然科学版),1993,14(1):186-190.
24 Cuevas P,Carceller F,Munoz-Willery I,et al .Intravenous fibroblast growth factor penetrates the blood-brain barrier and protects hippocampal neurons against ischemia-reperfersion injury.Surg Neurol,1998,49(1):77.
25. Zawada WM, Kirschman DL,Cchen JJ,et al.Growth factors rescue embryonic dopamine neurons from programmed cell death.Exp Neurol, 1996,140(1):60
26. Muthukrishnan L ,Warder E , Mcneil PL . Basic fibroblast growth factor is efficiently released from a cytolsolic storage site through plasmamembrane disruption of endothelial cells[J ] . J Cell Physiol ,1991 ,148 :11-20.
27.Zhan X , Hu X , Friesel R , et al . Long term growth factor exposure and differential tyrosine phosphorylation are required for DNA synthesis in B-ALBPc 3T3 cells[J ] . J Biol Chem,1993 ,268 :9611-9625.
28. Inoue K ,Sakai T , Hattori M . The cell adhesive effect of basic fibroblast growth factor on pituitary cells in vitro[J ] . Endocr J ,1991, 130 :381-387.
29.Framcki A,Uciechowski P,Floege J,et al .Autocrine growth regulation of human glomerular mesangial cells is primarily mediated by basic fibroblast growth factor .Am J Pathol,1995,147(5):1372
30.Sliutz G,Tempfer C,Obermain A,et al.Serum evaluation of basic FGF in breast cancer patients .Anti-cancer Res,1995;15(68):2675.
31. Tsuboi R,Rifkin DB. Recombinant basic fibroblast growth facror simulates wound healing in healing impaired mice [ J ] . J Exp Med , 1990 , 172 :245-251.
32. TStenberg BD , Phillips LG,Hokanson JA , et al . Effect of bFGF on the inhibition of contraction carsed by basteria [J] . J Surg Res, 1991 ,50 :47-52.
33.Aspenberg P ,Thorngren KG,Lohmander LS. Dose-dependent stimulation of bone induction by basic fibroblast growth factor in rats[J ] .Asta Orthop Scand ,1991 ,62:481-486.
34. Koshinage M , Anon HR , Whittemore SR . Altered acidic and basic fibroblast growth factor expression following spinal cord injury [ J ] . Exp Neurol ,1993 ,120 :32-38.
35. Unger E , Banai S ,Shou M, et al . Basic fibroblast growth factor enhances myocardial collateral flow in a canine model [J ] . AmJ Physiol ,1994 ,266: (4pt 2) :H1588-H1595.
36. Watanabe E,Smith DM,Sun J, et al. Effect of basic fibroblast growth factor on angiogenesis in the infarcted porcine heart [J]. Basic ResCaradiol, 1998, 93(1) :30-37.
37. 齐华阁,李兰更,刘建国,等 碱性成纤维细胞因子对急性前壁心肌梗死的影响[J ] . 中国危重病急救医学,1998 ,10 (1) :29-33.
38. 王楠,徐永华 佛波脂PMA促进碱性成纤维细胞生成长因子( bFGF )释放及机理的研究[J]1 实验生物学报,1998 ,31 :41-46.
39. 刘飞鹏,吴雄化,云涌等。PCR-四步两段扩增法分离人碱性成纤维细胞生长因子(hbfgf)基因「J」。生物工程进展,1997,17(1): 52-55.
40.Delrieu I . The high molecular weight isoforms of basic fibroblast growth factor (FGF2) : an insight into an intracrinemechanism「J」 FEBS Lett . 2000 ,468 (1) :6-10.
41. Koolwijk P, van Erck MG, de Vree WJ, et al . Cooperative effect of TNF-alpha , bFGF , and VEGF on the formation of tubular structures of human microvascular endothelial cells in a fibrin matrix . Role of urokinase acti-vity[J] . J CellBiol ,1996,132(6):1177-1188.
42. Seghezzi G, Patel S,Ren CJ,et al.Fibroblast growth factor2 (FGF2) induces vascular endothelial growth factor ( VEGF ) expression in the endothelial cells of forming capillaries : an autocrine mechanism contributing to angiogenesis[J].J CellBiol,1998,141(7):1659-1673.
43. Miyake H , Hara I ,Gohji K , et al . Expression of basic fibroblast growth factor is associated with resistance to cisplatin in a human blander cancer line[J ] . Cancer Lett ,1998 ,123 (2) :121-126.
44. Noda M, Hattori T, Kimura T, et al.Expression of fibroblast growth factor 2 mRNA in early and advanced gastric cancer [J]. Acta Oncol,1997 Ogata
45. Neuner Jehle M, Berghe LV, Bonnel S,et al. Ocular cell transferction with the human basic fibroblast growth factor gene delays photo receptor cell degeneration in RCS rats.Hum Gene Ther, 2000, 11 :1875-1890.
46. Akimoto M , Miyatake S , Kogishi J , et al . Adenovirally expressed basic fibroblast growth factor rescues photoreceptor cells in RCS rats . Invest Ophthalmol Vis Sci , 1999 ,40 : 273-279.
47. Lau D , McGee LH , Zhou S , et al . Retinal degeneration is slowed in transgenic rats by AAV2 mediated delivery of FGF2 . Invest Ophthalmol Vis Sci , 2000 , 41 :3622-3633.
48.Gospodarow ic1 D . F ibroblast growth factor : chem ical structure and biologic function. Clin Orthop Rel Res, 1990;257 (Aug): 231-248.
49. Martin P , Hopk insonWJ , McCluskey J . Growth factors and cutaneous wound repair. prog Grow th Factor R es, 1992;4 (1): 25-44.
50. Bennett N T,Schultz GS. Growth factors and wound heailing: Part Role in normal and chronic wound healing. Am J Surg ,1993;166 (1) : 74-81.
51.MarksM G,Doillon C,Silver FH.Effects of fibroblasts and basic fibroblast growth factor on facilitation of dermal wound healing by type collagen matrices. J Biom ed Mater Res, 1991;25 (5):683-696.
52. LawrenceW T, Diegelmann RF. Growth factors in wound healing.C lin D erm atol, 1994;12 (1) : 157-169.
53. HickeyMJ ,Wilson Y, Hurley JV et al . Mode of vascularization of control and basic fibroblast growth factor stimulated prefabricatedskin flaps. Plast Reconstr S u rg , 1998;101 (5) : 1296-1304
54. Ueno N, A Baird , Esch F, et al . Isolation of an am ino terminal extended form of basic fibroblast growth factor . Biochem Biophys Res Commun , 1986, 138:580-586.
55. 赵爱平, 徐林, 钱林法等. 人碱性成纤维细胞生长因子基因在大肠杆菌 中的克隆与表达. 中国生化药物杂志,1996, 17 (3)∶93-96.
56. Faktorovich EG , Steinberg RH , Yasumura D , et al . Photoreceptor Degeneration in inherited retinal dystrophy delayed by basic fibroblast growth factor. Nature , 1990 , 347 :83-86.
57.Moy F J , Seddon A P , Bohlen P , et al . High -resolution solution structure of basic fibroblast growth factor determined by multidimensional heteronuclear magnetic resonance spedtroscopy [J ] . Biochemistry , 1996, 35(42) :13552 -13561.
58.Aviezer D, Iozzo RV , Noonan DM , et al . Suppression of autocrine and paracrine functions of basic fibroblast growth factor by stable expression of perlecan antisense cDNA[J ] . Mol Cell Biol, 1997, 17(4) :1938-1946.
59. Jayson GC , Gallagher JT . Heparin oligo saccharides : inhibitors of the biological activity of bFGF on Caco-2 cells[ J ] . Br J Cancer, 1997 , 75 (1) :9-16.
60. Wagner M , Lopez ME , Cahn M , et al . Supression of fibroblast growth factor receptor signaling inhabits pancreatic cancer growth in vitro and in vivo[J] . Gastroenterology , 1998 ,114(4) :798-807 .
2.Trowell O A. Growth of tissues in vitro. VI. The effects of some tissue extracts on the growth of periosteal fibroblasts[J]. J Exp Biol, 1939,16:60-70.
3. Abraham JA,Whang JL.Tumolo A,et al Human basic fibroblast growth factor nucleotide sequence and genomic organization,EMBO J,1986,5;2523-2528
4. Muthukrishnan L,Warder E,Mcneil PL.Basic fibroblast growth factor is efficiently released from a eytolsolie storage site through plasma membrane disruption of endothelial cells.J Cell Physial ,1991,148:1-7.
5. Kurokawa T, Sasada R, Iwane M,et al . Cloning and expression of cDNA encoding human basic fibroblast growth factor . FEBSL ett, 1987, 213: 189 -194
6. Takayama H,Ray J,Raymon HK,et al. Basic fibroblast growth factor increase dopaminergic graft survival and function in a rat model of Parkinson's disease Nat Med,1995,1:53-58
7. Fawcett JW, Barker RA, Dunnett SB. Dopaminergic neuronal survival and the effects of bFGF in explant, three dimensional and monolayer cultures of embryonic rat ventral mesencephalon「J」.Exp Brain Res, 1995, 106(2):275-282
8.Abraham J A ,Mergia A Whang . Nucleotide sequence of a bovine clone encoding the angiogenic protein[J] . Basic Fibroblast Growth Factor Science , 1986 ,233 :545 -546.
9.Gospodarowicz D. Localisation of a fibroblast growth factor and its effect alone and with hydrocortisone on 3T3 cell growth [J]. Nature, 1974,249: 123-127.
10.Incue K,Sakai I,Hattori M.The cell-adhesive effect of basic fibroblast growth factor on piturtary cells in vitro. J Endocr,1991;130:381.
11. Battler A,Scheinowitz M,BorA.et al.Intoracoronary injection of bFGF enhances angiogenesis in infracted swine myocardium,J Coll Cardio (Am),1993:22(7):2001
12. Harada K,Grossman W,Friedman M,et al.bFGF improves myccardial function in chronically ischemic poreine hearts ,J Clin Invest,1994,94:623.
13. Gospodarowiez D,Ferrara N,Schwegerer L,et al.Structural characterization and Biological function of fibroblast growth factor .Endocrine Rev,1987;8(2):95-100.
14. Eriksson AE ,Cousens LS ,Weaver LH , et al . Three dimensional structure of human basic fibroblast growth factor [J]. Proc Natl Acad Sci USA,1991, 88 :34-41
15. Gospodarowicz D , Cheng J , Lui GM , et al . Isolation of brain fibroblast growth factor by heparinsepharose affinity chromat ography :identity with pituitary fibroblast growth factor [ J ] . Proc Natl Acad Sci USA, 1984, 81: 6963 -6967.
16. Kiefer MC , Stephans JC , Crawford K , et al . Ligand affinity cloning and structure of a cell surface heparan sulfate proteoglycan that binds basic fibroblast growth factor [J ] . Proc Natl Acad Sci USA 1990 ,87 :69-85
17. Hoffman R S. The growth -activating effect of extracs of adult and embryonic tissues of the rat on fibroblast coloies in culture[ J ] . Growth , 1940, 4:361 -371.
18. Luo Y, Lu W, Mohamedali KA , et al.The glycine box : a determine inant of specificity for fibroblast growth factor [J].Biochemistry, 1998,37:16-25.
19. Paulo M. et al. New observations on the intracellular localization and release of bFGF. J Cell Biol 1991,47:201-207.
20.Sommer A , Brewer M T, Thompson R C , et al. A form of human basic fibroblast growth factor with an extended amino terminus[J]. Biochem Biophys Res Commun, 1987 , 144 (2) : 543-550.
21. Heath W F , Cantrell A S , Mayne N G , et al . Mutation in the heparin domains of human basic fibroblast growth factor alter its biological activity[J ] .Biochemistry , 1991 , 30(22):5608-5615.
22.Estape D, Heuvel J , Rinas U. Susceptibility towards intramolecular disulphide-bond formation affects conformational stability and folding of human basic fibroblast growth factor[J ]. BiochemJ , 1998 , 335(Pt2) : 343 -349.
23. 向军俭 陈彤, 邹海燕等. 重组碱性成纤维细胞生长因子生物活性检测方法的研究[J] . 暨南大学学报(自然科学版),1993,14(1):186-190.
24 Cuevas P,Carceller F,Munoz-Willery I,et al .Intravenous fibroblast growth factor penetrates the blood-brain barrier and protects hippocampal neurons against ischemia-reperfersion injury.Surg Neurol,1998,49(1):77.
25. Zawada WM, Kirschman DL,Cchen JJ,et al.Growth factors rescue embryonic dopamine neurons from programmed cell death.Exp Neurol, 1996,140(1):60
26. Muthukrishnan L ,Warder E , Mcneil PL . Basic fibroblast growth factor is efficiently released from a cytolsolic storage site through plasmamembrane disruption of endothelial cells[J ] . J Cell Physiol ,1991 ,148 :11-20.
27.Zhan X , Hu X , Friesel R , et al . Long term growth factor exposure and differential tyrosine phosphorylation are required for DNA synthesis in B-ALBPc 3T3 cells[J ] . J Biol Chem,1993 ,268 :9611-9625.
28. Inoue K ,Sakai T , Hattori M . The cell adhesive effect of basic fibroblast growth factor on pituitary cells in vitro[J ] . Endocr J ,1991, 130 :381-387.
29.Framcki A,Uciechowski P,Floege J,et al .Autocrine growth regulation of human glomerular mesangial cells is primarily mediated by basic fibroblast growth factor .Am J Pathol,1995,147(5):1372
30.Sliutz G,Tempfer C,Obermain A,et al.Serum evaluation of basic FGF in breast cancer patients .Anti-cancer Res,1995;15(68):2675.
31. Tsuboi R,Rifkin DB. Recombinant basic fibroblast growth facror simulates wound healing in healing impaired mice [ J ] . J Exp Med , 1990 , 172 :245-251.
32. TStenberg BD , Phillips LG,Hokanson JA , et al . Effect of bFGF on the inhibition of contraction carsed by basteria [J] . J Surg Res, 1991 ,50 :47-52.
33.Aspenberg P ,Thorngren KG,Lohmander LS. Dose-dependent stimulation of bone induction by basic fibroblast growth factor in rats[J ] .Asta Orthop Scand ,1991 ,62:481-486.
34. Koshinage M , Anon HR , Whittemore SR . Altered acidic and basic fibroblast growth factor expression following spinal cord injury [ J ] . Exp Neurol ,1993 ,120 :32-38.
35. Unger E , Banai S ,Shou M, et al . Basic fibroblast growth factor enhances myocardial collateral flow in a canine model [J ] . AmJ Physiol ,1994 ,266: (4pt 2) :H1588-H1595.
36. Watanabe E,Smith DM,Sun J, et al. Effect of basic fibroblast growth factor on angiogenesis in the infarcted porcine heart [J]. Basic ResCaradiol, 1998, 93(1) :30-37.
37. 齐华阁,李兰更,刘建国,等 碱性成纤维细胞因子对急性前壁心肌梗死的影响[J ] . 中国危重病急救医学,1998 ,10 (1) :29-33.
38. 王楠,徐永华 佛波脂PMA促进碱性成纤维细胞生成长因子( bFGF )释放及机理的研究[J]1 实验生物学报,1998 ,31 :41-46.
39. 刘飞鹏,吴雄化,云涌等。PCR-四步两段扩增法分离人碱性成纤维细胞生长因子(hbfgf)基因「J」。生物工程进展,1997,17(1): 52-55.
40.Delrieu I . The high molecular weight isoforms of basic fibroblast growth factor (FGF2) : an insight into an intracrinemechanism「J」 FEBS Lett . 2000 ,468 (1) :6-10.
41. Koolwijk P, van Erck MG, de Vree WJ, et al . Cooperative effect of TNF-alpha , bFGF , and VEGF on the formation of tubular structures of human microvascular endothelial cells in a fibrin matrix . Role of urokinase acti-vity[J] . J CellBiol ,1996,132(6):1177-1188.
42. Seghezzi G, Patel S,Ren CJ,et al.Fibroblast growth factor2 (FGF2) induces vascular endothelial growth factor ( VEGF ) expression in the endothelial cells of forming capillaries : an autocrine mechanism contributing to angiogenesis[J].J CellBiol,1998,141(7):1659-1673.
43. Miyake H , Hara I ,Gohji K , et al . Expression of basic fibroblast growth factor is associated with resistance to cisplatin in a human blander cancer line[J ] . Cancer Lett ,1998 ,123 (2) :121-126.
44. Noda M, Hattori T, Kimura T, et al.Expression of fibroblast growth factor 2 mRNA in early and advanced gastric cancer [J]. Acta Oncol,1997 Ogata
45. Neuner Jehle M, Berghe LV, Bonnel S,et al. Ocular cell transferction with the human basic fibroblast growth factor gene delays photo receptor cell degeneration in RCS rats.Hum Gene Ther, 2000, 11 :1875-1890.
46. Akimoto M , Miyatake S , Kogishi J , et al . Adenovirally expressed basic fibroblast growth factor rescues photoreceptor cells in RCS rats . Invest Ophthalmol Vis Sci , 1999 ,40 : 273-279.
47. Lau D , McGee LH , Zhou S , et al . Retinal degeneration is slowed in transgenic rats by AAV2 mediated delivery of FGF2 . Invest Ophthalmol Vis Sci , 2000 , 41 :3622-3633.
48.Gospodarow ic1 D . F ibroblast growth factor : chem ical structure and biologic function. Clin Orthop Rel Res, 1990;257 (Aug): 231-248.
49. Martin P , Hopk insonWJ , McCluskey J . Growth factors and cutaneous wound repair. prog Grow th Factor R es, 1992;4 (1): 25-44.
50. Bennett N T,Schultz GS. Growth factors and wound heailing: Part Role in normal and chronic wound healing. Am J Surg ,1993;166 (1) : 74-81.
51.MarksM G,Doillon C,Silver FH.Effects of fibroblasts and basic fibroblast growth factor on facilitation of dermal wound healing by type collagen matrices. J Biom ed Mater Res, 1991;25 (5):683-696.
52. LawrenceW T, Diegelmann RF. Growth factors in wound healing.C lin D erm atol, 1994;12 (1) : 157-169.
53. HickeyMJ ,Wilson Y, Hurley JV et al . Mode of vascularization of control and basic fibroblast growth factor stimulated prefabricatedskin flaps. Plast Reconstr S u rg , 1998;101 (5) : 1296-1304
54. Ueno N, A Baird , Esch F, et al . Isolation of an am ino terminal extended form of basic fibroblast growth factor . Biochem Biophys Res Commun , 1986, 138:580-586.
55. 赵爱平, 徐林, 钱林法等. 人碱性成纤维细胞生长因子基因在大肠杆菌 中的克隆与表达. 中国生化药物杂志,1996, 17 (3)∶93-96.
56. Faktorovich EG , Steinberg RH , Yasumura D , et al . Photoreceptor Degeneration in inherited retinal dystrophy delayed by basic fibroblast growth factor. Nature , 1990 , 347 :83-86.
57.Moy F J , Seddon A P , Bohlen P , et al . High -resolution solution structure of basic fibroblast growth factor determined by multidimensional heteronuclear magnetic resonance spedtroscopy [J ] . Biochemistry , 1996, 35(42) :13552 -13561.
58.Aviezer D, Iozzo RV , Noonan DM , et al . Suppression of autocrine and paracrine functions of basic fibroblast growth factor by stable expression of perlecan antisense cDNA[J ] . Mol Cell Biol, 1997, 17(4) :1938-1946.
59. Jayson GC , Gallagher JT . Heparin oligo saccharides : inhibitors of the biological activity of bFGF on Caco-2 cells[ J ] . Br J Cancer, 1997 , 75 (1) :9-16.
60. Wagner M , Lopez ME , Cahn M , et al . Supression of fibroblast growth factor receptor signaling inhabits pancreatic cancer growth in vitro and in vivo[J] . Gastroenterology , 1998 ,114(4) :798-807 .