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CN1053698C - 细胞培养生产促红细胞生成素的方法 - Google Patents

细胞培养生产促红细胞生成素的方法 Download PDF

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CN1053698C
CN1053698C CN89105382.4A CN89105382A CN1053698C CN 1053698 C CN1053698 C CN 1053698C CN 89105382 A CN89105382 A CN 89105382A CN 1053698 C CN1053698 C CN 1053698C
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cell culture
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CN1044496A (zh
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金以丰
张鹤云
朱家珍
汤国枝
李敏薏
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Nanjing University
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Abstract

本发明属于人肾细胞培养技术领域，本发明的目的是通过细胞培养技术诱导意外死亡人肾细胞大量生产促红细胞生成素(EPO)，再经生化方法纯化后，作为肾性贫血及其它贫血病人的治疗用药。本发明的技术特征是从意外死亡胎儿肾脏进行细胞培养，从EPO分泌高峰期的换出液中经DEAE纤维素和phenyl-sepharose分离，再经Sephacryl S-200凝胶过滤精制，得到较高比活的天然的人的EPO。

Description

细胞培养生产促红细胞生成素的方法
本发明属于人肾细胞培养技术领域。
促红细胞生成素，(英文名Erythropoietin，简称EPO)是肾脏分泌的一种酸性糖蛋白激素，具有促进红细胞样干细胞分化，起始血红蛋白合成，刺激幼红系祖细胞增殖等功能，是红细胞生成的重要调节因子，被认为是治疗慢性肾衰病人贫血症的重要药物，是近年来肾病治疗中的一大突破。但由于从人尿中提取EPO价格十分昂贵，国内用基因工程方法生产EPO尚未投放市场，据悉价格也十分昂贵，影响了它的广泛应用。
在已有技术中，美国Amegen公司和日本Chugai药厂已先后通过基因工程手段在中国仓鼠卵母细胞(CHO)中表达生产EPO，纯度129000 IU/mg蛋白，在1989年已得到将rEPO用于肾透析病人治疗贫血症的许可，获利甚丰，但至今国内外均未见有利用正常人肾细胞培养生产天然的人的EPO的报导。
本发明的目的是通过细胞原代培养技术，加入复合生长因子及低氧等最佳条件，诱导意外死亡人肾细胞大量生产EPO，再经生化方法纯化后，作为肾性贫血及其它贫血病人的治疗用药。
本发明的技术方案是，取意外死亡胎儿肾脏进行原代组织块或细胞培养，从EPO分泌高峰期的换出液中经DEAE纤维素和phenyl-sepharose分离纯化得到一定比活的EPO，再经过Sephacryl凝胶分离精制，其制备流程为：无菌取出肾脏，剔除肾盂，制成肾组织块。
Figure C8910538200041
产品EPO的体外活性测定：取怀孕13天小鼠胎肝制成细胞悬液，在35mm直径的培养皿中加入1.2ml培养液(细胞2×105、胎牛血清20％、牛血清白蛋白1％、α-巯基乙醇10-5M)，0.4ml待测样品，甲基纤维素30％，培养皿置CO2培养箱中，37℃孵育3天，在倒置显微镜下计数整个培养皿，规定8个以上细胞组成的集落为一个红系集落(CFU-E)与WHO标准品集落数比较，测出其活性单位，根据Follin-酚法测得蛋白浓度，计算EPO的比活。
利用本发明所得EPO，保持了糖蛋白分子的完整性，含糖30％左右，比活60000IU/mg蛋白，以上体内外均具高生物活性、无热源、无过敏源、无毒性、一般药理无不良反应，且生产成本低，产品价格便宜，约为基因工程产品的1/10，而且产品是完全天然，未经任何改造。
下面是本发明的实施例：
4-6月龄胎肾组织块，加入培养液，在37℃培养箱中进行贴壁培养，6-7天换一次培养液，收集分泌高峰期(2-2.5个月以内)培养液51.26升，离心，去组织碎片，加蒸馏水25.63L，通过DEAE纤维素层析，收集pH6.5 0.25mol/L NaCl 10mol/L PBS洗脱的活性峰，加NaCl至4mol/L，经phenyl-sepharose CL-4B层析，依次以缓冲液A、B、C洗脱，收集峰C，对水透析，经浓缩后，经Sephaeryl S-200凝胶过滤，凝胶以50mmol/L PBS (pH6.8)平衡，洗脱，收集峰III，透析，共得1142mg促红细胞生成素蛋白质，比活为67000IU/ml，总活7654万单位，平均每升培养液可得部分纯化EPO 150万单位。

Claims (1)

  1. 一种细胞培养法生产红细胞生成素的方法，其特征是利用意外死亡的人肾组织块(或细胞)进行原代培养，加入RPMI1640培养液，在37℃恒温培养箱(室)中进行静止或转瓶培养，以诱导EPO的分泌，收集培养45-60天的EPO分泌高峰期的条件化培养液，离心去除细胞碎片后，上清对缓冲液A(pH6.8，4M NaCL，10mM PBS)透析平衡，经Phenyl SepherosCL-4B层析，依次以缓冲A、B(pH7.1，0.5M NaCL，10mM PBS)C(20％乙二醇，4M盐酸胍，10mM PBS)洗脱，收集c峰，将c峰对DEAE纤维素层析的起始缓冲液(pH6.5，0.1M NaCL，10mM PBS)透析平衡，层析时用缓冲液(pH6.5，0.25M NaCL，10mM PBS)洗脱，收集该洗脱峰，对蒸馏水透析后除菌，冻干。
CN89105382.4A 1989-01-19 1989-01-19 细胞培养生产促红细胞生成素的方法 Expired - Fee Related CN1053698C (zh)

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KR101632372B1 (ko) * 2007-06-08 2016-07-01 웨이크 포리스트 유니버시티 헬스 사이언시즈 신부전 치료를 위한 선택적 세포 치료법
US9580688B2 (en) 2007-06-08 2017-02-28 Wake Forest University Health Sciences Kidney structures and methods of forming the same
JP5974233B2 (ja) 2008-11-12 2016-08-24 リージェンメド（ケイマン）エルティーディー． 単離した腎細胞およびその使用
WO2010057013A1 (en) 2008-11-14 2010-05-20 Wake Forest University Health Sciences Selective cell therapy for the treatment of renal failure
EP2569418B1 (en) 2010-05-12 2016-11-02 RegenMed (Cayman) Ltd. Bioactive renal cells
US11123372B2 (en) 2016-07-29 2021-09-21 Prokidney Bioactive renal cells for the treatment of chronic kidney disease

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