重组EpoAB-NGF模拟肽在大肠杆菌中的表达、纯化及生物活性初步研究
摘要
红细胞生成素(Epo)最先作为一个促进红细胞生成的生长因子被确认,后来发现其在神经、血管等系统中具有不依赖于红细胞生成作用的细胞营养保护功能,并且这种营养保护作用已经被定位到了其序列中一个由17个氨基酸组成的多肽顺序上,称作EpoAB肽(AEHCSLNENITVPDTKV)。
神经生长因子(NGF)可通过结合靶细胞膜上的高/低亲和力受体(Trk/p75NTR)调控神经元的存活、增殖、分化、突触生长和凋亡等生命过程,有较好的临床应用前景。本室利用构建的表达p75NTR的R2L1凋亡细胞模型和噬菌体展示文库筛选出2个有抗凋亡作用的p75NTR的模拟配基,即NGF的模拟肽(NGF9和NGF12)。
本研究在前期工作的基础上,构建了原核表达GST-EpoAB-NGF9/12融合蛋白的重组质粒,筛选相应的表达菌株,优化了表达和纯化条件,获得融合蛋白,并通过刺激PC12细胞轴突生长和抑制R2L1细胞去血清凋亡的实验,初步证实了融合蛋白对神经细胞的营养保护作用。另外,尝试建立了EAE动物模型并用Epo和EpoAB进行了初步治疗,为进一步验证EpoAB-NGF9/12在动物体内的活性积累了经验。
本研究工作的开展,可为日后研究该重组蛋白的血脑屏障通透性,神经营养保护作用的多能性和可能的作用机制等打下基础,进而探索其在神经损伤、神经退行性病变动物模型或人类相应疾病的临床治疗上的应用前景。
Erythropoietin (Epo) was originally identified as the principal regulator of erythroid progenitor cells. It was newly found that Epo exerted cytoprotive and nutritional effects in neural system and blood vascular systerm which does not depend on erythopoiesis. Moreover, this nerval protective and nutritional action has been positioned in a polypeptide sequence composed of seventeen amino acids named EpoAB peptide (AEHCSLNENITVPDTKV).
Nerve grow factor (NGF) can bind to its receptors with high or low affinity (Trk A or p75NTR), and regulate series of vital processes in nerve cells such as survival, multiplication, differentiation, synaptic growth and apoptosis, exhibiting a promising clinical application perspective. In the previous study, we had screened out two peptides (NGF9/NGF12) that could bind to p75NTR and mimic anti-apoptosis function of NGF on R2L1 cultured with depriving serum medium by utilizing established R2L1 cell model which expressed p75NTR and phage display peptide library.
Based on the previous studies, our research focuses on obtaining the fusion protein of EpoAB-NGF9/12 and testing its biological activity in vitro and in vivo. The following major work has been done:
1. Constructed prokaryotic plasmids which can express GST-EpoAB-NGF9/12 in E.coli.;
2. Optimized conditions of protein expression and purification of GST-EpoAB-NGF9/12 with high yield (about 40 mg/L) and high purity (about 90%);
3. Verified NGF-like biological activity of GST-EpoAB-NGF9/12 in vitro on two kinds of cell line, tested neurite outgrowth induction activity of on PC 12 cell line and anti-apoptosis fuction on R2L1 cell model cultured with depriving serum medium;
4. Attempted to establish EAE animal model on C57BL/6 mice and treat them with Epo and EpoAB, and obtained much experience for the further study on biological activity of EpoAB-NGF9/12 in vivo.
Our study above has layed a foundation for the further study on blood-brain barrier permeability, nerval protective/nutritional pluripotency and mechanism of EpoAB-NGF9/12, and will benefit the clinical therapy of nerve injury, neurodegenerative disease in animal model or treatment for relevant human diseases.
神经生长因子(NGF)可通过结合靶细胞膜上的高/低亲和力受体(Trk/p75NTR)调控神经元的存活、增殖、分化、突触生长和凋亡等生命过程,有较好的临床应用前景。本室利用构建的表达p75NTR的R2L1凋亡细胞模型和噬菌体展示文库筛选出2个有抗凋亡作用的p75NTR的模拟配基,即NGF的模拟肽(NGF9和NGF12)。
本研究在前期工作的基础上,构建了原核表达GST-EpoAB-NGF9/12融合蛋白的重组质粒,筛选相应的表达菌株,优化了表达和纯化条件,获得融合蛋白,并通过刺激PC12细胞轴突生长和抑制R2L1细胞去血清凋亡的实验,初步证实了融合蛋白对神经细胞的营养保护作用。另外,尝试建立了EAE动物模型并用Epo和EpoAB进行了初步治疗,为进一步验证EpoAB-NGF9/12在动物体内的活性积累了经验。
本研究工作的开展,可为日后研究该重组蛋白的血脑屏障通透性,神经营养保护作用的多能性和可能的作用机制等打下基础,进而探索其在神经损伤、神经退行性病变动物模型或人类相应疾病的临床治疗上的应用前景。
Erythropoietin (Epo) was originally identified as the principal regulator of erythroid progenitor cells. It was newly found that Epo exerted cytoprotive and nutritional effects in neural system and blood vascular systerm which does not depend on erythopoiesis. Moreover, this nerval protective and nutritional action has been positioned in a polypeptide sequence composed of seventeen amino acids named EpoAB peptide (AEHCSLNENITVPDTKV).
Nerve grow factor (NGF) can bind to its receptors with high or low affinity (Trk A or p75NTR), and regulate series of vital processes in nerve cells such as survival, multiplication, differentiation, synaptic growth and apoptosis, exhibiting a promising clinical application perspective. In the previous study, we had screened out two peptides (NGF9/NGF12) that could bind to p75NTR and mimic anti-apoptosis function of NGF on R2L1 cultured with depriving serum medium by utilizing established R2L1 cell model which expressed p75NTR and phage display peptide library.
Based on the previous studies, our research focuses on obtaining the fusion protein of EpoAB-NGF9/12 and testing its biological activity in vitro and in vivo. The following major work has been done:
1. Constructed prokaryotic plasmids which can express GST-EpoAB-NGF9/12 in E.coli.;
2. Optimized conditions of protein expression and purification of GST-EpoAB-NGF9/12 with high yield (about 40 mg/L) and high purity (about 90%);
3. Verified NGF-like biological activity of GST-EpoAB-NGF9/12 in vitro on two kinds of cell line, tested neurite outgrowth induction activity of on PC 12 cell line and anti-apoptosis fuction on R2L1 cell model cultured with depriving serum medium;
4. Attempted to establish EAE animal model on C57BL/6 mice and treat them with Epo and EpoAB, and obtained much experience for the further study on biological activity of EpoAB-NGF9/12 in vivo.
Our study above has layed a foundation for the further study on blood-brain barrier permeability, nerval protective/nutritional pluripotency and mechanism of EpoAB-NGF9/12, and will benefit the clinical therapy of nerve injury, neurodegenerative disease in animal model or treatment for relevant human diseases.
引文
[1]Maiese K, Li F, Chong ZZ., New avenues of exploration for erythropoietin. JAMA.2005,293(1): 90-95.
[2]G. Grasso et al., The role of erythropoietin in neuroprotection:therapeutic perspectives. Drug news Perspect.2007,20(5):315-320.
[3]Quelle FW, Wang D, Nosaka T, et al. Erythropoietin induces activation of Stat5 through association with specific tyrosines on the receptor that are not required for a mitogenic response. Mol Cell Biol 1996,16(4):1622-31.
[4]Kilic E, Kilic U, Soliz J, Bassetti CL, Gassmann M, Hermann DM. Brain-derived erythropoietin protects from focal cerebral ischemia by dual activation of ERK-1/-2 and Akt pathways. FASEB J 2005,19(14):2026-8.
[5]Chong ZZ, Kang JQ, Maiese K. Erythropoietin fosters both intrinsic and extrinsic neuronal protection through modulation of microglia, Aktl, Bad, and caspase-mediated pathways. Br J Pharmacol 2003,138(6):1107-18.
[6]Digicaylioglu M, Lipton SA. Erythropoietin-mediated neuroprotection involves cross-talk between Jak2 and NF-kappaB signaling cascades. Nature 2001,412(6847):641-7
[7]Brines ML, Ghezzi P, Keenan S, et al. Erythropoietin crosses the blood-brain barrier to protect against experimental brain injury. Proc Natl Acad Sci USA.2000,97(19):10526-10531
[8]Ehrenreich H, Hasselblatt M, Dembowski C, et al. Erythropoietin therapy for acute stroke is both safe and beneficial. Mol Med,2002,8(8):495-505.
[9]Silverberg DS, Wexler D, Blum M, et al. The effect of correction of anaemia in diabetics and non-diabetics with severe resistant congestive heart failure and chronic renal failure by subcutaneous erythropoietin and intravenous iron.. Nephral Dial Transplant.2003,18:141-146.
[10]Erbayraktar S, Grasso G, Sfacteria A, et al. Asialoerythropoietin is a nonerythropoietic cytokine with broad neuroprotective activity in vivo. Proc Natl Acad Sci.2003,100:6741-6746
[11]Fiordaliso F, Chimenti S, Staszewsky L,et al. A nonerythropoietic derivative of erythropoietin protects the myocardium from ischemia-reperfusion injury. Proc Natl Acad Sci,2005; 102(6): 2046-2051.
[12]Brines M, Grasso G, Fiordaliso F, et al. Erythropoietin mediates tissue protection through an erythropoietin and common beta-sbunit heteroreceptor. Proc Natl Acad Sci,2004,101 (410): 14907-14912.
[13]Campana WM, Misasi R, O'Brien JS. Identification of a neurotrophic sequence in erythropoietin. Int J Mol Med.1998,1(1):235-241.
[14]Bothwell M. Functional interactions of neurotrophins and neurotrophin receptors. Annu Rev Neurosci.1995,18:223-53
[15]左明雪.细胞和分子神经生物学.北京：高等教育出版社&施普林格出版社,2000：289-296
[16]Anders Nykjaer, Thomas E Willnow, Claus Munck Petersen. p75NTR-live or let die. Current Opinion in Neurobiology 2005,15:49-57
[17]Hefti F, Weiner WJ. Nerve growth factor and Alzheimer's disease. Ann Neurol,1986,20(3): 275-81
[18]曹琳,杨恒文等.NGF对坐骨神经损伤后腰椎与损伤神经MBP含量变化的影响,第三军医大学学报,2002,24：326
[19]Colangelo et al. A new nerve growth factor-mimetic peptide cctive on neuropathic pain in rats. J. Neurosci.,2008,28(11):2698-2709
[20]Huang BR,Gu JJ, ming H, et al. Differential actions of neurotrophins on apoptosis mediated by the low affinity neurotrophin receptor p75NTR in immortalized neuronal cell lines. Neurochem Int., wooo,36(1):55-65.
[21]高鹏,王欣,陈虹,黄秉仁.神经生长因子低亲和力受体(p75NTR)模拟配基的筛选.忠国生物化学与分子生物学报，2003，19(6)：769-774。
[22]Kono DH, Urban JL, Horvath SJ, et al, et al Two minor deter minants of myelin basic protein induce experimental allergic encephalomyelitis in SJL/J mice. JExp Med,1988,168:213-227.
[23]Li W, Maeda Y, Yuan RR, et al.Beneficial effect of erythropoietin on experimental allergic encephalomyelitis. Ann Neurol.2004,56 (6):767-777
[24]郭立安,阎哲,张晓楠,武丽华.二氯乙酸对蛋白质结构蛋白性稳定性的影响.第西军医大学学报，2001,22(22)
[25]李志雄,魏麓云.多发性硬化血脑屏障破坏的研究进展.实用医药杂志.2008,25(11)：1391-1393
[26]Yuan R, Maeda Y, Li W, Lu W, Cook S, Dowling P. Erythropoietin:a potent inducer of peripheral immuno/inflammatory modulation in autoimmune EAE. PLoS ONE.2008,3(4):1924
[27]Triaca V, Tirassa, P Aloe L. Presence of nerve growth factor and TrkA expression in the SVZ of EAE rats:evidence for a possible functional significance. Exp Neurol,2005,191(1):53-64
[28]邢清和,王永铭,郑荣远.影响EAE动物模型建立的因素分析.中国临床神经科学.2000,8(4)：305-306
[29]陆正齐,朱灿胜,郑雪平,王敦敬,胡学强.不同抗原诱导不同动物多发性硬化模型的比较.中山大学学报(医学科学处版),2008,29(16)：684-689
[30]Pluchino S, Quattrini A, Brambilla E, et al. Injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis. Nature,2003,422 (6933):688-694
[1]Prass K, Scharff A, Ruscher K, et al. Hypoxia-induced stroke tolerance in the mouse is mediated by erythropoietin. Stroke 2003,34(8):1981-6.
[2]Warnecke C, Zaborowska Z, Kurreck J, et al. Differentiating the functional role of hypoxia-inducible factor (HIF)-l alpha and HIF-2alpha (EPAS-1) by the use of RNA interference: erythropoietin is a HIF-2alpha target gene in Hep3B and Kelly cells. FASEB J 2004; 18(12): 1462-4.
[3]Yu X, Lin CS, Costantini F, Noguchi CT. The human erythropoietin receptor gene rescues erythropoiesis and developmental defects in the erythropoietin receptor null mouse. Blood 2001, 98(2):475-7.
[4]Digicaylioglu M, Bichet S, Marti HH, et al. Localization of specific erythropoietin binding sites in defined areas of the mouse brain. Proc Natl Acad Sci USA 1995,92(9):3717-20.
[5]Chin K, Oda N, Shen K, Noguchi CT. Regulation of transcription of the human erythropoietin receptor gene by proteins binding to GATA-1 and Sp1 motifs. Nucleic Acids Res 1995,23(15): 3041-9.
[6]Pandolfi PP, Roth ME, Karis A, et al. Targeted disruption of the GATA3 gene causes severe abnormalities in the nervous system and in fetal liver haematopoiesis. Nat Genet 1995, 11(1):40-4.
[7]Quelle FW, Wang D, Nosaka T, et al. Erythropoietin induces activation of Stat5 through association with specific tyrosines on the receptor that are not required for a mitogenic response. Mol Cell Biol 1996,16(4):1622-31.
[8]Kilic E, Kilic U, Soliz J, Bassetti CL, Gassmann M, Hermann DM. Brain-derived erythropoietin protects from focal cerebral ischemia by dual activation of ERK-1/-2 and Akt pathways. FASEB J 2005,19(14):2026-8.
[9]Chong ZZ, Kang JQ, Maiese K. Erythropoietin fosters both intrinsic and extrinsic neuronal protection through modulation of microglia, Aktl, Bad, and caspase-mediated pathways. Br J Pharmacol 2003,138(6):1107-18.
[10]Digicaylioglu M, Lipton SA. Erythropoietin-mediated neuroprotection involves cross-talk between Jak2 and NF-kappaB signaling cascades. Nature 2001,412(6847):641-7
[11]Juul SE, Stallings SA, Christensen RD. Erythropoietin in the cerebrospinal fluid of neonates who sustained CNS injury. Pediatr Res 1999,46(5):543-7.
[12]KnabeW, Knerlich F,Washausen S, et al. Expression patterns of erythropoietin and its receptor in the developing midbrain. Anat Embryol (Berl) 2004,207(6):503-12.
[13]Iyer NV, Kotch LE, Agani F, et al. Cellular and developmental control of O2 homeostasis by hypoxia-inducible factor 1 alpha. Genes Dev 1998,12(2):149-62.
[14]Urao N, Okigaki M, Yamada H, et al. Erythropoietin-mobilized endothelial progenitors enhance reendothelialization via Aktendothelial nitric oxide synthase activation and prevent neointimal hyperplasia. Circ Res 2006,98(11):1405-13.
[15]Kertesz N, Wu J, Chen TH, Sucov HM, Wu H. The role of erythropoietin in regulating angiogenesis. Dev Biol 2004,276(1):101-10.
[16]Beleslin-Cokic BB, Cokic VP, Yu X, Weksler BB, Schechter AN, Noguchi CT. Erythropoietin and hypoxia stimulate erythropoietin receptor and nitric oxide production by endothelial cells. Blood 2004,104(7):2073-80.
[17]Kanagy NL, Perrine MF, Cheung DK, Walker BR. Erythropoietin ad ministration in vivo increases vascular nitric oxide synthase expression. JCardiovasc Pharmacol 2003, 42(4):527-33.
[18]Defouilloy C, Teiger E, Sediame S, et al. Polycythemia impairs vasodilator response to acetylcholine in patients with chronic hypoxemic lung disease. Am JRespir Crit Care Med 1998, 157(5Pt1):1452-60.
[19]Malhotra S, Savitz SI, Ocava L, Rosenbaum DM. Ischemic preconditioning is mediated by erythropoietin through PI-3 kinase signaling in an animal model of transient ischemic attack. J Neurosci Res 2006,83(1):19-27.
[20]Ruscher K, Freyer D, Karsch M, et al. Erythropoietin is a paracrine mediator of ischemic tolerance in the brain:evidence from an in vitro model. J Neurosci 2002,22(23):10291-301.
[21]Grimm C, Wenzel A, Stanescu D, et al. Constitutive overexpression of human erythropoietin protects the mouse retina against induced but not inherited retinal degeneration. J Neurosci 2004, 24(25):5651-8.
[22]Yatsiv I, Grigoriadis N, Simeonidou C, et al. Erythropoietin is neuroprotective, improves functional recovery, and reduces neuronal apoptosis and inflammation in a rodent model of experimental closed head injury. FASEB J2005,19(12):1701-3.
[23]Villa P, Bigini P, Mennini T, et al. Erythropoietin selectively attenuates cytokine production and inflammation in cerebral ischemia by targeting neuronal apoptosis. J Exp Med 2003, 198(6):971-5.
[24]Springborg JB, Ma X, Rochat P, et al. A single subcutaneous bolus of erythropoietin normalizes cerebral blood flow autoregulation after subarachnoid haemorrhage in rats. Br J Pharmacol 2002; 135 (3):823-9.
[25]Erbayraktar S, Grasso G, Sfacteria A, et al. Asialoerythropoietin is a nonerythropoietic cytokine with broad neuroprotective activity in vivo. Proc Natl Acad Sci USA 2003,100(11):6741-6.
[26]Leist M, Ghezzi P, Grasso G, et al. Derivatives of erythropoietin that are tissue protective but not erythropoietic. Science 2004,305(5681):239-42.
[27]Brines M, Grasso G, Fiordaliso F, et al. Erythropoietin mediates tissue protection through an erythropoietin and common betasubunit heteroreceptor. Proc Natl Acad Sci USA 2004,101(41): 14907-12.
[2]G. Grasso et al., The role of erythropoietin in neuroprotection:therapeutic perspectives. Drug news Perspect.2007,20(5):315-320.
[3]Quelle FW, Wang D, Nosaka T, et al. Erythropoietin induces activation of Stat5 through association with specific tyrosines on the receptor that are not required for a mitogenic response. Mol Cell Biol 1996,16(4):1622-31.
[4]Kilic E, Kilic U, Soliz J, Bassetti CL, Gassmann M, Hermann DM. Brain-derived erythropoietin protects from focal cerebral ischemia by dual activation of ERK-1/-2 and Akt pathways. FASEB J 2005,19(14):2026-8.
[5]Chong ZZ, Kang JQ, Maiese K. Erythropoietin fosters both intrinsic and extrinsic neuronal protection through modulation of microglia, Aktl, Bad, and caspase-mediated pathways. Br J Pharmacol 2003,138(6):1107-18.
[6]Digicaylioglu M, Lipton SA. Erythropoietin-mediated neuroprotection involves cross-talk between Jak2 and NF-kappaB signaling cascades. Nature 2001,412(6847):641-7
[7]Brines ML, Ghezzi P, Keenan S, et al. Erythropoietin crosses the blood-brain barrier to protect against experimental brain injury. Proc Natl Acad Sci USA.2000,97(19):10526-10531
[8]Ehrenreich H, Hasselblatt M, Dembowski C, et al. Erythropoietin therapy for acute stroke is both safe and beneficial. Mol Med,2002,8(8):495-505.
[9]Silverberg DS, Wexler D, Blum M, et al. The effect of correction of anaemia in diabetics and non-diabetics with severe resistant congestive heart failure and chronic renal failure by subcutaneous erythropoietin and intravenous iron.. Nephral Dial Transplant.2003,18:141-146.
[10]Erbayraktar S, Grasso G, Sfacteria A, et al. Asialoerythropoietin is a nonerythropoietic cytokine with broad neuroprotective activity in vivo. Proc Natl Acad Sci.2003,100:6741-6746
[11]Fiordaliso F, Chimenti S, Staszewsky L,et al. A nonerythropoietic derivative of erythropoietin protects the myocardium from ischemia-reperfusion injury. Proc Natl Acad Sci,2005; 102(6): 2046-2051.
[12]Brines M, Grasso G, Fiordaliso F, et al. Erythropoietin mediates tissue protection through an erythropoietin and common beta-sbunit heteroreceptor. Proc Natl Acad Sci,2004,101 (410): 14907-14912.
[13]Campana WM, Misasi R, O'Brien JS. Identification of a neurotrophic sequence in erythropoietin. Int J Mol Med.1998,1(1):235-241.
[14]Bothwell M. Functional interactions of neurotrophins and neurotrophin receptors. Annu Rev Neurosci.1995,18:223-53
[15]左明雪.细胞和分子神经生物学.北京：高等教育出版社&施普林格出版社,2000：289-296
[16]Anders Nykjaer, Thomas E Willnow, Claus Munck Petersen. p75NTR-live or let die. Current Opinion in Neurobiology 2005,15:49-57
[17]Hefti F, Weiner WJ. Nerve growth factor and Alzheimer's disease. Ann Neurol,1986,20(3): 275-81
[18]曹琳,杨恒文等.NGF对坐骨神经损伤后腰椎与损伤神经MBP含量变化的影响,第三军医大学学报,2002,24：326
[19]Colangelo et al. A new nerve growth factor-mimetic peptide cctive on neuropathic pain in rats. J. Neurosci.,2008,28(11):2698-2709
[20]Huang BR,Gu JJ, ming H, et al. Differential actions of neurotrophins on apoptosis mediated by the low affinity neurotrophin receptor p75NTR in immortalized neuronal cell lines. Neurochem Int., wooo,36(1):55-65.
[21]高鹏,王欣,陈虹,黄秉仁.神经生长因子低亲和力受体(p75NTR)模拟配基的筛选.忠国生物化学与分子生物学报，2003，19(6)：769-774。
[22]Kono DH, Urban JL, Horvath SJ, et al, et al Two minor deter minants of myelin basic protein induce experimental allergic encephalomyelitis in SJL/J mice. JExp Med,1988,168:213-227.
[23]Li W, Maeda Y, Yuan RR, et al.Beneficial effect of erythropoietin on experimental allergic encephalomyelitis. Ann Neurol.2004,56 (6):767-777
[24]郭立安,阎哲,张晓楠,武丽华.二氯乙酸对蛋白质结构蛋白性稳定性的影响.第西军医大学学报，2001,22(22)
[25]李志雄,魏麓云.多发性硬化血脑屏障破坏的研究进展.实用医药杂志.2008,25(11)：1391-1393
[26]Yuan R, Maeda Y, Li W, Lu W, Cook S, Dowling P. Erythropoietin:a potent inducer of peripheral immuno/inflammatory modulation in autoimmune EAE. PLoS ONE.2008,3(4):1924
[27]Triaca V, Tirassa, P Aloe L. Presence of nerve growth factor and TrkA expression in the SVZ of EAE rats:evidence for a possible functional significance. Exp Neurol,2005,191(1):53-64
[28]邢清和,王永铭,郑荣远.影响EAE动物模型建立的因素分析.中国临床神经科学.2000,8(4)：305-306
[29]陆正齐,朱灿胜,郑雪平,王敦敬,胡学强.不同抗原诱导不同动物多发性硬化模型的比较.中山大学学报(医学科学处版),2008,29(16)：684-689
[30]Pluchino S, Quattrini A, Brambilla E, et al. Injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis. Nature,2003,422 (6933):688-694
[1]Prass K, Scharff A, Ruscher K, et al. Hypoxia-induced stroke tolerance in the mouse is mediated by erythropoietin. Stroke 2003,34(8):1981-6.
[2]Warnecke C, Zaborowska Z, Kurreck J, et al. Differentiating the functional role of hypoxia-inducible factor (HIF)-l alpha and HIF-2alpha (EPAS-1) by the use of RNA interference: erythropoietin is a HIF-2alpha target gene in Hep3B and Kelly cells. FASEB J 2004; 18(12): 1462-4.
[3]Yu X, Lin CS, Costantini F, Noguchi CT. The human erythropoietin receptor gene rescues erythropoiesis and developmental defects in the erythropoietin receptor null mouse. Blood 2001, 98(2):475-7.
[4]Digicaylioglu M, Bichet S, Marti HH, et al. Localization of specific erythropoietin binding sites in defined areas of the mouse brain. Proc Natl Acad Sci USA 1995,92(9):3717-20.
[5]Chin K, Oda N, Shen K, Noguchi CT. Regulation of transcription of the human erythropoietin receptor gene by proteins binding to GATA-1 and Sp1 motifs. Nucleic Acids Res 1995,23(15): 3041-9.
[6]Pandolfi PP, Roth ME, Karis A, et al. Targeted disruption of the GATA3 gene causes severe abnormalities in the nervous system and in fetal liver haematopoiesis. Nat Genet 1995, 11(1):40-4.
[7]Quelle FW, Wang D, Nosaka T, et al. Erythropoietin induces activation of Stat5 through association with specific tyrosines on the receptor that are not required for a mitogenic response. Mol Cell Biol 1996,16(4):1622-31.
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