CN105664169A - Application of sodium pyruvate in preparation of storage liquid for improving stored erythrocyte quality and/or post-infusion tissue damage - Google Patents
Application of sodium pyruvate in preparation of storage liquid for improving stored erythrocyte quality and/or post-infusion tissue damage Download PDFInfo
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Abstract
The invention discloses a new use of sodium pyruvate, namely application in the preparation of a storage liquid for improving stored erythrocyte quality and/or post-infusion tissue damage.The sodium pyruvate is used as an active ingredient to prepare the storage liquid for improving stored erythrocyte quality and/or post-infusion tissue damage, it is possible to improve the stored erythrocyte quality and/or post-infusion tissue damage, the therapeutic effect is significant, no side effect occurs, safety is good, and the sodium pyruvate plays an important role in studying and developing this storage liquid and has promising application prospect and great social significance.
Description
Technical field
The present invention relates to field of medicaments, particularly relate to Sodium Pyruvate tissue injury after preparation improves stored red blood cell quality and/or infusion and store the application in liquid.
Background technology
In clinic, for the patient bled profusely caused in wound or operation, blood transfusion is a kind of basic treatment means, has saved countless life. Combining and the function of transports oxygen, carbon dioxide owing to erythrocyte has, it is possible to provide oxygen for tissue and transport the carbon dioxide that metabolism produces, therefore blood transfusion can improve the oxygen carrying capacity of blood, increase tissue oxygen supply, improves oxygenation status. But in clinical blood transfusion, not accomplishing that existing use now takes, the erythrocyte of patient's infusion mostly is the erythrocyte (i.e. stored red blood cell) storing a period of time.
At present, 4 DEG C of stored red blood cell time upper limits are 42 days clinically, erythrocyte is mainly saved in the CPDA-1 containing citrate, phosphate, glucose and adenine etc. and stores in liquid, or the SAGM containing sodium chloride, gland fat purine, glucose and mannitol etc. stores in liquid. Erythrocyte inevitably produces the change of a series of 26S Proteasome Structure and Function in storage process, namely " stores damage ". Such as, erythrocyte is in storage process, because energy is under-supply, ATP concentration reduces, peroxidating can be there is in its memebrane protein, membrane lipid, cellular oxidation stress level can raise, after occurring the stored red blood cell of " storing damage " to feed back, it is possible to can cause the untoward reaction such as body oxidative stress, inflammatory reaction and organ injury.
Although there are some researches show, improve the composition storing liquid and can improve red blood cells storage damage. energy metabolism of erythrocyte can be improved as added energy metabolism substrate glucose etc. in stored red blood cell, effectively reduce the hemolysis rate of stored red blood cell, but Oxidative Damages of Red Blood Cells can not be improved. Stowell etc. add vitamin C in stored red blood cell, stored red blood cell can be effectively improved and feed back survival rate, and its immunogenicity can be reduced, but the cytokine levels of body after it feeds back can not be improved, namely the inflammatory reaction level (Stowell of body after stored red blood cell feeds back can not be improved, S.R., Smith, N.H., Zimring, J.C., Fu, X., Palmer, A.F., Fontes, J., Banerjee, U.&Yazer, M.H. (2013) Additionofascorbicacidsolutiontostoredmurineredbloodcell sincreasesposttransfusionrecoveryanddecreasesmicropartic lesandalloimmunization.Transfusion, 53, 2248-2257.).
Therefore, find a kind of storage liquid for stored red blood cell, not only there is anti-oxidation function but also be provided that the energy supply of abundance, be this area problem demanding prompt solution with the tissue injury improving stored red blood cell quality and feedback causes.
Summary of the invention
It is an object of the invention to for the deficiencies in the prior art, and provide a kind of new application of Sodium Pyruvate, namely Sodium Pyruvate tissue injury after preparation improves stored red blood cell quality and/or improves infusion stores the application in liquid.
Described Sodium Pyruvate is pharmaceutical grade Sodium Pyruvate.
The active component of described storage liquid is Sodium Pyruvate.
The Sodium Pyruvate final concentration of 1-10mM in stored red blood cell, preferred final concentration of 1-2.49mM, 1-2mM, 2-2.49mM, 1-1.5mM, 1.5-2mM or 1.5-2.49mM.
The active component of described storage liquid is sodium pyruvate solution, and this solution is to be formed by Sodium Pyruvate and normal saline, or formulated by Sodium Pyruvate and anticoagulant for storage of whole blood; Described anticoagulant for storage of whole blood can be selected from the one in CPDA-1 solution, SAGM solution etc.
Sodium Pyruvate weight/mass percentage composition in described solution is 3.575-35.75%, it is preferable that 3.575-8.90%.
Described CPDA-1 solution comprises citrate, phosphate, glucose, adenine, containing 26.3g sodium citrate, 3.27g citric acid, 31.9g glucose, 0.275g adenine, 2.22g sodium dihydrogen phosphate and remaining aquesterilisa in every 1000mLCPDA-1 solution.
Described storage liquid stores after mixing with erythrocyte again.
Another object of the present invention is to provide a kind of red blood cell storage solution, this red blood cell storage solution stores after mixing with erythrocyte again, comprise Sodium Pyruvate and anticoagulant for storage of whole blood, the Sodium Pyruvate final concentration of 1-10mM in red blood cells storage process, it is preferred that final concentration of 1-2.49mM, 1-2mM, 2-2.49mM, 1-1.5mM, 1.5-2mM or 1.5-2.49mM; One in the preferred CPDA-1 solution of described anticoagulant for storage of whole blood, SAGM solution etc.
Wherein Sodium Pyruvate is solution, and this solution is to be formed by Sodium Pyruvate and normal saline, or formulated by Sodium Pyruvate and anticoagulant for storage of whole blood.
The invention provides a kind of new application of Sodium Pyruvate and improve tissue injury after stored red blood cell quality and infusion, be namely for improving tissue injury after stored red blood cell quality and infusion using Sodium Pyruvate as active component. First, in red blood cells storage process, add Sodium Pyruvate and can alleviate stored red blood cell oxidative stress level, increase the energy supply of stored red blood cell, retain the oxygen carrying capacity of stored red blood cell; Secondly, red blood cells storage process is added Sodium Pyruvate, after feedback, compared to matched group, the response to oxidative stress of body, inflammatory reaction and tissue injury all significantly reduce, illustrate that Sodium Pyruvate can alleviate stored red blood cell and feed back the tissue injury caused, reduce stored red blood cell and feed back untoward reaction. The present invention has expanded research and the therapeutic domain of Sodium Pyruvate, alleviates with it and improves tissue injury after stored red blood cell quality and infusion, evident in efficacy, and side effect is little, safety is good, plays a significant role in researching and developing this series products, having a extensive future, social meaning is huge.
Below in conjunction with drawings and the specific embodiments, the present invention is described in further details.
Accompanying drawing explanation
Fig. 1 show Sodium Pyruvate to the action effect figure of ATP concentration in stored red blood cell.
Fig. 2 show Sodium Pyruvate to the action effect figure of MDA content in stored red blood cell.
Fig. 3 show Sodium Pyruvate to the action effect figure of SOD activity in stored red blood cell.
Fig. 4 show Sodium Pyruvate to the action effect figure of P50 value in stored red blood cell.
Fig. 5 show Sodium Pyruvate to the action effect figure of plasma A ST, LDH activity and BUN content in stored red blood cell.
Fig. 6 show Sodium Pyruvate to the action effect figure of MDA content in hepatic tissue.
Fig. 7 show Sodium Pyruvate to the action effect figure of MPO activity in hepatic tissue.
Fig. 8 show the Sodium Pyruvate action effect figure to IL-6 in hepatic tissue and TNF-α content.
Fig. 9 show the action effect figure that hepatic tissue pathology is damaged by Sodium Pyruvate.
Figure 10 show the Sodium Pyruvate action effect figure to hemolysis rate.
Figure 11 show the Sodium Pyruvate of the different final concentration action effect figure to feeding back survival rate.
Detailed description of the invention
It is commonly used in the preservation liquid preserving whole blood at present consistent with the storage liquid composition of stored red blood cell; generally comprise glucose (providing energy for erythrocyte), citrate (anticoagulation), phosphate (buffer solution; maintain acid-base value), adenine (for ATP generate provide raw material); have possibly together with mannitol (increase osmotic pressure, protect erythrocyte membrane). But these preserve liquid and storage liquid all cannot fundamentally meet erythrocyte energy requirement in storage process, can not effectively reduce erythrocyte oxidative damage in storage process.
Research shows, acetone acid has antiinflammatory, antioxidation, is substrate and the key point of a kind of glycometabolic intermediate product tricarboxylic acid cycle (TricarboxylicAcidCycle, TCAcycle) of endogenous simultaneously. Sodium Pyruvate is the sodium salt of acetone acid, exists with acetone acid group (Pyruvate, Pyr) form in vivo, and Pyr is the common ion in people and animal and plant body, and is in the meet of three big materials (sugar, fat and protein) metabolism. Studies have found that, before stored red blood cell infusion with the addition of Sodium Pyruvate multiple shape liquid (multiple shape liquid be a kind of before stored red blood cell feeds back for Washed Red Blood Cells, recover the solution of red cell morphology to a certain extent) process stored red blood cell and can have its ATP level of efficient recovery and improve erythrocyte function of carrying oxygen. But, with the addition of the multiple shape liquid of Sodium Pyruvate only by processing stored red blood cell in short time, instant increase in erythrocyte 2,3-DPG content etc., can not fundamentally improve erythrocytic storage in storage process to damage, more can not effectively solve the damage to histoorgan after stored red blood cell feeds back. At present, it does not have in red blood cells storage process, add the pertinent literature report of Sodium Pyruvate, Sodium Pyruvate is also just unclear on what impact tissue has after stored red blood cell quality and feedback in storage process.
Does inventor courageously speculate, whether Sodium Pyruvate both can provide energy metabolism substrate for stored red blood cell, had again antioxidative effect concurrently simultaneously, it is possible to organ injury after red blood cells storage damage and infusion is had better protective effect?
Big quantity research through inventor finds, adds Sodium Pyruvate and can significantly improve the oxygen carrying capacity of stored red blood cell, increase ATP content and oxidation resistance in cell, reduce and store damage in storage process.
The erythrocyte of mouse red blood cell with the people storing 42 days owing to storing 14 days experienced by the change of similar morphological function, and has similar Physiology and biochemistry state. Therefore, the present invention adopts the C57BL/6 mouse red blood cell storing 14 days to check the Sodium Pyruvate impact on stored red blood cell quality and tissue injury.Following example are all divided into two groups by whether adding Sodium Pyruvate.
Embodiment is carried out under premised on technical solution of the present invention, gives detailed embodiment and concrete operating process, it will be appreciated that specific embodiment described herein only in order to explain the present invention, is not intended to limit the present invention.
The source of biomaterial used in embodiment is widely, and any biomaterial that can obtain with moral ethics that keeps on the right side of the law can be replaced according to the prompting in embodiment and use. Method therefor is conventional method if no special instructions. If no special instructions, in each embodiment, the material of same names or reagent content are identical.
Embodiment 1, preparation with the addition of the stored red blood cell of Sodium Pyruvate
1. the preparation of sodium pyruvate solution
With electronic analytical balance precise 35mg Sodium Pyruvate powder, it is dissolved in 1mL normal saline, preparation obtains the sodium pyruvate solution that Sodium Pyruvate concentration is 35mg/mL, treat that it dissolves completely, superclean bench filters with the bacterial filter in 0.22 μm of aperture, it is positioned in sterile centrifugation tube, stand-by.
2. the preparation of erythrocytic acquisition and stored red blood cell
Take the Nembutal sodium solution (its injection volume calculates) of mouse peritoneal injection 2.5% by 0.3mL/100g Mouse Weight, postanesthetic mice is fixed on 37 DEG C of hot plates, it is put on aseptic superclean bench, with cotton ball soaked in alcohol wiping mouse peritoneal and thoracic cavity outer surface, aseptic heart puncturing extracting blood, with CPDA-1 solution as anticoagulant and anticoagulant for storage of whole blood (purchased from American Sigma company, article No. C4431-50mL. Containing sodium citrate 26.3g, citric acid 3.27g, glucose 31.9g, adenine 0.275g, sodium dihydrogen phosphate 2.22g and aquesterilisa (supplying 1000mL) in 1000mLCPDA-1 solution). Remove leukocyte with leukocyte depletion filter, obtain white whole blood, and to make CPDA-1 solution final volume percentage composition in removing white whole blood be 14%. More than taking blood process is illustrative experiment process, must through step not as what obtain the present invention.
White whole blood will be gone to be divided into seven groups (n=8): SP group 1#-6#, i.e. Sodium Pyruvate group, add the sodium pyruvate solution prepared in step 1 and make final concentration of 1mM, 1.5mM, 2mM, 2.49mM, 5mM, 10mM (these six final concentrations correspondence SP group 1 respectively of Sodium Pyruvate in white whole blood#, SP group 2#..., SP group 6#); Blank group: add normal saline, the addition volume of normal saline is identical with the addition volume of SP group sodium pyruvate solution. Each group is gone white whole blood at 4 DEG C, and 400g is centrifuged 15min, removes supernatant, and making packed cell volume is 70-75%, 4 DEG C of blood preseration refrigerator storage 14 days, obtains stored red blood cell, stand-by.
ATP content in embodiment 2, mensuration stored red blood cell
With description method in ATP detection kit (ATP detection kit is purchased from green skies Bioisystech Co., Ltd, article No. S0026) measure that embodiment 1 obtains store 14 days after the ATP content of seven groups of stored red blood cells. With SP group 3#(in stored red blood cell the final concentration of 2mM of Sodium Pyruvate) is example, and result is as shown in Figure 1.
Fig. 1 result shows, blank group ATP content is 2.46 ± 0.19nmol/mg albumen, SP group 3#In stored red blood cell, ATP content is 2.82 ± 0.21nmol/mg albumen. Compare with blank group, SP group 3#ATP content in stored red blood cell is significantly raised (* represents p < 0.05). Illustrating that Sodium Pyruvate can improve ATP level in erythrocyte in erythrocytic storage process, provide substrate for stored red blood cell energy metabolism, alleviating the erythrocyte problem that energy is under-supply in storage process, thus improving the quality of stored red blood cell further.
SP group 1#、2#With 4#With SP group 3#Result is as good as, and repeats no more, SP group 5#With 6#ATP content be substantially less than SP group 3#ATP content (p < 0.05).
MDA content in embodiment 3, mensuration stored red blood cell
With description method in MDA detection kit (MDA detection kit builds up bio tech ltd, article No. A003-1 purchased from Nanjing) measure that embodiment 1 obtains store 14 days after lipid peroxidation product-malonaldehyde (MDA) content of seven groups of stored red blood cells. With SP group 2#For example, result is as shown in Figure 2.
One of most important end eventually metabolite produced by peroxidization is there is in the lipid material that MDA is cell membrane with ROS, its content reflects intensity and the speed that lipid within endothelial cells peroxidating occurs, therefore cell and the important indicator (KwiecienS of tissue oxidizing stress damage degree is reflected with MDA content, KonturekPC, SliwowskiZ, etal.Interactionbetweenselectivecyclooxygenaseinhibitors andcapsaicin-sensitiveafferentsensorynervesinpathogenesi sofstress-inducedgastriclesions.Roleofoxidativestress.JP hysiolPharmacol, 2012, 63:143-151). MDA content is more high, illustrates that the oxidative stress level of cell is more high, otherwise, illustrate that the oxidation level of cell is more low.
Fig. 2 result shows, blank group MDA content is 15.45 ± 0.33nmol/mg albumen, SP group 2#In stored red blood cell, MDA content is 14.66 ± 0.87nmol/mg albumen. Compare with blank group, SP group 2#MDA content in stored red blood cell substantially reduces (* represents p < 0.05). Illustrate that Sodium Pyruvate significantly reduces the MDA content in erythrocyte, reduce erythrocyte oxidative stress level, serve antioxidative effect, protect stored red blood cell membrane lipid, improve the quality of stored red blood cell.
SP group 1#-6#With SP group 3#Result is as good as, and repeats no more.
SOD activity in embodiment 4, mensuration stored red blood cell
With SOD measure that description method (test kit builds up bio tech ltd, article No. A001-1 purchased from Nanjing) in test kit measures that embodiment 1 obtains store 14 days after the SOD activity of seven groups of stored red blood cells. With SP group 1#For example, result is as shown in Figure 3.
SOD can generate H by catalysis ultra-oxygen anion free radical generation dismutation reaction2O2Remove the ultra-oxygen anion free radical of excessive concentrations in organism, its activity change can reflect the oxidated degree (Dong Liang coerced of organism, what is always cherished the memory of, the bright Dong Zhi of Wang Yuan raises the applied research progress of (2013) superoxide dismutase (SOD). Chinese agriculture science and technology Leader, 53-58.). SOD activity is more low, illustrates that the oxidation level of cell is more high; Otherwise, illustrate that the oxidation level of cell is more low.
Fig. 3 result shows, blank group SOD activity is 648.1 ± 10.0U/mg albumen, SP group 1#In stored red blood cell, SOD activity is 726.8 ± 12.3U/mg albumen. Compare with blank group, SP group 1#The notable rising (* represents p < 0.05) of SOD activity in stored red blood cell. Illustrate that Sodium Pyruvate significantly improves stored red blood cell SOD activity, reduce the oxidative stress level of stored red blood cell, strengthen the oxidation resistance of stored red blood cell, improve stored red blood cell quality.
SP group 1#-6#With SP group 3#Result is as good as, and repeats no more.
P in embodiment 5, mensuration stored red blood cell50Value
With blood oxygen analysis instrument HemoxAnalyzer (pennsylvania, USA TCS Science and Technology Ltd., model TCS-200 type) measure that embodiment 1 obtains store 14 days after the P of seven groups of stored red blood cells50Value.With SP group 4#For example, result is as shown in Figure 4.
The P of stored red blood cell50Value can reflect erythrocyte oxygen carrying capacity, in certain limit, and P50It is worth more big, more strong (the MehtaN of erythrocyte oxygen carrying capacity, WattsN.B, WelgeJ.A, etal.Comparisonofserumcalciumchangefollowingthyroidandno nthyroidnecksurgery.OtolaryngolHeadNeckSurg, 2006.134 (6): 901-906.). Compare with blank group, SP group 4#P in stored red blood cell50Value is apparently higher than blank group (* represents p < 0.05).
Fig. 4 result shows, blank group P50Value is 21.0 ± 0.7mmHg, SP group 4#P in stored red blood cell50Value is 23.9 ± 1.5mmHg. Illustrate that adding Sodium Pyruvate in storage process can significantly improve the oxygen carrying capacity of stored red blood cell, after feeding back body, improves the efficiency that erythrocyte is tissue oxygen supply, improve tissue oxygen and supply.
SP group 1#、2#With 4#With SP group 3#Result is as good as, and repeats no more, SP group 5#With 6#P50It is substantially less than SP group 3#P50Value (p < 0.05).
After embodiment 6, stored red blood cell feedback is internal, measure internal plasma A ST activity, BUN content and LDH activity
Feed back pre-treatment stored red blood cell: what Example 1 obtained stores the SP group 1 after 14 days#-6#With the stored red blood cell of blank group, respectively with the brine of its 10 times of volumes, 400g rotating speed is centrifuged, and abandons supernatant; Repeating 3 times, making packed cell volume is 70%-75%, finally obtains resuspended erythrocyte, stand-by.
Feed back stored red blood cell: be fixed on by mice on the special holder of mice, only expose tail for tail vein injection stored red blood cell. According to blood volume percentage of liveweight 7% estimates the total blood volume of mice, then distinguishes the SP group 1 after the process of the total blood volume 20% of tail venoclysis mice#-6#(process and feed back the process of stored red blood cell referring to the document: Yu, B., Lei with the resuspended erythrocyte of blank group, C., Baron, D.M., Steinbicker, A.U., Bloch, K.D.&Zapol, W.M. (2012) Diabetesaugmentsandinhalednitricoxidepreventstheadverseh emodynamiceffectsoftransfusingsyngeneicstoredbloodinmice .Transfusion, 52,1410-1422.).
After infusion 2h, heart puncturing extracting blood, 6000rpm, centrifugal 90s, take supernatant, stand-by.
According to Hod, E.A., Zhang, N., Sokol, S.A., Wojczyk, B.S., Francis, R.O., Ansaldi, D., Francis, K.P., Della-Latta, P., Whittier, S., Sheth, S., Hendrickson, J.E., Zimring, J.C., Brittenham, G.M.&Spitalnik, S.L. (2010) Transfusionofredbloodcellsafterprolongedstorageproducesh armfuleffectsthataremediatedbyironandinflammation.Blood, 115, 4284-4292. in record, AST activity, BUN content, LDH activity can react liver respectively, the degree of impairment of kidney and systemic organs, and its activity/content is more high, illustrate that the damage of organ is more serious, otherwise, the degree of injury of organ is more light.
Then by the SP group 1 after Hitachi 7180 Plasma Biochemical automatic analyzer coordinate the reagent bag (reagent bag purchased from Beijing Zhou Tianhua maple Medical Instruments company limited, the process that measures is by the assay method of analyser description) of this analyser to measure storage that embodiment 1 obtains 14 days#-6#After in blank group, stored red blood cell is fed back in Mice Body, the AST activity of blood plasma, BUN content and LDH activity in Mice Body, with SP group 3#For example, result is as shown in Figure 5.
Fig. 5 result shows, AST activity: blank group is 579 ± 96.5U/L, SP group 3#It is 332 ± 111.2U/L; BUN content: blank group is 14.6 ± 0.8mmol/L, SP group 3#It is 11.9 ± 1.0mmol/L; LDH activity: blank group is 479.0 ± 101.6U/L, SP group 3#It is 368.8 ± 72.5U/L. Compare with blank group, SP group 3#After feedback, in blood plasma, AST activity, BUN content and LDH activity all significantly reduce (* represents p < 0.05). Illustrating that the liver function of Sodium Pyruvate group receptor and renal function are apparently higher than blank group, Sodium Pyruvate can significantly reduce the hepatic tissue, the Renal tissues damage that cause after stored red blood cell feeds back body, is effectively protected the tissue of receptor and the function of organ.
SP group 1#、2#With 4#With SP group 3#Result is as good as, and repeats no more, SP group 5#With 6#AST activity, BUN content, LDH activity be significantly higher than SP group 3 all respectively#AST activity, BUN content, LDH activity (p < 0.05).
After embodiment 7, stored red blood cell feedback is internal, measure MDA content in hepatic tissue
Sacrifice after infusion stored red blood cell 2h in Example 6, (concrete grammar is referring to Hod to take murine liver tissue, E.A., Zhang, N., Sokol, S.A., Wojczyk, B.S., Francis, R.O., Ansaldi, D., Francis, K.P., Della-Latta, P., Whittier, S., Sheth, S., Hendrickson, J.E., Zimring, J.C., Brittenham, G.M.&Spitalnik, S.L. (2010) Transfusionofredbloodcellsafterprolongedstorageproducesh armfuleffectsthataremediatedbyironandinflammation.Blood, 115, 4284-4292.), by description method in MDA detection kit, (test kit builds up bio tech ltd purchased from Nanjing, article No. A003-1) measure that embodiment 1 obtains store 14 days after seven groups of stored red blood cells be fed back in Mice Body after, MDA content in murine liver tissue. with SP group 3#For example, result is as shown in Figure 6.
Fig. 6 result shows, blank group MDA content is 12.77 ± 1.11mmol/mg albumen, SP group 3#In murine liver tissue, MDA content is 11.69 ± 0.95mmol/mg albumen. Compare with blank group, SP group 3#MDA content in stored red blood cell significantly reduces (* represents p < 0.05). The MDA level of Sodium Pyruvate group hepatic tissue is substantially less than blank group, illustrate that the oxidation level of Sodium Pyruvate group hepatic tissue is substantially less than blank group, Sodium Pyruvate can significantly improve stored red blood cell and feed back the oxidative stress level of the hepatic tissue caused, alleviate the oxidative stress level of receptor, play the effect alleviating receptor untoward reaction.
SP group 1#、2#With 4#With SP group 3#Result is as good as, and repeats no more, SP group 5#With 6#MDA content be significantly higher than SP group 3#MDA content (p < 0.05).
After embodiment 8, stored red blood cell feedback is internal, measure myeloperoxidase (MPO) (MPO) activity in hepatic tissue
With embodiment 7, take murine liver tissue, by description method in MPO detection kit, (test kit builds up bio tech ltd purchased from Nanjing, article No. A044-1) measure that embodiment 1 obtains store 14 days after seven groups of stored red blood cells be fed back in Mice Body after, MPO activity in murine liver tissue. With SP group 3#For example, result is as shown in Figure 7.
MPO is the enzyme that one is both relevant with innate immune defence, relevant with tissue injury again. MPO derives from M7 (PMN), mononuclear cell and macrophage, and tissue inflammation level raises, and can bring out them and produce MPO, cause that in tissue, MPO content raises (Zhao, J.X., Wang, B., You, G.X., Wang, Y., Chen, G., Wang, Q., Zhang, X.G., Zhao, L., Zhou, H.&He, Y.Z. (2015) HypertonicSalineDextranAmelioratesOrganDamageinBeagleHem orrhagicShock.PLoSOne, 10, e0136012.).So, in tissue, MPO content is more high, illustrates that in tissue, neutrophilic granulocyte is more many, and namely infiltration is more many to the neutrophilic granulocyte of tissue, and tissue inflammation level is more high; Otherwise, tissue inflammation level is more low.
Fig. 7 result shows, blank group MPO content is 0.36 ± 0.04U/g wet tissue weight, SP group 3#In murine liver tissue, MPO content is 0.32 ± 0.02U/g wet tissue weight. Compare with blank group, SP group 3#MPO content in stored red blood cell significantly reduces (* represents p < 0.05). SP group 3 is described#Liver tissues inflammatory reaction level is substantially less than blank group, and Sodium Pyruvate can significantly improve stored red blood cell and feed back the inflammatory reaction level of the hepatic tissue caused, and reduces the inflammatory reaction level of body.
SP group 1#、2#With 4#With SP group 3#Result is as good as, and repeats no more, SP group 5#With 6#MPO content be significantly higher than SP group 3#MPO content (p < 0.05).
After embodiment 9, stored red blood cell feedback is internal, measure IL-6 content and TNF-α content in hepatic tissue
With embodiment 7, take murine liver tissue, with description method in ELISA kit (Elisa test kit purchased from American PeproTech) measure that embodiment 1 obtains store 14 days after seven groups of stored red blood cells be fed back in Mice Body after, IL-6 content in murine liver tissue and TNF-α content. With SP group 3#For example, result is as shown in Figure 8.
IL-6 is the T cell activated and the lymphokine of fibroblast generation, coordinate after injury damage defense reaction plays an important role, participate in one of important cytokine of immunomodulating and inflammatory reaction, be that host is to infecting and the main medium of reaction caused by tissue injury. TNF-α participates in inflammatory reaction and immunne response, at Organism immunoregulation, the aspect such as body physiological function and infection plays a significant role (Chen, G., You, G., Wang, Y., Lu, M., Cheng, W., Yang, J., Zhao, L.&Zhou, H. (2013) Effectsofsyntheticcolloidsonoxidativestressandinflammato ryresponseinhemorrhagicshock:comparisonofhydroxyethylsta rch130/0.4, hydroxyethylstarch200/0.5, andsuccinylatedgelatin.CritCare, 17, R141.). the rising of IL-6 and TNF-α level, illustrates that body inflammatory reaction level raises, otherwise, illustrate that organism immune response level reduces.
Fig. 8 result shows, IL-6 content: blank group is 389.7 ± 46.1pg/mg albumen, SP group 3#Murine liver tissue is 305.1 ± 26.5pg/mg albumen; TNF-α content: blank group is 42.59 ± 2.11pg/mg albumen, SP group 3#Murine liver tissue is 37.60 ± 3.41pg/mg albumen. Compare with blank group, SP group 3#IL-6 content and TNF-α content in stored red blood cell significantly reduce (* represents p < 0.05). After illustrating to be fed back in Mice Body by the erythrocyte that Sodium Pyruvate processed in storage process, IL-6 and the TNF-α of murine liver tissue significantly reduce, Sodium Pyruvate group inflammatory reaction level significantly reduces, and illustrates that Sodium Pyruvate can significantly reduce the body inflammatory reaction level after stored red blood cell feeds back.
SP group 1#、2#With 4#With SP group 3#Result is as good as, and repeats no more, SP group 5#With 6#IL-6 content and TNF-α content be significantly higher than SP group 3#IL-6 content and TNF-α content (p < 0.05).
After embodiment 10, stored red blood cell feedback is internal, observe the pathological change of hepatic tissue
With embodiment 7, take murine liver tissue, after pathological wax block embedding and HE dyeing process, (method step reference, Hod, E.A., Zhang, N., Sokol, S.A., Deng Blood, 115,4284-4292.) pathological change of hepatic tissue is observed, with SP group 3 in embodiment 1#Result be example, as shown in Figure 9.
Fig. 9 result shows, the white air bubble-shaped part of number in the figure 1 is hepatic tissue blood vessel, and the region of label 3 is normal liver tissue cell. Wherein blank group, the inflammatory cell that dark parts is infiltration of middle label 2, mainly based on lymphocyte and neutrophilic granulocyte, local hepatic tissue hole gap is dispersed in lymphocytic infiltration as seen on a small quantity. Sodium Pyruvate group does not observe obvious inflammatory cell infiltration. After showing to be fed back in Mice Body by the erythrocyte that Sodium Pyruvate processed in storage process, it is possible to effectively reduce hepatic tissue inflammatory reaction, alleviate pathology damage.
SP group 1#、2#With 4#With SP group 3#Result is as good as, and repeats no more, SP group 5#With 6#Pathological section in, occur infiltration inflammatory cell, hepatic tissue hole gap is dispersed in lymphocytic infiltration as seen on a small quantity, and its degree of tissue damage is significantly greater than 3#Degree of injury.
Embodiment 11, stored red blood cell feed back the mensuration of internal rear hemolysis rate
Use free hemoglobin to measure description method (test kit builds up bio tech ltd, article No. A071 purchased from Nanjing) in test kit measures that embodiment 1 obtains store 14 days after seven groups of stored red blood cell blood plasma in free hemoglobin concentration. With SP group 3#For example, embodiment 1 is obtained store 14 days after SP group 3#After being fed back in Mice Body respectively with stored red blood cell in blank group, with automatic blood cell calculating instrument (routine blood test), draw about 50 μ L Mouse whole blood and measure blood total hemoglobin concentration and packed cell volume (Hct).
According to formula: hemolysis rate=(free hemoglobin concentration × (1-Hct))/total hemoglobin concentration × 100%, obtain SP group 3#With the hemolysis rate of blank group, result is as shown in Figure 10.
Figure 10 result shows, blank group stored red blood cell hemolysis rate is 0.79 ± 0.03%, SP group 3#Stored red blood cell hemolysis rate is 0.77 ± 0.04%. Compare with blank group, SP group 3#The hemolysis rate of stored red blood cell has reduction trend, but two groups there was no significant difference (* represents p > 0.05). Illustrate that Sodium Pyruvate can reduce the hemolysis rate of stored red blood cell, but compare unknown significance difference with matched group. Meet the regulation of FDA Food and Drug Administration (FDA): it is 42 days that human erythrocyte stores the upper limit, and the stored red blood cell for feeding back meets hemolysis rate < 1%.
The infusion standard of stored red blood cell is by U.S. FDA: after external storage, hemolysis rate is less than 1%, erythrocytic survival rate (being namely present in the blood circulation of receptor) after infusion 24h however lower than 75%, the erythrocyte only reaching above standard could be used for feeding back.
SP group 1#-6#With SP group 3#Result is as good as, and repeats no more.
After embodiment 12, stored red blood cell feedback is internal, the impact on feeding back survival rate for 24 hours of the Sodium Pyruvate of the different final concentration of mensuration
Use Fluorescein isothiocyanate (FITC) (purchased from American Sigma company, article No. F-7250) fluorescently-labeled method, measure that embodiment 1 obtains store 14 days after SP group 1#、3#、5#、6#Feeding back in healthy mice body after 24h with stored red blood cell in blank group, stored red blood cell survival rate in body circulates, result is as shown in figure 11.
Fluorescein isothiocyanate (FITC) fluorescently-labeled method particularly as follows:
1. when lucifuge, accurately weigh 100mgFITC, be dissolved in the PBS (pH=7.4) of 10mL, obtain the FITC solution that concentration is 10mg/mL.
2. labelling stored red blood cell: carefully shaken up by stored red blood cell, takes erythrocyte 1 and manages (about 600 μ L) erythrocyte and, to 5mL centrifuge tube, add the PBS of 2.4mL, mix gently.Then being added thereto to the FITC solution 20 μ L that 1. step prepares, mixing, 37 DEG C of lucifuges hatch 20min.
3. centrifugal, washing: centrifugal 5min under 400g, abandons supernatant; Add the PBS washing of 2.4mL again, be centrifuged; Repeat 3 times, wash away FITC unnecessary in solution.
4., after centrifugal, erythrocyte and the FITC-erythrocyte of labelling at the bottom of pipe are taken, by tail vein injection, by 300 μ LFITC-red blood cell transfusions to Mice Body, and timing. Respectively at 10min, 24h, take blood through vena orbitalis posterior clump, take 5 μ L every time, be added in the little centrifuge tube of heparinization.
5. with 50,000 cells of Flow cytometry, distinguish fluorescence labeled cell, use CellQuestPro analysis of fluorescence labeled cell ratio. before using, it is calibrated with SpheroTM rainbow fluorescent grain flow cytometer, it is ensured that the setting of experiment is consistent every time. using the numerical value of 10min as basic point, the labeled rate of later time points and the ratio of the 10min mark rate red blood cell survival value as this time point, (concrete grammar is referring to Hod with time broken line graph to draw survival rate, E.A., Zhang, N., Sokol, S.A., Wojczyk, B.S., Francis, R.O., Ansaldi, D., Francis, K.P., Della-Latta, P., Whittier, S., Sheth, S., Hendrickson, J.E., Zimring, J.C., Brittenham, G.M.&Spitalnik, S.L. (2010) Transfusionofredbloodcellsafterprolongedstorageproducesh armfuleffectsthataremediatedbyironandinflammation.Blood, 115, 4284-4292.).
The relatively feedback survival rate after stored red blood cell 24h, measurement result is as shown in figure 11.
Figure 11 result shows, the feedback survival rate of blank group stored red blood cell is 75.63 ± 1.8%, it is 75.74 ± 5.4% that 1mMSP group stored red blood cell feeds back survival rate, it is 77.86 ± 0.4% that 2mMSP group stored red blood cell feeds back survival rate,, it is 73.41 ± 1.5% that 5mMSP group stored red blood cell feeds back survival rate, and it is 71.23 ± 2.0% that 10mMSP group stored red blood cell feeds back survival rate, compare between two, the optimal concentration that feedback survival rate is stored red blood cell of 2mMSP group stored red blood cell. When concentration is more than 5mM; rising with concentration; the protective effect of stored red blood cell is reduced by Sodium Pyruvate on the contrary; and in an experiment; observing and adopt the erythrocyte that 10mM Sodium Pyruvate stores to feed back, the animal mental status is poor, and liveness reduces; and mortality of animals is 50%, illustrates to store Sodium Pyruvate excessive concentration in liquid and can cause obvious side effect.
More than experiment proves that Sodium Pyruvate has dual-use function, both in storage process, energy metabolism substrate was provided for erythrocyte, serve again antioxidative effect, stored red blood cell can be alleviated after adding the stored red blood cell feedback body of Sodium Pyruvate simultaneously and feed back the tissue injury caused, this experiment by be not added with Sodium Pyruvate and compare, draw and add the tissue injury after Sodium Pyruvate can improve stored red blood cell quality and alleviate feedback.
The above is only the preferred embodiment of the present invention; it should be pointed out that, for those skilled in the art, under the premise without departing from the principles of the invention; can also making some improvements and modifications, these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (10)
1. Sodium Pyruvate application stored in liquid of tissue injury after preparation improves stored red blood cell quality and/or improves infusion.
2. application according to claim 1, it is characterised in that described Sodium Pyruvate is pharmaceutical grade Sodium Pyruvate.
3. application according to claim 1 and 2, it is characterised in that the active component of described storage liquid is Sodium Pyruvate.
4. application according to claim 3, it is characterised in that the Sodium Pyruvate final concentration of 1-10mM in stored red blood cell, it is preferred that final concentration of 1-2.49mM, 1-2mM, 2-2.49mM, 1-1.5mM, 1.5-2mM or 1.5-2.49mM.
5. application according to claim 3, it is characterised in that the active component of described storage liquid is sodium pyruvate solution, this solution is to be formed by Sodium Pyruvate and normal saline, or formulated by Sodium Pyruvate and anticoagulant for storage of whole blood; Described anticoagulant for storage of whole blood can be selected from the one in CPDA-1 solution, SAGM solution etc.
6. application according to claim 5, it is characterised in that Sodium Pyruvate weight/mass percentage composition in described solution is 3.575-35.75%, it is preferable that 3.575-8.90%.
7. the application according to claim 5 or 6, it is characterized in that, described CPDA-1 solution comprises citrate, phosphate, glucose, adenine, containing 26.3g sodium citrate, 3.27g citric acid, 31.9g glucose, 0.275g adenine, 2.22g sodium dihydrogen phosphate and remaining aquesterilisa in every 1000mLCPDA-1 solution.
8. according to the arbitrary described application of claim 3 to 8, it is characterised in that described storage liquid stores after mixing with erythrocyte again.
9. a red blood cell storage solution, this red blood cell storage solution stores after mixing with erythrocyte again, it is characterized in that, comprise Sodium Pyruvate and anticoagulant for storage of whole blood, the Sodium Pyruvate final concentration of 1-10mM in red blood cells storage process, it is preferred that final concentration of 1-2.49mM, 1-2mM, 2-2.49mM, 1-1.5mM, 1.5-2mM or 1.5-2.49mM; One in the preferred CPDA-1 solution of described anticoagulant for storage of whole blood, SAGM solution etc.
10. red blood cell storage solution according to claim 9, it is characterised in that wherein Sodium Pyruvate is solution, this solution is to be formed by Sodium Pyruvate and normal saline, or formulated by Sodium Pyruvate and anticoagulant for storage of whole blood.
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