逆转录病毒介导的人促红细胞生成素基因在淋巴细胞中的表达

本实验设计和构建了人促红细胞生成素（Epo）重组逆转录病毒载体（LXSN-Epo）,载体中小鼠白血病病毒长末端调控序列翻译调控人Epo基因的表达。通过重组逆转录病毒,将Epo基因转导到大鼠T和B淋巴细胞中,DNA-PCR分析证明带有人Epo基因的逆转录病毒已整合到T和B淋巴细胞基因中,RT-PCR分析显示了在两种细胞中均表达了 Epo mRNA。体外生物学活性测定显示,在转导的T淋巴细胞培养上清中Epo表达水平高达243mU/ml,在感染的B淋巴细胞培养上清中Epo活性达43mU/ml。在感染的B细胞中,Epo表达在mRNA和蛋白水平均证明了逆转录病毒能有效地和迅速地转导Epo基因到新培养的B细胞中,而不需要药物选择。这些研究开始了逆转录病毒介导的基因转移走向体内Epo基因治疗研究的过程。     

Gestörte alpha2-Adrenozeptorfunktion bei Hämodialysepatienten mit renaler Anämie — eine mögliche Ursache der Blutdrucksteigerung unter rekombinantem humanem Erythropoietin?

Summary Nine patients on maintenance hemodialysis and transfusion-demanding renal anemia (group A) were treated with rHuEPO 120 IU/kg i.v. three times per week. Hemoglobin-content was raised from 7.2±0.9 to 10.4±0.8 g/dl. In all patients blood pressure rose, three patients developed arterial hypertension. Mean diastoloic blood pressure was 66±12 and 78±16 mmHg (p<0.001) before and after rHuEPO. Rise in blood pressure was accompanied by a significant fall in plasma-noradrenaline-levels (from 498±100 to 383±75 pg/ml;p<0.05) and alpha₂-adrenoceptor-density (from 574±76 to 384±49;p<0.05). Compared to nine patients on maintenance hemodialysis and hematocrit over 30% (group B), patients with severe renal anemia (group A before treatment) had higher densities of alpha₂-adrenoceptors (574±76 vs. 218±32;p<0.001) despite higher plasma-noradrenaline-levels (498±100 vs. 399±63; n.s.). We suppose a anemia-related disturbance of alpha₂-receptor-function with the result of abolished receptor down-regulation and impaired vascular reagibility to vasoconstricting stimuli. With the correction of anemia receptor-function improves, receptor down-regulation as well as vascular reagibility is re-established resulting in augmented vascular resistance and higher blood pressure.

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Abkürzungen rHuEPO rekombinantes humanes Erythropoietin - teMAP mittlerer arterieller Blutdruck - RR Blutdrucknach RIVA-ROCCI - RBF regionaler Blutfluß - RPR regionaler peripherer Widerstand     

Erythropoietin as an angiogenic factor in gastric carcinoma

AIMS: Previous studies have shown that increased vascularity is associated with haematogenous metastasis and poor prognosis in gastric cancer. The role of erythropoietin (Epo) in angiogenesis has not been completely clarified, although its involvement has been reported. In this study we correlated microvascular density and Epo receptor (Epo-R) expression in endothelial and tumour cells with histopathological type in gastric cancer. METHODS AND RESULTS: Specimens of primary gastric adenocarcinomas obtained from 40 patients who had undergone curative gastrectomy were investigated immunohistochemically by using anti-CD31 and anti-Epo-R antibodies. Stage IV gastric carcinoma had a higher degree of vascularization than other stages, and Epo-R expression in both endothelial and tumour cells increased in parallel with malignancy grade and was highly correlated with the extent of angiogenesis. CONCLUSIONS: Epo-R level correlates with angiogenesis and progression of patients with gastric carcinoma and we suggest that Epo might have a trophic effect on the vasculature of the gastrointestinal tract. Understanding mechanisms of gastric cancer angiogenesis provides a basis for a rational approach to the development of an anti-angiogenic therapy in patients with gastric cancer.     

重组促红细胞生成素对血透患者NO的影响研究

目的 :探讨基因重组促红细胞生成素 (rHuEpo)对维持性血透患者血管活性介质一氧化氮 (NO)的影响。方法 :将长期规律性血透尿毒症患者 80例分为血液透析应用rHuEpo者 (HDepo组n =4 3例 ) ,血液透析不应用rHuEpo者 (HD组n =37例 ) ,根据血压情况两组再分为HDepo高血压亚组 (HDepo - 1组 ,n =2 3)、HDe po血压正常亚组 (HDepo - 2组 ,n =2 0 )和HD高血压亚组 (HD - 1组 ,n =19)、HD血压正常亚组 (HD - 2组 ,n =18)。另选尿毒症未透析患者 2 0例 (NHD组 )、原发性高血压患者 2 0例、健康对照组 35例 (CON组 )。HDepo组患者均接受rHuEpo治疗 3个月以上 ,用比色法测定血NO水平。结果 :尿毒症未透析患者血清NO水平明显高于正常对照组 ,p <0 0 0 1;尿毒症透析患者血清NO浓度较正常对照组高 ,p <0 0 0 1,但经透析治疗的尿毒症患者与尿毒症未透析者比较 ,血清NO水平较未透析者低 ,p <0 0 5 ;HDepo组经rHuEpo治疗三月后血NO浓度下降 ,与用药前及对照组比较 ,p<0 0 0 1,p <0 0 5。且用rHuEpo治疗后 ,HDepo组收缩压、舒张压、平均动脉压均较HD组高 ,p <0 0 0 1,血压增高与NO浓度的降低有相关性。结论 :rHuEpo可使尿毒症透析患者血NO浓度降低 ,rHuEpo致高血压与血NO浓度降低有一定的关系     

Effect of human recombinant erythropoietin on human hemopoietic progenitor cells in vitro

Summary Recombinant human erythropoietin (rhEpo), now available, might become increasingly more important for clinical use, e.g., in the treatment of anemia of chronic renal failure. As a prerequisite of clinical trials, we analyzed the stimulatory and suppressive effects of rhEpo on human hemopoiesis by adding rhEpo to in vitro cultures of nonadherent low-density bone marrow cells obtained from normal persons and from patients undergoing hemodialysis for chronic renal failure. rhEpo was shown to be an effective stimulus for erythroid and multilineage colony formation. The dose-response curve was similar for erythroid progenitors BFU-E from normal controls and patients with chronic renal failure. rhEpo had no effect on megakaryocytic colony formation nor on the megakaryocytic differentiation of multilineage stem cells. Because of a good stimulatory activity on erythroid and multilineage stem cells and lack of toxic effects, rhEpo might be useful in the treatment of certain kinds of anemia.Abbreviations BFU-E burst-forming unit-erythroid - CFU-E colony forming unit-erythroid - CFU-GEMM CFU-granulocyte, erythrocyte, macrophage, megakaryocyte - CFU-GM CFU-granulocyte, macrophage - CFU-Mk CFU-megakaryocyte - IMDM Iscove's modified Dulbecco's medium - Mo-CM Mo-cell line conditioned medium - rhEpo recombinant human erythropoietin     

Erythropoietin concentrations are elevated in the peritoneal fluid of women with endometriosis

Erythropoietin (Epo) is an important regulator of erythropoiesis and stimulates the proliferation of early erythroid precursors as well as the differentiation of late erythroid precursors of the erythroid lineage. However, recent studies have indicated that Epo also has angiogenic properties and plays an important role in the oestrogen-dependent cyclical angiogenesis within the mouse uterus. It was therefore postulated that Epo may be an important angiogenic factor in endometriosis. In order to address this hypothesis the concentration of Epo in peritoneal fluid (PF) was determined in patients with or without endometriosis. PF was collected from patients with endometriosis (n = 42) or without endometriosis (n = 18). Detectable concentrations of Epo were found in all PF samples analysed. The concentration of Epo in PF from patients with endometriosis was significantly higher than that in the control group (13.1 +/- 1.2 mIU/ml versus 7.2 +/- 0.7 mIU/ml, mean +/- SE respectively, P < 0.01). Furthermore, in patients with endometriosis the Epo concentrations in PF from patients with stage I disease (n = 17, 16.6 +/- 3.0 mIU/ml) were significantly higher than those with stage II (n = 8, 10.7 +/- 1.2 mIU/ml, P < 0.03), III (n = 13, 8.4 +/- 1.0 mIU/ml, P < 0.01), IV disease (n = 7, 7.5 +/- 1.0 mIU/ml, P < 0.01). These data suggest that Epo may play a role in the pathogenesis of endometriosis particularly in the initiation of the disease.     

还原型谷胱甘肽治疗尿毒症贫血的初步探讨

目的　观察外源性还原型谷胱甘肽对尿毒症贫血的治疗效果。方法　将 42例尿毒症贫血患者随机分为两组。两组患者均给予重组人红细胞生成素 (r- Hu EPO) 30 0 0 U,每周两次皮下注射 ,疗程 12周。治疗组患者同时静脉给予外源性还原型谷胱甘肽 (GSH) 12 0 0 mg,每周两次 ,疗程 12周。于治疗前和治疗后抽静脉血测定血红蛋白 (Hb) ,红细胞 (RBC)和红细胞压积 (HCT)水平。结果　 r- Hu EPO治疗后两组患者 HB,RBC和 HCT水平均显著上升 (P<0 .0 1)。 GSH治疗组 Hb,RBC和 HCT水平与对照组比较上升明显 (P<0 .0 5 ) ,治疗组 Hb,RBC和HCT的上升幅度高于对照组 (P<0 .0 5 )。结论　外源性还原型谷胱甘肽能显著提高 r- Hu EPO治疗肾性贫血的疗效 ,GSH可能是治疗尿毒症贫血的有效药物。     

妊娠晚期羊水粪染与胎儿促红细胞生成素的关系

目的研究羊水粪染的脐带动脉血的促红细胞生成素(EPO)是否发生改变,以及这种变化是否受血气分析指标和孕周等因素影响.方法妊娠晚期孕妇82例,于胎儿娩出检测胎儿脐带动脉血的pH、pco2、po2、、so、HCO3、Too、ABE和EPO.EPO采用ELISA方法.分娩后记录孕妇年龄、分娩孕周、新生儿出生体重、Apgar评分、羊水粪染.根据是否羊水粪染分成两组,比较两组之间的差别.采用应用Logistic逐步回归(Backward和Forward模式)分析各因素独立对羊水粪染的影响.羊水粪染为因变量,孕周、孕妇年龄、新生儿出生体重、EPO、pH、pco2、po2、HCO3-、Tco2、ABE、so为自变量.结果羊水粪染组24例,无羊水粪染组58例.羊水粪染组的EPO浓度是无羊水粪染对照组的2倍[(52.84±10.40)mIU/ml,n=24;(25.04±4.04)mIU/ml,n=58;P＜0.05].两组之间的HCO3-、Too、ABE存在统计学差别(P＜0.05),但pH、pco、po2等其他指标无明显差别.Logistic逐步回归分析结果显示EPO、HCO3-、Tco2为与羊水粪染有关的自变量.结论EPO浓度的升高是导致羊水粪染的危险因素,羊水粪染是胎儿长期、慢性缺氧的结果.EPO浓度的升高对预测围生儿远期预后有一定的临床意义.     

FK228抑制EPO介导的人红系前体细胞的增殖与分化

目的:探讨组蛋白脱乙酰化酶抑制剂FK228在红细胞生成素(EPO)介导的人红系前体细胞增殖与分化中的调节作用。方法:从经粒细胞集落刺激因子动员的肿瘤患者外周血单核细胞中分离CD34 细胞,用含干细胞生长因子(SCF)、EPO或SCF IL-3及不同浓度FK228的无血清培养基培养7d,分别用抗GPA及抗CD36单克隆抗体(mAb)染色并行流式细胞术检测;将CD34 细胞用含SCF IL-3的无血清培养基培养7d,分离CD36 GPA-细胞,将细胞用含有EPO FK228的无血清培养基培养7d,并行细胞计数;将CD36 GPAlow/-细胞用含EPO加或不加FK228的无血清培养基培养,并进行annexin V和PI染色。结果:FK228以一种剂量依赖方式抑制CD36 GPAhigh、CD36 GPAlow和CD36 GPA-细胞的产生;FK228可诱导CD36 GPAhigh和CD36 GPAlow/-细胞在含EPO的培养基中发生细胞凋亡。结论:FK228可抑制EPO介导的人红系前体细胞的增殖与分化。     

Pharmacokinetic and Pharmacological Profiles of Free and Liposomal Recombinant Human Erythropoietin After Intravenous and Subcutaneous Administrations in Rats

Purpose. Recombinant human erythropoietin (Epo) is used frequently through intravenous (i.v.) and subcutaneous (s.c.) administration for the clinical treatment of the last stage of renal anemia. We encapsulated Epo in liposomes to develop an alternative administration route. The purpose of our study was to evaluate the pharmacokinetics and the pharmacological effects of liposomal Epo in comparison with the Epo after i.v. and s.c. administration to rats. Methods. Epo was encapsulated in liposomes composed of dipalmitoylphosphatidylcholine (DPPC) and soybean-derived sterol mixture (SS) prepared by the reversed-phase evaporation vesicle method. After filtration through a 0.1 m polycarbonate membrane, liposomes were gel filtered (Epo/liposomes). Results. Epo/liposomes showed higher pharmacological activity than Epo/liposomes before gel filtration after i.v. administration to rats. Non-encapsulated Epo lost its activity, whereas encapsulated Epo in liposomes retained it. The pharmacological effects of Epo/liposomes were greater than those of Epo after i.v. administration. Epo/liposomes afforded 3–9 times higher AUC, lower clearance and lower steady-state volume of distribution than Epo after both i.v. and s.c. administrations. Epo/liposomes had an improved pharmacokinetic profile compared with Epo. S.c. administration of Epo/liposomes at 7 h may penetrate primarily (40% of dose) through the blood as a liposome and partly (7% of dose) in lymph. Conclusions. Epo/liposomes may reduce the frequency of injections required for a certain reticulocyte effect in comparison to Epo. The lower clearance of Epo/liposomes may increase the plasma concentrations of Epo, which increases the efficacy.