低氧应激促红细胞生成素生成效应及其影响因素

低氧应激促红细胞生成素生成效应及其影响因素大连大学医学院（大连，１１６０１２）孟宪法，于洪九青海医学院（西宁，８１０００１）许存和，耿排力天津医科大学（天津，３０００７０）姜如冈生理条件下，促红细胞生成素（ｅｒｙｔｈｒｏｐｏｉｅｔｉｎ，ＥＰＯ）是维持...     

用噬菌体表面表达技术筛选及表达抗重组人促红细胞 …

目的 获得抗重组人红细胞生成素（ｒｈＥＰＯ）的单链抗体（ＳｃＦｖ），为得到纯度高、活力强的重组人红细胞生成素（ｒｈＥＰＯ）产品和制备红细胞生成素（ＥＰＯ）检测试剂盒奠定基础。方法 采用噬菌体表达表达和重组抗体技术，从已建立的鼠抗ｒｈＥＰＯ杂交瘤细胞株Ｄ３中克隆出抗ｒｈＥＰＯ单链抗体ＳｃＦｖ基因片段，经噬菌体表面呈现，与固相抗原ｒｈＥＰＯ结合，“吸附－洗脱－富集”，酶联免疫吸附法（ＥＬＩＳＡ）检测，     

抗红细胞生成素单克隆抗体的制备及初步鉴定

红细胞生成素（ｅｒｙｔｈｒｏｐｏｉｅｔｉｎ，ＥＰＯ）是一种主要由肾小管内皮细胞产生的酸性糖蛋白，正常人血浆和尿中含量甚微。ＥＰＯ的主要生理功能是通过刺激骨髓多能干细胞进入红细胞系，并促进其增殖、分化而调节外周血液中红细胞的数量。ＥＰＯ用于治疗慢性肾衰...     

促红细胞生成素(EPO)阻止红系祖细胞凋亡的作用

促红细胞生成素（ＥＰＯ）阻止红系祖细胞凋亡的作用唐文秦省刘世广＊章静波＊（河南医科大学附属卫生生物教研室＊中国协和医科大学基础医学研究所）促红细胞生成素（ＥＰＯ）在红系发生过程中起着重要作用。ＥＰＯ可以直接作用于造血器官的红系祖细胞，促进红细胞的生成...     

rhuEPO治疗对血透病人的血液流变性及动静脉内瘘的影响

促红细胞生成素（ＥＰＯ）是肾脏分泌的一种激素，其作用是促进肾髓造血。ｒｈｕＥＰＯ是通过基因工程技术人工合成的ＥＰＯ，目前已广泛应用于肾性贫血的治疗，其副作用有高血压、栓塞等。对后者的研究各家报道不一，为此我们对５０例ｒｈｕＥＰＯ治疗的血透病人的血液流...     

肝细胞导向的促红细胞生成基因转移在肾性贫血基因 …

目的 探索用受体介导方法对肾性贫血基因治疗的可行性及疗效。方法 构建一种克隆了促红细胞生成素（ＥＰＯ）基因的ＥＢＶ复制子载体ＰＥＰＯ，该载体与半乳糖基化组蛋白结合形成可溶性的核酸蛋白复合物，以静脉注射的方式将复合物导入通过喂饲腺嘌呤造成肾性贫血大鼠，恢复正常饮食２周后，采血样检测血红蛋白 含量和红细胞数，并与对照组进行比较。结果 注射ＰＥＰＯ复合物组的红细胞数和血红蛋白含量都有明显升高，实验组和对     

血液透析对慢性肾衰血清红细胞生成素浓度无影响

用酶联免疫吸附分析测定了１５列慢性肾功能衰竭（ＣＲＦ）患者血透前、后血清促红细胞生成素（ＥＰＯ）浓度的变化。结果发现血透前慢性肾衰竭患者血清ＥＰＯ浓度在“正常范围”，透析后尽管贫血有所改善，但血清ＥＰＯ浓度无明显变化，与贫血改善无关。血透不影响ＣＲＦ血清ＥＰＯ浓度，血透对ＣＲＦ贫血的改善可能与透析清除某种红细胞生成抑制物质有关。     

肝细胞导向的促红细胞生成素基因转移在肾性贫血基因治疗的初步研究

目的探索用受体介导方法对肾性贫血基因治疗的可行性及疗效。方法构建一种克隆了促红细胞生成素（ＥＰＯ）基因的ＥＢＶ复制子载体ｐＥＰＯ，该载体与半乳糖基化组蛋白结合形成可溶性的核酸蛋白复合物，以静脉注射的方式将复合物导入通过喂饲腺嘌呤造成肾性贫血大鼠，恢复正常饮食２周后，采血样检测血红蛋白的含量和红细胞数，并与对照组进行比较。结果注射ｐＥＰＯ复合物组的红细胞数和血红蛋白含量都有明显升高，实验组和对照组的红细胞数分别为每毫升４９２个和４０７个；血红蛋白含量分别１１．４ｇ／ｍｌ和９．１ｇ／ｍｌ。两组比较，Ｐ＜０．０１。提示ＥＰＯ基因已被导入动物体内并表达出目的蛋白，但肾衰症状并不改善。结论以上结果证明通过受体介导的基因转移技术可将ＥＰＯ基因导入实验动物体内并进行表达，改善肾性贫血     

血小板生成因子的研究进展

骨髓抑制是癌症大剂量放 /化疗后的常见并发症。针对贫血及粒细胞减少症 ,目前已有促红细胞生成素 (ｅｒｙｔｈｒｏｐｏｉｅｔｉｎ ,ＥＰＯ)及粒细胞集落刺激因子(ｇｒａｎｕｌｏｃｙｔｅｃｏｌｏｎｙｓｔｉｍｕｌａｔｉｎｇｆａｃｔｏｒ ,Ｇ -ＣＳＦ)等药物广泛用于临床 ,并取得了较好的疗效。而对于放 /化疗后血小板减少症 ,主要靠输注血小板来进行治疗 ,但反复的血小板输注 ,会出现输血反应、血小板无效输注及输血相关性疾病等。近 10年来 ,人们发现了一些具有促血小板生长作用的细胞因子 ,经过系统的体内外研究 ,其中部分已进入了临床研…     

国内医讯

国内医讯我国基因工程人红细胞生成素研制成功基因工程人红细胞生成素（ＥＰＯ）已由南京军区军事医学研究所和海南亚龙生物医学研究所科技人员史江、方君等研制成功。日前，经卫生部批准，基因工程人红细胞生成素已用于临床。南京军区军事医学研究所和海南亚龙生物医学研...     

Measurement of Mouse Plasma Erythropoietin by an Improved ELISA

We have examined whether mouse plasma erythropoietin (EPO) can be measured by an improved enzyme-linked immunosorbent assay (ELISA) using milk proteins (Block Ace) both as a blocking reagent and as a diluent for standard recombinant human EPO (rHuEPO) and for plasma samples. Block Ace brought about high slope sensitivity of the standard curve, with a low background. The dose–response curves of normal or anaemic mouse plasma and of rHuEPO were linear and parallel to each other. The anaemic plasma had an additive effect with rHuEPO by increasing the absorbance at 405 nm. The coefficients of variation in the intra- and interassays ranged from 4.2% to 15.3%. The plasma EPO levels in 22 normal mice were 18.3 ± 10.3 mU/ml. An inverse relationship between the logarithm of plasma EPO concentrations and blood haemoglobin concentrations, red blood cell counts or packed cell volumes was found in normal mice and in mice with iron deficiency anaemia (IDA). These results show the validity for the use of the new improved ELISA method for measuring circulating murine EPO.     

EPO's alter ego: erythropoietin has multiple actions

Many cancer patients suffer from anemia, which has a major detrimental effect on their quality of life. Recombinant human erythropoietin (rHuEPO) is now widely used in cancer patients, as it improves hematocrit, lowers blood transfusion requirements, and improves quality of life. Recent research indicates that EPO has pleiotropic effects on the body well beyond the maintenance of red cell mass, but the mechanisms involved in relieving fatigue and improving quality of life in cancer patients are poorly understood. EPO receptors (EPO-Rs) have been detected in many different cells and tissues, providing evidence for autocrine, paracrine, and endocrine functions of EPO. Apart from its endocrine function, EPO may have a generalized role as an antiapoptotic agent that is associated with enhancement of muscle tone, mucosal status, and gonadal and cognitive function. The recent discovery of EPO-Rs in breast tumor vasculature, while raising important questions about the possible effects of pharmacological doses of rHuEPO on tumor cells, also suggests that the receptors could provide a useful target for drugs attached to EPO.     

Neonatal Anemia: Pathophysiology and Treatment

All neonates experience a decline in circulating red blood cell (RBC) mass due to diminished erythropoietin (EPO) levels. This effect is more pronounced in small, premature infants and can lead to severe anemia and need for RBC transfusions–particularly, if repeated phlebotomy is required to monitor acutely ill neonates. Although optimal RBC transfusion therapy has been a long-term challenge for neonatologists, the emergence of recombinant EPO as promising therapy for neonatal anemia is the major issue for 1994. Accordingly, this report for the 12th International Convocation on Immunology (Transfusion Immunology and Medicine) will focus on this aspect of neonatal transfusion medicine. Although several controlled trials to evaluate EPO as therapy have been completed, definitive answers to all questions regarding efficacy and possible toxicity have not been provided. However, therapy with EPO plus iron and adequate nutrition is likely to be proven effective for the relatively late anemia of stable prematures. To date, EPO has not been shown, convincingly, to alleviate the anemia present early in the life of acutely-ill, premature infants.     

Measurement of mouse plasma erythropoietin by an improved ELISA

We have examined whether mouse plasma erythropoietin (EPO) can be measured by an improved enzyme-linked immunosorbent assay (ELISA) using milk proteins (Block Ace) both as a blocking reagent and as a diluent for standard recombinant human EPO (rHuEPO) and for plasma samples. Block Ace brought about high slope sensitivity of the standard curve, with a low background. The dose–response curves of normal or anaemic mouse plasma and of rHuEPO were linear and parallel to each other. The anaemic plasma had an additive effect with rHuEPO by increasing the absorbance at 405 nm. The coefficients of variation in the intra- and interassays ranged from 4.2% to 15.3%. The plasma EPO levels in 22 normal mice were 18.3 ± 10.3 mU/ml. An inverse relationship between the logarithm of plasma EPO concentrations and blood haemoglobin concentrations, red blood cell counts or packed cell volumes was found in normal mice and in mice with iron deficiency anaemia (IDA). These results show the validity for the use of the new improved ELISA method for measuring circulating murine EPO.     

The detection of anti-erythropoietin antibodies in human serum and plasma: part II. Validation of a semi-quantitative 3H-thymidine uptake assay for neutralizing antibodies

Neutralizing antibodies to erythropoietin (EPO) can cause a loss of response to recombinant human EPO (rHuEPO) and lead to rare cases of sudden, unexplained, severe anemia in chronic renal failure patients treated with rHuEPO. An assay for neutralizing anti-EPO antibodies has been validated that is based on the inhibition of proliferation of human UT-7/EPO cells, an immortalized cell line, by neutralizing antibodies in serum test samples using 3H-thymidine as a marker for proliferation. The dependence of the human cell line on EPO for growth and proliferation in a concentration-dependent manner enabled the validation of a rHuEPO standard curve for cell proliferation that can be used to determine the presence of neutralizing anti-EPO antibodies in serum samples. Proliferation of the cells increases with increasing concentrations of EPO, forming an S-shaped standard curve, which is fit with a 4-parameter logistic model, between 2.5 and 50 mU/mL rHuEPO, with a percent coefficient of variation (% CV) from 8.7% to 22.1% and a % accuracy of 103.5% to 109.5%. Anti-EPO antibodies and serum with anti-EPO antibodies neutralize UT-7/EPO proliferation by 10 mU/mL rHuEPO in a concentration- or dilution-dependent manner with < or = 25% CV. Percent neutralization is calculated by determining the amount of EPO recovered from the original 10 mU/mL added using the formula [((10-concentration recovered)/10)x100%]. Stem cell factor (SCF) stimulated cell proliferation, but not as effectively as rHuEPO. Antibodies to SCF were not able to inhibit the proliferative response induced by EPO and vice versa, confirming the specificity of the assay for antibodies to EPO. High EPO levels can impact both the radioimmunoprecipitation and neutralization assays to produce a false negative result. However, the impact can be mitigated by the large dilutions used in the neutralization assay.     

Prolyl hydroxylase domain-2 (PHD2) inhibition may be a better therapeutic strategy in renal anemia

Recombinant human erythropoietin (rHuEPO) has revolutionized the life of dialysis patients with anemia of chronic kidney disease (CKD). Newer erythropoietin analogues with improved profile have been introduced recently. However, there are many concerns such as safety, economy and patient compliance with these newer rHuEPo analogues. Small molecules aimed to inhibit prolyl hydroxylase domain-2 (PHD2) may prevent degradation of hypoxia inducible factor-2 (HIF2) which leads to endogenous erythropoietin production. This therapeutic intervention may not only overcome the patient compliance and economic burden but also possibly overcome the safety issues related to rHuEPO and its analogues. Moreover, PHD2 inhibitors may increase the endogenous circulating iron availability via suppression of hepcidin, a master regulator of iron homeostasis which further reduces the need for exogenous intravenous iron administration for effective erythropoiesis in renal anemia patients. In conclusion, small molecule PHD2 inhibitors may have better therapeutic efficacy and potential to address clinical concerns associated with rHuEPO and rHuEPO mimetic peptides.     

Characterization and biological effects of recombinant human erythropoietin

Human recombinant erythropoietin (rHuEPO) has been purified to apparent homogeneity and compared to purified human urinary erythropoietin (EPO). Both the purified natural and recombinant EPO preparations were characterized in a competition radioimmunoassay (RIA), the exhypoxic polycythemic mouse bioassay, in vitro tissue culture bioassays using bone marrow cells, and by Western analysis. In the immunological and biological activity assays, the rHuEPO shows a dose response which parallels that of the natural hormone. By Western analysis, the recombinant and human urinary EPO migrate identically. Administration of rHuEPO increases the hematocrit of normal mice in a dose-dependent manner. Additionally, the rHuEPO is able to increase the hematocrit of rats made uremic as a result of subtotal nephrectomy. In summary, by all criteria examined, the rHuEPO is biologically active and equivalent to the natural hormone.     

The detection of anti-erythropoietin antibodies in human serum and plasma. Part I. Validation of the protocol for a radioimmunoprecipitation assay

Rare cases of unexplained sudden severe anemia or red cell aplasia and resistance to recombinant human erythropoietin (rHuEPO) in patients with chronic renal failure (CRF) have been attributed to the development of anti-EPO antibodies. The development and validation of a radioimmunoprecipitation (RIP) assay to detect human anti-EPO antibodies in serum or plasma has been hampered by the lack of purified antibody to fully characterize and validate the assay. We have prepared an affinity-purified human antibody to EPO and used the antibody to characterize and validate a sensitive and reproducible RIP assay that can qualitatively measure anti-EPO antibody in serum or plasma samples. The lower limit of detection of the assay is 8 ng/ml of purified antibody. The threshold for detecting antibody is > or =0.9% cpm bound. The precision of the assay using purified antibody standards ranges from 5.8% to 15.3% and the precision of the assay using dilutions of the positive control ranges from 15.9% to 18.7%. EPO in the samples did not interfere with detection of the anti-EPO antibody except at high concentrations.     

Clinical significance of red cell distribution width in polycythemia vera

We evaluated changes in red cell distribution width-standard deviation (RDW-SD) measured using a multiple parameter automated hematology analyzer E 4000 in patients with polycythemia vera (PV). Patients with iron deficiency anemia, those with chronic myelogenous leukemia, those with primary thrombocythemia, and normal subjects were examined as controls. In the patients with PV, as in those with the other 3 diseases, RDW-SD tended to be higher than in the normal controls when red blood cell counts were high. The RDW-SD in patients with PV transiently increased following administration of a myelosuppressive, which corresponded to the transition period from microcytes to normal blood cells. It was even higher during the polycythemic period than during the myelofibrotic period. This may be associated with hematopoietic abnormality due to extramedullary hematopoiesis. RDW-SD seems to well reflect the pathologic status of PV.     

Polycythemia and oxygen sensing

Polycythemias can be differentiated based on the responsiveness of erythroid progenitors to circulating cytokines. Primary polycythemias are characterized by an augmented response due to acquired somatic or inherited germ-line mutations that are expressed within hematopoietic progenitors causing increased proliferation or decreased apoptosis and resulting in accumulation of red blood cells. In terms of oxygen requirements, primary polycythemias can be viewed as the production of hemoglobin fully dissociated from the tissue oxygen needs and from the oxygen sensing pathway. Polycythemia vera (PV) is the most common primary polycythemia. PV bone marrow progenitors cells can form erythroid colonies in the absence of exogenous erythropoietin in vitro. These endogenous erythroid colonies (EEC) are useful in differentiating PV and secondary polycythemias. They also can differentiate PV where this feature is independent of Epo signalling from primary familial and congenital polycythemia. In this autosomal dominant primary polycythemia, at variance with PV, EEC formation is abolished by anti-Epo and anti-Epo receptor neutralising antibodies. Mutations of the EPOR have been described and resulted in nine cases in truncated EPORs lacking the cytoplasmic carboxy-terminal of the receptor which possesses a negative growth regulatory domain. However, recent data suggest that different mutations may cause PFCP in most cases. Secondary polycythemia can be viewed as either physiological response to satisfy the oxygen needs of the tissues, resulting for instance from high affinity hemoglobins or BPG mutase deficiency, or as the result of germ-line or somatic mutations disturbing the oxygen sensing pathway or its target: Epo. Chuvash polycythemia is a frequently symptomatic disorder with an autosomal recessive inheritance and inappropriately high Epo levels. The erythroid progenitors are hypersensitive to Epo linking this condition to both primary and secondary polycythemia. A germline missense mutation at nucleotide 598 in both alleles of the von Hippel-Lindau gene results in increased hypoxia inducible factor-1 (HIF-1) expression in normoxic conditions. HIF-1 controls the expression of many genes including Epo. Identifying causal defects in other situations like post-renal transplant erythrocytosis and cases of autosomal dominant polycythemia with high Epo levels will help further understanding of the regulation of erythropoiesis.