WO2023169249A1 - Red blood cell preservation solution and use thereof - Google Patents

Red blood cell preservation solution and use thereof Download PDF

Info

Publication number
WO2023169249A1
WO2023169249A1 PCT/CN2023/078627 CN2023078627W WO2023169249A1 WO 2023169249 A1 WO2023169249 A1 WO 2023169249A1 CN 2023078627 W CN2023078627 W CN 2023078627W WO 2023169249 A1 WO2023169249 A1 WO 2023169249A1
Authority
WO
WIPO (PCT)
Prior art keywords
red blood
blood cell
cell preservation
blood cells
preservation solution
Prior art date
Application number
PCT/CN2023/078627
Other languages
French (fr)
Chinese (zh)
Inventor
张磊
王文天
池颖
李慧媛
付荣凤
谷文静
Original Assignee
细胞生态海河实验室
中国医学科学院血液病医院(中国医学科学院血液学研究所)
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by 细胞生态海河实验室, 中国医学科学院血液病医院(中国医学科学院血液学研究所) filed Critical 细胞生态海河实验室
Publication of WO2023169249A1 publication Critical patent/WO2023169249A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This application belongs to the field of medical technology and specifically relates to a red blood cell preservation solution and its application.
  • Red blood cells are the main component cells of human blood.
  • the RBC content in adult blood reaches 5 ⁇ 10 12 /L (5 ⁇ 10 9 /mL, 5000million/mL).
  • Hematocrit (red blood cells in whole blood) percentage of volume) reaches 40-50%.
  • the main physiological function of red blood cells is to transport oxygen from the lungs and release it to the body for metabolism.
  • red blood cell transfusion has become a basic treatment method for clinically critical and severe patients.
  • red blood cells for emergency blood transfusion mainly come from social donations, which are then separated and stored in red blood cell preservation solutions.
  • red blood cells when blood is stored in a liquid matrix, red blood cells will undergo a series of biochemical and structural changes, that is, red blood cell storage damage, which will affect the survival of red blood cells after transfusion and cause changes in their functions.
  • red blood cell preservation solutions has become the focus and hot spot of many scholars' research.
  • CN1857312A discloses a red blood cell cryoprotective solution, including small molecule sugars (trehalose, glucose, sucrose, maltose, fructose or mannitol), sodium chloride, potassium dihydrogen phosphate and disodium hydrogen phosphate.
  • This protective solution can achieve deep cryopreservation of red blood cells, reduce the hemolysis rate of red blood cells, and greatly simplify the washing procedures of red blood cells after thawing.
  • the preservation time of red blood cells by this protective solution needs to be further improved.
  • CN111919835A discloses a preservation solution for maintaining the activity of red blood cells, including: heparin-poloxamer, glycine, potassium dihydrogen phosphate, basic fibroblast growth factor and trimethoprim, which can ensure the oxygen-carrying activity of red blood cells, but This preservation solution only maintains the activity of red blood cells by increasing the effective concentration of nitric oxide gas, and does not disclose research on the apoptosis and hemolysis of red blood cells.
  • CN104705287A discloses a red blood cell cryopreservation solution, which includes: tripotassium citrate monohydrate, sodium dihydrogen phosphate dihydrate, disodium hydrogen phosphate dodecahydrate and glycerin.
  • This preservation solution can achieve physiological balance inside and outside the red blood cell membrane and avoid rupture of the red blood cell membrane.
  • the presence of glycerol will cause the later washing process of red blood cells to be cumbersome and burden the medical staff.
  • red blood cell preservation solutions Although they can significantly extend the life of red blood cells, the key core technologies of anti-apoptosis and anti-hemolysis of red blood cells are still not mastered. It remains to be studied to further improve the ability of red blood cell preservation solutions to resist apoptosis, hemolysis, and natural disintegration of red blood cells and to extend the storage time of red blood cells.
  • the red blood cell preservation solution can strongly protect red blood cells and extremely significantly extend the life of red blood cells.
  • the present application provides a red blood cell preservation solution, which includes a red blood cell preservation base solution and an antioxidant, wherein the antioxidant includes uric acid.
  • the red blood cells can be further activated, extremely significantly enhance the ability of the red blood cells to resist hemolysis and apoptosis, and the in vitro storage time is significantly prolonged, which is suitable for the ultra-long-term in vitro low temperature of red blood cells for blood transfusion.
  • Preservation has great application value.
  • liver xanthine dehydrogenase is the rate-limiting enzyme for the production of uric acid
  • the lack of XDH enzyme in red blood cells means that even if adenine (a precursor to the production of uric acid) is added to the existing red blood cell preservation solution body), red blood cells cannot effectively produce uric acid. Therefore, red blood cells themselves are unable to store sufficient levels of uric acid unless added extracellularly.
  • Uric acid is a purine metabolite unique to primates (humans, etc.). In other mammals, uric acid is further metabolized to allantoin and excreted in the urine. Studies have proven that uric acid, as an antioxidant, can effectively increase the lifespan of model organisms. Therefore, uric acid is not only a metabolic end product in the human body, but also has important physiological functions.
  • the red blood cell preservation base solution may be a commercially available red blood cell preservation solution, including MAP preparation or Alderman's solution.
  • the MAP preparation contains trisodium citrate, citric acid, glucose, sodium dihydrogen phosphate, adenine, sodium chloride and mannitol;
  • the Aldrin's solution contains sodium chloride, sodium citrate, citric acid and glucose.
  • the concentration of uric acid in the red blood cell preservation solution is 10-1000 ⁇ M, for example, it can be 10 ⁇ M, 50 ⁇ M, 100 ⁇ M, 200 ⁇ M, 300 ⁇ M, 400 ⁇ M, 500 ⁇ M, 600 ⁇ M, 700 ⁇ M, 800 ⁇ M, 900 ⁇ M or 1000 ⁇ M, etc.
  • the concentration of uric acid in the red blood cell preservation solution is 200-800 ⁇ M.
  • the concentration of uric acid in the red blood cell preservation solution is 300-500 ⁇ M.
  • the concentration of uric acid in the red blood cell preservation solution is 10-1000 ⁇ M, which can ensure the effect on red blood cells.
  • the protective effect is preferably 200-800 ⁇ M, and further preferably 300-500 ⁇ M, because the normal value of uric acid level in the human body is 100-300 ⁇ M, and hyperuricemia can reach 500 ⁇ M.
  • 300-500 ⁇ M uric acid can extremely significantly protect red blood cells and resist vitamin C-induced hemolysis.
  • red blood cells will naturally hemolyze in MAP nutrient solution, and the addition of uric acid can significantly protect red blood cells and resist natural hemolysis.
  • the use temperature of the red blood cell preservation solution is 4-10°C, for example, it can be 4°C, 4.5°C, 5°C, 5.5°C, 6°C, 6.5°C, 7°C, 7.5°C, 8°C, 8.5°C , 9°C, 9.5°C or 10°C, etc.
  • the present application provides an application of the red blood cell preservation solution according to the first aspect in preserving red blood cells in vitro.
  • red blood cells can be activated, extremely significantly enhance the ability of red blood cells to resist hemolysis and apoptosis, and improve the membrane stability of red blood cells, thereby achieving long-term preservation of red blood cells in optimal condition.
  • This has great application value for the ultra-long-term in vitro cryogenic preservation of red blood cells for blood transfusion.
  • Figure 1 is a diagram showing the preservation results of red blood cells by the red blood cell preservation solution obtained in Example 1.
  • Figure 2 is a diagram showing the preservation results of red blood cells using the red blood cell preservation solution obtained in Example 2.
  • Figure 3 is a diagram showing the preservation results of red blood cells using the red blood cell preservation solution obtained in Example 3.
  • Figure 4 is a diagram showing the preservation results of red blood cells by the red blood cell preservation solution obtained in Example 4.
  • Figure 5 is a diagram showing the preservation results of red blood cells using the red blood cell preservation solution obtained in Example 5.
  • Figure 6 is a diagram showing the preservation results of red blood cells using the red blood cell preservation solution obtained in Example 6.
  • Figure 7 is a diagram showing the preservation results of red blood cells by the red blood cell preservation solution obtained in Comparative Example 1.
  • Figure 8 shows the hemolysis and apoptosis of red blood cells in different drug groups.
  • MAP reagent was purchased from Shanghai Yuanmu Biotechnology, model number is R18099;
  • Uric acid was purchased from Shanghai Yuanye Biotechnology, model number is S30640;
  • the remaining materials and raw materials can be obtained from other commercial sources without special instructions.
  • This embodiment provides a red blood cell preservation solution, including: 1mL MAP reagent, 500 ⁇ M uric acid;
  • 500 ⁇ M refers to the concentration of uric acid in the red blood cell preservation solution.
  • This embodiment provides a red blood cell preservation solution, including: 1mL MAP reagent, 200 ⁇ M uric acid;
  • 200 ⁇ M refers to the concentration of uric acid in the red blood cell preservation solution.
  • This embodiment provides a red blood cell preservation solution.
  • the only difference from Example 1 is that the concentration of uric acid in the red blood cell preservation solution is 10 ⁇ M.
  • the other parameters are consistent with Example 1.
  • This embodiment provides a red blood cell preservation solution.
  • the only difference from Example 1 is that the concentration of uric acid in the red blood cell preservation solution is 50 ⁇ M.
  • the other parameters are consistent with Example 1.
  • This embodiment provides a red blood cell preservation solution.
  • the only difference from Example 1 is that the concentration of uric acid in the red blood cell preservation solution is 100 ⁇ M.
  • the other parameters are consistent with Example 1.
  • This embodiment provides a red blood cell preservation solution.
  • the only difference from Example 1 is that the concentration of uric acid in the red blood cell preservation solution is 1000 ⁇ M.
  • the other parameters are consistent with Example 1.
  • This comparative example provides a red blood cell preservation solution.
  • the only difference from Example 1 is that the red blood cell preservation solution does not include uric acid, and the other parameters are consistent with Example 1.
  • red blood cell preservation solutions obtained in Examples 1-6 and Comparative Example 1 were used to preserve red blood cells, and the ability of red blood cells to resist hemolysis and apoptosis was observed.
  • the test method is as follows: Take 1mL of adult peripheral blood and count the red blood cells. According to the standard of 500,000 RBC/1mL red blood cell preservation solution, inoculate it into a 12-well plate. Incubate at 37°C. Observe under a microscope after 24 hours.
  • this application introduces uric acid as an antioxidant into the red blood cell preservation base solution, which can activate red blood cells, extremely significantly enhance the ability of red blood cells to resist hemolysis and apoptosis, and further improve the membrane stability of red blood cells, thereby achieving the best state of red blood cells.
  • the long-term preservation of red blood cells for blood transfusion has great application value and broad application prospects for the ultra-long-term in vitro cryogenic preservation of red blood cells for blood transfusion.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Dentistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Environmental Sciences (AREA)
  • Biophysics (AREA)
  • Physiology (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)

Abstract

The present invention relates to a red blood cell preservation solution and the use thereof. The red blood cell preservation solution contains a red blood cell preservation base solution and an antioxidant, wherein the antioxidant comprises uric acid. The red blood cell preservation solution can be used for long-term in-vitro preservation of red blood cells. The red blood cells can be further activated by introducing uric acid as an antioxidant into the red blood cell preservation base solution, thereby significantly enhancing the hemolysis and apoptosis resistance of red blood cells, and significantly prolonging the in-vitro preservation time, which has a great application value for ultra-long-term in-vitro low-temperature preservation of red blood cells for blood transfusion.

Description

一种红细胞保存液及其应用A kind of red blood cell preservation solution and its application 技术领域Technical field
本申请属于医疗技术领域，具体涉及一种红细胞保存液及其应用。This application belongs to the field of medical technology and specifically relates to a red blood cell preservation solution and its application.
背景技术Background technique
红细胞(Red blood cell，RBC)是人体血液的主要成分细胞，成人血液中RBC含量达5×1012/L(5×109/mL，5000million/mL)，血细胞比容(红细胞在全血中所占体积的百分比)达40-50％。红细胞的主要生理功能是从肺运输氧气并释放给机体以供新陈代谢。失血、贫血等紧急情况下，机体的红细胞数量严重降低，危及生命安全。因此，红细胞输血已成为临床危急重症患者的基础治疗手段。目前输血急救的红细胞主要来自于社会捐献，随后经红细胞分离，储存在红细胞保存液中。然而把血液储存在液体基质中时，红细胞会发生一系列生物化学与结构上的改变，即红细胞储存损伤，会影响输血后的红细胞生存并造成其功能改变。目前，针对红细胞保存液的开发已经成为众多学者研究的重点与热点。Red blood cells (RBCs) are the main component cells of human blood. The RBC content in adult blood reaches 5×10 12 /L (5×10 9 /mL, 5000million/mL). Hematocrit (red blood cells in whole blood) percentage of volume) reaches 40-50%. The main physiological function of red blood cells is to transport oxygen from the lungs and release it to the body for metabolism. In emergencies such as blood loss and anemia, the number of red blood cells in the body is severely reduced, endangering life. Therefore, red blood cell transfusion has become a basic treatment method for clinically critical and severe patients. At present, red blood cells for emergency blood transfusion mainly come from social donations, which are then separated and stored in red blood cell preservation solutions. However, when blood is stored in a liquid matrix, red blood cells will undergo a series of biochemical and structural changes, that is, red blood cell storage damage, which will affect the survival of red blood cells after transfusion and cause changes in their functions. At present, the development of red blood cell preservation solutions has become the focus and hot spot of many scholars' research.
CN1857312A公开了一种红细胞冷冻保护液，包括小分子糖(海藻糖、葡萄糖、蔗糖、麦芽糖、果糖或甘露醇)、氯化钠、磷酸二氢钾和磷酸氢二钠。该保护液可以实现对红细胞的深低温保存，降低红细胞的溶血率，大大简化解冻后红细胞的洗涤程序，但该保护液对红细胞的保存时间有待进一步提高。CN1857312A discloses a red blood cell cryoprotective solution, including small molecule sugars (trehalose, glucose, sucrose, maltose, fructose or mannitol), sodium chloride, potassium dihydrogen phosphate and disodium hydrogen phosphate. This protective solution can achieve deep cryopreservation of red blood cells, reduce the hemolysis rate of red blood cells, and greatly simplify the washing procedures of red blood cells after thawing. However, the preservation time of red blood cells by this protective solution needs to be further improved.
CN111919835A公开了一种维持红细胞活性的保存液，包括：肝素-泊洛沙姆、甘氨酸、磷酸二氢钾、碱性成纤维细胞生长因子和甲氧苄啶，可以保证红细胞的携氧活性，但该保存液仅仅是通过提高一氧化氮气体的有效浓度来维持红细胞的活性，没有公开对红细胞凋亡和溶血情况的研究。CN111919835A discloses a preservation solution for maintaining the activity of red blood cells, including: heparin-poloxamer, glycine, potassium dihydrogen phosphate, basic fibroblast growth factor and trimethoprim, which can ensure the oxygen-carrying activity of red blood cells, but This preservation solution only maintains the activity of red blood cells by increasing the effective concentration of nitric oxide gas, and does not disclose research on the apoptosis and hemolysis of red blood cells.
CN104705287A公开了一种红细胞冰冻保存液，包括：一水枸橼酸三钾、二水磷酸二氢钠、十二水磷酸氢二钠和甘油。该保存液可以实现红细胞膜内外的生理平衡，避免红细胞膜的破裂，但甘油的存在会导致后期的红细胞使用时的洗涤过程繁琐，给医护人员造成负担。CN104705287A discloses a red blood cell cryopreservation solution, which includes: tripotassium citrate monohydrate, sodium dihydrogen phosphate dihydrate, disodium hydrogen phosphate dodecahydrate and glycerin. This preservation solution can achieve physiological balance inside and outside the red blood cell membrane and avoid rupture of the red blood cell membrane. However, the presence of glycerol will cause the later washing process of red blood cells to be cumbersome and burden the medical staff.
基于以上研究，可以看到目前针对红细胞保存液的开发很多，尽管其可以明显延长红细胞的寿命，但仍然没有掌握到红细胞抗凋亡抗溶血关键核心技术， 对于进一步提高红细胞保存液对红细胞抗凋亡、抗溶血、抗自然崩解的能力以及延长红细胞的保存时间有待研究。Based on the above research, it can be seen that there are currently many developments for red blood cell preservation solutions. Although they can significantly extend the life of red blood cells, the key core technologies of anti-apoptosis and anti-hemolysis of red blood cells are still not mastered. It remains to be studied to further improve the ability of red blood cell preservation solutions to resist apoptosis, hemolysis, and natural disintegration of red blood cells and to extend the storage time of red blood cells.
发明内容Contents of the invention
本申请提供了一种红细胞保存液及其应用。所述红细胞保存液可以强力保护红细胞，极其显著地延长红细胞寿命。This application provides a red blood cell preservation solution and its application. The red blood cell preservation solution can strongly protect red blood cells and extremely significantly extend the life of red blood cells.
第一方面，本申请提供一种红细胞保存液，所述红细胞保存液包括红细胞保存基液和抗氧化剂，其中，所述抗氧化剂包括尿酸。In a first aspect, the present application provides a red blood cell preservation solution, which includes a red blood cell preservation base solution and an antioxidant, wherein the antioxidant includes uric acid.
本申请中，通过在红细胞保存基液中引入尿酸作为抗氧化剂，可以进一步活化红细胞，极其显著地增强红细胞抗溶血凋亡的能力，体外保存时间明显延长，这对于输血用红细胞的超长期体外低温保存，具有重大应用价值。In this application, by introducing uric acid as an antioxidant into the red blood cell preservation base solution, the red blood cells can be further activated, extremely significantly enhance the ability of the red blood cells to resist hemolysis and apoptosis, and the in vitro storage time is significantly prolonged, which is suitable for the ultra-long-term in vitro low temperature of red blood cells for blood transfusion. Preservation has great application value.
需要说明的是，肝脏黄嘌呤脱氢酶(xanthine dehydrogenase，XDH)是尿酸生成的限速酶，而红细胞中缺乏XDH酶，意味着即便现有红细胞保存液中添加了腺嘌呤(尿酸的生成前体)，红细胞也无法有效生成尿酸。因此，除非胞外添加，红细胞自身是无法储存足够水平的尿酸。而尿酸是灵长类动物(人类等)特有的嘌呤代谢产物。在其他哺乳动物中，尿酸会进一步代谢成尿囊素而随尿液排泄。已有研究证明，尿酸作为抗氧化剂，可以有效提高模式生物的寿命，因此，尿酸在人体中不仅仅是代谢终产物，其具有重要的生理功能。It should be noted that liver xanthine dehydrogenase (XDH) is the rate-limiting enzyme for the production of uric acid, and the lack of XDH enzyme in red blood cells means that even if adenine (a precursor to the production of uric acid) is added to the existing red blood cell preservation solution body), red blood cells cannot effectively produce uric acid. Therefore, red blood cells themselves are unable to store sufficient levels of uric acid unless added extracellularly. Uric acid is a purine metabolite unique to primates (humans, etc.). In other mammals, uric acid is further metabolized to allantoin and excreted in the urine. Studies have proven that uric acid, as an antioxidant, can effectively increase the lifespan of model organisms. Therefore, uric acid is not only a metabolic end product in the human body, but also has important physiological functions.
本申请中，所述红细胞保存基液可以是市售的红细胞保存液，包括MAP制剂或阿氏液。In this application, the red blood cell preservation base solution may be a commercially available red blood cell preservation solution, including MAP preparation or Alderman's solution.
其中，所述MAP制剂中含有枸橼酸三钠、枸橼酸、葡萄糖、磷酸二氢钠、腺嘌呤、氯化钠和甘露醇；Wherein, the MAP preparation contains trisodium citrate, citric acid, glucose, sodium dihydrogen phosphate, adenine, sodium chloride and mannitol;
所述阿氏液中含有氯化钠、柠檬酸钠、柠檬酸和葡萄糖。The Aldrin's solution contains sodium chloride, sodium citrate, citric acid and glucose.
本申请中，所述尿酸在红细胞保存液中的浓度为10-1000μM，例如可以是10μM、50μM、100μM、200μM、300μM、400μM、500μM、600μM、700μM、800μM、900μM或1000μM等。In this application, the concentration of uric acid in the red blood cell preservation solution is 10-1000 μM, for example, it can be 10 μM, 50 μM, 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, 800 μM, 900 μM or 1000 μM, etc.
上述数值范围内的其他点值均可选择，在此便不再一一赘述。Other point values within the above numerical range can be selected, so I will not go into details here.
优选地，所述尿酸在红细胞保存液中的浓度为200-800μM。Preferably, the concentration of uric acid in the red blood cell preservation solution is 200-800 μM.
进一步优选地，所述尿酸在红细胞保存液中的浓度为300-500μM。Further preferably, the concentration of uric acid in the red blood cell preservation solution is 300-500 μM.
所述尿酸在红细胞保存液中的浓度为10-1000μM，能够保证对红细胞起到 保护作用，优选为200-800μM，进一步优选为300-500μM，原因在于人体内的尿酸水平正常值在100-300μM，高尿酸血症可达500μM。根据发明人的体外红细胞培养实验，300-500μM尿酸即可极其显著的保护红细胞，抗维生素C诱导的溶血现象。另外，即便没有维生素C，红细胞在MAP营养液中也会自然溶血，而尿酸的添加则可以显著地保护红细胞，抗自然溶血反应。The concentration of uric acid in the red blood cell preservation solution is 10-1000 μM, which can ensure the effect on red blood cells. The protective effect is preferably 200-800 μM, and further preferably 300-500 μM, because the normal value of uric acid level in the human body is 100-300 μM, and hyperuricemia can reach 500 μM. According to the inventor's in vitro red blood cell culture experiments, 300-500 μM uric acid can extremely significantly protect red blood cells and resist vitamin C-induced hemolysis. In addition, even without vitamin C, red blood cells will naturally hemolyze in MAP nutrient solution, and the addition of uric acid can significantly protect red blood cells and resist natural hemolysis.
本申请中，所述红细胞保存液的使用温度为4-10℃，例如可以是4℃、4.5℃、5℃、5.5℃、6℃、6.5℃、7℃、7.5℃、8℃、8.5℃、9℃、9.5℃或10℃等。In this application, the use temperature of the red blood cell preservation solution is 4-10°C, for example, it can be 4°C, 4.5°C, 5°C, 5.5°C, 6°C, 6.5°C, 7°C, 7.5°C, 8°C, 8.5°C , 9℃, 9.5℃ or 10℃, etc.
上述数值范围内的其他点值均可选择，在此便不再一一赘述。Other point values within the above numerical range can be selected, so I will not go into details here.
第二方面，本申请提供一种根据第一方面所述的红细胞保存液在体外保存红细胞中的应用。In a second aspect, the present application provides an application of the red blood cell preservation solution according to the first aspect in preserving red blood cells in vitro.
相对于现有技术，本申请具有以下有益效果：Compared with the existing technology, this application has the following beneficial effects:
本申请中，通过在红细胞保存基液中引入尿酸作为抗氧化剂，可以活化红细胞，极其显著地增强红细胞抗溶血凋亡的能力，改善红细胞的膜稳态，从而实现最佳状态红细胞的长期保存，这对于输血用红细胞的超长期体外低温保存，具有重大应用价值。In this application, by introducing uric acid as an antioxidant into the red blood cell preservation base solution, red blood cells can be activated, extremely significantly enhance the ability of red blood cells to resist hemolysis and apoptosis, and improve the membrane stability of red blood cells, thereby achieving long-term preservation of red blood cells in optimal condition. This has great application value for the ultra-long-term in vitro cryogenic preservation of red blood cells for blood transfusion.
附图说明Description of the drawings
图1为实施例1得到的红细胞保存液对红细胞的保存结果图。Figure 1 is a diagram showing the preservation results of red blood cells by the red blood cell preservation solution obtained in Example 1.
图2为实施例2得到的红细胞保存液对红细胞的保存结果图。Figure 2 is a diagram showing the preservation results of red blood cells using the red blood cell preservation solution obtained in Example 2.
图3为实施例3得到的红细胞保存液对红细胞的保存结果图。Figure 3 is a diagram showing the preservation results of red blood cells using the red blood cell preservation solution obtained in Example 3.
图4为实施例4得到的红细胞保存液对红细胞的保存结果图。Figure 4 is a diagram showing the preservation results of red blood cells by the red blood cell preservation solution obtained in Example 4.
图5为实施例5得到的红细胞保存液对红细胞的保存结果图。Figure 5 is a diagram showing the preservation results of red blood cells using the red blood cell preservation solution obtained in Example 5.
图6为实施例6得到的红细胞保存液对红细胞的保存结果图。Figure 6 is a diagram showing the preservation results of red blood cells using the red blood cell preservation solution obtained in Example 6.
图7为对比例1得到的红细胞保存液对红细胞的保存结果图。Figure 7 is a diagram showing the preservation results of red blood cells by the red blood cell preservation solution obtained in Comparative Example 1.
图8为红细胞在不同加药组别中的溶血凋亡图。Figure 8 shows the hemolysis and apoptosis of red blood cells in different drug groups.
具体实施方式Detailed ways
下面通过具体实施方式来进一步说明本申请的技术方案。本领域技术人员应该明了，所述实施例仅仅是帮助理解本申请，不应视为对本申请的具体限制。The technical solutions of the present application will be further described below through specific implementations. Those skilled in the art should understand that the embodiments are only to help understand the present application and should not be regarded as specific limitations of the present application.
下述实施例、对比例和测试例中相应材料与原料的购买来源如下：The purchase sources of the corresponding materials and raw materials in the following examples, comparative examples and test examples are as follows:
MAP试剂购自上海远慕生物，型号为R18099； MAP reagent was purchased from Shanghai Yuanmu Biotechnology, model number is R18099;
阿氏液购自北京索莱宝，型号为R1016；Algren's solution was purchased from Beijing Solebao, model number is R1016;
尿酸购自上海源叶生物，型号为S30640；Uric acid was purchased from Shanghai Yuanye Biotechnology, model number is S30640;
其余材料和原料无特殊说明，均可从其他商业途径获得。The remaining materials and raw materials can be obtained from other commercial sources without special instructions.
实施例1Example 1
本实施例提供一种红细胞保存液，包括：1mL MAP试剂、500μM尿酸；This embodiment provides a red blood cell preservation solution, including: 1mL MAP reagent, 500 μM uric acid;
其中，500μM指尿酸在红细胞保存液中的浓度。Among them, 500 μM refers to the concentration of uric acid in the red blood cell preservation solution.
实施例2Example 2
本实施例提供一种红细胞保存液，包括：1mL MAP试剂、200μM尿酸；This embodiment provides a red blood cell preservation solution, including: 1mL MAP reagent, 200 μM uric acid;
其中，200μM指尿酸在红细胞保存液中的浓度。Among them, 200 μM refers to the concentration of uric acid in the red blood cell preservation solution.
实施例3Example 3
本实施例提供一种红细胞保存液，与实施例1的区别仅在于，所述尿酸在红细胞保存液中的浓度为10μM，其余参数与实施例1保持一致。This embodiment provides a red blood cell preservation solution. The only difference from Example 1 is that the concentration of uric acid in the red blood cell preservation solution is 10 μM. The other parameters are consistent with Example 1.
实施例4Example 4
本实施例提供一种红细胞保存液，与实施例1的区别仅在于，所述尿酸在红细胞保存液中的浓度为50μM，其余参数与实施例1保持一致。This embodiment provides a red blood cell preservation solution. The only difference from Example 1 is that the concentration of uric acid in the red blood cell preservation solution is 50 μM. The other parameters are consistent with Example 1.
实施例5Example 5
本实施例提供一种红细胞保存液，与实施例1的区别仅在于，所述尿酸在红细胞保存液中的浓度为100μM，其余参数与实施例1保持一致。This embodiment provides a red blood cell preservation solution. The only difference from Example 1 is that the concentration of uric acid in the red blood cell preservation solution is 100 μM. The other parameters are consistent with Example 1.
实施例6Example 6
本实施例提供一种红细胞保存液，与实施例1的区别仅在于，所述尿酸在红细胞保存液中的浓度为1000μM，其余参数与实施例1保持一致。This embodiment provides a red blood cell preservation solution. The only difference from Example 1 is that the concentration of uric acid in the red blood cell preservation solution is 1000 μM. The other parameters are consistent with Example 1.
对比例1Comparative example 1
本对比例提供一种红细胞保存液，与实施例1的区别仅在于，所述红细胞保存液不包括尿酸，其余参数与实施例1保持一致。This comparative example provides a red blood cell preservation solution. The only difference from Example 1 is that the red blood cell preservation solution does not include uric acid, and the other parameters are consistent with Example 1.
测试例1Test example 1
本测试例采用实施例1-6和对比例1得到的红细胞保存液对红细胞进行保存，观察红细胞抗溶血凋亡的能力。In this test example, the red blood cell preservation solutions obtained in Examples 1-6 and Comparative Example 1 were used to preserve red blood cells, and the ability of red blood cells to resist hemolysis and apoptosis was observed.
测试方法如下：取成人外周血1mL，对红细胞计数。按照50万RBC/1mL红细胞保存液的标准，接种于12孔板。置于37℃孵育培养。24h后在显微镜下进行观察。 The test method is as follows: Take 1mL of adult peripheral blood and count the red blood cells. According to the standard of 500,000 RBC/1mL red blood cell preservation solution, inoculate it into a 12-well plate. Incubate at 37°C. Observe under a microscope after 24 hours.
结果如图1-7所示，从图1-6可以看出，本申请提供的红细胞保存液在对红细胞保存时间为24h后，仍有大量RBC处于未溶血的完成状态，且细胞明亮；而图7(红细胞保存也中不含尿酸)中的红细胞在37℃孵育24h后，基本全部溶血，仅剩余红细胞膜结构。通过对比，可以充分表明尿酸对红细胞的特异性保护作用。The results are shown in Figures 1-7. It can be seen from Figures 1-6 that after the red blood cell preservation solution provided by the application has been stored for 24 hours, there are still a large number of RBCs in a complete state without hemolysis, and the cells are bright; and The red blood cells in Figure 7 (the red blood cells are preserved without uric acid) are basically completely hemolyzed after incubation at 37°C for 24 hours, leaving only the red blood cell membrane structure. Through comparison, the specific protective effect of uric acid on red blood cells can be fully demonstrated.
测试例2Test example 2
本测试例将100万个红细胞置于1640培养基中，分别在常氧(21％O2)和低氧(5％O2)下进行1mM维生素C(VC)、500μM尿酸(Uric acid，UA)、1mM VC+500μM UA双因子加药实验，并设置了空白对照组，3天后，在显微镜下进行观察。In this test example, 1 million red blood cells were placed in 1640 culture medium, and 1mM vitamin C (VC), 500μM uric acid (UA) were tested under normoxia (21% O 2 ) and hypoxia (5% O 2 ) respectively. ), 1mM VC+500μM UA double-factor dosing experiment, and set up a blank control group, and observed it under a microscope after 3 days.
实验结果如图8所示，加药3天后，与对照组(即，ctrl组)相比，1mM VC组内红细胞出现大量溶血凋亡，而添加500μM UA组内红细胞溶血凋亡现象得到了明显缓解。尤其是在5％O2下，近乎100％的红细胞均被VC诱导溶血，而至少50％以上的红细胞在UA的保护下还具有完整的细胞膜结构。由VC+UA组可见，UA可以强有力地保护红细胞，抵抗VC诱导的溶血凋亡。The experimental results are shown in Figure 8. Three days after adding the drug, compared with the control group (i.e., ctrl group), a large number of hemolysis and apoptosis of red blood cells occurred in the 1mM VC group, while the hemolysis and apoptosis of red blood cells in the 500 μM UA group was significantly improved. ease. Especially under 5% O2 , nearly 100% of red blood cells are induced to hemolysis by VC, and at least 50% of red blood cells still have an intact cell membrane structure under the protection of UA. It can be seen from the VC+UA group that UA can effectively protect red blood cells and resist hemolysis and apoptosis induced by VC.
综上所述，本申请通过在红细胞保存基液中引入尿酸作为抗氧化剂，可以活化红细胞，极其显著地增强红细胞抗溶血凋亡的能力，进一步改善红细胞的膜稳态，从而实现最佳状态红细胞的长期保存，这对于输血用红细胞的超长期体外低温保存，具有重大应用价值，具有广阔的应用前景。In summary, this application introduces uric acid as an antioxidant into the red blood cell preservation base solution, which can activate red blood cells, extremely significantly enhance the ability of red blood cells to resist hemolysis and apoptosis, and further improve the membrane stability of red blood cells, thereby achieving the best state of red blood cells. The long-term preservation of red blood cells for blood transfusion has great application value and broad application prospects for the ultra-long-term in vitro cryogenic preservation of red blood cells for blood transfusion.
申请人声明，以上所述仅为本申请的具体实施方式，但本申请的保护范围并不局限于此，所属技术领域的技术人员应该明了，任何属于本技术领域的技术人员在本申请揭露的技术范围内，可轻易想到的变化或替换，均落在本申请的保护范围和公开范围之内。 The applicant declares that the above are only specific implementation modes of the present application, but the protection scope of the present application is not limited thereto. Persons skilled in the technical field should understand that any person skilled in the technical field will not disclose any information disclosed in this application. Within the technical scope, changes or substitutions that can be easily imagined fall within the protection scope and disclosure scope of this application.

Claims (7)

  1. 一种红细胞保存液，其包括红细胞保存基液和抗氧化剂；A red blood cell preservation solution, which includes a red blood cell preservation base solution and antioxidants;
    其中，所述抗氧化剂包括尿酸。Wherein, the antioxidant includes uric acid.
  2. 根据权利要求1所述的红细胞保存液，其中，所述红细胞保存基液包括：MAP制剂或阿氏液。The red blood cell preservation solution according to claim 1, wherein the red blood cell preservation base solution includes: MAP preparation or Alderman's solution.
  3. 根据权利要求1或2所述的红细胞保存液，其中，所述尿酸在红细胞保存液中的浓度为10-1000μM。The red blood cell preservation solution according to claim 1 or 2, wherein the concentration of uric acid in the red blood cell preservation solution is 10-1000 μM.
  4. 根据权利要求1-3中任一项所述的红细胞保存液，其中，所述尿酸在红细胞保存液中的浓度为200-800μM。The red blood cell preservation solution according to any one of claims 1 to 3, wherein the concentration of uric acid in the red blood cell preservation solution is 200-800 μM.
  5. 根据权利要求1-4中任一项所述的红细胞保存液，其中，所述尿酸在红细胞保存液中的浓度为300-500μM。The red blood cell preservation solution according to any one of claims 1 to 4, wherein the concentration of uric acid in the red blood cell preservation solution is 300-500 μM.
  6. 根据权利要求1-5中任一项所述的红细胞保存液，其中，所述红细胞保存液的使用温度为4-10℃。The red blood cell preservation solution according to any one of claims 1 to 5, wherein the use temperature of the red blood cell preservation solution is 4-10°C.
  7. 根据权利要求1-6中任一项所述的红细胞保存液在体外保存红细胞中的应用。 Use of the red blood cell preservation solution according to any one of claims 1 to 6 for preserving red blood cells in vitro.
PCT/CN2023/078627 2022-03-07 2023-02-28 Red blood cell preservation solution and use thereof WO2023169249A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210215718.7A CN115039759B (en) 2022-03-07 2022-03-07 Erythrocyte preservation solution and application thereof
CN202210215718.7 2022-03-07

Publications (1)

Publication Number Publication Date
WO2023169249A1 true WO2023169249A1 (en) 2023-09-14

Family

ID=83157282

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/078627 WO2023169249A1 (en) 2022-03-07 2023-02-28 Red blood cell preservation solution and use thereof

Country Status (2)

Country Link
CN (1) CN115039759B (en)
WO (1) WO2023169249A1 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115039759B (en) * 2022-03-07 2023-11-03 细胞生态海河实验室 Erythrocyte preservation solution and application thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1987004072A1 (en) * 1986-01-08 1987-07-16 Shobhana Vora Method and additives for improving the quality and shelf life of stored blood
US6020120A (en) * 1997-10-22 2000-02-01 South Alabama Medical Science Foundation Use of N-acetylcysteine to store red blood cells and a method of aging red cells with nitrogen
CN113383769A (en) * 2021-06-29 2021-09-14 广州朗坤生物科技有限公司 Cell preservation solution and preparation method and application thereof
CN113557295A (en) * 2019-03-15 2021-10-26 株式会社美加细胞 Preservation solution for mammalian cells
CN115039759A (en) * 2022-03-07 2022-09-13 中国医学科学院血液病医院(中国医学科学院血液学研究所) Erythrocyte preservation solution and application thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1987004072A1 (en) * 1986-01-08 1987-07-16 Shobhana Vora Method and additives for improving the quality and shelf life of stored blood
US6020120A (en) * 1997-10-22 2000-02-01 South Alabama Medical Science Foundation Use of N-acetylcysteine to store red blood cells and a method of aging red cells with nitrogen
CN113557295A (en) * 2019-03-15 2021-10-26 株式会社美加细胞 Preservation solution for mammalian cells
CN113383769A (en) * 2021-06-29 2021-09-14 广州朗坤生物科技有限公司 Cell preservation solution and preparation method and application thereof
CN115039759A (en) * 2022-03-07 2022-09-13 中国医学科学院血液病医院(中国医学科学院血液学研究所) Erythrocyte preservation solution and application thereof

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BARDYN MANON, CHEN JINGKUI, DUSSIOT MICHAËL, CRETTAZ DAVID, SCHMID LUCAS, LÄNGST EMMANUEL, AMIREAULT PASCAL, TISSOT JEAN-DANIEL, J: "Restoration of Physiological Levels of Uric Acid and Ascorbic Acid Reroutes the Metabolism of Stored Red Blood Cells", METABOLITES, vol. 10, no. 6, 29 May 2020 (2020-05-29), pages 226, XP093089771, DOI: 10.3390/metabo10060226 *
J A KNIGHT, D A SEARLES: "The Effects of Various Antioxidants on Lipid Peroxidation in Stored Whole Blood", ANNALS OF CLINICAL AND LABORATORY SCIENCE., INSTITUTE FOR CLINICAL SCIENCE, PHILADELPHIA, PA., US, vol. 24, no. 4, 31 December 1994 (1994-12-31), US , pages 294 - 301, XP009548582, ISSN: 0091-7370 *
SONG YUNXIAO, TANG LI, HAN JIANPING, GAO YANTING, TANG BINGHUA, SHAO MINGXI, YUAN WENHUA, GE WEN, HUANG XIAOFENG, YAO TIANYUE, BIA: "Uric Acid Provides Protective Role in Red Blood Cells by Antioxidant Defense: A Hypothetical Analysis", OXIDATIVE MEDICINE AND CELLULAR LONGEVITY, HINDAWI PUBLISHING CORPORATION, US, vol. 2019, 27 March 2019 (2019-03-27), US , pages 1 - 12, XP093089775, ISSN: 1942-0900, DOI: 10.1155/2019/3435174 *
TZOUNAKAS VASSILIS L., ANASTASIADI ALKMINI T., ARVANITI VASILIKI-ZOI, LELLI VERONICA, FANELLI GIUSEPPINA, PARONIS EFTHYMIOS C., AP: "Supplementation with uric and ascorbic acid protects stored red blood cells through enhancement of non-enzymatic antioxidant activity and metabolic rewiring", REDOX BIOLOGY, ELSEVIER, NL, vol. 57, 1 November 2022 (2022-11-01), NL , pages 102477, XP093089776, ISSN: 2213-2317, DOI: 10.1016/j.redox.2022.102477 *
TZOUNAKAS VASSILIS L., GEORGATZAKOU HARA T., KRIEBARDIS ANASTASIOS G., PAPAGEORGIOU EFFIE G., STAMOULIS KONSTANTINOS E., FOUDOULAK: "Uric acid variation among regular blood donors is indicative of red blood cell susceptibility to storage lesion markers: A new hypothesis tested", TRANSFUSION, AMERICAN ASSOCIATION OF BLOOD BANKS, BETHESDA, MD., US, vol. 55, no. 11, 1 November 2015 (2015-11-01), US , pages 2659 - 2671, XP093089773, ISSN: 0041-1132, DOI: 10.1111/trf.13211 *

Also Published As

Publication number Publication date
CN115039759B (en) 2023-11-03
CN115039759A (en) 2022-09-13

Similar Documents

Publication Publication Date Title
Hamberger et al. Inverse enzymatic changes in neurons and glia during increased function and hypoxia
ROSEN et al. Failure of methylene blue treatment in toxic methemoglobinemia: association with glucose-6-phosphate dehydrogenase deficiency
Jacob et al. Oxidative hemolysis and erythrocyte metabolism in hereditary acatalasia
Lehot et al. Glucose homeostasis: comparison between hypothermic and normothermic cardiopulmonary bypass
Kager et al. Allopurinol in the treatment of visceral leishmaniasis
CN102349500B (en) Mesenchymal stem cell self-preserving liquid
WO2023169249A1 (en) Red blood cell preservation solution and use thereof
Peterson et al. Reactive oxygen species may cause myocardial reperfusion injury
Sia et al. Type B lactic acidosis associated with multiple myeloma
Searcy et al. Congenital hemolytic anemia in the Basenji dog due to erythrocyte pyruvate kinase deficiency
Zabucchi et al. Exocytosis in human polymorphonuclear leukocytes induced by A 23187 and calcium
Nakagawara et al. Action of cytochalasin E on polymorphonuclear leucocytes of guinea pig peritoneal exudates
CN111513059B (en) Erythrocyte preservation solution and application thereof
Soardo et al. Alcohol‐induced endothelial changes are associated with oxidative stress and are rapidly reversed after withdrawal
Morshed et al. Propylene glycol-mediated cell injury in a primary culture of human proximal tubule cells
Kelly et al. Vitamin E and dapsone-induced hemolysis
Steinberg et al. Erythrocyte glutathione peroxidase deficiency: biochemical studies on the mechanisms of drug-induced hemolysis
CN104056270A (en) Histone deacetylase inhibitor for preparing medicaments for treating multi-organ damage
El Ladha et al. The ABCCs of sepsis: A framework for understanding the pathophysiology of sepsis
Scott et al. The in vitro action of dapsone and its derivatives on normal and G6PD‐deficient red cells
Ghirmai et al. In vivo evaluation of electron mediators for the reduction of methemoglobin encapsulated in liposomes using electron energies produced by red blood cell glycolysis
Huang et al. Free radicals mediate amphetamine-induced acute pulmonary edema in isolated rat lung
Illner et al. Red blood cell sodium content and permeability changes in hemorrhagic shock
Truitt Jr et al. Anesthesia. LII. Effects of Alcohols and Acetaldehyde on Oxidative Phosphorylation in Brain
Utsunomiya et al. Myocardial protection with prostacyclin after lethal endotoxemia

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23765827

Country of ref document: EP

Kind code of ref document: A1