WO2021180221A1 - Application of low-molecular-weight hyaluronic acid fragments to induce string aggregation of red blood cells - Google Patents

Application of low-molecular-weight hyaluronic acid fragments to induce string aggregation of red blood cells Download PDF

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WO2021180221A1
WO2021180221A1 PCT/CN2021/080543 CN2021080543W WO2021180221A1 WO 2021180221 A1 WO2021180221 A1 WO 2021180221A1 CN 2021080543 W CN2021080543 W CN 2021080543W WO 2021180221 A1 WO2021180221 A1 WO 2021180221A1
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hyaluronic acid
molecular weight
low
red blood
molecular
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惠觅宙
宋琳
贾潇潇
惠鑫瑶
王凤舞
郭田田
王家麒
吴书音
崔家友
李鑫荣
姜媛媛
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青岛农业大学
青岛惠诺德生物科技有限公司
惠觅宙
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0641—Erythrocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/34—Sugars
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70585—CD44

Definitions

  • the invention relates to the field of biomedicine, in particular to an application of low molecular weight hyaluronic acid fragments in inducing the cluster-like aggregation of peripheral blood or venous red blood cells.
  • Human macromolecular hyaluronic acid subcutaneous tissue injection is mainly used for beauty, and there are many products, including Restylane, whose molecular weight is greater than 1.0x10 6 daltons.
  • Human cosmetic hyaluronic acid subcutaneous tissue injection can be used for various cosmetic purposes such as rhinoplasty, lip augmentation and wrinkle removal (1, 2, 3). Since Restylane has a large molecular weight and a large viscosity, its molecular weight is measured with a viscometer. Also due to the high molecular weight and viscosity of Restylane, there are many local adverse reactions of subcutaneous injection, including red, swollen, hard, and painful local injection inflammation. The more serious adverse reactions are local small blood vessel clogging and ulcers caused by injection (4).
  • the lower molecular weight hyaluronic acid subcutaneous injection product has less local adverse reactions, that is, the possibility of local small blood vessel blockage is less. Therefore, intravenous injection of hyaluronic acid with lower molecular weight is also possible, such as Legend Multi Dose for intravenous use in horse only (Bayer Corporation), which has a molecular weight of 3.0x10 5 daltons (www.equinelegend.com) (5).
  • the molecular weight of low-molecular-weight hyaluronic acid injection products is inaccurately determined with a viscometer.
  • gel electrophoresis and multi-angle laser methods are mainly used for determination (6, 7).
  • hyaluronic acid fragment B-HA commercial product (Medical device type 1, LUQIN Food Drug Medical Device registration number: 20190021) produced by the fully enzymatically hydrolyzed recombinant human hyaluronidase PH20 has clear human skin and mucosal resistance. Inflammatory activity, including a significant therapeutic effect on the clinical inflammation of human skin and mucous membranes (as shown in Figure 1). This clinical anti-inflammatory effect has been reported locally and a patent has been declared (14, 15, 16, 17, 18). , 19, 20, 21).
  • LEGEND Multi Dose for intravenous use in horse only (not for intra-articular use).
  • LEGEND INJECTABLE SOLUTION hyaluronate sodium
  • LEGEND Multi Dose for intravenous use in horse only (not for intra-articular use).
  • LEGEND INJECTABLE SOLUTION hyaluronate sodium
  • Hyaluronan 35kDa enhances epithelial barrier research and the development of the first of the coronavirus barriers and the first protection 9 .doi:10.1038/s41390-019-0563-9.
  • Zhao HD and Hui MZ Clinical study of bioactive hyaluronan fragments B-HA on chronic pharyngitis. Journal of Military Clinician, 2014, 42(8): 864-867.
  • Zhao HD and Hui MZ Clinical study of bioactive hyaluronan fragments B-HA on reflux laryngitis.Journal of Clinical Literatures(Chinese)2016,3(24):4898-4899.
  • the technical problem to be solved by the present invention is to study the biological activity mechanism of low molecular weight hyaluronic acid fragments, especially low molecular weight hyaluronic acid fragments with an average molecular weight of 35 ⁇ 8kDa, new biological activities and potential new clinical applications.
  • the present invention unexpectedly found that the hyaluronic acid fragment induces the red blood cell clusters of human and animal whole blood (peripheral blood and venous blood) (see the note below and refer to Figure 3), and the minimum concentration of the red blood cell clusters induced by the low molecular weight The range is negatively correlated with the molecular weight of hyaluronic acid fragments.
  • This article conducts an in-depth study of this discovery. Note: Erythrocytes aggregate is a reversible phenomenon of mammalian red blood cell aggregation. The aggregation of mammalian red blood cells like multiple coins stacked together is called erythrocyte rouleaux formation.
  • the present invention provides an application of low-molecular-weight hyaluronic acid fragments to induce money-like aggregation of red blood cells.
  • the use of low molecular weight hyaluronic acid fragments to induce a negative correlation between the minimum percentage concentration of red blood cell clusters in peripheral blood or venous red blood cells and the molecular weight is used to determine the molecular weight of low molecular weight hyaluronic acid fragments or to determine low molecular weight The range of molecular weight variation between batches of hyaluronic acid fragment products.
  • peripheral blood or venous blood is the peripheral blood or venous blood of humans, cats, dogs or rats.
  • the low-molecular-weight hyaluronic acid fragment is a hyaluronic acid fragment product with an average molecular weight of 35 ⁇ 8kDa; the minimum percentage concentration that induces a cluster of red blood cells in peripheral blood or venous blood is 0.15%.
  • low molecular weight hyaluronic acid fragments to induce different degrees of red blood cell aggregation in peripheral blood or venous red blood cells causes different red blood cell sedimentation rates to sensitively determine the small molecular weight variation range of low molecular weight hyaluronic acid fragments produced between batches.
  • the peripheral blood or venous blood is the peripheral blood or venous blood of humans, dogs or rats;
  • the low-molecular-weight hyaluronic acid fragment is a hyaluronic acid fragment product with an average molecular weight of 35 ⁇ 8kDa; the molecular weight produced between batches is determined In the variation range, the final concentration of the hyaluronic acid fragment product with an average molecular weight of 35 ⁇ 8kDa is 0.075%.
  • the present invention also provides a method for detecting the activity of low-molecular-weight hyaluronic acid fragments with an average molecular weight of 35 ⁇ 8kDa.
  • the low-molecular-weight hyaluronic acid fragments induce peripheral blood or venous red blood cell clusters by binding to CD44 on the surface of red blood cells.
  • the detection method is to measure the CD44 binding activity of low-molecular-weight hyaluronic acid fragments with an average molecular weight of 35 ⁇ 8kDa.
  • 10ug/ml anti-human CD44 antibody was used to inhibit the binding of hyaluronic acid fragments with an average molecular weight of 35 ⁇ 8kDa to CD44.
  • the present invention also provides an application of a low-molecular-weight hyaluronic acid fragment with an average molecular weight of 35 ⁇ 8kDa, the low-molecular-weight hyaluronic acid fragment rapidly binds to human neutrophils and enters neutrophils;
  • the application is used in the preparation of drugs for treating neutrophil-related inflammatory diseases.
  • the present invention also provides an application of a low-molecular-weight hyaluronic acid fragment with an average molecular weight of 35kDa.
  • the low-molecular-weight hyaluronic acid fragment directly and/or indirectly inhibits CD44 on the surface of red blood cells and/or white blood cells or the surface of white blood cells.
  • Human neutrophils activate and release various oxidizing substances; the application is as an inhibitor for inhibiting human neutrophil activation and releasing various oxidizing substances or in preparing treatments for neutrophil activation and releasing various oxidative substances.
  • Application in substance-related inflammatory disease drugs; the application has a different effect or degree of effectiveness on humans and other animals.
  • the present invention found that hyaluronic acid fragments induce money-like aggregation of red blood cells in human and animal peripheral blood and venous blood in the low molecular weight range. Negative correlation.
  • the present invention uses this negative correlation to determine the molecular weight of low-molecular-weight hyaluronic acid fragments and to determine the range of molecular weight variation produced between batches of low-molecular-weight hyaluronic acid fragments.
  • the venous blood of cats, dogs and rats and its induced red blood cell clustering can be used to determine the molecular weight and molecular weight variation of low molecular weight hyaluronic acid fragment products.
  • the present invention also found that hyaluronic acid fragments promote the sedimentation rate of red blood cells in the low molecular weight range.
  • the present invention uses the effect of this hyaluronic acid fragment on the sedimentation rate of red blood cells to determine the degree of molecular weight variation between batches of low-molecular-weight hyaluronic acid fragment products, forming a quality detection method for the molecular weight variation of hyaluronic acid fragment products between batches.
  • the present invention further discovered that the low-molecular-weight hyaluronic acid fragment B-HA with an average molecular weight of 35 ⁇ 8kDa induces the cluster-like aggregation of human red blood cells by binding to CD44 on the surface of human red blood cells, and its essence reflects that this low-molecular-weight hyaluronic acid fragment binds to CD44 on the surface of red blood cells.
  • Activity using the discovery to determine the biological activity of low molecular weight hyaluronic acid fragments with an average molecular weight of 35 ⁇ 8kDa.
  • the present invention also found that human neutrophils phagocytize the hyaluronic acid fragment B-HA with an average molecular weight of 35 ⁇ 8kDa, but it does not affect the function of human neutrophils to phagocytize exogenous fluorescent particles.
  • the present invention further discovered that low molecular weight hyaluronic acid fragments bind to the surface of human red blood cells and affect the hemodynamic behavior of human blood cells or the interaction between blood cells and white blood cells.
  • the hyaluronic acid fragment B-HA with an average molecular weight of 35 ⁇ 8kDa has an inhibitory effect on the activation of human neutrophils, combined with its good tissue permeability, suggesting that it has an inhibitory effect on human tissue inflammation.
  • the present invention further discovered that the low-molecular-weight hyaluronic acid fragment induces red blood cell clusters and promotes red blood cell sedimentation, which affects intravascular red blood cell hemodynamics and the interaction between red blood cells and white blood cells, which is species-specific, suggesting low The physiological functions, therapeutic effects and side effects of molecular weight hyaluronic acid fragments are species-specific.
  • Figure 1 is a diagram showing how recombinant human hyaluronidase PH20 fully enzymatically hydrolyzes macromolecular hyaluronic acid raw materials with an average molecular weight of 35 ⁇ 8kDa hyaluronic acid fragment B-HA to inhibit human skin redness, swelling and hard pain;
  • Figure 2 is the correlation curve of the minimum percentage concentration and molecular weight of the hyaluronic acid fragments of different molecular weights inducing the cluster-like aggregation of human peripheral red blood cells;
  • FIG. 3 (a) is a graph showing that the final concentration of 0.15% B-HA (or HA fragments 35kDa or HA35) induces human peripheral red blood cell clusters; (b) is 10ug/ml anti-human CD44 antibody inhibiting B-HA Induced human peripheral red blood cell money clusters; (c) non-specific rabbit IgG antibody did not inhibit B-HA induced human peripheral red blood cell money clusters; (d) is a recombinant human transparent added at a final concentration of 1927U/ml Plasmidase PH20 inhibits B-HA-induced human peripheral red blood cell money clusters;
  • Figure 4 is the correlation curve of the minimum percentage concentration and molecular weight of cat venous red blood cell clusters induced by hyaluronic acid fragments of different molecular weights
  • Figure 5 is a correlation curve of the minimum percentage concentration and molecular weight of the canine venous red blood cell clusters induced by hyaluronic acid fragments of different molecular weights;
  • Figure 6 is the correlation curve of the minimum percentage concentration and molecular weight of rat venous red blood cell clusters induced by hyaluronic acid fragments of different molecular weights
  • Figure 7 is a photo and percentage diagram of 50 multinucleated neutrophils containing fluorescent particles and clumps in the B-HA group and fluorescently labeled B-HA (Cy5.5 B-HA);
  • Figure 8 is a diagram showing the effect of B-HA and HA on human neutrophils phagocytosing fluorescent particles
  • Figure 9 shows the gel electrophoresis molecular weight determination results of different batches of B-HA (also known as HA35 or HA fragments 35kDa) products between batches;
  • Figure 10 is a graph showing the results of intra-assay variation in molecular weight of HA fragments 24kDa (FRD) by gel electrophoresis;
  • Figure 11 shows the effect of B-HA (also known as HA35 or HA fragments 35kDa) and HA raw material 1600kDa (FRD) (referred to in the figure as HA) on PMA-induced neutrophil activation of fresh human leukocytes.
  • B-HA also known as HA35 or HA fragments 35kDa
  • FPD HA raw material 1600kDa
  • Biological enzymatic production of low-molecular-weight hyaluronic acid fragments Preliminary experiments are used to determine that the biological enzyme PH20 is sufficient or slightly excessive to fully enzymatically hydrolyze and partially enzymatically hydrolyze hyaluronic acid raw materials to produce low-molecular-weight hyaluronic acid fragments.
  • Hyaluronidase is defined as: (1) In 10-20 minutes, the molecular weight and the molecular weight of the hyaluronic acid fragment produced by the sufficient or light excessive enzymatic hydrolysis and the high-molecular hyaluronic acid raw material In 1-6 hours, the molecular weight of hyaluronic acid fragments produced by full enzymatic hydrolysis and neutralization of high-molecular hyaluronic acid raw materials is basically the same (coefficient of variation CV ⁇ 15%); (2)>99% high or medium molecular weight The low-molecular-weight hyaluronic acid fragment products that are fully enzymatically hydrolyzed by the hyaluronic acid raw material in sufficient or light excess are all smoothly filtered by 0.22um filter membrane; (3) Hyaluronidase activity is fully enzymatically hydrolyzed in sufficient or light excess After the reaction, there is almost no residue or a small amount of residue ( ⁇ 15%), and all are inactivated at 80 degrees and 45 minutes. The time
  • the physicochemical production method for the production of hyaluronic acid fragments combines acid, alkali, and high-temperature production methods.
  • hyaluronic acid fragments were determined by gel electrophoresis and 18-angle laser. The raw material hyaluronic acid is measured with a viscometer and an 18-angle laser.
  • HA fragments 35kDa or HA35 or B-HA or B-HA (HH) are 6 different batches of B-HA equivalent mixture, 18-angle laser (GPC/SEC system equipped with an multi-angle laser light scattering( The result of the MALLS) detector was 35 ⁇ 8kDa (Table 16).
  • HA fragments (24kDa) manufactured by physicochemical combination of biological enzymes were originally Oligo HA from FRD. After repeated measurements of the present invention, its molecular weight and >90% molecular weight distribution are 24kDa and 10-40kDa, respectively.
  • Human peripheral blood and venous blood collection There are 8 healthy volunteers, aged 22 ⁇ 5 years old, 4 males and females. The collection of peripheral blood and venous blood was approved by the Medical Ethics Committee of Changchun Jiahe Surgical Hospital and I agree. The collection of animal peripheral blood and venous blood was approved by the Animal Hospital of Qingdao Agricultural University.
  • Bold type and italic type together represent the concentration of the lowest molecular weight hyaluronic acid fragment that causes the aggregation of red blood cell money strings; * represents severe cell deformation. Yes/Yes/Yes or No/No/No represents the results of three experiments using different human peripheral blood samples.
  • Fig. 2 shows the correlation curve of the minimum percentage concentration and molecular weight of human peripheral red blood cell clusters induced by hyaluronic acid fragments of different molecular weights.
  • the final concentration of B-HA (HA35) that induces red blood cell clustering is 0.15%, which is 1.5mg/ml.
  • the present invention uses B-HA to stimulate the function of freshly extracted human neutrophils at a concentration of 10ug/ml (Example 4).
  • a normal person with an average weight of 70 kg contains about 15 grams of hyaluronic acid, of which 1/3 is degraded every day (Stern R, Hyaluronan catabolism: a new metabolic pathway, 2004, Eur. J. Cell Biol. 83(7): 317– 25.doi:10.1078/0171-9335-00392).
  • the 28.5ng/ml hyaluronic acid in normal human serum has little effect on the blood concentration of 10ug/ml B-HA we use. Therefore, B-HA with a blood concentration of 10ug/ml binds to the surface of red blood cells in the serum, which may affect the hemodynamic behavior of human blood cells or the interaction between blood cells and white blood cells.
  • Human peripheral blood and venous blood collection There are 8 healthy volunteers, aged 22 ⁇ 5 years old, 4 males and females. The collection of peripheral blood and venous blood was approved by the Medical Ethics Committee of Changchun Jiahe Surgical Hospital and I agree. The collection of animal peripheral blood and venous blood was approved by the Animal Hospital of Qingdao Agricultural University.
  • hyaluronic acid fragment B-HA also known as HA fragments35kDa or HA35
  • Table 1 Use the low-molecular-weight hyaluronic acid fragment B-HA (also known as HA fragments35kDa or HA35) in Table 1 to mix with human peripheral blood or venous blood in a ratio of 1:2 to a final concentration of 0.15%, and finally reach human peripheral blood or venous blood The blood was diluted 3.5 times. Then, a microscope was used to observe and verify the red blood cell clusters caused by the low molecular weight hyaluronic acid fragment B-HA (also known as HA fragments35kDa or HA35).
  • FIG 3 (a) is a graph showing that the final concentration of 0.15% B-HA (or HA fragments 35kDa or HA35) induces human peripheral red blood cell clusters; (b) is 10ug/ml anti-human CD44 antibody inhibiting B-HA Induced human peripheral red blood cell money clusters; (c) non-specific rabbit IgG antibody did not inhibit B-HA induced human peripheral red blood cell money clusters; (d) is the final concentration of 1927U/ml recombinant human transparent Plasmidase PH20 inhibits B-HA-induced clusters of human peripheral red blood cells; Note: The results of using venous red blood cells are the same.
  • the low-molecular-weight hyaluronic acid fragment B-HA (HA35) induces red blood cell clusters.
  • the PH20 enzymatic hydrolysis added at a final concentration of 1927U/ml blocked the B-HA-induced red blood cell clustering. This result indicates that B-HA (HA35)-induced red blood cell clustering is achieved through B-HA molecules.
  • the present invention shows that the hyaluronic acid fragment B-HA (HA35) induces red blood cell clusters by binding to CD44 on the surface of red blood cells.
  • Hyaluronic acid fragments of different molecular weights and hyaluronic acid raw materials induce the minimum percentage concentration of cat venous red blood cell clusters.
  • Figure 4 is the correlation curve of the minimum percentage concentration and molecular weight of cat venous red blood cell clusters induced by hyaluronic acid fragments of different molecular weights.
  • Hyaluronic acid fragments of different molecular weights and hyaluronic acid raw materials induce the smallest percentage concentration of canine venous red blood cell money string aggregation.
  • Fig. 5 is the correlation curve of the minimum percentage concentration and molecular weight of the canine venous red blood cell clusters induced by hyaluronic acid fragments of different molecular weights.
  • Hyaluronic acid fragments of different molecular weights and hyaluronic acid raw materials induce the smallest percentage concentration of rat venous red blood cell clusters.
  • Bold type and italic type together represent the concentration of the lowest low-molecular-weight hyaluronic acid fragment that causes the aggregation of red blood cell money strings; * represents severe cell deformation.
  • Fig. 6 is a correlation curve of the minimum percentage concentration and molecular weight of rat venous red blood cell clusters induced by hyaluronic acid fragments of different molecular weights.
  • the present invention found that hyaluronic acid fragments of different molecular weights and hyaluronic acid raw materials induce money-string-like aggregation of venous red blood cells in cats, dogs and rats. This result is basically the same as the result of low-molecular-weight hyaluronic acid fragments inducing money clusters of human peripheral blood and venous red blood cells.
  • the present invention shows that the venous blood of cats, dogs and rats and its induced red blood cell cluster-like aggregation can be used to determine the molecular weight and molecular weight variation of low molecular weight hyaluronic acid fragment products.
  • results of the present invention showed that none of the 50 neutrophils in the B-HA group contained fluorescent particles or clumps.
  • the 50 neutrophils in the fluorescent-labeled B-HA (Cy5.5 B-HA) group 36 cells have strong fluorescent particles and clumps.
  • Figure 7 is a photograph and percentage of single cells containing fluorescent particles and clumps of 50 multinucleated neutrophils in the B-HA group and fluorescently labeled B-HA (Cy5.5 B-HA).
  • the present invention uses fluorescently labeled B-HA (Cy5.5 B-HA) to culture human red blood cells, and there is no phenomenon of fluorescent labeling on the surface of red blood cells. This result indicates that the luminescence intensity of fluorescently labeled B-HA (Cy5.5 B-HA) is not enough to detect the binding on the surface of red blood cells.
  • the present invention uses fluorescently labeled B-HA (Cy5.5 B-HA) to extract human neutrophils or whole blood cells for culture.
  • the present invention further explores the effect of 10ug/ml B-HA (HA fragments 35kDa or HA35) on the phagocytosis of foreign fluorescent particles by human neutrophils.
  • This experiment also used the sugar density gradient centrifugation method, that is, human venous blood leukocyte separation kit (endotoxin ⁇ 0.1EU) (Tianjin Haoyang Huake Biotechnology Co., Ltd.) to separate human venous blood.
  • human venous blood leukocyte separation kit endotoxin ⁇ 0.1EU
  • endotoxin ⁇ 0.1EU Tetranjin Haoyang Huake Biotechnology Co., Ltd.
  • RPMI-1640 medium containing 10% FBS for human neutrophils and adjust the cell density to 2 ⁇ 10 6 cells/ml.
  • B-HA HA fragments 35kDa or HA35
  • 10ug/mlHA or 1ng/mlLPS to stimulate neutrophils.
  • Figure 8 shows the effects of B-HA and HA on human neutrophils phagocytosing fluorescent particles.
  • 10ug/ml B-HA (HA fragments 35kDa or HA35) compared with Blank, P>0.05, no significant difference.
  • 10ug/ml HA raw material 1600kDa (FRD) (here referred to as HA) compared with Blank, P>0.05, no significant difference; compared with Blank, 0.01 ⁇ P ⁇ 0.05, significant difference, verifying the reliability of this experimental method .
  • B-HA (HA fragments 35kDa or HA35) has no effect on the phagocytosis of foreign fluorescent particles by human neutrophils.
  • the present invention shows that human neutrophils phagocytose B-HA (HA fragments 35kDa or HA35), suggesting that red blood cells and neutrophils that bind B-HA on the surface interact with each other (references in this paragraph).
  • OBJECTIVE To study the species specificity of different molecular weight hyaluronic acid fragments and hyaluronic acid raw materials inducing money-string aggregation of venous red blood cells in animals.
  • Bold type and italic type together represent the concentration of the lowest hyaluronic acid fragment that causes red blood cell aggregation; * represents severe cell deformation; # represents the formation of large red blood cell clusters.
  • HA Fragments 24kDa (FRD) has no red blood cell aggregation at the final concentration of 0.6%.
  • HA fragments 35kDa or B-HA or HA35 at the final concentration of 0.3%, 0.15%, although red blood cell aggregation is induced, but there is no obvious cluster-like aggregation, and the degree of aggregation is relatively light, which is comparable to human HA fragments 35kDa or B-HA or HA35
  • the induction of red blood cell clusters is significantly different.
  • Table 8 The results of different molecular weight hyaluronic acid fragments and hyaluronic acid raw materials induced money clusters of porcine venous red blood cells.
  • Bold type and italic type together represent the lowest concentration of hyaluronic acid fragments that cause the formation of red blood cell clusters; * represents severe cell deformation; # represents the formation of large red blood cell clusters.
  • Table 9 The results of different molecular weight hyaluronic acid fragments and hyaluronic acid raw materials inducing the cluster-like aggregation of bovine venous red blood cells.
  • Bold type and italic type together represent the lowest concentration of hyaluronic acid fragments that cause red blood cell aggregation; * represents severe cell deformation; # represents the formation of large red blood cell clusters; Yes/Yes or No/No or No /Yes represents the results of two experiments using different individual peripheral blood samples.
  • the red blood cells of the mink are not in good condition, and they immediately shrink and deform when added to the blood diluted by normal saline. Therefore, when the mink red blood cells are observed, there are many shrunken cells.
  • Yes/Yes or No/No or Yes/No represents the results of two experiments using peripheral blood samples of different individuals; alpaca red blood cells are oval in shape.
  • the present invention finds that hyaluronic acid fragments induce money clusters of human and animal peripheral blood and venous red blood cells and promote red blood cell sedimentation.
  • the present invention further discovered that the low-molecular-weight hyaluronic acid fragment B-HA with an average molecular weight of 35kDa induces the money-string aggregation of human erythrocytes by binding to CD44 on the surface of human erythrocytes ( Figure 3), which essentially reflects that this low-molecular-weight hyaluronic acid fragment binds to the surface of erythrocytes. CD44 activity.
  • Literature studies have shown that human leukocytes (including neutrophils, monocytes and lymphocytes) through their hemodynamic changes are removed from the blood vessel and exuded into the tissue inflammation zone to play an inflammatory role (references 1-8 in this section).
  • Literature studies also support that hyaluronic acid fragments affect the hemodynamic behavior of red blood cells and affect the function of white blood cells (references 9-14 in this section). For example, low-molecular-weight hyaluronic acid fragments of human tissues are produced in inflamed tissues and may enter the blood circulation to be cleared.
  • Literature studies also show that red blood cells bound by low-molecular-weight hyaluronic acid fragments are phagocytosed and cleared by neutrophils and macrophages in the liver and spleen (references 9-14 in this paragraph), suggesting that neutrophils and macrophages phagocytose low-molecular-weight transparent Fragments of uronic acid and red blood cells labeled with fragments of low-molecular-weight hyaluronic acid.
  • the low-molecular-weight hyaluronic acid fragments produced in inflamed tissues enter the blood circulation and combine with red blood cells, which may be related to the lifespan of red blood cells.
  • the present invention suggests that low-molecular-weight hyaluronic acid fragments induce red blood cell clusters and indicates that the physiological functions, therapeutic effects and side effects of low-molecular-weight hyaluronic acid fragments are species-specific.
  • the hyaluronic acid fragments HA fragments 35kDa or B-HA or HA35 which have anti-inflammatory effects on human skin and mucous membranes, have no effect on cattle, sheep or even monkeys.
  • Tables 2-5 and 6-12 show that cows and sheep are completely different from monkeys, horses, pigs, dogs, rats, cats and other animals. This result indicates that the low-molecular-weight hyaluronic acid fragments induce red blood cell clusters and red blood cell sedimentation, which affect the hemodynamics of red blood cells in blood vessels and the interaction between red blood cells and white blood cells, which are species-specific.
  • This species specificity indicates that the biological activity, therapeutic effects and side effects of low molecular weight hyaluronic acid fragments are species specific. In other words, this species-specificity indicates that the biological activity of low molecular weight hyaluronic acid fragments is not the same or effective to humans and other animals.
  • Purpose Use low-molecular-weight hyaluronic acid fragments to induce different red blood cell sedimentation rates caused by red blood cell clustering to determine the degree of molecular weight variation between batches of hyaluronic acid fragment B-HA (also known as HA35 or HA fragments 35kDa).
  • the present invention uses low molecular weight hyaluronic acid fragments to induce different red blood cell sedimentation rates caused by different degrees of red blood cell clustering to quantitatively determine the molecular weight variation of hyaluronic acid fragments produced between batches.
  • the difference in blood cell sedimentation rate caused by the different degree of red blood cell cluster-like aggregation is used to quantitatively determine the molecular weight variation of hyaluronic acid fragment products produced between batches.
  • Low molecular weight hyaluronic acid fragment B-HA also known as HA35 or HA fragments 35kDa
  • canine venous blood are mixed in a ratio of 1:2, plus anticoagulant EDTA and PBS buffer to the final concentration of 0.15%, respectively , 0.11%, 0.075%, and finally reach the 3.5-fold dilution of canine venous blood, draw 400ul of mixed blood into the erythrocyte sedimentation tube, let it stand for 25 minutes, then count the sedimentation distance of the blood in the erythrocyte sedimentation tube, and calculate the sedimentation rate of blood cells.
  • the low-molecular-weight hyaluronic acid fragment B-HA also known as HA35 or HA fragments 35kDa.
  • Table 13 Determine the effect of B-HA (also known as HA35 or HA fragments 35kDa) with final concentrations of 0.15%, 0.11% and 0.075% on the 25-minute sedimentation rate (cm/25minutes) of human venous red blood cells diluted by 3.5 times.
  • B-HA also known as HA35 or HA fragments 35kDa
  • Table 15 Determine the effect of B-HA (also known as HA35 or HA fragments 35kDa) with final concentrations of 0.15%, 0.11% and 0.075% on the 25-minute sedimentation rate (cm/25minutes) of rat venous red blood cells diluted by 3.5 times.
  • B-HA also known as HA35 or HA fragments 35kDa
  • Figure 9 Inter-batch measurement results of gel electrophoresis molecular weight of different batches of B-HA (also known as HA35 or HA fragments 35kDa) products.
  • Figure 10 shows the results of intra-assay variation in molecular weight of HA fragments 24kDa (FRD) by gel electrophoresis.
  • the present invention further uses low-molecular-weight hyaluronic acid fragments to induce different blood cell sedimentation rates caused by different degrees of red blood cell aggregation in rats, dogs and humans to quantitatively determine the molecular weight variation of hyaluronic acid fragments produced between batches.
  • the sensitivity of the determination is higher than that of observing the money-like clusters of red blood cells.
  • B-HA also known as HA35 or HA fragments 35kDa
  • commercial products require a sensitive method of measuring the molecular weight of lower molecular weight hyaluronic acid fragments for subcutaneous tissue injection products to monitor the variation of product molecular weight.
  • the present invention uses low-molecular-weight hyaluronic acid fragments for the first time to induce different blood cell sedimentation speeds caused by red blood cell cluster-like aggregation, and establishes a quality detection method for the production molecular weight variation of hyaluronic acid fragment products between batches.
  • hyaluronic acid fragments to induce rat, dog, and human red blood cell money clusters has different erythrocyte sedimentation rates, and its measurement sensitivity is high. It can be used to determine hyaluronic acid fragment B-HA (also known as HA35 or HA). Fragments 35kDa) The degree of slight variation in the molecular weight of the product between batches, the sensitivity is better than that of 18-angle laser measurement and gel electrophoresis measurement.
  • B-HA also known as HA35 or HA fragments 35kDa
  • neurotrophil activation To explore the biological and clinical significance of B-HA (also known as HA35 or HA fragments 35kDa) combined with the surface of human red blood cells on human leukocyte activation (neutrophil activation).
  • Inoculate 5x10 5 (24-well plate) or 1.2x10 7 (6-well plate) human neutrophils in each well first add 10ug/ml HA (also known as HA raw material 1600kDa) or B-HA (also known as HA35 or HA Fragments 35kDa) or saline for 30 minutes, then add Phorbol-12 myristate 13-acetate (PAM) at a final concentration of 25nM to activate for 30 minutes.
  • HA also known as HA raw material 1600kDa
  • B-HA also known as HA35 or HA Fragments 35kDa
  • PAM Phorbol-12 myristate 13-acetate
  • Flow cytometry was used to measure ROS (reactive oxygen species) released by neutrophils, and the result was expressed as Mean florescence intensity (Mean ⁇ SD).
  • FIG. 11 The effect of B-HA (also known as HA35 or HA fragments 35kDa) and HA raw material 1600kDa (FRD) (referred to in the figure as HA) on PMA-induced neutrophil activation of fresh human leukocytes.
  • B-HA also known as HA35 or HA fragments 35kDa
  • FPD HA raw material 1600kDa
  • Example 6 of the present application shows that the B-HA inhibits human leukocyte activation has species specificity, that is, the B-HA inhibits human leukocyte activation has different effects or degrees of effectiveness on humans and animals.
  • LYVE-1 a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan.J Cell BIol 144:789-801.
  • a serum-derived hyaluronan-associated protein is the heavy chain of the inter alpha-trypsin inhibitor. J Biol Chem 268:26725–26730.

Abstract

Disclosed is an application of low-molecular-weight hyaluronic acid fragments to induce string aggregation of red blood cells in peripheral blood or venous blood, for use in measuring the molecular weight of low-molecular-weight hyaluronic acid fragments or measuring the molecular weight variation range of products produced between batches. The low-molecular-weight hyaluronic acid fragments having an average molecular weight of 35 kDa induce string aggregation of human red blood cells by combining CD44 on the surface of human red blood cells, for use in activity detection of the low-molecular-weight hyaluronic acid fragments. Further disclosed is that the hyaluronic acid fragments having an average molecular weight of 35 kDa have an inhibitory effect on human neutrophil activation, and can prevent and treat inflammatory diseases related to neutrophil activation and releasing various oxidation substances.

Description

一种低分子量透明质酸片段诱发血红细胞钱串状聚集的应用Application of low-molecular-weight hyaluronic acid fragments in inducing string-like aggregation of red blood cells 技术领域Technical field
本发明涉及生物医学领域，特别涉及一种低分子量透明质酸片段诱发末梢血或静脉血红细胞钱串状聚集的应用。The invention relates to the field of biomedicine, in particular to an application of low molecular weight hyaluronic acid fragments in inducing the cluster-like aggregation of peripheral blood or venous red blood cells.
背景技术Background technique
人用高分子透明质酸皮下组织注射液主要用于美容，有多种产品，包括Restylane，其分子量大于1.0x10 6daltons。人美容用透明质酸皮下组织注射液可用于隆鼻、丰唇和除皱纹等多种美容用途(1、2、3)。由于Restylane分子量大，粘度也大，其分子量都使用粘度计测定。还由于，Restylane分子量和粘度大，皮下注射局部不良反应也较多，包括局部注射炎症反应红、肿、硬、痛。较严重的不良反应是注射局部小血管凝血堵塞和由其引发的溃疡(4)。 Human macromolecular hyaluronic acid subcutaneous tissue injection is mainly used for beauty, and there are many products, including Restylane, whose molecular weight is greater than 1.0x10 6 daltons. Human cosmetic hyaluronic acid subcutaneous tissue injection can be used for various cosmetic purposes such as rhinoplasty, lip augmentation and wrinkle removal (1, 2, 3). Since Restylane has a large molecular weight and a large viscosity, its molecular weight is measured with a viscometer. Also due to the high molecular weight and viscosity of Restylane, there are many local adverse reactions of subcutaneous injection, including red, swollen, hard, and painful local injection inflammation. The more serious adverse reactions are local small blood vessel clogging and ulcers caused by injection (4).
理论上，较低分子量的透明质酸皮下注射产品局部不良反应也较小，即局部小血管堵塞的可能性较小。因此，较低分子量的透明质酸静脉血管注射也成为可能，如马专用Legend Multi Dose for intravenous use in horse only(Bayer Corporation)，其分子量3.0x10 5daltons(www.equinelegend.com)(5)。低分子量的透明质酸注射产品分子量用粘度计测定不准，目前主要使用凝胶电泳和多角度激光方法测定(6、7)。 Theoretically, the lower molecular weight hyaluronic acid subcutaneous injection product has less local adverse reactions, that is, the possibility of local small blood vessel blockage is less. Therefore, intravenous injection of hyaluronic acid with lower molecular weight is also possible, such as Legend Multi Dose for intravenous use in horse only (Bayer Corporation), which has a molecular weight of 3.0x10 5 daltons (www.equinelegend.com) (5). The molecular weight of low-molecular-weight hyaluronic acid injection products is inaccurately determined with a viscometer. Currently, gel electrophoresis and multi-angle laser methods are mainly used for determination (6, 7).
文献研究(8、9、10、11)表明人初乳纯化出的透明质酸片段HA35的分子量平均35kDa。文献研究还表明人乳肪组织有透明质酸酶PH20(12)。本发明人使用重组人透明质酸酶PH20制造了平均分子量35±8kDa的透明质酸片段B-HA(13、14)。本发明人发现重组人透明质酸酶PH20充分酶解制造的透明质酸片段B-HA商业化产品(Medical device type 1，LUQIN Food Drug Medical Device registration number:20190021)有明确的人皮肤和粘膜抗炎活性，包括对人皮肤粘膜临床炎症表现红肿硬痛有显著治疗作用(如图1所示)，并在本地报道了这个临床抗炎作用和申报了专利(14、15、16、17、18、19、20、21)。Literature studies (8, 9, 10, 11) indicate that the hyaluronic acid fragment HA35 purified from human colostrum has an average molecular weight of 35kDa. Literature studies also show that human milk adipose tissue has hyaluronidase PH20 (12). The present inventors used recombinant human hyaluronidase PH20 to produce a hyaluronic acid fragment B-HA with an average molecular weight of 35±8 kDa (13, 14). The inventors found that the hyaluronic acid fragment B-HA commercial product (Medical device type 1, LUQIN Food Drug Medical Device registration number: 20190021) produced by the fully enzymatically hydrolyzed recombinant human hyaluronidase PH20 has clear human skin and mucosal resistance. Inflammatory activity, including a significant therapeutic effect on the clinical inflammation of human skin and mucous membranes (as shown in Figure 1). This clinical anti-inflammatory effect has been reported locally and a patent has been declared (14, 15, 16, 17, 18). , 19, 20, 21).
以上文献研究提示HA35或平均分子量35±8kDa的透明质酸片段B-HA的商业化产品，需要敏感的低分子量透明质酸皮下组织注射产品分子量测定方法，以控制商业化产品分子量在可以接受的范围内。另外，针对低分子量透明质酸片段，尤其是平均分子量35±8kDa的透明质酸片段的生物活性的作用机制和新的生物活性以及潜在新临床应用的研究还有限(22、23、24、25、26、27、28、29、30、31、32、33、34、35、36)，还需进一步研究和开发。The above literature studies suggest that the commercial products of HA35 or the hyaluronic acid fragment B-HA with an average molecular weight of 35±8kDa require a sensitive low molecular weight hyaluronic acid subcutaneous tissue injection product molecular weight determination method to control the molecular weight of the commercial product at an acceptable level Within range. In addition, research on the biological activity mechanism of low molecular weight hyaluronic acid fragments, especially hyaluronic acid fragments with an average molecular weight of 35±8kDa, new biological activities, and potential new clinical applications are still limited (22, 23, 24, 25 , 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36), further research and development are needed.
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发明内容Summary of the invention
本发明要解决的技术问题是研究低分子量透明质酸片段，尤其是平均分子量35±8kDa的低分子量透明质酸片段的生物活性的作用机制和新的生物活性以及潜在新临床应用。The technical problem to be solved by the present invention is to study the biological activity mechanism of low molecular weight hyaluronic acid fragments, especially low molecular weight hyaluronic acid fragments with an average molecular weight of 35±8kDa, new biological activities and potential new clinical applications.
本发明意外地发现透明质酸片段诱发人和动物全血(末梢血和静脉血)红细胞钱串状聚集(见下方注解和参考图3)，其诱发红细胞钱串状聚集的最小浓度在低分子范围内与透明质酸片段的分子量负相关。本文针对这个发现进行了深入研究。注：红细胞聚集(Erythrocytes aggregate)是一种可以逆转的哺乳动物红细胞聚集现象。哺乳动物红细胞象多个硬币摞在 一起形成的聚集叫红细胞钱串状聚集(erythrocyte rouleaux formation)。The present invention unexpectedly found that the hyaluronic acid fragment induces the red blood cell clusters of human and animal whole blood (peripheral blood and venous blood) (see the note below and refer to Figure 3), and the minimum concentration of the red blood cell clusters induced by the low molecular weight The range is negatively correlated with the molecular weight of hyaluronic acid fragments. This article conducts an in-depth study of this discovery. Note: Erythrocytes aggregate is a reversible phenomenon of mammalian red blood cell aggregation. The aggregation of mammalian red blood cells like multiple coins stacked together is called erythrocyte rouleaux formation.
基于上述研究获得如下技术方案：Based on the above research, the following technical solutions are obtained:
一方面，本发明提供了一种低分子量透明质酸片段诱发红细胞钱串状聚集的应用。In one aspect, the present invention provides an application of low-molecular-weight hyaluronic acid fragments to induce money-like aggregation of red blood cells.
进一步地，利用低分子量透明质酸片段诱发末梢血或静脉血红细胞钱串状聚集的最小百分比浓度和分子量大小之间呈负相关的关系，测定低分子量透明质酸片段的分子量大小或测定低分子量透明质酸片段产品批间生产的分子量变异范围。Further, the use of low molecular weight hyaluronic acid fragments to induce a negative correlation between the minimum percentage concentration of red blood cell clusters in peripheral blood or venous red blood cells and the molecular weight is used to determine the molecular weight of low molecular weight hyaluronic acid fragments or to determine low molecular weight The range of molecular weight variation between batches of hyaluronic acid fragment products.
进一步地，所述末梢血或静脉血为人、猫、犬或大鼠的末梢血或静脉血。Further, the peripheral blood or venous blood is the peripheral blood or venous blood of humans, cats, dogs or rats.
进一步地，所述低分子量透明质酸片段为平均分子量35±8kDa的透明质酸片段产品；其诱发末梢血或静脉血红细胞钱串状聚集的最小百分比浓度为0.15％。Further, the low-molecular-weight hyaluronic acid fragment is a hyaluronic acid fragment product with an average molecular weight of 35±8kDa; the minimum percentage concentration that induces a cluster of red blood cells in peripheral blood or venous blood is 0.15%.
进一步地，利用低分子量透明质酸片段诱发末梢血或静脉血红细胞钱串状聚集程度不同造成的血红细胞沉降率不同，敏感测定低分子量透明质酸片段产品批间生产的分子量微小变异范围。Furthermore, the use of low molecular weight hyaluronic acid fragments to induce different degrees of red blood cell aggregation in peripheral blood or venous red blood cells causes different red blood cell sedimentation rates to sensitively determine the small molecular weight variation range of low molecular weight hyaluronic acid fragments produced between batches.
进一步地，所述末梢血或静脉血为人、犬或大鼠的末梢血或静脉血；所述低分子量透明质酸片段为平均分子量35±8kDa的透明质酸片段产品；测定批间生产的分子量变异范围时，所述平均分子量35±8kDa的透明质酸片段产品终浓度为0.075％。Further, the peripheral blood or venous blood is the peripheral blood or venous blood of humans, dogs or rats; the low-molecular-weight hyaluronic acid fragment is a hyaluronic acid fragment product with an average molecular weight of 35±8kDa; the molecular weight produced between batches is determined In the variation range, the final concentration of the hyaluronic acid fragment product with an average molecular weight of 35±8kDa is 0.075%.
另一方面，本发明还提供了一种平均分子量35±8kDa的低分子量透明质酸片段的活性检测方法，所述低分子量透明质酸片段通过结合红细胞表面CD44诱发末梢血或静脉血红细胞钱串状聚集；所述检测方法为对平均分子量35±8kDa的低分子量透明质酸片段进行CD44结合活性测定。On the other hand, the present invention also provides a method for detecting the activity of low-molecular-weight hyaluronic acid fragments with an average molecular weight of 35±8kDa. The low-molecular-weight hyaluronic acid fragments induce peripheral blood or venous red blood cell clusters by binding to CD44 on the surface of red blood cells. The detection method is to measure the CD44 binding activity of low-molecular-weight hyaluronic acid fragments with an average molecular weight of 35±8kDa.
进一步地，利用10ug/ml的抗人CD44抗体抑制平均分子量35±8kDa的透明质酸片段结合CD44。Furthermore, 10ug/ml anti-human CD44 antibody was used to inhibit the binding of hyaluronic acid fragments with an average molecular weight of 35±8kDa to CD44.
再一方面，本发明还提供了一种平均分子量35±8kDa的低分子量透明质酸片段的应用，所述低分子量透明质酸片段与人中性粒细胞快速结合并进入中性粒细胞；所述应用为在制备治疗中性粒细胞相关炎症性疾病药物中应用。In another aspect, the present invention also provides an application of a low-molecular-weight hyaluronic acid fragment with an average molecular weight of 35±8kDa, the low-molecular-weight hyaluronic acid fragment rapidly binds to human neutrophils and enters neutrophils; The application is used in the preparation of drugs for treating neutrophil-related inflammatory diseases.
再一方面，本发明还提供了一种平均分子量35kDa的低分子量透明质 酸片段的应用，所述低分子量透明质酸片段通过结合红细胞和/或白细胞表面CD44或白细胞表面直接和/或间接抑制人中性粒细胞活化和释放各种氧化物质；所述应用为作为抑制人中性粒细胞活化和释放各种氧化物质的抑制剂的应用或在制备治疗中性粒细胞活化和释放各种氧化物质相关炎症性疾病药物中应用；所述应用对人和其它动物效果不一样或有效的程度不一样。In another aspect, the present invention also provides an application of a low-molecular-weight hyaluronic acid fragment with an average molecular weight of 35kDa. The low-molecular-weight hyaluronic acid fragment directly and/or indirectly inhibits CD44 on the surface of red blood cells and/or white blood cells or the surface of white blood cells. Human neutrophils activate and release various oxidizing substances; the application is as an inhibitor for inhibiting human neutrophil activation and releasing various oxidizing substances or in preparing treatments for neutrophil activation and releasing various oxidative substances. Application in substance-related inflammatory disease drugs; the application has a different effect or degree of effectiveness on humans and other animals.
综上所述，本发明发现透明质酸片段在低分子量范围内诱发人和动物末梢血和静脉血红细胞钱串状聚集，其诱发红细胞钱串状聚集的最小浓度与透明质酸片段的分子量大小负相关关系。本发明利用这个负相关关系测定低分子量透明质酸片段的分子量和测定低分子量透明质酸片段产品批间生产的分子量变异范围。具体可以使用猫、犬和大鼠静脉血和其诱发红细胞钱串状聚集来测定低分子量透明质酸片段产品的分子量和分子量变异情况。本发明也发现透明质酸片段在低分子量范围内促进血红细胞沉降速度。本发明首次利用这个透明质酸片段对血红细胞沉降速度的作用测定低分子量透明质酸片段产品批间生产的分子量变异程度，形成了透明质酸片段产品批间生产分子量变异质量检测方法。本发明进一步发现平均分子量35±8kDa的低分子量透明质酸片段B-HA诱发人红细胞钱串状聚集是通过结合人红细胞表面CD44，其本质反映了这个低分子量透明质酸片段结合红细胞表面CD44的活性，利用发现测定平均分子量35±8kDa的低分子量透明质酸片段的生物活性。本发明还发现人中性粒细胞吞噬平均分子量35±8kDa的透明质酸片段B-HA，但不影响人中性粒细胞吞噬外源荧光颗粒的功能。本发明进一步发现低分子量的透明质酸片段与人血红细胞表面结合和影响人血细胞血液动力学行为或血细胞和白细胞互相间作用。平均分子量35±8kDa的透明质酸片段B-HA对人中性粒细胞活化有抑制作用，结合其组织渗透性好的特点，提示其对人体组织炎症有抑制作用。本发明还进一步发现低分子量透明质酸片段诱发红细胞钱串状聚集和促进血红细胞沉降的这种影响血管内血红细胞血液动力学和红细胞与白细胞互相间反应的作用有种属特异性，提示低分子量透明质酸片段的生理功能、治疗作用和副作用有种属特异性。In summary, the present invention found that hyaluronic acid fragments induce money-like aggregation of red blood cells in human and animal peripheral blood and venous blood in the low molecular weight range. Negative correlation. The present invention uses this negative correlation to determine the molecular weight of low-molecular-weight hyaluronic acid fragments and to determine the range of molecular weight variation produced between batches of low-molecular-weight hyaluronic acid fragments. Specifically, the venous blood of cats, dogs and rats and its induced red blood cell clustering can be used to determine the molecular weight and molecular weight variation of low molecular weight hyaluronic acid fragment products. The present invention also found that hyaluronic acid fragments promote the sedimentation rate of red blood cells in the low molecular weight range. The present invention uses the effect of this hyaluronic acid fragment on the sedimentation rate of red blood cells to determine the degree of molecular weight variation between batches of low-molecular-weight hyaluronic acid fragment products, forming a quality detection method for the molecular weight variation of hyaluronic acid fragment products between batches. The present invention further discovered that the low-molecular-weight hyaluronic acid fragment B-HA with an average molecular weight of 35±8kDa induces the cluster-like aggregation of human red blood cells by binding to CD44 on the surface of human red blood cells, and its essence reflects that this low-molecular-weight hyaluronic acid fragment binds to CD44 on the surface of red blood cells. Activity, using the discovery to determine the biological activity of low molecular weight hyaluronic acid fragments with an average molecular weight of 35±8kDa. The present invention also found that human neutrophils phagocytize the hyaluronic acid fragment B-HA with an average molecular weight of 35±8kDa, but it does not affect the function of human neutrophils to phagocytize exogenous fluorescent particles. The present invention further discovered that low molecular weight hyaluronic acid fragments bind to the surface of human red blood cells and affect the hemodynamic behavior of human blood cells or the interaction between blood cells and white blood cells. The hyaluronic acid fragment B-HA with an average molecular weight of 35±8kDa has an inhibitory effect on the activation of human neutrophils, combined with its good tissue permeability, suggesting that it has an inhibitory effect on human tissue inflammation. The present invention further discovered that the low-molecular-weight hyaluronic acid fragment induces red blood cell clusters and promotes red blood cell sedimentation, which affects intravascular red blood cell hemodynamics and the interaction between red blood cells and white blood cells, which is species-specific, suggesting low The physiological functions, therapeutic effects and side effects of molecular weight hyaluronic acid fragments are species-specific.
附图说明Description of the drawings
上述仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，以下结合附图与具体实施方式对本发明作进一步的详细说明。The foregoing is only an overview of the technical solutions of the present invention. In order to better understand the technical means of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.
图1为重组人透明质酸酶PH20充分酶解大分子透明质酸原料制造的平均分子量35±8kDa透明质酸片段B-HA抑制人皮肤红肿硬痛图解；Figure 1 is a diagram showing how recombinant human hyaluronidase PH20 fully enzymatically hydrolyzes macromolecular hyaluronic acid raw materials with an average molecular weight of 35±8kDa hyaluronic acid fragment B-HA to inhibit human skin redness, swelling and hard pain;
图2为不同分子量的透明质酸片段诱发人末梢血红细胞钱串状聚集的最小百分比浓度和分子量大小的相关曲线；Figure 2 is the correlation curve of the minimum percentage concentration and molecular weight of the hyaluronic acid fragments of different molecular weights inducing the cluster-like aggregation of human peripheral red blood cells;
图3中，(a)是终浓度0.15％B-HA(或HA fragments 35kDa或HA35)诱发人末梢血红细胞钱串状聚集图；(b)是10ug/ml的抗人CD44抗体抑制B-HA诱发人末梢血红细胞钱串状聚集图；(c)是非特异性兔IgG抗体没有抑制B-HA诱发人末梢血红细胞钱串状聚集图；(d)是终浓度1927U/ml加入的重组人透明质酸酶PH20抑制B-HA诱发人末梢血红细胞钱串状聚集图；In Figure 3, (a) is a graph showing that the final concentration of 0.15% B-HA (or HA fragments 35kDa or HA35) induces human peripheral red blood cell clusters; (b) is 10ug/ml anti-human CD44 antibody inhibiting B-HA Induced human peripheral red blood cell money clusters; (c) non-specific rabbit IgG antibody did not inhibit B-HA induced human peripheral red blood cell money clusters; (d) is a recombinant human transparent added at a final concentration of 1927U/ml Plasmidase PH20 inhibits B-HA-induced human peripheral red blood cell money clusters;
图4为不同分子量的透明质酸片段诱发猫静脉血红细胞钱串状聚集的最小百分比浓度和分子量大小的相关曲线；Figure 4 is the correlation curve of the minimum percentage concentration and molecular weight of cat venous red blood cell clusters induced by hyaluronic acid fragments of different molecular weights;
图5为不同分子量的透明质酸片段诱发犬静脉血红细胞钱串状聚集的最小百分比浓度和分子量大小的相关曲线；Figure 5 is a correlation curve of the minimum percentage concentration and molecular weight of the canine venous red blood cell clusters induced by hyaluronic acid fragments of different molecular weights;
图6为不同分子量的透明质酸片段诱发大鼠静脉血红细胞钱串状聚集的最小百分比浓度和分子量大小的相关曲线；Figure 6 is the correlation curve of the minimum percentage concentration and molecular weight of rat venous red blood cell clusters induced by hyaluronic acid fragments of different molecular weights;
图7为B-HA组和荧光标记B-HA(Cy5.5 B-HA)50个多核中性粒细胞含有荧光颗粒和团块的单细胞照片和百分率图；Figure 7 is a photo and percentage diagram of 50 multinucleated neutrophils containing fluorescent particles and clumps in the B-HA group and fluorescently labeled B-HA (Cy5.5 B-HA);
图8为B-HA和HA对人中性粒细胞吞噬荧光颗粒的效果图；Figure 8 is a diagram showing the effect of B-HA and HA on human neutrophils phagocytosing fluorescent particles;
图9为不同批次的B-HA(又称HA35或HA fragments 35kDa)产品的凝胶电泳分子量批间测定结果；Figure 9 shows the gel electrophoresis molecular weight determination results of different batches of B-HA (also known as HA35 or HA fragments 35kDa) products between batches;
图10为HA fragments 24kDa(FRD)的凝胶电泳分子量批内变异测定结果图；Figure 10 is a graph showing the results of intra-assay variation in molecular weight of HA fragments 24kDa (FRD) by gel electrophoresis;
图11为B-HA(又称HA35或HA fragments 35kDa)和HA原料1600kDa(FRD)(图中称HA)对PMA诱导的新鲜人白细胞活化(neutrophil activation)的作用。Figure 11 shows the effect of B-HA (also known as HA35 or HA fragments 35kDa) and HA raw material 1600kDa (FRD) (referred to in the figure as HA) on PMA-induced neutrophil activation of fresh human leukocytes.
具体实施方式Detailed ways
以下通过具体的实施例对本发明进行说明，此处所描述的实施例仅用于说明和解释本发明，并不用于限定本发明。The following describes the present invention through specific embodiments, and the embodiments described here are only used to illustrate and explain the present invention, and are not used to limit the present invention.
实施例1Example 1
目的：本发明使用的不同分子量的透明质酸片段和原料透明质酸的制备和分子量测定。Purpose: Preparation and molecular weight determination of hyaluronic acid fragments with different molecular weights and raw hyaluronic acid used in the present invention.
方法：method:
生物酶法制造低分子量透明质酸片段：采用预实验确定生物酶PH20足量或轻微过量充分酶解和非足量部分酶解透明质酸原料制造低分子量透明质酸片段。透明质酸酶足量或轻度过量充分酶解的定义为：(1)10-20分钟足量或轻度过量充分酶解中和高分子透明质酸原料制造的透明质酸片段的分子量和1-6小时足量或轻度过量充分酶解中和高分子透明质酸原料制造的透明质酸片段的分子量基本一致(变异系数CV<15％)；(2)>99％高或中分子透明质酸原料被足量或轻度过量充分酶解的低分子透明质酸片段产物被0.22um滤膜全部顺利滤过；(3)透明质酸酶活性经足量或轻度过量充分酶解反应后基本没有残留或少量残留(<15％)，80度45分钟全部灭活。透明质酸酶解制造的时间点包括：10分钟、20分钟、40分钟、1小时、2小时、3小时、4小时、5小时、6小时。Biological enzymatic production of low-molecular-weight hyaluronic acid fragments: Preliminary experiments are used to determine that the biological enzyme PH20 is sufficient or slightly excessive to fully enzymatically hydrolyze and partially enzymatically hydrolyze hyaluronic acid raw materials to produce low-molecular-weight hyaluronic acid fragments. Hyaluronidase is defined as: (1) In 10-20 minutes, the molecular weight and the molecular weight of the hyaluronic acid fragment produced by the sufficient or light excessive enzymatic hydrolysis and the high-molecular hyaluronic acid raw material In 1-6 hours, the molecular weight of hyaluronic acid fragments produced by full enzymatic hydrolysis and neutralization of high-molecular hyaluronic acid raw materials is basically the same (coefficient of variation CV<15%); (2)>99% high or medium molecular weight The low-molecular-weight hyaluronic acid fragment products that are fully enzymatically hydrolyzed by the hyaluronic acid raw material in sufficient or light excess are all smoothly filtered by 0.22um filter membrane; (3) Hyaluronidase activity is fully enzymatically hydrolyzed in sufficient or light excess After the reaction, there is almost no residue or a small amount of residue (<15%), and all are inactivated at 80 degrees and 45 minutes. The time points of hyaluronic acid enzymatic production include: 10 minutes, 20 minutes, 40 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, and 6 hours.
理化生产方法制造透明质酸片段结合了酸、碱、高温制造方法。The physicochemical production method for the production of hyaluronic acid fragments combines acid, alkali, and high-temperature production methods.
分子量测定：透明质酸片段采用凝胶电泳和18角度激光测定。原料透明质酸采用粘度计和18角度激光测定。Molecular weight determination: hyaluronic acid fragments were determined by gel electrophoresis and 18-angle laser. The raw material hyaluronic acid is measured with a viscometer and an 18-angle laser.
结果：result:
表1.本实验使用透明质酸片段和原料透明质酸平均分子量和分子量分布。Table 1. This experiment uses hyaluronic acid fragments and raw hyaluronic acid average molecular weight and molecular weight distribution.
Figure PCTCN2021080543-appb-000001
Figure PCTCN2021080543-appb-000001
Figure PCTCN2021080543-appb-000002
Figure PCTCN2021080543-appb-000002
注1：HA fragments 35kDa或HA35或B-HA或B-HA(HH)是6个不同批次的B-HA等量混合物，18角度激光(GPC/SEC system equipped with an multi angle laser light scattering(MALLS)detector)测定结果为35±8kDa(表16)。Note 1: HA fragments 35kDa or HA35 or B-HA or B-HA (HH) are 6 different batches of B-HA equivalent mixture, 18-angle laser (GPC/SEC system equipped with an multi-angle laser light scattering( The result of the MALLS) detector was 35±8kDa (Table 16).
注2：理化结合生物酶制造HA fragments(24kDa)原本是FRD公司的Oligo HA，经本发明反复测定，其分子量和>90％分子量分布分别为24kDa和10-40kDa。Note 2: HA fragments (24kDa) manufactured by physicochemical combination of biological enzymes were originally Oligo HA from FRD. After repeated measurements of the present invention, its molecular weight and >90% molecular weight distribution are 24kDa and 10-40kDa, respectively.
结论：表1中本实验使用的透明质酸片段和透明质酸原料的平均分子量和分子量分布是经2-3种方法多次评估的可靠结果。Conclusion: The average molecular weight and molecular weight distribution of the hyaluronic acid fragments and hyaluronic acid raw materials used in this experiment in Table 1 are the reliable results of multiple evaluations by 2-3 methods.
实施例2Example 2
目的：研究不同分子量的透明质酸片段和透明质酸原料对人新鲜末梢血和静脉血的作用。Objective: To study the effects of hyaluronic acid fragments and raw materials of different molecular weights on fresh human peripheral blood and venous blood.
方法：method:
人末梢血和静脉血采集：健康志愿者共有8人，年龄22±5岁，男女各4名，末梢血和静脉血采集经长春嘉和外科医院的医学伦理委员会批准和本人同意。动物末梢血和静脉血采集均经青岛农业大学动物医院许可。Human peripheral blood and venous blood collection: There are 8 healthy volunteers, aged 22±5 years old, 4 males and females. The collection of peripheral blood and venous blood was approved by the Medical Ethics Committee of Changchun Jiahe Surgical Hospital and I agree. The collection of animal peripheral blood and venous blood was approved by the Animal Hospital of Qingdao Agricultural University.
使用不同分子量的透明质酸片段或透明质酸原料(表1)与人末梢血或静脉血按照1:2的比例混合，加上抗凝剂EDTA和PBS缓冲液至终浓度分别 为1.2％、0.6％、0.3％、0.15％、0.075％、0.0375％，既最终达到人末梢血和静脉血3.5倍稀释。然后，使用显微镜观察不同分子量的透明质酸片段和透明质酸原料(表1)引发的红细胞钱串状聚集情况，得出其诱发人末梢血红细胞钱串状聚集的最小百分比浓度。Use different molecular weight hyaluronic acid fragments or hyaluronic acid raw materials (Table 1) and human peripheral blood or venous blood in a ratio of 1:2, plus anticoagulant EDTA and PBS buffer to the final concentration of 1.2%, respectively, 0.6%, 0.3%, 0.15%, 0.075%, 0.0375%, and finally reached 3.5 times dilution of human peripheral blood and venous blood. Then, use a microscope to observe the red blood cell money-like aggregation caused by hyaluronic acid fragments of different molecular weights and hyaluronic acid raw materials (Table 1), and obtain the minimum percentage concentration that induces the money-like aggregation of human peripheral red blood cells.
结果：result:
表2.不同分子量的透明质酸片段和透明质酸原料诱发人末梢血红细胞钱串状聚集的最小百分比浓度。注：使用静脉血红细胞结果相同。Table 2. The minimum percentage concentration of hyaluronic acid fragments of different molecular weights and hyaluronic acid raw materials to induce money clusters of human peripheral red blood cells. Note: The results are the same when using venous red blood cells.
Figure PCTCN2021080543-appb-000003
Figure PCTCN2021080543-appb-000003
注：黑体字和斜体字共同代表引起血红细胞钱串样聚集形成的最低分子量透明质酸片段的浓度；*代表严重细胞变形。Yes/Yes/Yes或No/No/No代表三次使用不同人末梢血标本的实验结果。Note: Bold type and italic type together represent the concentration of the lowest molecular weight hyaluronic acid fragment that causes the aggregation of red blood cell money strings; * represents severe cell deformation. Yes/Yes/Yes or No/No/No represents the results of three experiments using different human peripheral blood samples.
图2表示不同分子量的透明质酸片段诱发人末梢血红细胞钱串状聚集的最小百分比浓度和分子量大小的相关曲线。Fig. 2 shows the correlation curve of the minimum percentage concentration and molecular weight of human peripheral red blood cell clusters induced by hyaluronic acid fragments of different molecular weights.
讨论：discuss:
诱发红细胞钱串状聚集的B-HA(HA35)终浓度为0.15％,既1.5mg/ml。本发明使用刺激新鲜提取人中性粒细胞功能的B-HA浓度为10ug/ml(实施例4)。一个平均体重70kg正常人体内含有约15克透明质酸，其中每天1/3被降解(Stern R，Hyaluronan catabolism:a new metabolic pathway，2004，Eur.J.Cell Biol.83(7):317–25.doi:10.1078/0171-9335-00392)。文献报道正常人血清透明质酸含量为28.5ng/ml，既0.028ug/ml(Mi-Soon Han,Yongjung Park,Hyon-suk Kim,Evaluation of automated assays for measuring serum hyaluronic acid:for the diagnosis of rheumatoid arthritis,Lab Med Online,2014,4(2):98-104.)，比10ug/ml低375倍。正常人血清中28.5ng/ml的透明质酸对我们使用的10ug/ml血浓度的B-HA影响不大。因此，10ug/ml血浓度的B-HA在血清中结合红细胞表面有影响人血细胞血液动力学行为或血细胞和白细胞互相间作用的可能性。The final concentration of B-HA (HA35) that induces red blood cell clustering is 0.15%, which is 1.5mg/ml. The present invention uses B-HA to stimulate the function of freshly extracted human neutrophils at a concentration of 10ug/ml (Example 4). A normal person with an average weight of 70 kg contains about 15 grams of hyaluronic acid, of which 1/3 is degraded every day (Stern R, Hyaluronan catabolism: a new metabolic pathway, 2004, Eur. J. Cell Biol. 83(7): 317– 25.doi:10.1078/0171-9335-00392). The literature reports that the serum hyaluronic acid content of normal people is 28.5ng/ml, which is 0.028ug/ml (Mi-Soon Han, Yongjung Park, Hyon-suk Kim, Evaluation of automated assays for measuring serum hyaluronic acid: for the diagnosis of rheumatoid arthritis ,Lab Med Online,2014,4(2):98-104.), which is 375 times lower than 10ug/ml. The 28.5ng/ml hyaluronic acid in normal human serum has little effect on the blood concentration of 10ug/ml B-HA we use. Therefore, B-HA with a blood concentration of 10ug/ml binds to the surface of red blood cells in the serum, which may affect the hemodynamic behavior of human blood cells or the interaction between blood cells and white blood cells.
结论：本发明发现低分子量的透明质酸片段诱发人末梢血和静脉血红细胞钱串状聚集(图3)，其诱发人红细胞钱串状聚集的最小浓度与透明质酸片段的分子量负相关，可以用来测定低分子量透明质酸片段的分子量和监测低分子量透明质酸片段产品分子量变异情况。这个结果还表明低分子量的透明质酸片段与人血红细胞表面结合和影响人血细胞血液动力学行为或血细胞和白细胞互相间作用。Conclusion: The present invention found that low-molecular-weight hyaluronic acid fragments induce money-string aggregation of human peripheral blood and venous red blood cells (Figure 3). The minimum concentration that induces money-string aggregation of human red blood cells is negatively correlated with the molecular weight of hyaluronic acid fragments. It can be used to determine the molecular weight of low-molecular-weight hyaluronic acid fragments and monitor the molecular weight variation of low-molecular-weight hyaluronic acid fragments. This result also indicates that low molecular weight hyaluronic acid fragments bind to the surface of human red blood cells and affect the hemodynamic behavior of human blood cells or the interaction between blood cells and white blood cells.
实施例3Example 3
目的：研究低分子量透明质酸片段HA fragments35kDa或HA35或B-HA诱发人末梢血和静脉血红细胞钱串状聚集的分子机制。Objective: To study the molecular mechanism of low molecular weight hyaluronic acid fragments HA fragments 35kDa or HA35 or B-HA inducing the clustering of human peripheral blood and venous red blood cells.
方法：method:
人末梢血和静脉血采集：健康志愿者共有8人，年龄22±5岁，男女各4名，末梢血和静脉血采集经长春嘉和外科医院的医学伦理委员会批准和本人同意。动物末梢血和静脉血采集均经青岛农业大学动物医院许可。Human peripheral blood and venous blood collection: There are 8 healthy volunteers, aged 22±5 years old, 4 males and females. The collection of peripheral blood and venous blood was approved by the Medical Ethics Committee of Changchun Jiahe Surgical Hospital and I agree. The collection of animal peripheral blood and venous blood was approved by the Animal Hospital of Qingdao Agricultural University.
使用表1中的低分子量透明质酸片段B-HA(又称HA fragments35kDa或HA35)与人末梢血或静脉血按照1:2的比例混合至终浓度为0.15％，最终达到人末梢血或静脉血3.5倍稀释。然后，使用显微镜观察和验证低分子量透明质酸片段B-HA(又称HA fragments35kDa或HA35)引发的红细胞钱串状聚集。Use the low-molecular-weight hyaluronic acid fragment B-HA (also known as HA fragments35kDa or HA35) in Table 1 to mix with human peripheral blood or venous blood in a ratio of 1:2 to a final concentration of 0.15%, and finally reach human peripheral blood or venous blood The blood was diluted 3.5 times. Then, a microscope was used to observe and verify the red blood cell clusters caused by the low molecular weight hyaluronic acid fragment B-HA (also known as HA fragments35kDa or HA35).
使用小体积的抗人CD44抗体(0.5mg/ml,ab157107,Abcam)和对照非特异性兔IgG抗体(0.5mg/ml,ab171870，Abcam)或重组人透明质酸酶PH20(27000U/ml，HH Technology)与人末梢血或静脉血混合并37℃孵育25分钟，再加低分子量透明质酸片段B-HA，最终达到诱发人末梢血红细胞钱串状聚集的最小百分比浓度和人末梢血或静脉血稀释3.5倍。然后，使用显微镜观察相关红细胞钱串状聚集程度。Use a small volume of anti-human CD44 antibody (0.5mg/ml, ab157107, Abcam) and control non-specific rabbit IgG antibody (0.5mg/ml, ab171870, Abcam) or recombinant human hyaluronidase PH20 (27000U/ml, HH Technology) ) Mix with human peripheral blood or venous blood and incubate at 37°C for 25 minutes, then add low-molecular-weight hyaluronic acid fragment B-HA, and finally achieve the minimum percentage concentration that induces the cluster-like aggregation of human peripheral red blood cells and human peripheral blood or venous blood Dilute 3.5 times. Then, use a microscope to observe the degree of cluster-like clusters of related red blood cells.
结果：result:
图3中，(a)是终浓度0.15％B-HA(或HA fragments 35kDa或HA35)诱发人末梢血红细胞钱串状聚集图；(b)是10ug/ml的抗人CD44抗体抑制B-HA诱发人末梢血红细胞钱串状聚集图；(c)是非特异性兔IgG抗体没有抑制B-HA诱发人末梢血红细胞钱串状聚集图；(d)是终浓度1927U/ml加入的重组人透明质酸酶PH20抑制B-HA诱发人末梢血红细胞钱串状聚集图；注：使用静脉血红细胞结果相同。In Figure 3, (a) is a graph showing that the final concentration of 0.15% B-HA (or HA fragments 35kDa or HA35) induces human peripheral red blood cell clusters; (b) is 10ug/ml anti-human CD44 antibody inhibiting B-HA Induced human peripheral red blood cell money clusters; (c) non-specific rabbit IgG antibody did not inhibit B-HA induced human peripheral red blood cell money clusters; (d) is the final concentration of 1927U/ml recombinant human transparent Plasmidase PH20 inhibits B-HA-induced clusters of human peripheral red blood cells; Note: The results of using venous red blood cells are the same.
讨论：discuss:
低分子透明质酸片段B-HA(HA35)诱发红细胞钱串状聚集。终浓度1927U/ml加入的PH20酶解阻断B-HA诱发红细胞钱串状聚集。这个结果表明B-HA(HA35)诱发的红细胞钱串状聚集是通过B-HA分子实现。另有实验结果表明终浓度1927U/ml加入的PH20在完全酶解B-HA(HA35)。The low-molecular-weight hyaluronic acid fragment B-HA (HA35) induces red blood cell clusters. The PH20 enzymatic hydrolysis added at a final concentration of 1927U/ml blocked the B-HA-induced red blood cell clustering. This result indicates that B-HA (HA35)-induced red blood cell clustering is achieved through B-HA molecules. Another experimental result showed that PH20 added at a final concentration of 1927U/ml was completely enzymatically hydrolyzing B-HA (HA35).
10ug/ml的抗人CD44抗体阻断B-HA(HA35)诱发红细胞钱串状聚集。这个结果表明B-HA诱发红细胞钱串状聚集是通过结合红细胞表面CD44。这个实验也是透明质酸片段和透明质酸结合CD44活性检测实验。10ug/ml anti-human CD44 antibody blocked B-HA (HA35) induced red blood cell clusters. This result indicates that B-HA induces red blood cell clusters by binding to CD44 on the surface of red blood cells. This experiment is also an experiment to detect the activity of hyaluronic acid fragments and hyaluronic acid combined with CD44.
结论：本发明表明透明质酸片段B-HA(HA35)通过与红细胞表面CD44结合诱发红细胞钱串状聚集。Conclusion: The present invention shows that the hyaluronic acid fragment B-HA (HA35) induces red blood cell clusters by binding to CD44 on the surface of red blood cells.
实施例4Example 4
目的:研究不同分子量的透明质酸片段和透明质酸原料诱发猫、犬和大鼠静脉血红细胞钱串状聚集的作用。Objective: To study the effect of different molecular weight hyaluronic acid fragments and hyaluronic acid raw materials in inducing the cluster-like aggregation of venous red blood cells in cats, dogs and rats.
方法：method:
大鼠、犬和猫静脉血采集均经青岛农业大学动物医院许可。使用不同分子量的透明质酸片段和透明质酸原料(表1)与大鼠、犬或猫静脉血按照1:2的比例，加上抗凝剂EDTA和PBS缓冲液至终浓度分别为1.2％、0.6％、0.3％、0.15％、0.075％、0.0375％，最终达到大鼠、犬和猫静脉血3.5倍稀释。然后，使用显微镜观察不同分子量的透明质酸片段和透明质酸原料(表1)引发的红细胞钱串状聚集情况，得出其诱发大鼠、犬和猫静脉血红细胞钱串状聚集的最小百分比浓度。The venous blood collection of rats, dogs and cats was approved by the Animal Hospital of Qingdao Agricultural University. Use different molecular weight hyaluronic acid fragments and hyaluronic acid raw materials (Table 1) and rat, dog or cat venous blood in a ratio of 1:2, plus anticoagulant EDTA and PBS buffer to the final concentration of 1.2%, respectively , 0.6%, 0.3%, 0.15%, 0.075%, 0.0375%, and finally reach 3.5 times dilution of rat, dog and cat venous blood. Then, use a microscope to observe the red blood cell money-like aggregation caused by hyaluronic acid fragments of different molecular weights and hyaluronic acid raw materials (Table 1), and obtain the minimum percentage of red blood cell money-like aggregation induced by rat, dog and cat venous red blood cells concentration.
结果：result:
表3.不同分子量的透明质酸片段和透明质酸原料诱发猫静脉血红细胞钱串状聚集的最小百分比浓度。Table 3. Hyaluronic acid fragments of different molecular weights and hyaluronic acid raw materials induce the minimum percentage concentration of cat venous red blood cell clusters.
Figure PCTCN2021080543-appb-000004
Figure PCTCN2021080543-appb-000004
注：黑体字和斜体字共同代表引起血红细胞钱串样聚集形成的最低低分子量透明质酸片段的浓度；*代表严重细胞变形；#代表大块血红细胞钱串样聚集形成。Note: Bold and italic together represent the concentration of the lowest low-molecular-weight hyaluronic acid fragments that cause the aggregation of red blood cell clusters; * represents severe cell deformation; # represents the formation of large red blood cell clusters.
图4是不同分子量的透明质酸片段诱发猫静脉血红细胞钱串状聚集的最小百分比浓度和分子量大小的相关曲线。Figure 4 is the correlation curve of the minimum percentage concentration and molecular weight of cat venous red blood cell clusters induced by hyaluronic acid fragments of different molecular weights.
表4.不同分子量的透明质酸片段和透明质酸原料诱发犬静脉血红细胞钱串状聚集的最小百分比浓度。Table 4. Hyaluronic acid fragments of different molecular weights and hyaluronic acid raw materials induce the smallest percentage concentration of canine venous red blood cell money string aggregation.
Figure PCTCN2021080543-appb-000005
Figure PCTCN2021080543-appb-000005
注：黑体字和斜体字共同代表引起血红细胞钱串样聚集形成的最低低分子量透明质酸片段的浓度；*代表严重细胞变形；#代表大块血红细胞钱串样聚集形成。Note: Bold and italic together represent the concentration of the lowest low-molecular-weight hyaluronic acid fragments that cause the aggregation of red blood cell clusters; * represents severe cell deformation; # represents the formation of large red blood cell clusters.
图5为不同分子量的透明质酸片段诱发犬静脉血红细胞钱串状聚集的最小百分比浓度和分子量大小的相关曲线。Fig. 5 is the correlation curve of the minimum percentage concentration and molecular weight of the canine venous red blood cell clusters induced by hyaluronic acid fragments of different molecular weights.
表5.不同分子量的透明质酸片段和透明质酸原料诱发大鼠静脉血红细胞钱串状聚集的最小百分比浓度。Table 5. Hyaluronic acid fragments of different molecular weights and hyaluronic acid raw materials induce the smallest percentage concentration of rat venous red blood cell clusters.
Figure PCTCN2021080543-appb-000006
Figure PCTCN2021080543-appb-000006
Figure PCTCN2021080543-appb-000007
Figure PCTCN2021080543-appb-000007
注：黑体字和斜体字共同代表引起血红细胞钱串样聚集形成的最低低分子量透明质酸片段的浓度；*代表严重细胞变形。Note: Bold type and italic type together represent the concentration of the lowest low-molecular-weight hyaluronic acid fragment that causes the aggregation of red blood cell money strings; * represents severe cell deformation.
图6为不同分子量的透明质酸片段诱发大鼠静脉血红细胞钱串状聚集的最小百分比浓度和分子量大小的相关曲线。Fig. 6 is a correlation curve of the minimum percentage concentration and molecular weight of rat venous red blood cell clusters induced by hyaluronic acid fragments of different molecular weights.
结论：本发明发现不同分子量的透明质酸片段和透明质酸原料诱发猫、犬和大鼠静脉血红细胞钱串状聚集。这个结果和低分子量的透明质酸片段诱发人末梢血和静脉血红细胞钱串状聚集的结果基本相同。本发明表明可以使用猫、犬和大鼠静脉血和其诱发红细胞钱串状聚集来测定低分子量透明质酸片段产品的分子量和分子量变异情况。Conclusion: The present invention found that hyaluronic acid fragments of different molecular weights and hyaluronic acid raw materials induce money-string-like aggregation of venous red blood cells in cats, dogs and rats. This result is basically the same as the result of low-molecular-weight hyaluronic acid fragments inducing money clusters of human peripheral blood and venous red blood cells. The present invention shows that the venous blood of cats, dogs and rats and its induced red blood cell cluster-like aggregation can be used to determine the molecular weight and molecular weight variation of low molecular weight hyaluronic acid fragment products.
实施例5Example 5
目的：研究B-HA(HA fragments 35kDa或HA35)与新鲜人血中性粒细胞的互相间作用。Purpose: To study the interaction between B-HA (HA fragments 35kDa or HA35) and fresh human blood neutrophils.
方法：采用新鲜采集的人指尖血或静脉血与抗凝混匀，去除上层血清，用红细胞裂解液对红细胞进行裂解2次，PBS清洗一次，最后用PBS重悬，加入B-HA组和荧光标记B-HA(Cy5.5 B-HA)15分钟内随机使用激光共聚焦显微镜观察50个多核中性粒细胞。Method: Mix freshly collected human fingertip blood or venous blood with anticoagulation, remove the upper layer of serum, lyse the red blood cells twice with red blood cell lysis solution, wash once with PBS, and finally resuspend in PBS, add B-HA group and Fluorescence-labeled B-HA (Cy5.5 B-HA) was randomly used to observe 50 multinucleated neutrophils using a laser confocal microscope within 15 minutes.
结果：本发明结果表明B-HA组50个中性粒细胞里边没有一个细胞内有荧光颗粒和团块。荧光标记B-HA(Cy5.5 B-HA)组50个中性粒细胞里边有36个细胞有强荧光颗粒和团块。Results: The results of the present invention showed that none of the 50 neutrophils in the B-HA group contained fluorescent particles or clumps. Among the 50 neutrophils in the fluorescent-labeled B-HA (Cy5.5 B-HA) group, 36 cells have strong fluorescent particles and clumps.
图7为B-HA组和荧光标记B-HA(Cy5.5 B-HA)50个多核中性粒细胞含有荧光颗粒和团块的单细胞照片和百分率。本发明使用荧光标记B-HA(Cy5.5 B-HA)与人红细胞培养没有发现红细胞表面荧光标记现象。这个结果表明荧光标记B-HA(Cy5.5 B-HA)的发光强度不足以探测其红细胞表面的结合。本发明使用荧光标记B-HA(Cy5.5 B-HA)与提取人中性粒细胞或全血细胞培养。研究结果表明人中性粒细胞胞内颗粒有荧光标记现象(图 7)。这个结果表明中性粒细胞吞噬和胞内浓缩了荧光标记B-HA(Cy5.5 B-HA)或人中性粒细胞快速结合并摄入荧光标记B-HA(Cy5.5 B-HA)。Figure 7 is a photograph and percentage of single cells containing fluorescent particles and clumps of 50 multinucleated neutrophils in the B-HA group and fluorescently labeled B-HA (Cy5.5 B-HA). The present invention uses fluorescently labeled B-HA (Cy5.5 B-HA) to culture human red blood cells, and there is no phenomenon of fluorescent labeling on the surface of red blood cells. This result indicates that the luminescence intensity of fluorescently labeled B-HA (Cy5.5 B-HA) is not enough to detect the binding on the surface of red blood cells. The present invention uses fluorescently labeled B-HA (Cy5.5 B-HA) to extract human neutrophils or whole blood cells for culture. The results of the study indicate that the intracellular granules of human neutrophils are fluorescently labeled (Figure 7). This result indicates that neutrophils phagocytosed and concentrated intracellularly with fluorescent-labeled B-HA (Cy5.5 B-HA) or human neutrophils quickly combined and ingested fluorescent-labeled B-HA (Cy5.5 B-HA) .
结论：1.本研究表明低分子量透明质酸片段B-HA(HA fragments 35kDa或HA35)快速结合人中性粒细胞和进入人中性粒细胞；2.本研究表明人中性粒细胞吞噬低分子量透明质酸片段B-HA(HA fragments 35kDa或HA35)。Conclusion: 1. This study shows that the low molecular weight hyaluronic acid fragment B-HA (HA fragments 35kDa or HA35) quickly binds to human neutrophils and enters human neutrophils; 2. This study shows that human neutrophils have low phagocytosis Molecular weight hyaluronic acid fragment B-HA (HA fragments 35kDa or HA35).
因此，本发明进一步探索了10ug/ml的B-HA(HA fragments 35kDa或HA35)对人中性粒细胞吞噬异物荧光颗粒的作用。Therefore, the present invention further explores the effect of 10ug/ml B-HA (HA fragments 35kDa or HA35) on the phagocytosis of foreign fluorescent particles by human neutrophils.
方法：method:
本实验还使用糖密度梯度离心法，即人静脉血白细胞分离试剂盒(内毒素<0.1EU)(天津灏洋华科生物科技有限公司)，分离人静脉血。常温下采集人静脉血1800rpm离心25分钟，吸取单个核细胞层(淋巴细胞和少部分单核细胞)和多核细胞层(中性粒细胞为主)混合，充分裂解红细胞，反复清洗两次，用3ml含10％FBS的1640培养基重悬备用。采用白细胞分类染色液染色观察细胞形态，调整密度至1×10 6个/ml，再备用。每次采集不同志愿者的血液排除个体差异和确保实验可重复。 This experiment also used the sugar density gradient centrifugation method, that is, human venous blood leukocyte separation kit (endotoxin <0.1EU) (Tianjin Haoyang Huake Biotechnology Co., Ltd.) to separate human venous blood. Collect human venous blood at room temperature by centrifugation at 1800 rpm for 25 minutes. Mix the mononuclear cell layer (lymphocytes and a small part of monocytes) and the multinucleated cell layer (mainly neutrophils), fully lyse the red blood cells, and repeatedly wash twice with 3ml 1640 medium containing 10% FBS was resuspended for later use. Use leukocyte classification staining solution to observe the cell morphology, adjust the density to 1×10 6 cells/ml, and then set aside. Collect blood from different volunteers each time to eliminate individual differences and ensure that the experiment is repeatable.
人中性粒细胞用含有10％FBS的RPMI-1640培养基，调整细胞密度至2×10 6个/ml。每孔加入200ul细胞，接种于24孔板中，加入10ug/ml B-HA(HA fragments 35kDa或HA35)或10ug/mlHA或1ng/mlLPS刺激中性粒细胞。直径2um乳胶珠，羧酸盐改性聚苯乙烯(L3030Sigma-Aldrich)每孔加入3.5ul，调节荧光颗粒密度为2×10 7个/ml，建立中性粒细胞与荧光颗粒的最佳吞噬模型，37℃吞噬培养1h后，流式细胞仪(FACSCalibur美国PE公司)488nm波长激光器，收集波长为575nm的红色荧光，得出中性粒细胞的吞噬率，从而研究中性粒细胞的吞噬能力。实验完成后，再新鲜提取另外一人静脉血白细胞重复实验，确保实验结果可重复。 Use RPMI-1640 medium containing 10% FBS for human neutrophils, and adjust the cell density to 2×10 6 cells/ml. Add 200ul cells to each well, inoculate it in a 24-well plate, add 10ug/ml B-HA (HA fragments 35kDa or HA35) or 10ug/mlHA or 1ng/mlLPS to stimulate neutrophils. 2um diameter latex beads, carboxylate modified polystyrene (L3030Sigma-Aldrich), add 3.5ul per well, adjust the density of fluorescent particles to 2×10 7 /ml, establish the best phagocytosis model of neutrophils and fluorescent particles After phagocytosis and culture at 37°C for 1 hour, a flow cytometer (FACSCalibur, USA PE company) 488nm wavelength laser collects red fluorescence with a wavelength of 575nm to obtain the phagocytic rate of neutrophils to study the phagocytic ability of neutrophils. After the experiment is completed, another person's venous white blood cells are freshly extracted to repeat the experiment to ensure that the experimental results are repeatable.
图8为B-HA和HA对人中性粒细胞吞噬荧光颗粒的效果。Figure 8 shows the effects of B-HA and HA on human neutrophils phagocytosing fluorescent particles.
10ug/ml的B-HA(HA fragments 35kDa或HA35)与Blank相比较，P>0.05，无显著性差异。10ug/ml的HA原料1600kDa(FRD)(这里简称HA)与Blank相比较，P>0.05，无显著性差异；LPS与Blank相比较，0.01<P<0.05，显著性差异，验证本实验方法可靠。10ug/ml B-HA (HA fragments 35kDa or HA35) compared with Blank, P>0.05, no significant difference. 10ug/ml HA raw material 1600kDa (FRD) (here referred to as HA) compared with Blank, P>0.05, no significant difference; compared with Blank, 0.01<P<0.05, significant difference, verifying the reliability of this experimental method .
结论：10ug/ml的B-HA(HA fragments 35kDa或HA35)对人中性粒细胞吞噬异物荧光颗粒的作用没有影响。本发明表明人中性粒细胞吞噬B-HA(HA fragments 35kDa或HA35)，提示表面结合B-HA的红细胞和中性粒细胞有互相间作用(本段参考文献)。Conclusion: 10ug/ml B-HA (HA fragments 35kDa or HA35) has no effect on the phagocytosis of foreign fluorescent particles by human neutrophils. The present invention shows that human neutrophils phagocytose B-HA (HA fragments 35kDa or HA35), suggesting that red blood cells and neutrophils that bind B-HA on the surface interact with each other (references in this paragraph).
本段参考文献：References for this paragraph:
1.Anel Lizcano,Ismael Secunodino,Simon Dohrmann,Ross Corriden,Cristina Rohena,Sandra Diaz,Pradipta Ghosh,Lingquan Deng,Victor Nizet and Ajit Varki(2017),Erythrocyte sialoglycoproteins engage Siglec-9 on neutrophils to supress activation,Blood,129(23):3100-3110.doi:10.1182/blood-2016-11-751636.1. Anel Lizcano, Ismael Seconodino, Simon Dohrmann, Ross Corriden, Cristina Rohena, Sandra Diaz, Pradipta Ghosh, Lingquan Deng, Victor Nizet and Ajit Varki (2017), Erythrocytes 129, activation, activation, activation, organic Sigle protein 129 (23):3100-3110.doi:10.1182/blood-2016-11-751636.
2.Ismael Secundino1,Anel Lizcano1,Markus Roupe,Xiaoxia Wang,Jason N.Cole,Joshua Olson,Raza Ali1,Samira Dahesh2 & Lenah K.Amayreh,& Anna Henningham1,Ajit Varki,Victor Nizet(2016),Host and pathogen hyaluronan signal through human Siglec-9 to suppress neutrophil activation,J Mol Med(2016)94:219–233.DOI 10.1007/s00109-015-1341-8.2.Ismael Secundino1, Anel Lizcano1, Markus Roupe, Xiaoxia Wang, Jason N. Cole, Joshua Olson, Raza Ali1, Samira Dahesh2 & Lenah K. Amayreh, & Anna Henningham1, Ajit (2016) Pathogen Hyt, Ajit (2016) signal through human Siglec-9 to suppress neutrophil activation, J Mol Med(2016)94:219–233.DOI 10.1007/s00109-015-1341-8.
实施例6Example 6
目的：研究不同分子量透明质酸片段和透明质酸原料诱发动物静脉血红细胞钱串状聚集的种属特异性。OBJECTIVE: To study the species specificity of different molecular weight hyaluronic acid fragments and hyaluronic acid raw materials inducing money-string aggregation of venous red blood cells in animals.
方法：采集猴、羊、猪、牛、马、水貂、羊驼等不同动物的静脉血采集均经青岛农业大学动物医院许可。使用不同分子量的透明质酸片段和透明质酸原料(表1)与猴、羊、猪、牛、马、水貂、羊驼等不同动物的静脉血按照1:2的比例混合，加上抗凝剂EDTA和PBS缓冲液至终浓度分别为1.2％、0.6％、0.3％、0.15％、0.075％、0.0375％，既最终达到猴、羊、猪、牛、马、水貂、羊驼等不同动物的静脉血3.5倍稀释。然后，使用显微镜观察不同分子量的透明质酸片段和透明质酸原料(表1)引发的红细胞钱串状聚集情况，得出其诱发猴、羊、猪、牛、马、水貂、羊驼等不同动物的静脉血红细胞钱串状聚集的最小百分比浓度。Methods: The venous blood collection of monkeys, sheep, pigs, cattle, horses, minks, alpacas and other animals was approved by the Animal Hospital of Qingdao Agricultural University. Use different molecular weight hyaluronic acid fragments and hyaluronic acid raw materials (Table 1) to mix with the venous blood of monkeys, sheep, pigs, cattle, horses, minks, alpacas and other animals in a ratio of 1:2, plus anticoagulation EDTA and PBS buffer solution to the final concentration of 1.2%, 0.6%, 0.3%, 0.15%, 0.075%, 0.0375%, and finally reached the monkey, sheep, pig, cow, horse, mink, alpaca and other animals The venous blood was diluted 3.5 times. Then, using a microscope to observe the clusters of red blood cells caused by hyaluronic acid fragments of different molecular weights and hyaluronic acid raw materials (Table 1), it is concluded that it induces different types of monkeys, sheep, pigs, cows, horses, minks, alpaca, etc. The minimum percentage concentration of venous red blood cell clusters in an animal.
结果：result:
表6.不同分子量透明质酸片段和透明质酸原料诱发猴静脉血红细胞钱串状聚集的结果。Table 6. The results of different molecular weight hyaluronic acid fragments and hyaluronic acid raw materials induced money clusters of monkey venous red blood cells.
Figure PCTCN2021080543-appb-000008
Figure PCTCN2021080543-appb-000008
Figure PCTCN2021080543-appb-000009
Figure PCTCN2021080543-appb-000009
注：黑体字和斜体字共同代表引起血红细胞聚集形成的最低透明质酸片段的浓度；*代表严重细胞变形；#代表大块血红细胞钱串样聚集形成。Note: Bold type and italic type together represent the concentration of the lowest hyaluronic acid fragment that causes red blood cell aggregation; * represents severe cell deformation; # represents the formation of large red blood cell clusters.
讨论：猴血液红细胞在正常状态下也会存在红细胞两两相连的现象。HA fragments 24kDa(FRD)在终浓度0.6％时没有红细胞聚集现象。HA fragments 35kDa或B-HA或HA35在终浓度0.3％、0.15％虽然诱发红细胞聚集，但是没有明显的钱串状聚集存在，其聚集程度也比较轻，与人HA fragments 35kDa或B-HA或HA35诱发红细胞钱串状聚集明显不同。Discussion: Under normal conditions, monkey blood red blood cells will also have red blood cells connected in pairs. HA Fragments 24kDa (FRD) has no red blood cell aggregation at the final concentration of 0.6%. HA fragments 35kDa or B-HA or HA35 at the final concentration of 0.3%, 0.15%, although red blood cell aggregation is induced, but there is no obvious cluster-like aggregation, and the degree of aggregation is relatively light, which is comparable to human HA fragments 35kDa or B-HA or HA35 The induction of red blood cell clusters is significantly different.
表7.不同分子量透明质酸片段和透明质酸原料诱发羊静脉血红细胞钱串状聚集的结果。Table 7. Results of different molecular weight hyaluronic acid fragments and hyaluronic acid raw materials inducing the cluster-like aggregation of red blood cells in sheep veins.
Figure PCTCN2021080543-appb-000010
Figure PCTCN2021080543-appb-000010
Figure PCTCN2021080543-appb-000011
Figure PCTCN2021080543-appb-000011
表8.不同分子量透明质酸片段和透明质酸原料诱发猪静脉血红细胞钱串状聚集的结果。Table 8. The results of different molecular weight hyaluronic acid fragments and hyaluronic acid raw materials induced money clusters of porcine venous red blood cells.
Figure PCTCN2021080543-appb-000012
Figure PCTCN2021080543-appb-000012
注：黑体字和斜体字共同代表引起血红细胞钱串状聚集形成的最低透明质酸片段的浓度；*代表严重细胞变形；#代表大块血红细胞钱串样聚集形成。Note: Bold type and italic type together represent the lowest concentration of hyaluronic acid fragments that cause the formation of red blood cell clusters; * represents severe cell deformation; # represents the formation of large red blood cell clusters.
表9.不同分子量透明质酸片段和透明质酸原料诱发牛静脉血红细胞钱串状聚集的结果。Table 9. The results of different molecular weight hyaluronic acid fragments and hyaluronic acid raw materials inducing the cluster-like aggregation of bovine venous red blood cells.
Figure PCTCN2021080543-appb-000013
Figure PCTCN2021080543-appb-000013
Figure PCTCN2021080543-appb-000014
Figure PCTCN2021080543-appb-000014
注：Yes/Yes或No/No代表两次使用不同个体末梢血标本的实验结果。Note: Yes/Yes or No/No represents the results of two experiments using different individual peripheral blood samples.
表10.不同分子量透明质酸片段和透明质酸原料诱发马静脉血红细胞钱串状聚集的结果。Table 10. Results of different molecular weight hyaluronic acid fragments and hyaluronic acid raw materials inducing money clusters of horse venous red blood cells.
Figure PCTCN2021080543-appb-000015
Figure PCTCN2021080543-appb-000015
注：黑体字和斜体字共同代表引起血红细胞聚集形成的最低透明质酸片段的浓度；*代表严重细胞变形；#代表大块血红细胞钱串样聚集形成；Yes/Yes或No/No或No/Yes代表两次使用不同个体末梢血标本的实验结果。Note: Bold type and italic type together represent the lowest concentration of hyaluronic acid fragments that cause red blood cell aggregation; * represents severe cell deformation; # represents the formation of large red blood cell clusters; Yes/Yes or No/No or No /Yes represents the results of two experiments using different individual peripheral blood samples.
表11.不同分子量透明质酸片段和透明质酸原料诱发水貂静脉血红细胞钱串状聚集的结果。Table 11. Results of different molecular weight hyaluronic acid fragments and hyaluronic acid raw materials inducing the clusters of red blood cells in mink veins.
Figure PCTCN2021080543-appb-000016
Figure PCTCN2021080543-appb-000016
注：*代表严重细胞变形；#代表大块血红细胞钱串样聚集形成。Note: * represents severe cell deformation; # represents the formation of large red blood cell clusters.
讨论：水貂血红细胞状态不好，加入被生理盐水稀释的血液中立刻皱缩变形。所以水貂血红细胞观察时，存在许多皱缩细胞。Discussion: The red blood cells of the mink are not in good condition, and they immediately shrink and deform when added to the blood diluted by normal saline. Therefore, when the mink red blood cells are observed, there are many shrunken cells.
表12.不同分子量透明质酸片段和透明质酸原料诱发羊驼静脉血红细胞钱串状聚集的结果。Table 12. Results of different molecular weight hyaluronic acid fragments and hyaluronic acid raw materials inducing the cluster-like aggregation of red blood cells in alpaca veins.
Figure PCTCN2021080543-appb-000017
Figure PCTCN2021080543-appb-000017
Figure PCTCN2021080543-appb-000018
Figure PCTCN2021080543-appb-000018
注：Yes/Yes或No/No或Yes/No代表两次使用不同个体末梢血标本的实验结果；羊驼红细胞为椭圆形。Note: Yes/Yes or No/No or Yes/No represents the results of two experiments using peripheral blood samples of different individuals; alpaca red blood cells are oval in shape.
讨论：discuss:
本发明发现透明质酸片段诱发人和动物末梢血和静脉血红细胞钱串状聚集和促进血红细胞沉降。本发明进一步发现平均分子量35kDa的低分子量透明质酸片段B-HA诱发人红细胞钱串状聚集是通过结合人红细胞表面CD44(图3)，其本质反映了这个低分子量透明质酸片段结合红细胞表面CD44的活性。文献研究表明人白细胞(包括中性粒细胞、单核细胞和淋巴细胞)通过其血液动力学改变从血管内移走渗出进入组织炎症区发挥炎症作用(本节参考文献1-8)。文献研究还支持透明质酸片段影响红细胞的血液动力学行为和影响白细胞功能(本节参考文献9-14)。例如，人体组织的低分子量透明质酸片段在炎症组织中产生并可能进入血循环被清理。文献研究还表明低分子量透明质酸片段结合的红细胞被肝和脾中性粒细胞和巨噬细胞吞噬清理(本段参考文献9-14)，提示中性粒细胞和巨噬细胞吞噬低分子量透明质酸片段和被低分子量透明质酸片段标记的红细胞。炎症组织中产生的低分子量透明质酸片段进入血循环与红细胞结合，可能与红细胞寿命有关。The present invention finds that hyaluronic acid fragments induce money clusters of human and animal peripheral blood and venous red blood cells and promote red blood cell sedimentation. The present invention further discovered that the low-molecular-weight hyaluronic acid fragment B-HA with an average molecular weight of 35kDa induces the money-string aggregation of human erythrocytes by binding to CD44 on the surface of human erythrocytes (Figure 3), which essentially reflects that this low-molecular-weight hyaluronic acid fragment binds to the surface of erythrocytes. CD44 activity. Literature studies have shown that human leukocytes (including neutrophils, monocytes and lymphocytes) through their hemodynamic changes are removed from the blood vessel and exuded into the tissue inflammation zone to play an inflammatory role (references 1-8 in this section). Literature studies also support that hyaluronic acid fragments affect the hemodynamic behavior of red blood cells and affect the function of white blood cells (references 9-14 in this section). For example, low-molecular-weight hyaluronic acid fragments of human tissues are produced in inflamed tissues and may enter the blood circulation to be cleared. Literature studies also show that red blood cells bound by low-molecular-weight hyaluronic acid fragments are phagocytosed and cleared by neutrophils and macrophages in the liver and spleen (references 9-14 in this paragraph), suggesting that neutrophils and macrophages phagocytose low-molecular-weight transparent Fragments of uronic acid and red blood cells labeled with fragments of low-molecular-weight hyaluronic acid. The low-molecular-weight hyaluronic acid fragments produced in inflamed tissues enter the blood circulation and combine with red blood cells, which may be related to the lifespan of red blood cells.
本发明提示低分子量透明质酸片段诱发红细胞钱串状聚集和提示表明低分子量透明质酸片段的生理功能、治疗作用和副作用有种属特异性。换 句话说，对人皮肤粘膜有抗炎作用的透明质酸片段HA fragments 35kDa或B-HA或HA35，对牛和羊甚至猴没有作用。The present invention suggests that low-molecular-weight hyaluronic acid fragments induce red blood cell clusters and indicates that the physiological functions, therapeutic effects and side effects of low-molecular-weight hyaluronic acid fragments are species-specific. In other words, the hyaluronic acid fragments HA fragments 35kDa or B-HA or HA35, which have anti-inflammatory effects on human skin and mucous membranes, have no effect on cattle, sheep or even monkeys.
本段参考文献：References for this paragraph:
1.Wright,H.L.,R.J.Moots,R.C.Bucknall,and S.W.Edwards.2010.Neutrophil function in inflammation and inflammatory diseases(review).Rheumatol.49:1618-1631.1. Wright, H.L., R.J. Moots, R.C. Bucknall, and S.W. Edwards. 2010. Neutrophil function in inflammation and inflammatory diseases(review). Rheumatol.49:1618-1631.
2.Pardo,A.,R.Barrios,M.Gaxiola,I.Segura-Valdez,G.Carrillo,M.Mejia,and M.Selman.2000.Increase of lung neutrophils in hypersensitivity pneumonitis is associated with lung fibrosis.Am.J.Respir.Crit.Care Med.161:1698-1704.2. Pardo, A., R. Barrios, M. Gaxiola, I. Segura-Valdez, G. Carrillo, M. Mejia, and M. Selman. 2000. Increase of lung neutrophils in hypersensitivity pneumonitis is associated with lung fibrosis. Am .J.Respir.Crit.Care Med.161:1698-1704.
3.Butterfield,T.A.,T.M.Best,and M.A.Merrick.2006.The dual roles of neutrophils and macrophages in inflammation:A critical balance between tissue damage and repair(Review).J.Athl.Train.41(4):457-465.3.Butterfield, TA, TMBest, and MAMerrick. 2006. The dual roles of neutrophils and macrophages in inflammation: A critical balance between tissue damage and repair (Review). J. Athl.Train.41(4):457- 465.
4.Wright,H.L.,R.J.Moots,R.C.Bucknall,and S.W.Edwards.2010.Neutrophil function in inflammation and inflammatory diseases(review).Rheumatol.49:1618-1631.4. Wright, H.L., R.J. Moots, R.C. Bucknall, and S.W. Edwards. 2010. Neutrophil function in inflammation and inflammatory diseases(review). Rheumatol. 49:1618-1631.
5.Butterfield,T.A.,T.M.Best,and M.A.Merrick.2006.The dual roles of neutrophils and macrophages in inflammation:A critical balance between tissue damage and repair(Review).J.Athl.Train.41(4):457-465.5.Butterfield, TA, TMBest, and MAMerrick. 2006. The dual roles of neutrophils and macrophages in inflammation: A critical balance between tissue damage and repair (Review). J. Athl.Train.41(4):457- 465.
6.Kleijn,S.D.,M.Kox,I.E.Sama,J.Pillay,A.V.Diepen,M.A.Huijnen,J.G.Hoeven,G.Ferwerda,P.W.M.Hermans,and P.Pickkers.2012.Transcriptome kinetics of circulating neutrophils during human experiental endotoxemia.PLoS One7(6):e38255.6.Kleijn,SD,M.Kox,IESama,J.Pillay,AVDiepen,MAHuijnen,JGHoeven,G.Ferwerda,PWMHermans,and P.Pickkers.2012.Transcriptome kinetics of circulating neutrophils during human experiental endotoxemia. PLoS One7(6): e38255.
7.McDonald,B.,and P.Kubes.2015.Interactions between CD44 and hyaluonan in leukocyte trafficking.Front.Immunol.6(68):1-6.7.McDonald,B.,and P.Kubes.2015.Interactions between CD44 and hyaluonan in leukocyte trafficking.Front.Immunol.6(68):1-6.
8.Wright,H.L.,R.J.Moots,R.C.Bucknall,and S.W.Edwards.2010.Neutrophil function in inflammation and inflammatory diseases(review).Rheumatol.49:1618-1631.8.Wright,H.L.,R.J.Moots,R.C.Bucknall,and S.W.Edwards.2010.Neutrophil function in inflammation and inflammatory diseases(review).Rheumatol.49:1618-1631.
9.Luquita A,Urli L,Svetaz MJ,Gennaro AM,Giorgetti ME,Pistone G,Volpintesta R,Palatnik S,Rasia M:In vitro and ex vivo effect  of hyaluronic acid on erythrocyte flow properties.J Biomed Sci 2010,17:1-8.9.Luquita A, Urli L, Svetaz MJ, Gennaro AM, Giorgetti ME, Pistone G, Volpintesta R, Palatnik S, Rasia M: In vitro and ex vivo effect of hyaluronic acid on erythrocyte 17 properties. J 2010, Scimed flow: 1-8.
10.Kerfoot SM，McRea K,Lam F,McAvoy EF,Clark S,Brain M,Lalor PF,Adams DH,Kubes P:A noval mechanism of erythrocyte capture from circulation in humans.Exp Hematol 2008,36:111-118.10.Kerfoot SM, McRea K, Lam F, McAvoy EF, Clark S, Brain M, Lalor PF, Adams DH, Kubes P: A novel mechanism of erythrocyte capture from circulation in humans. Exp Hematol 2008, 36: 111-118.
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13.Vachon E,Martin R,Kwok V,Cherepanov V,Chow CW,Doerschuk CM,Plumb J,Grinstein S,Downey GP:CD44-mediated phagocytosis induces inside-out activation of complement receptor-3 in murine macrophages.Blood 2007,110(13):4492-4502.13.Vachon E, Martin R, Kwok V, Cherepanov V, Chow CW, Doerschuk CM, Plumb J, Grinstein S, Downey GP: CD44-mediated phagocytosis induces inside-out activation of complement receiver-3 pha, murderBlood 2007. 110(13):4492-4502.
14.Melder RJ,Yuan J,Munn LL,Jain RK：Erythrocytes Enhance Lymphocyte Rolling and Arrest in Vivo.Microvascular Research 2000,59:316–322.14. Melder RJ, Yuan J, Munn LL, Jain RK: Erythrocytes Enhance Lymphocyte Rolling and Arrest in Vivo. Microvascular Research 2000, 59: 316-322.
结论：表2-5和6-12表明牛和羊与猴、马、猪、犬、大鼠、猫等其它动物完全不一样。这个结果表明低分子量透明质酸片段诱发红细胞钱串状聚集和红细胞沉降的这种影响血管内血红细胞血液动力学和红细胞与白细胞互相间行为的作用有种属特异性。这个种属特异性表明低分子量透明质酸片段的生物活性、治疗作用和副作用有种属特异性。换句话说，这个种属特异性表明低分子量透明质酸片段的生物活性对人和其它动物效果不一样或有效的程度不一样。Conclusion: Tables 2-5 and 6-12 show that cows and sheep are completely different from monkeys, horses, pigs, dogs, rats, cats and other animals. This result indicates that the low-molecular-weight hyaluronic acid fragments induce red blood cell clusters and red blood cell sedimentation, which affect the hemodynamics of red blood cells in blood vessels and the interaction between red blood cells and white blood cells, which are species-specific. This species specificity indicates that the biological activity, therapeutic effects and side effects of low molecular weight hyaluronic acid fragments are species specific. In other words, this species-specificity indicates that the biological activity of low molecular weight hyaluronic acid fragments is not the same or effective to humans and other animals.
实施例7Example 7
目的：利用低分子量透明质酸片段诱发红细胞钱串状聚集造成的红细胞沉降率不同来测定透明质酸片段B-HA(又称HA35或HA fragments 35kDa)产品的批间生产分子量变异程度。Purpose: Use low-molecular-weight hyaluronic acid fragments to induce different red blood cell sedimentation rates caused by red blood cell clustering to determine the degree of molecular weight variation between batches of hyaluronic acid fragment B-HA (also known as HA35 or HA fragments 35kDa).
方法：本发明利用低分子量透明质酸片段诱发血红细胞钱串状聚集的程度不同造成的红细胞沉降率不同来定量测定批间生产的透明质酸片段产品 的分子量变异。利用血红细胞钱串状聚集程度不同造成的血球沉降率不同来定量测定批间生产的透明质酸片段产品的分子量变异。低分子量透明质酸片段B-HA(又称HA35或HA fragments 35kDa)与新鲜采集的犬静脉血按照1:2的比例混匀加上抗凝剂EDTA和PBS缓冲液至终浓度分别为0.15％、0.11％、0.075％，既最终达到犬静脉血3.5倍稀释，吸取400ul混合好的血液至血沉管中，静置25min，过后计数血沉管中血液的下沉距离，计算血球的沉降率。Method: The present invention uses low molecular weight hyaluronic acid fragments to induce different red blood cell sedimentation rates caused by different degrees of red blood cell clustering to quantitatively determine the molecular weight variation of hyaluronic acid fragments produced between batches. The difference in blood cell sedimentation rate caused by the different degree of red blood cell cluster-like aggregation is used to quantitatively determine the molecular weight variation of hyaluronic acid fragment products produced between batches. Low molecular weight hyaluronic acid fragment B-HA (also known as HA35 or HA fragments 35kDa) and freshly collected canine venous blood are mixed in a ratio of 1:2, plus anticoagulant EDTA and PBS buffer to the final concentration of 0.15%, respectively , 0.11%, 0.075%, and finally reach the 3.5-fold dilution of canine venous blood, draw 400ul of mixed blood into the erythrocyte sedimentation tube, let it stand for 25 minutes, then count the sedimentation distance of the blood in the erythrocyte sedimentation tube, and calculate the sedimentation rate of blood cells.
具体还包括18角度激光测定和凝胶电泳测定低分子量透明质酸片段B-HA(又称HA35或HA fragments 35kDa)的分子量。Specifically, it also includes 18-angle laser measurement and gel electrophoresis to determine the molecular weight of the low-molecular-weight hyaluronic acid fragment B-HA (also known as HA35 or HA fragments 35kDa).
表13.测定终浓度0.15％、0.11％和0.075％的B-HA(又称HA35或HA fragments 35kDa)对3.5倍稀释的人静脉血红细胞25分钟沉降率(cm/25minutes)的影响。Table 13. Determine the effect of B-HA (also known as HA35 or HA fragments 35kDa) with final concentrations of 0.15%, 0.11% and 0.075% on the 25-minute sedimentation rate (cm/25minutes) of human venous red blood cells diluted by 3.5 times.
Figure PCTCN2021080543-appb-000019
Figure PCTCN2021080543-appb-000019
结论：1.使用B-HA(又称HA35或HA fragments 35kDa)的终浓度(0.15％)高时批间变异小和测定灵敏度低；2.使用B-HA或HA35或HA fragments 35kDa的终浓度(0.075％)低时批间变异大和测定灵敏度也高。Conclusion: 1. When the final concentration (0.15%) of B-HA (also known as HA35 or HA fragments 35kDa) is high, the inter-assay variability is small and the determination sensitivity is low; 2. The final concentration of B-HA or HA35 or HA fragments 35kDa is used. (0.075%) When it is low, the inter-assay variability is large and the determination sensitivity is also high.
表14.测定终浓度0.15％、0.11％和0.075％的B-HA(又称HA35或HA fragments 35kDa)对3.5倍稀释的犬静脉血红细胞25分钟沉降率(cm/25minutes)的影响。Table 14. Determination of the effect of B-HA (also known as HA35 or HA fragments 35kDa) with final concentrations of 0.15%, 0.11% and 0.075% on the 25-minute sedimentation rate (cm/25minutes) of canine venous red blood cells diluted by 3.5 times.
Figure PCTCN2021080543-appb-000020
Figure PCTCN2021080543-appb-000020
结论：1.使用B-HA(又称HA35或HA fragments 35kDa)的终浓度(0.15％)高时批间变异小和测定灵敏度低；2.使用B-HA(又称HA35或HA fragments 35kDa)的终浓度(0.075％)低时批间变异大和测定灵敏度也高。Conclusion: 1. When the final concentration (0.15%) of B-HA (also known as HA35 or HA fragments 35kDa) is high, the inter-assay variability is small and the determination sensitivity is low; 2. Using B-HA (also known as HA35 or HA fragments 35kDa) When the final concentration (0.075%) is low, the inter-assay variability is large and the determination sensitivity is also high.
表15.测定终浓度0.15％、0.11％和0.075％的B-HA(又称HA35或HA fragments 35kDa)对3.5倍稀释的大鼠静脉血红细胞25分钟沉降率(cm/25minutes)的影响。Table 15. Determine the effect of B-HA (also known as HA35 or HA fragments 35kDa) with final concentrations of 0.15%, 0.11% and 0.075% on the 25-minute sedimentation rate (cm/25minutes) of rat venous red blood cells diluted by 3.5 times.
Figure PCTCN2021080543-appb-000021
Figure PCTCN2021080543-appb-000021
Figure PCTCN2021080543-appb-000022
Figure PCTCN2021080543-appb-000022
结论：1.使用B-HA(又称HA35或HA fragments 35kDa)的终浓度(0.15％)高时批间变异小和测定灵敏度低；2.使用B-HA(又称HA35或HA fragments 35kDa)的终浓度(0.075％)低时批间变异大和测定灵敏度也高。Conclusion: 1. When the final concentration (0.15%) of B-HA (also known as HA35 or HA fragments 35kDa) is high, the inter-assay variability is small and the determination sensitivity is low; 2. Using B-HA (also known as HA35 or HA fragments 35kDa) When the final concentration (0.075%) is low, the inter-assay variability is large and the determination sensitivity is also high.
图9.不同批次的B-HA(又称HA35或HA fragments 35kDa)产品的凝胶电泳分子量批间测定结果。Figure 9. Inter-batch measurement results of gel electrophoresis molecular weight of different batches of B-HA (also known as HA35 or HA fragments 35kDa) products.
结果表明不同批次生产的B-HA(又称HA35或HA fragments 35kDa)产品的凝胶电泳分子量批内测定变异小于30％。The results showed that the intra-assay variation in gel electrophoresis molecular weight of B-HA (also known as HA35 or HA fragments 35kDa) produced in different batches was less than 30%.
表16.不同批次的B-HA(又称HA35或HA fragments 35kDa)产品的18角度激光(GPC/SEC system equipped with an multi angle laser light scattering(MALLS)detector)测定结果。Table 16. 18-angle laser (GPC/SEC system equipped with an multi-angle laser light scattering (MALLS) detector) measurement results of different batches of B-HA (also known as HA35 or HA fragments 35kDa) products.
Figure PCTCN2021080543-appb-000023
Figure PCTCN2021080543-appb-000023
结果表明批间变异CV＝22％。The results showed that the inter-assay variation CV=22%.
图10为HA fragments 24kDa(FRD)的凝胶电泳分子量批内变异测定结果。Figure 10 shows the results of intra-assay variation in molecular weight of HA fragments 24kDa (FRD) by gel electrophoresis.
结果表明同一HA fragments 24kDa(FRD)样品的凝胶电泳分子量批内测定变异几乎观测不到。这个结果还表明凝胶电泳测定低分子量透明质酸片段方法稳定。The results showed that the intra-assay variation of the gel electrophoresis molecular weight of the same HA fragments 24kDa (FRD) sample was almost unobservable. This result also shows that the method of gel electrophoresis for the determination of low molecular weight hyaluronic acid fragments is stable.
讨论：本发明进一步利用低分子量透明质酸片段诱发大鼠、犬和人血红细胞钱串状聚集程度不同造成的血球沉降率不同来定量测定批间生产的透明质酸片段产品的分子量变异，其测定灵敏度比观察红细胞钱串状聚集高。B-HA(又称HA35或HA fragments 35kDa)商业化产品需要敏感的测定较低分子量的透明质酸片段皮下组织注射产品分子量的方法来监测产品分子量的变异情况。本发明首次利用低分子量透明质酸片段引发红细胞钱串状聚集造成的血球沉降速度不同，建立透明质酸片段产品批间生产分子量变异质量检测方法。Discussion: The present invention further uses low-molecular-weight hyaluronic acid fragments to induce different blood cell sedimentation rates caused by different degrees of red blood cell aggregation in rats, dogs and humans to quantitatively determine the molecular weight variation of hyaluronic acid fragments produced between batches. The sensitivity of the determination is higher than that of observing the money-like clusters of red blood cells. B-HA (also known as HA35 or HA fragments 35kDa) commercial products require a sensitive method of measuring the molecular weight of lower molecular weight hyaluronic acid fragments for subcutaneous tissue injection products to monitor the variation of product molecular weight. The present invention uses low-molecular-weight hyaluronic acid fragments for the first time to induce different blood cell sedimentation speeds caused by red blood cell cluster-like aggregation, and establishes a quality detection method for the production molecular weight variation of hyaluronic acid fragment products between batches.
结论：利用低分子量透明质酸片段诱发大鼠、犬和人红细胞钱串状聚集造成的红细胞沉降率不同，其测定灵敏度高，可以用来测定透明质酸片段B-HA(又称HA35或HA fragments 35kDa)产品的批间生产分子量微小变异程度，其灵敏度比18角度激光测定和凝胶电泳测定的灵敏度更好。Conclusion: Using low molecular weight hyaluronic acid fragments to induce rat, dog, and human red blood cell money clusters has different erythrocyte sedimentation rates, and its measurement sensitivity is high. It can be used to determine hyaluronic acid fragment B-HA (also known as HA35 or HA). Fragments 35kDa) The degree of slight variation in the molecular weight of the product between batches, the sensitivity is better than that of 18-angle laser measurement and gel electrophoresis measurement.
实施例8Example 8
目的：探索B-HA(又称HA35或HA fragments 35kDa)结合人红细胞表面对人白细胞活化(neutrophil activation)的生物学和临床意义。Objective: To explore the biological and clinical significance of B-HA (also known as HA35 or HA fragments 35kDa) combined with the surface of human red blood cells on human leukocyte activation (neutrophil activation).
方法：先使用含有Ca 2+和Mg 2+和5.5mM glucose的HBSS(Thermo Scientific)将2x10 6新鲜提取的人中性粒细胞混合均匀。加入10ug/ml的OxyBURST Green H2HFF BSA(Molecular Probes)培育30分钟。每井接种5x10 5(24-well plate)或1.2x10 7(6-well plate)人中性粒细胞，先加入10ug/ml HA(又称HA原料1600kDa)或B-HA(又称HA35或HA fragments 35kDa)或盐水培养30分钟，再加入终浓度25nM的Phorbol-12 myristate 13-acetate(PAM)活化30分钟。采用流式细胞仪测定中性粒细胞释放的ROS(reactive oxygen species)，结果表达为Mean florescence intensity(Mean±SD)。 Method: First use HBSS (Thermo Scientific) containing Ca 2+ and Mg 2+ and 5.5 mM glucose to mix 2×10 6 freshly extracted human neutrophils uniformly. Add 10ug/ml OxyBURST Green H2HFF BSA (Molecular Probes) and incubate for 30 minutes. Inoculate 5x10 5 (24-well plate) or 1.2x10 7 (6-well plate) human neutrophils in each well, first add 10ug/ml HA (also known as HA raw material 1600kDa) or B-HA (also known as HA35 or HA Fragments 35kDa) or saline for 30 minutes, then add Phorbol-12 myristate 13-acetate (PAM) at a final concentration of 25nM to activate for 30 minutes. Flow cytometry was used to measure ROS (reactive oxygen species) released by neutrophils, and the result was expressed as Mean florescence intensity (Mean±SD).
结果result
图11.B-HA(又称HA35或HA fragments 35kDa)和HA原料1600kDa(FRD)(图中称HA)对PMA诱导的新鲜人白细胞活化(neutrophil activation)的作用。Figure 11. The effect of B-HA (also known as HA35 or HA fragments 35kDa) and HA raw material 1600kDa (FRD) (referred to in the figure as HA) on PMA-induced neutrophil activation of fresh human leukocytes.
结果表明10ug/ml的B-HA和HA原料1600kDa(又称HA原料1600kDa,FRD)(图中称HA)都抑制了PMA诱导的新鲜人白细胞活化(neutrophil activation)。换句话说，低分子量的B-HA和高分子量的HA都抑制了能够移走到有病组织中性粒细胞的活化(neutrophil activation)和其释放ROS自由基(free radicals)破坏人体组织。考虑到低分子量的B-HA渗透性远比高分子量的HA的渗透性好，低分子量的B-HA的临床应用价值更高。另外，即使高分子量的HA进入人体组织，也因其分子太大难于接近细胞和结合细胞相关受体产生作用。The results show that 10ug/ml B-HA and HA raw material 1600kDa (also known as HA raw material 1600kDa, FRD) (referred to as HA in the figure) both inhibit PMA-induced neutrophil activation of fresh human leukocytes. In other words, both low-molecular-weight B-HA and high-molecular-weight HA inhibit neutrophil activation (neutrophil activation) that can be removed to diseased tissues and the release of ROS free radicals (free radicals) that damage human tissues. Considering that the permeability of low-molecular-weight B-HA is much better than that of high-molecular-weight HA, the clinical application value of low-molecular-weight B-HA is higher. In addition, even if high molecular weight HA enters human tissues, it is too large for its molecules to get close to cells and bind to cell-related receptors.
人血液中红细胞数量比白细胞多1000倍。人血液中红细胞数量和白细胞表面保持密切接触。文献研究表明红细胞表面的Sialic acid结合白细胞表面的Ig-like lectins(Siglecs)抑制白细胞活化制造炎症。本发明意料之外地发现B-HA诱发红细胞钱串状聚集，参与了白细胞静止和活化的调 解。不适当的白细胞活化诱发炎症性疾病，抑制不适当的白细胞活化可能是治疗炎症性疾病的一个有效途径。进一步使用新鲜人全血做B-HA ex vivo临床研究很有必要。There are 1,000 times more red blood cells in human blood than white blood cells. The number of red blood cells in human blood is in close contact with the surface of white blood cells. Literature studies have shown that Sialic acid on the surface of red blood cells combined with Ig-like lectins (Siglecs) on the surface of white blood cells inhibits the activation of white blood cells to produce inflammation. The present invention unexpectedly discovered that B-HA induces red blood cell clusters and participates in the mediation of leukocyte quiescence and activation. Inappropriate leukocyte activation induces inflammatory diseases, and inhibition of inappropriate leukocyte activation may be an effective way to treat inflammatory diseases. It is necessary to further use fresh human whole blood for B-HA ex vivo clinical research.
有文献表明sialic acid或表达sialic acid的细菌Group B steptococcus和透明质酸或表达透明质酸的细菌Group A steptococcus都与人中性粒细胞表面的Siglec-9结合，抑制人中性粒细胞oxydative burst,既产生强氧化物ROS或自由基参与人中性粒细胞相关的炎症反应(见本段参考文献9)。除了Siglec-9,本发明不排除透明质酸和透明质酸片段还可能通过其它受体和结合蛋白产生功能(见本段参考文献1-9)。There are literatures showing that sialic acid or sialic acid-expressing bacteria Group B steptococcus and hyaluronic acid or hyaluronic acid-expressing bacteria Group A steptococcus both bind to Siglec-9 on the surface of human neutrophils and inhibit the oxydative burst of human neutrophils. , Both produce strong oxide ROS or free radicals and participate in the inflammatory response related to human neutrophils (see reference 9 in this paragraph). In addition to Siglec-9, the present invention does not exclude that hyaluronic acid and hyaluronic acid fragments may also function through other receptors and binding proteins (see References 1-9 in this paragraph).
另外，本申请实施例6的研究表明这个B-HA抑制人白细胞活化的作用有种属特异性，既这个B-HA抑制人白细胞活化的作用对人和动物效果不同或有效的程度不同。In addition, the study in Example 6 of the present application shows that the B-HA inhibits human leukocyte activation has species specificity, that is, the B-HA inhibits human leukocyte activation has different effects or degrees of effectiveness on humans and animals.
本段参考文献：References for this paragraph:
1.Jaworski DM,Kelly GM,Piepmeier JM,Hockfield S(1996)BEHAB(brain enriched hyaluronan binding)is expressed in surgical samples of glioma and in intracranial grafts of invasive glioma cell lines.Cancer Res 56:2293–2298.1. Jaworski DM, Kelly GM, Piepmeier JM, Hockfield S (1996) BEHAB (brain enriched hyaluronan binding) is expressed in surgical samples of glioma and in intracranial grafts of invasive glioma cells: Cancer2293--Res 2298.
2.Deepa SS,Carulli D,Galtrey C,Rhodes K,Fukuda J,Mikami T,Sugahara K,Fawcett JW(2006)Composition of perineuronal net extracellular matrix in rat brain:a different disaccharide composition for the net-associated proteoglycans.J Biol Chem 281:17789–17800.2.Deepa SS, Carulli D, Galtrey C, Rhodes K, Fukuda J, Mikami T, Sugahara K, Fawcett JW (2006) Composition of periodontal net extracellular matrix in rat brain: a different disaccharide compositionoglycated for the net-associated. Biol Chem 281: 17789-17800.
3.Matsumoto K,Shionyu M,Go M,Shimizu K,Shinomura T,Kimata K,Watanabe H(2003)Distinct interaction of versican/PG-M with hyaluronan and link protein.J Biol Chem 278:41205–41212.3.Matsumoto K, Shionyu M, Go M, Shimizu K, Shinomura T, Kimata K, Watanabe H (2003) Distinct interaction of versican/PG-M with hyaluronan and link protein. J Biol Chem 278:41205-41212.
4.Seyfried NT,McVey GF,Almond A,Mahoney DJ,Dudhia J,Day AJ(2005)Expression and purification of functionally active hyaluronan-binding domains from human cartilage link protein,aggrecan and versican:formation of ternary complexes with defined hyaluronan oligosaccharides.J Biol Chem 280:5435–5448.4. Seyfried NT, McVey GF, Almond A, Mahoney DJ, Dudhia J, Day AJ (2005) Expression and Purification of Functionally Active Hyaluronan-binding Domains from Human Cartilage Link Protein, Aggrecan and versican: Formation Hypophysical Complexes .J Biol Chem 280:5435-5448.
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8.Huang L,Yoneda M,Kimata K(1993)A serum-derived hyaluronan-associated protein(SHAP)is the heavy chain of the inter alpha-trypsin inhibitor.J Biol Chem 268:26725–26730.8. Huang L, Yoneda M, Kimata K (1993) A serum-derived hyaluronan-associated protein (SHAP) is the heavy chain of the inter alpha-trypsin inhibitor. J Biol Chem 268:26725–26730.
9.Lizcano A,Secundino I,
Figure PCTCN2021080543-appb-000024
S,Corriden R,Rohena C,Diaz S,Ghosh P,Deng L,Nizet V,Varki A(2017)Erythrocyte sialoglycoproteins engage Siglec-9 on neutrophils to suppress activation.Blood 129(23):3100-3110. 9.Lizcano A, Secundino I,
Figure PCTCN2021080543-appb-000024
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结论：1.本发明结果提示渗透性好的低分子量的B-HA或HA35或HA fragments 35kDa注射液有利于控制白细胞活化(neutrophil activation)程度在一个适当的水平范围内，有可能成为一个副作用小的抗炎(anti-inflammation)候选药物；2.这个B-HA抑制人白细胞活化的作用有种属特异性，既这个B-HA抑制人白细胞活化的作用对人和动物效果不同或有效的程度不同。Conclusion: 1. The results of the present invention suggest that B-HA or HA35 or HA fragments 35kDa injection with good permeability and low molecular weight is beneficial to control the degree of white blood cell activation (neutrophil activation) within an appropriate level, which may become a side effect. The anti-inflammation drug candidate; 2. This B-HA inhibits the activation of human leukocytes with species specificity, that is, the effect of this B-HA inhibits the activation of human leukocytes is different or effective to humans and animals. different.
以上所述，仅是本发明的较佳实施例而已，并非对本发明作任何形式上的限制，本领域技术人员利用上述揭示的技术内容做出些许简单修改、等同变化或修饰，均落在本发明的保护范围内。The above are only preferred embodiments of the present invention, and are not intended to limit the present invention in any form. Those skilled in the art use the technical content disclosed above to make some simple modifications, equivalent changes or modifications, which fall within the present invention. Within the scope of protection of the invention.

Claims (10)

  1. 一种低分子量透明质酸片段诱发红细胞钱串状聚集的应用。An application of low-molecular-weight hyaluronic acid fragments in inducing red blood cell clusters.
  2. 根据权利要求1所述的应用，其特征在于，利用低分子量透明质酸片段诱发末梢血或静脉血红细胞钱串状聚集的最小百分比浓度和分子量大小之间呈负相关的关系，测定低分子量透明质酸片段的分子量大小或测定低分子量透明质酸片段产品批间生产的分子量变异范围。The application according to claim 1, wherein the low molecular weight hyaluronic acid fragments are used to induce a negative correlation between the minimum percentage concentration of the peripheral blood or venous red blood cell clusters and the molecular weight to determine the low molecular weight transparent The molecular weight of the fragments of hyaluronic acid or the measurement of the molecular weight variation range of low-molecular-weight hyaluronic acid fragments produced between batches.
  3. 根据权利要求2所述的应用，其特征在于，所述末梢血或静脉血为人、猫、犬或大鼠的末梢血或静脉血。The application according to claim 2, wherein the peripheral blood or venous blood is the peripheral blood or venous blood of humans, cats, dogs or rats.
  4. 根据权利要求2所述的应用，其特征在于，所述低分子量透明质酸片段为平均分子量35±8kDa的透明质酸片段产品；其诱发末梢血或静脉血红细胞钱串状聚集的最小百分比浓度为0.15％。The application according to claim 2, wherein the low-molecular-weight hyaluronic acid fragment is a hyaluronic acid fragment product with an average molecular weight of 35±8kDa; it induces a minimum percentage concentration of red blood cell clusters in peripheral blood or venous blood It is 0.15%.
  5. 根据权利要求1所述的应用，其特征在于，利用低分子量透明质酸片段诱发末梢血或静脉血红细胞钱串状聚集程度不同造成的血红细胞沉降率不同，敏感测定低分子量透明质酸片段产品批间生产的分子量微小变异范围。The application according to claim 1, characterized in that low molecular weight hyaluronic acid fragments are used to induce different degrees of red blood cell aggregation in peripheral blood or venous red blood cells, resulting in different red blood cell sedimentation rates, and sensitive determination of low molecular weight hyaluronic acid fragment products The small variation range of molecular weight produced from batch to batch.
  6. 根据权利要求5所述的应用，其特征在，所述末梢血或静脉血为人、犬或大鼠的末梢血或静脉血；所述低分子量透明质酸片段为平均分子量35±8kDa的透明质酸片段产品；测定批间生产的分子量变异范围时，所述平均分子量35±8kDa的透明质酸片段产品终浓度为0.075％。The application according to claim 5, wherein the peripheral blood or venous blood is the peripheral blood or venous blood of human, dog, or rat; the low molecular weight hyaluronic acid fragment is hyaluronic acid with an average molecular weight of 35±8kDa Acid fragment product; when determining the molecular weight variation range produced between batches, the final concentration of the hyaluronic acid fragment product with an average molecular weight of 35±8kDa is 0.075%.
  7. 一种平均分子量35±8kDa的低分子量透明质酸片段的活性检测方法，其特征在于，所述低分子量透明质酸片段通过结合红细胞表面CD44诱发末梢血或静脉血红细胞钱串状聚集；A method for detecting the activity of low-molecular-weight hyaluronic acid fragments with an average molecular weight of 35±8kDa, which is characterized in that the low-molecular-weight hyaluronic acid fragment induces cluster-like aggregation of peripheral blood or venous red blood cells by binding to CD44 on the surface of red blood cells;
    所述活性检测方法为对平均分子量35±8kDa的低分子量透明质酸片段进行CD44结合活性测定。The activity detection method is to measure the CD44 binding activity of low-molecular-weight hyaluronic acid fragments with an average molecular weight of 35±8kDa.
  8. 根据权利要求7所述的平均分子量35±8kDa的低分子量透明质酸片段的活性检测方法，其特征在于，利用10ug/ml的抗人CD44抗体抑制平均 分子量35±8kDa的透明质酸片段结合CD44。The method for detecting the activity of low-molecular-weight hyaluronic acid fragments with an average molecular weight of 35±8kDa according to claim 7, wherein 10ug/ml of anti-human CD44 antibody is used to inhibit the binding of hyaluronic acid fragments with an average molecular weight of 35±8kDa to CD44. .
  9. 一种平均分子量35±8kDa的低分子量透明质酸片段的应用，其特征在于，所述低分子量透明质酸片段与人中性粒细胞快速结合并进入中性粒细胞；An application of a low-molecular-weight hyaluronic acid fragment with an average molecular weight of 35±8kDa, characterized in that the low-molecular-weight hyaluronic acid fragment quickly binds to human neutrophils and enters neutrophils;
    所述应用为在制备治疗中性粒细胞相关炎症性疾病药物中应用；The application is in the preparation of a medicine for treating neutrophil-related inflammatory diseases;
    所述应用对人和其它动物效果不一样或有效的程度不一样。The application has a different effect or degree of effectiveness on humans and other animals.
  10. 一种平均分子量35±8kDa的低分子量透明质酸片段的应用，其特征在于，所述低分子量透明质酸片段通过结合红细胞和/或白细胞表面CD44或白细胞表面直接和/或间接抑制人中性粒细胞活化和释放各种氧化物质；An application of a low-molecular-weight hyaluronic acid fragment with an average molecular weight of 35±8kDa, characterized in that the low-molecular-weight hyaluronic acid fragment directly and/or indirectly inhibits human neutrality by binding to CD44 on the surface of red blood cells and/or white blood cells or the surface of white blood cells Granulocyte activation and release of various oxidizing substances;
    所述应用为作为抑制人中性粒细胞活化和释放各种氧化物质的抑制剂的应用或在制备治疗中性粒细胞活化和释放各种氧化物质相关炎症性疾病药物中应用；The application is an application as an inhibitor for inhibiting the activation of human neutrophils and the release of various oxidizing substances, or an application in the preparation of drugs for the treatment of inflammatory diseases related to the activation and release of various oxidizing substances of neutrophils;
    所述应用对人和其它动物效果不一样或有效的程度不一样。The application has a different effect or degree of effectiveness on humans and other animals.
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