中等纯度重组人表皮生长因子（rhEGF）的分离纯化及在美容产品中的应用
|
摘要
基因重组人表皮生长因子(recombinant human Epidermal Growth Factor,rhEGF)在医学上有显著的创伤修复、愈合伤口等功效。近年来发现，rhEGF在美容产品领域也有广阔的应用前景，具有促进皮肤细胞更新、防止皮肤衰老等功能。采用分泌型表达的基因工程大肠杆菌培养获得rhEGF，根据美容等日化行业对rhEGF的质量与纯度要求，设计了一条成本低廉、简便快速、能满足日化产品活性添加纯度要求的rhEGF分离工艺，得到了中等纯度的rhEGF冻干粉产品，并试制出含rhEGF的美容化妆品，观察了使用效果。
通过启发式方法设计了分离纯化rhEGF的工艺路线，包括基因工程大肠杆菌发酵液离心得到上清液、上清液盐析、透析脱盐、离子交换树脂层析、再脱盐、脱色和冻干等过程。通过正交和单因素试验考察了各因素的影响程度和最佳的水平。研究了中等纯度rhEGF冻干粉中rhEGF活性在不同温度、pH和稳定保护剂等条件下的变化，开发了在美容化妆品中添加rhEGF的关键工艺，并利用随机双盲法结合统计学分析研究了含rhEGF美容化妆品使用疗效的显著性。
中等纯度rhEGF冻干粉生产工艺的最佳操作条件为：1)rhEGF发酵液在4℃，6000rpm离心25min获得上清液(0.16-0.21g rhEGF／L)；2)上清液在冰浴中缓慢加入361g(NH_4)_2SO-4固体／L(60％饱和度)盐析，溶液pH控制在6.0，低速(60rpm)搅拌2h后静置4h，离心得到沉淀；3)沉淀用pH 7.4的磷酸盐缓冲液(PBS)溶解得rhEGF溶液，装满2／3透析袋脱盐，对透析液(水)的比例为1：40，每次透析2h，更换5次透析液后得到rhEGF盐析透析液(1.30-1.60grhEGF／L)；4)rhEGF盐析透析液经层高10cm(XK16／20)的凝胶型弱碱阴离子DEAE Sephadex-A50层析，流速控制在150mL／h，一次进杆体积75mL，用含0.5mol／L NaCl的PBS(pH7.4)洗脱得到rhEGF离交液(4.42-5.44g rhEGF／L)；5)经再次透析脱盐的rhEGF离交液流经层高7.5cm(XK16／20)的大孔型弱碱阴离子D314树脂脱色，溶液pH5.0，流速控制60mL／h，一次进样体积120mL，收集的流出液即rhEGF脱色液(3.54-4.35g rhEGF／L)；6)rhEGF脱色液冻干得到中等纯度的rhEGF冻干粉。通过以上分离提纯工艺，冻干粉产品rhEGF的纯度达到32％以上，整个工艺rhEGF得率53％以上。
实验表明rhEGF冻干粉中加入10％甘露醇、10％丝蛋白或1％低分子透明质酸钠可以减少rhEGF活性损失的50％以上(25℃室温下储存30天)，而且与rhEGF有正协同美容效果。1％透明质酸(HA)与1％丝蛋白同时存在能使rhEGF水剂的有效治疗的浓度从300μg／mL降低到100μg／mL。统计学分析显示，rhEGF冻干粉水剂(含终浓度1％HA和1％丝蛋白)对治疗皮肤的粉刺、皱纹和衰老等
浙江大学硕士学位论文＼自动少 摘要
效果较好，使用前后差别有显著和高度显著性叩4．05刀．ofL
含 rhEGF美容化妆品中 rhEGF最佳添加量为终浓度 200pg rhEGF／mL，在生
产结束前温度降至40℃左右时采用“微量稀释法”添加到中性的化妆品液体或
营体中。含rhEGF美容化妆品使用30天后与使用不含rhEGF的化妆品对照组比
较有显著差异，rhEGF美容化妆品总平均有效率为84．9％。
以上研究结果表明中等纯度rhEGF冻干粉符合美容化妆品的添加要求，分
离操作简便，产品成本低廉。试制的含甘露醇、HA与丝蛋白保护和增效的rhEGF
美容化妆品达到了预期的去痘修复。去现、＃L袁老与大田等效果，为fhEGF人
规模用于R化产品打下了基础。
The objectives of this thesis are: to design a process of purification of recombinant human epidermal growth factor(rhEGF) with low cost, easy operation and meeting the request of purity used in daily chemicals; to manufacture the cosmetics containing an active additive of rhEGF, and to evaluate the curative effect of cosmetics on the skin regeneration and senescence.
The separation and purification process of rhEGF from fermentation broth including following steps: centrifuging fermentation broth to get supernatant containing rhEGF, salting-out of rhEGF from supernatant, desalting, separating rhEGF by ion exchange chromatography, desalting again, decoloring, and vacuum freeze-drying. The factors affecting separation and purificntion were evaluated rind the optimal operation conditions were obtained.
Fermentation broth was centrifugated at 40癈, 6000 rpm, for 25 min, to obtain supernatant, which was salted-out by 361 g( NH4)2 SO4 solid/ L(60% saturated degree) under pH6.0 and low agitation speed(60 rpm) for 2 hours and standing for 4 hours. Then, centrifuged sedimentation was dissolved with PBS at pH7.4. Obtained rhFGF solution was dialyzed by dialysis bag, in which, the rhEGF solution was 2/3 in full and the ratio of rhEGF to water was 1:40, each dialysis lasted for 2 hours and the operation was repeated for 5 times. After dialysis, solution was ion-exchanged in a DEAE Sephadex?A50 column with a height of 10 cm( XK16/20). The column was operated with volume velocity of 150mL/h, volume injection of 75mL, and \\as eluted by PBS solution containing 0.5 mol/L NaCl.. The eluant contained 4.42-5.44 g/L of rhEGF and was decolored by a D314 resin column with the height of 7.5 cm( XK16/20), and operated at pH5.0. volume velocity of 60 mL/h. and volume injection of 120 mL. The collection of rhEGF (3.54-4.35 g rhEGF/L) was freeze-dried in Freezone?4.5 apparatus to obtain rhEGF powder with the purity of above 32%. The overall yield of rhEGF was above 53%.
The experimental results indicate that the addition of 10%mannitol, 10%silk protein and 1%HA helps not only to reduce the loss of activity of rhEGF up to 50% (25 癈,after 30 days), but also to multiply the effect of beautying. The effective concentration of rhEGF is l00ug/mL with 1% HA and 1% silk protein. The statistic analysis showed that freezing-dried powder of rhEGF demonstrated good effect to cure acne, to reduce wrinkle and caducity, and the distinct difference was observed between with and without treatment of rhEGF( P<0.05-0.01). The optimized
ABSTRACT
concentration of rhEGF in cosmetics was 200jig/mL, which should added when temperature of the neutral liquid or ointment was decreased to about 40 癈. The average ratio of effectiveness by using of cosmetics containing rhFGF was 84.9% after 30 days from the time of use.
The freezing-dried powder of rhEGF with moderate-purity met the requirement of daily chemicals used in cosmetic industry. Cosmetics containing rhEGF. together with manntiol, HA and silk protein, had the anticipative effect of anti-acne, anti-wrinkle, and whitening.
通过启发式方法设计了分离纯化rhEGF的工艺路线，包括基因工程大肠杆菌发酵液离心得到上清液、上清液盐析、透析脱盐、离子交换树脂层析、再脱盐、脱色和冻干等过程。通过正交和单因素试验考察了各因素的影响程度和最佳的水平。研究了中等纯度rhEGF冻干粉中rhEGF活性在不同温度、pH和稳定保护剂等条件下的变化，开发了在美容化妆品中添加rhEGF的关键工艺，并利用随机双盲法结合统计学分析研究了含rhEGF美容化妆品使用疗效的显著性。
中等纯度rhEGF冻干粉生产工艺的最佳操作条件为：1)rhEGF发酵液在4℃，6000rpm离心25min获得上清液(0.16-0.21g rhEGF／L)；2)上清液在冰浴中缓慢加入361g(NH_4)_2SO-4固体／L(60％饱和度)盐析，溶液pH控制在6.0，低速(60rpm)搅拌2h后静置4h，离心得到沉淀；3)沉淀用pH 7.4的磷酸盐缓冲液(PBS)溶解得rhEGF溶液，装满2／3透析袋脱盐，对透析液(水)的比例为1：40，每次透析2h，更换5次透析液后得到rhEGF盐析透析液(1.30-1.60grhEGF／L)；4)rhEGF盐析透析液经层高10cm(XK16／20)的凝胶型弱碱阴离子DEAE Sephadex-A50层析，流速控制在150mL／h，一次进杆体积75mL，用含0.5mol／L NaCl的PBS(pH7.4)洗脱得到rhEGF离交液(4.42-5.44g rhEGF／L)；5)经再次透析脱盐的rhEGF离交液流经层高7.5cm(XK16／20)的大孔型弱碱阴离子D314树脂脱色，溶液pH5.0，流速控制60mL／h，一次进样体积120mL，收集的流出液即rhEGF脱色液(3.54-4.35g rhEGF／L)；6)rhEGF脱色液冻干得到中等纯度的rhEGF冻干粉。通过以上分离提纯工艺，冻干粉产品rhEGF的纯度达到32％以上，整个工艺rhEGF得率53％以上。
实验表明rhEGF冻干粉中加入10％甘露醇、10％丝蛋白或1％低分子透明质酸钠可以减少rhEGF活性损失的50％以上(25℃室温下储存30天)，而且与rhEGF有正协同美容效果。1％透明质酸(HA)与1％丝蛋白同时存在能使rhEGF水剂的有效治疗的浓度从300μg／mL降低到100μg／mL。统计学分析显示，rhEGF冻干粉水剂(含终浓度1％HA和1％丝蛋白)对治疗皮肤的粉刺、皱纹和衰老等
浙江大学硕士学位论文＼自动少 摘要
效果较好，使用前后差别有显著和高度显著性叩4．05刀．ofL
含 rhEGF美容化妆品中 rhEGF最佳添加量为终浓度 200pg rhEGF／mL，在生
产结束前温度降至40℃左右时采用“微量稀释法”添加到中性的化妆品液体或
营体中。含rhEGF美容化妆品使用30天后与使用不含rhEGF的化妆品对照组比
较有显著差异，rhEGF美容化妆品总平均有效率为84．9％。
以上研究结果表明中等纯度rhEGF冻干粉符合美容化妆品的添加要求，分
离操作简便，产品成本低廉。试制的含甘露醇、HA与丝蛋白保护和增效的rhEGF
美容化妆品达到了预期的去痘修复。去现、＃L袁老与大田等效果，为fhEGF人
规模用于R化产品打下了基础。
The objectives of this thesis are: to design a process of purification of recombinant human epidermal growth factor(rhEGF) with low cost, easy operation and meeting the request of purity used in daily chemicals; to manufacture the cosmetics containing an active additive of rhEGF, and to evaluate the curative effect of cosmetics on the skin regeneration and senescence.
The separation and purification process of rhEGF from fermentation broth including following steps: centrifuging fermentation broth to get supernatant containing rhEGF, salting-out of rhEGF from supernatant, desalting, separating rhEGF by ion exchange chromatography, desalting again, decoloring, and vacuum freeze-drying. The factors affecting separation and purificntion were evaluated rind the optimal operation conditions were obtained.
Fermentation broth was centrifugated at 40癈, 6000 rpm, for 25 min, to obtain supernatant, which was salted-out by 361 g( NH4)2 SO4 solid/ L(60% saturated degree) under pH6.0 and low agitation speed(60 rpm) for 2 hours and standing for 4 hours. Then, centrifuged sedimentation was dissolved with PBS at pH7.4. Obtained rhFGF solution was dialyzed by dialysis bag, in which, the rhEGF solution was 2/3 in full and the ratio of rhEGF to water was 1:40, each dialysis lasted for 2 hours and the operation was repeated for 5 times. After dialysis, solution was ion-exchanged in a DEAE Sephadex?A50 column with a height of 10 cm( XK16/20). The column was operated with volume velocity of 150mL/h, volume injection of 75mL, and \\as eluted by PBS solution containing 0.5 mol/L NaCl.. The eluant contained 4.42-5.44 g/L of rhEGF and was decolored by a D314 resin column with the height of 7.5 cm( XK16/20), and operated at pH5.0. volume velocity of 60 mL/h. and volume injection of 120 mL. The collection of rhEGF (3.54-4.35 g rhEGF/L) was freeze-dried in Freezone?4.5 apparatus to obtain rhEGF powder with the purity of above 32%. The overall yield of rhEGF was above 53%.
The experimental results indicate that the addition of 10%mannitol, 10%silk protein and 1%HA helps not only to reduce the loss of activity of rhEGF up to 50% (25 癈,after 30 days), but also to multiply the effect of beautying. The effective concentration of rhEGF is l00ug/mL with 1% HA and 1% silk protein. The statistic analysis showed that freezing-dried powder of rhEGF demonstrated good effect to cure acne, to reduce wrinkle and caducity, and the distinct difference was observed between with and without treatment of rhEGF( P<0.05-0.01). The optimized
ABSTRACT
concentration of rhEGF in cosmetics was 200jig/mL, which should added when temperature of the neutral liquid or ointment was decreased to about 40 癈. The average ratio of effectiveness by using of cosmetics containing rhFGF was 84.9% after 30 days from the time of use.
The freezing-dried powder of rhEGF with moderate-purity met the requirement of daily chemicals used in cosmetic industry. Cosmetics containing rhEGF. together with manntiol, HA and silk protein, had the anticipative effect of anti-acne, anti-wrinkle, and whitening.
引文
[1] 马大龙．生物技术药物[M]．北京：科学出版社，2001：11-12，21．
[2] S. Cohen. Purification of a nerve-growth promoting from mouse salivary gland and its neurocytotoxic antiserum[J]. Proc.Natl.Acad. Sci. USA, 1960,46: 302-310.
[3] J.Taylor, W.M.Michell,S.Cohen. Epidermal growth factor:physical and chemical properties[J]. J.Bioc.Chem, 1972.247:5928-5934.
[4] Gregory H.. Isolation and structure of urogastore and its relationship to epidermal growth factor[J]. Nature, 1975,257:325-327.
[5] 席全胜，钱欣国，甘人宝．EGF家族研究进展[J]．生命的科学，1999，19 (3)：135-137．
[6] Bell G.I.,Fong N.M.,Stempien M.M.,et al..Human epidermal growth factor precursor:cDNA sequence,expression in vitro and gene organiation[J]. Nucleic Acid Research, 1986,14:8427-8446.
[7] Pessonen. Characterization of material with epidermal growth factor immunoreactivity in human serum and platets[J]. Clin.Endo.Matab, 1989,68:486-488.
[8] G. Carpenter, S.Cohen. Epidermal growth factor[J]. Annu.Rev.Biochem., 1979.48:193-196.
[9] U.Shiro H.,Cohen S.. Affinity labeling of the protein kinase associated with the epidermal growth factor receptor in membrane vesicles from A431 cells[J]. Biol. Chem., 1980,255: 8363-8365.
[10] Savage C.R.,Hasb J.H.,Cohen S.. Epidermal growth factor:location of disulfide bonds[J]. J.Biol. Chem., 1973,247:7612-7622.
[11] Cooke R.M.,Wilkinson A.J.,Baron M.,et al.. The solution structure of human epidermal growth factor[J]. Nature, 1987,327:339-341.
[12] Kohda D.,Go N.,Hayashi K.,et al.. Tertiary structure of mouse epidermal growth factor: determination by two-climensinal proton NMR[J]. The Journal of Biochemistry, 1988, 103:741-743.
[13] Fisher D.A.,Lakshmanan J.J.. Metabolism and effects of epidermal growth factor and related growth factor in mammals[J]. Endocrine Review, 1990,11 (3):418-442.
[14] Martin P.,Hopkinson-Woolley J.,Mccluskey J.. Growth factors and cutaneous repair[J]. Progr.Growth Factor Res., 1992,4:25-44.
[15] Gospodarowicz D.. Epidermal and nerve growth factors in mammalian development[J]. Ann.Rev. Biochem., 1981,43:251-265.
[16] S.Cohen. Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the new-born animal[J]. J.Bioc.Chem., 1962,237:1555-1556.
[17] 傅小兵，孙国柱，王亚平，等．表皮细胞生长因子与碱性成纤维细胞生长因子促进创面修复效应的比较性研究[J]．中国修复重建外科杂志，1999，13 (5)：278-282．
[18] Downwand J.. Close similamity of epidermal growth factor oncogene protein sequence[J]. Nature, 1984,307:521-527.
[19] Yarden Y.,Schlessinger J.. Epidermal growth factor induced rapid,reversible aggregation of the purified epidermal growth factor receptor[J]. Biochemistry, 1986, 26:1443-1451.
[20] Helin K.,Velu T.,Martin P.,et al. The biological activity of the human epidermal growth factor receptor is positively regulated by its C-terminal tyrosines[J]. Oncogene, 1991:625-832.
[21] Merlino G.T.. Epidermal growth factor receptor relation and funtion[J]. Semin Cancer Biol.,1990,(1):277-284.
[22] Hudson L.G.,Santon J.B.,Gill G.N. Regulation of epidermal growth factor gene expression[J]. Molecular Endocrinology, 1989,(3):400-408.
[23] May K.H.. J.Biol.Chem.,1989,264:17838-17844.
[24] Well S.A.. Proc.Natl.Acad. Sci. USA,1988,85:7597-7601.
[25] Chen W.S.. Nature, 1987,328:820-823.
[26] Wedegaertuner P.B.,Gill G.N.. J.Biol.Chem., 1989,264:11346-11353.
[27] Breyer M.D. Jacobson H.R.,Breyer J.A.J.Clin.Invest, 1988,82(10):1313-1320.
[28] Masayuki K.. Development of a sensitive enzyme immuoassay for human epidermal growth factor[J]. Clin.Chim.Acta, 1986,156:51-53.
[29] kasselberg Ag,Orth D.N.,Gray M.E.,et al.. Immuocy to chemical localization of human epidermal growth factor/urogastrone in several human tissues[J]. J.Histochem. Cytochem, 1985,33:315.
[30] Fuse H.. Epidermal growth factor contents in seminal plasma as a marker of prostatic function[J]. Arch Androl, 1992,29:79-81.
[31] Olsen P.S.. Role of epidermal growth factor in gastroduodenal mucosal protection[J]. J.Clin. Gastroenterol, 1988,suppl 1:146-147.
[32] Orsinib B.,Brocchi A.,Calabro A., et al.. radioimmunoassay of epidermal growth factor in human saliva and gastric juice[J]. Clin. Biochem., 1991,24:135-137.
[33] Sugimoto Kaname. Process for the production of human epidermal growth factor[P].US Patent: 19840619708,1986-11-04.
[34] March D.,Osborne E.,Roberts E.,et al.. Chemical synthesis of human epidermal growth factor and human type α-TGF[R]. Proceeding 10th American Peptide Symposium, 1988:202-203.
[35] 袁汉英，闵永洁，石松，等．人表皮生长因子基因的化学合成和克隆及其在酵母中的表达[J]．生物工程学报，1991，7(4)：300-306．
[36] Smith J.,Derbyshine R.B.,Dunthomel L.,et al. Gene, 1984,32:321-322.
[37] Oka T.,sakamoto S.,Miyoshi K.,et al.. Synthesis and secretion of human epidermal growth factor by scherichia coli.[J]. Proc.Natl.Acad.Sci.USA, 1985, 82:7212-7216.
[38] Kishimoto F.,Gomi H.,Kanaoka M.,et al.. Gene, 1986,45:311.
[39] Oka T.,Sumi S.,Fuwa T.,et al. Agric.Biol. Chem., 1987,51:1099.
[40] Batchikova N.V.,Altman I.B.,Sinyagina E.A.,et al.. Bioeng. Khirm, 1992,18:766-777.
[41] 甘人宝，黄培勇，袁宇，等．人表皮生长因子在大肠杆菌中高效表达和分泌[J]．生物化学与生物物理学报，1992，24 (6)：587-589．
[42] Fujimoto K.M.,Marumoto R.,Fukuda T.. J.Biol Chem., 1991,266:1728-1730.
[43] W.K.R. Wong,Sutherland M.L.. Excretion of heterologous proteins from E.coli[P].EP patent:EP0357391,1990-03-07.
[44] Yadward V.B.,Wison S.,Ward O.P..Production of human epidermal growth factor by an ampicillin resistant E. coli strain[J]. Biotechnology Letter, 1994,16:885-890.
[45] Xu Zhi-nan, Liu Gang,Cen Peilin,et al.. Factors influencing excretive production of human epidermal growth factor(hEGF) with recombinant Escherichia coli k12 system[J]. Bioprocess and Biosystem Engineering.2000,23(6):669-674.
[46] Flock J.I.,Fotheringham I.,Light J.,et al.. Mol.Gen.Genet., 1984:195:246-247.
[47] Yamagata H.,Nakahawa K.,Suzuki Y.,et al.. Proc.NatI.Acad.Sci.USA, 1989,86: 8589-8591.
[48] Ebisu S.,Takagi H.,Kadowaki K.,et al.. Production of human epidermal growth factor by Bacillus brevis increased with use of a stable plasmid from B. brevis 481[J]. Biosci. Biotech. Biochem., 1992,56:812-813.
[49] Brake A.J.,Merryweather J.P.,Coit D.G.,et al.. Proc.Natl.acad. Sci.USA, 1984,81: 4642-4644.
[50] Clements J.M.,Catlin G.H.,Price M.J.,et al.. Secretion of human epidermal growth factor from Saccharomyces cervisiae using synthetic sequence[J]. Gene, 1991,106:267-268.
[51] Barr. US patent:US5096825,1992.
特殊上标未编改
[52] Edwards. WO patent:WO9216626,1993.
[53] Clare J.J.,Romanos M.A.,Rayment F.B.,et al..Production of mouse epidermal growth factor in yeast:High-level secretion using Pichia pastoris strains containing multiple copy genes[J].Gene, 1991,105:205-207.
[54] 黄秉仁，蔡良婉，廖洪涛，等．人工合成表皮生长因子基因在甲基型酵母中的高效表达[J]．中国生物化学与分子生物学报，1998，14(5)：512-517．
[55] P.V. Hamsa, P. Kachroo,B.B.Chattoo..Production and secretion of biologically active human epidermal growth factor in Yarrowia lipolytica[J].Current Genetics,1998,33(3):231-237.
[56] Joo-Hyung Heo,Hye Soon Won,Hyun Ah Kang, et al..Purification of recombinant human epidermal growth factor secreted from the methylotrophic yeast Hansenula polymorpha[J].Protein Expression and Purification,2002,24:117-122.
[57] Mroczkowshi B.. Methods Enzymol,1991,198:175-177.
[58] 胡大海，陈璧，汤朝武，等．人表皮生长因子基因真核表达质粒pcDNA_3-hEGF的构建[J]．西北国防医学杂志，2000，21(1)：18-22．
[59] 董肇杨，陈玉林，戴方平，等．hEGF转基因表皮细胞的制备及其EGF蛋白表达[J]．中华整形外科杂志，2000，16(3)：177-179．
[60] Gao Jianwei,Dai Ziyu,Hooker Brian S.,et al..Method of producing human epidermal growth factor from whole plants or plant cell cultures[P].WO patent: WO9821348,1998-05-22.
[61] 戴激，施定基，张卉，等．人表皮生长因子(hEGF)基因在蓝藻中的表达[J]．植物学报，2001，43(12)：1260-1261．
[62] 李守芬，苏钟浦，王立坤，等．人表皮生长因子的三种分离方法及其生物学活性[J]．中国医科大学学报，1992，21(3)：172-174．
[63] R.C.Huang,E.Lam,Y.H.Chen,et al.. Human epidermal growth factor excreted by recombinant Escherichia coli k-12 has the correct N-terminus and its fully bioactive[J],process Biochemistry, 1999,35:1-5.
[64] 黄秉仁，蔡良婉，王欣，等．甲基营养型酵母系统表达的重组人表皮生长因子的纯化及其性质[J]．中国医学科学院学报，2001，23(2)：106-110．
[65] 单继贵，秦德安，何学民，等．孕妇尿液中表皮生长因子的分离、纯化及活性检测[J]．华东师范大学学报(自然科学版)，1996，(4)：89-94．
[66] Geory-Nascimento C.,Gyenes A.,Halloran S.M.,et al.. Biochemistory,1988,27: 797-799.
[67] 张伟，麦荫乔．人尿表皮生长因子的提取、纯化[J]．白求恩医科大学学报，1993，19(2)：210-211．
[68] 阮文权，李玉忠，杨邱明，等．重组大肠杆菌生产人表皮生长因子的发酵条件[J]．无锡轻工大学学报，2001，20(5)：489-492．
[69] Vazquez J.,Freyre M.,Duarte,et al..Biotechol.Apl.,1990,7:42-45.
[70] 赵明，王会信，邱宝成，等．小鼠额下腺表皮生长因子的分离、纯化与鉴定[J]．生物化学杂志，1992，8(1)：61-66．
[71] 贺师鹏，黄天贵，黄力新，等．表皮生长因子的lodogen标记法与放射性比度测定[J]．北京医科大学学报，1992，24(5)：416．
[72] 叶春婷，李斯明，查健群，等．结晶紫比色法检测表皮生长因子生物活性的探讨[J]．中华微生物和免疫学杂志，1997，17(2)：154-155．
[73] 赵忠良，路凡，陈南春，等．人表皮生长因子在大肠杆菌中的融合表达与活性分析[J]．第四军医大学学报，1997，18(5)：497-498．
[74] Tim Mosmann. Rapid colorimetric assay for celluar growth and surrival:application to proliferation and proliferation and cytoxicity assays[J].J.Immunological Methods,1983,63:55-57.
[75] 何海琼，王金发，陈妍，等．重组人表皮生长因子生物活性的测定[J]．广东医学，1998，
19(5)：324-325．
[76] 周志荣，管樱．小鼠颚下腺提取液中表皮生长因子的含量测定[J]．南京医科大学学报，1994，14(2)：174-176．
[77] G.Carpenter, Stanly Cohen. A biological assay for epidermal growth factor/urogastrone and related polypeptides[J]. Anal. Biochem.,1985,153:279-281.
[78] Pesonen K.,Alfthan H.,Stenman U.H.,et al. Anal. Biochem.,1986,157:208.
[79] 刘农乐，蒋滋慧，赵明，等．人表皮生长因子的发光免疫测定法——吖啶酯标记抗体的应用[J]．生物化学与生物物理进展，1993，20(1)：43-45．
[80] Kashimata M.,Hiramatsu M.,Kumegawa M.et al..Shika Kiso Lgakkai Zasshi, 1985,7:1260-1263.
[81] Nishimuro Satoshi,Hayashi Kyozo,Hiratana Hajime.et al.. Enzyme imunoassay method for epidermal growth factor[P]. EP Patent:EP022810,1987-07-15.
[82] 孙国柱，吴志谷，周宝桐．重组人表皮生长因子对片皮体外培养生长的影响[J]．中华整形烧伤外科杂志，1996，12(6)：431-432．
[83] Singh G.,Foster C.S..Epidermal growth factor in alkaliburned corneal epitheliar Wound healing[J]. Am.J.Ophthalmol,1987,103:802-804.
[84] Noshimura. Heterogeneity of human epidermal growth factor/urogastrone from human urine[J]. Chem. Pharm. Bull.,1985,33(9),4037-4040.
[85] Okumura.Improvement in wound healing by epidermal growth factor intment.Pharmaceutical Research,1990,7:1289-1293.
[86] Savage C.R.,Harper R..Human epidermal growth factor/urogastrone:rapid purification procedure and partial characterization[J]. Analytical Biochemistry, 1981,111:195-196.
[87] Nishamura,Toyohiko,Uchida,et al..Method of producing human epidermal growth factor[P].US Patent:US4528186,1983-06-09.
[88] Yamazaki Yoshio. Production of human epidermal growth factor[P]. JP Patent: JP62030718,1987-02-09.
[89] Harper R.A.,Pierce J.,Savage C.R.. Purification of human growth factor by monoclonal antibody chromatography[J]. Methods in Enzynology, 1987,146:1-13.
[90] Shing Y.,Davidson S.,Klagsbrun M.. Purification of polypeptide growth factors from milk[J].Method in Enzynology,1987,146,42-48.
[91] Sindrey, Dennis R.,Dwivedi,et al.. Method of purifying human epidermal growth factor[P].US Patent:US5290920,1994-03-01.
[92] Bykov V.A.,Nikitina Z.K.,Spivak S.M.,et al. Method of isolation and purification of human epidermal growth factor(h-EGF)[P].RU Patent:RU2108341,1998-04-10.
[93] Diff. Methods in purification of hEGF, BICH366,Biot Sem..
[94] Noh Kyu-seung,Jung Joo-young,Ko Ryeu-wook,et al.. Puritification process of recombinant human epidermal growth factor[P]. KR Patent:KR9701508,1997-02-11.
[95] 童望宇．人表皮生长因子工艺的应用基础研究[D]．杭州：浙江大学，2001，3．
[96] Jo Jung-Myong, Kang Ⅱ-Ku. Method for purification of epidermal growth factor[P]. KR Patent:KR9208370,1992-09-26.
[97] Kim Kyu-Don,Kim Soon-Ha. Elimination of pigment substances during purification of human growth factor[P].KR Patent:KR9702490,1997-03-05.
[98] 陆兵，谢英华，汤国营，等．重组人表皮细胞生长因子的稳定性[J]．生物技术通讯，2001，12(3)：194-197．
[99] 王世玲，周亮，马建丽，等．外用重组人表皮生长因子对健康人体及烧伤患者不良反应试验[J]．中国新药杂志，2000，9(1)：34-36．
[100] Skokan S.J.,Davis R.H.. principle of wound healing and growth factor consideration[J].J.Am. Prodia. Med.associ.,1993,83(4):223.
[101] Madeleine bastide,Elisabeth Papaconstantin,Jean-Jacques Etienne,et al..Compsn.Contg.Infinitesimal dilution of epidermal growth factor stimulates normal cells but inhibits malignant ones and is useful in cosmetic and pharmaceutical copsns.[P].FR Patent:FR2695556,1994-03-18.
[102] Urea M.S.,Merryweath J.P.,Mallenbach G.T.,et al.. Proc. Natl. Acad. Sci. USA, 1983,80:7461-7465.
[103] 林跃生，王敏华，陈家祺，等．重组人表皮生长因子促进角膜上皮损伤修复的研究[J]．中国实用眼科杂志，2000，18(11)：700-703．
[104] 刘兰，饶春明，丁有学，等．重组人表皮生长因子生物活性国家标准品的研制[J]．中国生物制品杂志，2002，15(3)：175-176．
[105] 陆兵，谢英华，李京京，等．重组人表皮生长因子不同剂型用于创伤治疗的实验研究[J]．中华临床医药，2002，3(3)：6-7．
[106] Finkenaur, Amyl.. Stabilized compositions containing epidermal growth factor[P]. US Paten:U S4717717,1988-01-05.
[107] 张许昌，毛红．表皮生长因子的性质及其在化妆品中的应[J]．日用化学工业，1994，(1)：22-28．
[108] Brown G.L.,Nanney L.B.,Giffen J.,et al.. Enhancement of wound healing by topical treament with epidermal growth factor[J].New.Eng.J.Med.,1989,321:76-79.
[109] Ham Seung Hee,Kang Jin Seok,Chung Jong Keun,et al..Topital composition containing human epidermal growth factor[P].WO Patent:WO9944631,1999-09-01.
[110] Kim young-Jun,Shon Mi-Young,Lee Jang-Won,rt al.. Astable composition comprising epidermal growth factor as an active ingredient[P]. WO Patent:WO0162276.2001-08-30.
[111] 周建来，宋业光，王英书．表皮生长因子促进皮肤创面愈合的研究[J]．中国修复重建外科杂志，1997，11(5)：267-268．
[112] 廖镇江，黄伯高，向军，等．人烧伤创面应用表皮生长因子的临床评价[J]．上海第二医科大学学报，1996，16(4)：266-268．
[113] 王世玲，郭振朵，周一平，等．重组人表皮生长因子促进慢性创面的愈合[J]．中华创伤杂志，1998，14(6)：348-349．
[114] 衣承尔，陈玉林，韦多，等．重组人表皮生长因子对Ⅱ度烧伤创面愈合的促进作用[J]．中华创伤杂志，1998，14(6)：350-352．
[115] 王世玲，郭振荣，周亮，等．重组人表皮生长因子对促进烧伤创面的愈合作用[J]．中国临床药理学杂志，1998，14(3)：150-154．
[116] 姜国辉，唐刚华，苗小春，等．重组人表皮生长因子透皮吸收实验研究[J]．中国药学杂志，1999，134(11)：746-748．
[117] 张祥明，周和平．细胞活素(rhEGF)Ⅱ期临床研究总结报告[J]．数理医药学杂志，1999，12(1)：22-23．
[118] 周亮，王世玲，马建丽，等．重组人表皮生长因子治疗烧伤100例[J]．中国新药杂志，2000，9(6)：399-401．
[119] 王世玲，周亮，李利根，等．重组人表皮生长因子软膏治疗烧伤创面的安全性研究[J]．中国临床药理学杂志，2001，(4)：268-271．
[120] Latlo M.,Kahari V.M.,Niinikoshi J.,et al.. Epidermal growth factor increases collage production in granulation tissue by stimulation of fibroblast proliferation and not by activivation of procollagen genes[J]. Biochem.J.,1987,247:385-388.
[121] Rian A.M.,Leo T.F.. Invest Ophthalmol Vis. Scr., 1995,36:2120-2121.
[122] Konturek S.J.,Brzozowaki T.,Drozdowicz D.,et al..Role of growth factor in gastroduodenal
protection and healing of peptie ulcer[J]. Gastroenterol clin. North. Am., 1990,19:41-44.
[123] Szabo S.,Kusstatscher S.,Sakoulas G.,et al,, Growth factors:new "endogenous drugs" for ulcer healing[J]. Scand J.Gastroenterol suppl.,1995,210(suppl):15-18.
[124] 许春娣，奚容平，丁红珍，等．基因重组人表皮生长因子对胃黏膜保护的实验研究[J]．中华儿科杂志，1997，35(12)：635-638．
[125] 杨琨，田红燕，吕平，等．表皮生长因子对肝损害动物的保护作用[J]．实用美容整形外科杂志，2000，11(1)：14-15．
[126] 王世玲，张皖清，周亮，等．重组人表皮生长因子凝胶随机对照治疗口腔溃疡临床研究[J]．中国新药杂志，2002，11(5)：396-398．
[127] 张烽，顾玉东，徐建光，等．外源性表皮生长因子对大鼠坐骨神经损伤后神经元保护的实验研究[J]．上海医学，2000，23(7)：411-413．
[128] Tong J.X.,Keith M.R.. Diphenyliperazine enhance regeneration after facial nerve injury[J].J.Neurocytol.,1997,26:339-347.
[129] Richard,Morrison H.I.,Kornbluma F.M.,et al..Trophic stimulation of cultured neuros from neonatal rat brain by epidermal growth factor[J].Science, 1987.238:72-75.
[130] Yoichi M..Ann.Surg.,1990,211:263-265.
[131] Gazzaniga Annibale,Stroppl Federico,Gianesllo Valter.A pharmaeceutical composition in the form of a cream for dermal and ophthalmic use[P].EP Patent:EP0205051,1986-12-17.
[132] Gonzales-Lopez Tania, Rodriguez-Machado Joxse,Fernandez-Aragon Guillermo,et al..Use of human recombinant epidermal growth factor in the manufacture of a medicament for treating ance[P].WO Patent:WO9616669,1996-06-06.
[133] Wu Wenjun,Wu Wutong, Yao Wenbing. EGF essence for protecting skin and special conditioning liquid[P].CN Patent:CN 1175402,1998-03-11.
[134] Chery Guttman.Biorngineered cosmetics join bayyle vs.aging skin[J].Dermatogy Times,1999,(7):67-68.
[135] Detsina A.N.,Vjazovaja E.A.,Vachinskinj A.G.. Method of production of regenerating cosmetics cream for skin care[P].RU Patent:RU2144815,2000-01-27.
[136] Jo Byon KII. Cosmetic composition for protecting skin comprising retinal and epidermal growth factor[P].JP Patent:JP2001181176,2001-07-03.
[137] Nishigama, A.Cutive. Compositon in use of oral[P]. J P Patent:JP263115, 1987-05-23.
[138] Tran Toan,Greff Dariel. New processes and compositons for slowing down hair regrowth after depilation[P].FR Patent:FR2571615,1986-04-18.
[139] Moore G.P.M.,Panaretto B.A.,Robertson D.. Inhibition of wool growth in merina sheep by epidermal growth factor and a derivative[J]. Aust. J.Biol. Sci., 1982,35:163-172.
[140] Na kagawa S.,Yoshida S.,Mirao Y.,et al. Biological effects of biosynthetic human EGF on the growth of mammalian ceils in vitro[J]. Differentiation. 1985,29:294-298.
[2] S. Cohen. Purification of a nerve-growth promoting from mouse salivary gland and its neurocytotoxic antiserum[J]. Proc.Natl.Acad. Sci. USA, 1960,46: 302-310.
[3] J.Taylor, W.M.Michell,S.Cohen. Epidermal growth factor:physical and chemical properties[J]. J.Bioc.Chem, 1972.247:5928-5934.
[4] Gregory H.. Isolation and structure of urogastore and its relationship to epidermal growth factor[J]. Nature, 1975,257:325-327.
[5] 席全胜，钱欣国，甘人宝．EGF家族研究进展[J]．生命的科学，1999，19 (3)：135-137．
[6] Bell G.I.,Fong N.M.,Stempien M.M.,et al..Human epidermal growth factor precursor:cDNA sequence,expression in vitro and gene organiation[J]. Nucleic Acid Research, 1986,14:8427-8446.
[7] Pessonen. Characterization of material with epidermal growth factor immunoreactivity in human serum and platets[J]. Clin.Endo.Matab, 1989,68:486-488.
[8] G. Carpenter, S.Cohen. Epidermal growth factor[J]. Annu.Rev.Biochem., 1979.48:193-196.
[9] U.Shiro H.,Cohen S.. Affinity labeling of the protein kinase associated with the epidermal growth factor receptor in membrane vesicles from A431 cells[J]. Biol. Chem., 1980,255: 8363-8365.
[10] Savage C.R.,Hasb J.H.,Cohen S.. Epidermal growth factor:location of disulfide bonds[J]. J.Biol. Chem., 1973,247:7612-7622.
[11] Cooke R.M.,Wilkinson A.J.,Baron M.,et al.. The solution structure of human epidermal growth factor[J]. Nature, 1987,327:339-341.
[12] Kohda D.,Go N.,Hayashi K.,et al.. Tertiary structure of mouse epidermal growth factor: determination by two-climensinal proton NMR[J]. The Journal of Biochemistry, 1988, 103:741-743.
[13] Fisher D.A.,Lakshmanan J.J.. Metabolism and effects of epidermal growth factor and related growth factor in mammals[J]. Endocrine Review, 1990,11 (3):418-442.
[14] Martin P.,Hopkinson-Woolley J.,Mccluskey J.. Growth factors and cutaneous repair[J]. Progr.Growth Factor Res., 1992,4:25-44.
[15] Gospodarowicz D.. Epidermal and nerve growth factors in mammalian development[J]. Ann.Rev. Biochem., 1981,43:251-265.
[16] S.Cohen. Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the new-born animal[J]. J.Bioc.Chem., 1962,237:1555-1556.
[17] 傅小兵，孙国柱，王亚平，等．表皮细胞生长因子与碱性成纤维细胞生长因子促进创面修复效应的比较性研究[J]．中国修复重建外科杂志，1999，13 (5)：278-282．
[18] Downwand J.. Close similamity of epidermal growth factor oncogene protein sequence[J]. Nature, 1984,307:521-527.
[19] Yarden Y.,Schlessinger J.. Epidermal growth factor induced rapid,reversible aggregation of the purified epidermal growth factor receptor[J]. Biochemistry, 1986, 26:1443-1451.
[20] Helin K.,Velu T.,Martin P.,et al. The biological activity of the human epidermal growth factor receptor is positively regulated by its C-terminal tyrosines[J]. Oncogene, 1991:625-832.
[21] Merlino G.T.. Epidermal growth factor receptor relation and funtion[J]. Semin Cancer Biol.,1990,(1):277-284.
[22] Hudson L.G.,Santon J.B.,Gill G.N. Regulation of epidermal growth factor gene expression[J]. Molecular Endocrinology, 1989,(3):400-408.
[23] May K.H.. J.Biol.Chem.,1989,264:17838-17844.
[24] Well S.A.. Proc.Natl.Acad. Sci. USA,1988,85:7597-7601.
[25] Chen W.S.. Nature, 1987,328:820-823.
[26] Wedegaertuner P.B.,Gill G.N.. J.Biol.Chem., 1989,264:11346-11353.
[27] Breyer M.D. Jacobson H.R.,Breyer J.A.J.Clin.Invest, 1988,82(10):1313-1320.
[28] Masayuki K.. Development of a sensitive enzyme immuoassay for human epidermal growth factor[J]. Clin.Chim.Acta, 1986,156:51-53.
[29] kasselberg Ag,Orth D.N.,Gray M.E.,et al.. Immuocy to chemical localization of human epidermal growth factor/urogastrone in several human tissues[J]. J.Histochem. Cytochem, 1985,33:315.
[30] Fuse H.. Epidermal growth factor contents in seminal plasma as a marker of prostatic function[J]. Arch Androl, 1992,29:79-81.
[31] Olsen P.S.. Role of epidermal growth factor in gastroduodenal mucosal protection[J]. J.Clin. Gastroenterol, 1988,suppl 1:146-147.
[32] Orsinib B.,Brocchi A.,Calabro A., et al.. radioimmunoassay of epidermal growth factor in human saliva and gastric juice[J]. Clin. Biochem., 1991,24:135-137.
[33] Sugimoto Kaname. Process for the production of human epidermal growth factor[P].US Patent: 19840619708,1986-11-04.
[34] March D.,Osborne E.,Roberts E.,et al.. Chemical synthesis of human epidermal growth factor and human type α-TGF[R]. Proceeding 10th American Peptide Symposium, 1988:202-203.
[35] 袁汉英，闵永洁，石松，等．人表皮生长因子基因的化学合成和克隆及其在酵母中的表达[J]．生物工程学报，1991，7(4)：300-306．
[36] Smith J.,Derbyshine R.B.,Dunthomel L.,et al. Gene, 1984,32:321-322.
[37] Oka T.,sakamoto S.,Miyoshi K.,et al.. Synthesis and secretion of human epidermal growth factor by scherichia coli.[J]. Proc.Natl.Acad.Sci.USA, 1985, 82:7212-7216.
[38] Kishimoto F.,Gomi H.,Kanaoka M.,et al.. Gene, 1986,45:311.
[39] Oka T.,Sumi S.,Fuwa T.,et al. Agric.Biol. Chem., 1987,51:1099.
[40] Batchikova N.V.,Altman I.B.,Sinyagina E.A.,et al.. Bioeng. Khirm, 1992,18:766-777.
[41] 甘人宝，黄培勇，袁宇，等．人表皮生长因子在大肠杆菌中高效表达和分泌[J]．生物化学与生物物理学报，1992，24 (6)：587-589．
[42] Fujimoto K.M.,Marumoto R.,Fukuda T.. J.Biol Chem., 1991,266:1728-1730.
[43] W.K.R. Wong,Sutherland M.L.. Excretion of heterologous proteins from E.coli[P].EP patent:EP0357391,1990-03-07.
[44] Yadward V.B.,Wison S.,Ward O.P..Production of human epidermal growth factor by an ampicillin resistant E. coli strain[J]. Biotechnology Letter, 1994,16:885-890.
[45] Xu Zhi-nan, Liu Gang,Cen Peilin,et al.. Factors influencing excretive production of human epidermal growth factor(hEGF) with recombinant Escherichia coli k12 system[J]. Bioprocess and Biosystem Engineering.2000,23(6):669-674.
[46] Flock J.I.,Fotheringham I.,Light J.,et al.. Mol.Gen.Genet., 1984:195:246-247.
[47] Yamagata H.,Nakahawa K.,Suzuki Y.,et al.. Proc.NatI.Acad.Sci.USA, 1989,86: 8589-8591.
[48] Ebisu S.,Takagi H.,Kadowaki K.,et al.. Production of human epidermal growth factor by Bacillus brevis increased with use of a stable plasmid from B. brevis 481[J]. Biosci. Biotech. Biochem., 1992,56:812-813.
[49] Brake A.J.,Merryweather J.P.,Coit D.G.,et al.. Proc.Natl.acad. Sci.USA, 1984,81: 4642-4644.
[50] Clements J.M.,Catlin G.H.,Price M.J.,et al.. Secretion of human epidermal growth factor from Saccharomyces cervisiae using synthetic sequence[J]. Gene, 1991,106:267-268.
[51] Barr. US patent:US5096825,1992.
特殊上标未编改
[52] Edwards. WO patent:WO9216626,1993.
[53] Clare J.J.,Romanos M.A.,Rayment F.B.,et al..Production of mouse epidermal growth factor in yeast:High-level secretion using Pichia pastoris strains containing multiple copy genes[J].Gene, 1991,105:205-207.
[54] 黄秉仁，蔡良婉，廖洪涛，等．人工合成表皮生长因子基因在甲基型酵母中的高效表达[J]．中国生物化学与分子生物学报，1998，14(5)：512-517．
[55] P.V. Hamsa, P. Kachroo,B.B.Chattoo..Production and secretion of biologically active human epidermal growth factor in Yarrowia lipolytica[J].Current Genetics,1998,33(3):231-237.
[56] Joo-Hyung Heo,Hye Soon Won,Hyun Ah Kang, et al..Purification of recombinant human epidermal growth factor secreted from the methylotrophic yeast Hansenula polymorpha[J].Protein Expression and Purification,2002,24:117-122.
[57] Mroczkowshi B.. Methods Enzymol,1991,198:175-177.
[58] 胡大海，陈璧，汤朝武，等．人表皮生长因子基因真核表达质粒pcDNA_3-hEGF的构建[J]．西北国防医学杂志，2000，21(1)：18-22．
[59] 董肇杨，陈玉林，戴方平，等．hEGF转基因表皮细胞的制备及其EGF蛋白表达[J]．中华整形外科杂志，2000，16(3)：177-179．
[60] Gao Jianwei,Dai Ziyu,Hooker Brian S.,et al..Method of producing human epidermal growth factor from whole plants or plant cell cultures[P].WO patent: WO9821348,1998-05-22.
[61] 戴激，施定基，张卉，等．人表皮生长因子(hEGF)基因在蓝藻中的表达[J]．植物学报，2001，43(12)：1260-1261．
[62] 李守芬，苏钟浦，王立坤，等．人表皮生长因子的三种分离方法及其生物学活性[J]．中国医科大学学报，1992，21(3)：172-174．
[63] R.C.Huang,E.Lam,Y.H.Chen,et al.. Human epidermal growth factor excreted by recombinant Escherichia coli k-12 has the correct N-terminus and its fully bioactive[J],process Biochemistry, 1999,35:1-5.
[64] 黄秉仁，蔡良婉，王欣，等．甲基营养型酵母系统表达的重组人表皮生长因子的纯化及其性质[J]．中国医学科学院学报，2001，23(2)：106-110．
[65] 单继贵，秦德安，何学民，等．孕妇尿液中表皮生长因子的分离、纯化及活性检测[J]．华东师范大学学报(自然科学版)，1996，(4)：89-94．
[66] Geory-Nascimento C.,Gyenes A.,Halloran S.M.,et al.. Biochemistory,1988,27: 797-799.
[67] 张伟，麦荫乔．人尿表皮生长因子的提取、纯化[J]．白求恩医科大学学报，1993，19(2)：210-211．
[68] 阮文权，李玉忠，杨邱明，等．重组大肠杆菌生产人表皮生长因子的发酵条件[J]．无锡轻工大学学报，2001，20(5)：489-492．
[69] Vazquez J.,Freyre M.,Duarte,et al..Biotechol.Apl.,1990,7:42-45.
[70] 赵明，王会信，邱宝成，等．小鼠额下腺表皮生长因子的分离、纯化与鉴定[J]．生物化学杂志，1992，8(1)：61-66．
[71] 贺师鹏，黄天贵，黄力新，等．表皮生长因子的lodogen标记法与放射性比度测定[J]．北京医科大学学报，1992，24(5)：416．
[72] 叶春婷，李斯明，查健群，等．结晶紫比色法检测表皮生长因子生物活性的探讨[J]．中华微生物和免疫学杂志，1997，17(2)：154-155．
[73] 赵忠良，路凡，陈南春，等．人表皮生长因子在大肠杆菌中的融合表达与活性分析[J]．第四军医大学学报，1997，18(5)：497-498．
[74] Tim Mosmann. Rapid colorimetric assay for celluar growth and surrival:application to proliferation and proliferation and cytoxicity assays[J].J.Immunological Methods,1983,63:55-57.
[75] 何海琼，王金发，陈妍，等．重组人表皮生长因子生物活性的测定[J]．广东医学，1998，
19(5)：324-325．
[76] 周志荣，管樱．小鼠颚下腺提取液中表皮生长因子的含量测定[J]．南京医科大学学报，1994，14(2)：174-176．
[77] G.Carpenter, Stanly Cohen. A biological assay for epidermal growth factor/urogastrone and related polypeptides[J]. Anal. Biochem.,1985,153:279-281.
[78] Pesonen K.,Alfthan H.,Stenman U.H.,et al. Anal. Biochem.,1986,157:208.
[79] 刘农乐，蒋滋慧，赵明，等．人表皮生长因子的发光免疫测定法——吖啶酯标记抗体的应用[J]．生物化学与生物物理进展，1993，20(1)：43-45．
[80] Kashimata M.,Hiramatsu M.,Kumegawa M.et al..Shika Kiso Lgakkai Zasshi, 1985,7:1260-1263.
[81] Nishimuro Satoshi,Hayashi Kyozo,Hiratana Hajime.et al.. Enzyme imunoassay method for epidermal growth factor[P]. EP Patent:EP022810,1987-07-15.
[82] 孙国柱，吴志谷，周宝桐．重组人表皮生长因子对片皮体外培养生长的影响[J]．中华整形烧伤外科杂志，1996，12(6)：431-432．
[83] Singh G.,Foster C.S..Epidermal growth factor in alkaliburned corneal epitheliar Wound healing[J]. Am.J.Ophthalmol,1987,103:802-804.
[84] Noshimura. Heterogeneity of human epidermal growth factor/urogastrone from human urine[J]. Chem. Pharm. Bull.,1985,33(9),4037-4040.
[85] Okumura.Improvement in wound healing by epidermal growth factor intment.Pharmaceutical Research,1990,7:1289-1293.
[86] Savage C.R.,Harper R..Human epidermal growth factor/urogastrone:rapid purification procedure and partial characterization[J]. Analytical Biochemistry, 1981,111:195-196.
[87] Nishamura,Toyohiko,Uchida,et al..Method of producing human epidermal growth factor[P].US Patent:US4528186,1983-06-09.
[88] Yamazaki Yoshio. Production of human epidermal growth factor[P]. JP Patent: JP62030718,1987-02-09.
[89] Harper R.A.,Pierce J.,Savage C.R.. Purification of human growth factor by monoclonal antibody chromatography[J]. Methods in Enzynology, 1987,146:1-13.
[90] Shing Y.,Davidson S.,Klagsbrun M.. Purification of polypeptide growth factors from milk[J].Method in Enzynology,1987,146,42-48.
[91] Sindrey, Dennis R.,Dwivedi,et al.. Method of purifying human epidermal growth factor[P].US Patent:US5290920,1994-03-01.
[92] Bykov V.A.,Nikitina Z.K.,Spivak S.M.,et al. Method of isolation and purification of human epidermal growth factor(h-EGF)[P].RU Patent:RU2108341,1998-04-10.
[93] Diff. Methods in purification of hEGF, BICH366,Biot Sem..
[94] Noh Kyu-seung,Jung Joo-young,Ko Ryeu-wook,et al.. Puritification process of recombinant human epidermal growth factor[P]. KR Patent:KR9701508,1997-02-11.
[95] 童望宇．人表皮生长因子工艺的应用基础研究[D]．杭州：浙江大学，2001，3．
[96] Jo Jung-Myong, Kang Ⅱ-Ku. Method for purification of epidermal growth factor[P]. KR Patent:KR9208370,1992-09-26.
[97] Kim Kyu-Don,Kim Soon-Ha. Elimination of pigment substances during purification of human growth factor[P].KR Patent:KR9702490,1997-03-05.
[98] 陆兵，谢英华，汤国营，等．重组人表皮细胞生长因子的稳定性[J]．生物技术通讯，2001，12(3)：194-197．
[99] 王世玲，周亮，马建丽，等．外用重组人表皮生长因子对健康人体及烧伤患者不良反应试验[J]．中国新药杂志，2000，9(1)：34-36．
[100] Skokan S.J.,Davis R.H.. principle of wound healing and growth factor consideration[J].J.Am. Prodia. Med.associ.,1993,83(4):223.
[101] Madeleine bastide,Elisabeth Papaconstantin,Jean-Jacques Etienne,et al..Compsn.Contg.Infinitesimal dilution of epidermal growth factor stimulates normal cells but inhibits malignant ones and is useful in cosmetic and pharmaceutical copsns.[P].FR Patent:FR2695556,1994-03-18.
[102] Urea M.S.,Merryweath J.P.,Mallenbach G.T.,et al.. Proc. Natl. Acad. Sci. USA, 1983,80:7461-7465.
[103] 林跃生，王敏华，陈家祺，等．重组人表皮生长因子促进角膜上皮损伤修复的研究[J]．中国实用眼科杂志，2000，18(11)：700-703．
[104] 刘兰，饶春明，丁有学，等．重组人表皮生长因子生物活性国家标准品的研制[J]．中国生物制品杂志，2002，15(3)：175-176．
[105] 陆兵，谢英华，李京京，等．重组人表皮生长因子不同剂型用于创伤治疗的实验研究[J]．中华临床医药，2002，3(3)：6-7．
[106] Finkenaur, Amyl.. Stabilized compositions containing epidermal growth factor[P]. US Paten:U S4717717,1988-01-05.
[107] 张许昌，毛红．表皮生长因子的性质及其在化妆品中的应[J]．日用化学工业，1994，(1)：22-28．
[108] Brown G.L.,Nanney L.B.,Giffen J.,et al.. Enhancement of wound healing by topical treament with epidermal growth factor[J].New.Eng.J.Med.,1989,321:76-79.
[109] Ham Seung Hee,Kang Jin Seok,Chung Jong Keun,et al..Topital composition containing human epidermal growth factor[P].WO Patent:WO9944631,1999-09-01.
[110] Kim young-Jun,Shon Mi-Young,Lee Jang-Won,rt al.. Astable composition comprising epidermal growth factor as an active ingredient[P]. WO Patent:WO0162276.2001-08-30.
[111] 周建来，宋业光，王英书．表皮生长因子促进皮肤创面愈合的研究[J]．中国修复重建外科杂志，1997，11(5)：267-268．
[112] 廖镇江，黄伯高，向军，等．人烧伤创面应用表皮生长因子的临床评价[J]．上海第二医科大学学报，1996，16(4)：266-268．
[113] 王世玲，郭振朵，周一平，等．重组人表皮生长因子促进慢性创面的愈合[J]．中华创伤杂志，1998，14(6)：348-349．
[114] 衣承尔，陈玉林，韦多，等．重组人表皮生长因子对Ⅱ度烧伤创面愈合的促进作用[J]．中华创伤杂志，1998，14(6)：350-352．
[115] 王世玲，郭振荣，周亮，等．重组人表皮生长因子对促进烧伤创面的愈合作用[J]．中国临床药理学杂志，1998，14(3)：150-154．
[116] 姜国辉，唐刚华，苗小春，等．重组人表皮生长因子透皮吸收实验研究[J]．中国药学杂志，1999，134(11)：746-748．
[117] 张祥明，周和平．细胞活素(rhEGF)Ⅱ期临床研究总结报告[J]．数理医药学杂志，1999，12(1)：22-23．
[118] 周亮，王世玲，马建丽，等．重组人表皮生长因子治疗烧伤100例[J]．中国新药杂志，2000，9(6)：399-401．
[119] 王世玲，周亮，李利根，等．重组人表皮生长因子软膏治疗烧伤创面的安全性研究[J]．中国临床药理学杂志，2001，(4)：268-271．
[120] Latlo M.,Kahari V.M.,Niinikoshi J.,et al.. Epidermal growth factor increases collage production in granulation tissue by stimulation of fibroblast proliferation and not by activivation of procollagen genes[J]. Biochem.J.,1987,247:385-388.
[121] Rian A.M.,Leo T.F.. Invest Ophthalmol Vis. Scr., 1995,36:2120-2121.
[122] Konturek S.J.,Brzozowaki T.,Drozdowicz D.,et al..Role of growth factor in gastroduodenal
protection and healing of peptie ulcer[J]. Gastroenterol clin. North. Am., 1990,19:41-44.
[123] Szabo S.,Kusstatscher S.,Sakoulas G.,et al,, Growth factors:new "endogenous drugs" for ulcer healing[J]. Scand J.Gastroenterol suppl.,1995,210(suppl):15-18.
[124] 许春娣，奚容平，丁红珍，等．基因重组人表皮生长因子对胃黏膜保护的实验研究[J]．中华儿科杂志，1997，35(12)：635-638．
[125] 杨琨，田红燕，吕平，等．表皮生长因子对肝损害动物的保护作用[J]．实用美容整形外科杂志，2000，11(1)：14-15．
[126] 王世玲，张皖清，周亮，等．重组人表皮生长因子凝胶随机对照治疗口腔溃疡临床研究[J]．中国新药杂志，2002，11(5)：396-398．
[127] 张烽，顾玉东，徐建光，等．外源性表皮生长因子对大鼠坐骨神经损伤后神经元保护的实验研究[J]．上海医学，2000，23(7)：411-413．
[128] Tong J.X.,Keith M.R.. Diphenyliperazine enhance regeneration after facial nerve injury[J].J.Neurocytol.,1997,26:339-347.
[129] Richard,Morrison H.I.,Kornbluma F.M.,et al..Trophic stimulation of cultured neuros from neonatal rat brain by epidermal growth factor[J].Science, 1987.238:72-75.
[130] Yoichi M..Ann.Surg.,1990,211:263-265.
[131] Gazzaniga Annibale,Stroppl Federico,Gianesllo Valter.A pharmaeceutical composition in the form of a cream for dermal and ophthalmic use[P].EP Patent:EP0205051,1986-12-17.
[132] Gonzales-Lopez Tania, Rodriguez-Machado Joxse,Fernandez-Aragon Guillermo,et al..Use of human recombinant epidermal growth factor in the manufacture of a medicament for treating ance[P].WO Patent:WO9616669,1996-06-06.
[133] Wu Wenjun,Wu Wutong, Yao Wenbing. EGF essence for protecting skin and special conditioning liquid[P].CN Patent:CN 1175402,1998-03-11.
[134] Chery Guttman.Biorngineered cosmetics join bayyle vs.aging skin[J].Dermatogy Times,1999,(7):67-68.
[135] Detsina A.N.,Vjazovaja E.A.,Vachinskinj A.G.. Method of production of regenerating cosmetics cream for skin care[P].RU Patent:RU2144815,2000-01-27.
[136] Jo Byon KII. Cosmetic composition for protecting skin comprising retinal and epidermal growth factor[P].JP Patent:JP2001181176,2001-07-03.
[137] Nishigama, A.Cutive. Compositon in use of oral[P]. J P Patent:JP263115, 1987-05-23.
[138] Tran Toan,Greff Dariel. New processes and compositons for slowing down hair regrowth after depilation[P].FR Patent:FR2571615,1986-04-18.
[139] Moore G.P.M.,Panaretto B.A.,Robertson D.. Inhibition of wool growth in merina sheep by epidermal growth factor and a derivative[J]. Aust. J.Biol. Sci., 1982,35:163-172.
[140] Na kagawa S.,Yoshida S.,Mirao Y.,et al. Biological effects of biosynthetic human EGF on the growth of mammalian ceils in vitro[J]. Differentiation. 1985,29:294-298.