重组人成纤维细胞生长因子8a生产工艺及药效学初探
|
摘要
成纤维细胞生长因子8a (fibroblast growth factor 8,FGF8)作为成纤维细胞生长因子家族(fibroblast growth factor family,FGFs家族)的第8个成员的a亚型,它具有一般家族成员的作用:促进软骨形成、活化上皮细胞、促进伤口愈合等,同该家族的其他成员相比,FGF8a有望成为既刺激正常细胞分化,又无诱导癌变之虞的新一代细胞生长因子。FGF8a用药的有效性和安全性成为全球研究的焦点。
本研究首先在摇瓶实验中摸索发酵基本条件,从培养基pH值、诱导时机、IPTG诱导终浓度和诱导温度四个影响因素做“四因素四水平”正交实验设计,找出最适发酵工艺参数。然后根据摇瓶实验结果和以往经验控制发酵参数,进行发酵罐发酵。发酵结束后离心收菌,进行SDS-PAGE分析目的蛋白含量。纯化过程中对前期的方法进行优化,简化蛋白复性操作。然后采用MTT法检测rhFGF8a蛋白活性,计算出蛋白生物学活性。在药效学方面,首先进行鸡胚尿囊膜(CAM)实验,观察rhFGF8a在诱发CAM血管生成的能力;然后研究rhFGF8a与间隙连接蛋白抑癌基因Cx43的关系,通过western blotting印迹分析观察rhFGF8a能否独立上调Hela细胞Cx43蛋白的表达。
研究表明,摇瓶正交实验得出最优参数组合为培养基pH值7.0,IPTG诱导时机OD600值为0.5, IPTG终浓度为0.5mM、诱导温度30℃。在发酵过程中,参数控制为:(1)空气流量初始设定为10L/min,诱导后升为15L/min,溶氧控制在40%;(2)温度初始为37℃,诱导时降到30℃;(3)pH值设定为7.0;(4)搅拌速度:设定搅拌速度下限为105rpm,上限为500 rpm;(5)补料:在发酵培养基中培养3h后开始匀速补料250ml/h。发酵结束收获菌体20g/L,经SDS-PAGE电泳分析目的蛋白rhFGF8a表达量占菌体总蛋白的30%左右。优化蛋白纯化条件,蛋白复性所需的氧化型谷胱甘肽与还原型谷胱甘肽用量减少至原来的1/10,尿素浓度从4M梯度开始,降低了成本,rhFGF8a纯化过程蛋白收率为57%。MTT法检测结果表明发酵纯化后收集的蛋白对成纤维3T3细胞有一定的促增长作用,生物学活性为22000IU/mg。药效学方面,CAM实验表明,rhFGF8a较bFGF在诱发CAM血管生成能力上相对较弱;Western blotting印迹表明rhFGF8a能独立上调Hela细胞抑癌基因Cx43的表达。
Fibroblast Growth Factor8 isoform a(FGF8a),a member of Fibroblast Growth Factor family, has a general role of other family members: promoting cartilage formation, activating the epithelial cells,promoting wound healing and so on. Comparing with other family members, FGF8a is expected to become a new cell growth factor, which not only stimulates normal cell differentiation, but also evades the risk of cancer. The efficiency and safety of FGF8a will be focused amomg the research.
In this study, "four factors four level" orthogonal experiment was designed to explore the fermentation conditions, in which the culture medium pH, induction time, final concentration of IPTG and induct temperature would be considered to identify the optimal fermentation parameters. Then fermentation parameters are controlled according to the results of shake flask experiments. Protein content is analyzed by SDS-PAGE. To simplify the process of protein refolding, pre-experimental purification methods are optimized. Then rhFGF8a protein activity is calculated through MTT method. Chickembryo chorioallantoic membrane (CAM) experiment is used to observing the angiogenesis in CAM capabilities in FGF8a’s pharmacodynamics. Then the relationship between rhFGF8a and gap junction protein Cx43 (tumor suppressor gene) was researched. Whether rhFGF8a up-regulates the expression of Cx43 gene is analyzed by the western blotting method.
The results showed that, the optimal parameters are medium pH value is 7.0, OD600 value is 0.5, IPTG eventually concentration is 0.5 mM, inducing temperature is 30 degrees by orthogonal experimen. In the fermentation process parameters are controlled: (1) the air flow is set 10L/min first, 15L/min is set after induced.Dissolved oxygen is controlled at 40%. (2) Temperature is controlled at 37 degrees first, 30 degrees is set after induced. (3) pH value is set at 7.0.(4) Stirring speed is controlled between 105rpm and 500rpm. (5) After 3 hours the fermentation cultivation are joined at 250ml/h speed.20 grams wet bacteria per liter fermentation solution could be obtained, and the yield rate of expressed products was about 30% of total host proteins which is identified through SDS-PAGE. The dosage of oxidized glutathione and reduced glutathione are decreased to 0.1 times after optimizing purification conditions, and the yield of purified rhFGF8a is 57%. By MTT method, the purified rhFGF8a plays a role in promoting the growth of fibroblast 3T3 cells, and its biological activity is 22000IU/mg. CAM experiments show that the rhFGF8a has weak capacity on angiogenesis. And compared with bFGF and EGF, the rhFGF8a can up-regulate expression of Cx43 gene through western blotting.
本研究首先在摇瓶实验中摸索发酵基本条件,从培养基pH值、诱导时机、IPTG诱导终浓度和诱导温度四个影响因素做“四因素四水平”正交实验设计,找出最适发酵工艺参数。然后根据摇瓶实验结果和以往经验控制发酵参数,进行发酵罐发酵。发酵结束后离心收菌,进行SDS-PAGE分析目的蛋白含量。纯化过程中对前期的方法进行优化,简化蛋白复性操作。然后采用MTT法检测rhFGF8a蛋白活性,计算出蛋白生物学活性。在药效学方面,首先进行鸡胚尿囊膜(CAM)实验,观察rhFGF8a在诱发CAM血管生成的能力;然后研究rhFGF8a与间隙连接蛋白抑癌基因Cx43的关系,通过western blotting印迹分析观察rhFGF8a能否独立上调Hela细胞Cx43蛋白的表达。
研究表明,摇瓶正交实验得出最优参数组合为培养基pH值7.0,IPTG诱导时机OD600值为0.5, IPTG终浓度为0.5mM、诱导温度30℃。在发酵过程中,参数控制为:(1)空气流量初始设定为10L/min,诱导后升为15L/min,溶氧控制在40%;(2)温度初始为37℃,诱导时降到30℃;(3)pH值设定为7.0;(4)搅拌速度:设定搅拌速度下限为105rpm,上限为500 rpm;(5)补料:在发酵培养基中培养3h后开始匀速补料250ml/h。发酵结束收获菌体20g/L,经SDS-PAGE电泳分析目的蛋白rhFGF8a表达量占菌体总蛋白的30%左右。优化蛋白纯化条件,蛋白复性所需的氧化型谷胱甘肽与还原型谷胱甘肽用量减少至原来的1/10,尿素浓度从4M梯度开始,降低了成本,rhFGF8a纯化过程蛋白收率为57%。MTT法检测结果表明发酵纯化后收集的蛋白对成纤维3T3细胞有一定的促增长作用,生物学活性为22000IU/mg。药效学方面,CAM实验表明,rhFGF8a较bFGF在诱发CAM血管生成能力上相对较弱;Western blotting印迹表明rhFGF8a能独立上调Hela细胞抑癌基因Cx43的表达。
Fibroblast Growth Factor8 isoform a(FGF8a),a member of Fibroblast Growth Factor family, has a general role of other family members: promoting cartilage formation, activating the epithelial cells,promoting wound healing and so on. Comparing with other family members, FGF8a is expected to become a new cell growth factor, which not only stimulates normal cell differentiation, but also evades the risk of cancer. The efficiency and safety of FGF8a will be focused amomg the research.
In this study, "four factors four level" orthogonal experiment was designed to explore the fermentation conditions, in which the culture medium pH, induction time, final concentration of IPTG and induct temperature would be considered to identify the optimal fermentation parameters. Then fermentation parameters are controlled according to the results of shake flask experiments. Protein content is analyzed by SDS-PAGE. To simplify the process of protein refolding, pre-experimental purification methods are optimized. Then rhFGF8a protein activity is calculated through MTT method. Chickembryo chorioallantoic membrane (CAM) experiment is used to observing the angiogenesis in CAM capabilities in FGF8a’s pharmacodynamics. Then the relationship between rhFGF8a and gap junction protein Cx43 (tumor suppressor gene) was researched. Whether rhFGF8a up-regulates the expression of Cx43 gene is analyzed by the western blotting method.
The results showed that, the optimal parameters are medium pH value is 7.0, OD600 value is 0.5, IPTG eventually concentration is 0.5 mM, inducing temperature is 30 degrees by orthogonal experimen. In the fermentation process parameters are controlled: (1) the air flow is set 10L/min first, 15L/min is set after induced.Dissolved oxygen is controlled at 40%. (2) Temperature is controlled at 37 degrees first, 30 degrees is set after induced. (3) pH value is set at 7.0.(4) Stirring speed is controlled between 105rpm and 500rpm. (5) After 3 hours the fermentation cultivation are joined at 250ml/h speed.20 grams wet bacteria per liter fermentation solution could be obtained, and the yield rate of expressed products was about 30% of total host proteins which is identified through SDS-PAGE. The dosage of oxidized glutathione and reduced glutathione are decreased to 0.1 times after optimizing purification conditions, and the yield of purified rhFGF8a is 57%. By MTT method, the purified rhFGF8a plays a role in promoting the growth of fibroblast 3T3 cells, and its biological activity is 22000IU/mg. CAM experiments show that the rhFGF8a has weak capacity on angiogenesis. And compared with bFGF and EGF, the rhFGF8a can up-regulate expression of Cx43 gene through western blotting.
引文
[1]纪华,李泽桂. FGF与中枢神经系统[J].解剖科学进展,2004,10:185-188.
[2] Ornitz DM, Itoh N. fibroblast growth factors[J]. gene boilogy, 2001, 2(3): 3005.1-3005.12.
[3] Mateeki J, Wesche J. Translocation of FGE-1 and FGF-2 across vesicular membranes occurs during G1-phase by a common mechanism[J]. Molecular Biology of the Cell, 2004, 15:801-814.
[4] Itoh N. FGFs as multifunctional signaling molecules: diversity of structure and function [J]. seikagaku, 2001,73(7): 525-535.
[5]付小兵,孙同柱,孙晓庆等. aFGF和bFGF在大鼠不同发育阶段肠道定位和表达特征的比较研究[J].中国危重病急救医学, 2001, 13(7):407-409.
[6] Miller D L, Ortega S, Bashayan O, et al. Compensation by fibroblast growth factor 1 (FGF1) does not account for the mild phenotypic defects observed in FGF2 null mice[J]. Mol Cell Biol,2000, 20(6): 2260– 2268.
[7] Li JJ, Friedman-Kien AE, Cockerell C,et al. Evaluation of the tumor igenic and angiogenic potential of human fibroblast growth factor FGF3 in nude mice[J]. Cancer ResClin Oncol , 1998,124(5):259-264.
[8] Galdemard C, Yamagata H, Brison O,et al. Regulation of FGF-3 gene expression in tumorigenic and nontumorigenic clones of a human colon carcinoma cell line[J]. Biol Chem, 2000, 275(23):17364-17373.
[9] Uematsu F, Kan M, Wang F,et al. Ligand binding properties of binary complexes of heparin andimmunoglobulin-like modules of FGF receptor [J]. Biochem Biophys Res Commun, 2000,272(3): 830- 836.
[10] Steiner MS. Review of peptide growth factors in benign prostatic hyperplasia and urological maliganancy[J]. Urol, 1995, 1085-1096.
[11] Hu MC, Wang YP, Qiu WR,et al. Hematopoietic progenitor kinase-1(HPK1)stress response singnaling pathway activates IkapoaB kianses(IKK-alpha/beta)and IKK-beta is a development ally regulated proteinkinase[J].Oncogene,1999,18:2635-2642.
[12] Firth JD, Putnins EE. Keratinocyte growth factor 1 inhibits wound edge epithelial cell apoptos is in vitro[J]. Invest Dermatol 2004, 122(1): 222-231.
[13] Jeschke MG, Richter G, Hemdon DN, et al. Non-viral liposomal keratinocyte growth factor (KGF) cDNA gene transfer improves dermal and epidermal regeneration through stimulation of epithelial and mesenchymal factors[J]. Gen Ther , 2002,9(16): 1065-1074.
[14] Werner S, Peters KG, Longaker MT, et al. Large induction ofkeratinocyte growth factor expression in the dermis during wound healing[J]. Proc Natl Acad Sci USA, 1992,89(15): 6896-6900.
[15] Baskin LS, Sutherland RS, Thomson AA, et al. Growth factors in bladder wound healing[J]. Urol, 1997, 157(6): 2388-2395.
[16] Brauchle M, Madlener M, Wagner AD, et al. Keratinocyte growth factor is highly over expressed ininflammatory bowel disease[J]. Am J Pathol, 1996,149(2): 521-529.
[17] Johnson WF, Dipalma CR, Ziegler TR, et al. Keratinocyte growth factor enhances early gut adaptation in a rat model of short bowel syndrome[J].Vet Surg, 2000, 29(1): 17-27.
[18] Carrington LM, Boulton M. Hepatocyte growth factor and keratinocyte growth factor regulation of epithelial and stromal corneal wound healing[J]. Cataract Refract Surg, 2005, 31(2): 412-423.
[19] Xu JF, Qu JM, He LX, et al. Impaired upregulation of keratinocyte growth factor in injured lungs induced by Pseudomonas aeruginosa in immunosuppres sed rats[J] . Chin Med J (Engl),
[20] Plotnikov AN, Eliseenkova AV, Ibrahimi OA, et al.Crystal structure of fibroblast growth factor 9 reveals regions implicated in dimerization an autoinhibition[J]. Biol Chem, 2001, 276:4322-4329.
[21] Colvin JS, Green RP, Schmanl J, et al. Male-to-female sex reversal in mice lacking Fibroblast growth factor 9[J].Cell, 2001, 104: 875-880.
[22] Tsai SJ, Wu MH, Chen HM,et al.Fibroblast growth factor 9 is an endometrial stromal growth factor[J].Endocrinology, 2002,143:2715-2721. 2006, 119(17): 421-429.
[23] Aaronson SA, Bottaro DP, Miki Tet ,et al. Keratinocyte growth factor A fibroblast growth factor family member with unusual target cell specificity[J].AnnN Y Acad Sci, 1991, 638(1): 62-67.
[24] Tao H, YoshimotoY, YoshiokaHet,et al.FGF10 isamesenchymally derived stimulator for epidermal development in the chick embryonic skin[J]. Mech Dev, 2002, 116(1-2): 39.
[25] Jimenez PA, Rampy MA. Keatinocyte growth factor-2 accelerates wound healing in incisional wounds[J]. Surg Res, 1999, 81(2): 238.
[26] Xia YP, Zhao Y, Marcus Jet ,et al. Effects of keratinocyte growth factor-2(KGF-2) on wound healing in an ischemia-impaired rabbit ear model and onscar formations[J]. Pathol, 1999, 188(4): 431.
[27] Mann A, Breuhahn K, Schirmacher Pet al. Keratinocyte-derived granulocy temacrophaolony stimulating factor accelerates wound healing: Stimulation of keratinocyte proliferation, granulation tissue formation, and vascularization[J]. Invest Dermatol, 2001, 117(6):1382
[28] Alderson R, Gohari Fritsh S, Olsen H, et al.In vitro and in vivo effects of repifermin (keratinocyte growth factor 2, KGF2)on human carcinoma cells[J]. Cancer Chemother Parmacol, 2002, 50: 202-212.
[29] Coulier F, Pontarotti P, Rollbin R, et al.Of worms and men: an evolutionary perspective on the fibroblast growth factor (FGF) and FGF receptor families[J]. Mol Evol, 1997, 44(1):43-56.
[30]孙蒙,于永利.成纤维细胞生长因子13基因在酵母细胞中的表达[J].分子与细胞免疫学,2006,8(16):401-403.
[31] Mcwhirte JR, Goulding M, Weiner JA,et al.A novel fibroblast growth factor gene expressed in the developingnervous system is a downstream target of the chimeric homeodomain oncoproteinE2A-Pbx1[J].Devolopment,1997,24:3221-3232.
[32] Munekazu Komada, Hirotomo Saitsu, Kohei Shiota,et al.Expression of Fgf15 is regulated by bothactivator and repressor forms of Gli2 in vitro[J], Biochemical and Biophysical Research Communications, 2008, 2(369):350-356.
[33] Yuhei Hotta , Sayaka Saki , Morichika Konishi,et al. Fgf16 is required for cardiomyocyte proliferation in the mouse embryonic heart[J].Developmental Dynamics, 2008,237(10):2947-54
[34] Jeremy A. Cholfin , John L.R. Rubenstein, Frontal cortex subdivision patterning is coordinately regulated by Fgf8, Fgf17, and Emx2 [J].Interscience,2008,2(509): 144- 155.
[35] Ohbayashi N, Shibayama M, Kurotaki Y.FgF18 is required for normal cell proliferation and differentiation duringosteogenesis and chondrogenesis[J].Genes Dev,2002,16(7):870
[36] Haque T, Nakada S, Hamdy RC. A review of FGF18: Its expression, signaling pathways and possible functions during embryogenesis and post-natal development[J]. Histol Histopathol, 2007 ,22(1):97-105.
[37] Benjamin C. Lin, Manping Wang, Craig Blackmore,et al. Liver-specific Activities of FGF19 Require Klotho beta[J]. Biological Chemistry,2007,37(282): 27277-27284.
[38] Gao X, Scott WK, Wang G,et al.Gene-Gene Interaction Between FGF20 and MAOB in Parkinson Disease[J]. Annals of Human Genetics, 2008,2(72) :157-162.
[39] Wider C, Dachsel JC, Soto AI,et al.FGF20 and Parkinson's disease: No evidence of association or pathogenicity via alphasynuclein expression[J]. Movement disorders,2009, 24(3):455-459.
[40] Moyers JS, Shyanova TL, Mehrbod F,etal. Molecular determinants of FGF 21 activity energy and cross-talk with PPAR gamma signaling [J]. cellular physiology,2007,210(1):l-6.
[41] Huang X , Yu C , Jin CForced expression of hepatocyte-specific fibroblast growth factor 21 delaysinitiation of chemically induced hepatocarcinogenesis[J]. Molecular Carcinogenesis, 2006,45(12):934-942.
[43] Linhoff, David M, Joshua R, et al. FGF22 and Its Close Relatives Are Presynaptic Organizing Molecules in the Mammalian Brain[J]. Cell, 2004,2(118): 257-270.
[44] Jonnaon KB. Zahraadnik R, Iatsson T, et al. Fibroblast growth factor 23 in oncogenic osteomalacia and X-linked hypophosphatemia[J]. N Engl J Med,2003,348:1656-1663.
[45]郭晓强.成纤维细胞生长因子23和血磷代谢[J].生命的化学,2007,(2)27:149-151.
[46] Mattei M. G., deLapeyriere O, Bresnick J. ,et al.Mouse Fgf7 (fibroblast growth factor 7) and Fgf8 (fibroblast growth factor 8) genes map to chromosomes 2 and 19 respectively[J]. Mammalian Genome,1995, 6: 196-197.
[47] Yoshiura K., Leysens N. J, Chang J ,et al. Genomic structure, sequence, and mapping of human FGF8 with no evidence for its role in craniosynostosis/limb defect syndromes[J]. Am. J. Med. Genet,1997,72: 354-362.
[48] Tanaka A, Miyamoto K, Minamino Netal. Cloning and charaxterization of an androgen-dependent growth of mouse mummary carcinoma cells[J]. Proc Natl Acad Sci USA, 1992,77: 89:8928.
[49] Sangwoo Shim, Narina Bae, Sang Yoon Park, et al. Isolation of Xenopus FGF-8b and Comparison withFGF-8a [J]. Molecules and Cells, 2005, 19(3): 310-317.
[50] Akira Tanaka, Tomoko Kamiakito, Y Hakamata, et al. Extensive neuronal localization and neurotrophic function of fibroblast growth factor 8 in the nervous system [J]. Brain Research, 2001, 912: 105-115.
[51] B Kwabi-Addo, M Ozen and M Ittmann. The role of fibroblast growth factors and their receptors in prostate cancer[J]. Endocrine-Related Cancer, 2004, 11: 709-724.
[52] Sonia Garel, Kelly J. Huffman and John L. R. Rubenstein. Molecular regionalization of the neocortex is disrupted in FGF8 hypomorphic mutants [J]. Development, 2003, 130: 1903-1914.
[53] Fukuchi-Shimogori T, Grove E A. Neocortex patterning by the secreted signaling molecule FGF8 [J]. Science, 2001, 294: 1071-1074.
[54] Steffen Scholpp, Claudia Lohs and Michael Brand. Engrailed and FGF8 act synergistically to maintain the boundary between diencephalon and mesencephalon [J]. Development 2003, 130: 4881-4893.
[55] Tatsuya Sato, Isato Araki and Harukazu Nakamura. Inductive signal and tissue responsiveness defining the tectum and the cerebellum[J]. Development, 2001, 128: 2461-2469.
[56] EL Wiellette and H Sive. Early requirement for FGF8 function during hindbrain pattern formation in zebrafish [J]. Dev Dyn, 2004, 229(2): 393-9.
[57] Lewandoski M., Sun X., Martin G. R. : FGF8 signalling from the AER is essential for normal limb developmen[J]t. Nature Genet. 2000,26: 460-463.
[58] Sabine Fischer, Bruce W. Draper and Carl J. Neumann:The zebrafish FGF24 mutant identifies an additional level of FGF signaling involved in vertebrate forelimb initiation[J]. Development 2003, 130:3515-3524.
[59] Sun X., Mariani F. V., Martin G. R. Functions of FGF signalling from the apical ectodermal ridge in limb development [J]. Nature, 2002, 418: 501-508.
[60]王春梅,姜涛,周春燕.诱导心脏发生的早期信号通路[J].中国生物化学与分子生物学报, 2004, 10, 20(5): 572～577.
[61] Jung J, Zheng M., Goldfarb M., Zaret K. S.. Initiation of mammalian liver development from endoderm by fibroblast growth factors [J]. Science, 1999, 284: 1998-2003.
[62] Hitoo N and Hideki H., Mesencephalic. Neural stem cells develop to dopaminergic neurons more strongly in dopamine-depleted striatum than in intact striatum [J]. Exp Neurol, 2000, 28: 31-40.
[63] Ye W, Shimamura K, Rubenstein JL, et al. FGF and Shh signals control dopaminergic and serotonergic cell fate in the anterior neural plate [J]. Cell, 1998, 93: 755–766.
[64] Creuzet Sophie, Bernadette Schuler, Gérard Couly and Nicole M. Le Douarin:Reciprocal relationships between Fgf8 and neural crest cells in facial and forebrain development[J]. PNAS 2004, 101 (14):4843-4847.
[65] Dong Liu, Hsin Chu, Lisa Maves, et al:Fgf3 and Fgf8 dependent and independent transcription factorsare required for otic placode specification[J]. Development 2003, 130:2213-2224.
[66] Lèger Sophie, Michael Brand:Fgf8 and Fgf3 are required for zebrafish ear placode induction, maintenance and inner ear patterning[J]. Mechanisms of Development 2002,119:91–108.
[67] Jaskoll Tina, Dan Witcher, Leo Toreno, et al. FGF8 dose-dependent regulation of embryonic bmandibular salivary gland morphogenesis[J]. Developmental Biology 2004,268: 457– 469
[68] Emily A. Morgan, Susan B. Nguyen, Virginia Scott and H. Scott Stadler. Loss of Bmp7 and Fgf8 signaling in Hoxa13-mutant mice causes hypospadia[J]. Development 2003,130:3095-3109
[69] Jean M. Hébert, Mary Lin, Juha Partanen, et al:FGF signaling through FGFR1 is required for olfactory bulb morphogenesis[J]. Development 2003,130:1101-1111
[70] Radwan Abu-Issa, Graham Smyth, Ida Smoak, et al:Fgf8 is required for pharyngeal arch and cardiovascular development in the mouse[J]. Development 2002,129:4613-4625
[71] R Huang, D Stolte, H Kurz, et al:Ventral axial organs regulate expression of myotomal Fgf-8 that influences rib development[J]. Dev Biol, March 1, 2003, 255(1): 30-47.
[72] Dubrulle J., McGrew M. J., Pourquie O. : FGF signaling controls somite boundary position and regulates segmentation clock control of spatiotemporal Hox gene activation[J]. Cell 106: 219-232, 2001.
[73] Streit A., Berliner A. J., Papanayotou C. , et al : Initiation of neural induction by FGF signalling before gastrulation[J]. Nature 2000,406: 74-78.
[74] Payson R. A.,Wu J,Liu Y.,Chiu I.-M. : The human FGF-8 gene localizes on chromosome 10q24 and is subjected to induction by androgen in breast cancer cells[J]. Oncogene 1996,13: 47-53.
[75] Tatsuya Sato and Harukazu Nakamura:The Fgf8 signal causes cerebellar differentiation by activating the Ras-ERK signaling pathway[J]. Development 2004,131:4275-4285.
[76] Johanna K. Ruohola, Tiina P. Viitanen, Eeva M. Valve,et al:Enhanced Invasion and Tumor Growth of Fibroblast Growth Factor 8b-overexpressing MCF-7 Human Breast Cancer Cells[J]. Cancer Research 2001,61(5), 4229-4237.
[77] Revilla A, Bennett MV, Barrio LC, eta.l Moleculardeterminants of membrane potential dependence in vertebrate gap junction channels[J].Proc natlAcad SciUSA,2000, 97(26): 14760-14765.
[78] CalderMD, Caveney AN, Smith LC, et a.l Responsiveness of bovine cumulus oocyte complexes (COC) to porcine and recombinanthuman FSH, and the effect ofCOC quality on gonadotropin receptorand Cx43 marker gene mRNAs during maturation in vitro [ J].Reprod BiolEndocrinol,2003, 1(1): 14-19.
[79] Ai Z, FischerA, SprayDC, eta.l Wnt-1 regulation ofconnexin43 in cardiac- myocytes [J].American Society for Clinical Investigation,2000, 105(2): 161-171.
[80] KrutovskikhVA,TroyanovskySM, PiccoliC, etal. Differential effect of subcellular localization of communication impairing gap junction protein connexin43 on tumor cell growth in vivo[J]. Oncogen,2000, 19(4): 505-513.
[81] Su YA, BittnerML,Chen Y, et a.l Identification of tumor suppressor genes using human melanoma celllines UACC903, UACC903 ( +6), and SRS3 by comparison of expression profiles [ J].MolCarcinog,2000, 28(2): 119-127.
[82] Ilvesaro J, Tavi P, Tuukkanen J. Connexin-mimetic peptide Gap 27 decreases osteoclastic activity [ J].BMC Musculoskelet Disord,2001, 2(1): 1500-1513.
[83] Samoilova M, Li J. Gap Junctions in Epileptogenesis: Chicken orEgg[J]. Epilepsy Curr,2004, 4(2): 80-81.
[84] Contreras JE, SánchezHA, Eugenin EA, et a.lMetabolic inhibition induces opening of unapposed connexin 43 gap junction hemichannels and reduces gap junctional communication in cortical astrocytes in culture[J].ProcNatlAcad SciUSA,2002, 99(1): 495-500.
[85]冯纬樱.重组人成纤维细胞生长因子-8a的纯化研究[D].桂林:广西师范大学,2007:35-43.
[86]黄明,蒋寅.改造稀有密码子对rhFGF8a原核表达的影响[J].细胞与分子免疫学杂志, 2008.25(6):101-104.
[2] Ornitz DM, Itoh N. fibroblast growth factors[J]. gene boilogy, 2001, 2(3): 3005.1-3005.12.
[3] Mateeki J, Wesche J. Translocation of FGE-1 and FGF-2 across vesicular membranes occurs during G1-phase by a common mechanism[J]. Molecular Biology of the Cell, 2004, 15:801-814.
[4] Itoh N. FGFs as multifunctional signaling molecules: diversity of structure and function [J]. seikagaku, 2001,73(7): 525-535.
[5]付小兵,孙同柱,孙晓庆等. aFGF和bFGF在大鼠不同发育阶段肠道定位和表达特征的比较研究[J].中国危重病急救医学, 2001, 13(7):407-409.
[6] Miller D L, Ortega S, Bashayan O, et al. Compensation by fibroblast growth factor 1 (FGF1) does not account for the mild phenotypic defects observed in FGF2 null mice[J]. Mol Cell Biol,2000, 20(6): 2260– 2268.
[7] Li JJ, Friedman-Kien AE, Cockerell C,et al. Evaluation of the tumor igenic and angiogenic potential of human fibroblast growth factor FGF3 in nude mice[J]. Cancer ResClin Oncol , 1998,124(5):259-264.
[8] Galdemard C, Yamagata H, Brison O,et al. Regulation of FGF-3 gene expression in tumorigenic and nontumorigenic clones of a human colon carcinoma cell line[J]. Biol Chem, 2000, 275(23):17364-17373.
[9] Uematsu F, Kan M, Wang F,et al. Ligand binding properties of binary complexes of heparin andimmunoglobulin-like modules of FGF receptor [J]. Biochem Biophys Res Commun, 2000,272(3): 830- 836.
[10] Steiner MS. Review of peptide growth factors in benign prostatic hyperplasia and urological maliganancy[J]. Urol, 1995, 1085-1096.
[11] Hu MC, Wang YP, Qiu WR,et al. Hematopoietic progenitor kinase-1(HPK1)stress response singnaling pathway activates IkapoaB kianses(IKK-alpha/beta)and IKK-beta is a development ally regulated proteinkinase[J].Oncogene,1999,18:2635-2642.
[12] Firth JD, Putnins EE. Keratinocyte growth factor 1 inhibits wound edge epithelial cell apoptos is in vitro[J]. Invest Dermatol 2004, 122(1): 222-231.
[13] Jeschke MG, Richter G, Hemdon DN, et al. Non-viral liposomal keratinocyte growth factor (KGF) cDNA gene transfer improves dermal and epidermal regeneration through stimulation of epithelial and mesenchymal factors[J]. Gen Ther , 2002,9(16): 1065-1074.
[14] Werner S, Peters KG, Longaker MT, et al. Large induction ofkeratinocyte growth factor expression in the dermis during wound healing[J]. Proc Natl Acad Sci USA, 1992,89(15): 6896-6900.
[15] Baskin LS, Sutherland RS, Thomson AA, et al. Growth factors in bladder wound healing[J]. Urol, 1997, 157(6): 2388-2395.
[16] Brauchle M, Madlener M, Wagner AD, et al. Keratinocyte growth factor is highly over expressed ininflammatory bowel disease[J]. Am J Pathol, 1996,149(2): 521-529.
[17] Johnson WF, Dipalma CR, Ziegler TR, et al. Keratinocyte growth factor enhances early gut adaptation in a rat model of short bowel syndrome[J].Vet Surg, 2000, 29(1): 17-27.
[18] Carrington LM, Boulton M. Hepatocyte growth factor and keratinocyte growth factor regulation of epithelial and stromal corneal wound healing[J]. Cataract Refract Surg, 2005, 31(2): 412-423.
[19] Xu JF, Qu JM, He LX, et al. Impaired upregulation of keratinocyte growth factor in injured lungs induced by Pseudomonas aeruginosa in immunosuppres sed rats[J] . Chin Med J (Engl),
[20] Plotnikov AN, Eliseenkova AV, Ibrahimi OA, et al.Crystal structure of fibroblast growth factor 9 reveals regions implicated in dimerization an autoinhibition[J]. Biol Chem, 2001, 276:4322-4329.
[21] Colvin JS, Green RP, Schmanl J, et al. Male-to-female sex reversal in mice lacking Fibroblast growth factor 9[J].Cell, 2001, 104: 875-880.
[22] Tsai SJ, Wu MH, Chen HM,et al.Fibroblast growth factor 9 is an endometrial stromal growth factor[J].Endocrinology, 2002,143:2715-2721. 2006, 119(17): 421-429.
[23] Aaronson SA, Bottaro DP, Miki Tet ,et al. Keratinocyte growth factor A fibroblast growth factor family member with unusual target cell specificity[J].AnnN Y Acad Sci, 1991, 638(1): 62-67.
[24] Tao H, YoshimotoY, YoshiokaHet,et al.FGF10 isamesenchymally derived stimulator for epidermal development in the chick embryonic skin[J]. Mech Dev, 2002, 116(1-2): 39.
[25] Jimenez PA, Rampy MA. Keatinocyte growth factor-2 accelerates wound healing in incisional wounds[J]. Surg Res, 1999, 81(2): 238.
[26] Xia YP, Zhao Y, Marcus Jet ,et al. Effects of keratinocyte growth factor-2(KGF-2) on wound healing in an ischemia-impaired rabbit ear model and onscar formations[J]. Pathol, 1999, 188(4): 431.
[27] Mann A, Breuhahn K, Schirmacher Pet al. Keratinocyte-derived granulocy temacrophaolony stimulating factor accelerates wound healing: Stimulation of keratinocyte proliferation, granulation tissue formation, and vascularization[J]. Invest Dermatol, 2001, 117(6):1382
[28] Alderson R, Gohari Fritsh S, Olsen H, et al.In vitro and in vivo effects of repifermin (keratinocyte growth factor 2, KGF2)on human carcinoma cells[J]. Cancer Chemother Parmacol, 2002, 50: 202-212.
[29] Coulier F, Pontarotti P, Rollbin R, et al.Of worms and men: an evolutionary perspective on the fibroblast growth factor (FGF) and FGF receptor families[J]. Mol Evol, 1997, 44(1):43-56.
[30]孙蒙,于永利.成纤维细胞生长因子13基因在酵母细胞中的表达[J].分子与细胞免疫学,2006,8(16):401-403.
[31] Mcwhirte JR, Goulding M, Weiner JA,et al.A novel fibroblast growth factor gene expressed in the developingnervous system is a downstream target of the chimeric homeodomain oncoproteinE2A-Pbx1[J].Devolopment,1997,24:3221-3232.
[32] Munekazu Komada, Hirotomo Saitsu, Kohei Shiota,et al.Expression of Fgf15 is regulated by bothactivator and repressor forms of Gli2 in vitro[J], Biochemical and Biophysical Research Communications, 2008, 2(369):350-356.
[33] Yuhei Hotta , Sayaka Saki , Morichika Konishi,et al. Fgf16 is required for cardiomyocyte proliferation in the mouse embryonic heart[J].Developmental Dynamics, 2008,237(10):2947-54
[34] Jeremy A. Cholfin , John L.R. Rubenstein, Frontal cortex subdivision patterning is coordinately regulated by Fgf8, Fgf17, and Emx2 [J].Interscience,2008,2(509): 144- 155.
[35] Ohbayashi N, Shibayama M, Kurotaki Y.FgF18 is required for normal cell proliferation and differentiation duringosteogenesis and chondrogenesis[J].Genes Dev,2002,16(7):870
[36] Haque T, Nakada S, Hamdy RC. A review of FGF18: Its expression, signaling pathways and possible functions during embryogenesis and post-natal development[J]. Histol Histopathol, 2007 ,22(1):97-105.
[37] Benjamin C. Lin, Manping Wang, Craig Blackmore,et al. Liver-specific Activities of FGF19 Require Klotho beta[J]. Biological Chemistry,2007,37(282): 27277-27284.
[38] Gao X, Scott WK, Wang G,et al.Gene-Gene Interaction Between FGF20 and MAOB in Parkinson Disease[J]. Annals of Human Genetics, 2008,2(72) :157-162.
[39] Wider C, Dachsel JC, Soto AI,et al.FGF20 and Parkinson's disease: No evidence of association or pathogenicity via alphasynuclein expression[J]. Movement disorders,2009, 24(3):455-459.
[40] Moyers JS, Shyanova TL, Mehrbod F,etal. Molecular determinants of FGF 21 activity energy and cross-talk with PPAR gamma signaling [J]. cellular physiology,2007,210(1):l-6.
[41] Huang X , Yu C , Jin CForced expression of hepatocyte-specific fibroblast growth factor 21 delaysinitiation of chemically induced hepatocarcinogenesis[J]. Molecular Carcinogenesis, 2006,45(12):934-942.
[43] Linhoff, David M, Joshua R, et al. FGF22 and Its Close Relatives Are Presynaptic Organizing Molecules in the Mammalian Brain[J]. Cell, 2004,2(118): 257-270.
[44] Jonnaon KB. Zahraadnik R, Iatsson T, et al. Fibroblast growth factor 23 in oncogenic osteomalacia and X-linked hypophosphatemia[J]. N Engl J Med,2003,348:1656-1663.
[45]郭晓强.成纤维细胞生长因子23和血磷代谢[J].生命的化学,2007,(2)27:149-151.
[46] Mattei M. G., deLapeyriere O, Bresnick J. ,et al.Mouse Fgf7 (fibroblast growth factor 7) and Fgf8 (fibroblast growth factor 8) genes map to chromosomes 2 and 19 respectively[J]. Mammalian Genome,1995, 6: 196-197.
[47] Yoshiura K., Leysens N. J, Chang J ,et al. Genomic structure, sequence, and mapping of human FGF8 with no evidence for its role in craniosynostosis/limb defect syndromes[J]. Am. J. Med. Genet,1997,72: 354-362.
[48] Tanaka A, Miyamoto K, Minamino Netal. Cloning and charaxterization of an androgen-dependent growth of mouse mummary carcinoma cells[J]. Proc Natl Acad Sci USA, 1992,77: 89:8928.
[49] Sangwoo Shim, Narina Bae, Sang Yoon Park, et al. Isolation of Xenopus FGF-8b and Comparison withFGF-8a [J]. Molecules and Cells, 2005, 19(3): 310-317.
[50] Akira Tanaka, Tomoko Kamiakito, Y Hakamata, et al. Extensive neuronal localization and neurotrophic function of fibroblast growth factor 8 in the nervous system [J]. Brain Research, 2001, 912: 105-115.
[51] B Kwabi-Addo, M Ozen and M Ittmann. The role of fibroblast growth factors and their receptors in prostate cancer[J]. Endocrine-Related Cancer, 2004, 11: 709-724.
[52] Sonia Garel, Kelly J. Huffman and John L. R. Rubenstein. Molecular regionalization of the neocortex is disrupted in FGF8 hypomorphic mutants [J]. Development, 2003, 130: 1903-1914.
[53] Fukuchi-Shimogori T, Grove E A. Neocortex patterning by the secreted signaling molecule FGF8 [J]. Science, 2001, 294: 1071-1074.
[54] Steffen Scholpp, Claudia Lohs and Michael Brand. Engrailed and FGF8 act synergistically to maintain the boundary between diencephalon and mesencephalon [J]. Development 2003, 130: 4881-4893.
[55] Tatsuya Sato, Isato Araki and Harukazu Nakamura. Inductive signal and tissue responsiveness defining the tectum and the cerebellum[J]. Development, 2001, 128: 2461-2469.
[56] EL Wiellette and H Sive. Early requirement for FGF8 function during hindbrain pattern formation in zebrafish [J]. Dev Dyn, 2004, 229(2): 393-9.
[57] Lewandoski M., Sun X., Martin G. R. : FGF8 signalling from the AER is essential for normal limb developmen[J]t. Nature Genet. 2000,26: 460-463.
[58] Sabine Fischer, Bruce W. Draper and Carl J. Neumann:The zebrafish FGF24 mutant identifies an additional level of FGF signaling involved in vertebrate forelimb initiation[J]. Development 2003, 130:3515-3524.
[59] Sun X., Mariani F. V., Martin G. R. Functions of FGF signalling from the apical ectodermal ridge in limb development [J]. Nature, 2002, 418: 501-508.
[60]王春梅,姜涛,周春燕.诱导心脏发生的早期信号通路[J].中国生物化学与分子生物学报, 2004, 10, 20(5): 572～577.
[61] Jung J, Zheng M., Goldfarb M., Zaret K. S.. Initiation of mammalian liver development from endoderm by fibroblast growth factors [J]. Science, 1999, 284: 1998-2003.
[62] Hitoo N and Hideki H., Mesencephalic. Neural stem cells develop to dopaminergic neurons more strongly in dopamine-depleted striatum than in intact striatum [J]. Exp Neurol, 2000, 28: 31-40.
[63] Ye W, Shimamura K, Rubenstein JL, et al. FGF and Shh signals control dopaminergic and serotonergic cell fate in the anterior neural plate [J]. Cell, 1998, 93: 755–766.
[64] Creuzet Sophie, Bernadette Schuler, Gérard Couly and Nicole M. Le Douarin:Reciprocal relationships between Fgf8 and neural crest cells in facial and forebrain development[J]. PNAS 2004, 101 (14):4843-4847.
[65] Dong Liu, Hsin Chu, Lisa Maves, et al:Fgf3 and Fgf8 dependent and independent transcription factorsare required for otic placode specification[J]. Development 2003, 130:2213-2224.
[66] Lèger Sophie, Michael Brand:Fgf8 and Fgf3 are required for zebrafish ear placode induction, maintenance and inner ear patterning[J]. Mechanisms of Development 2002,119:91–108.
[67] Jaskoll Tina, Dan Witcher, Leo Toreno, et al. FGF8 dose-dependent regulation of embryonic bmandibular salivary gland morphogenesis[J]. Developmental Biology 2004,268: 457– 469
[68] Emily A. Morgan, Susan B. Nguyen, Virginia Scott and H. Scott Stadler. Loss of Bmp7 and Fgf8 signaling in Hoxa13-mutant mice causes hypospadia[J]. Development 2003,130:3095-3109
[69] Jean M. Hébert, Mary Lin, Juha Partanen, et al:FGF signaling through FGFR1 is required for olfactory bulb morphogenesis[J]. Development 2003,130:1101-1111
[70] Radwan Abu-Issa, Graham Smyth, Ida Smoak, et al:Fgf8 is required for pharyngeal arch and cardiovascular development in the mouse[J]. Development 2002,129:4613-4625
[71] R Huang, D Stolte, H Kurz, et al:Ventral axial organs regulate expression of myotomal Fgf-8 that influences rib development[J]. Dev Biol, March 1, 2003, 255(1): 30-47.
[72] Dubrulle J., McGrew M. J., Pourquie O. : FGF signaling controls somite boundary position and regulates segmentation clock control of spatiotemporal Hox gene activation[J]. Cell 106: 219-232, 2001.
[73] Streit A., Berliner A. J., Papanayotou C. , et al : Initiation of neural induction by FGF signalling before gastrulation[J]. Nature 2000,406: 74-78.
[74] Payson R. A.,Wu J,Liu Y.,Chiu I.-M. : The human FGF-8 gene localizes on chromosome 10q24 and is subjected to induction by androgen in breast cancer cells[J]. Oncogene 1996,13: 47-53.
[75] Tatsuya Sato and Harukazu Nakamura:The Fgf8 signal causes cerebellar differentiation by activating the Ras-ERK signaling pathway[J]. Development 2004,131:4275-4285.
[76] Johanna K. Ruohola, Tiina P. Viitanen, Eeva M. Valve,et al:Enhanced Invasion and Tumor Growth of Fibroblast Growth Factor 8b-overexpressing MCF-7 Human Breast Cancer Cells[J]. Cancer Research 2001,61(5), 4229-4237.
[77] Revilla A, Bennett MV, Barrio LC, eta.l Moleculardeterminants of membrane potential dependence in vertebrate gap junction channels[J].Proc natlAcad SciUSA,2000, 97(26): 14760-14765.
[78] CalderMD, Caveney AN, Smith LC, et a.l Responsiveness of bovine cumulus oocyte complexes (COC) to porcine and recombinanthuman FSH, and the effect ofCOC quality on gonadotropin receptorand Cx43 marker gene mRNAs during maturation in vitro [ J].Reprod BiolEndocrinol,2003, 1(1): 14-19.
[79] Ai Z, FischerA, SprayDC, eta.l Wnt-1 regulation ofconnexin43 in cardiac- myocytes [J].American Society for Clinical Investigation,2000, 105(2): 161-171.
[80] KrutovskikhVA,TroyanovskySM, PiccoliC, etal. Differential effect of subcellular localization of communication impairing gap junction protein connexin43 on tumor cell growth in vivo[J]. Oncogen,2000, 19(4): 505-513.
[81] Su YA, BittnerML,Chen Y, et a.l Identification of tumor suppressor genes using human melanoma celllines UACC903, UACC903 ( +6), and SRS3 by comparison of expression profiles [ J].MolCarcinog,2000, 28(2): 119-127.
[82] Ilvesaro J, Tavi P, Tuukkanen J. Connexin-mimetic peptide Gap 27 decreases osteoclastic activity [ J].BMC Musculoskelet Disord,2001, 2(1): 1500-1513.
[83] Samoilova M, Li J. Gap Junctions in Epileptogenesis: Chicken orEgg[J]. Epilepsy Curr,2004, 4(2): 80-81.
[84] Contreras JE, SánchezHA, Eugenin EA, et a.lMetabolic inhibition induces opening of unapposed connexin 43 gap junction hemichannels and reduces gap junctional communication in cortical astrocytes in culture[J].ProcNatlAcad SciUSA,2002, 99(1): 495-500.
[85]冯纬樱.重组人成纤维细胞生长因子-8a的纯化研究[D].桂林:广西师范大学,2007:35-43.
[86]黄明,蒋寅.改造稀有密码子对rhFGF8a原核表达的影响[J].细胞与分子免疫学杂志, 2008.25(6):101-104.