WO2018103723A1 - 甘露糖在用于提高Treg细胞数量及其Foxp3因子表达水平的新用途 - Google Patents

甘露糖在用于提高Treg细胞数量及其Foxp3因子表达水平的新用途 Download PDF

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WO2018103723A1
WO2018103723A1 PCT/CN2017/115136 CN2017115136W WO2018103723A1 WO 2018103723 A1 WO2018103723 A1 WO 2018103723A1 CN 2017115136 W CN2017115136 W CN 2017115136W WO 2018103723 A1 WO2018103723 A1 WO 2018103723A1
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autoimmune
group
mannose
cells
treg
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高尚先
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高尚先
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7004—Monosaccharides having only carbon, hydrogen and oxygen atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/60—Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the present application relates to the fields of medicines, foods, health products and cosmetics, and in particular to new uses of mannose for increasing the number of Treg cells and the expression levels of Foxp3.
  • the immunologic function is performed by the body under the interaction of lymphocytes, monocytes, macrophages, NK cells and other related cells and their products; immune function is the role of the immune system based on immune recognition.
  • the immune function of the body is mainly manifested in three aspects: immune prevention, immune stabilization and immune surveillance.
  • Immunooligic defence refers to the function of the body to resist and eliminate pathogenic microorganisms or other foreign bodies. Abnormalities in immune prevention can cause diseases. If the reaction is too high, hypersensitivity may occur; if the reaction is too low, immunodeficiency may occur.
  • immune stability refers to the body to clear the damaged or aging cells, maintaining their physiological balance. Immune stability dysfunction can lead to autoimmune diseases.
  • immunological surveillance refers to the body to identify and remove mutant cells appearing in the body to prevent tumor function.
  • the immune surveillance function is low and it is prone to malignant tumors.
  • immune homeostasis refers to the interaction between the cellular composition of the immune system, immune cells, cells and cytokine networks, and maintains its functional state relatively stable.
  • Autoimmune diseases are diseases caused by the body's immune response to its own antigen and causing damage to its own tissues.
  • Treg regulatory T cells
  • nTreg natural regulatory T cells
  • inducedTreg induced regulatory T cells
  • Treg is a subpopulation of CD4 + T cells with immunosuppressive activity.
  • Treg cells inhibit the production of T cells and antigen presenting cells by releasing cytokines IL-10 and TGF- ⁇ , and reduce the production of inflammatory cytokines and antibody secretion.
  • Treg exerts immunomodulatory effects by up-regulating the expression of inhibitory immune cell surface molecules and down-regulating the expression of genes involved in activated T cells.
  • the initial CD4 + T cells are activated by the costimulatory signal after being stimulated by the foreign antigen, and differentiate into different subtypes of effector T cells.
  • Initial CD4 + T cells can differentiate into Treg cells under the induction of TGF- ⁇ alone. When IL-6 is present, it can induce the expression of ROR ⁇ t, inhibit the production of Treg cells, and promote the differentiation of primary CD4 + T cells into Th17 cells.
  • the level of Treg cells is imbalanced with other immune cells such as Th17 cells.
  • the levels of Treg cells and other immune cells are adjusted to treat immune homeostasis. Provides new ways. In view of this, the present application is specifically filed.
  • the primary object of the present application is to propose the use of mannose in the preparation of a medicament, food, nutraceutical or cosmetic for the treatment of an autoimmune disease.
  • a second object of the present application is to propose the use of mannose in the preparation of a medicament, food, nutraceutical or cosmetic for maintaining an immune homeostasis.
  • a third object of the present application is to propose the use of mannose in the preparation of a medicament, a food, a health supplement or a cosmetic for increasing the number of Treg cells and the expression level of Foxp3.
  • the present application relates to the use of mannose in the manufacture of a medicament, food, nutraceutical or cosmetic for the treatment of an autoimmune disease.
  • the autoimmune disease is an autoimmune disease caused by a decrease in the number of Treg cells.
  • the autoimmune disease is selected from the group consisting of lupus erythematosus, hyperthyroidism, type I or type II diabetes and its complications, IgA nephritis, dry mouth syndrome, rheumatoid arthritis, ankylosing spondylitis, simple sexual obesity, bronchial asthma, neurodermatitis, oral ulcers, ulcerative colitis, psoriasis, vitiligo, Behcet's disease, autoimmune eczema, autoimmune uveitis, autoimmune ocular iridocyclitis, Autoimmune conjunctivitis, autoimmune dry eye, autoimmune cataract, autoimmune glaucoma, allergic rhinitis, irritable bowel syndrome, itchy skin.
  • the application also relates to the use of mannose in the manufacture of a medicament, food, nutraceutical or cosmetic for the maintenance of immune homeostasis.
  • the disease caused by the steady-state imbalance of the immune system is selected from the group consisting of lupus erythematosus, hyperthyroidism, type I or type II diabetes and its complications, IgA nephritis, dry mouth syndrome, rheumatoid arthritis, and rigidity.
  • Spondylitis simple obesity, bronchial asthma, neurodermatitis, oral ulcers, ulcerative colitis, psoriasis, vitiligo, Behcet's disease, autoimmune eczema, autoimmune uveitis, autoimmune eye iris Ciliary body inflammation, autoimmune conjunctivitis, autoimmune dry eye syndrome, autoimmune cataract, autoimmune glaucoma, allergic rhinitis, irritable bowel syndrome, itchy skin.
  • autoimmune dry eye syndrome refers to dry eye caused by autoimmune factors or caused by autoimmune diseases
  • autoimmune cataract refers to autoimmune factors Cataract caused by or caused by autoimmune diseases
  • autoimmune glaucoma refers to glaucoma caused by autoimmune factors or caused by autoimmune diseases.
  • the present application also relates to the use of mannose in the manufacture of a medicament, food, nutraceutical or cosmetic for increasing the number of Treg cells and their Foxp3 factor expression levels.
  • the mannose in the above application is preferably D-mannose.
  • mannose has an increase in the number of Treg cells and its Foxp3 factor expression level, and thus the level of Treg cells and other immune cells such as Th17 cells, when the levels of Treg cells are imbalanced with other immune cells such as Th17 cells.
  • a certain role which can be used for drugs, foods, health products or cosmetics with imbalanced levels of Treg cells and other immune cells such as Th17 cells, autoimmunity caused by imbalance in the level of Treg cells and other immune cells such as Th17 cells.
  • sexual diseases and immune homeostasis imbalances caused by imbalances in the levels of Treg cells and other immune cells such as Th17 cells have therapeutic and adjunctive therapeutic effects.
  • Figure 1 shows the preparation process of antigenic proteins used in the animal model of dry syndrome.
  • the present application relates to the use of mannose in the manufacture of a medicament, food, nutraceutical or cosmetic for the treatment of an autoimmune disease.
  • the mannose in the present application is preferably D-mannose.
  • autoimmune diseases refer to diseases caused by the body's immune response to autoantigens, resulting in damage to the tissues, including lupus erythematosus, hyperthyroidism, and type I due to decreased Treg cells. Or type II diabetes and its complications, IgA nephritis, dry mouth syndrome, rheumatoid arthritis, ankylosing spondylitis, simple obesity due to decreased Treg cells, bronchial asthma, neurodermatitis, oral ulcers, Ulcerative colitis, psoriasis, vitiligo, Behcet's disease, autoimmune eczema, autoimmune uveitis, autoimmune ocular iridocyclitis, autoimmune conjunctivitis, allergic rhinitis.
  • the present application finds that when mannose has a stimulating effect on Treg cells under pathological conditions, it can promote the increase of Treg cells, thereby adjusting the balance between Treg cells and Th17 cells, and maintaining immune stability. Therefore, it can treat and adjuvant the autoimmune diseases.
  • mannose in the preparation of a medicament for treating an autoimmune disease, mannose can be prepared into various dosage forms, such as an oral preparation, an injection preparation and an external preparation, and the oral preparation is selected from the group consisting of a tablet, a buccal tablet, a disintegrating agent, an oral solution,
  • the injection preparation may be selected from the group consisting of a water needle or a lyophilized powder injection
  • the external preparation may be selected from the group consisting of external water, lotion, gel, spray, patch, eye drop, ointment or tincture.
  • mannose In the preparation of foods for treating autoimmune diseases, mannose can be prepared into various foods such as soft candy, hard candy, cakes, jellies, beverages and the like.
  • mannose In the preparation of a health care product for treating an autoimmune disease, mannose can be prepared into various dosage forms of health care products such as tablets, buccal tablets, disintegrating agents, oral liquids, capsules, oral liquids and the like.
  • mannose in the preparation of a cosmetic for treating an autoimmune disease, can be prepared into various forms of cosmetics such as a liquid, a lotion, a gel, a spray, a patch, a paste, and the like.
  • the amount of mannose is 0.001 to 200 g/day.
  • the application also relates to the use of mannose in the manufacture of a medicament, food, nutraceutical or cosmetic for the maintenance of immune homeostasis.
  • lupus erythematosus, hyperthyroidism, type I or type II diabetes and its complications chronic ulcerative colitis, IgA nephritis, dry mouth syndrome, rheumatoid arthritis, ankylosing spondylitis, simple sexual obesity, bronchial asthma, neurodermatitis, oral ulcers, ulcerative colitis, psoriasis, vitiligo, Behcet's disease, autoimmune eczema, autoimmune uveitis, autoimmune ocular iridocyclitis, Autoimmune conjunctivitis, autoimmune uveitis, autoimmune iridocyclitis, autoimmune conjunctivitis, autoimmune dry eye, autoimmune cataract, autoimmune glaucoma, allergic rhinitis, Irritable
  • autoimmune dry eye syndrome refers to dry eye caused by autoimmune factors or caused by autoimmune diseases
  • autoimmune cataract refers to caused by autoimmune factors, or Cataract caused by autoimmune diseases
  • autoimmune glaucoma refers to glaucoma caused by autoimmune factors or caused by autoimmune diseases.
  • These diseases are caused by disorders of immune homeostasis. Mannose can protect and treat the disease by regulating the level of Treg cells and maintaining immune homeostasis.
  • mannose in the preparation of a medicament for maintaining an immune homeostasis, can be prepared into various dosage forms, such as an oral preparation, an injection preparation and an external preparation, and the oral preparation is selected from the group consisting of a tablet, a buccal tablet, a disintegrating agent, and an oral solution.
  • the dosage form of the injection preparation may be selected from the group consisting of a water needle or a lyophilized powder injection
  • the external preparation may be selected from the group consisting of external water, lotion, gel, spray, patch, eye drop, ointment or tincture.
  • mannose In preparing foods for maintaining immune homeostasis, mannose can be prepared into various foods such as soft candy, hard candy, cakes, jellies, beverages and the like.
  • mannose In the preparation of a health care product for maintaining immune homeostasis, mannose can be prepared into various dosage forms of health care products such as tablets, buccal tablets, disintegrating agents, oral liquids, capsules, oral liquids and the like.
  • mannose In the preparation of a cosmetic for maintaining an immune homeostasis, mannose can be prepared into various forms of cosmetics such as a liquid, a lotion, a gel, a spray, a patch, a paste, and the like.
  • the amount of mannose is 0.001 to 200 g/day.
  • the present application also relates to the use of mannose in the manufacture of a medicament, food, nutraceutical or cosmetic for increasing the number of Treg cells and their Foxp3 factor expression levels.
  • the Foxp3 factor is a member of the forkhead transcription factor family and is a key regulator of Treg cell development and function. It can regulate the development and function of Treg cells by binding to chromosomes and regulating the expression and function of various genes. Studies have shown that Foxp3 is critical CD4 + CD 25 + Treg development and function of the molecule, the CD4 + Treg can be suppressed conventional C TCR mediated D4 + T cell proliferation and cytokine production, in the maintenance of peripheral tolerance to prevent autoimmune disease Play an important role. The gain of function experimental study directly demonstrates the relationship between Foxp3 and native Treg cells. The expression of Foxp3 is essential for natural T cells to acquire Treg cell phenotype and function, through loss of function experiments.
  • Foxp3 is indispensable for Treg cell development. Foxp3 is required for the development and function of Treg cells, and Foxp3 is expressed only in CD4 + CD25 + T cells, not in native CD4 + CD 25 - T cells and immature thymocytes, in B cells and CD8 + T cells and no significant expression, may be considered as Foxp3 Treg cells specific markers, the Foxp3 Treg cells considered positive T cells. Mannose can maintain the immune homeostasis by increasing the number of Treg cells and its Foxp3 factor expression level, thereby treating and adjuvanting the diseases caused by the decrease in the number of Treg cells and the expression level of Foxp3.
  • mannose in the preparation of a medicament for increasing the number of Treg cells and the expression level of Foxp3 factor, mannose can be prepared into various dosage forms, such as oral preparations, injection preparations and external preparations, and the oral preparation is selected from the group consisting of tablets, oral liquids, capsules and the like.
  • the injectable preparation may be selected from a water needle or a lyophilized powder injection
  • the external preparation may be selected from an external water preparation, an ointment or a stimulant.
  • mannose can be prepared into various foods such as soft candy, hard candy, cakes, jellies, beverages and the like.
  • mannose in the preparation of a health supplement for increasing the number of Treg cells and the expression level of Foxp3 factor, mannose can be prepared into various health care products such as tablets, buccal tablets, disintegrating agents, oral liquids, capsules, oral liquids and the like.
  • mannose in the preparation of a cosmetic for increasing the number of Treg cells and the expression level of Foxp3 factor, mannose can be prepared into various forms of cosmetics such as a liquid, a lotion, a gel, a spray, a patch, a paste, and the like.
  • the amount of mannose is 0.001 to 200 g/day.
  • D-mannose has the effect of increasing Treg cell levels and maintaining immune homeostasis in a variety of animal models that cause imbalances in Th17/Treg cell levels.
  • rheumatoid arthritis lupus erythematosus, bronchial asthma, diabetes, obesity, hyperthyroidism, IgA nephritis, dry mouth syndrome, neurodermatitis, oral ulcers, ulcerative colitis, psoriasis, autoimmune eczema
  • animal models such as autoimmune uveitis, immunoconjunctivitis, dry eye, allergic rhinitis, intestinal stress syndrome, cataract, Treg cells after 7 to 28 days of D-mannose administration to model animals The level is improved and the clinical symptoms of the model animals are alleviated to some extent.
  • the technical effect of the present application will be further described below by taking an animal model as an example.
  • Example 1 Experimental animal model of bronchial asthma
  • mice were randomly divided into control group, asthma group and treatment group (10 in each group):
  • OVA ovarian protein
  • alum adjuvant Sigma
  • mice in the treatment group were tested with the asthma group. After successful modeling in the asthma group, D-mannose water (1.0 M) was fed freely for 21 days, and cell statistics were performed.
  • control group was given a simple alum adjuvant (without OVA) sensitization and PBS atomization inhalation at the same time during the modeling experiment.
  • mice were anesthetized and sacrificed.
  • the trachea was fixed in the supine position.
  • the trachea was intubated and fixed with an indwelling needle.
  • the rats were lavaged with cold PBS, 0.5 ml each time, a total of 1 ml.
  • the cells of the lavage fluid were counted, centrifuged, and the cell sediment was taken for Wright. Sorted counts of stained cells.
  • Pulmonary vascular blood was perfused from the right ventricle of mice with PBS.
  • the excised lung tissue was cut and incubated in 1 ml of PBS containing 0.002 g (0.2%) collagenase I (Sigma-Aldrich) for 15 min, and the lung tissue was placed at 300 mesh.
  • the stainless steel mesh was cut with an ophthalmic scissors.
  • the single cell suspension in the culture dish was collected, centrifuged at 2500 r/min for 5 min, the supernatant was removed, 2 ml of red blood cell lysate was added and incubated for 8 min at room temperature, and the supernatant was centrifuged twice to wash the supernatant.
  • 1640 medium containing 10% fetal calf serum The cells were made into a single cell suspension, 50 ng/ml PMA (Sigma-Aldrich), 500 ng/ml ionomycin (Sigma-Aldrich) and 0.7 ⁇ l/ml GolgiPlug (BD Biosciences) were added. Incubate for 4-6 h in a 37 ° C, 5% CO 2 incubator.
  • the cells after the incubation were collected and centrifuged to remove the supernatant. After mixing, the fluorescently labeled antibody was added, and Hamster antimouse CD3-Pecy7 (BD Biosciences) and Ratantimouse CD4-FITC (BD Biosciences) were added, and the cells were incubated at room temperature for 30 minutes in the dark, and centrifuged twice with PBS. Remove the supernatant, fix the fixative for 30 min, centrifuge for 1 time to remove the supernatant, add the permeate and incubate for 5 min at room temperature, centrifuge to remove the supernatant, mix and add Rat antimouse IL-4-APC (BD Biosciences), Rat antimouse IL-17.
  • Rat antimouse IL-4-APC Rat antimouse IL-17.
  • Detection of Foxp3 + Treg cells Add Rat antimouse CD4-FITC (BD Biosciences) Rat antimouse CD25-PE (BD Biosciences) for 30 min at room temperature in the dark, wash twice with PBS, fix the fixative for 30 min, wash once with PBS, The permeate was added for 5 min, and antimouse Foxp3-PEcy5 (eBBiosciences) was added, and the cells were incubated at room temperature for 30 min in the dark, and the labeled cells were assayed by flow cytometry (FAC-SClibur, BD Biosciences).
  • the results are shown in Table 1.
  • the bronchoalveolar lavage fluid of normal mice is mainly macrophages and lymphocytes, especially in the former, while the content of eosinophils and neutrophils is very small.
  • the total number of cells, lymphocytes, eosinophils and neutrophils in the bronchoalveolar lavage fluid of the asthma group were significantly higher than those of the control group (P ⁇ 0.001), suggesting that the airway inflammation in the OVA group was significantly higher than that in the control group. .
  • the model is displayed successfully.
  • Table 1 Total number of cells in bronchoalveolar lavage fluid and white blood cell classification
  • Treg cells 7.67 ⁇ 0.44)% (4.87 ⁇ 0.35)% (6.87 ⁇ 0.65)% ⁇ Th17 cell percentage (1.07 ⁇ 0.07)% (1.74 ⁇ 0.17)%* (1.21 ⁇ 0.08)% ⁇ Treg/Th17 7.38 ⁇ 0.71 3.02 ⁇ 0.49** 5.68 ⁇ 0.67 ⁇
  • the level of Treg cells was improved after the application of D-mannose to the animal model, and the clinical symptoms of the treatment group were also improved to some extent, and the white blood cell level was also significantly decreased.
  • Wistar rats male
  • weighing about 100 g, aged 4 weeks were purchased from the Experimental Animal Center of Shandong University.
  • the feeding conditions were the same as in Example 1, and the entire experimental procedure was carried out in accordance with the guidelines for animal protection and use. Rats were randomly divided into control group, CIA model group and treatment group (20 in each group):
  • CIA model group 7 ml of bovine type II collagen (collagen type II, CII containing acetic acid, concentration of 2rng/ml) was slowly added dropwise to an equal volume of complete freund's adjuvant (CFA, before the experiment). Fully emulsified in Sigma), the final concentration of type II collagen is 1 mg/ml.
  • the prepared collagen and adjuvant mixture were injected into the tail of the rat, intradermally, and 200 ⁇ g (0.2 ml) was initially immunized on the 0th day, and 200 ⁇ g (0.2 ml) was boosted on the contralateral tail root on the 7th day.
  • the RA model group was given 0.2 ml of an emulsion containing bovine type II collagen 1 mg/ml, and the normal control group was intradermally injected with an equal amount of physiological saline.
  • Rats were intraperitoneally injected with an excess of sodium pentobarbital.
  • the spleens were taken under sterile conditions, washed with PBS buffer, and then cut into small pieces by ophthalmology. Milled on a 200-mesh steel sieve and centrifuged at 2000 r/min for 5 min. Discard the supernatant, add 5 mL of red blood cell lysate, mix, let stand for 5 min, centrifuge at 2000 r / min for 5 min, discard the supernatant, wash PBS buffer for 1 to 2 times, and obtain spleen cells.
  • the detection was carried out by flow cytometry as in Example 1.
  • the level of Treg cells was improved, and the clinical symptoms of the treatment group were also improved to some extent.
  • D-mannose also has a certain therapeutic effect on ankylosing spondylitis (rheumatoid spondylitis).
  • mice 40 weighing (20.0 ⁇ 2.0) g were purchased from Shanghai Slack Laboratory Animal Center. Raised in a special disinfection breeding box, constant room temperature (21 ⁇ 25 ° C), filtered air, humidity 50% ⁇ 65%, light 10 ⁇ 12h / d, feed, water are disinfected, replacement of feed and other operations are super clean Conducted in the station, the entire experimental process was carried out in accordance with animal protection and use guidelines.
  • NOD mice were randomly divided into control group and treatment group (20 in each group): the treatment group was fed with D-mannose water (1.2 M), and after 21 consecutive days, cell statistics were performed.
  • mice spleen tissue cells were taken by the method of Example 2 and detected by flow cytometry.
  • ⁇ is P ⁇ 0.01 compared to the model group.
  • the level of Treg cells was improved, and the clinical symptoms of the treatment group were also improved to some extent.
  • Rats were randomly divided into control group, UC model group and treatment group (20 in each group):
  • DSS sodium dextran sulfate
  • mice spleen tissue cells were taken for detection by the method of Example 3, and detected by flow cytometry.
  • the level of Treg cells was improved, and the clinical symptoms of the treatment group were also improved to some extent.
  • Example 5 Animal model experiment of lupus erythematosus
  • SPF-class MRL/lpr spontaneous systemic lupus erythematosus-like lupus-like model mice 40 females, weighing about 20g, 3 months old, purchased from Shandong University Experimental Animal Center. The whole experiment was closed in the SPF class animal room. Mice were randomly divided into control group and treatment group (20 in each group):
  • Treatment group Mice were fed D-mannose water (1.0 M) to drink freely, and after 21 consecutive days, cell statistics were performed.
  • mice spleen tissue cells were taken for detection by the method of Example 3, and detected by flow cytometry.
  • ⁇ is P ⁇ 0.01 compared to the model group.
  • the level of Treg cells was improved after D-mannose was applied to the animal model, and the clinical symptoms of the treatment group were also improved to some extent.
  • mice were randomly divided into control group, model group and treatment group (20 in each group):
  • mice spleen tissue cells were taken for detection by the method of Example 3, and detected by flow cytometry.
  • the level of Treg cells was improved, and the clinical symptoms of the treatment group were also improved to some extent.
  • Wistar rats, half male and half female, weighing 180-200 g, were kept in the same manner as in Example 1.
  • the whole experimental procedure was carried out according to the guidelines for animal protection and use.
  • tissue homogenate-60 ° C low temperature refrigerator for storage.
  • the above-mentioned tissue homogenate is mixed with Freund's adjuvant in an equal ratio, and an antigen emulsifier is prepared according to the instructions of the adjuvant (no delamination after standing).
  • Rat spleen tissue cells were taken for detection by the method of Example 3, and detected by flow cytometry.
  • the level of Treg cells was improved, and the clinical symptoms of the treatment group were also improved to some extent.
  • Model group 50 rats were given high-energy and high-fat diet for 14 weeks. The body weight of the rats was measured every Monday, and the body weight of each group was observed. After 14 weeks, 20 rats with the highest body weight in the high-energy diet group were set as DIO group; 10 DIO rats were used for cell statistics.
  • High-energy feed ingredients 30% fat, 40% carbohydrate, 15% protein, 4% ash, 3% cellulose.
  • the feed heat was 4.76 kcal/g.
  • Rat spleen tissue cells were taken for detection by the method of Example 3, and detected by flow cytometry.
  • the level of Treg cells was improved, and the clinical symptoms of the treatment group were also improved to some extent.
  • Example 9 Animal model experiment of IgA nephropathy
  • Model group 50 rats were given oral administration of bovine serum albumin (BSA), 25% subcutaneous injection of carbon tetrachloride (CCL4) solution and 0.25 g/L lipopolysaccharide. (Lipopolysaccharide, LPS) The method of tail vein injection established a rat model of IgA nephropathy for 9 weeks.
  • BSA bovine serum albumin
  • CCL4 carbon tetrachloride
  • LPS Lipopolysaccharide
  • Rat spleen tissue cells were taken for detection by the method of Example 3, and detected by flow cytometry.
  • the lanolin is heated and melted, and the liquid paraffin is added in a ratio of 1:3 (v:v). After 8 lbs of 20 minutes autoclaving, it is Freund's incomplete adjuvant (IFA), and BCG (final concentration 6 mg/ml) The cooled IFA is added and fully emulsified to obtain Freund's complete adjuvant (CFA).
  • IFA Freund's incomplete adjuvant
  • BCG final concentration 6 mg/ml
  • the submandibular glands of Balb/c mice were removed, the lymph nodes were removed, placed in PBS buffer, and homogenized. After homogenization, the component separated at 10 5 g from 1000 g to 10 5 g was designated as component P5 by differential centrifugation according to the procedure shown in FIG.
  • the P5 antigen protein content was determined by a Coomassie blue colorimetric method using a spectrophotometer.
  • the P5 protein was emulsified with CFA to obtain an emulsified P5 antigen having a final antigen concentration of 0.75 mg/ml. Rats were randomly divided into control group and model group:
  • Model group subcutaneous injection of emulsified P5 protein antigen in the hind paw and bilateral inguinal hernia, 0.1ml/foot, containing BCG 3mg/ml, on the first day, 7th day and 21st day after the first immunization, abdominal cavity Injecting pertussis vaccine 0.2ml (the concentration of pertussis is 2.9 ⁇ 10 10 /ml).
  • subcutaneous injection of emulsified P5 protein antigen boosts the immune system, resulting in SS-like salivary gland changes and clinical manifestations. .
  • mice spleen tissue cells were taken for detection by the method of Example 3, and detected by flow cytometry.
  • the level of Treg cells was improved, and the clinical symptoms of the treatment group were also improved to some extent.
  • Hyperthyroidism model group 200 ⁇ g of levothyroxine sodium (LT 4 ) (trade name, excellent Jiale, produced by Merkelon Pharmaceutical Group, Germany, each containing 100 ⁇ g) was ground and dissolved in 3 to 5 ml of distilled water. Once, after 21 days, changes in heart rate, blood pressure, heart weight, myocardial cell size, heart-to-weight ratio, and other changes in the cardiovascular system of patients with hyperthyroidism were consistent, indicating successful modeling.
  • LT 4 levothyroxine sodium
  • Rat spleen tissue cells were taken for detection by the method of Example 3, and detected by flow cytometry.
  • mice were randomly divided into control group, model group and treatment group (20 in each group):
  • FITC isothiocyanate
  • mice spleen tissue cells were taken for detection by the method of Example 3, and detected by flow cytometry.
  • Model group The day before sensitization, the back 3cm ⁇ 3cm area was depilated with hair removal cream, and the next day hair removal area was coated with 5% 2,4-dinitrochlorobenzene (DNCB) 50 ⁇ L for the first time; 2 weeks After the back hair removal 3cm ⁇ 3cm, the next day, coated with 0.1% 2,4-dinitrochlorobenzene (DNCB) 100 ⁇ L excitation, 1 time / week, for 4 weeks, confirmed by pathological section successfully.
  • DNCB 2,4-dinitrochlorobenzene
  • Rat spleen tissue cells were taken for detection by the method of Example 3, and detected by flow cytometry.
  • Lewis male rats weighing about 160-180 g, were purchased from the Experimental Animal Center of Shandong University. The feeding conditions were the same as in Example 1. The whole experimental procedure was carried out according to the guidelines for animal protection and use.
  • Model group 30 ⁇ g of photoreceptor vitamin A-type binding protein (IRBP, Shanghai Shenggong) was mixed with 30 ⁇ l of PBS, and then added with 0.5 mg of Mycobacterium tuberculosis (H37Ra, Difco, USA) and 30 ⁇ l of complete adjuvant (CFA). Fully emulsified and injected into the unilateral hind paw of the rat for immunization to make an experimental autoimmune uveitis (EAU) model; 8 to 9 days after immunization, there was inflammation in the anterior segment of the eye, which was characterized by iris vasodilation.
  • EAU autoimmune uveitis
  • Treatment group 10 rats of model rats were fed with D-mannose water (1.0 M) for free drinking, and eye drops (106.5 mM NaCl, 26.1 mM NaHCO 3 , 18.7 mM KCl, 1.0 mM MgCl). 2 , 0.5 mM NaH 2 PO 4 , 1.1 mM CaCl 2 10 mM HEPE, 0.2 M D-mannose), 3 to 5 drops three times a day; after 7 consecutive days, cell statistics were performed.
  • Rat spleen tissue cells were taken for detection by the method of Example 3, and detected by flow cytometry.
  • mice were randomly divided into control group, model group and treatment group (20 in each group):
  • mice of model mice were fed with D-mannose water (1.0 M) for free drinking, and eye drops (106.5 mM NaCl, 26.1 mM NaHCO 3 , 18.7 mM KCl, 1.0 mM MgCl). 2 , 0.5 mM NaH 2 PO 4 , 1.1 mM CaCl 2 10 mM HEPE, 0.2 M D-mannose), 3 to 5 drops three times a day; after 7 consecutive days, cell statistics were performed.
  • Rat spleen tissue cells were taken for detection by the method of Example 3, and detected by flow cytometry.
  • (1) Model group After rabbit anesthesia, the left lower lacrimal gland was removed from the animal clean bench, and the lacrimal gland was transferred to a centrifuge tube containing 5 ml of Ham's solution to separate rabbit lacrimal gland epithelial cells; autologous lacrimal gland epithelial cells were transferred to 96-well plate.
  • the cells were cultured in a 24-well plate in DMEM for 2 days, and cells cultured in a 96-well plate (cell density: 3*10 6 /ml, 100 ⁇ l per well) were used for detection of proliferation by BrdU kit; 24-well plates were used ( The cells cultured at a cell density of 1.8 ⁇ 10 7 /ml and 400 ⁇ l per well were used for mixed culture.
  • Irradiation with gamma rays maintains antigenicity.
  • Rabbit peripheral blood lymphocytes were isolated, and the separated lymphocytes were collected into a centrifuge tube, resuspended after centrifugation, and the cell density was adjusted to prepare cell fluids corresponding to the density of lacrimal gland cells. 3 ⁇ 10 5 lymphocytes were added to each well of a 96-well plate, and 1.8 ⁇ 10 6 lymphocytes were added to each well of the well plate. After 4 days of mixed co-cultivation, the BrdU method (BrdU kit, product of Roche) detected a lymphocyte proliferation rate of 3.71. The model group was transfused with autologous lymphocytes 5 ⁇ 10 4 through the ear vein.
  • tear secretion was significantly reduced in the dry eye model group, and tear film rupture time was shortened.
  • Sodium fluorescein staining showed diffuse punctate coloration of the cornea.
  • lacrimal gland and conjunctival tissue staining showed lymphocytic infiltration, indicating successful modeling.
  • Treatment group 10 rabbits were successfully fed with D-mannose water (1.0M) for free drinking, and after 7 days, eye drops (106.5 mM NaCl, 26.1 mM NaHCO 3 , 18.7 mM KCl, 1.0 mM MgCl 2 ) , 0.5 mM NaH 2 PO 4 , 1.1 mM CaCl 2 10 mM HEPE, 0.2 M D-mannose), 3 to 5 drops per day 3 times; cell statistics were performed.
  • the spleen was isolated. The removed spleen was placed in a cell culture dish containing RPMI-1640 medium and ground. The Pasteur pipette was evenly blown, filtered, and centrifuged at 2000 rpm for 8 min. The supernatant was discarded, and the cells were resuspended in 20 ml of RPMI-1640 medium, added to 20 ml of Ficoll, and centrifuged at 2000 rpm for 20 min. The intermediate misty lymphocyte layer was aspirated, 20 ml of RPMI-1640 medium, 2000 rpm, 20 min, washed twice, and the cells were resuspended in RPMI-1640 medium and counted.
  • Example 17 Animal model experiment of allergic rhinitis
  • mice were randomly divided into control group, model group and treatment group (20 in each group):
  • Model group The model group was sensitized with ovalbumin supplemented with aluminum hydroxide adjuvant. Mice with 40% ovalbumin aluminum hydroxide PBS suspension were intraperitoneally injected with 200 ⁇ L (containing 20 mg of ovalbumin), and the same day was used for the 14th day. The method is again immunized. The blank group mice were intraperitoneally injected with PBS solution at the same time. On the 21st day of immunization, 20 ⁇ L of 1% ovalbumin PBS solution (containing 100 ⁇ g of ovalbumin) was intranasally challenged for 6 days to establish a mouse model of allergic rhinitis.
  • Rat spleen tissue cells were taken for detection by the method of Example 3, and detected by flow cytometry.
  • Example 18 Animal model test of intestinal stress syndrome
  • Model group The model group rats were administered with a physiological saline suspension containing 350 to 400 Trichinella spiralis larvae. After 2 weeks, the model was successfully confirmed by pathological section.
  • Rat venous blood was taken 2ml, anticoagulated with EDTA, and CD4 and CD25 on the surface of peripheral blood mononuclear cells (PBMC) were detected by fluorescence direct labeling and flow cytometry.
  • PBMC peripheral blood mononuclear cells
  • the supporting software analyzed the data, recorded the percentage of positive cells, and subtracted the non-specific control value from the statistical analysis of the percentage of peripheral blood Treg.
  • Model group 10-day-old rats were injected subcutaneously with sodium selenite at a dose of 3.46 mg/kg body weight (ie, 20 ⁇ mol/kg body weight), once every other day for a total of 3 injections. Within 30 minutes after the first injection of sodium selenite, physiological saline was intraperitoneally injected at 0.1 mg/10 g for 6 days. Observation by a slit lamp microscope showed successful modeling.
  • Treatment group 10 rats of model rats were fed D-mannose water (1.0 M) freely from the 2nd day of modeling, while eye drops (106.5 mM NaCl, 26.1 mM NaHCO 3 , 18.7 mM KCl, 1.0 mM) MgCl 2 , 0.5 mM NaH 2 PO 4 , 1.1 mM CaCl 2 10 mM HEPE, 0.2 M D-mannose), 3 to 5 drops three times a day; for 14 consecutive days, cell statistics were performed.
  • Rat spleen tissue cells were taken for detection by the method of Example 3, and detected by flow cytometry.
  • Vitiligo is a common multiple pigmented skin disease. The exact pathogenesis of vitiligo has not been fully elucidated. There are various hypotheses. Among them, autoimmune theory has received more and more attention. Most researchers believe that CD4 + CD25 + Treg cells are lower in vitiligo patients than in normal people, especially in patients with generalized vitiligo compared with normal people, CD4 + CD25 + Treg cells are significantly reduced.
  • RT-PCR reverse transcription polymerase chain reaction
  • Oral administration taking 40g/day 2 times, 28 days is a course of treatment.
  • the flow cytometer is a product of the American BD company (model: FACSCalibur).
  • PE-labeled murine anti-human CD4 IgG-PE
  • FITC-labeled murine anti-human CD25 IgG-PE
  • isotype control ⁇ 1/ ⁇ 2 were purchased from BD, USA.
  • Ficoll separation solution, TRIZOL, Ex-Taq DNA polymerase, dNTP, MML-V reverse transcriptase, M2MLV reverse transcriptase, etc. were purchased from Jingmei Biotech Co., Ltd., and the primers were synthesized by Shanghai Shenggong Biological Company.
  • MNCS peripheral blood mononuclear cells
  • RNA extraction of MNCs was performed according to the TRIzol (Shanghai Shenggong Company) reagent product specification. The quality of RNA was determined by agarose gel electrophoresis. The total RNA concentration was calculated by absorbance scanning, and the ratio of A260/A280 was calculated. It is 1.8 to 2.0. Take 5 ⁇ g of total RNA, 50 ⁇ mol/LOligo (dT) 162 ⁇ l, 2.5 mmol/L dNTP 3 ⁇ l, add DEPC-treated double distilled water to 34 ⁇ l, place it in ice water at 65 ° C for 5 min, centrifuge, and then add M2MLV5 ⁇ reverse transcriptase.
  • dT ⁇ mol/LOligo
  • the Foxp3 primer sequences are as follows:
  • the expected product length is 443 bp
  • the internal reference ⁇ -actin sequence is as follows:
  • the product is expected to be 1126 bp in length.
  • the reaction conditions were denaturation at 94 ° C for 40 s, annealing at 60 ° C for 40 s, extension at 72 ° C for 60 s, and 35 cycles.
  • the product was electrophoresed on 2% agarose and scanned by a gel imaging system. Foxp3 mRNA expression levels were expressed as Foxp3/ ⁇ -actin.
  • SPSS 10.0 software was used for data analysis. The data were expressed as x ⁇ s. The differences between groups were compared by two independent samples t test. Paired t-test was used before and after treatment between the same group. P ⁇ 0.05 was considered statistically. significance.
  • Table 21 Comparison of the number of CD4 + CD25 + Treg cells in peripheral blood of patients and normal controls (x ⁇ s%)
  • Table 22 Changes in the number of CD4 + CD25 + Treg cells and the expression level of Foxp3 mRNA in peripheral blood before and after treatment (x ⁇ s)

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Abstract

药物、食品、保健品和化妆品领域，具体讲，涉及甘露糖的用途。甘露糖在用于制备治疗自身免疫性疾病的药物、食品、保健品或化妆品中的应用；还涉及甘露糖在用于制备用于维护免疫稳态的药物、食品、保健品或化妆品中的应用；还涉及甘露糖在用于制备提高Treg细胞水平的药物、食品、保健品或化妆品中的应用。经研究发现，甘露糖在Treg细胞水平发生失衡时，对Treg细胞水平的调整具有一定的作用，从而可将其用于Treg细胞水平失衡的药物、食品、保健品或化妆品，对由Treg细胞水平失衡引发的自身免疫性疾病以及由Treg细胞水平失衡引发的免疫稳态失衡具有治疗及辅助治疗的效果。

Description

甘露糖在用于提高Treg细胞数量及其Foxp3因子表达水平的新用途 技术领域
本申请涉及药物、食品、保健品和化妆品领域，具体讲，涉及甘露糖在用于提高Treg细胞数量及Foxp3其因子表达水平的新用途。
背景技术
免疫功能(immunologic function)是机体在淋巴细胞、单核细胞、巨噬细胞、NK细胞和其他有关细胞及其产物的相互作用下完成的；免疫功能是免疫系统根据免疫识别而发挥的作用。机体的免疫功能主要表现在三个方面即免疫预防、免疫稳定和免疫监视。
1、免疫预防(immunoligic defence)指机体抵抗和清除病原微生物或其它异物的功能。免疫预防功能发生异常可引起疾病，如反应过高可出现超敏反应；反应过低可导致免疫缺陷病。
2、免疫稳定(immunoligic hemeostasis)：指机体清除损伤或衰老的细胞，维持其生理平衡的功能。免疫稳定功能失调可导致自身免疫病。
3、免疫监视(immunoligic surveillance)：指机体识别和清除体内出现的突变细胞，防止发生肿瘤的功能。免疫监视功能低下，易患恶性肿瘤。
其中，免疫稳定(immune homeostasis)是指免疫系统的细胞组成、免疫细胞间、细胞和细胞因子网络之间的相互作用，维持其功能状态的相对稳定。自身免疫病(autoimmune diseases)是指机体对自身抗原发生免疫反应而导致自身组织损害所引起的疾病。
免疫系统紊乱在发病中起着重要作用，免疫调节逐渐成为自身免疫病的中心环节。越来越多的研究发现，调节性T细胞(regulatory T cell，Treg)在生理和病理生理条件下对免疫调节起到关键作用，在感染、肿瘤、移植免疫及自身免疫病中均发挥着重要作用。Treg主要分为两大类：自然调节性T细胞(natural Treg，nTreg)和诱导性调节性T细胞(inducedTreg，iTreg)。Treg是一种具有免疫抑制活性的CD4 +T细胞亚群，Treg细胞通过释放细胞因子IL-10和TGF-β发挥抑制T细胞及抗原呈递细胞的功能，降低炎 性细胞因子的产生及抗体分泌而发挥免疫效应。Foxp3是Treg的重要转录因子，其持续表达是维持Treg抑制活性的关键因素。Treg通过上调抑制性免疫细胞表面分子表达和下调活化T细胞的相关基因表达，发挥免疫调节作用。初始CD4 +T细胞在受到外来抗原刺激后，在共刺激信号的作用下活化，分化成不同亚型的效应T细胞。初始CD4 +T细胞在TGF-β的单独诱导下，可以分化为Treg细胞，当IL-6同时存在时，能够诱导RORγt的表达，抑制Treg细胞产生，促进初始CD4 +T细胞分化成Thl7细胞。
当免疫稳态发生紊乱时，Treg细胞与其他免疫细胞如Thl7细胞数量水平失衡，通过提高Treg细胞数量及其Foxp3因子表达水平调整Treg细胞与其他免疫细胞如Thl7细胞数量水平为治疗免疫稳态紊乱提供了新途径。鉴于此，特提出本申请。
发明内容
本申请的首要发明目的在于提出甘露糖在用于制备治疗自身免疫性疾病的药物、食品、保健品或化妆品中的应用。
本申请的第二发明目的在于提出甘露糖在用于制备用于维护免疫稳态的药物、食品、保健品或化妆品中的应用。
本申请的第三发明目的在于提出甘露糖在用于制备提高Treg细胞数量及其Foxp3因子表达水平的药物、食品、保健品或化妆品中的应用。
为了完成本申请的目的，采用的技术方案为：
本申请涉及甘露糖在用于制备治疗自身免疫性疾病的药物、食品、保健品或化妆品中的应用。
优选的，所述自身免疫性疾病为由于Treg细胞数量降低引起的自身免疫性疾病。
优选的，所述自身免疫性疾病选自红斑狼疮、甲状腺功能亢进、I型或II型糖尿病及其并发症、IgA肾炎、口眼干燥综合征、类风湿性关节炎、强直性脊柱炎、单纯性肥胖、支气管哮喘、神经性皮炎、口腔溃疡、溃疡性结肠炎、牛皮藓、白癜风、白塞氏病、自身免疫性湿疹、自身免疫性葡萄膜炎、自身免疫性眼虹膜睫状体炎、自身免疫性结膜炎、自身免疫性干眼症、自身免疫性白内障、自身免疫性青光眼、过敏性鼻炎、肠易激综合征、皮肤瘙痒。
本申请还涉及甘露糖在用于制备用于维护免疫稳态的药物、食品、保健品或化妆品中的应用。
优选的，由所述免疫系统稳态失衡引起的疾病选自红斑狼疮、甲状腺功能亢进、I 型或II型糖尿病及其并发症、IgA肾炎、口眼干燥综合征、类风湿性关节炎、强直性脊柱炎、单纯性肥胖、支气管哮喘、神经性皮炎、口腔溃疡、溃疡性结肠炎、牛皮藓、白癜风、白塞氏病、自身免疫性湿疹、自身免疫性葡萄膜炎、自身免疫性眼虹膜睫状体炎、自身免疫性结膜炎、自身免疫性干眼症、自身免疫性白内障、自身免疫性青光眼、过敏性鼻炎、肠易激综合征、皮肤瘙痒。
其中，在本申请中自身免疫性干眼症是指由自身免疫性因素影响所致的、或者由自身免疫性疾病所引起的干眼症；自身免疫性白内障是指由自身免疫性因素影响所致的、或者由自身免疫性疾病所引起的白内障；自身免疫性青光眼是指由自身免疫性因素影响所致的、或者由自身免疫性疾病所引起的青光眼。
本申请还涉及甘露糖在用于制备提高Treg细胞数量及其Foxp3因子表达水平的药物、食品、保健品或化妆品中的应用。
上述应用中的甘露糖优选为D-甘露糖。
本申请的技术方案至少具有以下有益的效果：
本申请经研究发现，甘露糖在Treg细胞与其他免疫细胞如Thl7细胞数量水平发生失衡时，对增加Treg细胞数量及其Foxp3因子表达水平、进而调整Treg细胞与其他免疫细胞如Thl7细胞数量水平具有一定的作用，从而可将其用于Treg细胞与其他免疫细胞如Thl7细胞数量水平失衡的药物、食品、保健品或化妆品，对由Treg细胞与其他免疫细胞如Thl7细胞数量水平失衡引发的自身免疫性疾病以及由Treg细胞与其他免疫细胞如Thl7细胞数量水平失衡引发的免疫稳态失衡具有治疗及辅助治疗的效果。
附图说明：
图1为干燥综合症动物模型中所用抗原蛋白的制备流程。
下面结合具体实施例，进一步阐述本申请。应理解，这些实施例仅用于说明本申请而不用于限制本申请的范围。
具体实施方式
本申请涉及甘露糖在用于制备治疗自身免疫性疾病的药物、食品、保健品或化妆品中的应用。本申请中的甘露糖优选为D-甘露糖。
在本申请中，自身免疫性疾病(autoimmune diseases)是指机体对自身抗原发生免 疫反应而导致自身组织损害所引起的疾病，包括有红斑狼疮、甲状腺功能亢进、由于Treg细胞数量降低引起的I型或II型糖尿病及其并发症、IgA肾炎、口眼干燥综合征、类风湿性关节炎、强直性脊柱炎、由于Treg细胞数量降低引起的单纯性肥胖、支气管哮喘、神经性皮炎、口腔溃疡、溃疡性结肠炎、牛皮藓、白癜风、白塞氏病、自身免疫性湿疹、自身免疫性葡萄膜炎、自身免疫性眼虹膜睫状体炎、自身免疫性结膜炎、过敏性鼻炎。而当发生自身免疫性疾病时，而本申请通过发现，当甘露糖对病理状态下的Treg细胞具有刺激作用，可促进Treg细胞水平提高，从而可以调整Treg细胞与Th17细胞的平衡，维护免疫稳定，从而对自身免疫性疾病起到治疗及辅助治疗作用。
在用于制备治疗自身免疫性疾病的药物时，甘露糖可制备成多种剂型，例如口服制剂、注射制剂及外用制剂，口服制剂选自片剂、口含片、崩解剂、口服液、胶囊等剂型，注射制剂可选自水针或冻干粉针剂，外用制剂可选自外用水剂、洗液、凝胶、喷剂、贴剂、滴眼剂、膏剂或汀剂。
在用于制备治疗自身免疫性疾病的食品时，甘露糖可制备成多种食品，如软糖、硬糖、糕点、果冻、饮料等。
在用于制备治疗自身免疫性疾病的保健品时，甘露糖可制备成多种剂型保健品，如片剂、口含片、崩解剂、口服液、胶囊、口服液等。
在用于制备治疗自身免疫性疾病的化妆品时，甘露糖可制备成多种形态的化妆品，如水剂、洗液、凝胶、喷剂、贴剂、膏剂等。
制备治疗自身免疫性疾病的药物、食品、保健品或化妆品中，甘露糖的用量为0.001～200g/天。
本申请还涉及甘露糖在用于制备用于维护免疫稳态的药物、食品、保健品或化妆品中的应用。经近期研究发现，红斑狼疮、甲状腺功能亢进、I型或II型糖尿病及其并发症、慢性溃疡性结肠炎、IgA肾炎、口眼干燥综合征、类风湿性关节炎、强直性脊柱炎、单纯性肥胖、支气管哮喘、神经性皮炎、口腔溃疡、溃疡性结肠炎、牛皮藓、白癜风、白塞氏病、自身免疫性湿疹、自身免疫性葡萄膜炎、自身免疫性眼虹膜睫状体炎、自身免疫性结膜炎、自身免疫性葡萄膜炎、自身免疫性眼虹膜睫状体炎、自身免疫性结膜炎、自身免疫性干眼症、自身免疫性白内障、自身免疫性青光眼、过敏性鼻炎、肠易激综合征、皮肤瘙痒。其中，自身免疫性干眼症是指由自身免疫性因素影响所致的、或者由自身免疫性疾病所引起的干眼症；自身免疫性白内障是指由自身免疫性因素影响所致的、或者由自身免疫性疾病所引起的白内障；自身免疫性青光眼是 指由自身免疫性因素影响所致的、或者由自身免疫性疾病所引起的青光眼。这些疾病均是由免疫稳态发生紊乱造成的。甘露糖可通过调节Treg细胞的水平，维护免疫稳态，从而对这些疾病起到治疗及辅助治疗作用。
在用于制备用于维护免疫稳态的药物时，甘露糖可制备成多种剂型，例如口服制剂、注射制剂及外用制剂，口服制剂选自片剂、口含片、崩解剂、口服液、胶囊等剂型，注射制剂可选自水针或冻干粉针剂，外用制剂可选自外用水剂、洗液、凝胶、喷剂、贴剂、滴眼剂、膏剂或汀剂。在用于制备用于维护免疫稳态的食品时，甘露糖可制备成多种食品，如软糖、硬糖、糕点、果冻、饮料等。
在用于制备维护免疫稳态的保健品时，甘露糖可制备成多种剂型保健品，如片剂、口含片、崩解剂、口服液、胶囊、口服液等。
在用于制备维护免疫稳态的化妆品时，甘露糖可制备成多种形态的化妆品，如水剂、洗液、凝胶、喷剂、贴剂、膏剂等。
制备治疗用于维护免疫稳态的药物、食品、保健品或化妆品中，甘露糖的用量为0.001～200g/天。
本申请还涉及甘露糖在用于制备提高Treg细胞数量及其Foxp3因子表达水平的药物、食品、保健品或化妆品中的应用。
Foxp3因子是叉头状转录因子家族的成员，为Treg细胞发育和发挥功能的关键调节因子。可以通过与染色体结合后，调节多种基因的表达和功能，进而控制Treg细胞的发育和功能。研究表明Foxp3是CD4 +CD 25 +Treg发育和功能的关键分子，该CD4 +Treg能够抑制TCR介导的传统C D4 +T细胞增殖和产生细胞因子，在维持外周耐受、防止自身免疫性疾病中发挥重要作用。通过一些功能获得(gain of function)实验研究，直接证明了Foxp3与天然Treg细胞的关系，Foxp3的表达对于天然T细胞获得Treg细胞表型和功能是必需的，通过功能失去(loss of function)实验表明Foxp3是Treg细胞发育不可缺少的。Foxp3在Treg细胞的发育和功能上是必需的，且Foxp3仅在CD4 +CD25 +T细胞中表达，在天然CD4 +CD 25 -T细胞和非成熟的胸腺细胞中不表达，在B细胞和CD8 +T细胞中也无明显表达，可将Foxp3看作是Treg细胞的特异性标志物，将Foxp3阳性T细胞看作Treg细胞。甘露糖可通过提高Treg细胞数量及其Foxp3因子表达水平，维护免疫稳态，从而对由Treg细胞数量及其Foxp3因子表达水平下降引起的疾病起到治疗及辅助治疗作用。
在用于制备提高Treg细胞数量及其Foxp3因子表达水平的药物时，甘露糖可制备 成多种剂型，例如口服制剂、注射制剂及外用制剂，口服制剂选自片剂、口服液、胶囊等剂型，注射制剂可选自水针或冻干粉针剂，外用制剂可选自外用水剂、膏剂或汀剂。
在用于制备提高Treg细胞数量及Foxp3因子的表达水平的食品时，甘露糖可制备成多种食品，如软糖、硬糖、糕点、果冻、饮料等。
在用于制备提高Treg细胞数量及Foxp3因子的表达水平的保健品时，甘露糖可制备成多种保健品，如片剂、口含片、崩解剂、口服液、胶囊、口服液等。
在用于制备提高Treg细胞数量及Foxp3因子的表达水平的化妆品时，甘露糖可制备成多种形态的化妆品，如水剂、洗液、凝胶、喷剂、贴剂、膏剂等。
制备治疗提高Treg细胞水平的药物、食品、保健品或化妆品中，甘露糖的用量为0.001～200g/天。
申请人发现，D-甘露糖在多种造成Th17/Treg细胞水平失衡的动物模型中，具有提高Treg细胞水平，维持免疫稳态的效果。例如在类风湿性关节炎、红斑狼疮、支气管哮喘、糖尿病、肥胖、甲状腺功能亢进、IgA肾炎、口眼干燥综合征、神经性皮炎、口腔溃疡、溃疡性结肠炎、牛皮藓、自身免疫性湿疹、自身免疫性葡萄膜炎、免疫性结膜炎、干眼症、过敏性鼻炎、肠应激综合征、白内障等动物模型中，在给予模型动物7～28天的D-甘露糖后，Treg细胞水平得到提高，并且模型动物的临床症状得到了一定程度的缓解。下面以动物模型为例进一步说明本申请的技术效果。
实施例1：支气管哮喘动物模型实验
1材料与方法
1.1动物分组及哮喘模型的建立
清洁级Balb/c小鼠购自山东大学实验动物中心，饲养于专用消毒饲养盒内，恒定室温(21～25℃)，过滤空气，湿度50％～65％，光照10～12h/d，饲料、水皆经消毒处理，更换饲料等操作均在超净台内进行，整个实验过程按动物保护和使用指导方针进行。
将小鼠随机分为对照组、哮喘组和治疗组(每组10只)：
(1)哮喘组于小鼠第6周、第8周给予卵蛋白(OVA)明矾佐剂(Sigma公司)200μl腹腔注射致敏，第9周(即第2次致敏后1周)给予1％卵蛋白(OVA)高频雾化吸入进行激发，每天1次，每次30min，连续7d。进行模型验证和细胞统计。
(2)治疗组小鼠造模实验同哮喘组，在经哮喘组验证造模成功后，喂D-甘露糖水(1.0M)自由饮用，连续21天后，进行细胞统计。
(3)对照组在造模实验过程中的相同时间给予单纯明矾佐剂(不含OVA)致敏及PBS雾化吸入激发。
1.2支气管肺泡灌洗
将小鼠麻醉处死，仰卧位固定暴露气管，用留置针进行气管插管并固定，用冷PBS灌洗，每次0.5ml，共1ml，灌洗液细胞计数后离心，取细胞沉渣进行瑞氏染色细胞分类计数。
1.3肺组织Foxp3 +Treg及Th17细胞检测
用PBS从小鼠右心室灌注，去除肺血管血液，离体肺组织剪碎后在1ml含0.002g(0.2％)胶原蛋白酶I(Sigma-Aldrich)的PBS中孵育15min，将肺组织置于300目不锈钢筛网上并用眼科剪将其剪碎，收集培养皿中单细胞悬液，2500r/min离心5min，去上清，加入2ml红细胞裂解液室温孵育8min，PBS洗2次离心去上清，再用含10％胎牛血清的1640培养基将细胞制成单细胞悬液，加入50ng/ml PMA(Sigma-Aldrich)，500ng/ml离子霉素(Sigma-Aldrich)及0.7μl/ml GolgiPlug(BD Biosciences)在37℃、5％CO 2孵箱中共同孵育4～6h。
收集孵育后的细胞离心去上清，混匀后加入荧光标记抗体，加入Hamster antimouse CD3-Pecy7(BD Biosciences)、Ratantimouse CD4-FITC(BD Biosciences)，室温避光孵育30min，用PBS洗2次离心去上清，固定剂固定30min，PBS洗1次离心去上清，加入通透液室温孵育5min离心去上清，混匀后加Rat antimouse IL-4-APC(BDBiosciences)、Rat antimouse IL-17-PE(BDBiosciences)、Rat antimouse IFN-γ-PerCPcy5.5(BDBioscience)，室温避光孵育30min，离心去上清后加入200μl PBS混匀后用流式细胞仪(FACSClibur,BDBiosciences)检测。
Foxp3 +Treg细胞的检测：加入Rat antimouse CD4-FITC(BDBiosciences)Rat antimouse CD25-PE(BDBiosciences)室温避光孵育30min，用PBS洗2次去上清，固定剂固定30min，用PBS洗1次，加入通透液5min，加antimouse Foxp3-PEcy5(eBBiosciences)，室温避光孵育30min，标记后的细胞上流式细胞仪型(FAC-SClibur,BDBiosciences)检测。
1.3统计学分析数据用均数±标准差表示，采用Graphad prism5.0统计软件，两组间均数比较采用t检验，以P<0.05为差异有统计学意义。
2结果
2.1小鼠支气管肺泡灌洗液细胞计数和分类计数比较结果
结果如表1所示，正常小鼠支气管肺泡灌洗液中主要为巨噬细胞和淋巴细胞，尤以前者为主，而嗜酸性粒细胞和中性粒细胞含量甚少。哮喘组小鼠支气管肺泡灌洗液中细胞总数、淋巴细胞、嗜酸性粒细胞、中性粒细胞比例均明显高于对照组(P<0.001)，提示OVA组小鼠气道炎症较对照组明显。显示造模成功。
表1：支气管肺泡灌洗液细胞总数和白细胞分类
Figure PCTCN2017115136-appb-000001
*与对照组相比P<0.001。
2.2小鼠肺组织Foxp3 +Treg、Th17细胞、Foxp3 +Treg/Th17检测结果
结果如表2所示：
表2：
细胞 对照组 哮喘组 治疗组
Treg细胞百分比 (7.67±0.44)％ (4.87±0.35)％ (6.87±0.65)％ Δ
Th17细胞百分比 (1.07±0.07)％ (1.74±0.17)％* (1.21±0.08)％ Δ
Treg/Th17 7.38±0.71 3.02±0.49** 5.68±0.67 Δ
*与对照组相比P<0.01；**与对照组相比P<0.01；Δ与哮喘组相比P<0.01。
根据支气管哮喘动物模型发现，在对动物模型应用D-甘露糖后，Treg细胞水平得到提高，同时治疗组的临床症状也得到一定程度改善，同时其白细胞水平也显著下降。
实验例2：关节炎动物模型实验
1材料与方法
1.1动物分组及CIA动物模型(胶原诱导的大鼠关节炎模型)的建立
Wistar大鼠(雄性)，体重100g左右，鼠龄4周，购自山东大学实验动物中心。饲养条件同实施例1，整个实验过程按动物保护和使用指导方针进行。将大鼠随机分为对照组、CIA模型组和治疗组(每组20只)：
(1)CIA模型组：实验前将7ml牛II型胶原(collagen type II，CII含乙酸，浓度 为2rng/ml)，缓慢滴加至等体积的弗氏完全佐剂(complete freund's adjuvant，CFA，Sigma公司)中充分乳化，II型胶原终浓度为1mg/ml。将制备好的胶原和佐剂混合物于大鼠尾根部、皮内注射，第0天初次免疫200μg(0.2ml)，第7天于对侧尾根部加强免疫200μg(0.2ml)。
初次免疫：RA模型组给均予含有牛II型胶原1mg/ml的乳化液0.2ml，正常对照组大鼠皮内注射等量的生理盐水。
加强免疫：初次免疫动物1周后，RA模型组给予含有牛II型胶原Img/ml的乳化液0.2ml，正常对照组大鼠皮内注射等量的生理盐水。
初次免疫后每周进行关节炎指数(arthritis index，AI)评定：0分＝无肿胀或有红斑等关节炎表现；1分＝1-2个祉间关节受累；2分＝3-4个祉间关节或1个更大关节受累；3分＝大于4个关节受累；4分＝全爪严重的关节炎；每爪4分，共计16分。因为病变主要累及后足,所以6-8分即为严重关节炎，初次免疫前观察1次，初次免疫后急性期前6周每3日观察一次，此后慢性期每周观察1次。初次免疫大鼠后9～21天，实验组的45只wistar大鼠中有42只造模成功，用于后续实验。
(2)治疗组：将造模成功的大鼠20只，喂D-甘露糖水(1.0M)自由饮用，连续21天后，进行细胞统计。
(3)对照组在造模实验过程中的相同时间给予生理盐水。
1.2脾组织Foxp3 +Treg及Th17细胞检测
将大鼠腹腔注射过量戊巴比妥钠处死，无菌条件下取脾脏，用PBS缓冲液冲洗后用眼科剪剪成小块，于200目钢筛上研磨滤过，2000r/min离心5min，弃上清，加入红细胞裂解液5mL混匀，静置5min，2000r/min离心5min，弃上清，PBS缓冲液洗1～2遍，得到脾细胞。
按实施例1中方法采用流式细胞仪进行检测。
1.3统计学分析数据用均数±标准差表示，采用Graphad prism5.0统计软件，两组间均数比较采用t检验，以P<0.05为差异有统计学意义。
2结果 检测结果如表3所示：
表3：
细胞 对照组 CIA模型组 治疗组
Treg细胞百分比 (0.45±0.04)％ (0.32±0.32)％** (0.40±0.03)％ Δ
Th17细胞百分比 (0.50±0.03)％ (0.98±0.03)％* (0.71±0.03)％ Δ
Treg/Th17 0.91±0.01 0.33±0.21** 0.56±0.04 Δ
注：*与对照组相比P<0.01；**与对照组相比P<0.01；Δ与模型组相比P<0.01。
根据CIA动物模型发现，在对动物模型应用D-甘露糖后，Treg细胞水平得到提高，治疗组的临床症状也得到一定程度改善。
通过该动物实验模型同时也可证实，D-甘露糖对强直性脊柱炎(类风湿性脊柱炎)也具有一定治疗作用。
实验例3：NOD动物模型实验
1材料与方法
1.1动物分组
8周龄雌性非肥胖型糖尿病(nonobese diabetic，NOD)小鼠(40只)，体重(20.0±2.0)g，从上海斯莱克实验动物中心购买。饲养于专用消毒饲养盒内，恒定室温(21～25℃)，过滤空气，湿度50％～65％，光照10～12h/d，饲料、水皆经消毒处理，更换饲料等操作均在超净台内进行，整个实验过程按动物保护和使用指导方针进行。
将NOD小鼠随机分为对照组、治疗组(每组20只)：治疗组喂D-甘露糖水(1.2M)，连续21天后，进行细胞统计。
1.2脾组织Foxp3 +Treg及Th17细胞检测
采用实施例2中方法取小鼠脾组织细胞，采用流式细胞仪进行检测。
1.3统计学分析数据用均数±标准差表示，采用Graphad prism5.0统计软件，两组间均数比较采用t检验，以P<0.05为差异有统计学意义。
2结果 检测结果如表4所示：
表4：
细胞 NOD模型组 治疗组
Treg细胞百分比 (0.34±0.03)％ (0.48±0.12)％ Δ
Th17细胞百分比 (0.90±0.15)％ (0.70±0.21)％ Δ
Treg/Th17 0.38±0.12 0.68±0.18 Δ
注：Δ与模型组相比P<0.01。
根据NOD动物模型发现，在对动物模型应用D-甘露糖后，Treg细胞水平得到提高，治疗组的临床症状也得到一定程度改善。
实施例4：溃疡性结肠炎动物模型实验
1材料与方法
1.1动物分组及UC动物模型(溃疡性结肠炎模型)的建立
健康成年Sprague-Dawley(SD)大鼠，雌雄各半，200g左右，购自山东大学实验动物中心，饲养条件同实施例1，整个实验过程按动物保护和使用指导方针进行。
将大鼠随机分为对照组、UC模型组和治疗组(每组20只)：
(1)模型组：
50g DSS(葡聚糖硫酸酯钠)加入1000ml蒸馏水中，充分溶解，配成5％DSS溶液，每日根据实际饮用量新鲜配制，供大鼠自由饮用1周。大鼠毛色干枯，精神欠活跃，反应欠灵活，均在第2～4天开始出现腹泻、血便、体质量减轻等症状。
(2)治疗组：将造模成功的大鼠20只，喂D-甘露糖水(1.0M)自由饮用，连续21天后，进行细胞统计。
(3)对照组在造模实验过程中的相同时间给予生理盐水。
1.2脾组织Foxp3 +Treg及Th17细胞检测
采用实施例3中方法取小鼠脾组织细胞进行检测，采用流式细胞仪进行检测。
1.3统计学分析数据用均数±标准差表示，采用Graphad prism5.0统计软件，两组间均数比较采用t检验，以P<0.05为差异有统计学意义。
2结果 检测结果如表5所示：
表5：
细胞 对照组 UC模型组 治疗组
Treg细胞百分比 (0.43±0.05)％ (0.31±0.23)％** (0.39±0.12)％ Δ
Th17细胞百分比 (0.51±0.03)％ (0.89±0.12)％** (0.65±0.21)％ Δ
Treg/Th17 0.84±0.01 0.35±0.14** 0.60±0.18 Δ
注：*与对照组相比P<0.01；**与对照组相比P<0.01；Δ与模型组相比P<0.01。
根据UC动物模型发现，在对动物模型应用甘露糖后，Treg细胞水平得到提高，治疗组的临床症状也得到一定程度改善。
实施例5：红斑狼疮动物模型实验
1材料与方法
1.1动物分组
SPF级MRL/lpr自发系统性红斑狼疮狼疮样模型小鼠40只，雌性，体重20g左右，3月龄，购自山东大学实验动物中心。实验全程于SPF级验动物室封闭饲养。将小鼠随机分为对照组和治疗组(每组20只)：
(1)治疗组：将小鼠喂D-甘露糖水(1.0M)自由饮用，连续21天后，进行细胞统计。
(2)对照组在造模实验过程中的相同时间给予生理盐水。
1.2脾组织Foxp3 +Treg及Th17细胞检测
采用实施例3中方法取小鼠脾组织细胞进行检测，采用流式细胞仪进行检测。
1.3统计学分析数据用均数±标准差表示，采用Graphad prism5.0统计软件，两组间均数比较采用t检验，以P<0.05为差异有统计学意义。
2结果 检测结果如表6所示：
表6：
细胞 模型组 治疗组
Treg细胞百分比 (0.32±0.04)％ (0.41±0.10)％ Δ
Th17细胞百分比 (0.91±0.07)％ (0.70±0.22)％ Δ
Treg/Th17 0.35±0.13 0.58±0.16 Δ
注：Δ与模型组相比P<0.01。
根据自发系统性红斑狼疮狼疮样模型发现，在对动物模型应用D-甘露糖后，Treg细胞水平得到提高，同时治疗组的临床症状也得到一定程度改善。
实施例6：银屑病动物模型实验
1材料与方法
1.1动物分组及银屑病动物模型的建立
清洁级Balb/c小鼠购自山东大学实验动物中心，饲养于专用消毒饲养盒内，饲养条件同实施例1，整个实验过程按动物保护和使用指导方针进行。
将小鼠随机分为对照组、模型组和治疗组(每组20只)：
(1)模型组：
将小鼠电动剃须刀剪毛后，用5％咪喹莫特乳膏涂抹于除空白组外其余每只小鼠背部裸露皮肤，50mg/只，1/日建立银屑病动物模型。与正常对照组相比，咪喹莫特乳膏涂抹1d后模型组皮肤即出现红斑；2～3d出现鳞屑，4～5d最严重；6d皮损稍好转，7～ 8d皮肤再次加重并增厚。病理切片示皮肤出现角化过度、角化不全、棘层肥厚和角质层内多形核细胞浸润等类似人类银屑病的组织学改变。
(2)治疗组：将造模成功小鼠喂D-甘露糖水(1.0M)自由饮用，连续21天后，进行细胞统计。
(3)对照组在造模实验过程中的相同时间给予生理盐水。
1.2脾组织Foxp3 +Treg及Th17细胞检测
采用实施例3中方法取小鼠脾组织细胞进行检测，采用流式细胞仪进行检测。
1.3统计学分析数据用均数±标准差表示，采用Graphad prism5.0统计软件，两组间均数比较采用t检验，以P<0.05为差异有统计学意义。
2结果 检测结果如表7所示：
表7：
细胞 对照组 模型组 治疗组
Treg细胞百分比 (0.40±0.05)％ (0.30±0.23)％** (0.38±0.12)％ Δ
Th17细胞百分比 (0.50±0.03)％ (0.90±0.12)％** (0.67±0.21)％ Δ
Treg/Th17 0.80±0.04 0.30±0.19** 0.57±0.18 Δ
注：*与对照组相比P<0.01；**与对照组相比P<0.01；Δ与模型组相比P<0.01。
根据银屑病模型发现，在对动物模型应用D-甘露糖后，Treg细胞水平得到提高，治疗组的临床症状也得到一定程度改善。
实施例7：复发性口疮动物模型实验
1材料与方法
1.1动物分组及RAU(recurrent aphthous ulcertion，复发性口疮)动物模型的建立
Wistar大鼠，雌雄各半，体重180～200g，饲养条件同实施例1，整个实验过程按动物保护和使用指导方针进行。
将大鼠随机分为对照组、模型组和治疗组(每组20只)：
(1)模型组：
将30只Wistar大鼠麻醉后剥取口腔黏膜，加入0.1mol/L，pH7.4的PBS缓冲液制成组织匀浆-60℃低温冰箱保存备用。免疫动物时，将上述组织匀浆与弗氏佐剂等比例混合，按佐剂说明书操作制成抗原乳化剂(放置后不分层)。
将40只大鼠(雌雄各半)脊柱两侧剪去鼠毛，碘伏消毒后每侧皮下注射0.1mL抗原 乳化剂，每周注射1次，共注射6次。观察大鼠口腔粘膜变化。实验大鼠于最后一次注射后第2天口腔黏膜开始局部充血，注射后第6天所有实验大鼠均出现了溃疡，溃疡多发生在唇、颊黏膜、口底，溃疡呈椭圆形或圆形，边缘整齐，表面有灰黄色假膜，直径1～2mm，一般3～5d自行愈合，后又复发。显示造模成功，
(2)治疗组：将造模成功大鼠喂D-甘露糖水(1.2M)自由饮用，连续21天后，进行细胞统计。
(3)另20只未注射抗原大鼠为正常对照组。
1.2脾组织Foxp3 +Treg及Th17细胞检测
采用实施例3中方法取大鼠脾组织细胞进行检测，采用流式细胞仪进行检测。
1.3统计学分析数据用均数±标准差表示，采用Graphad prism5.0统计软件，两组间均数比较采用t检验，以P<0.05为差异有统计学意义。
2结果 检测结果如表8所示：
表8：
细胞 对照组 RAU模型组 治疗组
Treg细胞百分比 (0.46±0.04)％ (0.30±0.23)％** (0.41±0.14)％ Δ
Th17细胞百分比 (0.53±0.05)％ (0.97±0.11)％** (0.70±0.13)％ Δ
Treg/Th17 0.87±0.01 0.31±0.07* 0.59±0.06 Δ
注：*与对照组相比P<0.01；**与对照组相比P<0.01；Δ与模型组相比P<0.01。
根据RAU模型发现，在对动物模型应用D-甘露糖后，Treg细胞水平得到提高，治疗组的临床症状也得到一定程度改善。
实施例8：食源性肥胖动物模型实验
1材料与方法
1.1动物分组及食源性肥胖(DIO)动物模型的建立
50只刚断乳的健康SPF级雄性SD大鼠，体重40～50g，购自山东大学实验动物中心。饲养条件同实施例1，整个实验过程按动物保护和使用指导方针进行。
将大鼠随机分为对照组、模型组：
(1)模型组：50只，给予高能量高脂肪饲料，喂养14周，每周一测量大鼠体重，观察各组大鼠体重变化。14周后，高能饲料组动物体重最高的20只大鼠，设为DIO组；取10只DIO大鼠进行细胞统计。
(2)治疗组：将造模成功大鼠10只喂D-甘露糖水(1.0M)自由饮用，连续21天后，进行细胞统计。
(3)对照组10只大鼠给予基础大鼠饲料。
其中，标准动物饲料成份：5％脂肪，55％碳水化合物，22％蛋白质，7％灰分，5％纤维素。饲料热量为3.80kcal/g。
高能饲料成份：30％脂肪，40％碳水化合物，15％蛋白质，4％灰分，3％纤维素。饲料热量为4.76kcal/g。
1.2脾组织Foxp3 +Treg及Th17细胞检测
采用实施例3中方法取大鼠脾组织细胞进行检测，采用流式细胞仪进行检测。
1.3统计学分析
数据用均数±标准差表示，采用Graphad prism5.0统计软件，两组间均数比较采用t检验，以P<0.05为差异有统计学意义。
2结果 检测结果如表9所示：
表9：
细胞 对照组 DIO模型组 治疗组
Treg细胞百分比 (0.44±0.03)％ (0.38±0.23)％** (0.40±0.35)％ Δ
Th17细胞百分比 (0.49±0.02)％ (0.92±0.15)％** (0.76±0.32)％ Δ
Treg/Th17 0.90±0.03 0.41±0.02* 0.52±0.23 Δ
注：*与对照组相比P<0.01；**与对照组相比P<0.01；Δ与模型组相比P<0.01。
根据DIO模型发现，在对动物模型应用D-甘露糖后，Treg细胞水平得到提高，治疗组的临床症状也得到一定程度改善。
实施例9：IgA肾病动物模型实验
1材料与方法
1.1动物分组及IgA肾病动物模型的建立
选取80只雄性健康清洁级Wistar大鼠，体重200±20g饲养条件同实施例1，整个实验过程按动物保护和使用指导方针进行。适应性喂1周后，随机将大鼠分为对照组、模型组：
(1)模型组：50只，造模组给予牛血清白蛋白(Bevine serum albumin，BSA)口服灌胃，25％四氯化碳(Carbon tetrachloride，CCL4)溶液皮下注射以及0.25g/L脂多 糖(Lipopolysaccharide，LPS)尾静脉注射的方法建立IgA肾病大鼠模型，共9周。
(2)治疗组：将造模成功大鼠10只喂D-甘露糖水(1.0M)自由饮用，连续21天后，进行细胞统计。
(3)对照组10只大鼠给予生理盐水。
1.2脾组织Foxp3 +Treg及Th17细胞检测
采用实施例3中方法取大鼠脾组织细胞进行检测，采用流式细胞仪进行检测。
1.3统计学分析数据用均数±标准差表示，采用Graphad prism5.0统计软件，两组间均数比较采用t检验，以P<0.05为差异有统计学意义。
2结果 检测结果如表10所示：
表10：
细胞 对照组 IgA模型组 治疗组
Treg细胞百分比 (0.45±0.03)％ (0.35±0.23)％** (0.42±0.32)％ Δ
Th17细胞百分比 (0.50±0.02)％ (0.96±0.15)％** (0.72±0.31)％ Δ
Treg/Th17 0.90±0.03 0.36±0.21** 0.58±0.35 Δ
注：*与对照组相比P<0.01；**与对照组相比P<0.01；Δ与模型组相比P<0.01。
根据动物模型实验发现，在对动物模型应用D-甘露糖后，Treg细胞水平得到提高，治疗组的临床症状也得到一定程度改善。
实施例10：口眼干燥综合症动物模型实验
1材料与方法
1.1弗氏完全佐剂与抗原制备
清洁级Balb/c小鼠购自山东大学实验动物中心，饲养于专用消毒饲养盒内饲养条件同实施例1，整个实验过程按动物保护和使用指导方针进行。
将羊毛脂加热融化，加入液体石蜡按1:3(v:v)的比例配制后，8磅20分钟高压灭菌即为弗氏不完全佐剂(IFA)，将BCG(终浓度6mg/ml)加入冷却后的IFA,充分乳化，即得弗氏完全佐剂(CFA)。
1.2干燥综合症动物模型的制备
取出Balb/c小鼠的颌下腺，剔除淋巴结，放入PBS缓冲液中，匀浆。匀浆后用差速离心法，按照图1所示流程从1000g到10 5g将在10 5g时分离得到的组分设为组分 P5。用分光光度计按考马斯亮蓝比色法测定P5抗原蛋白含量。将P5蛋白与CFA乳化，得到最终抗原浓度为0.75mg/ml的乳化P5抗原。随机将大鼠分为对照组、模型组：
(1)模型组：小鼠两后脚掌及两侧腹股沟皮下注射乳化P5蛋白抗原，0.1ml/只脚掌，含BCG 3mg/ml，首次免疫后第1天、第7天和第21天,腹腔注射百日咳疫苗0.2ml(百日咳死菌体浓度为2.9×10 10个/毫升)，首次免疫后第21天与第35天，皮下注射乳化P5蛋白抗原加强免疫，产生类似SS的唾液腺改变及临床表现。
(2)治疗组：将造模成功小鼠10只喂D-甘露糖水(1.0M)自由饮用，连续21天后，进行细胞统计。
(3)对照组10只小鼠不做任何处理。
1.2脾组织Foxp3 +Treg及Th17细胞检测
采用实施例3中方法取小鼠脾组织细胞进行检测，采用流式细胞仪进行检测。
1.3统计学分析
数据用均数±标准差表示，采用Graphad prism5.0统计软件，两组间均数比较采用t检验，以P<0.05为差异有统计学意义。
2结果 检测结果如表11所示：
表11：
细胞 对照组 模型组 治疗组
Treg细胞百分比 (0.50±0.03)％ (0.40±0.21)％** (0.46±0.25)％ Δ
Th17细胞百分比 (0.52±0.02)％ (0.95±0.17)％** (0.82±0.22)％ Δ
Treg/Th17 0.96±0.03 0.42±0.20* 0.56±0.23 Δ
注：*与对照组相比P<0.01；**与对照组相比P<0.01；Δ与模型组相比P<0.01。
根据模型发现，在对动物模型应用D-甘露糖后，Treg细胞水平得到提高，治疗组的临床症状也得到一定程度改善。
实施例11：甲亢动物模型实验
1材料与方法
1.1动物分组及甲亢动物模型的建立
SD雄性大鼠，体重350～390g，购自山东大学实验动物中心，饲养条件同实施例1，整个实验过程按动物保护和使用指导方针进行。
(1)甲亢模型组：将左甲状腺素钠(L-T 4)200μg(商品名优甲乐，德国默克里昂制药集团生产，每片含100μg)研磨后溶解于3～5ml蒸馏水中灌胃，每日一次，21天后心率、血压、心脏重量、心肌细胞大小、心重体重比值等指标的变化与甲亢病人的心血管系统的变化基本一致，显示造模成功。
(2)治疗组：将造模成功大鼠10只喂D-甘露糖水(1.0M)自由饮用，连续21天后，进行细胞统计。
(3)对照组10只大鼠给予生理盐水。
1.2脾组织Foxp3 +Treg及Th17细胞检测
采用实施例3中方法取大鼠脾组织细胞进行检测，采用流式细胞仪进行检测。
1.3统计学分析
数据用均数±标准差表示，采用Graphad prism5.0统计软件，两组间均数比较采用t检验，以P<0.05为差异有统计学意义。
2结果 检测结果如表12所示：
表12：
细胞 对照组 模型组 治疗组
Treg细胞百分比 (0.50±0.03)％ (0.37±0.21)％** (0.42±0.23)％ Δ
Th17细胞百分比 (0.55±0.02)％ (0.90±0.13)％** (0.80±0.32)％ Δ
Treg/Th17 0.91±0.01 0.41±0.02* 0.53±0.26 Δ
注：*与对照组相比P<0.01；**与对照组相比P<0.01；Δ与模型组相比P<0.01。
根据动物模型实验发现，在对动物模型应用D-甘露糖后，Treg细胞水平得到提高，治疗组的临床症状也得到一定程度改善。
实施例12：接触性皮炎动物模型实验
1材料与方法
1.1动物分组及接触性皮炎动物模型的建立
清洁级Balb/c小鼠购自山东大学实验动物中心，饲养于专用消毒饲养盒内，饲养条件同实施例1，整个实验过程按动物保护和使用指导方针进行。
将小鼠随机分为对照组、模型组和治疗组(每组20只)：
(1)模型组：将地塞米松片先用酒精溶解后，配置成0.2mg/ml的溶液。实验第0天小鼠背部剃毛，第1d和第2d用5-异硫氰酸荧光素(FITC)1％(丙酮：邻苯二甲酸 二丁酯＝1：1)涂于小鼠背部剃毛部位进行致敏，第7d用FITC0.5％(丙酮：邻苯二甲酸二丁酯＝1：1)涂于小鼠左耳激发。
(2)治疗组：从实验第3天将造模小鼠10只喂D-甘露糖水(1.0M)自由饮用，连续21天后，进行细胞统计。
(3)对照组10只小鼠不做任何处理。
1.2脾组织Foxp3 +Treg及Th17细胞检测
采用实施例3中方法取小鼠脾组织细胞进行检测，采用流式细胞仪进行检测。
1.3统计学分析数据用均数±标准差表示，采用Graphad prism5.0统计软件，两组间均数比较采用t检验，以P<0.05为差异有统计学意义。
2结果 检测结果如表13所示：
表13：
细胞 对照组 模型组 治疗组
Treg细胞百分比 (0.45±0.03)％ (0.38±0.23)％** (0.42±0.24)％ Δ
Th17细胞百分比 (0.50±0.02)％ (0.96±0.17)％** (0.72±0.31)％ Δ
Treg/Th17 0.90±0.03 0.40±0.22* 0.58±0.28 Δ
注：*与对照组相比P<0.01；**与对照组相比P<0.01；Δ与模型组相比P<0.01。
根据动物模型实验发现，在对动物模型应用D-甘露糖后，Treg细胞水平得到提高，治疗组的临床症状也得到一定程度改善。
实施例13：湿疹动物模型实验
1材料与方法
1.1动物分组及湿疹动物模型的建立
SD雄性大鼠，体重350～390g，购自山东大学实验动物中心，饲养条件同实施例1，整个实验过程按动物保护和使用指导方针进行。
将大鼠随机分为对照组、模型组和治疗组(每组20只)：
(1)模型组：致敏前一天，背部3cm×3cm区域用脱毛膏脱毛，次日脱毛区外涂5％2,4-二硝基氯苯(DNCB)50μL致敏第1次；2周后，背部脱毛3cm×3cm，次日，外涂0.1％2,4-二硝基氯苯(DNCB)100μL激发，1次/周，连续4周，经病理切片验证造模成功。
(2)治疗组：从实验第3天将造模小鼠10只喂D-甘露糖水(1.0M)自由饮用， 连续21天后，进行细胞统计。
(3)对照组10只小鼠不做任何处理。
1.2脾组织Foxp3 +Treg及Th17细胞检测
采用实施例3中方法取大鼠脾组织细胞进行检测，采用流式细胞仪进行检测。
1.3统计学分析数据用均数±标准差表示，采用Graphad prism5.0统计软件，两组间均数比较采用t检验，以P<0.05为差异有统计学意义。、
2结果 检测结果如表14所示：
表14：
细胞 对照组 模型组 治疗组
Treg细胞百分比 (0.45±0.03)％ (0.39±0.23)％** (0.42±0.31)％ Δ
Th17细胞百分比 (0.50±0.02)％ (0.90±0.16)％** (0.73±0.20)％ Δ
Treg/Th17 0.90±0.03 0.43±0.21* 0.57±0.24 Δ
注：*与对照组相比P<0.01；**与对照组相比P<0.01；Δ与模型组相比P<0.01。
根据动物模型实验发现，在对动物模型应用D-甘露糖后，Treg细胞水平得到提高，治疗组的临床症状也得到一定程度改善。
实施例14：自身免疫性葡萄膜炎动物模型实验
1材料与方法
1.1动物分组及自身免疫性葡萄膜炎(EAU)动物模型的建立
Lewis雄性大鼠，体重约160～180g，购自山东大学实验动物中心，饲养条件同实施例1，整个实验过程按动物保护和使用指导方针进行。
将大鼠随机分为对照组、模型组和治疗组(每组20只)：
(1)模型组：将光感受器间维生素A类结合蛋白(IRBP，上海生工)30μg与PBS30μl混合，然后加入结核分枝杆菌(H37Ra，美国Difco公司)0.5mg、完全佐剂(CFA)30μl充分乳化后注射至大鼠单侧后足塾皮下进行免疫，制作实验性自身免疫性葡萄膜炎(EAU)模型；免疫后8到9天大鼠眼前节有炎症出现，表现为虹膜血管扩张，瞳孔异常收缩，前房轻度或中度混池；免疫后9天实验大鼠眼前节炎症达到高峰，表现为前房重度混池，瞳孔可见或不可见，伴或不伴积脓。验证造模成功。
(2)治疗组：从实验第3天将造模大鼠10只喂D-甘露糖水(1.0M)自由饮用，同时滴眼(106.5mM NaCl、26.1mM NaHCO 3、18.7mM KCl、1.0mM MgCl 2、0.5mM  NaH 2PO 4、1.1mM CaCl 2 10mM HEPE、0.2M D-甘露糖)，每日3次，每次3～5滴；连续7天后，进行细胞统计。
(3)对照组20只大鼠不做任何处理。
1.2脾组织Foxp3 +Treg及Th17细胞检测
采用实施例3中方法取大鼠脾组织细胞进行检测，采用流式细胞仪进行检测。
1.3统计学分析数据用均数±标准差表示，采用Graphad prism5.0统计软件，两组间均数比较采用t检验，以P<0.05为差异有统计学意义。、
2结果 检测结果如表15所示：
表15：
细胞 对照组 模型组 治疗组
Treg细胞百分比 (0.44±0.03)％ (0.37±0.16)％** (0.40±0.21)％ Δ
Th17细胞百分比 (0.52±0.02)％ (0.91±0.18)％** (0.71±0.25)％ Δ
Treg/Th17 0.84±0.03 0.40±0.21* 0.56±0.24 Δ
注：*与对照组相比P<0.01；**与对照组相比P<0.01；Δ与模型组相比P<0.01。
根据动物模型实验发现，在对动物模型应用D-甘露糖后，Treg细胞水平得到提高，治疗组的临床症状也得到一定程度改善。
实施例15：免疫性结膜炎动物模型实验
1材料与方法
1.1动物分组及免疫性结膜炎动物模型的建立
清洁级Balb/c小鼠购自山东大学实验动物中心，饲养条件同实施例1，整个实验过程按动物保护和使用指导方针进行。
将小鼠随机分为对照组、模型组和治疗组(每组20只)：
(1)模型组：致敏当天将含有致敏原鸡卵白蛋白OVA(美国sigma公司)100μg PBS缓冲液共0.5ml的混合物经腹腔注射使组小鼠致敏。在实验第10天，将OVA(5mg/ml)溶于Ph7.2的PBS液中滴入小鼠的双眼进行致敏原攻击以诱发免疫性结膜炎的临东症状。经病理切片验证造模成功。
(2)治疗组：从实验第3天将造模小鼠10只喂D-甘露糖水(1.0M)自由饮用，同时滴眼(106.5mM NaCl、26.1mM NaHCO 3、18.7mM KCl、1.0mM MgCl 2、0.5mM NaH 2PO 4、1.1mM CaCl 2 10mM HEPE、0.2M D-甘露糖)，每日3次，每次3～5滴；连 续7天后，进行细胞统计。
(3)对照组20只小鼠不做任何处理。
1.2脾组织Foxp3 +Treg及Th17细胞检测
采用实施例3中方法取大鼠脾组织细胞进行检测，采用流式细胞仪进行检测。
1.3统计学分析数据用均数±标准差表示，采用Graphad prism5.0统计软件，两组间均数比较采用t检验，以P<0.05为差异有统计学意义。、
2结果
检测结果如表16所示：
表16：
细胞 对照组 模型组 治疗组
Treg细胞百分比 (0.48±0.02)％ (0.42±0.16)％** (0.45±0.21)％ Δ
Th17细胞百分比 (0.64±0.05)％ (0.92±0.18)％** (0.71±0.35)％ Δ
Treg/Th17 0.75±0.03 0.46±0.21* 0.63±0.28 Δ
注：*与对照组相比P<0.01；**与对照组相比P<0.01；Δ与模型组相比P<0.01。
根据动物模型实验发现，在对动物模型应用D-甘露糖后，Treg细胞水平得到提高，治疗组的临床症状也得到一定程度改善。
实施例16：干眼症动物模型实验
1材料与方法
1.1动物分组及干眼症动物模型的建立
釆用成年雌性新西兰白兔，体重在2.5～3kg，实验前所有新西兰白兔均接受临床眼科检查，排除已有眼病动物。
将兔随机分为对照组、模型组和治疗组(每组10只)：
(1)模型组：兔麻醉后于动物超净台内摘取左侧下泪腺，将泪腺转移到盛有5mlHam’s液的离心管中,分离兔泪腺上皮细胞；自体泪腺上皮细胞转入96孔板和24孔板采用DMEM全培养基独立培养2天，采用96孔板(细胞密度3*10 6个/ml，每孔加100μl)培养的细胞用于BrdU试剂盒检测增殖；采用24孔板(细胞密度1.8×10 7个/ml，每孔400μl)培养的细胞用于混合培养。γ射线照射，使其保持抗原性。分离兔外周血淋巴细胞，将分离的淋巴细胞回收放入离心管，离心后重悬，调整细胞密度，分别配制与泪腺细胞相对应密度的细胞液。96孔板每孔加入淋巴细胞3×10 5个，孔板每孔加入 淋巴细胞1.8×10 6个。混合共培养4天后，BrdU法(BrdU试剂盒，roche公司产品)检测淋巴细胞增殖比率为3.71。模型组通过耳缘静脉回输激活的自体淋巴细胞5×10 4。与正常组相比，干眼模型组泪液分泌显著减少，泪膜破裂时间缩短荧光素钠染色显示角膜弥漫点状着色。静脉注射激活的淋巴细胞周后，泪腺和结膜组织染色均显示淋巴细胞浸润，显示造模成功。
(2)治疗组：将造模成功兔10只喂D-甘露糖水(1.0M)自由饮用，连续7天后，同时滴眼(106.5mM NaCl、26.1mM NaHCO 3、18.7mM KCl、1.0mM MgCl 2、0.5mM NaH 2PO 4、1.1mM CaCl 2 10mM HEPE、0.2M D-甘露糖)，每日3次，每次3～5滴；进行细胞统计。
(3)对照组10只兔不做任何处理。
1.2脾组织Foxp3 +Treg及Th17细胞检测
肌肉注射麻醉后，分离脾脏。取出的脾脏置于盛有RPMI-1640培养基的细胞培养皿中，碾磨。巴斯德吸管吹打均匀，过滤，2000rpm离心8min。弃上清，20ml RPMI-1640培养基重悬细胞，加入到20mlFicoll上，离心，2000rpm，20min。吸取中间雾状淋巴细胞层，20ml RPMI-1640培养基，2000rpm，20min，洗两次，l RPMI-1640培养基重悬细胞，计数。取10 5细胞，加入缓冲液30～50μl，FITC-antiRabbit CD4，PE-antihuman-FOXP3各加入2μl染色，避光放入4度冰箱，解育60分钟。取出流式管，离心1500rpm,10min，轻轻弃上清。加入缓冲液离心1500rpm，10min，重复此步骤一次，缓冲液重悬，上FACS(流式细胞仪，美国BD公司)检测。
1.3统计学分析
数据用均数±标准差表示，采用Graphad prism5.0统计软件，两组间均数比较采用t检验，以P<0.05为差异有统计学意义。
2结果 检测结果如表17所示：
表17：
细胞 对照组 模型组 治疗组
Treg细胞百分比 (6.5±0.03)％ (3.8±0.23)％** (5.2±0.31)％ Δ
注：*与对照组相比P<0.01；**与对照组相比P<0.01；Δ与模型组相比P<0.01。
根据动物模型实验发现，在对动物模型应用D-甘露糖后，Treg细胞水平得到提高，治疗组的临床症状也得到一定程度改善。
实施例17：过敏性鼻炎动物模型实验
1材料与方法
1.1动物分组及过敏性鼻炎动物模型的建立
清洁级Balb/c小鼠购自山东大学实验动物中心，饲养条件同实施例1，整个实验过程按动物保护和使用指导方针进行。
将小鼠随机分为对照组、模型组和治疗组(每组20只)：
(1)模型组：模型组用卵白蛋白辅以氢氧化铝佐剂致敏，用40％卵白蛋白氢氧化铝PBS混悬液小鼠腹腔注射200μL(含卵白蛋白20mg)，第14天用相同方法再次免疫。空白组小鼠在同一时间腹腔注射PBS溶液。免疫第21天，用1％卵白蛋白PBS溶液20μL(含卵白蛋白100μg)滴鼻激发，连续重复6d，建立小鼠过敏性鼻炎模型。
(2)治疗组：从实验第3天将造模小鼠10只喂D-甘露糖水(1.0M)自由饮用，连续14天后，进行细胞统计。
(3)对照组20只小鼠不做任何处理。
1.2脾组织Foxp3 +Treg及Th17细胞检测
采用实施例3中方法取大鼠脾组织细胞进行检测，采用流式细胞仪进行检测。
1.3统计学分析
数据用均数±标准差表示，采用Graphad prism5.0统计软件，两组间均数比较采用t检验，以P<0.05为差异有统计学意义。
2结果 检测结果如表18所示：
表18：
细胞 对照组 模型组 治疗组
Treg细胞百分比 (0.54±0.03)％ (0.32±0.27)％** (0.45±0.31)％ Δ
Th17细胞百分比 (0.76±0.02)％ (0.97±0.19)％** (0.86±0.20)％ Δ
Treg/Th17 0.71±0.03 0.33±0.24* 0.52±0.27 Δ
注：*与对照组相比P<0.01；**与对照组相比P<0.01；Δ与模型组相比P<0.01。
根据动物模型实验发现，在对动物模型应用D-甘露糖后，Treg细胞水平得到提高，治疗组的临床症状也得到一定程度改善。
实施例18：肠应激综合征动物模型实验
1材料与方法
1.1动物分组及肠应激综合征动物模型的建立
成年雄性SD大鼠60只，体重250～350g，购自山东大学实验动物中心，饲养条件同实施例1，整个实验过程按动物保护和使用指导方针进行。
将大鼠随机分为对照组、模型组和治疗组(每组20只)：
(1)模型组：模型组大鼠给予含有350～400条旋毛虫幼虫囊胞的生理盐水悬液灌胃。2周后经经病理切片验证造模成功。
(2)治疗组：从造模第2天将造模大鼠10只喂D-甘露糖水(1.0M)自由饮用，连续14天后，进行细胞统计。
(3)对照组给予等量生理盐水灌胃。
1.2外周血Foxp3+Treg细胞检测
取大鼠静脉血2ml，使用EDTA抗凝,应用荧光直接标记法及流式细胞仪检测外周血单个核细胞(PBMC)膜表面CD4、CD25。取不同荧光标记的单克隆抗体各20μl，分别加入100μl肝素抗凝的外周血，室温下孵育15min，加入红细胞裂解液，PBS洗涤2次，用流式细胞仪(BD公司，FACS Cali2bur)检测，配套软件分析数据，记录阳性细胞百分率，减去非特异对照值外周血Treg百分比测定统计分析。
1.3统计学分析
数据用均数±标准差表示，采用Graphad prism5.0统计软件，两组间均数比较采用t检验，以P<0.05为差异有统计学意义。
2结果 检测结果如表19所示：
表19：
细胞 对照组 模型组 治疗组
Treg细胞百分比 (3.90±0.15)％ (2.26±0.13)％** (3.34±0.21)％ Δ
注：*与对照组相比P<0.01；**与对照组相比P<0.01；Δ与模型组相比P<0.01。
根据动物模型实验发现，在对动物模型应用D-甘露糖后，Treg细胞水平得到提高，治疗组的临床症状也得到一定程度改善。
实施例19：白内障动物模型实验
1材料与方法
1.1动物分组及白内障动物模型的建立
未睁眼的7天龄、健康、清洁级SD大鼠60只，体重20～22g，常规母乳喂养,， 购自山东大学实验动物中心，饲养条件同实施例1，整个实验过程按动物保护和使用指导方针进行。
将大鼠随机分为对照组、模型组和治疗组(每组20只)：
(1)模型组：给10天龄大鼠按3.46mg/kg体重(即20μmol/kg体重)颈部皮下注射亚硒酸钠，隔日一次，共注射3次。首次注射亚硒酸钠30分钟内，按0.1mg/10g体腹腔注射生理盐水，共注射6天。经裂隙灯显微镜观察显示造模成功。
(2)治疗组：从造模第2天将造模大鼠10只喂D-甘露糖水(1.0M)自由饮用，同时滴眼(106.5mM NaCl、26.1mM NaHCO 3、18.7mM KCl、1.0mM MgCl 2、0.5mM NaH 2PO 4、1.1mM CaCl 2 10mM HEPE、0.2M D-甘露糖)，每日3次，每次3～5滴；连续14天后，进行细胞统计。
(3)对照组给予等量生理盐水灌胃。
1.2脾组织Foxp3 +Treg及Th17细胞检测
采用实施例3中方法取大鼠脾组织细胞进行检测，采用流式细胞仪进行检测。
1.3统计学分析数据用均数±标准差表示，采用Graphad prism5.0统计软件，两组间均数比较采用t检验，以P<0.05为差异有统计学意义。
2结果 检测结果如表20所示：
表20：
细胞 对照组 模型组 治疗组
Treg细胞百分比 (0.50±0.03)％ (0.41±0.24)％** (0.46±0.22)％ Δ
Th17细胞百分比 (0.65±0.02)％ (0.77±0.32)％** (0.72±0.32)％ Δ
Treg/Th17 0.77±0.03 0.53±0.24* 0.63±0.27 Δ
注：*与对照组相比P<0.01；**与对照组相比P<0.01；Δ与模型组相比P<0.01。
根据动物模型实验发现，在对动物模型应用D-甘露糖后，Treg细胞水平得到提高，治疗组的临床症状也得到一定程度改善。
实验例1
白癜风(vitiligo)是一种常见多发性色素脱失性皮肤病，白癜风的确切发病机制尚未完全阐明，目前有多种假说。其中自身免疫学说受到越来越多的关注。多数学者研究认为白癜风患者与正常人相比CD4 +CD25 +Treg细胞降低，尤其是泛发性白癜风患者与正常人相比CD4 +CD25 +Treg细胞显著降低。
本实验例采用流式细胞术检测白癜风患者外周血中CD4 +CD25 +Treg细胞的数量，并用逆转录聚合酶链反应(RT-PCR)方法对治疗前后白癜风患者CD4 +CD25 +Treg细胞中Foxp3mRNA的表达情况进行检测。
1材料和方法
1.1标本来源：24例白癜风患者，为皮肤科门诊患者，均符合全国色素病学组2003年12月制定的疗效判断标准。
痊愈为白斑全部消退，恢复正常肤色；显效为白斑部分消退或缩小，恢复正常肤色的面积占皮损面积≥50％；好转为白斑部分消退或缩小；无效为白斑无色素再生或范围扩大；有效＝痊愈+显效+好转；显效＝痊愈+显效。以16例正常自愿者的外周血作为正常对照。患者及家属、志愿者均对实验知情同意。
口服给药，40g/天分2次服用，28天为一疗程。
1.2仪器和试剂：流式细胞仪为美国BD公司产品(型号：FACSCalibur)。PE标记的鼠抗人CD4(IgG-PE)，FITC标记的鼠抗人CD25(IgG-PE)及同型对照γ1/γ2购于美国BD公司。Ficoll分离液、TRIZOL、Ex-Taq DNA聚合酶、dNTP、MML-V逆转录酶、M2MLV逆转录酶等购于晶美生物公司，引物由上海生工生物公司合成。
1.3方法
1.3.1外周血CD4 +CD25 +Treg细胞的检测：所有标本取自于清晨空腹静脉血，肝素抗凝。取100μL全血，分别加入抗CD4、抗CD25抗体各10μl，另设对照管，加入同型阴性对照γ1/γ2，室温孵育20～30min，加入红细胞裂解液，振荡混匀，室温孵育10min。2ml PBS以1000r/min洗涤5min，弃上清，将细胞悬浮于0.5ml PBS洗涤液中，振荡混匀后上机检测淋巴细胞亚群及CD4+CD25+Treg细胞亚群水平。
1.3.2外周血单个核细胞(MNCS)的分离：取患者及对照者肝素抗凝外周血3～5ml，PBS稀释，淋巴细胞分离液(Ficoll)分离出单个核细胞，PBS洗涤2次，用含10％小牛血清的RPMI-1640将调细胞至1×10 9/L。
1.3.3总RNA提取及cDNA的合成：按TRIzol(上海生工公司产品)试剂产品说明书进行MNCs总RNA提取，琼脂糖凝胶电泳鉴定RNA的质量，吸光度扫描计算总RNA浓度，A260/A280比值为1.8～2.0。取细胞总RNA5μg，50pmol/LOligo(dT)162μl，2.5mmol/L dNTP 3μl，DEPC处理的双蒸水补充至34μl，于65℃5min后放入冰水中并离心，然后依次加入M2MLV5×逆转录酶缓冲液10μl，0.1mmol/L DDT 4μl，M2MLV逆转录酶200U(Invitrogen公司产品)，DEPC处理的双蒸水补充至50μl，37℃60min，70℃15min后中止反应。所得cDNA于-20℃保存。
1.3.4RT-PCR检测Foxp3mRNA表达水平：参照文后文献[9]设计引物，由上海生工公司合成，用无RNA酶的灭菌双蒸水溶解为20μmol/L，-20℃保存备用。
Foxp3引物序列如下：
上游：5'-AGG CTT CAT CT G TGG CAT CAT-3'，(SEQ ID NO:1)；
下游：5'-CTT GCGGAA CTC CAG CTC AT-3'，(SEQ ID NO:2)；
预计产物长度443bp；
内参照β-actin序列如下：
上游：5'-ATG GAT GAT GAT ATC GCG CG-3'，(SEQ ID NO:3)；
下游：5'-CATGAA GCA TTTGCG GTG GAC GAT GGA GGGGCC-3'，(SEQ ID NO:4)；
预计产物长度1126bp。
反应条件为94℃变性40s、60℃退火40s、72℃延伸60s，35个循环。产物2％琼脂糖电泳，凝胶成像系统扫描。Foxp3mRNA表达水平以Foxp3/β-actin表达。
1.4统计学方法：采用SPSS 10.0软件进行数据分析，数据以x±s表示，组间差异比较采用两独立样本t检验，同组间治疗前后比较采用配对t检验，P<0.05为差异有统计学意义。
2实验结果实验结果如表21～23所示。
表21：患者与正常对照组外周血中CD4 +CD25 +Treg细胞数量的比较(x±s％)
组别 例数 CD4+T CD4 +CD25 +T CD4 +CD25 +T/CD4 +T
健康对照组 16 39.32±8.21 7.89±2.87 6.8±3.87
患者组 24 25.54±7.32 2.87±3.54* 2.3±2.76*
*P<0.05。
表22：治疗前后外周血中CD4 +CD25 +Treg细胞数量以及Foxp3mRNA的表达水平的变化(x±s)
组别 例数 CD4 +CD25 +T(％) Foxp3/β-actin(A)
治疗前 24 2.67±3.53 0.25±0.15
治疗后 24 5.54±4.12* 0.75±0.32*
*P<0.05。
表23：治疗前后白斑部分的变化(x±s)
组别 例数 有效率
患者组 24 58％
结果显示：治疗后患者CD4 +CD25 +Treg细胞的数量与治疗前相比明显增加(P<0.05)，细胞中Foxp3mRNA的表达水平较治疗前有显著升高(P<0.05)。说明经过有效治疗，随着CD4 +CD25 +Treg细胞中Foxp3mRNA的表达水平的升高，白癜风患者CD4 +CD25 +Treg细胞的免疫调节功能得到恢复，抑制自身反应性T细胞活性和功能加强，使患者的病情得到缓解。
实验例2白塞氏病
1材料和方法
1.1研究对象
2013年1月～2015年5月已明确诊断的白塞氏病患者共44例，其中女38例，男6例，年龄18～52岁。全部符合1995年第五届国际白塞氏病讨论会提出的诊断标准。其中临床表现有复发性口腔溃疡(＞3次/年)者44例，生殖器及外阴溃疡36例，结节性红斑27例，关节痛19例，眼虹膜睫状体炎或结膜炎20例。按患者就诊顺序，随机分成两组，每组22例。两组患者性别、年龄、病程及皮损等方面差异均无统计学意义(P＞0.05)，具有可比性。所有患者不伴有全身系统性疾病，就诊前未接受过免疫调节治疗，且均无既往史及家族史，全部自愿参加本研究。
1.2研究方法
患者均给予D-甘露糖40g/天分2次服用，8周为一疗程。8周后停药，停药3个月后进行疗效评判。
1.3观察疗效指标
痊愈为临床症状及体征完全消失，且实验室检查无异常，停药3个月内无症状复发；有效为临床症状及体征消失或减轻，实验室检查大部分正常，但停药3个月内有症状的复发；无效为临床症状及体征无明显改善，实验室检查全部异常。以痊愈率及有效率相加计算有效率。
2结果 实验结果如表24所示。
表24：
组别 例数 有效率
患者组 24 56％
实验例3青光眼
1材料和方法
1.1研究对象
2014年8月-2016年8月,确诊为POAG患者49例(90眼),均符合1987年中华医学会青光眼学组制定的青光眼诊断标准。年龄26～68岁,中位年龄53岁,病程为3个月～5年(中位数2年),视力为0.4～1.0。
1.2研究方法
随机分为两组，实验组患者均给予D-甘露糖40g/天分2次服用，同时滴眼(106.5mM NaCl、26.1mM NaHCO 3、18.7mM KCl、1.0mM MgCl 2、0.5mM NaH 2PO 4、1.1mM CaCl 2 10mM HEPE、0.2M D-甘露糖)，每日3次，每次3～5滴；8周后进行疗效评判。
1.3观察疗效指标:10:00或14:00单次眼压不超过15mmHg为有效。
2结果 实验结果如表25所示。
表25：
组别 例数 有效率
患者组 90 65％
本申请虽然以较佳实施例公开如上，但并不是用来限定权利要求，任何本领域技术人员在不脱离本申请构思的前提下，都可以做出若干可能的变动和修改，因此本申请的保护范围应当以本申请权利要求所界定的范围为准。

Claims (7)

  1. 甘露糖在用于制备治疗自身免疫性疾病的药物、食品、保健品或化妆品中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述自身免疫性疾病为由于Treg细胞数量降低引起的自身免疫性疾病。
  3. 根据权利要求1所述的应用，其特征在于，所述自身免疫性疾病选自红斑狼疮、甲状腺功能亢进、I型或II型糖尿病及其并发症、IgA肾炎、口眼干燥综合征、类风湿性关节炎、强直性脊柱炎、单纯性肥胖、支气管哮喘、神经性皮炎、口腔溃疡、溃疡性结肠炎、牛皮藓、白癜风、白塞氏病、自身免疫性湿疹、自身免疫性葡萄膜炎、自身免疫性眼虹膜睫状体炎、自身免疫性结膜炎、自身免疫性干眼症、自身免疫性白内障、自身免疫性青光眼、过敏性鼻炎、肠易激综合征、皮肤瘙痒。
  4. 甘露糖在用于制备用于维护免疫稳态的药物、食品、保健品或化妆品中的应用。
  5. 根据权利要求4所述的应用，其特征在于，由所述免疫系统稳态失衡引起的疾病选自红斑狼疮、甲状腺功能亢进、I型或II型糖尿病及其并发症、IgA肾炎、口眼干燥综合征、类风湿性关节炎、强直性脊柱炎、单纯性肥胖、支气管哮喘、神经性皮炎、口腔溃疡、溃疡性结肠炎、牛皮藓、白癜风、白塞氏病、自身免疫性湿疹自身免疫性葡萄膜炎、自身免疫性眼虹膜睫状体炎、自身免疫性结膜炎、自身免疫性干眼症、自身免疫性因素白内障、自身免疫性青光眼、过敏性鼻炎、肠易激综合征、皮肤瘙痒。
  6. 甘露糖在用于制备提高Treg细胞数量及其Foxp3因子表达水平的药物、食品、保健品或化妆品中的应用。
  7. 根据权利要求1～6任一权利要求所述的应用，所述甘露糖为D-甘露糖。
PCT/CN2017/115136 2016-12-10 2017-12-08 甘露糖在用于提高Treg细胞数量及其Foxp3因子表达水平的新用途 WO2018103723A1 (zh)

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