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 PDFInfo
- Publication number
- 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
- Authority
- CN
- China
- Prior art keywords
- pbs
- epo
- erythropoietin
- nacl
- peak
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- OXCMYAYHXIHQOA-UHFFFAOYSA-N potassium;[2-butyl-5-chloro-3-[[4-[2-(1,2,4-triaza-3-azanidacyclopenta-1,4-dien-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol Chemical compound [K+].CCCCC1=NC(Cl)=C(CO)N1CC1=CC=C(C=2C(=CC=CC=2)C2=N[N-]N=N2)C=C1 OXCMYAYHXIHQOA-UHFFFAOYSA-N 0.000 title claims abstract description 24
- 102000003951 Erythropoietin Human genes 0.000 title claims abstract description 12
- 108090000394 Erythropoietin Proteins 0.000 title claims abstract description 12
- 229940105423 erythropoietin Drugs 0.000 title claims abstract description 11
- 238000004113 cell culture Methods 0.000 title claims abstract description 6
- 230000028327 secretion Effects 0.000 claims abstract description 6
- 102100031939 Erythropoietin Human genes 0.000 claims description 13
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 235000015097 nutrients Nutrition 0.000 claims description 7
- 238000004587 chromatography analysis Methods 0.000 claims description 5
- 238000000502 dialysis Methods 0.000 claims description 5
- 238000000034 method Methods 0.000 claims description 4
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 claims description 3
- 239000001913 cellulose Substances 0.000 claims description 3
- 229920002678 cellulose Polymers 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 210000005084 renal tissue Anatomy 0.000 claims description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims 3
- 238000013016 damping Methods 0.000 claims 2
- 239000012530 fluid Substances 0.000 claims 2
- 239000012980 RPMI-1640 medium Substances 0.000 claims 1
- 230000003139 buffering effect Effects 0.000 claims 1
- 230000003750 conditioning effect Effects 0.000 claims 1
- 238000010828 elution Methods 0.000 claims 1
- 238000004108 freeze drying Methods 0.000 claims 1
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 claims 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims 1
- 238000005096 rolling process Methods 0.000 claims 1
- 230000003068 static effect Effects 0.000 claims 1
- 239000006228 supernatant Substances 0.000 claims 1
- 210000003734 kidney Anatomy 0.000 abstract description 5
- 210000004027 cell Anatomy 0.000 abstract description 4
- 229920002684 Sepharose Polymers 0.000 abstract description 3
- 239000003814 drug Substances 0.000 abstract description 3
- 210000003292 kidney cell Anatomy 0.000 abstract description 3
- 206010058116 Nephrogenic anaemia Diseases 0.000 abstract description 2
- 208000007502 anemia Diseases 0.000 abstract description 2
- 238000002306 biochemical method Methods 0.000 abstract description 2
- 230000000694 effects Effects 0.000 abstract description 2
- 210000003754 fetus Anatomy 0.000 abstract description 2
- 239000007788 liquid Substances 0.000 abstract description 2
- GUBGYTABKSRVRQ-WFVLMXAXSA-N DEAE-cellulose Chemical compound OC1C(O)C(O)C(CO)O[C@H]1O[C@@H]1C(CO)OC(O)C(O)C1O GUBGYTABKSRVRQ-WFVLMXAXSA-N 0.000 abstract 1
- 210000003013 erythroid precursor cell Anatomy 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 102000003886 Glycoproteins Human genes 0.000 description 2
- 108090000288 Glycoproteins Proteins 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- GHKCSRZBNZQHKW-UHFFFAOYSA-N 1-sulfanylethanol Chemical compound CC(O)S GHKCSRZBNZQHKW-UHFFFAOYSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241000699802 Cricetulus griseus Species 0.000 description 1
- 206010055690 Foetal death Diseases 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 101000878457 Macrocallista nimbosa FMRFamide Proteins 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 206010067482 No adverse event Diseases 0.000 description 1
- 239000012506 Sephacryl® Substances 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000004115 adherent culture Methods 0.000 description 1
- 239000013566 allergen Substances 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 208000020832 chronic kidney disease Diseases 0.000 description 1
- 208000022831 chronic renal failure syndrome Diseases 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000012407 engineering method Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000000913 erythropoietic effect Effects 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 208000018875 hypoxemia Diseases 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 208000017169 kidney disease Diseases 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 210000003360 nephrocyte Anatomy 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 210000000287 oocyte Anatomy 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
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
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。
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)
- 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。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN89105382.4A CN1053698C (zh) | 1989-01-19 | 1989-01-19 | 细胞培养生产促红细胞生成素的方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN89105382.4A CN1053698C (zh) | 1989-01-19 | 1989-01-19 | 细胞培养生产促红细胞生成素的方法 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1044496A CN1044496A (zh) | 1990-08-08 |
| CN1053698C true CN1053698C (zh) | 2000-06-21 |
Family
ID=4856144
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN89105382.4A Expired - Fee Related CN1053698C (zh) | 1989-01-19 | 1989-01-19 | 细胞培养生产促红细胞生成素的方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1053698C (zh) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101804287B1 (ko) | 2007-06-08 | 2017-12-04 | 웨이크 포리스트 유니버시티 헬스 사이언시즈 | 신부전 치료를 위한 선택적 세포 치료법 |
| US10590391B2 (en) | 2007-06-08 | 2020-03-17 | Wake Forest University Health Sciences | Selective cell therapy for the treatment of renal failure |
| US9580688B2 (en) | 2007-06-08 | 2017-02-28 | Wake Forest University Health Sciences | Kidney structures and methods of forming the same |
| EP2662440B1 (en) | 2008-11-12 | 2018-04-25 | inRegen | Isolated renal cells and uses thereof |
| CN106214700B (zh) | 2010-05-12 | 2021-09-07 | 因瑞金公司 | 生物活性肾细胞 |
| US11123372B2 (en) | 2016-07-29 | 2021-09-21 | Prokidney | Bioactive renal cells for the treatment of chronic kidney disease |
-
1989
- 1989-01-19 CN CN89105382.4A patent/CN1053698C/zh not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN1044496A (zh) | 1990-08-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Takahashi et al. | Recombinant human transforming growth factor-alpha stimulates the formation of osteoclast-like cells in long-term human marrow cultures. | |
| TW516962B (en) | A human TNFR1-IgG1 preparation | |
| Steeg et al. | Regulation of murine macrophage Ia antigen expression by an immune interferon-like lymphokine: inhibitory effect of endotoxin. | |
| KR860000897B1 (ko) | 인체 에리트로포이에틴의 제조방법 | |
| Chen et al. | Further studies on the thymocyte stimulating factor | |
| CN1699408A (zh) | 高效抑制血管生成多肽及其制备方法和应用 | |
| CN1053698C (zh) | 细胞培养生产促红细胞生成素的方法 | |
| Jyonouchi et al. | Humoral factors in very young NZB mice that enhance the maturation of normal B cell precursors. Partial purification and characterization. | |
| KR20170089924A (ko) | 줄기세포 재료 및 그 제조방법 | |
| Lipschitz et al. | Role of colony-stimulating factor in myelopoiesis in murine long-term bone marrow cultures | |
| Tsao et al. | Expression of the functional erythropoietin receptors on interleukin 3-dependent murine cell lines. | |
| Choppin et al. | Characterization of erythropoietin produced by IW32 murine erythroleukemia cells | |
| Metcalf et al. | Selective stimulation by mouse spleen cell conditioned medium of human eosinophil colony formation | |
| Keith et al. | The effect of diphenylhydantoin on fibroblasts in vitro | |
| Kawakita et al. | Thrombopoiesis‐and Megakaryocyte Colony‐stimulating Factors in the Urine of Patients with Idiopathic Thrombocytopenic Purpura | |
| Wise et al. | Effects of transforming growth factor-β1 on cultured dental follicle cells from rat mandibular molars | |
| Okabe et al. | Growth factor (s) produced by a human leukemic cell line growing in a protein-free chemically defined medium | |
| EP0672684A1 (en) | Novel megakaryocyte colony stimulating factor and production thereof | |
| CN116425857B (zh) | 一种无糖基化修饰的il-15及制备方法 | |
| US5034513A (en) | Avian interleukin-2 | |
| Sateesh | Biotechnology-5: Animal Cells, Immunology & Plant Biotechnology | |
| CN1221754A (zh) | 血小板生成素/粒、巨噬细胞集落刺激因子融合蛋白及用途 | |
| Ohta et al. | Induction of proliferation of human circulating monocytes in vitro by lectin-induced factor (s) from lymphocytes | |
| Kovacevic et al. | Erythroid progenitor cells from pig bone marrow and peripheral blood | |
| JPH03505732A (ja) | 移動刺激因子 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C06 | Publication | ||
| PB01 | Publication | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C19 | Lapse of patent right due to non-payment of the annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |