rhEPO-Fc融合蛋白抗贫血作用及其机制研究

【作者】 刘元元；

【导师】 姚明辉；

【作者基本信息】 复旦大学 ， 药理学， 2008， 博士

【摘要】 重组人促红细胞生成素(rhEPO)是最早用于临床的重组基因工程药物之一,目前已广泛用于各种贫血患者的治疗,包括肾性贫血、肿瘤放、化疗所致的贫血等,但由于其半衰期较短,需要频繁给药,从而限制了其临床应用。长效重组人促红细胞生成素融合蛋白rhEPO-Fc是由rhEPO与IgG的Fc片段融合而成,这种新型的rhEPO-Fc融合蛋白表现为血浆半衰期延长,生物活性增加,因此在治疗贫血时可以减少用药量并减少注射的频率。本研究采用C57小鼠γ-辐射后骨髓抑制性贫血模型、SD大鼠肾切除后肾性贫血模型、及猕猴环磷酰胺所致骨髓抑制性贫血模型,研究rhEPO-Fc对动物不同贫血模型的长效治疗作用及量效关系,并确定rhEPO-Fc长期使用是否会产生抗体。同时在整体动物实验基础上进行体外研究,采用人骨髓来源的UT-7/EPO细胞株,用CCK-8、流式细胞仪、光镜、透射电镜等方法观察rhEPO-Fc抑制γ-射线辐射诱导UT-7/EPO细胞凋亡的作用,用免疫荧光、western blotting等技术研究rhEPO-Fc抑制γ-射线辐射诱导UT-7/EPO细胞凋亡时凋亡相关蛋白Bcl-2、Bax及caspase3表达水平的变化,以探讨rhEPO-Fc抑制γ-射线辐射所诱导UT-7/EPO细胞凋亡的分子机制。第一部分rhEPO-Fc对不同动物贫血模型治疗作用的研究1 rhEPO-Fc对γ辐射所致C57小鼠骨髓抑制性贫血的影响C57小鼠70只,经137Csγ-射线4Gy单次全身辐射,造成骨髓抑制性贫血,于辐射前及辐射后不同时间采血,测定红细胞计数、血红蛋白量、红细胞压积及网织红细胞计数等,小鼠随机分为5组,分别为溶媒对照组,rhEPO-Fc 3个剂量组(剂量分别为7.5,15,30μg·kg-1,每周给药1次)和rhEPO组(阳性对照组,剂量为7.5μg·kg-1,每周给药3次),均为皮下注射给药,连续给药25天,停药后观察5天。结果显示rhEPO组每周3次、rhEPO-Fc 7.5,15,30μg·kg-1组每周1次皮下注射,C57小鼠红细胞计数、血红蛋白量、红细胞压积从给药后第11天到25天与溶媒对照组比较明显升高(p<0.01),rhEPO-Fc 30μg·kg-1组每周1次皮下注射,C57小鼠从给药后第7天开始到停药后5天,rhEPO组每周3次皮下注射,C57小鼠在药后第18及25天网织红细胞计数均明显升高,与溶媒对照组比较差别有显著意义(p<0.01)。rhEPO-Fc 7.5,15,30μg·kg-1组每周1次皮下注射,C57小鼠脾脏重量和脾脏系数与对照组比较无明显差异(p>0.05)。其中rhEPO-Fc 7.5,15,30μg·kg-1三个剂量组在升高红细胞计数、血红蛋白量、红细胞压积及网织红细胞计数时呈现剂量依赖性。结果提示rhEPO-Fc能有效治疗γ-辐射所致C57小鼠骨髓抑制性贫血,rhEPO-Fc 15、30μg·kg-1每周1次皮下注射疗效与rhEPO 7.5μg·kg-1每周3次皮下注射疗效相似,而且rhEPO-Fc 7.5、15、30μg·kg-1三个剂量组之间存在剂量依赖关系。2 rhEPO-Fc对部分肾切除所致SD大鼠肾性贫血的影响SD大鼠70只,经2次手术后切除5/6肾脏,造成肾性贫血模型,于肾切除前及切除后不同时间采血,测定红细胞计数、血红蛋白量、红细胞压积、网织红细胞计数及血尿素氮等。SD大鼠随机分为6组,分别为模型组、假手术组、rhEPO-Fc3个剂量组(剂量分别为5.4、10.7、21.4μg·kg-1,每周1次给药)和rhEPO组(阳性对照组,剂量为5.4μg·kg-1,每周3次给药),均为皮下注射,连续用药4周,停药后观察2周。结果显示rhEPO组(药后第1周到停药后1周)、rhEPO-Fc5.4μg·kg-1组(药后第1周到药后第2周)、10.7μg·kg-1组(药后第1周到药后第4周)、21.4μg·kg-1组(药后第1周到停药后2周)红细胞计数、血红蛋白量、红细胞压积及网织红细胞计数(用药后1周)与模型组比较明显升高,差别有显著意义(p<0.01)。肾切除后的模型组、rhEPO-Fc5.4、10.7、21.4μg·kg-1组及rhEPO组血尿素氮在整个实验过程中均维持在较高水平,与假手术组比较,差异均有显著意义(P<0.01)。其中rhEPO-Fc 5.4、10.7、21.4μg·kg-1三个剂量组在治疗部分肾切除所致SD大鼠肾性贫血时呈现剂量依赖关系。结果提示rhEPO-Fc能有效治疗部分肾切除所致SD大鼠肾性贫血,rhEPO-Fc 10.7、21.4μg·kg-1每周1次与rhEPO5.4μg·kg-1每周3次皮下注射疗效相似,而且rhEPO-Fc5.4、10.7、21.4μg·kg-1三个剂量组之间存在剂量依赖关系。3 rhEPO-Fc对环磷酰胺所致猕猴骨髓抑制性贫血的影响猕猴16只(雌9雄7),静脉注射环磷酰胺50mg·kg-1×2天造成骨髓抑制,在用环磷酰胺前及以后不同时间采血,测定红细胞计数、血红蛋白量、红细胞压积、网织红细胞计数、白细胞计数及血小板计数等。猕猴分为4组,每组4只,分别为溶媒对照组、2个rhEPO-Fc治疗组(剂量分别为5、10μg·kg-1,每周1次给药)、rhEPO组(剂量为2.5μg·kg-1,每周3次给药),均为皮下注射,连续用药49天,停药后观察35天。结果显示rhEPO组、rhEPO-Fc10μg·kg-1组药后42及49天猕猴红细胞计数、红细胞压积、血红蛋白量(药后35天到49天)、网织红细胞计数(药后49天)与溶媒对照组比较明显升高,差别有显著意义(p<0.01)。rhEPO组、rhEPO-Fc10μg·kg-1组从药后第28天到停药后21天猕猴红细胞计数、血红蛋白量(药后第28天到停药后14天)、红细胞压积(药后第28天到药后49天)、网织红细胞计数(药后49天)与实验前比较明显升高,差别有显著意义(p<0.01)。rhEPO-Fc5μg·kg-1组(药后第12及14天)、rhEPO-Fc 10μg·kg-1组(药后第14、16、42天)猕猴血小板明显升高,与溶媒对照组比较差别有显著意义(p<0.05)；rhEPO组、rhEPO-Fc5、10μg·kg-1组猕猴的外周血白细胞计数在整个实验过程中与溶媒对照组及实验前比较,差别无显著意义(p>0.05)。结果提示皮下注射rhEPO-Fc能有效治疗由环磷酰胺所致猕猴骨髓抑制性贫血,rhEPO-Fc10μg·kg-1每周1次与rhEPO2.5μg·kg-1每周3次皮下注射疗效相似。4 Elisa法检测猕猴rhEPO-Fc抗体用药前各只猕猴血清中均未检测到抗体,并且rhEPO-Fc小、大剂量(5μg·kg-1、10μg·kg-1,1次·周-1sc.)在连续使用49天及停药后35天内均未检测到rhEPO-Fc抗体；而rhEPO组一只猕猴用药后12天开始产生rhEPO抗体,直至停药后21天血清中仍有抗体存在。提示rhEPO-Fc与rhEPO不同,长期使用不易产生抗体。第二部分rhEPO-Fc抑制γ-辐射诱导UT-7/EPO细胞凋亡的作用及机制研究1 rhEPO-Fc抑制γ-辐射诱导UT-7/EPO细胞凋亡的作用UT-7/EPO细胞用8Gyγ-射线照射后,用细胞因子治疗或不治疗,并把细胞分为5组：对照组(不加rhEPO)、rhEPO-Fc 0.7U/ml组、rhEPO-Fc 7U/ml组、rhEPO-Fc 70U/ml组、rhEPO 7U/ml组。各组细胞用CCK-8、流式细胞仪、光镜、透射电镜等方法观察rhEPO-Fc抑制γ-射线辐射诱导UT-7/EPO细胞凋亡的作用。结果显示γ-辐射后分别给予rhEPO-Fc 0.7、7、70U/ml及rhEPO 7U/ml能显著提高UT-7/EPO细胞存活率,明显抑制凋亡率,在24h、48h、72h与辐射对照组细胞比较有显著性差异；且rhEPO-Fc 0.7、7、70U/ml及rhEPO 7U/ml明显减少γ-辐射后G0/G1期细胞数,增加G2/M期、S期的细胞数,在24h与辐射对照组比较有显著性差异；细胞形态学观察可见γ-辐射后的UT-7/EPO细胞体积变小,核碎裂,核染色质浓缩,聚集于周边形成新月状,呈现出细胞凋亡典型的变化,γ-辐射后分别给予rhEPO-Fc 0.7、7、70U/ml及rhEPO 7U/ml的UT-7/EPO细胞表现出正常形态,未见凋亡典型特征。结果提示rhEPO-Fc 0.7、7、70U/ml可抑制γ-射线辐射诱导UT-7/EPO细胞凋亡2 rhEPO-Fc抑制γ-辐射诱导UT-7/EPO细胞凋亡的机制经不同处理的各组UT-7/EPO细胞用免疫荧光及western blotting技术观察凋亡相关蛋白Bcl-2、Bax及caspase 3表达。免疫荧光结果显示UT-7/EPO细胞γ-辐射后24h抗凋亡蛋白Bcl-2表达较弱,γ-辐射后分别给予rhEPO-Fc0.7、7、70U/ml及rhEPO 7U/ml则Bcl-2表达增强,各组细胞促凋亡蛋白Bax表达未见差异。western blotting结果显示γ-辐射后24h及48h的UT-7/EPO细胞,抗凋亡蛋白Bcl-2表达基本消失,相对Bcl-2/Bax蛋白比值较低,caspase 3前体(pro-caspase 3)表达较弱,caspase 3活性片段p17和p12被诱导；而γ-辐射后分别给予rhEPO-Fc0.7、7、70U/ml,及rhEPO 7U/ml,与辐射对照组比较,抗凋亡蛋白Bcl-2表达增强,Bcl-2/Bax蛋白比值与辐射对照组比较明显增加(p<0.01),caspase 3的表达也与正常培养的细胞接近,未见活性片段被诱导,促凋亡蛋白Bax的表达在各组细胞之间无显著差异。结果提示rhEPO-Fc可能通过增加抗凋亡蛋白Bcl-2的表达、增加Bcl-2/Bax蛋白比值及抑制caspase 3的激活而对抗γ-辐射所诱导的UT-7/EPO细胞凋亡。结论：1. rhEPO-Fc每周1次皮下注射在纠正γ-辐射所导致的C57小鼠骨髓抑制性贫血、5/6肾切除所导致的SD大鼠肾性贫血及环磷酰胺所诱导的猕猴骨髓抑制性贫血时与rhEPO每周3次皮下注射等效。2. rhEPO-Fc每周1次皮下注射,在连续使用49天及停药后35天内未检测到猕猴血清中抗rhEPO-Fc抗体。3. rhEPO-Fc可能通过增加抗凋亡蛋白Bcl-2的表达、增加Bcl-2/Bax蛋白比值及抑制caspase 3的激活而对抗γ-辐射所诱导的UT-7/EPO细胞凋亡更多还原

【Abstract】 Recombinant human erythropoietin (rhEPO) has been extensively used in the treatment of anemia associated with chronic renal failure, cancer chemotherapy and radiotherapy. However, rhEPO has a shorter serum half-life and the treatment often requires frequent injections, which results in limitation of this treatment. The long-acting recombinant human rhEPO-Fc fusion proteins consist of rhEPO and the Fc part of a human IgG molecule, which prolonged serum half-life time and increased biological activity, allowing for reduced dosing frequency in the treatment of anemia.In present studies, we first investigated the long-acting effect and dose-effect relationship of rhEPO-Fc on anemia of myelosuppression induced byγirradiation in C57 mice, anemia in partially nephrectomized rats, and anemia of myelosuppression induced by cyclophosphamide in rhesus monkeys, and Elisa assay was performed to detect the rhEPO-Fc antibody in rhesus monkey serum. Furthermore, we employed UT-7/EPO cell line which was derived from a patient’s bone marrow with leukemia to observe the inhibitive effect of rhEPO-Fc on apoptosis induced byγirradiation with CCK-8 assay, flow cytometry, light microscopy and transmission electron microscopy. Immunofluorescence and western blotting were used to detect the changes of expression of apoptosis-related proteins Bcl-2, Bax and caspase 3 to investigate the molecular mechanisms of rhEPO-Fc inhibiting apoptosis induced byγirradiation in human UT-7/EPO cells.PartⅠEffect of rhEPO-Fc on anemia in different animal models1. Effect of rhEPO-Fc on anemia of myelosuppression induced byγirradiation in C57 mice.The anemia was induced by total body irradiation with a single 4Gy dose using a 137Csγ-rays source in C57 mice. The blood samples were collected before irradiation and different indicated periods after irradiation. The RBCs, hemoglobin hematocrit and reticulocyte were determined. C57 mice were randomly divided into five groups: solvent control group, rhEPO-Fc three dose groups (7.5,15,30μg·kg-1 once weekly sc.), and rhEPO group(7.5μg·kg-1 three times weekly sc.). All drugs were successively given for 25 days, and the experiment was continued until the 5th day of post-treatment. It was observed the increase in RBCs, hemoglobin, and hematocrit, they were significantly higher (p<0.01) in rhEPO, rhEPO-Fc 7.5,15,30μg·kg-1 groups than that in control group on the 11th～25th day of treatment, and reticulocyte were significantly higher (p<0.01) in rhEPO-Fc 30μg·kg-1 group from the 7th day of treatment to the end of experiment, and rhEPO group on the 18th～25th day of treatment than that in control group. There was no significant difference in spleen weight and spleen index among rhEPO-Fc 7.5,15,30μg·kg-1 groups and control group (p>0.05). Furthermore, rhEPO-Fc 7.5,15,30μg·kg-1 promote the increase in RBCs, hemoglobin, hematocrit and reticulocyte in dosing-dependent manner. Our results indicated that rhEPO-Fc could effectively correct the anemia of myelosuppression induced byγirradiation in C57 mice. The effects of rhEPO-Fc 15μg·kg-1 and 30μg·kg-1 once weekly sc.were the same as the effect of 7.5μg·kg-1 three times weekly sc. There is a dose-dependent relationship among rhEPO-Fc 7.5,15,30μg·kg-1.2. Effect of rhEPO-Fc on anemia caused by partially nephrectomy in ratsAnemia was caused by a two-step 5/6 nephrectomy in rats. The blood samples were collected before nephrectomy and different indicated periods after nephrectomy. The RBCs, hemoglobin, hematocrit, reticulocyte and BUN were determined. SD rats were randomly divided into six groups:model group, sham operated group, rhEPO-Fc three dose groups (5.4,10.7,21.4μg·kg-1 once weekly sc.), and rhEPO group(5.4μg·kg-1 three times weekly sc.). All drugs were successively given for 4 weeks, and the experiment was continued until the 2nd week of post-treatment. It was observed the increase in RBCs, hemoglobin and hematocrit, they were significantly higher (p<0.01) in rhEPO group (on the 1st～4th week of treatment), rhEPO-Fc 5.4μg·kg-1 group (on the 1st and 2nd week of treatment), rhEPO-Fc 10.7μg·kg-1 group(on the 1st～3rd week of treatment) and rhEPO-Fc 21.4μg·kg-1 group(on the 1st～4th week of treatment) than that in model group, and reticulocyte were significantly higher (p<0.01) in rhEPO-Fc three dose groups and rhEPO group (on the 1st week of treatment) than that in model group. The levels of BUN were significantly higher in model group, rhEPO-Fc 5.4,10.7,21.4μg·kg-1 groups, and rhEPO group than that in sham operated group. Furthermore, rhEPO-Fc 5.4,10.7,21.4μg·kg-1 promote the increase in RBCs, hemoglobin, hematocrit and reticulocyte in dosing-dependent manner. These results indicated that rhEPO-Fc could effectively correct the anemia caused by partially nephrectomy in rats. The effects of rhEPO-Fc 10.7 and 21.4μg·kg-1 once weekly sc.were the same as the effect of rhEPO 5.4μg·kg-1 three times weekly sc. There is a dose-dependent relationship among rhEPO-Fc 5.4,10.7,21.4μg·kg-1.3. Effect of rhEPO-Fc on anemia of myelosuppression induced by cyclophosphamide in rhesus monkeys.Anemia of myelosuppression was induced by administration of 50mg·kg-1 cyclophosphamide iv. for successive 2 days in rhesus monkeys. The blood samples were collected before administration of cyclophosphamide and different indicated periods after administration of cyclophosphamide. The RBCs, hemoglobin, hematocrit, reticulocyte, platelets and WBCs were determined. Rhesus monkeys were randomly divided into four groups:solvent control group, rhEPO-Fc two dose groups (5,10μg·kg-1 once weekly sc.), and rhEPO group(2.5μg·kg-1 three times weekly sc.). All drugs were successively given for 49 days, the experiment was continued until the 35th day of post-treatment. It was observed the increase in RBCs, hemoglobin, and hematocrit, they were significantly higher (p<0.01) in rhEPO group and rhEPO-Fc 10μg·kg-1 group (on the 35th-49th day of treatment) than that in control group, and reticulocyte were significantly higher (p<0.01) in rhEPO-Fc 10μg·kg-1 group and rhEPO group (on the 49th day of treatment) than that in model group. Furthermore, an increase in RBCs (from the 28th day of treatment to the 21th day of post-treatment), hemoglobin (from the 28th day of treatment to the 14th day of post-treatment), hematocrit (on the 28th～49th day of treatment and the 49th day of treatment) were significantly increased (p<0.01) in rhEPO group and rhEPO-Fc 10μg·kg-1 group, comparing with baseline. The increase in platelet counts were significantly higher (p<0.05) in rhEPO-Fc 5μg·kg-1 group (on the 12th and the 14th day of treatment) and rhEPO-Fc 10μg·kg-1 group (on the 14th,16th,42th day of treatment). There was no significant difference in WBCs among rhEPO-Fc 5, 10μg·kg-1, rhEPO and group. These results showed that rhEPO-Fc could effectively correct the anemia of myelosuppression induced by cyclophosphamide in rhesus monkeys. The effects of rhEPO-Fc 10μg·kg-1 once weekly sc. were the same as the effect of rhEPO 2.5μg·kg-1 three times weekly sc.4 The detection of rhEPO-Fc antibody by Elisa assay in serum of rhesus monkeys.It was observed that no rhEPO-Fc antibodies in serum of rhesus monkeys could be detected by Elisa assay before treatment, and in rhEPO-Fc 5 and 10μg·kg-1 group, and there were no rhEPO-Fc antibodies in serum of rhesus monkeys to be detected by Elisa assay throughout the study period. However, rhEPO antibody could be detected by Elisa assay in serum of one rhesus monkey in rhEPO group throughout the study period.PartⅡThe inhibitive effect and mechanism of rhEPO-Fc on apoptosis induced byγirradiation in human UT-7/EPO cell line1. The effect of rhEPO-Fc on apoptosis induced byγirradiation in UT-7/EPO cellsUT-7/EPO cells were irradiated with a single 8Gy dose using a 137Csγ-rays source. Irradiated UT-7/EPO cells were treated with or without cytokines and randomly divided into five groups:irradiation control group, rhEPO-Fc 0.7U/ml group, rhEPO-Fc 7U/ml group, rhEPO-Fc 70U/ml group and rhEPO group. The effect of rhEPO-Fc on apoptosis induced byγirradiation in UT-7/EPO cells were observed by CCK-8 assay, flow cytometry, light microscopy and transmission electron microscopy. Results showed that 24h,48h and 72h afterγirradiation, the cell viabilities were significantly increased (p<0.01) and apoptotic rates of irradiated UT-7/EPO cells were significantly decreased (p<0.01) in rhEPO-Fc 0.7,7,70U/ml and rhEPO 7 U/ml group, compared with irradiation control group, and 24h afterγirradiation, the percent of UT-7/EPO cells in G0/G1 phase were significantly decreased (p<0.01) and the percent of UT-7/EPO cells in G2/M and S phase were significantly increased (p<0.01) in rhEPO-Fc 0.7,7,70U/ml and rhEPO 7 U/ml group compared to irradiation control group. Furthermore, the irradiated UT-7/EPO cells exhibited characteristic morphology of apoptosis, including cell shrunk, nucleus fragmented, chromatin condensation and margination forming the shape of crescents or lump, cytoplasmic vacuolization, on the contrary, the irradiated UT-7/EPO cells appeared morphologically normal and showed no sign of apoptosis in rhEPO-Fc 0.7,7,70U/ml and rhEPO 7 U/ml group. These results indicated that rhEPO-Fc 0.7,7,70U/ml could inhibit the apoptosis induced byγirradiation in UT-7/EPO cells.2. The mechanisms of rhEPO-Fc inhibiting apoptosis induced byγirradiation in human UT-7/EPO cells.Immunofluorescence and western blotting were used to detected the changes of expression of apoptosis-related proteins Bcl-2, Bax and caspase 3 in different treated UT-7/EPO cells. The results of immunofluorescence showed that the expression level of anti-apoptotic protein Bcl-2 was lower at 24h followingγirradiation, however, the expression level of Bcl-2 was higher in rhEPO-Fc 0.7,7,70U/ml and rhEPO 7 U/ml group, the expression of the Bax showed no difference among variable. The results of western blotting showed that the expression level of Bcl-2 protein and the relative Bcl-2/Bax protein ratio significantly decreased, the expression levels of pro-caspase 3 significantly reduced and the cleavage of caspase 3 to catalytically active fragments were also induced at 24h and 48h followingγirradiation, however, the expression of the Bcl-2 and caspase 3 in rhEPO-Fc 0.7,7,70U/ml and rhEPO 7 U/ml group showed no significant difference compared with the normal cultured UT-7/EPO cells, the relative Bcl-2/Bax protein ratio significantly increased compared with irradiation control group. But the expression of the Bax protein showed no difference among variable. These results indicate that rhEPO-Fc could exerts anti-apoptotic effect on irradiated human UT-7/EPO cells via increase the expression of Bcl-2 and the relative Bcl-2/Bax ratio, and inhibition the activation of caspase 3.Summary1. The effects of rhEPO-Fc once weekly sc. exert the same effect as the rhEPO three times weekly sc. on anemia of myelosuppression induced byγirradiation in C57 mice, anemia caused by partially nephrectomy in rats and anemia of myelosuppression induced by cyclophosphamide in rhesus monkeys. 2. No rhEPO-Fc antibodies in serum of rhesus monkeys could be detected by Elisa assay throughout the study period in rhEPO-Fc 5 and 10μg·kg-1 group.3. rhEPO-Fc exerts anti-apoptotic effects on irradiated human UT-7/EPO cells via increase the expression of Bcl-2 and the relative Bcl-2/Bax ratio, and inhibition the activation of caspase 3.更多还原