WO2024083176A1 - 治疗或预防巨噬细胞介导的疾病的药物及其应用 - Google Patents

治疗或预防巨噬细胞介导的疾病的药物及其应用 Download PDF

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WO2024083176A1
WO2024083176A1 PCT/CN2023/125342 CN2023125342W WO2024083176A1 WO 2024083176 A1 WO2024083176 A1 WO 2024083176A1 CN 2023125342 W CN2023125342 W CN 2023125342W WO 2024083176 A1 WO2024083176 A1 WO 2024083176A1
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eggs
diseases
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钱进
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安徽楚沅生物科技有限公司
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/50—Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/57—Birds; Materials from birds, e.g. eggs, feathers, egg white, egg yolk or endothelium corneum gigeriae galli
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • the present invention relates to a drug for treating or preventing macrophage-mediated diseases and its application.
  • Macrophages show different types under different stimuli, and they express more inducible nitric oxide synthase (iNOS) or arginase, and are called M1 and M2 macrophages accordingly.
  • M1 macrophages are enriched in the early inflammatory sites and activated by proinflammatory factors such as LPS, TNF ⁇ , and IFN ⁇ , and then promote the occurrence and development of inflammation by secreting inflammatory factors such as IL12, protecting the body from foreign substances.
  • M2 cells play a role in inhibiting inflammation, repairing tissues, and reconstructing tissue structures.
  • the ratio of the number of M1/M2 macrophage groups changes over time, and eventually completely eliminates the impact of inflammation.
  • M2 macrophages are closely related to anti-inflammatory response and immune homeostasis, participate in tissue repair, tissue and organ remodeling, scar formation and injury healing, and have immunosuppressive effects.
  • TLR4/NF- ⁇ B signaling pathway plays a core regulatory role in the inflammatory response.
  • NF- ⁇ B activity is inhibited by I ⁇ B; after being stimulated by cytokine receptors such as TLR and TNF receptor, I ⁇ B kinase activation will phosphorylate I ⁇ B, thereby degrading the proteasome and releasing NF- ⁇ B to transport into the nucleus, activating the corresponding gene transcription.
  • NF- ⁇ B activation can lead to the synthesis and release of inflammatory cytokines.
  • diabetic ulcers have become one of the most common chronic and refractory wounds in clinical practice. Persistent chronic inflammation is a typical feature of diabetic skin wounds. Due to the complex microenvironment of diabetic wounds, such as hypoxia, infection, ischemia, inflammation, oxidative stress, etc., diabetic ulcers tend to be prolonged and recurrent, while traditional debridement and dressing therapy has limited effect on diabetic wounds. Therefore, how to effectively and economically treat diabetic ulcers has become an urgent problem to be solved.
  • M1 macrophages are responsible for engulfing necrotic tissue and cell debris, while M2 macrophages are involved in inhibiting inflammation and promoting tissue regeneration.
  • Diabetic wounds are usually in a state of excessive inflammation, with a large number of M1 macrophages in the wound tissue, and the transformation of macrophages to M2 macrophages is blocked. It is worth noting that previous studies have shown that increasing the expression of M2 macrophages can inhibit inflammation and promote tissue regeneration. Type may be an important factor in diabetic wound repair.
  • the first aspect of the present invention provides the use of amniotic fluid in the preparation of a medicament for treating and/or preventing macrophage-mediated diseases, wherein the amniotic fluid comes from eggs with an embryonic age of 5-12 days, preferably eggs with an embryonic age of 6-11 days, more preferably eggs with an embryonic age of 7-9 days, more preferably eggs with an embryonic age of 7-8 days, or from eggs of other birds other than chickens whose developmental period corresponds to the developmental period of the eggs of the embryonic age; or from embryos of rodents with a gestational age of 8-14 days, or from embryos of other non-human mammals other than rodents whose developmental period corresponds to the developmental period of rodents with a gestational age of 8-14 days.
  • the present invention also provides the use of amniotic fluid in the preparation of a drug for treating and/or preventing diseases mediated by M1 macrophages, wherein the amniotic fluid comes from eggs with an embryonic age of 5-12 days, preferably eggs with an embryonic age of 6-11 days, more preferably eggs with an embryonic age of 7-9 days, more preferably eggs with an embryonic age of 7-8 days, or from eggs of other birds other than chickens whose developmental period corresponds to the developmental period of the eggs with the embryonic age; or from embryos of rodents with a gestational age of 8-14 days, or from embryos of other non-human mammals other than rodents whose developmental period corresponds to the developmental period of rodents with a gestational age of 8-14 days.
  • the present invention also provides the use of amniotic fluid in the preparation of a drug for treating and/or preventing diseases mediated by M2 macrophages, wherein the amniotic fluid comes from eggs with an embryonic age of 5-12 days, preferably eggs with an embryonic age of 6-11 days, more preferably eggs with an embryonic age of 7-9 days, more preferably eggs with an embryonic age of 7-8 days, or from eggs of other birds other than chickens whose developmental period corresponds to the developmental period of the eggs with the embryonic age; or from embryos of rodents with a gestational age of 8-14 days, or from embryos of other non-human mammals other than rodents whose developmental period corresponds to the developmental period of rodents with a gestational age of 8-14 days.
  • the present invention also provides the use of amniotic fluid in preparing a preparation for treating and/or preventing diseases mediated by the TLR4/NF- ⁇ B signaling pathway, wherein the amniotic fluid comes from eggs with an embryonic age of 5-12 days, preferably eggs with an embryonic age of 6-11 days, more preferably eggs with an embryonic age of 7-9 days, more preferably eggs with an embryonic age of 7-8 days, or from eggs of other birds other than chickens whose developmental period corresponds to the developmental period of the eggs with the embryonic age; or from embryos of rodents with a gestational age of 8-14 days, or from embryos of other non-human mammals other than rodents whose developmental period corresponds to the developmental period of rodents with a gestational age of 8-14 days.
  • the present invention also provides the use of amniotic fluid in preparing a preparation for inhibiting the number of M1 macrophage groups and increasing the proportion of M2 macrophage types, or in preparing a preparation for promoting the transformation of M1 macrophages into M2 macrophages, wherein the amniotic fluid is from eggs with an embryonic age of 5-12 days, preferably eggs with an embryonic age of 6-11 days, more preferably eggs with an embryonic age of 7-9 days, more preferably eggs with an embryonic age of 7-8 days, or from eggs of other birds other than chickens whose developmental period corresponds to the developmental period of the eggs of the embryonic age; or from embryos of rodents with a gestational age of 8-14 days, or Embryos from non-human mammals other than rodents whose developmental period corresponds to that of rodents with a gestational age of 8-14 days.
  • the medicament or formulation is a cell culture comprising the amniotic fluid and/or embryonic stem cells.
  • the drug or preparation is a pharmaceutical composition comprising the amniotic fluid and/or chicken embryonic stem cells and a pharmaceutically acceptable excipient.
  • the macrophage-mediated disease is selected from: hypertrophic scar; chronic obstructive pulmonary disease; tumors, such as breast cancer, liver cancer; metabolic diseases, such as severe obesity, insulin resistance, type II diabetes; inflammatory diseases, such as acute pancreatitis, atherosclerosis; cardiovascular diseases, such as myocarditis, myocardial infarction, arrhythmia; neuropathy, such as Alzheimer's disease; brain diseases, such as cerebral infarction, brain injury; ophthalmic diseases, such as autoimmune uveitis, retinopathy, keratitis, corneal transplantation, Sjögren's syndrome, uveal melanoma, Myopia; immune inflammatory diseases, such as inflammatory bowel disease, autoimmune hepatitis, asthma, alcoholic liver disease, colitis, multiple sclerosis, periodontitis; arthritis, such as rheumatoid arthritis, bone erosion, synovitis, osteoarthritis; nephritis, such as
  • the M1 macrophage-mediated disease is selected from: hypoplastic scar; chronic obstructive pulmonary disease; tumors, such as breast cancer and liver cancer; metabolic diseases, such as severe obesity, insulin resistance, and type II diabetes; inflammatory diseases, such as acute pancreatitis; coronary artery disease, such as atherosclerosis; kidney disease; obesity; cardiovascular diseases, such as myocarditis and myocardial infarction; brain diseases, such as cerebral infarction and brain injury; ophthalmic diseases, such as autoimmune uveitis, retinopathy, keratitis, corneal transplantation, Sjögren's syndrome, and uveal melanoma; immune inflammatory diseases, such as inflammatory bowel disease, autoimmune hepatitis, asthma, alcoholic liver disease, colitis, multiple sclerosis, periodontitis, and osteoarthritis; diabetes, gestational diabetes, and diabetic nephropathy.
  • hypoplastic scar such as chronic obstructive pulmonary disease
  • tumors such as breast
  • the M2 macrophage-mediated disease is selected from: hyperplastic scars in the proliferative phase; tumors, such as breast cancer and liver cancer; metabolic diseases, such as insulin resistance and type II diabetes; inflammatory diseases, such as acute pancreatitis; cardiovascular diseases, such as myocardial infarction, myocardial failure, atherosclerosis, coronary artery disease, and myocarditis; cerebral infarction; ophthalmic diseases, such as autoimmune uveitis, retinopathy, keratitis, corneal transplantation, Sjögren's syndrome, and uveal melanoma; immune inflammatory diseases, such as inflammatory bowel disease, autoimmune hepatitis, asthma, alcoholic liver disease, colitis, multiple sclerosis, periodontitis, and osteoarthritis; diabetes, gestational diabetes, and diabetic nephropathy.
  • hyperplastic scars in the proliferative phase such as breast cancer and liver cancer
  • metabolic diseases such as insulin resistance and type II diabetes
  • inflammatory diseases
  • the disease mediated by the TLR4/NF- ⁇ B signaling pathway is selected from: systemic lupus erythematosus; vascular inflammation, such as atherosclerosis, coronary heart disease; myocarditis, such as myocardial ischemia tissue inflammation, myocardial injury; hepatitis, such as liver failure, alcoholic liver injury, inflammatory immune response during alcohol metabolism; fatty liver; pneumonia, such as Acute lung injury, chronic obstructive pulmonary disease, silicosis; nephritis, such as acute kidney injury, lupus nephritis; inflammatory bowel disease, such as acute enteritis, ulcerative colitis, radiation proctitis; gastritis, such as chronic atrophic gastritis; acute respiratory tract infection, such as pneumonia, bronchitis, pharyngitis, sinusitis, otitis media; periodontitis, hyperuricemia; allergic rhinitis, allergic rhin
  • Figure 1 is a schematic diagram of ceAF alleviating LPS-stimulated RAW 264.7 cell inflammation through the TLR4/NF- ⁇ B signaling pathway.
  • each group of 4 columns of bar graphs is a group, and each group of bar graphs corresponds to CD206, Arg-1, iNOS, TNF- ⁇ , IL-6, IL-1 ⁇ , TLR4, NF- ⁇ B, and pl ⁇ B from left to right.
  • FIG2 is a schematic diagram of ceAF inducing RAW264.7 polarization toward M2 macrophages in vitro.
  • FIG3 is a schematic diagram showing how ceAF promotes wound healing in STZ-induced diabetic mice.
  • Figure 4 is a schematic diagram of ceAF improving the histological parameters of wounds in STZ-induced diabetic mice.
  • FIG5 is a schematic diagram showing that ceAF improves indicators related to wound healing in STZ-induced diabetic mice.
  • Figure 6 is a schematic diagram showing that ceAF can regulate wound-related inflammatory factors in STZ-induced diabetic mice.
  • every two columns of bar graphs form a group, and each group of bar graphs corresponds to CD206, Arg-1, iNOS, TNF- ⁇ , IL-6, and IL-1 ⁇ from left to right.
  • the present invention finds that chicken embryo amniotic fluid (ceAF) can induce macrophages to transform from M1 type to M2 type through TLR4/NF- ⁇ B signaling pathway, thereby completing the present invention.
  • ceAF chicken embryo amniotic fluid
  • the amniotic fluid can come from fowl eggs and non-human mammals.
  • Fowl eggs refer to eggs of fowl.
  • Preferred fowl are poultry, such as chickens, ducks and geese.
  • the present invention uses fowl eggs with an embryonic age of 5-20 days, preferably 6-15 days. It should be understood that the suitable embryonic age may not be the same for different fowl eggs.
  • eggs whose developmental period corresponds to the developmental period in which the eggs of the above-mentioned embryonic age are located can be used.
  • eggs with an embryonic age of 8-10 days, especially 8-9 days, may be the best.
  • ceAF refers to amniotic fluid extracted from chicken embryos at 6-8 days of incubation.
  • the amniotic fluid of poultry eggs can be obtained by conventional methods. For example, the blunt end of an egg of the corresponding embryonic age can be tapped to break the eggshell, and the eggshell can be peeled open to form a hole with a diameter of about 2 cm. Then, the shell membrane and the yolk membrane are carefully torn apart with tweezers, and care is taken not to damage the amnion. The amnion and the connected tissues enveloping the embryo are poured from the shell into a culture dish, and the amnion is pierced with a syringe to extract the amnion until the amnion is close to the embryo, thereby obtaining the amniotic fluid used in the present invention.
  • amniotic fluid can also be from non-human mammals, especially rodents, such as from mice.
  • Other non-human mammals can be common livestock, such as cattle, sheep, dogs, cats, pigs, etc.
  • amniotic fluid is from the embryo of a rodent with a gestational age of 8-14 days, or from the embryo of a non-human mammal corresponding to the developmental period of a rodent with a gestational age of 8-14 days. Conventional methods can be used to obtain amniotic fluid.
  • the abdominal cavity of a mouse with a gestational age of 8-14 days is cut open with surgical scissors, the uterus is carefully removed and cut open, and the amniotic membrane is pierced with a syringe to extract amniotic fluid until the amniotic membrane is close to the embryo, thus obtaining the amniotic fluid used for the present invention.
  • amniotic fluid can be centrifuged to separate possible impurities, such as egg yolk, etc., to obtain as pure amniotic fluid as possible.
  • the supernatant obtained after centrifugation is the amniotic fluid used in the present invention.
  • the "amniotic fluid” shown herein should refer to "pure" amniotic fluid, that is, amniotic fluid separated from poultry eggs or non-human mammal embryos that does not contain other components in poultry eggs or non-human mammal embryos and is not contaminated by exogenous substances. Pure amniotic fluid can be stored in a refrigerator below -60°C and used after thawing.
  • amniotic fluid described herein can be used as an active ingredient of a drug for in vivo administration to a subject in need thereof to promote cell growth and tissue repair in vivo.
  • an effective amount of the amniotic fluid described herein, or a pharmaceutical composition containing the amniotic fluid can be administered to a subject in need thereof.
  • the amniotic fluid from various sources described in the present invention has the same or similar biological activity, and when used in the present invention, can be used to regulate the TLR4/NF- ⁇ B signaling pathway, inhibit M1 macrophages, increase M2 macrophages, thereby treating or preventing diseases that benefit from the regulation of the TLR4/NF- ⁇ B signaling pathway and macrophage-mediated diseases.
  • the animal may be a mammal, especially a human.
  • TLRs Toll-like receptors
  • PAMPs pathogen-associated molecular patterns
  • LPS lipopolysaccharide
  • TLRs can also regulate pathological processes such as cholesterol metabolism, plate stability, cell apoptosis, inflammation and vascular remodeling, and immune response.
  • TLRs bind to ligands, they ultimately lead to the activation of NF- ⁇ B and regulate the expression of inflammation and immune-related genes.
  • NF- ⁇ B can play a role in multiple links in the formation of diseases, such as inflammatory response, foam cell formation, vascular smooth muscle proliferation and apoptosis. It regulates enzymes (such as cyclooxygenase 2) involved in early low-density lipoprotein (LDL) modification and inflammatory lipid formation. Activated NF- ⁇ B can also promote the transcription of adhesion molecules, proinflammatory factors, chemokines, growth factors and other factors, which play an important role in the occurrence and development of inflammatory diseases.
  • diseases such as inflammatory response, foam cell formation, vascular smooth muscle proliferation and apoptosis. It regulates enzymes (such as cyclooxygenase 2) involved in early low-density lipoprotein (LDL) modification and inflammatory lipid formation.
  • Activated NF- ⁇ B can also promote the transcription of adhesion molecules, proinflammatory factors, chemokines, growth factors and other factors, which play an important role in the occurrence and development of inflammatory diseases.
  • diseases mediated by the TLR4/NF- ⁇ B signaling pathway include but are not limited to: inflammatory reactions caused by neuropathy and/or vascular lesions, selected from systemic lupus erythematosus (Ji Juan. The role of abnormal activation of TLR4 signaling leading to aging of bone marrow mesenchymal stem cells in the occurrence of systemic lupus erythematosus [D]. Nantong University, 2017.); vascular inflammation, atherosclerosis (Li Hongmei, Wang Xian. Research progress on the correlation between TLR4/MyD88/NF- ⁇ B signaling pathway and atherosclerotic cardiovascular disease [J].
  • TLR4/NF- ⁇ B signaling pathway activates LncRNA RP11-20G6 regulates airway inflammation and remodeling in chronic obstructive pulmonary disease [J]. Journal of Anhui Medical University, 2022, 57(4): 586-593), silicosis (Zhu Lili.
  • nephritis such as acute kidney injury (Zhan Yun, Zhang Yingjie, Leng Bin. Astragaloside I improves lipopolysaccharide-induced acute vascular endothelial injury through the TLR4/NF- ⁇ B pathway [J]. Pharmacology and Clinic, 2018, 34(03): 77-80.DOI: 10.13412/j.cnki.zyyl.2018.03.019.), lupus nephritis (Chen Ning. Expression and significance of HMGB1/TLR/NF- ⁇ B signaling pathway in renal tissue of lupus nephritis mice [D].
  • the amniotic fluid described in this article can inhibit the activation of the TLR4/NF- ⁇ B signaling pathway, thereby treating or preventing various diseases mediated by the TLR4/NF- ⁇ B signaling pathway.
  • macrophage mediation refers to the role of the dynamic balance and imbalance of cell subsets with different functional characteristics in macrophages in the pathological process of the disease.
  • Macrophage-mediated diseases include but are not limited to hypertrophic scars (Li Zhenjiang, Li Shujun, Zhou Jian, et al. Study on macrophage activation-related factors in hypertrophic scar tissue at different stages [J]. Journal of Zunyi Medical University, 2022, 45(1): 87-91); chronic obstructive pneumonia (Xing Shigang. Study on the role of macrophages in the pathogenesis of chronic obstructive pulmonary disease [J].
  • Practical Geriatrics, 2021, 35(12): 1217-1218) brain injury (Diao Zhongji, Lei Rui, Yin Shi, Liu Hongling, Liu Qing. Analysis of the correlation between M1 macrophage levels and the severity and prognosis of coronary heart disease [J].
  • ophthalmic diseases such as autoimmune uveitis, retinopathy, corneal inflammation, corneal transplantation, Sjögren's syndrome, uveal melanoma, myopia (Qu Ruyi, Zhou Mengxian, Bi Hongsheng, Guo Dadong. Research progress on the mechanism of macrophage polarization in the occurrence and development of ophthalmic diseases [J]. New Progress in Ophthalmology, 2022, 42(3): 239-243); immune inflammatory diseases, such as inflammatory bowel disease, autoimmune hepatitis, asthma (Jia Rui, Hui Yi, Yan Shuguang (guidance), Li Jingtao.
  • nephritis such as acute kidney injury, chronic kidney disease, end-stage renal disease, proliferative glomerulonephritis, membranous nephropathy, diabetic nephropathy, Henoch-Schonlein purpura nephritis, ANCA-associated small vessel vasculitis, urinary tract infection, autosomal dominant polycystic kidney disease (Ding Na, Wang Bo, Huang Bintao, Hao Jian. Research progress on macrophage migration inhibitory factor in the pathogenesis of renal inflammatory diseases [J].
  • Macrophages include two subtypes, M1 and M2.
  • M1 macrophages are also called pro-inflammatory phenotype macrophages. They mainly phagocytize foreign substances such as bacteria and endogenous substances such as apoptotic cell fragments in the body, protecting tissues and organs from invasion by foreign substances.
  • M1 macrophages are enriched in the early inflammation site and activated by pro-inflammatory factors. They promote the occurrence and development of inflammation by secreting inflammatory factors and protect the body from invasion by foreign substances.
  • M2 cells inhibit inflammation, repair tissues, and reconstruct tissue structures. Therefore, M2 macrophages are also called anti-inflammatory phenotype or immunoregulatory macrophages.
  • the ratio of the number of M1/M2 macrophage populations changes over time, and eventually completely eliminates the effects of inflammation.
  • M1 macrophage-mediated refers to the effects of M1 macrophages in diseases, such as being activated by proinflammatory factors, secreting inflammatory factors, and protecting the body from invasion by foreign substances.
  • the diseases mediated by M1 macrophages include, but are not limited to, immune inflammatory diseases, metabolic diseases, diabetes, and tumors.
  • the disease mediated by M1 macrophages described herein is persistent chronic inflammation of diabetic wounds.
  • M2 macrophage-mediated refers to the effects of M2 macrophages in inhibiting inflammation and inflammatory factors, promoting wound healing and repair, etc. in diseases.
  • Diseases mediated by M2 macrophages include but are not limited to hypertrophic scars in the hyperplastic phase (Li Zhenjiang, Li Shujun, Zhou Jian, et al. Study on macrophage activation-related factors in hypertrophic scar tissue at different stages [J].
  • tumors such as breast cancer and liver cancer
  • metabolic diseases such as insulin resistance and type II diabetes
  • inflammatory diseases such as acute pancreatitis (Wang Yongkang, Li Jiayi, Guan Fei, Lei Jiahui. Macrophage polarization mechanism and its role in common diseases [J].
  • cardiovascular diseases such as myocardial infarction, myocardial failure, atherosclerosis, and coronary artery disease (Zhang Xiangning, Dang Guohui, Feng Juan (guidance).
  • ophthalmic diseases such as autoimmune uveitis, retinopathy, keratitis, corneal transplantation, Sjögren's syndrome, uveal melanoma (Qu Ruyi, Zhou Mengxian, Bi Hongsheng, Guo Dadong. Research progress on the mechanism of macrophage polarization in the occurrence and development of ophthalmic diseases [J]. New Progress in Ophthalmology, 2022, 42(3): 239-243); immune inflammatory diseases, such as inflammatory bowel disease, autoimmune hepatitis, asthma (Jia Rui, Hui Yi, Yan Shuguang (guidance) , Li Jingtao.
  • the diseases mediated by M2 macrophages include but are not limited to immune inflammatory diseases, metabolic diseases, diabetes and tumors.
  • the diseases mediated by M2 macrophages described herein are persistent chronic inflammation of diabetic wounds.
  • the amniotic fluid described herein treats and prevents macrophage-mediated diseases by reducing the number of M1 macrophages and increasing the proportion of M2 macrophages.
  • amniotic fluid especially avian egg amniotic fluid as described herein, more preferably chicken egg amniotic fluid is used to prevent or treat macrophage-mediated diseases.
  • the present invention also provides a pharmaceutical composition, which contains the amniotic fluid described herein, especially the amniotic fluid in poultry eggs, more preferably the amniotic fluid of eggs with an embryonic age of 5-12 days, more preferably 6-11 days, more preferably 6-9 days, and more preferably 7-8 days.
  • the pharmaceutical composition can be amniotic fluid or a lyophilized agent thereof frozen at below -60°C, such as lyophilized amniotic fluid.
  • the pharmaceutical composition may also contain other pharmaceutically acceptable carriers or excipients, such as physiological saline for injection, water for injection, or glucose injection.
  • the pharmaceutical composition contains 5-40% (v/v) or 10%-35% of amniotic fluid, preferably 15-30%.
  • composition containing the amniotic fluid described herein generally also contains a pharmaceutically acceptable excipient.
  • pharmaceutically acceptable excipient refers to a carrier, diluent and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, including but not limited to: antibiotics, humectants, pH adjusters, surfactants, carbohydrates, adjuvants, anti- Oxidants, chelating agents, ionic strength enhancers, preservatives, carriers, glidants, sweeteners, dyes/colorants, flavor enhancers, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents or emulsifiers.
  • pharmaceutically acceptable excipients may include one or more inactive ingredients, including but not limited to: stabilizers, preservatives, additives, adjuvants, sprays, compressed air or other suitable gases, or other suitable inactive ingredients used in combination with pharmacological compounds. More specifically, suitable pharmaceutically acceptable excipients may be excipients commonly used in the art for diabetic foot. In one or more embodiments, suitable pharmaceutically acceptable excipients suitable for sprays are selected from one or more of the following: water, gluconolactone, sodium benzoate, arbutin, sodium hyaluronate, niacinamide, glycerol.
  • suitable pharmaceutically acceptable excipients suitable for coatings or dressings are selected from one or more of the following: water, glycerol, panthenol, magnesium ascorbyl phosphate, niacinamide, sodium hyaluronate, phenoxyethanol, caprylyl glycol, sorbic acid.
  • the content of pharmaceutically acceptable excipients can be determined according to actual conditions in this field.
  • dressings When spraying, coating or applying drugs to wounds, dressings are generally added.
  • Local dressings are wound dressings, materials used to cover sores, wounds or other lesions.
  • passive dressings There are passive dressings, interactive dressings and bioactive dressings.
  • Wound dressings suitable for the use of the present invention are known in the art.
  • the pharmaceutical composition described herein may also contain other active ingredients that help the patient's wound recovery, including but not limited to Centella asiatica extract, rose hydrosol, licorice root extract, olive leaf extract, calendula flower extract, white willow bark extract, lavender extract, lemon fruit extract, hydrolyzed soy protein, mugwort extract, tea extract, thyme extract, purple coneflower extract, Hypericum perforatum flower/leaf extract, aloe barbadensis leaf juice powder, and yeast extract.
  • active ingredients including but not limited to Centella asiatica extract, rose hydrosol, licorice root extract, olive leaf extract, calendula flower extract, white willow bark extract, lavender extract, lemon fruit extract, hydrolyzed soy protein, mugwort extract, tea extract, thyme extract, purple coneflower extract, Hypericum perforatum flower/leaf extract, aloe barbadensis leaf juice powder, and yeast extract.
  • the present invention also provides the use of the amniotic fluid described in any embodiment of the present invention in the preparation of a preparation for treating and/or preventing diseases mediated by the TLR4/NF- ⁇ B signaling pathway.
  • the present invention also provides the use of the amniotic fluid described in any embodiment of the present invention in the preparation of a preparation for treating and/or preventing macrophage-mediated diseases.
  • the present invention also provides the use of the amniotic fluid described in any embodiment of the present invention in the preparation of a preparation for treating and/or preventing M1 macrophage-mediated diseases.
  • the present invention also provides the use of the amniotic fluid described in any embodiment of the present invention in the preparation of a preparation for treating and/or preventing M2 macrophage-mediated diseases.
  • the present invention also provides the use of the amniotic fluid described in any embodiment of the present invention in the preparation of an agent for inhibiting the number of M1 macrophage populations and increasing the proportion of M2 macrophage types.
  • the present invention also provides the use of the amniotic fluid described in any embodiment of the present invention in the preparation of a preparation that promotes the transformation of M1 macrophages to M2 macrophages.
  • the present invention provides the use of the amniotic fluid described in any embodiment herein in the preparation of a reagent for one or more of the following purposes: (1) improving the healing speed of a patient's wound; (2) promoting the growth of new blood vessels in a patient's wound; (3) reducing the pro-inflammatory factors IL-6 and TNF- ⁇ in patients with inflammation, and increasing TGF- ⁇ 1 and IL-10 in patients with inflammation; (4) promoting the formation of type III collagen in the wound surface of the patient, or increasing the ratio of type III collagen to type I collagen in the wound surface of the patient; and (5) reducing the transcription and translation levels of iNOS, TNF- ⁇ , IL-6 and IL-1 ⁇ in the inflammatory patient, and increasing the transcription and translation levels of CD206 and Arg-1 in the inflammatory patient.
  • the present invention also provides the amniotic fluid or a composition containing the amniotic fluid described in any embodiment of the present invention for treating and/or preventing diseases mediated by the TLR4/NF- ⁇ B signaling pathway, for treating and/or preventing macrophage-mediated diseases, for treating and/or preventing M1 macrophage-mediated diseases, for treating and/or preventing M2 macrophage-mediated diseases, for inhibiting the number of M1 macrophage populations, increasing the proportion of M2 macrophage types, and/or promoting the transformation of M1 macrophages to M2 macrophages.
  • Also provided herein is a method for treating and/or preventing a disease mediated by the TLR4/NF- ⁇ B signaling pathway, comprising the step of administering a therapeutically effective amount of the amniotic fluid described herein or a composition containing the amniotic fluid to a subject in need thereof.
  • the present invention also provides methods for treating and/or preventing macrophage-mediated diseases.
  • the present invention also provides methods for treating and/or preventing M1 macrophage-mediated diseases.
  • the present invention also provides methods for treating and/or preventing M2 macrophage-mediated diseases. These methods include the step of administering to a subject in need thereof a therapeutically effective amount of the amniotic fluid described herein or a composition containing the amniotic fluid.
  • the present invention also provides a method for promoting the transformation of M1 macrophages to M2 macrophages, comprising the step of administering an effective amount of the amniotic fluid described herein or a composition containing the amniotic fluid to a subject in need.
  • the method can be used to treat or prevent diseases that benefit from an increase in M2 macrophages, including but not limited to the various diseases described above.
  • the method can be an in vivo method or an in vitro method.
  • the present invention also provides a method for tissue repair in an inflammatory subject, the method comprising using the amniotic fluid described herein or a composite dressing prepared with the amniotic fluid as the main raw material, or a cell culture medium containing the amniotic fluid described herein to culture tissue cells of interest in vitro, and after forming a tissue matrix, implanting the tissue matrix into the tissue damage or defect site.
  • the present invention also provides a method for improving the healing speed of a patient's wound, the method comprising applying the amniotic fluid described herein or a pharmaceutical composition containing the amniotic fluid to the patient's wound, or applying a composite dressing prepared with the amniotic fluid of the present invention as the main raw material.
  • the present invention also provides a method for promoting the growth of new blood vessels in a patient's wound, the method comprising applying the amniotic fluid described herein or a pharmaceutical composition containing the amniotic fluid to the patient's wound, or applying a composite dressing prepared with the amniotic fluid of the present invention as the main raw material.
  • the present invention also provides a method for reducing the pro-inflammatory factors IL-6 and TNF- ⁇ in inflammatory patients, and increasing TGF- ⁇ 1 and IL-10 in inflammatory patients, the method comprising administering an effective amount of amniotic fluid described herein or a pharmaceutical composition containing the amniotic fluid to the patient.
  • the present invention also provides a method for promoting the formation of type III collagen in a patient's wound, or a method for increasing the ratio of type III collagen to type I collagen at a patient's wound, the method comprising applying the amniotic fluid described herein or a pharmaceutical composition containing the amniotic fluid to the patient's wound, or applying a composite dressing prepared with the amniotic fluid of the present invention as the main raw material.
  • the present invention also provides a method for reducing the transcription and translation of iNOS, TNF- ⁇ , IL-6 and IL-1 ⁇ in patients with inflammation.
  • a method for improving the transcription and translation levels of CD206 and Arg-1 in an inflammatory patient the method comprising administering to the patient an effective amount of the amniotic fluid described herein or a pharmaceutical composition containing the amniotic fluid.
  • a therapeutically effective amount refers to a dose that can achieve treatment, prevention, alleviation and/or relief of a disease or condition in a subject.
  • the therapeutically effective amount can be determined based on factors such as the patient's age, gender, the disease and its severity, and other physical conditions of the patient.
  • a subject or patient generally refers to a mammal, particularly a human.
  • the dosage and frequency of administration can be determined by medical staff according to the specific condition, the age and gender of the patient, etc.
  • a therapeutically effective amount refers to a dosage sufficient to improve or alleviate the symptoms associated with the disease in some way.
  • Such a dosage can be administered as a single dose, or can be administered according to an effective treatment regimen.
  • the dosage may cure the disease, but the administration is usually to improve the symptoms of the disease. Repeated administration is generally required to achieve the desired symptom improvement.
  • the dosage given to a person it can usually be 1-200ml/time, and can be injected daily or weekly.
  • the frequency of administration can be multiple times a day, twice a day, every two days, every three days, every four days, every five days or every six days, or once every half a month, or once a month.
  • a cell culture medium which contains an appropriate amount of amniotic fluid as described herein.
  • the content of amniotic fluid in the cell culture medium can be determined according to the type of cells to be cultured.
  • the amount of amniotic fluid added can be 0.1-30% by weight of the cell culture medium, such as 1-25% or 3-20%.
  • a suitable cell culture medium can be selected according to the cells to be cultured.
  • Exemplary cell culture media include, but are not limited to, various commercially available culture media, such as DMEM, RPMI 1640, MEM, DMEM/F12, etc.
  • PE CD206 antibody (12-2069-42) and FITC F4/80 antibody (11-4801-82) were purchased from eBioscience.
  • Arginase1 (Arg-1; 93668), ⁇ -SMA (19245) and CD206 (24595) were purchased from CST.
  • GAPDH (ab181602), iNOS (ab178945), TNF- ⁇ (ab183218), IL-6 (ab290735), IL-1 ⁇ (ab254360) and CD31 (ab281583) were purchased from Abcam.
  • TLR4 (A5258), NF- ⁇ B-p65 (A19653) and p-IKB (AP0707) were purchased from ABclonal.
  • Trizol reagent and SYBRgreen were purchased from Vazyme Biotech. STZ (S0130) and glucose (D9434) were purchased from Sigma-Aldrich.
  • Fertilized chicken eggs were incubated at 38°C and 50% humidity. ceAFs were isolated from them between days 6 and 8 of incubation. After centrifugation (2500 ⁇ g, 20 min), the supernatant was filtered using a 0.22 ⁇ m sterile filter (Millipore, USA), and the filtered samples were aliquoted and kept at ⁇ 80°C.
  • RAW264.7 cells were provided by the Cell Bank of the Chinese Academy of Sciences and cultured in high-glucose DMEM medium containing 10% FBS and 1% double antibody at 37°C and 5% CO2 .
  • High glucose conditions were DMEM with 40 mM glucose.
  • concentrations of ceAF 0%, 1%, 5%, 10%, and 20%, respectively were added to the culture medium for subsequent experiments.
  • mice Male, 8 weeks old were obtained from the Model Animal Research Center of Nanjing University and housed in a specific pathogen-free environment with unrestricted access to water and food. Eighteen mice in each group were intraperitoneally injected with 50 mg/kg streptozotocin (STZ, in sodium citrate buffer) daily for 5 days to construct the STZ-induced diabetes model. Blood glucose was measured in mice three weeks later, and mice with blood glucose levels >16.7 mM were classified as diabetic. To establish the excisional wound model, an 8-mm circular biopsy punch was performed on the dorsal skin of mice after hair removal.
  • STZ streptozotocin
  • the wound edge tissue was fixed, dehydrated, embedded in paraffin, and sliced at 5 ⁇ m thickness.
  • Masson trichrome (MT), hematoxylin-eosin (H&E), and Sirius red staining were performed according to standardized histological procedures.
  • MT masson trichrome
  • H&E hematoxylin-eosin
  • Sirius red staining were performed according to standardized histological procedures.
  • CD206, iNOS, CD31, and ⁇ -SMA monoclonal antibodies (1 ⁇ g/ml) were stained at 4°C overnight. Then, specific fluorescent secondary antibodies were incubated, followed by DAPI staining.
  • RAW264.7 cells were washed with PBS, fixed in paraformaldehyde (4%), permeabilized with 0.1% TritonX-100 and blocked with BSA (3%). Then, cells were incubated with corresponding primary and secondary antibodies according to the instructions. All photos were taken using an Olympus FluoView FV3000 confocal microscope (Tokyo, Japan).
  • RNA samples and wound edge tissue were treated with Trizol reagent to isolate total RNA.
  • RT-qPCR was performed by SYBR green dye using the StepOne RT-qPCR system (Applied Biosystems, USA). After normalization with GAPDH, the relative gene levels were determined using the 2 - ⁇ CT method.
  • the primer sequences are shown in Table 1.
  • Protein samples were isolated from lysed skin tissues and cells using RIPA lysis buffer (KeyGEN, China). BCA assay was performed to determine the total protein concentration after centrifugation. Protein samples were separated by 10% SDS-PAGE gel and transferred to PDVF membrane (Millipore, USA). After blocking with 5% BSA, the membrane was incubated with the corresponding primary antibody overnight and the secondary antibody for 1 hour. ECL substrate kit (Vazyme, China) was used for visualization of protein bands.
  • RAW264.7 cell viability was assessed using the CCK-8 assay (Beyotime, China). After starvation for 12 h, cells were exposed to ceAF at concentrations of 0%, 1%, 5%, 10%, or 20%, and then incubated for 24 h. The cells were rinsed 3 times with PBS and then covered with 200 ⁇ L of incomplete medium (10 ⁇ L) containing a CCK-8 mixture and incubated at 37 ° C. The absorbance was measured at 450 nm using a microplate reader.
  • RAW264.7 cells were exposed to 10% ceAF for 48 h, and the cell supernatants were collected for testing.
  • the secreted IL-6, IL-10, TGF- ⁇ 1 and TNF- ⁇ were measured by ELISA kits according to the kit instructions (Elabscience, China).
  • Example 1 ceAF alleviates lipopolysaccharide-stimulated RAW264.7 cell inflammation via TLR4/NF- ⁇ B signaling pathway
  • this example used RAW264.7 as experimental cells and stimulated with LPS (100 ng/ml) for 48 hours to induce cellular inflammation.
  • Example 2 ceAF induces RAW264.7 cells to polarize into M2 macrophages in vitro
  • Example 6 ceAF can regulate wound-related inflammatory factors in STZ-induced diabetic mice

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Abstract

羊水在制备治疗和/或预防巨噬细胞介导的疾病的药物，治疗和/或预防M1型巨噬细胞介导的疾病的药物，治疗和/或预防M2型巨噬细胞介导的疾病的药物，治疗和/或预防TLR4/NF-κB信号通路介导的疾病的制剂，以及用于抑制M1型巨噬细胞类群数目、提高M2型巨噬细胞类型比例的制剂，或促进M1型巨噬细胞向M2型巨噬细胞转化的制剂中的应用。所述羊水来自胚龄为5-12天的鸡蛋，或者来自发育时期与所述胚龄的鸡蛋所处的发育时期相对应的鸡以外的其它禽类的蛋；或来自胎龄为8-14天的啮齿类动物的胚胎，或来自发育时期与胎龄为8-14天的啮齿类动物的发育时期相对应的啮齿类动物以外的其它非人哺乳动物的胚胎。

Description

治疗或预防巨噬细胞介导的疾病的药物及其应用 技术领域
本发明涉及治疗或预防巨噬细胞介导的疾病的药物及其应用。
背景技术
巨噬细胞在不同刺激下表现为不同类型，诱导性表达更多的一氧化氮合酶(inducible nitric oxide synthase，iNOS)或精氨酸酶，并相应地被称为M1型及M2型巨噬细胞。在炎症性疾病中，M1型巨噬细胞在早期炎症部位富集，并被LPS、TNFα、IFNγ等促炎因子激活，并随后通过分泌IL12等炎症因子的方式促进炎症的发生发展，保护机体免受外来物质侵袭。而在炎症后期，M2细胞则起到了抑制炎症、修复组织及组织结构重建等作用。在炎症发生发展中M1/M2巨噬细胞类群数目比例随着时间的推移不断变化，最终完全消除炎症的影响。而在某些慢性炎症及特定急性验证进程中，M1/M2细胞类群比例失调，过度M1细胞类群激活会导致组织严重损伤，并导致后续更严重的炎症因子风暴等不良症状。因此，M2型巨噬细胞与抗炎反应和免疫稳态密切相关，参与组织修复、组织器官重塑、瘢痕形成和损伤愈合过程，具有免疫抑制作用。
来源于外泌体的不同种类miRNA、转录因子及其他非编码RNA通过不同信号通路调节M2型巨噬细胞极化，其中，Toll样受体4(Toll-like receptor 4，TLR4)/NF-κB信号通路在炎症反应中发挥核心调控作用。非激活状态下，NF-κB活性受IκB抑制；而受到TLR及TNF受体等细胞因子受体刺激后，IκB激酶激活将使IκB磷酸化，进而使蛋白酶体降解并释放NF-κB向核内转运，激活相应基因转录。NF-κB的激活可导致炎性细胞因子的合成和释放。
随着糖尿病发病率的上升，糖尿病溃疡已成为临床上最常见的慢性难治性创面之一，持续性慢性炎症是糖尿病皮肤创面的典型特征。由于糖尿病创面的微环境复杂，如缺氧、感染、缺血状态、炎症、氧化应激等，糖尿病溃疡往往会延长和复发，而传统的清创换药疗法对糖尿病伤口的效果有限。因此，如何有效、经济地治疗糖尿病溃疡成为一个亟待解决的问题。
在伤口愈合过程中，M1巨噬细胞负责吞噬坏死组织和细胞碎片，而M2巨噬细胞则参与抑制炎症和促进组织再生。糖尿病伤口通常处于过度炎症状态，创面组织中存在大量M1型巨噬细胞，巨噬细胞向M2型的转化受阻。值得注意的是，先前的研究表明，增加M2表 型可能是糖尿病伤口修复的重要因素。
发明内容
本发明第一方面提供羊水在制备治疗和/或预防巨噬细胞介导的疾病的药物中的应用，其中，所述羊水来自胚龄为5-12天的鸡蛋，优选胚龄为6-11天的鸡蛋，更优选胚龄为7-9天的鸡蛋，更优选胚龄为7-8天的鸡蛋，或者来自发育时期与所述胚龄的鸡蛋所处的发育时期相对应的鸡以外的其它禽类的蛋；或来自胎龄为8-14天的啮齿类动物的胚胎，或来自发育时期与胎龄为8-14天的啮齿类动物的发育时期相对应的啮齿类动物以外的其它非人哺乳动物的胚胎。
本发明还提供羊水在制备治疗和/或预防M1型巨噬细胞介导的疾病的药物中的应用，其中，所述羊水来自胚龄为5-12天的鸡蛋，优选胚龄为6-11天的鸡蛋，更优选胚龄为7-9天的鸡蛋，更优选胚龄为7-8天的鸡蛋，或者来自发育时期与所述胚龄的鸡蛋所处的发育时期相对应的鸡以外的其它禽类的蛋；或来自胎龄为8-14天的啮齿类动物的胚胎，或来自发育时期与胎龄为8-14天的啮齿类动物的发育时期相对应的啮齿类动物以外的其它非人哺乳动物的胚胎。
本发明还提供羊水在制备治疗和/或预防M2型巨噬细胞介导的疾病的药物中的应用，其中，所述羊水来自胚龄为5-12天的鸡蛋，优选胚龄为6-11天的鸡蛋，更优选胚龄为7-9天的鸡蛋，更优选胚龄为7-8天的鸡蛋，或者来自发育时期与所述胚龄的鸡蛋所处的发育时期相对应的鸡以外的其它禽类的蛋；或来自胎龄为8-14天的啮齿类动物的胚胎，或来自发育时期与胎龄为8-14天的啮齿类动物的发育时期相对应的啮齿类动物以外的其它非人哺乳动物的胚胎。
本发明还提供羊水在制备治疗和/或预防TLR4/NF-κB信号通路介导的疾病的制剂中的应用，其中，所述羊水来自胚龄为5-12天的鸡蛋，优选胚龄为6-11天的鸡蛋，更优选胚龄为7-9天的鸡蛋，更优选胚龄为7-8天的鸡蛋，或者来自发育时期与所述胚龄的鸡蛋所处的发育时期相对应的鸡以外的其它禽类的蛋；或来自胎龄为8-14天的啮齿类动物的胚胎，或来自发育时期与胎龄为8-14天的啮齿类动物的发育时期相对应的啮齿类动物以外的其它非人哺乳动物的胚胎。
本发明还提供羊水在制备用于抑制M1型巨噬细胞类群数目、提高M2型巨噬细胞类型比例的制剂中的应用，或在制备促进M1型巨噬细胞向M2型巨噬细胞转化的制剂中的应用，其中，所述羊水来自胚龄为5-12天的鸡蛋，优选胚龄为6-11天的鸡蛋，更优选胚龄为7-9天的鸡蛋，更优选胚龄为7-8天的鸡蛋，或者来自发育时期与所述胚龄的鸡蛋所处的发育时期相对应的鸡以外的其它禽类的蛋；或来自胎龄为8-14天的啮齿类动物的胚胎，或 来自发育时期与胎龄为8-14天的啮齿类动物的发育时期相对应的啮齿类动物以外的其它非人哺乳动物的胚胎。
在一个或多个实施方案中，所述药物或制剂是包含所述羊水的细胞培养物和/或胚胎干细胞。
在一个或多个实施方案中，所述药物或制剂是包含所述羊水和/或鸡的胚胎干细胞以及药学上可接受辅料的药物组合物。
在一个或多个实施方案中，所述巨噬细胞介导的疾病选自：增生性瘢痕；慢性阻塞性肺炎；肿瘤，如乳腺癌、肝癌；代谢性疾病，如重度肥胖、胰岛素抵抗、II型糖尿病；炎症性疾病，如急性胰腺炎、动脉粥样硬化；心血管疾病，如心肌炎、心肌梗死、心率失常；神经病变，如阿兹海默症；脑部疾病，如脑梗死、脑损伤；眼科疾病，如自身免疫性葡萄膜炎、视网膜病变、角膜炎、角膜移植、干燥综合征、葡萄膜黑色素瘤、近视；免疫炎症性疾病，如炎症性肠炎、自身免疫性肝炎、哮喘、酒精性肝病、结肠炎、多发性硬化、牙周炎；关节炎，如类风湿性关节炎、骨侵蚀、滑膜炎、骨关节炎；肾炎，如急性肾损伤、慢性肾脏病、终末期肾病、增值性肾小球肾炎、膜性肾病、糖尿病肾病、过敏性紫癜肾炎、ANCA相关小血管炎、尿路感染、常染色体显性多囊肾；细菌感染性疾病，如脓毒症；妊娠期高血压；糖尿病、妊娠期糖尿病、糖尿病肾病。
在一个或多个实施方案中，所述M1型巨噬细胞介导的疾病选自：减退期增生性瘢痕；慢性阻塞性肺炎；肿瘤，如乳腺癌、肝癌；代谢性疾病，如重度肥胖、胰岛素抵抗、II型糖尿病；炎症性疾病，如急性胰腺炎；冠状动脉疾病，如动脉粥样硬化；肾病；肥胖；心血管疾病，如心肌炎、心肌梗死；脑部疾病，如脑梗死、脑损伤；眼科疾病，如自身免疫性葡萄膜炎、视网膜病变、角膜炎、角膜移植、干燥综合征、葡萄膜黑色素瘤；免疫炎症性疾病，如炎症性肠炎、自身免疫性肝炎、哮喘、酒精性肝病、结肠炎、多发性硬化、牙周炎、骨关节炎；糖尿病、妊娠期糖尿病、糖尿病肾病。
在一个或多个实施方案中，所述M2型巨噬细胞介导的疾病选自：增生期增生性瘢痕；肿瘤，如乳腺癌、肝癌；代谢性疾病，如胰岛素抵抗、II型糖尿病；炎症性疾病，如急性胰腺炎；心血管疾病，如心肌梗死、心肌衰竭、动脉粥样硬化、冠状动脉疾病、心肌炎；脑梗死；眼科疾病，如自身免疫性葡萄膜炎、视网膜病变、角膜炎、角膜移植、干燥综合征、葡萄膜黑色素瘤；免疫炎症性疾病，如炎症性肠炎、自身免疫性肝炎、哮喘、酒精性肝病、结肠炎、多发性硬化、牙周炎、骨关节炎；糖尿病、妊娠期糖尿病、糖尿病肾病。
在一个或多个实施方案中，所述TLR4/NF-κB信号通路介导的疾病选自：选自系统性红斑狼疮；血管炎症，如动脉粥样硬化、冠心病；心肌炎，如心肌缺血组织炎症、心肌损伤；肝炎，如肝衰竭，酒精性肝损伤、酒精代谢过程中的炎症免疫反应；脂肪肝；肺炎，如 急性肺损伤、慢性阻塞性肺疾病、矽肺；肾炎，如急性肾损伤、狼疮性肾炎；炎症性肠病，如急性肠炎、溃疡性结肠炎、放射性直肠炎；胃炎，如慢性萎缩性胃炎；急性呼吸道感染，如肺炎、支气管炎、咽炎、鼻窦、中耳炎；牙周炎、高尿酸血症；变应性鼻炎、过敏性鼻炎；乳腺炎；关节炎，如急性痛风性关节炎、慢性关节炎、类风湿性关节炎；创面组织炎症、增生性瘢痕；多囊卵巢综合征；肿瘤，如垂体泌乳素腺瘤、促肾上腺皮质激素腺瘤、颅内动脉瘤；感染性疾病，如细菌性感染、真菌性感染、病毒感染；过敏性疾病，如皮肤过敏性疾病、支气管哮喘、过敏性鼻炎、过敏性紫癜。
附图说明
图1为ceAF通过TLR4/NF-κB信号通路减轻脂多糖刺激的RAW 264.7细胞炎症的示意图。D中，每4列柱状图为一组，每组柱状图从左到右依次对应于：CD206、Arg-1、iNOS、TNF-α、IL-6、IL-1β、TLR4、NF-κB、plκB。
图2为ceAF体外诱导RAW264.7向M2巨噬细胞极化的示意图。
图3为ceAF促进STZ诱导的糖尿病小鼠创面愈合的示意图。
图4为ceAF改善STZ诱导的糖尿病小鼠创面组织学参数的示意图。
图5为ceAF改善STZ诱导的糖尿病小鼠创面愈合相关指标的示意图。
图6为ceAF可调节STZ诱导的糖尿病小鼠创面相关炎症因子的示意图。B中，每2列柱状图为一组，每组柱状图从左到右依次对应于CD206、Arg-1、iNOS、TNF-α、IL-6、IL-1β。
具体实施方式
应理解，在本发明范围中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成优选的技术方案。
本发明发现，鸡胚羊水(ceAF)可以通过TLR4/NF-κB信号通路诱导巨噬细胞由M1型向M2型转化，由此完成本发明。
羊水
羊水可来自禽蛋和非人哺乳动物。禽蛋指禽类的蛋。优选的禽类为家禽，如鸡、鸭和鹅。优选的是，本发明使用胚龄在5-20天、优选6-15天的禽蛋。应理解，不同禽蛋，合适的胚龄未必相同。例如，当使用鸡蛋时，优选使用胚龄为5-12天的鸡蛋，更优选使用胚龄为6-11天的鸡蛋，更优选使用胚龄为7-9天的鸡蛋、更优选使用胚龄为7-8天的鸡蛋。当使用其它禽类的蛋时，可使用其发育时期与上述胚龄的鸡蛋所处的发育时期相对应的蛋。 例如，当使用鸭蛋时，胚龄为8-10天、尤其是8-9天的鸭蛋可能是最好的。在一些具体的实施方案中，ceAF是指从孵化6-8天的鸡胚中提取的羊水。
可采用常规的方法获得禽蛋羊水。例如，可敲击相应胚龄的蛋的钝端，使蛋壳碎裂，将蛋壳剥开形成一个直径约为2厘米的口子。然后用镊子小心撕开壳膜和卵黄膜，注意不要破坏羊膜。将包裹着胚胎的羊膜和相连组织从壳中倾倒至培养皿中，用注射器刺入羊膜抽取羊水，直至羊膜紧贴胚胎，由此即可获得用于本发明的羊水。
本文中，羊水还可来自非人哺乳动物，尤其是啮齿类动物，如来自小鼠。其它非人哺乳动物可以是常见的家畜，例如牛、羊、狗、猫、猪等。在某些实施方案中，羊水来自胎龄为8-14天的啮齿类动物的胚胎，或来自其发育时期与胎龄为8-14天的啮齿类动物所处的发育时期相对应的非人哺乳动物的胚胎。可采用常规的方法获得羊水。例如，用手术剪剪开怀孕8-14天的小鼠腹腔，小心取出并剪开子宫，用注射器刺入羊膜抽取羊水，直至羊膜紧贴胚胎，由此即可获得用于本发明的羊水。
应理解，必要时，可对羊水进行离心，以分离出可能含有的杂质，例如卵黄等，尽可能获得纯的羊水。离心后获得的上清液即为用于本发明的羊水。应理解的是，获取羊水的所有步骤都需在无菌条件下进行；另外，本文所示的“羊水”应指“纯”的羊水，即分离自禽蛋或非人哺乳动物胚胎的不含有禽蛋内或非人哺乳动物胚胎内其它成分、且也未被外源物质污染的羊水。纯的羊水可储存于-60℃以下的冰箱中，解冻后再使用。
本文所述的羊水可作为药物的活性成分，用于体内给予需要的对象，体内促进细胞生长和组织修复。例如，可给予需要的对象有效量的本文所述的羊水，或含有所述羊水的药物组合物。
应理解，结合CN 201810911038.2、CN 201810909485.4、CN 201810909193.0以及CN 201910887556.X可知，本发明所述的各种来源的羊水具有相同或类似的生物学活性，用于本发明时，均可用于调节TLR4/NF-κB信号通路，抑制M1型巨噬细胞、增加M2型巨噬细胞，从而治疗或预防受益于TLR4/NF-κB信号通路调控的疾病以及巨噬细胞介导的疾病。
本文中，动物可以是哺乳动物，尤其是人。
疾病
Toll样受体(Toll-like receptor，TLRs)是一类介导天然免疫的受体家族，尤其是TLR4，能够识别多种病原相关分子模式(PAMPs)并激活炎症细胞，即可识别外源性配体-脂多糖(lipopolysaccharide，LPS)，同时也能识别动脉损伤过程中表达的内源性配体，并能调控胆固醇代谢、板块稳定性、细胞凋亡、炎症和血管重塑以及免疫反应等病理过程，TLRs与配体结合后最终导致NF-κB的活化，调控炎症和免疫相关基因的表达。
NF-κB可在疾病形成中的多个环节，如炎症反应、泡沫细胞形成血管平滑肌的增殖和细胞凋亡中起作用。调控参与早期低密度脂蛋白(LDL)修饰和炎症脂质形成过程的酶类(如环氧化酶2)。活化的NF-κB还可促进黏附分子、促炎因子、趋化因子、生长因子等因子的转录，这些因子在炎症类疾病的发生和发展中起到了重要作用。
本文中，TLR4/NF-κB信号通路介导的疾病包括但不限于：神经病变和/或血管病变引起的炎症反应，选自系统性红斑狼疮(季娟.TLR4信号异常激活导致骨髓间充质干细胞衰老在系统性红斑狼疮发生中的作用[D].南通大学，2017.)；血管炎症，动脉粥样硬化(李红梅，王显.TLR4/MyD88/NF-κB信号通路与动脉粥样硬化性心血管疾病的相关性研究进展[J].中国循证心血管医学杂志，2017，9(09)：1132-1134.)、冠心病(林勇军等.银杏叶滴丸对冠心病患者TLR4/NF-κB信号通路及免疫指标的影响[J].海峡药学，2017，29(03)：89-92.)；心肌炎，如心肌缺血组织炎症(周雪玲.基于TLR4/NF-κB信号通路探讨活心丸抗急性心肌缺血炎症反应的作用机制[D].福建中医药大学，2020.DOI：10.27021/d.cnki.gfjzc.2020.000334.)、心肌损伤(韦皓等.HIF-1α通过TLR4/NF-κB信号通路对心肌缺血-再灌注大鼠心肌损伤的保护机制分析[J].临床和实验医学杂志，2019，18(10)：1017-1020.)；肝炎，如肝衰竭(刘巧红，刘江凯，李素领.解毒化瘀通腑颗粒治疗亚急性和慢加急性肝衰竭患者的疗效及其对TLR4/NF-κB炎性通路的影响[J].中西医结合肝病杂志，2018，28(05)：264-267.)，酒精性肝损伤(朱晓宁，汪静，张玉蓉，尹玥，彭孟云，曾勇.祛痰活血方上调SOCS1抑制TLR4/NF-κB信号通路改善非酒精性脂肪性肝炎小鼠肝损伤[J].世界科学技术-中医药现代化，2020，22(12)：4293-4299.)、酒精代谢过程中的炎症免疫反应(殷小磊，卢伟娜，冯丽英.LPS/TLR4信号途径在非酒精性脂肪性肝病中的作用.世界华人消化杂志2013；21(28)：2957-2962.DOI：10.11569/wcjd.v21.i28.2957)；脂肪肝(殷小磊，卢伟娜，冯丽英.LPS/TLR4信号途径在非酒精性脂肪性肝病中的作用.世界华人消化杂志2013；21(28)：2957-2962.DOI：10.11569/wcjd.v21.i28.2957)；肺炎，如急性肺损伤(牛泽群，王立明，冯辉，孙江利，裴红红，潘龙飞.TLR4/NF-κB信号通路在急性重症胰腺炎相关肺损伤大鼠中的作用研究[J].现代消化及介入诊疗，2019，24(12)：1404-1407.)、慢性阻塞性肺疾病(陈训春，李名兰，潘碧云，王燕英，丁毅鹏.TLR4/NF-κB信号通路激活LncRNA RP11-20G6调控慢性阻塞性肺疾病气道炎症和重塑[J].安徽医科大学学报，2022，57(4)：586-593)、矽肺(祝丽丽.TLR4和RAGE介导的炎性因子在矽肺发病中的作用研究[D].山西医科大学，2014.)；肾炎，如急性肾损伤(战云，张英杰，冷彬.黄芪甲苷通过TLR4/NF-κB通路改善脂多糖诱导的急性血管内皮损伤[J].中药药理与临床，2018，34(03)：77-80.DOI：10.13412/j.cnki.zyyl.2018.03.019.)、狼疮性肾炎(陈宁.HMGB1/TLR/NF-κB信号途径在狼疮性肾炎小鼠肾组织中的表达及意义[D].河北医科大学，2010.)；炎症性肠病，如急性肠炎(李学慧等.CD11b激动剂leukadherin-1对小鼠实验 性结肠炎发病的影响及其机制研究[J].中华微生物学和免疫学杂志，2019(12)：904-905-906-907-908-909-910.)、溃疡性结肠炎(蔺晓源，刘杰民.TLR4/MyD88/NF-κB信号通路与溃疡性结肠炎[J].胃肠病学，2013，18(04)：244-246.)、放射性直肠炎(祝朝富，安佰平，黄洪婕，杜驰，吴勇俊，蓝兰，李丹，雷冬梅，李世杰，敖睿.基于TLR4/NF-κB信号通路探讨托里消毒散治疗放射性直肠炎的作用机制[J].安徽医科大学学报，2020，55(09)：1367-1373.DOI：10.19405/j.cnki.issn1000-1492.2020.09.010.)；胃炎，如慢性萎缩性胃炎(周威，袁星星.美洲大蠊提取物对慢性萎缩性胃炎大鼠TLR4/NF-κB信号通路的影响[J].世界华人消化杂志，2017，25(21)：1945-1951.)；急性呼吸道感染，如肺炎(郭沙沙.TLR-MYD88-NF-κBP65依赖信号通路在支原体肺炎小鼠肺组织中的作用[D].青岛大学，2017.)、支气管炎(吴宁，黄雨霄，石雪，陈晋伦，彭凌峰，杨露露，徐红，孙见飞，刘华.苗药“杆努尽烟”对慢性支气管炎大鼠TLR4-MyD88-NF-κB信号通路的影响[J].贵州医科大学学报，2020，45(11)：1283-1288.DOI：10.19367/j.cnki.2096-8388.2020.11.009.)、咽炎(徐金红.基于TLR4/NF-κB信号通路探讨紫正地黄汤治疗急性咽炎的机制研究[D].安徽中医药大学，2020.DOI：10.26922/d.cnki.ganzc.2020.000216.)、鼻窦炎(肖佳宁，薛珊珊，倪平敏，王卓，吴拥军.基于NF-κB信号通路探讨鼻渊合剂对急性鼻窦炎的作用机制[J].南京中医药大学学报，2022，38(3)：247-253.doi：10.14148/j.issn.1672-0482.2022.0247)、中耳炎(CN201180025442.6，罗伦才，童妍，张兴国等.基于TLR4/MyD88/NF-κB通路研究愈疡胶囊抗中耳炎的作用[J].西北药学杂志，2021，36(01)：66-71.)；牙周炎、高尿酸血症(郭竹玲，汤涵，黄妙，等.TLR4/NF-κB信号通路在调控牙周炎及高尿酸血症及其相互作用的研究进展[J].中华口腔医学研究杂志：电子版，2021，15(1)：5.)；哮喘(李鸿佳.槲皮素通过TLR4/NF-κB信号调节哮喘气道炎症机制研究[D].山东大学，2015.)；变应性鼻炎(尹丽丽.TREM-1、TLR4、TNF-a及NF-κB在变应性鼻炎小鼠鼻粘膜中的表达及作用机制[D].华中科技大学，2013.)、过敏性鼻炎(胡琛，梁晨阳，周维国.炎症介导TLR4/NF-kB通路在过敏性鼻炎患者中的表达和作用[J].标记免疫分析与临床，2018，25(06)：788-790+838.)；乳腺炎(卢劲晔，顾蓓蓓.TLR4/My D88/NF-κB信号通路与乳腺炎的研究进展[J].畜牧与兽医，2018，50(11)：127-129.)；关节炎，如急性痛风性关节炎(LUOFei，MEI Yan.Effects of procyanidins on TLR4/NF-κB signaling pathway in rats with acute gouty arthritis.Chinese Journal of Clinical Pharmacology and Therapeutics.2018，(1)：41-46.DOI：10.12092/j.issn.1009-2501.2018.01.008)，慢性关节炎(俞碧君.TLR信号通路在慢性炎症性关节炎中的作用[J].国际免疫学杂志，2007，30(06)：443-447.)，类风湿性关节炎(白琳，杨雨欣，万巧凤，等.黄芩苷经TLR2/NF-κB途径减轻类风湿关节炎大鼠滑膜炎[J].中国药理学通报，2017，33(11)：1569-73.)；创面组织炎症、增生性瘢痕(李强.TRAIL、DR5及NF-κB p65在人难愈合创面肉芽组织和增生 性瘢痕中的表达及其意义研究[D].山东大学，2008.)；多囊卵巢综合征(姚知林，黄映红，徐晓娟.补肾化痰方对多囊卵巢综合征雌鼠TLR4/NF-κB炎性信号通路，氧化应激和胰岛素抵抗的影响[J].四川中医，2019，37(11)：6.)；垂体泌乳素腺瘤(郭润竹.大麦芽碱对高泌乳素血症和泌乳素腺瘤的药效作用及机制研究[D].湖北中医药大学，2019.DOI：10.27134/d.cnki.ghbzc.2019.000015.)、促肾上腺皮质激素腺瘤(Wu Q，Feng Y，Liu L，Liu Y，Liu X，Zhang L，Li Y，Wang L.Corticotropin-Releasing Factor Aggravates Ischemic Stroke Injury by the Inflammatory Activation of Microglia.Endocrinology.2022 Mar 1；163(3)：bqac013.doi：10.1210/endocr/bqac013.PMID：35137012.)、颅内动脉瘤(Wang Y，Jin J.Roles of macrophages in formation and progression of intracranial aneurysms.Zhejiang Da Xue Xue Bao Yi Xue Ban.2019 Apr 25；48(2)：204-213.Chinese.doi：10.3785/j.issn.1008-9292.2019.04.13.PMID：31309760；PMCID：PMC8800668.)等肿瘤的发生、发展；感染性疾病，如细菌性感染、真菌性感染和病毒感染(单佳铃，程虹毓，文乐，钟国跃，朱继孝.TLR/MyD 88/NF-κB信号通路参与不同疾病作用机制研究进展[J].中国药理学通报，2019，35(4)：451-455)；过敏性疾病，如皮肤过敏性疾病、支气管哮喘、过敏性鼻炎(单佳铃，程虹毓，文乐，钟国跃，朱继孝.TLR/MyD 88/NF-κB信号通路参与不同疾病作用机制研究进展[J].中国药理学通报，2019，35(4)：451-455)和过敏性紫癜(王子威，杨丽君.TLR9、MyD88及NF-κB在过敏性紫癜中的表达及意义[J].中国现代医生，2016，54(29)：9-12)。
本文所述的羊水可抑制TLR4/NF-κB信号通路的活化，从而治疗或预防各种TLR4/NF-κB信号通路介导的疾病。
本文中，巨噬细胞介导是指巨噬细胞中功能特性各不相同的细胞亚群的动态平衡及失衡在疾病病理过程中的作用。巨噬细胞介导的疾病包括但不限于增生性瘢痕(李镇江，李书俊，周健等.不同时期增生性瘢痕组织中巨噬细胞活化相关因子的研究[J].遵义医科大学学报，2022，45(1)：87-91)；慢性阻塞性肺炎(邢士刚.巨噬细胞在慢性阻塞性肺疾病发病机制中的作用研究[J].中国实用医药，2019，14(12)：196-197)；肿瘤，如乳腺癌、肝癌；代谢性疾病，如重度肥胖、胰岛素抵抗、II型糖尿病；炎症性疾病，如急性胰腺炎、动脉粥样硬化(王永康，李佳怡，关飞，雷家慧.巨噬细胞极化机制及其在常见疾病中的作用[J].热带病与寄生虫学，2022，20(2)：103-108112)；心血管疾病，如心肌炎、心肌梗死、心率失常(董经纬，苗柳.巨噬细胞功能及其在心脏疾病中作用的研究进展[J].中国当代医药，2022，29(16)：49-52)；神经病变，如阿兹海默症；脑部疾病，如脑梗死(王佳贺.前言——巨噬细胞与相关疾病的研究进展[J].实用老年医学，2021，35(12)：1217-1218)、脑损伤(刁中极，雷锐，殷实，刘鸿玲，刘擎.M1型巨噬细胞水平与冠心病疾病严重程度及预后的相关性分析[J].中国医学创新，2022，19(4)：161-165)；眼科疾病，如自身免疫性葡萄膜炎、视网膜病变、角膜 炎、角膜移植、干燥综合征、葡萄膜黑色素瘤、近视(屈如意，周梦贤，毕宏生，郭大东.巨噬细胞极化在眼科疾病发生发展过程中的作用机制研究进展[J].眼科新进展，2022，42(3)：239-243)；免疫炎症性疾病，如炎症性肠炎、自身免疫性肝炎、哮喘(贾瑞，惠毅，闫曙光(指导)，李京涛.巨噬细胞M1/M2型极化与免疫炎症性疾病关系的研究进展[J].中国免疫学杂志，2021，37(22)：2791-2797)、酒精性肝病、结肠炎(吴燕，张定然，王新慧，许宏扬，刘沛尧，齐智利.巨噬细胞极化及其对炎性疾病作用的研究进展[J].中国畜牧杂志，2021，57(7)：22-26)、多发性硬化(李杏，王丹丹，唐琪，刘杰，顾中一，赵欢，孙宏晨.巨噬细胞极化的转录调控及其对相关疾病影响的研究进展[J].吉林大学学报：医学版，2016，0(3)：622-625)、牙周炎(周琦，孙慧娟，于栋华，刘树民.巨噬细胞M1/M2型极化在不同疾病中的作用机制[J].中国药理学通报，2020，36(11)：1502-1506)；关节炎，如类风湿性关节炎、骨侵蚀、滑膜炎(王东轶，沈俊逸，陆乐，蔡辉.巨噬细胞极化失衡与类风湿关节炎疾病活动及骨侵蚀的相关性[J].医学研究生学报，2021，34(8)：823-828)、骨关节炎(周琦，孙慧娟，于栋华，刘树民.巨噬细胞M1/M2型极化在不同疾病中的作用机制[J].中国药理学通报，2020，36(11)：1502-1506)；肾炎，如急性肾损伤、慢性肾脏病、终末期肾病、增值性肾小球肾炎、膜性肾病、糖尿病肾病、过敏性紫癜肾炎、ANCA相关小血管炎、尿路感染、常染色体显性多囊肾(丁娜，王博，黄彬涛，郝健.巨噬细胞移动抑制因子在肾炎性疾病发病机制中的研究进展[J].河北医学，2021，27(1)：170-174)；细菌感染性疾病，如脓毒症(黄雪超，沈诗洋，莫然.巨噬细胞膜仿生化纳米递药系统用于炎症性疾病治疗的研究进展[J].中国药物评价，2021，38(4)：279-283)；妊娠期高血压(赵彩珍，乔福元.巨噬细胞与妊娠期高血压疾病[J].中国优生与遗传杂志，2006，14(3)：126-128)；糖尿病、妊娠期糖尿病、糖尿病肾病(周琦，孙慧娟，于栋华，刘树民.巨噬细胞M1/M2型极化在不同疾病中的作用机制[J].中国药理学通报，2020，36(11)：1502-1506)。
巨噬细胞包含M1和M2两个亚型。M1型巨噬细胞又称为促炎表型巨噬细胞，其在体内主要起到吞噬细菌等外来物质及凋亡细胞碎片等内源性物质，起到保护组织器官免受外来物质侵袭。在炎症性疾病中，M1型巨噬细胞在早期炎症部位富集，并被促炎因子激活，通过分泌炎症因子促进炎症的发生发展，保护机体免受外来物质侵袭；而在炎症后期，M2细胞则起到了抑制炎症、修复组织及组织结构重建等作用，因此M2型巨噬细胞又称为抗炎表型或免疫调节型巨噬细胞。在炎症发生发展中M1/M2巨噬细胞类群数目比例随着时间的推移不断变化，最终完全消除炎症的影响。
本文中，M1型巨噬细胞介导是指疾病中M1型巨噬细胞产生的被促炎因子激活、分泌炎症因子、保护机体免受外来物质侵袭等作用。M1型巨噬细胞介导的疾病包括但不限于减退期增生性瘢痕(李镇江，李书俊，周健等.不同时期增生性瘢痕组织中巨噬细胞活化相关因 子的研究[J].遵义医科大学学报，2022，45(1)：87-91)；慢性阻塞性肺炎(邢士刚.巨噬细胞在慢性阻塞性肺疾病发病机制中的作用研究[J].中国实用医药，2019，14(12)：196-197)；肿瘤，如乳腺癌、肝癌；代谢性疾病，如重度肥胖、胰岛素抵抗、II型糖尿病；炎症性疾病，如急性胰腺炎(王永康，李佳怡，关飞，雷家慧.巨噬细胞极化机制及其在常见疾病中的作用[J].热带病与寄生虫学，2022，20(2)：103-108112)；冠状动脉疾病，如动脉粥样硬化；肾病；肥胖(刁中极，雷锐，殷实，刘鸿玲，刘擎.M1型巨噬细胞水平与冠心病疾病严重程度及预后的相关性分析[J].中国医学创新，2022，19(4)：161-165)；心血管疾病，如心肌炎、心肌梗死(董经纬，苗柳.巨噬细胞功能及其在心脏疾病中作用的研究进展[J].中国当代医药，2022，29(16)：49-52)；脑部疾病，如脑梗死(王佳贺.前言——巨噬细胞与相关疾病的研究进展[J].实用老年医学，2021，35(12)：1217-1218)、脑损伤(刁中极，雷锐，殷实，刘鸿玲，刘擎.M1型巨噬细胞水平与冠心病疾病严重程度及预后的相关性分析[J].中国医学创新，2022，19(4)：161-165)；眼科疾病，如自身免疫性葡萄膜炎、视网膜病变、角膜炎、角膜移植、干燥综合征、葡萄膜黑色素瘤(屈如意，周梦贤，毕宏生，郭大东.巨噬细胞极化在眼科疾病发生发展过程中的作用机制研究进展[J].眼科新进展，2022，42(3)：239-243)；免疫炎症性疾病，如炎症性肠炎、自身免疫性肝炎、哮喘(贾瑞，惠毅，闫曙光(指导)，李京涛.巨噬细胞M1/M2型极化与免疫炎症性疾病关系的研究进展[J].中国免疫学杂志，2021，37(22)：2791-2797)、酒精性肝病、结肠炎(吴燕，张定然，王新慧，许宏扬，刘沛尧，齐智利.巨噬细胞极化及其对炎性疾病作用的研究进展[J].中国畜牧杂志，2021，57(7)：22-26)、多发性硬化(李杏，王丹丹，唐琪，刘杰，顾中一，赵欢，孙宏晨.巨噬细胞极化的转录调控及其对相关疾病影响的研究进展[J].吉林大学学报：医学版，2016，0(3)：622-625)、牙周炎、骨关节炎；糖尿病、妊娠期糖尿病、糖尿病肾病(周琦，孙慧娟，于栋华，刘树民.巨噬细胞M1/M2型极化在不同疾病中的作用机制[J].中国药理学通报，2020，36(11)：1502-1506)。
在一些实施方案中，M1型巨噬细胞介导的疾病包括但不限于免疫性炎症疾病、代谢性疾病、糖尿病和肿瘤。在示例性的实施方案中，本文所述的M1型巨噬细胞介导的疾病是糖尿病创面持续性慢性炎症。
本文中，M2型巨噬细胞介导是指在疾病中M2型巨噬细胞产生的抑制炎症及炎症因子、促进创面愈合修复等作用。M2型巨噬细胞介导的疾病包括但不限于增生期增生性瘢痕(李镇江，李书俊，周健等.不同时期增生性瘢痕组织中巨噬细胞活化相关因子的研究[J].遵义医科大学学报，2022，45(1)：87-91)；肿瘤，如乳腺癌、肝癌；代谢性疾病，如胰岛素抵抗、II型糖尿病；炎症性疾病，如急性胰腺炎(王永康，李佳怡，关飞，雷家慧.巨噬细胞极化机制及其在常见疾病中的作用[J].热带病与寄生虫学，2022，20(2)：103-108112)；心血管疾病，如心肌梗死、心肌衰竭、动脉粥样硬化、冠状动脉疾病(张祥凝，党国徽，冯娟(指导).外泌体对 M2型巨噬细胞的调控及其在心血管疾病中的作用[J].中国免疫学杂志，2022，38(10)：1257-1262)、心肌炎(董经纬，苗柳.巨噬细胞功能及其在心脏疾病中作用的研究进展[J].中国当代医药，2022，29(16)：49-52)；脑梗死(王佳贺.前言——巨噬细胞与相关疾病的研究进展[J].实用老年医学，2021，35(12)：1217-1218)；眼科疾病，如自身免疫性葡萄膜炎、视网膜病变、角膜炎、角膜移植、干燥综合征、葡萄膜黑色素瘤(屈如意，周梦贤，毕宏生，郭大东.巨噬细胞极化在眼科疾病发生发展过程中的作用机制研究进展[J].眼科新进展，2022，42(3)：239-243)；免疫炎症性疾病，如炎症性肠炎、自身免疫性肝炎、哮喘(贾瑞，惠毅，闫曙光(指导)，李京涛.巨噬细胞M1/M2型极化与免疫炎症性疾病关系的研究进展[J].中国免疫学杂志，2021，37(22)：2791-2797)、酒精性肝病、结肠炎(吴燕，张定然，王新慧，许宏扬，刘沛尧，齐智利.巨噬细胞极化及其对炎性疾病作用的研究进展[J].中国畜牧杂志，2021，57(7)：22-26)、多发性硬化(李杏，王丹丹，唐琪，刘杰，顾中一，赵欢，孙宏晨.巨噬细胞极化的转录调控及其对相关疾病影响的研究进展[J].吉林大学学报：医学版，2016，0(3)：622-625)、牙周炎、骨关节炎；糖尿病、妊娠期糖尿病、糖尿病肾病(周琦，孙慧娟，于栋华，刘树民.巨噬细胞M1/M2型极化在不同疾病中的作用机制[J].中国药理学通报，2020，36(11)：1502-1506)。
在一些实施方案中，M2型巨噬细胞介导的疾病包括但不限于免疫性炎症疾病、代谢性疾病、糖尿病和肿瘤。在示例性的实施方案中，本文所述的M2型巨噬细胞介导的疾病是糖尿病创面持续性慢性炎症。
在一些实施方案中，本文所述的羊水通过减少M1型巨噬细胞数目、提高M2型巨噬细胞比例的方式对巨噬细胞介导的疾病展开治疗和预防。
本发明特别优选的实施方案中，使用羊水，尤其是本文所述的禽蛋羊水，更优选是鸡蛋羊水预防或治疗巨噬细胞介导的疾病。
药物组合物
本文还提供一种药物组合物，该药物组合物含有本文所述的羊水，尤其是家禽的蛋中的羊水，更优选为胚龄为5-12天、更优选为6-11天、更优选6-9天、更优选7-8天的鸡蛋的羊水。药物组合物可以是-60℃以下冷冻保存羊水或其冻干试剂，例如冻干羊水。药物组合物中还可含有其它药学上可接受的载体或赋形剂，例如注射用生理盐水、注射用水或葡萄糖注射液等。优选地，药物组合物含有5-40％(v/v)或10％-35％的羊水，优选15-30％。
含有本文所述羊水的药物组合物通常还含有药学上可接受的辅料。本文中，“药学上可接受的辅料”是指在药理学和/或生理学上与受试者和活性成分相容的载体、稀释剂和/或赋形剂，包括但不限于：抗生素，保湿剂，pH调节剂，表面活性剂，碳水化合物，佐剂，抗 氧化剂，螯合剂，离子强度增强剂、防腐剂、载剂、助流剂、甜味剂、染料/着色剂、增味剂、润湿剂、分散剂、悬浮剂、稳定剂、等渗剂、溶剂或乳化剂。在一些实施方案中，药学上可接受的辅料可以包括一种或多种非活性成分，包括但不限于：稳定剂、防腐剂、添加剂、佐剂、喷雾剂、压缩空气或其它适宜的气体，或其它适宜的与药效化合物合用的非活性成分。更具体而言，合适的药学上可接受的辅料可以是本领域常用于糖尿病足的辅料。在一个或多个实施方案中，适用于喷剂的合适的药学上可接受的辅料选自以下一种或多种：水、葡糖酸内酯、苯甲酸钠、熊果苷、透明质酸钠、烟酰胺、甘油。在一个或多个实施方案中，适用于涂敷剂或敷料的合适的药学上可接受的辅料选自以下一种或多种：水、甘油、泛醇、抗坏血酸磷酸酯镁、烟酰胺、透明质酸钠、苯氧乙醇、辛甘醇、山梨酸。本领域可根据实际情况确定药学上可接受的辅料的含量。
将药物喷、涂、敷于伤口一般需添加敷料。“敷料”是包扎伤口的用品，用以覆盖疮、伤口或其他损害的材料。伤口敷料的种类有被动型敷料、相互作用型敷料和生物活性敷料。本领域知晓适用于本发明用途的伤口敷料。
本文所述药物组合物除含有羊水之外，还可含有其他有助于患者伤口恢复的活性成分，包括但不限于积雪草提取物、玫瑰纯露、甘草根提取物、油橄榄叶提取物、金盏花花提取物、白柳树皮提取物、薰衣草提取物、柠檬果提取物、水解大豆蛋白、艾叶提取物、茶叶提取物、百里香提取物、紫松果菊提取物、贯叶连翘花/叶提取物、库拉索芦荟叶汁粉、酵母提取物。
用途和治疗方法
本文还提供本文任一实施方案所述的羊水在制备治疗和/或预防TLR4/NF-κB信号通路介导的疾病的制剂中的应用。本文另一方面还提供本文任一实施方案所述的羊水在制备治疗和/或预防巨噬细胞介导的疾病的制剂中的应用。本文另一方面还提供本文任一实施方案所述的羊水在制备治疗和/或预防M1型巨噬细胞介导的疾病的制剂中的应用。本文另一方面还提供本文任一实施方案所述的羊水在制备治疗和/或预防M2型巨噬细胞介导的疾病的制剂中的应用。本文另一方面还提供本文任一实施方案所述的羊水在制备用于抑制M1型巨噬细胞类群数目、提高M2型巨噬细胞类型比例的试剂中的应用。本文另一方面还提供本文任一实施方案所述的羊水在制备促进M1型巨噬细胞向M2型巨噬细胞转化的制剂中的应用。
在一些实施方案中，本发明提供本文任一实施方案所述的羊水在制备用于以下一个或多个用途的试剂中的应用：(1)提高患者创面愈合速度；(2)促进患者创面新生血管生长；(3)降低炎症患者的促炎因子IL-6和TNF-α，提高该炎症患者的TGF-β1和IL-10； (4)促进患者创面的III型胶原形成，或提高患者创面处III型胶原与I型胶原比例；和(5)降低炎症患者的iNOS、TNF-α、IL-6和IL-1β的转录和翻译水平，提高该炎症患者的CD206和Arg-1的转录和翻译水平。
在一些实施方案中，本文还提供用于治疗和/或预防TLR4/NF-κB信号通路介导的疾病，用于治疗和/或预防巨噬细胞介导的疾病，用于治疗和/或预防M1型巨噬细胞介导的疾病，用于治疗和/或预防M2型巨噬细胞介导的疾病，用于抑制M1型巨噬细胞类群数目、提高M2型巨噬细胞类型比例，和/或用于促进M1型巨噬细胞向M2型巨噬细胞转化的本文任一实施方案所述的羊水或含有该羊水的组合物。
本文还提供治疗和/或预防TLR4/NF-κB信号通路介导的疾病的方法，包括给予有需要的对象治疗有效量的本文所述的羊水或含有所述羊水的组合物的步骤。
本发明还提供治疗和/或预防巨噬细胞介导的疾病的方法。本发明还提供治疗和/或预防M1型巨噬细胞介导的疾病的方法。本发明还提供治疗和/或预防M2型巨噬细胞介导的疾病的方法。这些方法包括给予有需要的对象治疗有效量的本文所述的羊水或含有所述羊水的组合物的步骤。
本发明还提供促进M1型巨噬细胞向M2型巨噬细胞转化的方法，包括给予需要的对象有效量的本文所述的羊水或含有所述羊水的组合物的步骤。在一些实施方案中，该方法可用于治疗或预防受益于M2型巨噬细胞增加的疾病，包括但不限于前文所述的各类疾病。该方法可以是体内方法，也可以是体外方法。
本文还提供一种炎症对象组织修复方法，所述方法包括使用本文所述的羊水或由其为主要原料配制成的复合敷料，或含有本文所述的羊水的细胞培养基体外培养感兴趣的组织细胞，形成组织基质后，将该组织基质植入组织损伤或缺损部位。
在一些实施方案中，本文还提供一种提高患者创面愈合速度的方法，该方法包括给予在该患者创面外涂本文所述的羊水或含有该羊水的药物组合物，或外敷由本发明的羊水为主要原料配制成的复合敷料。在一些实施方案中，本文还提供一种促进患者创面新生血管生长的方法，该方法包括给予在该患者创面外涂本文所述的羊水或含有该羊水的药物组合物，或外敷由本发明的羊水为主要原料配制成的复合敷料。在一些实施方案中，本发明还提供一种降低炎症患者的促炎因子IL-6和TNF-α，提高该炎症患者的TGF-β1和IL-10的方法，该方法包括给予该患者有效量的本文所述的羊水或含有该羊水的药物组合物。在一些实施方案中，本发明还提供一种促进患者创面的III型胶原形成的方法，或提高患者创面处III型胶原与I型胶原比例的方法，该方法包括给予在该患者创面外涂本文所述的羊水或含有该羊水的药物组合物，或外敷由本发明的羊水为主要原料配制成的复合敷料。在一些实施方案中，本发明还提供一种降低炎症患者的iNOS、TNF-α、IL-6和IL-1β的转录和翻 译水平，提高该炎症患者的CD206和Arg-1的转录和翻译水平的方法，该方法包括给予该患者有效量的本文所述的羊水或含有该羊水的药物组合物。
本文中，治疗有效量是指可在受试者中实现治疗、预防、减轻和/或缓解疾病或病症的剂量。可根据患者年龄、性别、所患病症及其严重程度、患者的其它身体状况等因素确定治疗有效量。本文中，受试者或患者通常指哺乳动物，尤其指人。
本文中，给药的剂量和频次可根据具体的病情，患者的年龄和性别等情况由医护人员确定。通常，对于特定疾病的治疗，治疗有效量是指足以改善或以某些方式减轻与疾病有关的症状的药量。这样的药量可作为单一剂量施用，或者可依据有效的治疗方案给药。给药量也许可治愈疾病，但是给药通常是为了改善疾病的症状。一般需要反复给药来实现所需的症状改善。例如，对于给予人的剂量，通常可在1-200ml/次，可每天或每周注射给予。在某些实施方案中，给药频次可以是每天多次、每天二次、每二天、每三天、每四天、每五天或每六天给药一次，或每半个月给药一次，或者每月给药一次。
细胞培养基
在某些实施方案中，本文还提供一种细胞培养基，该细胞培养基含有适量的本文所述的羊水。细胞培养基中羊水的含量可根据所培养的细胞的类型加以确定，例如，羊水的添加量可以是该细胞培养基重量的0.1～30％，如1～25％或3～20％不等。可根据待培养的细胞选择合适的细胞培养基，示例性的细胞培养基包括但不限于可市售获得的各种培养基，如DMEM、RPMI 1640、MEM、DMEM/F12等。
以下将以具体实施例的方式对本发明作进一步说明。应理解，这些实施例仅仅是阐述性的，并非用于限制本发明的范围。实施例中所用到的方法和试剂，除非另有说明，否则为本领域的常规方法和试剂。
材料与方法
抗体和试剂
PE CD206抗体(12-2069-42)和FITC F4/80抗体(11-4801-82)购自eBioscience。Arginase1(Arg-1；93668)、α-SMA(19245)和CD206(24595)购自CST。GAPDH(ab181602)、iNOS(ab178945)、TNF-α(ab183218)、IL-6(ab290735)、IL-1β(ab254360)和CD31(ab281583)购自Abcam。TLR4(A5258)、NF-κB-p65(A19653)和p-IKB(AP0707)购自ABclonal。Trizol试剂和SYBRgreen购自Vazyme Biotech。STZ(S0130)和葡萄糖(D9434)购自Sigma-Aldrich。
ceAF的制备
受精鸡蛋在38℃和50％湿度下孵化。在孵化的第6天和第8天之间，从中分离出ceAF。离心(2500×g，20分钟)后，使用0.22μm无菌过滤器(Millipore，美国)过滤上清液，将过滤后的样本等分并保持在-80℃。
细胞培养
RAW264.7细胞由中国科学院细胞库提供，在含有10％FBS和1％双抗体的高糖DMEM培养基中37℃、5％CO2培养。高葡萄糖条件为具有40mM葡萄糖的DMEM。在培养基中加入各种浓度(分别为0％、1％、5％、10％、20％)的ceAF用于后续实验。
动物和伤口处理
实验方案经南京鼓楼医院动物保护与伦理委员会批准。C57BL/6小鼠(雄性，8周龄)取自南京大学模型动物研究中心，饲养在无特定病原体的环境中，不受限制地获得水和食物。每组18只小鼠每天腹腔注射50mg/kg链脲佐菌素(STZ，在柠檬酸钠缓冲液中)，持续5天，以构建STZ诱导的糖尿病模型。三周后对小鼠进行血糖测量，血糖水平＞16.7mM的小鼠被归类为糖尿病。为了建立切除伤口模型，在小鼠脱毛后的背部皮肤上进行了8毫米圆形活检穿孔。造模后，每天将10％ceAF局部涂抹于创面，对照小鼠给予等体积的PBS。在第0、3、5、7、11天拍摄伤口图像，并用ImageJ软件(美国国立卫生研究院，National Institutes of Health)测量伤口面积。在受伤后第5天和第10天收集伤口组织样本用于后续实验。
组织学和免疫荧光染色
将伤口边缘组织固定、脱水、包埋在石蜡中，并以5μm厚度切片。根据标准化组织学程序进行马松三色(MT)、苏木精-伊红(H&E)和天狼星红染色。为了评估巨噬细胞极化和血管生成，CD206、iNOS、CD31和α-SMA单克隆抗体(1μg/ml)染色在4℃过夜。然后，孵育特异性荧光二抗，然后进行DAPI染色。
用PBS依次冲洗RAW264.7细胞，固定在多聚甲醛(4％)中，用0.1％TritonX-100穿孔并用BSA(3％)封闭。然后，根据说明将细胞与相应的一抗和二抗一起孵育。所有照片均使用Olympus FluoView FV3000共聚焦显微镜(东京，日本)拍摄。
RNA分离和RT-qPCR
按照制造商的说明，用Trizol试剂处理细胞和伤口边缘组织以分离总RNA。RT-qPCR由SYBR green染料使用StepOne RT-qPCR系统(Applied Biosystems，USA)进行。用GAPDH标准化后，使用2-ΔΔCT方法确定相对基因水平。引物序列如表1所示。
表1：RT-qPCR所用的引物序列(SEQ ID NO：1-14)
蛋白印迹(Western blotting，WB)分析
使用RIPA裂解缓冲液(KeyGEN，中国)从裂解的皮肤组织和细胞中分离蛋白质样品。进行BCA测定以确定离心后的总蛋白质浓度。通过10％SDS-PAGE凝胶分离蛋白质样品并转移到PDVF膜(Millipore，USA)上。用5％BSA封闭后，将膜与相应的一抗孵育过夜、二抗孵育l小时。使用ECL底物试剂盒(Vazyme，中国)进行蛋白质条带的可视化。
流式细胞术
为了确定RAW264.7巨噬细胞的极化趋势，将细胞与FITC缀合的抗小鼠F4/80抗体和PE缀合的抗小鼠CD206抗体在4℃避光预孵育30分钟。使用流式细胞仪(FACSCanto II，BD，USA)确定细胞表型，并使用FlowJo软件进行数据分析。
细胞活力测试
使用CCK-8测定法(Beyotime，中国)评估RAW264.7细胞活力。饥饿12小时后，将细胞暴露于浓度为0％、1％、5％、10％或20％的ceAF，然后孵育24小时。将细胞用PBS冲洗3次，然后用200μL含有CCK-8混合物的不完全培养基(10μL)覆盖并在37℃下孵育。使用酶标仪在450nm处测量吸光度。
酶联免疫吸附试验(ELISA)
将RAW264.7细胞在10％ceAF中暴露48h，收集细胞上清液进行测试。根据试剂盒说明书(Elabscience，China)通过ELISA试剂盒测量分泌的IL-6、IL-10、TGF-β1和TNF-α。
统计分析
实验数据用Graphpad Prism v8.0软件进行分析，并以Mean±SEM表示。参数检验用于符合正态分布的数据(Shapiro-Wilk检验)。如果数据呈正态分布，则采用单因素方差分析和Newman-Keuls事后检验比较多组间的统计学差异。如果数据通过正态性检验，则使用双尾学生t检验对两组进行比较。使用双向ANOVA分析两个因素的组合效应，然后进行Tukey后测。至少进行了三个独立的测定，并且P＜0.05被定义为具有统计学意义。
实施例1：ceAF通过TLR4/NF-κB信号通路减轻脂多糖刺激的RAW264.7细胞炎症
为了评估ceAF对脂多糖(LPS)诱导的细胞炎症的调节作用，本实施例使用RAW264.7作为实验细胞，LPS(100ng/ml)刺激48小时诱导细胞炎症。
首先用不同浓度ceAF对细胞增殖存活能力进行检测，CCK8结果显示，随ceAF浓度升高，RAW264.7增殖存活能力提升，在10％浓度时达到峰值(图1，A)。接着将RAW264.7分成空白对照(NC)、LPS刺激组(LPS)、LPS和ceAF联合干预组(LPS+ceAF)以及ceAF干预组(ceAF)。qPCR结果显示LPS刺激后，在转录水平M2巨噬细胞相关基因(CD206)降低，M1巨噬细胞相关基因(iNOS)升高，炎症相关基因(TNF-α、IL-6、IL-1β)升高，这些趋势在ceAF干预后被逆转(图1，B)。进一步在翻译水平进行Western blotting(WB)实验，得到了相同的趋势结果(图1，C-D)。
实施例2：ceAF体外诱导RAW264.7向M2巨噬细胞极化
用流式细胞术和细胞免疫荧光对ceAF诱导巨噬细胞向M2极化进行验证。流式细胞术用CD86标记巨噬细胞，用CD206定位M2巨噬细胞。细胞免疫荧光用Arg-1定位M2巨噬细胞。结果显示ceAF干预48小时和72小时均有效提高了M2巨噬细胞的比例(图2，A-D)。收集RAW264.7细胞上清液，ELISA检测炎症相关细胞因子，结果显示促炎因子IL-6和TNF-α在ceAF干预后明显下降，M2相关分泌因子TGF-β1和IL-10在ceAF干预后明显上升(图2，E)。
实施例3：ceAF促进STZ诱导的糖尿病小鼠创面愈合
为了验证ceAF对链脲佐菌素(STZ)诱导的糖尿病小鼠创面愈合作用，选用8周龄雄 性小鼠分两组，STZ(50mg/kg)小鼠腹腔连续注射五天，一周后测量血糖，血糖＞16.7mM视为糖尿病造模成功。小鼠背部中央皮肤剪刀锐性制造直径8mm皮肤全层创面，对照组(DM组)每日常规换药，实验组每日予以10％ceAF外用换药处理，在第0、3、5、7、11天相机记录创面照片。结果显示自第五天起，ceAF组愈合速度明显快于对照组(图3，A-B)，第11天测量小鼠体重和血糖两组无明显差异(图3，C-D)，体重和血糖两项混淆因素可排除。
实施例4：ceAF改善STZ诱导的糖尿病小鼠创面组织学参数
本实施例通过组织病理学进一步证实了ceAF对STZ诱导的糖尿病小鼠创面的影响。创面造模后第5天进行H&E染色观察创缘组织中炎症细胞浸润情况，发现ceAF组炎症细胞明显减少(图4，A-B)。创面造模后第10天进行MT染色观察创缘组织中胶原沉积情况，发现ceAF组胶原沉积明显增多(图4，C-D)。两组创面均愈合后取愈合区域皮肤组织进行天狼猩红染色评估愈合质量(图4，E)，结果显示ceAF组III型胶原居多，对照组I型胶原居多，表明ceAF组瘢痕生成少，愈合质量较好。
实施例5：ceAF改善STZ诱导的糖尿病小鼠创面愈合相关指标
通过组织免疫荧光法，进一步证实了ceAF对STZ诱导的糖尿病小鼠愈合相关指标的影响。取第5天皮肤创缘组织进行F4/80和CD206共染定位M2巨噬细胞，用F4/80和iNOS共染定位M1巨噬细胞，发现ceAF组创缘M2巨噬细胞明显增多(图5，A)，M1巨噬细胞明显减少(图5，B)。取第10天皮肤创缘组织进行CD31和α-SMA染色(图5，C-D)，结果表明ceAF组新生血管明显增多。
实施例6：ceAF可调节STZ诱导的糖尿病小鼠创面相关炎症因子
为了在体内验证ceAF对STZ诱导的糖尿病小鼠创面相关炎症因子的影响，取创面造模后第5天创缘组织进行WB和qPCR检测，发现ceAF组M2巨噬细胞基因CD206和Arg-1在转录和翻译水平均升高(图6，A、C-D)，M1巨噬细胞标记基因iNOS，炎症相关基因TNF-α，IL-6和IL-1β在转录和翻译水平均降低(图6，A、E-H)。
综上所述，结果表明ceAF在体外能改善LPS诱导的RAW264.7细胞炎症反应，在体内能促进STZ诱导的糖尿病小鼠创面愈合。这一功能通过调节TLR4/NF-κB信号通路实现。

Claims (10)

  1. 羊水在制备治疗和/或预防巨噬细胞介导的疾病的药物中的应用，
    其中，所述羊水来自胚龄为5-12天的鸡蛋，优选胚龄为6-11天的鸡蛋，更优选胚龄为7-9天的鸡蛋，更优选胚龄为7-8天的鸡蛋，或者来自发育时期与所述胚龄的鸡蛋所处的发育时期相对应的鸡以外的其它禽类的蛋；或来自胎龄为8-14天的啮齿类动物的胚胎，或来自发育时期与胎龄为8-14天的啮齿类动物的发育时期相对应的啮齿类动物以外的其它非人哺乳动物的胚胎。
  2. 羊水在制备治疗和/或预防M1型巨噬细胞介导的疾病的药物中的应用，
    其中，所述羊水来自胚龄为5-12天的鸡蛋，优选胚龄为6-11天的鸡蛋，更优选胚龄为7-9天的鸡蛋，更优选胚龄为7-8天的鸡蛋，或者来自发育时期与所述胚龄的鸡蛋所处的发育时期相对应的鸡以外的其它禽类的蛋；或来自胎龄为8-14天的啮齿类动物的胚胎，或来自发育时期与胎龄为8-14天的啮齿类动物的发育时期相对应的啮齿类动物以外的其它非人哺乳动物的胚胎。
  3. 羊水在制备治疗和/或预防M2型巨噬细胞介导的疾病的药物中的应用，
    其中，所述羊水来自胚龄为5-12天的鸡蛋，优选胚龄为6-11天的鸡蛋，更优选胚龄为7-9天的鸡蛋，更优选胚龄为7-8天的鸡蛋，或者来自发育时期与所述胚龄的鸡蛋所处的发育时期相对应的鸡以外的其它禽类的蛋；或来自胎龄为8-14天的啮齿类动物的胚胎，或来自发育时期与胎龄为8-14天的啮齿类动物的发育时期相对应的啮齿类动物以外的其它非人哺乳动物的胚胎。
  4. 羊水在制备治疗和/或预防TLR4/NF-κB信号通路介导的疾病的制剂中的应用，
    其中，所述羊水来自胚龄为5-12天的鸡蛋，优选胚龄为6-11天的鸡蛋，更优选胚龄为7-9天的鸡蛋，更优选胚龄为7-8天的鸡蛋，或者来自发育时期与所述胚龄的鸡蛋所处的发育时期相对应的鸡以外的其它禽类的蛋；或来自胎龄为8-14天的啮齿类动物的胚胎，或来自发育时期与胎龄为8-14天的啮齿类动物的发育时期相对应的啮齿类动物以外的其它非人哺乳动物的胚胎。
  5. 羊水在制备用于抑制M1型巨噬细胞类群数目、提高M2型巨噬细胞类型比例的制剂中的应用，或在制备促进M1型巨噬细胞向M2型巨噬细胞转化的制剂中的应用，
    其中，所述羊水来自胚龄为5-12天的鸡蛋，优选胚龄为6-11天的鸡蛋，更优选胚龄为7-9天的鸡蛋，更优选胚龄为7-8天的鸡蛋，或者来自发育时期与所述胚龄的鸡蛋所处的发育时期相对应的鸡以外的其它禽类的蛋；或来自胎龄为8-14天的啮齿类动物的胚胎，或来自发育时期与胎龄为8-14天的啮齿类动物的发育时期相对应的啮齿类动物以外的其 它非人哺乳动物的胚胎。
  6. 如权利要求1-5中任一项所述的应用，其特征在于，所述药物或制剂是包含所述羊水的细胞培养物和/或胚胎干细胞，
    优选地，所述药物或制剂是包含所述羊水和/或鸡的胚胎干细胞以及药学上可接受辅料的药物组合物。
  7. 如权利要求1所述的应用，其特征在于，所述巨噬细胞介导的疾病选自：增生性瘢痕；慢性阻塞性肺炎；肿瘤，如乳腺癌、肝癌；代谢性疾病，如重度肥胖、胰岛素抵抗、II型糖尿病；炎症性疾病，如急性胰腺炎、动脉粥样硬化；心血管疾病，如心肌炎、心肌梗死、心率失常；神经病变，如阿兹海默症；脑部疾病，如脑梗死、脑损伤；眼科疾病，如自身免疫性葡萄膜炎、视网膜病变、角膜炎、角膜移植、干燥综合征、葡萄膜黑色素瘤、近视；免疫炎症性疾病，如炎症性肠炎、自身免疫性肝炎、哮喘、酒精性肝病、结肠炎、多发性硬化、牙周炎；关节炎，如类风湿性关节炎、骨侵蚀、滑膜炎、骨关节炎；肾炎，如急性肾损伤、慢性肾脏病、终末期肾病、增值性肾小球肾炎、膜性肾病、糖尿病肾病、过敏性紫癜肾炎、ANCA相关小血管炎、尿路感染、常染色体显性多囊肾；细菌感染性疾病，如脓毒症；妊娠期高血压；糖尿病、妊娠期糖尿病、糖尿病肾病。
  8. 如权利要求2所述的应用，其特征在于，所述M1型巨噬细胞介导的疾病选自：减退期增生性瘢痕；慢性阻塞性肺炎；肿瘤，如乳腺癌、肝癌；代谢性疾病，如重度肥胖、胰岛素抵抗、II型糖尿病；炎症性疾病，如急性胰腺炎；冠状动脉疾病，如动脉粥样硬化；肾病；肥胖；心血管疾病，如心肌炎、心肌梗死；脑部疾病，如脑梗死、脑损伤；眼科疾病，如自身免疫性葡萄膜炎、视网膜病变、角膜炎、角膜移植、干燥综合征、葡萄膜黑色素瘤；免疫炎症性疾病，如炎症性肠炎、自身免疫性肝炎、哮喘、酒精性肝病、结肠炎、多发性硬化、牙周炎、骨关节炎；糖尿病、妊娠期糖尿病、糖尿病肾病。
  9. 如权利要求3所述的应用，其特征在于，所述M2型巨噬细胞介导的疾病选自：增生期增生性瘢痕；肿瘤，如乳腺癌、肝癌；代谢性疾病，如胰岛素抵抗、II型糖尿病；炎症性疾病，如急性胰腺炎；心血管疾病，如心肌梗死、心肌衰竭、动脉粥样硬化、冠状动脉疾病、心肌炎；脑梗死；眼科疾病，如自身免疫性葡萄膜炎、视网膜病变、角膜炎、角膜移植、干燥综合征、葡萄膜黑色素瘤；免疫炎症性疾病，如炎症性肠炎、自身免疫性肝炎、哮喘、酒精性肝病、结肠炎、多发性硬化、牙周炎、骨关节炎；糖尿病、妊娠期糖尿病、糖尿病肾病。
  10. 如权利要求4所述的应用，其特征在于，所述TLR4/NF-κB信号通路介导的疾病选自：选自系统性红斑狼疮；血管炎症，如动脉粥样硬化、冠心病；心肌炎，如心肌缺血组织炎症、心肌损伤；肝炎，如肝衰竭，酒精性肝损伤、酒精代谢过程中的炎症免疫反 应；脂肪肝；肺炎，如急性肺损伤、慢性阻塞性肺疾病、矽肺；肾炎，如急性肾损伤、狼疮性肾炎；炎症性肠病，如急性肠炎、溃疡性结肠炎、放射性直肠炎；胃炎，如慢性萎缩性胃炎；急性呼吸道感染，如肺炎、支气管炎、咽炎、鼻窦、中耳炎；牙周炎、高尿酸血症；变应性鼻炎、过敏性鼻炎；乳腺炎；关节炎，如急性痛风性关节炎、慢性关节炎、类风湿性关节炎；创面组织炎症、增生性瘢痕；多囊卵巢综合征；肿瘤，如垂体泌乳素腺瘤、促肾上腺皮质激素腺瘤、颅内动脉瘤；感染性疾病，如细菌性感染、真菌性感染、病毒感染；过敏性疾病，如皮肤过敏性疾病、支气管哮喘、过敏性鼻炎、过敏性紫癜。
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