CN113508806B - Application of amphiphilic compound in preparation of blood erythrocyte cryoprotectant - Google Patents
Application of amphiphilic compound in preparation of blood erythrocyte cryoprotectant Download PDFInfo
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- 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
- A01N1/10—Preservation of living parts
- A01N1/12—Chemical aspects of preservation
- A01N1/122—Preservation or perfusion media
- A01N1/125—Freeze protecting agents, e.g. cryoprotectants or osmolarity regulators
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Abstract
Description
技术领域technical field
本发明属于细胞冻存领域,具体涉及一种两亲性化合物用于制备血液红细胞冷冻保护剂的应用。The invention belongs to the field of cell cryopreservation, in particular to the application of an amphiphilic compound for preparing a blood red blood cell cryoprotectant.
背景技术Background technique
冷冻保存是指在超低温状态下,使细胞、组织或器官的新陈代谢极低甚至停止,而让组织细胞的生理活性得以延长的技术。如:全血在2-6℃可保存30天左右,造血干细胞在-80℃超低温冰箱中可保存一年,在-196℃液氮中可长期保存。冷冻保存保证了被保存对象的形态和遗传稳定性,具有非常重要的科学和市场价值。因此,冷冻保存已广泛应用于生物医学研究、食品科学和农业育种等领域。然而在这个过程中,冰的结晶及其重结晶是造成细胞损伤及死亡的主要原因之一。为了避免冰结晶带来的风险,常常采用高浓度的有机溶剂如二甲基亚砜(DMSO)、乙二醇(EG)和甘油等被广泛用于在冷冻保存过程中实现低温保存介质的玻璃化和无冰凝固。Cryopreservation refers to a technology that prolongs the physiological activity of tissue cells by making the metabolism of cells, tissues or organs extremely low or even stopping under ultra-low temperature. For example, whole blood can be stored for about 30 days at 2-6°C, hematopoietic stem cells can be stored for one year in -80°C ultra-low temperature refrigerator, and long-term storage in -196°C liquid nitrogen. Cryopreservation ensures the morphological and genetic stability of the preserved objects, and has very important scientific and market value. Therefore, cryopreservation has been widely used in fields such as biomedical research, food science, and agricultural breeding. However, in this process, ice crystallization and its recrystallization are one of the main causes of cell damage and death. In order to avoid the risk of ice crystallization, high concentrations of organic solvents such as dimethyl sulfoxide (DMSO), ethylene glycol (EG), and glycerol, which are widely used to achieve cryopreservation during cryopreservation, are often used. melting and ice-free freezing.
血红细胞可以在2–6℃保存30天,也可以在-196℃长期保存。对于患有白血病、溶血性贫血和严重失血的创伤患者来说,输血是挽救生命的手段。在血细胞的冷冻保存过程中,冰的再结晶是细胞损伤的主要原因。传统的低温保护剂,如二甲基亚砜(DMSO)和甘油虽然能在冻存复苏后取得较好的存活率,但是残留的有机溶剂的使用会对输入的患者造成溶血的危险。目前甘油广泛地应用于冷冻保存血红细胞,但是采用甘油作为冷冻保护剂的红细胞,在输血时血管内的甘油残留量能达到1%,这种残留量已足够对接受输血的患者产生严重的副作用。因此,在临床应用之前,必须先将甘油从红细胞中去除。然而,从红细胞中去除甘油是一个耗时的过程,并且需要特殊的设备,因此开发新的红细胞的冷冻保护剂对红细胞的应用有着巨大的推动作用。Red blood cells can be stored at 2–6°C for 30 days, or at -196°C for long-term storage. Blood transfusions are life-saving for trauma patients with leukemia, hemolytic anemia, and severe blood loss. During cryopreservation of blood cells, ice recrystallization is a major cause of cell damage. Although traditional cryoprotectants such as dimethyl sulfoxide (DMSO) and glycerol can achieve better survival rates after cryopreservation and resuscitation, the use of residual organic solvents poses the risk of hemolysis in transfused patients. At present, glycerol is widely used in cryopreservation of red blood cells. However, when glycerol is used as a cryoprotectant for red blood cells, the residual amount of glycerin in blood vessels can reach 1% during blood transfusion, which is enough to cause serious side effects to patients receiving blood transfusion. . Therefore, glycerol must be removed from red blood cells before clinical application. However, removing glycerol from red blood cells is a time-consuming process and requires special equipment, so the development of new red blood cell cryoprotectants has a huge push for red blood cell applications.
发明内容SUMMARY OF THE INVENTION
本发明的目的是提供一种小分子化合物用于红细胞的冷冻保存的应用,不需使用有机溶剂。这种小分子化合物具有良好的水溶性及两亲性(亲冰性和亲水性),可以在复温过程中有效地抑制冰的重结晶,从而避免细胞因冰晶生长过快而导致的损伤。本发明进一步提供一种用于血细胞冷冻保存的冷冻保存试剂。The purpose of the present invention is to provide the application of a small molecule compound for cryopreservation of red blood cells without using an organic solvent. This small molecule compound has good water solubility and amphiphilicity (ice-philic and hydrophilic), which can effectively inhibit ice recrystallization during rewarming, thereby avoiding cell damage caused by excessive ice crystal growth. . The present invention further provides a cryopreservation reagent for cryopreservation of blood cells.
本发明提供一种两亲性化合物作为抗冻剂的用途,所述抗冻剂抑制水溶液中的冰晶生长,所述化合物如式(I)所示:The present invention provides the use of an amphiphilic compound as an antifreeze agent, the antifreeze agent inhibits the growth of ice crystals in an aqueous solution, and the compound is represented by formula (I):
根据本发明,式(I)所示的两亲性化合物可以采用葡萄糖酸内酯与丝氨酸反应制备而成。在一个实施方案中,所述葡萄糖酸内酯与丝氨酸在有机溶剂中反应制备而成,例如有机溶剂为甲醇。在一个具体实施方案中,所述葡萄糖内酯与丝氨酸的反应温度为30-70℃,反应时间为24h–48h,投料比葡萄糖酸内酯比丝氨酸摩尔比为1:(1-5)。According to the present invention, the amphiphilic compound represented by formula (I) can be prepared by reacting gluconolactone with serine. In one embodiment, the gluconolactone is prepared by reacting with serine in an organic solvent, for example, the organic solvent is methanol. In a specific embodiment, the reaction temperature of the gluconolactone and serine is 30-70°C, the reaction time is 24h-48h, and the molar ratio of feeding ratio gluconolactone to serine is 1:(1-5).
在另一个实施方案中,式(I)所示的两亲性化合物是葡萄糖内酯与丝氨酸经固相合成法合成,包括:树脂溶胀、将一种氨基保护的氨基酸共价键连接在溶胀的树脂上、脱保护、加入糖类化合物(例如葡萄糖内酯)缩合反应、切割、纯化等步骤合成式(I)所示的化合物。In another embodiment, the amphiphilic compound represented by formula (I) is synthesized by solid-phase synthesis of glucolactone and serine, including: resin swelling, covalently linking an amino-protected amino acid to the swollen The compound represented by the formula (I) is synthesized by the steps of on-resin, deprotection, addition of a saccharide compound (such as glucolactone), condensation reaction, cleavage, and purification.
本发明进一步提供式(I)所示的两亲性化合物在制备红细胞冷冻保存试剂中的应用。The present invention further provides the use of the amphiphilic compound represented by formula (I) in the preparation of a red blood cell cryopreservation reagent.
根据本发明,所述红细胞冷冻保存试剂包括式(I)所示的两亲性化合物。在一个实施方案中,所述红细胞冷冻保存试剂包括式(I)所示的两亲性的化合物和缓冲液。According to the present invention, the red blood cell cryopreservation reagent includes the amphiphilic compound represented by formula (I). In one embodiment, the red blood cell cryopreservation reagent includes an amphiphilic compound of formula (I) and a buffer.
本发明提供一种红细胞冷冻保存试剂,所述红细胞冷冻保存试剂包括式(I)所示的两亲性的化合物。在一个实施方案中,所述红细胞冷冻保存试剂包括式(I)所示的两亲性化合物和缓冲液。The present invention provides a red blood cell cryopreservation reagent, which comprises the amphiphilic compound represented by formula (I). In one embodiment, the red blood cell cryopreservation reagent includes an amphiphilic compound of formula (I) and a buffer.
根据本发明的优选技术方案,所述冷冻保存试剂不含有有机溶剂,所述有机溶剂包括但不限于二甲基甲酰胺(DMF)、乙二醇、丙二醇、丙三醇等。According to a preferred technical solution of the present invention, the cryopreservation reagent does not contain an organic solvent, and the organic solvent includes, but is not limited to, dimethylformamide (DMF), ethylene glycol, propylene glycol, glycerin, and the like.
根据本发明的优选技术方案,所述冷冻保存试剂由式(I)所示的两亲性化合物和缓冲液组成。According to a preferred technical solution of the present invention, the cryopreservation reagent is composed of the amphiphilic compound represented by formula (I) and a buffer.
本发明还提供一种红细胞的冷冻保存方法,包括:配制包括式(I)所示的两亲性化合物的溶液,将红细胞的悬浮液与所配制的溶液混合,制得红细胞冻存溶液,液氮冷冻保存。The present invention also provides a method for cryopreserving erythrocytes, comprising: preparing a solution comprising the amphiphilic compound represented by formula (I), mixing the suspension of erythrocytes with the prepared solution to prepare a cryopreservation solution for erythrocytes; Nitrogen cryopreservation.
根据本发明,所述血细胞冻存溶液中,所述两亲性化合物摩尔浓度为18-520mM,优选100-400mM,例如360mM。According to the present invention, in the blood cell cryopreservation solution, the molar concentration of the amphiphilic compound is 18-520 mM, preferably 100-400 mM, such as 360 mM.
根据本发明,所述包括两亲性化合物的溶液为所述化合物溶解于缓冲液所形成的溶液。According to the present invention, the solution comprising the amphiphilic compound is a solution formed by dissolving the compound in a buffer.
本发明中,所述缓冲液可以选自本领域已知的细胞培养缓冲液,例如PBS缓冲液、DPBS缓冲液、hepes-buffered HTF缓冲液或其他细胞培养缓冲液中的任一种,优选PBS缓冲液。在一个实施方案中,所述PBS缓冲液pH7.0-7.4。In the present invention, the buffer can be selected from cell culture buffers known in the art, such as any of PBS buffer, DPBS buffer, hepes-buffered HTF buffer or other cell culture buffers, preferably PBS buffer. In one embodiment, the PBS buffer is pH 7.0-7.4.
本发明中,所述红细胞来源于动物血液,例如温血哺乳动物血液,包括但不限于人类、非人类灵长类动物、家畜(例如牛、绵羊、猪)、其他哺乳动物(例如狗、猫)。所述红细胞可经本领域已知的临床应用方法从血液中进行分离而得到。In the present invention, the red blood cells are derived from animal blood, such as warm-blooded mammalian blood, including but not limited to humans, non-human primates, livestock (such as cattle, sheep, pigs), other mammals (such as dogs, cats) ). The red blood cells can be isolated from blood by methods known in the art for clinical application.
本发明中,“两亲性”是指具有亲水性和亲冰性。所述亲水性为可与水分子形成非共价作用,例如可与水形成氢键、范德华尔斯作用、静电作用、疏水作用或者π-π作用等;亲冰性是指为可与冰形成非共价作用,例如可与冰形成氢键、范德华尔斯作用、静电作用、疏水作用或者π-π作用等。In the present invention, "amphiphilic" means having hydrophilicity and ice-philicity. The hydrophilicity means that it can form a non-covalent interaction with water molecules, for example, it can form hydrogen bonds with water, van der Waals interaction, electrostatic interaction, hydrophobic interaction or π-π interaction, etc.; Form non-covalent interactions, such as hydrogen bonds with ice, van der Waals interactions, electrostatic interactions, hydrophobic interactions, or π-π interactions.
有益效果:Beneficial effects:
本发明的化合物具有多羟基结构,亲水性良好,与此同时有具有较高的IRI活性(图4),在冷冻保存过程的复温过程中能够有效的抑制冰晶的重结晶,因此在冷冻保存血红细胞有良好的效果。血细胞在本发明的冷冻保存试剂中冷冻保存后可以达到40%-60%的细胞恢复率,无需加入有机溶剂。本发明提供的红细胞保存材料制备简单,原材料丰富,细胞相容性好,具有良好的冷冻保存效果。The compound of the present invention has a polyhydroxy structure, good hydrophilicity, and at the same time has a high IRI activity (Fig. 4), and can effectively inhibit the recrystallization of ice crystals during the rewarming process of the cryopreservation process. Preservation of red blood cells has a good effect. After the blood cells are cryopreserved in the cryopreservation reagent of the present invention, a cell recovery rate of 40%-60% can be achieved without adding an organic solvent. The red blood cell preservation material provided by the invention is simple in preparation, rich in raw materials, good in cell compatibility, and has a good cryopreservation effect.
附图说明Description of drawings
图1为实施例1制备的化合物G-S的核磁氢谱(上)和碳谱(下)(溶剂:重水);Fig. 1 is the hydrogen nuclear magnetic spectrum (top) and carbon spectrum (bottom) of compound G-S prepared in Example 1 (solvent: deuterium);
图2为实施例1所述的纯PBS缓冲液和G-S的PBS缓冲液抑制重结晶的光学图片;Fig. 2 is the optical picture of the pure PBS buffer described in Example 1 and the PBS buffer of G-S inhibiting recrystallization;
图3为实施例1中不同浓度的G-S的PBS缓冲液溶液中平均最大冰晶尺寸占比PBS(%);Fig. 3 is the average maximum ice crystal size ratio PBS (%) in the PBS buffer solution of different concentrations of G-S in Example 1;
图4为实施例3所述的纯PBS缓冲液和G-S的PBS缓冲液的细胞冷冻保存恢复率。FIG. 4 is the cell cryopreservation recovery rate of pure PBS buffer described in Example 3 and G-S PBS buffer.
具体实施方式Detailed ways
下面结合具体实施例对本发明作进一步阐述,但本发明并不限于以下实施例。所述方法如无特别说明均为常规方法。所述材料如无特别说明均能从公开商业途径得到。The present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments. The methods are conventional methods unless otherwise specified. The materials are available from open commercial sources unless otherwise specified.
实施例1式(I)化合物的制备Example 1 Preparation of compound of formula (I)
将1mmol的的葡萄糖酸内酯溶解在30mL甲醇中,充分搅拌溶解,然后加入10mmol的丝氨酸,在60℃条件下搅拌反应48h,然后将反应液在冰水浴中冷却5h,过滤,将滤液旋蒸后得到产物。Dissolve 1 mmol of gluconolactone in 30 mL of methanol, stir to dissolve, then add 10 mmol of serine, stir at 60 °C for 48 h, then cool the reaction solution in an ice-water bath for 5 h, filter, and spin the filtrate. After the product is obtained.
所得产物的核磁氢谱和碳谱(AVANCEⅡ400M NMR)如图1所示,根据图1可以确定所制备的产物具有如式(I)所示结构,记为化合物G-S。The hydrogen nuclear magnetic spectrum and carbon spectrum (AVANCE II 400M NMR) of the obtained product are shown in Figure 1. According to Figure 1, it can be determined that the prepared product has the structure shown in formula (I), which is denoted as compound G-S.
将所得到的产物进行IRI活性分析评估其抑冰性能。IRI活性分析是通过Splat-Cooling方法进行的。用于研究IRI活性的实验装置为尼康偏振光学显微镜(AZ100)和Linkman(LTS420)冷台。所有样品以所需的浓度溶解在PBS溶液中。将10μL样品溶液从1.5米处滴到预先降温到-60℃的冷台上。液滴瞬间结成一层薄冰。然后将冷台以10℃/分钟的升温速率升到-6℃。然后,将结冰的样品在此温度下退火30分钟。之后通过显微镜上的相机对冰晶进行拍照,图像的处理是使用Image J软件,结果如图2所示。每张照片中选取25个最大冰晶,并统计其中最大轴的长度。这个过程重复三次,计算出平均值,并且用平均值比上PBS缓冲溶液中最大冰晶尺寸的平均值,即为平均最大冰晶尺寸占比PBS(%)。不同浓度的化合物G-S溶液中平均最大冰晶尺寸占比PBS(%)如图3所示。根据图3可以看出,G-S溶液的平均最大冰晶尺寸占比PBS(%)为20-55%左右,表明化合物G-S显著减小了PBS缓冲液中冰晶生长尺寸,显著抑制了溶液中的冰晶生长。当溶液中G-S化合物的浓度为360mM时最小,平均最大冰晶尺寸占比PBS(%)仅为20%-25%左右,取得了优异的抑冰效果。The obtained product was subjected to IRI activity assay to evaluate its ice suppression performance. IRI activity analysis was performed by the Splat-Cooling method. The experimental setup used to study the IRI activity was a Nikon polarizing optical microscope (AZ100) and a Linkman (LTS420) cold stage. All samples were dissolved in PBS solution at the desired concentration. Drop 10 μL of the sample solution from 1.5 m onto a cold stage cooled to -60 °C in advance. The droplets instantly formed a thin layer of ice. The cold stage was then raised to -6°C at a ramp rate of 10°C/min. Then, the frozen samples were annealed at this temperature for 30 minutes. The ice crystals were then photographed by the camera on the microscope, and the images were processed using Image J software. The results are shown in Figure 2. Select 25 largest ice crystals in each photo, and count the length of the largest axis. This process is repeated three times, the average value is calculated, and the average value is compared with the average value of the maximum ice crystal size in the PBS buffer solution, that is, the average maximum ice crystal size ratio in PBS (%). The average maximum ice crystal size ratio in PBS (%) in compound G-S solutions with different concentrations is shown in Fig. 3 . According to Figure 3, it can be seen that the average maximum ice crystal size of G-S solution accounts for about 20-55% of PBS (%), indicating that compound G-S significantly reduces the size of ice crystal growth in PBS buffer and significantly inhibits ice crystal growth in solution. . When the concentration of G-S compound in the solution is 360mM, it is the smallest, and the average maximum ice crystal size accounts for only about 20%-25% of PBS (%), and an excellent ice suppression effect is achieved.
实施例2冷冻保存试剂的制备Example 2 Preparation of cryopreservation reagents
将实施例1所制得的产物溶解在水中,充分搅拌溶解之后冷冻干燥。取干燥后的产物溶解在PBS缓冲液(NaCl(136.9mmol L-1),KCl(2.7mmol L-1),Na2HPO4(10.0mmol L-1),KH2PO4(2.0mmol L-1),pH7.4)中备用。The product prepared in Example 1 was dissolved in water, fully stirred to dissolve, and then freeze-dried. The dried product was dissolved in PBS buffer (NaCl (136.9mmol L -1 ), KCl (2.7mmol L -1 ), Na 2 HPO 4 (10.0mmol L -1 ), KH 2 PO 4 (2.0mmol L - 1 ) 1 ), pH 7.4) for use.
实施例3绵羊血红细胞冷冻保存实验Example 3 Sheep red blood cell cryopreservation experiment
本次实验所用细胞为绵羊血细胞。选取5ml的血细胞,转速4000r/min,离心5min,离心洗涤数次,直到悬浮液澄清。将得到的血细胞保存在4℃冰箱中备用。取50μL预先准备好的血细胞,然后加入同体积的G-S溶液,G-S溶液中化合物G-S的摩尔浓度为720mM。最终得到含有细胞的冷冻保存溶液,其中化合物G-S浓度为360mM。然后在液氮中冷冻保存2h,将冷冻的细胞在45℃水浴温度复温,然后测试细胞恢复率,在图4中可以看出纯的PBS缓冲液冷冻保存细胞恢复率基本上为0%,而实施例2制备的冷冻保存试剂冷冻血细胞的细胞恢复率可达50%以上甚至60%,无需加入有机溶剂,取得了良好的冷冻保存效果。The cells used in this experiment were sheep blood cells. Select 5 ml of blood cells, spin at 4000 r/min, centrifuge for 5 min, and wash by centrifugation several times until the suspension is clear. The obtained blood cells were stored in a 4°C refrigerator for later use. Take 50 μL of pre-prepared blood cells, and then add the same volume of G-S solution. The molar concentration of compound G-S in the G-S solution is 720 mM. A cryopreservation solution containing cells was finally obtained, in which the concentration of compound G-S was 360 mM. Then cryopreserved in liquid nitrogen for 2 hours, rewarmed the frozen cells at a water bath temperature of 45 °C, and then tested the cell recovery rate. It can be seen in Figure 4 that the cell recovery rate of pure PBS buffer cryopreservation is basically 0%. On the other hand, the cell recovery rate of the frozen blood cells of the cryopreservation reagent prepared in Example 2 can reach more than 50% or even 60%, without adding an organic solvent, and a good cryopreservation effect is achieved.
以上,对本发明的实施方式进行了说明。但是,本发明不限定于上述实施方式。凡在本发明的精神和原则之内,所做的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。The embodiments of the present invention have been described above. However, the present invention is not limited to the above-described embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.
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