WO2023241717A1 - 促进哺乳动物器官再生修复的物质及其应用 - Google Patents

促进哺乳动物器官再生修复的物质及其应用 Download PDF

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WO2023241717A1
WO2023241717A1 PCT/CN2023/100868 CN2023100868W WO2023241717A1 WO 2023241717 A1 WO2023241717 A1 WO 2023241717A1 CN 2023100868 W CN2023100868 W CN 2023100868W WO 2023241717 A1 WO2023241717 A1 WO 2023241717A1
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regeneration
repair
organs
tissue
inhibitor
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PCT/CN2023/100868
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English (en)
French (fr)
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李伟
周琪
何正泉
卢宗宝
王帅
王馨
袁雪薇
王柳
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中国科学院动物研究所
北京干细胞与再生医学研究院
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Publication of WO2023241717A1 publication Critical patent/WO2023241717A1/zh

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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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/196—Carboxylic acids, e.g. valproic acid having an amino group the amino group being directly attached to a ring, e.g. anthranilic acid, mefenamic acid, diclofenac, chlorambucil
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/202—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having three or more double bonds, e.g. linolenic
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/203—Retinoic acids ; Salts thereof
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
    • A61K31/381—Heterocyclic compounds having sulfur as a ring hetero atom having five-membered rings
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A61K31/423—Oxazoles condensed with carbocyclic rings
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/45—Non condensed piperidines, e.g. piperocaine having oxo groups directly attached to the heterocyclic ring, e.g. cycloheximide
    • A—HUMAN NECESSITIES
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4741—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having oxygen as a ring hetero atom, e.g. tubocuraran derivatives, noscapine, bicuculline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/05—Dipeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
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    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/14—Drugs for dermatological disorders for baldness or alopecia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • 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
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/80—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members
    • C07D211/84—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen directly attached to ring carbon atoms
    • C07D211/86—Oxygen atoms
    • C07D211/88—Oxygen atoms attached in positions 2 and 6, e.g. glutarimide

Definitions

  • the present invention relates to the field of biotechnology, and in particular to a method for promoting mammalian organ regeneration and repair and related applications.
  • Regeneration refers to the repair process in which the whole body or an organ is partially lost due to trauma, and a structure that is the same in form and function as the lost part is grown based on the remaining part. Failure of regeneration can lead to loss of tissue or organ functionality, ultimately leading to various diseases and even death. Different species in nature have different regeneration abilities, which can be divided into: 1. Individual level regeneration. For example, lower plants can regenerate a plant from a single cell, and some higher plants can use roots, stems, leaves and other tissues to regenerate new ones. Plants and lower organisms such as planarians can use any part of the body to regenerate a complete individual; 2.
  • Regeneration from excision, tailed amphibians such as salamanders, geckos and some fish can regenerate tails, limbs and fins.
  • Regeneration at the tissue level such as liver cell proliferation and regeneration after liver resection; 4.
  • Regeneration at the cellular level such as regrowth of neuron axons after rupture.
  • mammals, including humans have greatly lost their ability to regenerate and have almost no ability to regenerate.
  • Regeneration is limited to the fetal period and specific periods such as the liver and skin, and specific tissues or organs. .
  • the general trend is: as the evolutionary level increases, the regeneration ability becomes weaker or even lost.
  • Fibrosis is a non-regenerative way of repairing damage. Promoting the regeneration of damaged tissue (regenerative therapy) can not only control fibrosis, but also restore the original function of the tissue. Therefore, regenerative therapy is the most effective way to prevent and treat fibrosis-related diseases. ideal means.
  • Pulmonary fibrosis is a pathological change characterized by the proliferation of fibroblasts and the accumulation of large amounts of extracellular matrix, accompanied by inflammatory damage and tissue structure destruction. That is, the normal alveolar tissue is damaged and undergoes abnormal repair resulting in structural abnormalities (scar formation). Pulmonary fibrosis will seriously affect the human respiratory function, manifesting as various dyspnea, which will continue to worsen with the aggravation of the condition and lung damage and the patient's respiratory function. It is reported that the morbidity and mortality of idiopathic pulmonary fibrosis are increasing year by year worldwide, and the average survival time after diagnosis is less than 3 years, which is higher than most tumors, so it is also called a "tumor-like disease" . Therefore, promoting the regeneration of damaged tissue is the most fundamental means to treat and prevent pulmonary fibrosis-related diseases and has important application value.
  • this application provides a small molecule compound with the ability to promote the regeneration and repair of mammals, and has achieved technological, Unexpected technical effects.
  • the technical solution of this application is as follows:
  • the ability to promote the regeneration and repair of mammalian tissues or complex structures or organs described in this application is achieved by inducing activation of the TBK1-IRF3 pathway, preferably by inhibiting protein synthesis.
  • the present application also provides the application of the substance capable of upregulating ISG gene expression in preparing drugs or reagents for promoting the regeneration and repair ability of mammalian tissues or complex structures or organs.
  • the tissue described in this application is skin, fat, muscle, bone, hair follicle, blood vessel or nerve.
  • the complex structure described in this application is at least two or more body structures including skin, hair follicles, glands, cartilage, muscles, fat, blood vessels, nerves or limbs.
  • the organ described in this application is lung, liver, heart, pancreatic islet or kidney.
  • the complex structure is an ear, a limb, a finger, an eye or a nose.
  • the regenerative repair described in this application is regeneration after the ear has been removed.
  • the MAPK inhibitor provided in this application may be one or more of a P38 inhibitor and a selective P38 ⁇ inhibitor.
  • the P38 inhibitor is Doramapimod
  • the selective P38 ⁇ inhibitor is MAPK13-IN-1.
  • retinoic acid receptor-related orphan receptor inhibitors provided by this application are effective in promoting tissue and organ
  • the application of regenerative repair ability is achieved through the reverse activation of retinoic acid receptor-related orphan receptor ⁇ (ROR ⁇ ).
  • the protein synthesis inhibitor provided in this application is used to promote tissue and organ regeneration and repair capabilities by activating the STING-TBK1-IRF3 signal.
  • the protein synthesis inhibitor provided by this application can be cycloheximide (CHX), anisomycin (Ani), Didemnin B (DIDB), Bofanomycin One or more of Bouvardin (BVD), Narciclasine or Pancratisatin.
  • the present application also provides a composition containing the substance that can promote the regeneration and repair ability of mammalian tissue or complex structure or organ, and is prepared to promote the regeneration and repair ability of mammalian tissue or complex structure or organ. or the application of drugs or reagents with organ regeneration and repair capabilities and the preparation of drugs or reagents for the treatment of diseases related to the regeneration and repair of mammalian tissues or complex structures or organs.
  • the substance capable of up-regulating ISG gene expression or the composition containing a substance capable of up-regulating ISG gene expression in the method is the substance capable of up-regulating ISG gene expression provided in this application. Substances or compositions provided in this application.
  • substances capable of upregulating ISG gene expression or containing substances capable of upregulating ISG gene expression can be administered to the subject in need by administration methods such as intraperitoneal injection, intravenous injection, intragastric administration, oral administration, or skin application. composition of matter.
  • Figure 1I is a schematic diagram of the healing effect of mouse ear holes after 3 weeks of treatment with different administration methods (21 days after injury).
  • Figure 1J shows the healing effect of 2mm ear piercing in Nsun2 knockout mice (21 days after injury).
  • Figure 4B is a schematic diagram of interferon-responsive genes and their enrichment.
  • Figures 4C and 4D are schematic diagrams of the experimental results of quantitative qPCR detection of CHX-induced ISG gene expression.
  • Figure 4E is a schematic diagram of the results of immunofluorescence staining to identify CHX activation of the STING/TBK1/IRF3 signaling pathway.
  • FIGS. 7A and 7B are schematic diagrams of experimental results of quantitative qPCR detection of interferon (IFN) ⁇ -induced ISG gene expression.
  • Figures 8A and 8B are schematic diagrams of the experimental results of quantitative qPCR detection of ISG gene expression induced by S100A8/A9.
  • Figure 8C is a statistical graph showing the effects of S100A8/A9 drug treatment for 35 days on mice with ear piercing trauma caused by a 2mm diameter ear hole punch.
  • Figures 10B and 10C are schematic diagrams of the experimental results of quantitative qPCR detection of SR3335-induced ISG gene expression.
  • Figure 11B shows the healing effect of a 4mm ear hole in mice treated with Vehicle/CRB for 30 days.
  • Figure 11D is a picture of ⁇ -SMA immunofluorescence staining of mouse auricle tissue after being treated with Vehicle/CRB for 7 days.
  • Figure 11E shows the 4mm ear piercing of mice after being treated with Vehicle/CRB for more than 180 days.
  • Figure 12A is a schematic diagram of extremity modeling.
  • Figure 12E shows the regeneration results of bone tissue after 30 days of drug CR treatment.
  • Figure 13 is a schematic diagram of activating ISG gene expression to promote mammalian regeneration ability and promote regeneration and repair.
  • the substances capable of up-regulating ISG gene expression include compounds, cytokines, proteins and inhibitors.
  • the present application also relates to the use of the substance capable of upregulating ISG gene expression in the preparation of drugs or reagents for treating diseases related to the regeneration and repair of mammalian tissues or complex structures or organs.
  • the ability to promote the regeneration and repair of mammalian tissues or complex structures or organs is achieved by inducing activation of the TBK1-IRF3 pathway.
  • the substance capable of upregulating ISG gene expression is a MAPK inhibitor.
  • the MAPK (mitogen-activated protein kinase) pathway has a three-level signaling process: MAPK, MAPK kinase (MEK or MKK), and MAPK kinase kinase (MEKK or MKKK). These three kinases can be activated in sequence and jointly regulate a variety of important physiological and pathological effects such as cell growth, differentiation, stress, and inflammatory responses.
  • MAPK pathway There are four main branch routes of the MAPK pathway: ERK, JNK, p38/MAPK and ERK5. Among them, ERK regulates cell growth and differentiation, and JNK and p38MAPK signaling pathways play an important role in stress responses such as inflammation and apoptosis.
  • MAPK inhibitors can regulate the signal transduction of various MAPK pathways.
  • the substance capable of upregulating ISG gene expression is a retinoic acid receptor-related orphan receptor inhibitor, specifically, a retinoic acid receptor-related orphan receptor alpha (ROR ⁇ ) inhibitor. agent.
  • a retinoic acid receptor-related orphan receptor inhibitor specifically, a retinoic acid receptor-related orphan receptor alpha (ROR ⁇ ) inhibitor. agent.
  • the ability to promote tissue and organ regeneration and repair is achieved through reverse activation of retinoic acid receptor-related orphan receptor ⁇ (RORa).
  • RORa retinoic acid receptor-related orphan receptor ⁇
  • the ROR ⁇ inhibitor is a selective ROR ⁇ inverse agonist, preferably SR3335.
  • the substance capable of upregulating ISG gene expression is a protein synthesis inhibitor.
  • Protein synthesis inhibitors are a class of substances that affect protein biosynthesis. They can act on DNA replication and RNA transcription, and have an indirect effect on protein biosynthesis. They can act on all aspects of protein synthesis, including inhibiting initiation factors and elongation factors. and the role of ribonucleosomes, etc. Among them, common protein synthesis inhibitors are mainly blockers that can inhibit the protein biosynthesis and translation process.
  • the ability to promote tissue and organ regeneration and repair is achieved by inhibiting protein synthesis.
  • the protein synthesis inhibitor can be cycloheximide (Cycloheximide (CHX), Didemnin B (DIDB) or plant antibiotics (Bouvardin (BVD)), Narciclasine or Pancratistatin.
  • CHX Cycloheximide
  • DIDB Didemnin B
  • BVD plant antibiotics
  • Narciclasine Narciclasine or Pancratistatin.
  • the present application also provides a composition containing the substance capable of upregulating ISG gene expression in promoting the regeneration and repair ability of mammalian tissues or complex structures or organs, or preparing drugs for promoting the regeneration and repair ability of mammalian tissues or complex structures or organs, or Use in agents or preparations for the treatment of diseases associated with the regenerative repair of mammalian tissues or complex structures or organs.
  • the protein synthesis inhibitor in the composition is cycloheximide (CHX), and the BMP activator is BMP signaling agonist sb4.
  • all-trans retinoic acid is 0.25-8 parts by weight
  • BMP signaling agonist sb4 is 0.25-4 parts by weight. parts by weight.
  • the regenerative repair refers to the partial loss of the whole body, organ or local tissue of a mammalian organism due to trauma, and the growth of new parts with the same morphology and function as the lost part based on the remaining parts. Structural repair process.
  • the regenerative repair is to promote the regeneration of tissue or complex structures or organs after tissue or organ removal or damage.
  • the complex structure is at least two or more body structures including skin, hair follicles, glands, cartilage, muscles, fat, blood vessels, nerves, or limbs.
  • the organ is lung, skin, heart, liver, kidney, stomach, intestine, etc.
  • the complex structure described in this application is a body structure component composed of different tissues or capable of completing Functional parts of the body with specific physiological functions or functional activities, such as ears, organs, limbs, eyes, nose, etc.
  • the regenerative repair is to promote regeneration after the ear is partially removed.
  • the regenerative repair is to promote regeneration and repair after skin damage, hair regeneration after hair loss, regeneration and repair of cartilage and muscle damage, regeneration of lungs, liver, skin, heart, kidney, muscle fibrosis and blood vessels. , Regeneration after nerve and limb injury.
  • the regeneration and repair is to promote the regeneration and repair of burnt skin.
  • the diseases related to the regeneration and repair of tissues and organs include but are not limited to skin burns, fibrosis of organs, muscle/cartilage damage or neurological diseases, etc., preferably skin burns/scalds/wounds, Hair loss, cartilage and muscle damage, pulmonary fibrosis, liver fibrosis, renal fibrosis, myocardial fibrosis, limb trauma or various neurological diseases, etc.
  • pharmaceutically acceptable carriers or excipients may also be added to the medicine or reagent.
  • the medicament or agent can be prepared in the following form: the protein synthesis inhibitor or a composition containing the same is mixed with a pharmaceutically acceptable carrier, for example, to obtain an oral preparation, such as a tablet (including sugar-coated tablet, Film-coated tablets, sublingual tablets, orally disintegrating tablets), capsules (including soft capsules, microcapsules), granules, powders, lozenges, syrups, emulsions, suspensions, films (e.g., orally disintegrating films), etc., parenteral preparations such as injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections, infusions), external preparations (e.g., skin preparations, ointments), suppositories (such as rectal suppositories, vaginal suppositories), pills, nasal drops, respiratory preparations (inhalants), eye drops, etc.
  • these formulations can be used as controlled release formulations (eg.,
  • examples of the above-mentioned pharmaceutically acceptable carriers include excipients (for example, starch, lactose, sucrose, calcium carbonate, calcium phosphate, etc.), binders (for example, starch, gum arabic, carboxymethylcellulose, hydroxymethylcellulose, etc.).
  • excipients for example, starch, lactose, sucrose, calcium carbonate, calcium phosphate, etc.
  • binders for example, starch, gum arabic, carboxymethylcellulose, hydroxymethylcellulose, etc.
  • lubricant e.g., magnesium stearate, calcium stearate, talc, etc.
  • disintegrant e.g., carboxymethylcellulose Calcium, talc, etc.
  • diluents e.g., water for injection, saline, etc.
  • additives e.g., stabilizers, preservatives, color
  • the administration method of the substance, composition, drug or reagent capable of up-regulating ISG gene expression can be intraperitoneal injection, intravenous injection, gastric administration, oral administration or skin application.
  • the amount of administration to a subject varies according to the route of administration, symptoms, age of the patient, etc., and can be determined practically by the clinician.
  • MRL mice 1 and P21-/- mutant mice 2 can close 2 mm (mm) ear holes, but wild-type experimental mice cannot close them. Therefore, ear hole closure can be used as a good model to evaluate regeneration ability.
  • the main screening targets are important signaling pathways that regulate morphogenesis, organ development, immunity and stress responses (metabolism, translation, etc.) during individual development; they also include genes and signaling pathways involved in tumorigenesis and regeneration of lower organisms.
  • auricle At the center of the mouse's auricle, use an ear hole punch with a diameter of 2 mm to punch holes in the left and right auricles of the mouse respectively, and administer the drug to the post-traumatic mice through intraperitoneal, intravenous injection or intragastric administration (targeted screening target Dotted small molecules or growth factors were dissolved in physiological saline or DMSO), and drug-free solvents were used as controls.
  • the DMSO-dissolved drug delivery system is: 2-5% DMSO + 30-40% PEG400 + 2-5% Tween 80, added in sequence according to the final concentration (volume ratio). Administration was given every 2 days, and observations were made every 7 days.
  • the above-mentioned ear piercing trauma mouse model is used in the following examples of the present application for ear piercing regeneration experiments, in which 7-week-old C57BL/6 mice were purchased from Beijing Vitong Lever Experimental Animal Technology Co., Ltd.; Nsun2 knockout The mice were prepared by the applicant's laboratory, and CRISPR/Cas9-mediated gene knockout technology was used to obtain gene knockout embryos by injecting Cas9mRNA and Nsun2 sgRNA into mouse fertilized eggs, and further bred to obtain stable knockout mice. ; CHX: (i.e.
  • Example 1 Translation inhibition (Cycloheximide (hereinafter abbreviated as CHX or C); Anisomycin (abbreviated as Ani)) promotes the regeneration of mouse ear piercings with a diameter of 2mm.
  • CHX Cycloheximide
  • Ani Anisomycin
  • the schematic diagram in Figure 1C shows the closure of mouse ear holes after treatment with different concentrations of Vehicle/CHX: CHX at each concentration has a promoting effect on the closure of ear holes with a diameter of 2 mm, and the concentration is greater than 8 mg/kg. After three weeks of treatment, the mouse ear piercing wounds were completely closed. n ⁇ 8.
  • Figure 1D is a photo of the healing status of the 2 mm ear piercing in mice treated with Vehicle/CHX (20 mg/kg) for 30 days. The ear piercing wound treated with CHX has been completely closed.
  • the KI67 immunohistochemical staining of mouse auricle tissue in Figure 1G shows that after 7 days of CHX (20 mg/kg) treatment, the mouse basal layer cells express a large number of KI67, a marker protein for cell proliferation. As shown by the arrow, the expression in the control group is relatively less.
  • the HE staining picture of mouse auricle tissue in Figure 1H shows that after 180 days of closure of mouse ear holes treated with CHX (20 mg/kg), the wound part contains hair follicles, glands, cartilage, muscles, blood vessels and other tissues and tissue derivatives. regeneration.
  • FIG. 1I The schematic diagram in Figure 1I shows that mice with ear piercing trauma were treated with different administration methods such as intragastric administration and intraperitoneal injection, and the healing effect was produced after 3 weeks (21 days after injury).
  • n ⁇ 6 mice with ear piercing trauma were treated with different administration methods such as intragastric administration and intraperitoneal injection, and the healing effect was produced after 3 weeks (21 days after injury).
  • ns no significant difference, t test.
  • FIG. 2A shows the effect of different doses of CHX on the closure of ear holes with a diameter of 4 mm. It was found that 20 mg/kg has a similar effect to MRL/lpr in super-healing mice. It can promote the ear hole wound to become smaller, but cannot completely close it. When the dose Greater than 20mg/kg (125,175mg/kg) can achieve closure of the ear hole.
  • Figure 2B is a photo of ear piercing healing after 90 days of DMSO/CHX drug treatment. It shows that the area of the 4mm ear piercing of mice treated with CHX (125 mg/kg) was significantly reduced.
  • Example 4 Cycloheximide CHX activates the STING-TBK1-IRF3-interferon-stimulated genes (ISGs) pathway, and ISG gene expression is necessary for CHX-induced regeneration.
  • CHX CHX was used to treat mouse primary fibroblasts and macrophages respectively, and large-scale RNA-seq (transcription level) and Ribo-seq (ribosome imprint sequencing, translatomics) were performed. ).
  • RNA-seq transcription level
  • Ribo-seq ribosome imprint sequencing, translatomics
  • Example 5 Narciclasine activates ISG to promote regeneration in mice.
  • Narcissus is found in various Amaryllidaceae plants and has translation elongation inhibitory effects.
  • puromycin PURO
  • Figure 5A Further quantitative qPCR found that it significantly promoted the expression of ISG genes, as shown in Figures 5B and 5C.
  • Figure 5B shows the expression of ISG genes in fibroblasts
  • Figure 5C shows the expression of ISG genes in macrophages.
  • Figure 5D shows the healing status of mouse ear holes with 2 mm diameter ear punch wounds treated with Narciclasine for 30 days.
  • Figure 5E shows different doses of Narciclasine Regenerative effects on mouse ear piercings.
  • Figures 5F and 5G show histochemistry and Masson section staining data respectively, illustrating the regeneration structure of cartilage (long black arrow), hair follicle (asterisk), gland/sebaceous gland (triangular arrow) and other structures. From the figure, we can see the structure of cartilage Multiple generation centers, it is speculated that such multiple starting points for regeneration will greatly speed up the regeneration speed.
  • mice The mouse model construction method is the same as Example 2.
  • DMSO/NRB N-(n-(n-(n-(n-(n-(n-(n-(n-(n-(N-(N-(DPD)) once every 2 days, and the mice were anesthetized every 7 days.
  • DPD proximal–distal
  • DAP anterior–posterior
  • the experimental results are shown in Figures 5H to 5J.
  • Figure 5H shows the healing effect of a 4mm ear piercing in mice after 30 days of NRB treatment, and shows that it has a healing-promoting effect. After 30 days of treatment, the wound in the mouse ear piercing was completely closed, and it was identified as a regeneration event.
  • Figures 5I and 5J show that the HE section staining data well indicates the regenerative structures of cartilage (long black arrow), hair follicles (asterisks), glands/sebaceous glands (triangular arrows), muscles (dotted line selection area) and other structures. .
  • Example 6 Pancrastatin activates ISG to promote regeneration of ear piercing sites in mice.
  • Pancrastatin activates ISG.
  • Puromycin like narcissus, is an Amaryllidaceae alkaloid. It was first verified through the incorporation experiment of puromycin (PURO) that it can significantly inhibit the overall protein translation, as shown in Figure 6A. Furthermore, through quantitative qPCR experimental results, it was found that puromycin can significantly inhibit the overall protein translation. Pancratistatin can also significantly promote ISG gene expression, as shown in Figures 6B and 6C. Figure 6B shows ISG gene expression in fibroblasts, and Figure 6C shows ISG gene expression in macrophages.
  • Figure 6D shows the effect of pancratistatin administration on promoting the healing of the ear piercing in the treatment group 21 days after the ear piercing injury, showing that the ear piercing is completely closed.
  • Figure 6E shows the closure of the ear holes after Pancratistatin treatment for 21-28 days.
  • the results of Figure 6F (HE staining) and 6G (Masson staining) show that the mouse ear piercing wound is completely closed and identified as a regeneration event.
  • the staining data better shows multiple cartilage development centers (long black arrows), hair follicles ( Regenerative structures of structures such as asterisks), glands/sebaceous glands (triangular arrows), and muscles (areas selected by dotted lines).
  • Pancratistatin small molecule can promote ear hole closure and promote regeneration.
  • Example 7 Interferon activates ISG to promote ear piercing regeneration.
  • ISG is an interferon-stimulated gene.
  • interferon (IFN) ⁇ interferon ⁇
  • IFN interferon
  • Figure 7A shows the ISG gene expression of fibroblasts
  • Figure 7B shows the ISG gene expression of macrophages.
  • IFN ⁇ 50 ⁇ g/kg
  • Example 8 S100A8/A9 activates ISG to promote ear hole regeneration.
  • Alarmin is an inducing molecule that enhances expression after body injury.
  • S100A8/A9 5ng/ml
  • Figure 8A shows the expression of ISG genes in fibroblasts
  • Figure 8B shows the expression of ISG genes in macrophages.
  • the regeneration-promoting effect was further verified through the mouse ear piercing model.
  • PBS Vehicle group
  • S100A8/A9 heterodimer Biolegend, 765502
  • mALB mouse albumin
  • the injection doses were 25 and 12.5 ⁇ g/kg respectively.
  • the regeneration effect of the ear piercing was detected on the 35th day after injury. The results are shown in Figure 8C.
  • S100A8/A9 (12.5 ⁇ g/kg) significantly promoted the healing of the ear piercings, and one of them was completely closed.
  • Further HE staining and Masson staining were used to identify the regeneration of various tissue structures, such as epidermis, dermis, hair follicles, glands, cartilage, etc., as shown in Figure 8D.
  • Example 9 Inhibiting MAPK13 (p38) activates ISG to promote ear piercing regeneration.
  • the experimental method is: administer the following reagents to the mouse model of ear piercing trauma, and detect the healing of the ear piercing 21 days after the trauma.
  • MAPK13-IN-1 (MCE, HY-12839, 5mg/kg) selective P38 ⁇ inhibitor; Doramapimod (MCE, HY-10320, 5mg/kg), P38 inhibitor; SB203580 (MCE, HY-10256, 5mg/kg ), a selective P38 ⁇ / ⁇ inhibitor; BIX02189 (MCE, HY-12839, 5mg/kg), a selective MEK5, ERK5 inhibitor; SP600125 (MCE, HY-12041, 5mg/kg) a selective JNK inhibitor. n ⁇ 5, *p ⁇ 0.05, ***p ⁇ 0.001, ns: no significant difference, t test. The experimental results are shown in Figure 9A.
  • Example 10 Inhibiting retinoic acid receptor-related orphan receptors activates ISG to promote ear piercing regeneration.
  • SR3335 (MCE, HY-14413, 10mg/kg), selective ROR ⁇ inverse agonist; SR1078 (MCE, HY-10320, 1mg/kg), ROR ⁇ agonist; n ⁇ 7, **p ⁇ 0.01, * **p ⁇ 0.001, ns: no significant difference, t test.
  • Rora knockout mice (introduced from Shanghai Southern Model Animal Center) were used to further verify that homozygous knockout of Rora can significantly promote the closure of mouse ear holes and promote the regeneration of various tissues (Figure 10F).
  • Example 11 Combination of CRB (C: protein synthesis inhibitor CHX; R: RARs activator all-trans retinoic acid (All-trans retinoic acid); B: BMP activator BMP (signaling agonist sb4)) promotes the growth of mice The 4mm ear hole is closed and the cut area is regenerated.
  • CRB protein synthesis inhibitor CHX
  • R RARs activator all-trans retinoic acid (All-trans retinoic acid)
  • B BMP activator BMP (signaling agonist sb4)
  • DMSO/CRB DMSO/CRB
  • CB dosage CHX 20mg/kg, full ATRA 20mg/kg, BMP signaling agonist sb4 10-20mg/kg
  • the mice were anesthetized every 7 days, and vernier calipers were used to measure the proximal–distal (DPD) and anterior–posterior (DAP) axes of the mouse’s ear piercings, and the time of the mice’s pierced ears was calculated.
  • Figure 11A shows the closure of mouse ear holes after DMSO/CRB treatment. The results showed that CRB promoted the closure of ear holes with a diameter of 4 mm. After 30 days of administration, the mouse ear holes were completely closed.
  • Figure 11B shows the closure of a 4mm ear hole in mice after 30 days of drug treatment. It can be seen that in mice treated with the drug, the ear hole trauma has been closed.
  • Figure 11E shows the regeneration of various tissues after drug (CRB) induction for more than 90 days, showing the regeneration of epidermis, dermis, glands, hair follicles, muscles, cartilage, fat, and muscle.
  • CRB drug
  • mice 8-week-old ICR mice as a model, the mice were anesthetized with 5% chloral hydrate, injected intraperitoneally with 10 mL/kg, and anesthetized according to body weight. The anesthetized mice were bound, and 75% ethanol was used to sterilize the mouse's left upper limb and surgical instruments.
  • 75% ethanol was used to sterilize the mouse's left upper limb and surgical instruments.
  • the distance from the elbow joint to the radius and ulna is measured, 10 mm is retained, and the rest to the palm, fingers and other segments are removed and modeled (the ulna and radius are two bones of the forearm. The simplest way to distinguish them is thumb The radius is on the finger side and the ulna is on the little finger side).
  • FIG. 12A is a schematic diagram of extremity modeling.
  • Figure 12B shows the induced regeneration phenomenon of the limbs after drug treatment for 20, 40, and 120 days. It can be seen that outgrowth occurs at the amputation site of mice treated with the drug. Scale bar is 2mm.
  • Figure 12C shows the statistical results of the extremity outgrowth length.
  • FIG. 12D shows that after 20 and 40 days of drug CR treatment, computerized tomography (CT) was used to obtain high-definition images of the bone tissue reconstruction process.
  • CT computerized tomography

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Abstract

一种能够上调ISG基因表达的物质在制备用于促进哺乳动物组织或复杂结构或器官再生修复能力的药物或试剂中的应用，上调ISG基因表达的物质选自MAPK抑制剂、视黄酸受体相关孤儿受体抑制剂、蛋白质合成抑制剂、干扰素(IFNγ，β，λ)或警报素(S100A8/A9)蛋白中的一种或两种以上，再生修复为促进组织或器官切除或损伤后的组织或复杂结构或器官的再生。

Description

促进哺乳动物器官再生修复的物质及其应用 技术领域
本发明涉及生物技术领域，具体涉及一种促进哺乳动物器官再生修复的方法及其相关应用。
背景技术
再生是指生物体的整体或器官发生创伤而部分丢失,在剩余部分的基础上又生长出与丢失部分在形态与功能上相同的结构的修复过程。再生失败会导致组织或器官功能性丧失，最终导致各类疾病甚至死亡。自然界不同物种拥有不同的再生能力，再生能力可以分为：一、个体水平再生，如低等植物能从单个细胞再生出一个植株，一些高等植物可利用根、茎、叶等组织再生出新的植株，低等生物如涡虫等可以利用身体任何部分再生出完整个体；二、割除再生，有尾两栖类如蝾蚺、壁虎及某些鱼类等可以进行断尾再生、肢体再生及鱼鳍再生；三、组织水平再生，如肝切除后可以通过肝细胞增殖再生、皮肤组织的再生；四、细胞水平再生，如神经元轴突断裂重新生长等。然而，相比于植物和低等动物，哺乳动物包括人在内，极大地丧失了再生能力，几乎没有割除再生能力，再生仅局限发生于胎儿时期以及肝脏、皮肤等特定时期、特定组织或器官。总体趋势为：随着进化等级越高，再生能力越弱，甚至丧失。
包括人在内的哺乳动物面临各种损伤的时候，会发生疤痕形成的修复方式，这种方式会直接引起组织器官丧能，如截肢运动丧失等；或纤维化相关疾病，如心血管疾病、退行性神经系统疾病、肺部疾病、肝部疾病、糖尿病、皮肤病等。在全世界范围内，组织、器官的损伤导致的疤痕形成是许多疾病致残、致死的主要原因。因此提高哺乳动物原位再生修复能力是理解生命本质及治疗一系列相关疾病的重要手段。
对哺乳动物而言，重度和慢性损伤通常通过形成疤痕而不是组织再生来修复，其特点是用大量结缔组织增生和细胞外基质沉积的纤维化组织取代功 能性组织。研究表明，纤维化是许多慢性疾病致残、致死的主要原因。许多COVID-19患者在出院后都有炎症后肺纤维化，这极大地影响病人的预后生活质量。目前在临床上，尽管少数药物或细胞治疗手段可以用来缓解特定器官、特定类型的纤维化进程，但无法恢复损伤的组织，并且极度缺乏广泛而有效的抗纤维化手段，因此亟待开发广谱的抗纤维化并促进再生的候选靶点及药物。纤维化是损伤的一种非再生的修复方式，促进损伤组织的再生(再生疗法)不仅可以控制纤维化，还能恢复组织的原有的功能，因此再生疗法是预防和治疗纤维化相关疾病最理想的手段。
肺纤维化是由成纤维细胞增殖及大量细胞外基质堆积并伴炎症损伤、组织结构破坏为特征病理性改变，即是正常的肺泡组织被损坏后经过异常修复导致结构异常(疤痕形成)。肺纤维化会严重影响人体呼吸功能，表现为各种呼吸困难并随着病情、肺部损伤的加重及患者呼吸功能不断恶化。据报道特发性肺纤维化在世界范围内的发病率和死亡率逐年增加，诊断后的平均生存期不到3年，高于大多数肿瘤，因此又被称为一种“类肿瘤疾病”。因此促进损伤组织的再生是治疗和预防肺纤维化相关疾病最根本的手段，具有重要的应用价值。
发明内容
为了实现哺乳动物组织、器官的再生修复，为与之相关疾病的预防和诊疗开发更多技术途径，本申请提供了一种具有促进哺乳动物再生修复能力的小分子化合物，取得了开创性的、预料不到的技术效果。本申请的技术方案如下：
本申请提供了一种能够上调ISG基因表达的物质，及其在促进哺乳动物组织或复杂结构或器官再生修复能力中的应用。
进一步的，所述能够上调ISG基因表达的物质选自MAPK抑制剂、视黄酸受体相关孤儿受体抑制剂、蛋白质合成抑制剂、干扰素(IFNγ，β，λ)或警报素(S100A8/A9)蛋白中的一种或两种以上。
ISG基因即STING-TBK1-IRF3-干扰素刺激基因(Interferon-stimulated genes,ISGs)通路。干扰素(Interferon,IFN)作用于靶细胞表面受体后，通过一系列信号传导激活干扰素刺激基因的表达。干扰素刺激基因及其表达产物具有抗病毒、免疫调控等多种生物学功能，是干扰素发挥功能的重要效应分子。
进一步的，本申请中所述促进哺乳动物组织或复杂结构或器官再生修复能力是通过诱导TBK1-IRF3通路激活实现的，优选的，通过对蛋白合成抑制实现的。
本申请还提供了所述能够上调ISG基因表达的物质在制备用于促进哺乳动物组织或复杂结构或器官再生修复能力的药物或试剂中的应用。
本申请还提供了所述能够上调ISG基因表达的物质在制备用于治疗与哺乳动物组织或复杂结构或器官的再生修复相关的疾病的药物或试剂中的应用。
进一步的，本申请中所述再生修复为促进组织或器官切除或损伤后的组织或复杂结构或器官的再生。
优选地，本申请所述组织为皮肤、脂肪、肌肉、骨骼、毛囊、血管或神经。
优选地，本申请所述复杂结构为至少包括皮肤、毛囊、腺体、软骨、肌肉、脂肪、血管、神经或与肢体中的两种以上的机体结构。
优选地，本申请所述器官为肺、肝、心、胰岛或肾。
进一步优选地，所述复杂结构为耳朵、肢体、手指、眼或鼻。
优选地，本申请所述再生修复为耳朵被切除后的再生。
优选地，本申请所述再生修复为促进皮肤损伤后的再生修复，脱发后的毛发再生，软骨肌肉损伤再生修复，肺、肝、皮肤、心、肾、肌肉纤维化的再生以及血管、神经与肢体损伤后的再生。
优选地，本申请所述再生修复为促进烫伤皮肤的再生修复。
优选地，本申请所述与组织或复杂结构或器官的再生修复相关的疾病为皮肤烫伤、皮肤创伤、皮肤烧伤、脱发、软骨肌肉损伤、肝纤维化、肺纤维化或肢体损伤。
本申请所提供的所述MAPK抑制剂，在促进组织器官再生修复能力的应用中，是通过对p38MAPK通路的抑制实现的。优选地，通过对P38δ的抑制实现。
本申请所提供的所述MAPK抑制剂可以为P38抑制剂、选择性P38δ抑制剂的一种或两种以上。优选地，所述P38抑制剂为达马莫德(Doramapimod)，所述选择性P38δ抑制剂为MAPK13-IN-1。
本申请所提供的所述视黄酸受体相关孤儿受体抑制剂，在促进组织器官 再生修复能力的应用中，是通过对视黄酸受体相关孤儿受体α(RORα)的反向激活实现的。
本申请所提供的所述视黄酸受体相关孤儿受体抑制剂可以为选择性的RORα反向激动剂。优选地，可以为SR3335。
本申请所提供的所述蛋白质合成抑制剂，在促进组织器官再生修复能力的应用中，是通过诱导生命静息实现的。
本申请所提供的所述蛋白质合成抑制剂，在促进组织器官再生修复能力的应用中，是通过激活STING-TBK1-IRF3信号实现的。
本申请所提供的所述蛋白质合成抑制剂可以为环己酰亚胺(Cycloheximide(CHX))、茴香霉素(Anisomycin(Ani))、膜海鞘素B(Didemnin B(DIDB))、波凡霉素(Bouvardin(BVD))、水仙环素(Narciclasine)或水鬼蕉碱(Pancratistatin)中的一种或两种以上。
本申请还提供了包含所述可以促进哺乳动物组织或复杂结构或器官再生修复能力的物质的组合物在促进哺乳动物组织或复杂结构或器官再生修复能力、制备用于促进哺乳动物组织或复杂结构或器官再生修复能力的药物或试剂以及制备用于治疗与哺乳动物组织或复杂结构或器官的再生修复相关的疾病的药物或试剂中的应用。
优选地，本申请提供的一种组合物包括蛋白质合成抑制剂、全反式视黄酸和BMP激活剂。
优选地，所述组合物中的蛋白质合成抑制剂为环己酰亚胺(Cycloheximide(CHX))，BMP激活剂为BMP signaling agonist sb4。
优选地，在所述组合物中，以所述环己酰亚胺为1重量份计，全反式视黄酸为0.25～8重量份，BMP signaling agonist sb4为0.25～4重量份。
本申请还提供了所述可以促进哺乳动物组织或复杂结构或器官再生修复能力的物质或其组合物的给药方式可以为腹腔注射、静脉注射、灌胃、口服或皮肤涂抹。
本申请还提供了一种促进哺乳动物组织或复杂结构或器官再生修复的方法，其包括向有需要的受试者施用能够上调ISG基因表达的物质或包含能够上调ISG基因表达的物质的组合物。
进一步的，所述方法中的能够上调ISG基因表达的物质或包含能够上调ISG基因表达的物质的组合物即为本申请中提供的能够上调ISG基因表达的 物质或本申请中提供的组合物。
进一步的，所述方法中，可以以腹腔注射、静脉注射、灌胃、口服或皮肤涂抹等给药方式向有需要的受试者施用能够上调ISG基因表达的物质或包含能够上调ISG基因表达的物质的组合物。
附图说明
图1A为再生的非洲刺鼠与非再生的小鼠转录组之间的差异基因分析及其功能富集结果。
图1B为Vehicle与不同翻译抑制剂Ani、CHX药物处理后，直径2mm的小鼠耳洞愈合情况示意图。
图1C为经Vehicle与不同剂量的CHX药物处理后，直径2mm的小鼠耳洞闭合情况示意图。
图1D为直径2mm耳洞打孔器创伤的小鼠耳洞经CHX药物处理30天的闭合情况示意图，比例尺为1mm。
图1E为经CHX药物处理后，小鼠耳廓组织HE染色结果，比例尺为200um。
图1F为经CHX药物处理后，小鼠耳廓组织HE染色结果，比例尺为1mm。
图1G为经CHX药物处理7天后，小鼠耳廓组织的KI67免疫组化染色结果示意图，比例尺为100um。
图1H为经CHX药物处理耳洞闭合180天后，小鼠耳廓组织HE染色结果示意图，比例尺为1mm。
图1I为不同给药方式处理3周后小鼠耳洞愈合效果(损伤后21天)示意图。
图1J为Nsun2敲除小鼠2mm耳洞愈合效果(损伤后21天)。
图2A为经DMSO/CHX处理后，直径4mm的小鼠耳洞闭合情况示意图。
图2B为直径4mm耳洞打孔器创伤的小鼠耳洞经CHX处理50天的闭合情况示意图，比例尺为1mm。
图3为蛋白质与RNA合成、铁死亡和自噬的抑制剂处理小鼠耳洞愈合效果(损伤后21天)。
图4A为为CHX导致转录和翻译水平共同上调基因分析示意图。
图4B为干扰素响应基因及富集情况示意图。
图4C、4D为定量qPCR检测CHX诱导ISG基因表达实验结果示意图。
图4E为免疫荧光染色鉴定CHX激活STING/TBK1/IRF3信号通路结果示意图。
图4F为定量qPCR检测GSK86126与Des抑制STING/TBK1/IRF3通路活性抑制CHX诱导ISG基因表达结果示意图。
图4G为GSK86126与Des抑制STING/TBK1/IRF3通路活性抑制CHX诱导的再生结果示意图。
图4H为蛋白Westernblot实验揭示Des抑制STING/TBK1/IRF3通路活性并不抑制CHX蛋白合成抑制功能结果示意图。
图5A为Puromycin(PURO)参入实验验证水仙环素的蛋白翻译抑制作用实验结果示意图。
图5B、5C为定量qPCR检测水仙环素诱导ISG基因表达实验结果示意图。
图5D为直径2mm耳洞打孔器创伤的小鼠耳洞经Nar药物处理30天的闭合情况示意图，比例尺为1mm。
图5E为经Vehicle与不同剂量的Nar药物处理后，直径2mm的小鼠耳洞闭合情况示意图。
图5F为经Nar药物处理后的免疫组化结果示意图。
图5G为经Nar药物处理后的Masson切片染色结果示意图。
图5H为经Vehicle/NRB处理后，小鼠4mm耳洞的愈合情况示意图。
图5I为经NRB处理后的免疫组化结果示意图。
图5J为经NRB处理后的，HE切片染色结果示意图。
图6A为Puromycin参入实验验证水鬼蕉碱的的蛋白翻译抑制作用实验结果示意图。
图6B、6C为定量qPCR检测水鬼蕉碱诱导ISG基因表达实验结果示意图。
图6D为直径2mm耳洞打孔器创伤的小鼠耳洞经Pan药物处理21天的闭合情况示意图，比例尺为1mm。
图6E为直径2mm耳洞打孔器创伤的小鼠耳洞经Pan药物处理21天效果统计图，DMSO替代Pan处理为对照。
图6F为经Pan药物处理后的HE切片结果示意图。
图6G为经Pan药物处理后的HE切片结果示意图。
图7A、7B为定量qPCR检测干扰素(IFN)γ诱导ISG基因表达实验结果示意图。
图7C为直径2mm耳洞打孔器创伤的小鼠耳洞经干扰素(IFN)γ药物处理35天后效果统计图
图8A、8B为定量qPCR检测S100A8/A9诱导ISG基因表达实验结果示意图。
图8C为直径2mm耳洞打孔器创伤的小鼠耳洞经S100A8/A9药物处理35天后效果统计图。
图8D为直径2mm耳洞打孔器创伤的小鼠耳洞经S100A8/A9药物处理HE染色(左)和Masson染色(右)结果。
图9A为不同MAPKs抑制剂促进耳洞愈合效果示意图，结果显示P38或P38δ抑制剂显著促进再生。
图9B为定量qPCR检测MAPK13-IN-1诱导ISG基因表达实验结果示意图。
图9C为MAPK13-IN-1诱导再生耳廓组织HE染色图结果。
图9D为MAPK13-IN-1诱导再生耳廓组织Masson染色图结果。
图10A为RORα反向激动(抑制)剂与激动剂促进耳洞愈合效果示意图。
图10B、10C为定量qPCR检测SR3335诱导ISG基因表达实验结果示意图。
图10D为SR3335诱导再生耳廓组织HE染色图结果。
图10E为SR3335诱导再生耳廓组织Masson染色图结果。
图10F为Rora纯合敲除小鼠再生耳廓组织HE染色图结果。
图11A为经Vehicle/CRB处理后，小鼠耳洞的愈合情况示意图。
图11B为小鼠4mm耳洞经Vehicle/CRB处理30天后的愈合效果图。
图11C为经Vehicle/CRB处理后小鼠耳廓组织HE染色图。
图11D为经Vehicle/CRB处理7天后小鼠耳廓组织α-SMA免疫荧光染色图。
图11E为小鼠4mm耳洞经Vehicle/CRB处理大于180天后，小鼠耳廓 组织HE染色结果示意图，比例尺为100μm。
图11F免疫荧光染色血管标记物CD31染色结果，比例尺为20μm。
图12A为肢端造模的模式图。
图12B显示经药物处理20、40、120天后的肢端诱导再生现象。比例尺为2mm。
图12C为肢端outgrowth长度的统计结果。n≥3，***p<0.001，t检验。
图12D显示经药物CR处理20天和40天后，利用电子计算机断层扫描(CT)获取骨组织重建的过程高清图。
图12E为药物CR处理30后骨组织的再生结果。
图12F为HE染色发现该组合可以促进截肢肢端多组织类型的再生。
图12G显示经药物处理21、50、120天后，小鼠肢端再生组织Masson染色结果。比例尺为2mm，n≥3，t检验。
图13为激活ISG基因表达促进哺乳动物再生能力，促进再生修复的示意图。
具体实施方式
以下通过具体实施例来详细阐述和说明本发明的实施方式，但以下内容不应理解为对本发明作任何限制。
本申请主要涉及一种能够促进哺乳动物组织或复杂结构或器官再生修复能力的物质，所述物质为能够上调ISG基因表达的物质。
在本申请中，所述能够上调ISG基因表达的物质包括化合物、细胞因子、蛋白质及抑制剂。
在一个具体的实施方式中，所述能够上调ISG基因表达的物质选自MAPK抑制剂、视黄酸受体相关孤儿受体抑制剂、蛋白质合成抑制剂、干扰素(IFNγ，β，λ)或警报素(S100A8/A9)蛋白中的一种或两种以上。
本申请还涉及所述能够上调ISG基因表达的物质在制备用于促进哺乳动物组织或复杂结构或器官再生修复能力的药物或试剂中的应用。
本申请还涉及所述能够上调ISG基因表达的物质在制备用于治疗与哺乳动物组织或复杂结构或器官的再生修复相关的疾病的药物或试剂中的应用。
在本申请的实施方式中，所述促进哺乳动物组织或复杂结构或器官再生修复能力是通过诱导TBK1-IRF3通路激活实现的。
在本申请一个具体的实施方式中，所述能够上调ISG基因表达的物质为MAPK抑制剂。MAPK(丝裂原活化蛋白激酶)通路有三级的信号传递过程：MAPK，MAPK激酶(MEK或MKK)以及MAPK激酶的激酶(MEKK或MKKK)。这三种激酶能依次激活，共同调节着细胞的生长、分化、应激、炎症反应等多种重要的生理、病理效应。MAPK通路有4种主要的分支路线：ERK、JNK、p38/MAPK和ERK5。其中ERK调控细胞生长和分化，JNK和p38MAPK信号通路在炎症和细胞凋亡等应激反应中发挥重要作用。MAPK抑制剂可对MAPK各通路信号的转导进行调控。
在本申请的具体实施方式中，所述促进组织器官再生修复能力是通过对p38MAPK通路的抑制实现的，进一步的，是通过对P38δ通路的抑制实现。
在一些具体的实施方式中，所述MAPK抑制剂选自特异性P38抑制剂或选择性P38δ抑制剂。优选的，所述P38抑制剂为达马莫德(Doramapimod)，所述选择性P38δ抑制剂为MAPK13-IN-1。
在本申请一个具体的实施方式中，所述能够上调ISG基因表达的物质为视黄酸受体相关孤儿受体抑制剂，具体的，为视黄酸受体相关孤儿受体α(RORα)抑制剂。
在本申请的具体实施方式中，所述促进组织器官再生修复能力是通过对视黄酸受体相关孤儿受体α(RORα)的反向激活实现的。在一些具体的实施方式中，所述RORα抑制剂为选择性的RORα反向激动剂，优选的，为SR3335。
在本申请一个具体的实施方式中，所述能够上调ISG基因表达的物质为蛋白质合成抑制剂。蛋白质合成抑制剂是一类影响蛋白质生物合成的物质，它们可以作用于DNA复制和RNA转录，对蛋白质的生物合成起间接作用，可作用于蛋白质合成的各个环节，包括抑制起始因子，延长因子及核糖核蛋白体的作用等等。其中，常见的蛋白质合成抑制剂主要为可以抑制蛋白质生物合成翻译过程的阻断剂等。
在本申请的具体实施方式中，所述促进组织器官再生修复能力是通过对蛋白合成抑制实现的。
在一些具体的实施方式中，所述蛋白质合成抑制剂可以为环己酰亚胺 (Cycloheximide(CHX)、膜海鞘素B(Didemnin B(DIDB))或植物抗生素(Bouvardin(BVD))、水仙环素(Narciclasine)或水鬼蕉碱(Pancratistatin)。
在本申请一个具体的实施方式中，所述蛋白质合成抑制剂为环己酰亚胺，缩写为CHX，其化学式为：4-((R)-2-((1S,3S,5S)-3,5-二甲基-2-氧基环己基)-2-羟基乙基)哌啶-2,6-二酮(4-((R)-2-((1S,3S,5S)-3,5-dimethyl-2-oxocyclohexyl)-2-hydroxyethyl)piperidine-2,6-dione)
本申请还提供了包含所述能够上调ISG基因表达的物质的组合物在促进哺乳动物组织或复杂结构或器官再生修复能力或制备用于促进哺乳动物组织或复杂结构或器官再生修复能力的药物或试剂或制备用于治疗与哺乳动物组织或复杂结构或器官的再生修复相关的疾病的药物或试剂中的应用。
在一个具体的实施方式中，所述组合物包括蛋白质合成抑制剂、全反式视黄酸和BMP激活剂。
在一个优选的实施方式中，所述组合物中的蛋白质合成抑制剂为环己酰亚胺(Cycloheximide(CHX))，BMP激活剂为BMP signaling agonist sb4。
在一个优选的实施方式中，在所述组合物中，以所述环己酰亚胺为1重量份计，全反式视黄酸为0.25～8重量份，BMP signaling agonist sb4为0.25～4重量份。
在本申请的实施方式中，所述再生修复是指哺乳动物生物体的整体、器官或局部组织发生创伤而部分丢失，在剩余部分的基础上又生长出与丢失部分在形态与功能上相同的结构的修复过程。
在一个具体的实施方式中，所述再生修复为促进组织或器官切除或损伤后的组织或复杂结构或器官的再生。
在一些具体的实施方式中，所述组织为表皮、真皮、肌肉、骨骼、脂肪、毛囊、血管或神经。
在一些具体的实施方式中，所述复杂结构为至少包括皮肤、毛囊、腺体、软骨、肌肉、脂肪、血管、神经或与肢体中的两种以上的机体结构。
在一些具体的实施方式中，所述器官为肺、皮肤、心脏、肝脏、肾、胃、肠等。
本申请所述的复杂结构，是由不同组织构成的机体结构部件或能够完成 特定生理功能或功能活动的机体功能部件，如耳朵、各脏器、肢体、眼睛、鼻等。
在一个优选的实施方式中，所述复杂结构为耳洞。
在一个具体的实施方式中，所述再生修复为促进耳朵被部分切除后的再生。
在一个具体的实施方式中，所述再生修复为促进皮肤损伤后的再生修复，脱发后的毛发再生，软骨肌肉损伤再生修复，肺、肝、皮肤、心、肾、肌肉纤维化的再生以及血管、神经与肢体损伤后的再生。
在一个具体的实施方式中，所述再生修复为促进烫伤皮肤的再生修复。
在本申请的实施方式中，所述与组织器官的再生修复相关的疾病包括但不限于皮肤烫伤、器官的纤维化、肌肉/软骨损伤或神经系统疾病等，优选为皮肤烧/烫/创伤、脱发、软骨肌肉损伤、肺纤维化、肝纤维化、肾纤维化、心肌纤维化、肢体创伤或多种神经系统疾病等。
在本申请的实施方式中，所述药物或试剂中还可以添加药物学上可接受的载体或辅料。
具体的，所述药物或试剂可以以如下形式制备：将所述蛋白质合成抑制剂或包含其的组合物与药学上可接受的载体混合，例如得到口服制剂，诸如片剂(包括糖衣片剂、薄膜包衣片剂、舌下片剂、口腔崩解片剂)、胶囊剂(包括软胶囊剂、微囊剂)、颗粒剂、粉末剂、锭剂、糖浆剂、乳剂、混悬剂、薄膜(例如、口服崩解性的薄膜)等、肠胃外制剂如注射剂(例如皮下注射剂、静脉内注射剂、肌内注射剂、腹腔注射剂、滴注剂)、外用制剂(例如皮肤制剂、软膏剂)、栓剂(例如直肠栓剂、阴道栓剂)、丸剂、滴鼻剂、呼吸制剂(吸入剂)、眼药水等。除此之外，这些制剂可作为控释制剂(例如持续释放微囊剂)、诸如立即释放制剂、持续释放制剂等。这样的制剂可通过本技术领域中常规使用的制备方法获得。
具体的，上述药学上可接受的载体的例子包括赋形剂(例如，淀粉，乳糖，蔗糖，碳酸钙，磷酸钙等)，粘合剂(例如，淀粉，阿拉伯胶，羧甲纤维素，羟丙基纤维素，结晶纤维素，海藻酸，凝胶，聚乙烯吡咯烷酮等)，润滑剂(例如，硬脂酸镁，硬脂酸钙，滑石粉等)，崩解剂(例如，羧甲纤维素钙，滑石粉等)，稀释剂(例如，注射用水，盐水等)，添加剂(例如，稳定剂，防腐剂，着色剂，调味剂，溶解助剂，乳化剂，缓冲剂，等渗剂等)， 等等。
在本申请的实施方式中，所述能够上调ISG基因表达的物质、组合物、药物或试剂的给药方式可以为腹腔注射、静脉注射、灌胃、口服或皮肤涂抹。具体的，向受试者的给药量根据给药途径、症状、患者年龄等等而不同，临床医生可以实际确定。
实施例
近年来研究显示，如MRL小鼠1及P21-/-突变鼠2可以闭合2mm(毫米)耳洞，而野生型实验小鼠均不能闭合，因此耳洞闭合可作为评估再生能力良好的模型来筛选促进提高再生能力的细胞、信号通路、基因。主要筛选靶点为调控个体发育过程中形态发生、器官发育、免疫与应激发应(代谢、翻译等)等重要信号通路；同时也包括参与肿瘤发生及低等生物再生的基因及信号通路。
耳洞创伤小鼠模型的建立：选用7周龄C57BL/6小鼠，使用5％水合氯醛对小鼠实施麻醉，腹腔注射10mL/kg，按体重进行麻醉。将麻醉好的小鼠进行绑定，使用75％的乙醇对小鼠耳朵和器械进行消毒。以小鼠的耳廓中心，使用直径为2mm的耳洞打孔器对小鼠左右耳廓分别打孔，对创伤后小鼠通过腹腔、静脉注射或灌胃的方式给药(靶向筛选靶点的小分子或生长因子溶于生理盐水或DMSO)，以不含药物的溶剂作为对照。DMSO溶解的给药体系为：2-5％DMSO+30-40％PEG400+2-5％Tween 80按终浓度(体积比)依次加入。每2天给药一次，每7天进行观察，使用游标卡尺对小鼠耳洞的近-远轴(proximal–distal，DPD)和前后轴(anterior–posterior，DAP)进行测量，并计算小鼠被割除耳洞的面积，面积的计算公式为S＝πDPD×DAP/4，对小鼠的耳洞闭合情况进行记录。
如无特别说明，本申请以下实施例中均采用上述耳洞创伤小鼠模型进行耳洞再生实验，其中7周龄C57BL/6小鼠购自北京维通利华实验动物技术有限公司；Nsun2敲除小鼠由申请人实验室制备，利用CRISPR/Cas9介导基因敲除技术，通过将注射Cas9mRNA与Nsun2的sgRNA注射到小鼠受精卵获得基因敲除胚胎，进一步繁殖获得稳定敲除的小鼠；CHX:(即4-((R)-2-((1S,3S,5S)-3,5-dimethyl-2-oxocyclohexyl)-2-hydroxyethyl)piperidine-2,6-dione)购自MedCamExpress。其他材料、试剂等，如无特殊说明，均可 从商业途径得到。
实施例一：翻译抑制(环己酰亚胺(Cycloheximide，后缩写为CHX或C)；茴香霉素(Anisomycin，缩写为Ani))促进小鼠直径2mm耳洞割处再生。
实验方法：对耳洞创伤小鼠以不同剂量CHX(溶于生理盐水或DMSO)分别采取腹腔注射给药(图1I实验中增加灌胃给药)，生理盐水或DMSO作为对照组(标记为Vehicle)。对药物处理过的小鼠创伤耳廓进行鉴定。具体实验结果如图1所示。
图1A转录组分析再生的非洲刺鼠与非再生的小鼠(C57BL/6小鼠)之间差异基因，并通过功能富集分析，结果显示再生非洲刺鼠相比于非再生小鼠相比显著下调是核糖体亚基及翻译相关。
图1B显示，基于图1A分析结果，对非再生小鼠利用翻译的小分子抑制剂Ani,CHX分别进行药物处理21天后，观察其对2mm耳洞愈合效果，结果显示相比较于对照组(Vehicle)，翻译抑制剂Ani(10mg/kg),CHX(20mg/kg)均能显著促进耳洞愈合，CHX愈合效果更佳，由此发现翻译抑制能显著促进非再生物种的再生。后续实验基于CHX开展。
图1C的示意图显示了经不同浓度的Vehicle/CHX进行处理后，小鼠耳洞的闭合情况：各浓度的CHX对于直径2mm的耳洞闭合都有促进作用，其中浓度大于8mg/kg以上，药物处理三周后，小鼠耳洞创口完全闭合。n≥8。
图1D为经Vehicle/CHX(20mg/kg)处理耳洞创伤小鼠2mm耳洞30天后的愈合情况照片，其中以CHX处理的耳洞创伤已经完全闭合。
图1E的小鼠耳廓组织HE染色图显示，经CHX(20mg/kg)处理后，耳洞创伤小鼠耳洞部分的皮肤，结缔组织等多种组织结构生长连接在一起，创伤已经完全愈合。
图1F的小鼠耳廓组织HE染色图显示，耳洞创伤小鼠经DMSO/CHX(20mg/kg)处理后，创伤后第1天(D1组)两组织均局部可见灶性坏死，坏死灶内炎性细胞弥漫性浸润，如箭头①所示；组织内真皮层内可见炎性细胞弥散性浸润，如箭头②所示；其中CHX组中部分细胞内可见含铁血黄素沉积，如箭头③所示。第15天DMSO组的组织中部分细胞内可见含铁血黄素沉积，如箭头③所示；可见组织水肿，真皮下结缔组织间隙增大，组织结 构疏松，如箭头④所示。第15天CHX组的组织损伤后愈合形成的肉芽组织，肉芽组织内成纤维细胞和血管大量增生，如箭头⑤所示；并可见炎性细胞弥散性分布，如箭头②所示。
图1G的小鼠耳廓组织的KI67免疫组化染色图显示，CHX(20mg/kg)处理7天后，小鼠基底层细胞大量表达细胞增殖的标志性蛋白KI67，如箭头显示，对照组表达相对较少。
图1H的小鼠耳廓组织HE染色图显示，经CHX(20mg/kg)处理的小鼠耳洞闭合180天后，伤口部分有毛囊，腺体，软骨，肌肉与血管等组织和组织衍生物的再生。
图1I的示意图显示，采用灌胃和腹腔注射等不同给药方式对耳洞创伤小鼠进行处理，3周后均产生愈合效果(损伤后21天)。其中n≥6。***p<0.001，ns：无显著性差异，t检验。
此外，Nsun2敲除被报道可以通过调控tRNA稳定性来抑制翻译，因此用Nsun2敲除小鼠作为遗传学模型来验证翻译抑制对再生作用。具体方法为：分别以野生型小鼠(WT)与Nsun2敲除小鼠(KO)建立耳洞创伤小鼠模型，制造直径2mm耳洞(方法同上述实施例)，3周后分别观察并测量耳洞愈合情况。结果显示Nsun2敲除的小鼠相比野生型小鼠，耳洞面积明显减小。这也表明翻译抑制对再生具有作用(图1J)。
以上实验结果显示：不同剂量的蛋白质合成抑制剂CHX都能显著促进2mm耳洞愈合，且这种促进效果具有剂量依赖性，大于8mg/kg剂量可以促进闭合。闭合耳洞可以再生处毛囊，腺体，软骨和肌肉等组织和组织衍生物。同时也证实不同给药方式均具有促进再生的效果。
实施例二：环己酰亚胺CHX促进小鼠直径4mm耳洞割处再生修复。
研究发现极少数的哺乳动物如非洲刺鼠具有较强再生能力，能够再生4mm耳洞，同时还发现4mm耳洞闭合模型可以区分再生能力强弱，目前没有任何人工实现4mm耳洞闭合再生，即使是被报道“超级再生”的MRL小鼠也不能完全闭合。因此4mm耳洞可以作为评价哺乳动物割处再生的良好模型，其模型建立采取实施例1的方法，其中以直径为4mm的耳洞打孔器对小鼠左右耳廓分别打孔。
实验方法：对4mm耳洞创伤小鼠以不同剂量的CHX(20,125,175mg/kg, 溶于DMSO或生理盐水)采取腹腔注射给药，DMSO作为对照组。对药物处理过的小鼠创伤耳廓进行鉴定。具体实验结果如图2所示。
图2A的示意图显示，不同剂量的CHX对于直径4mm的耳洞闭合效果，发现20mg/kg与超级愈合小鼠MRL/lpr效果类似，有促进耳洞伤口变小作用，但不能完全闭合，当剂量大于20mg/kg(125,175mg/kg)能实现耳洞的闭合。
图2B为DMSO/CHX药物处理90天后的耳洞愈合照片，其显示经CHX(125mg/kg)处理的小鼠4mm耳洞，创伤的面积显著减小。
实施例三：环己酰亚胺CHX促进耳洞再生不依赖于铁死亡与自噬的抑制。
CHX作为一种抗真菌的抗生素，除了抑制真核生物蛋白质合成和RNA合成外，还可抑制铁死亡和细胞自噬。为了验证其在再生过程中通过哪个靶点发挥作用，分别针对铁死亡和自噬进行小分子验证。
实验方法：采用上述相同的耳洞创伤小鼠模型进行实验，分别用铁死亡和自噬的抑制剂代替CHX观察其促进耳洞愈合的效果。DMSO替代小分子的对照组。Auto:Autophinib(MCE，HY-101920)，抑制自噬；3BDO：3BDO(MCE，HY-U00434)，抑制自噬；UAMC：UAMC-3203(MCE，MCE，HY-112909A)，抑制铁死亡；EBSE：Ebselen(MCE，HY-13750)，抑制铁死亡。使用浓度为10-20mg/kg，给药方式同CHX，n≥8，**p<0.01，***p<0.001，ns：无显著性差异，t检验。鉴定不同抑制剂对2mm耳洞愈合效果(损伤后21天测定)。结果如图3所示：分别用铁死亡和自噬的抑制剂均不能像CHX一样促进耳洞愈合，这说明CHX促进耳洞再生不依赖于其对铁死亡或自噬的抑制活性。
实施例四：环己酰亚胺CHX激活STING-TBK1-IRF3-干扰素刺激基因(Interferon-stimulated genes,ISGs)通路，且ISG基因表达是CHX诱导再生所必需的。
为了进一步验证CHX诱导再生的机制，利用CHX分别处理小鼠原代成纤维细胞和巨噬细胞，并进行大样本量RNA-seq(转录水平)和Ribo-seq(核糖体印记测序，翻译组学)。通过生信息学分析发现CHX导致两类细胞转录水平和翻译水平共同上调基因32个(G1，图4A)，功能分析发现这些基 因为干扰素响应基因，功能主要富集到响应干扰素和病毒(图4B)。进一步通过qPCR实验验证CHX确实能够上调经典ISG表达，如Mx2,Ifit1,Cxcl10,Ifih1等(图4C显示成纤维细胞ISG基因表达，图4D显示巨噬细胞ISG基因表达)。为了验证ISG基因表达是否是CHX诱导再生所必需的，我们首先在体外发现CHX通过激活STING-TBK1-IRF3信号通路激活ISG，主要体现STING蛋白核周富集，IRF3蛋白入核(图4E)，且利用小分子抑制剂GSK8612(5μM)或Dexamethasone(Dex,10μM)抑制TBK1能显著抑制能显著CHX(1μg/ml)诱导的ISG基因的表达(图4F)，在体内抑制TBK1(GSK8612(25mg/kg)，Dexamethasone(Dex,10mg/kg))能显著抑制CHX(20mg/kg)诱导的耳洞再生(图4G)。此外通过Puromycin(PURO)参入实验(见参考文献《Kearse,et al.Ribosome queuing enables non-AUG translation to be resistant to multiple protein synthesis inhibitors,2019,Genes&Development》)发现Dexamethasone并不影响CHX对整体翻译的抑制，预示CHX介导的翻译抑制位于诱导ISG的上游(图4H)。以上说明ISG基因表达是CHX诱导再生所必需的。
实施例五：水仙环素(Narciclasine)激活ISG促进小鼠再生。
(1)水仙环素(Narciclasine)的激活ISG基因表达。
水仙环素存在于各种石蒜科植物中，具有翻译延伸抑制作用。首先通过Puromycin(PURO)参入实验验证其可显著抑制整体的蛋白翻译，如图5A所示。进一步定量qPCR发现其显著促进ISG基因的表达，如图5B、5C所示，其中图5B显示成纤维细胞ISG基因表达，图5C显示巨噬细胞ISG基因表达。
(2)水仙环素促进小鼠2mm耳洞割处再生。
实验方法：对2mm耳洞创伤小鼠以不同剂量(1-3mg/kg)Narciclasine(溶于DMSO，给药体系2-5％DMSO+30-40％PEG400+2-5％Tween80+生理盐水)分别采取腹腔注射给药，不含药物的DMSO作为对照组(标记为Vehicle)。对药物处理过的小鼠创伤耳廓进行鉴定。具体实验结果如图5D～5G所示。
其中，图5D的示意图显示了直径2mm耳洞打孔器创伤的小鼠耳洞经Narciclasine药物处理30天的愈合情况。图5E显示了不同剂量的Narciclasine 对于小鼠耳洞的再生效果。图5F、5G分别为组化与Masson切片染色数据表征了软骨(黑色长箭头)、毛囊(星号)、腺体/皮脂腺(三角形箭头)等结构的再生结构，从图中可看到软骨的多个发生中心，推测这样多起点再生大大加快再生速度。
以上实验结果显示，不同剂量的Narciclasine均可显著促进2mm耳洞愈合，闭合耳洞可以再生出毛囊，腺体，软骨和肌肉等组织和组织衍生物，证实了Narciclasine的再生促进作用。
(3)组合NRB促进小鼠4mm耳洞割处再生。
实验方法：小鼠模型构建方法同实施例二，对4mm耳洞创伤后的小鼠，采用腹腔注射DMSO/NRB(NRB：Narciclasine 3mg/kg、ATRA 20mg/kg，BMP signaling agonist sb4 10-20mg/kg)的方式每2天给药一次，并且每7天对小鼠进行麻醉，使用游标卡尺对小鼠耳洞的近-远轴(proximal–distal，DPD)和前后轴(anterior–posterior，DAP)进行测量，并计算小鼠被割除耳洞的面积，面积的计算公式为S＝π×DPD×DAP/4。对药物处理过的小鼠创伤耳廓进行进一步鉴定。实验结果如图5H～5J所示。
其中，图5H显示了小鼠4mm耳洞经NRB处理30天后的愈合效果，并显示具有愈合促进作用，在给药处理30天后，小鼠耳洞完创口全闭合，且鉴定为再生事件。图5I，5J显示，HE切片染色数据很好地指征了软骨(黑色长箭头)、毛囊(星号)、腺体/皮脂腺(三角形箭头)、肌肉(虚线框选区域)等结构的再生结构。
以上实验结果显示，NRB小分子组合物也可以促进4mm耳洞闭合并促进再生。
实施例六：水鬼蕉碱(Pancratistatin)激活ISG促进小鼠耳洞割处再生。
(1)水鬼蕉碱(Pancratistatin)激活ISG。
水鬼蕉碱与水仙环素一样也是石蒜科植物生物碱，首先通过Puromycin(PURO)参入实验验证其可显著抑制整体的蛋白翻译，如图6A所示，进一步，通过定量qPCR实验结果发现水鬼蕉碱(Pancratistatin)也可显著促进ISG基因表达，如图6B、6C所示，其中图6B显示成纤维细胞ISG基因表达，图6C显示巨噬细胞ISG基因表达。
(2)水鬼蕉碱(Pancratistatin)促进小鼠2mm耳洞割处再生。
实验方法：对2mm耳洞创伤小鼠以2mg/kg水鬼蕉碱(Pancratistatin)(溶于DMSO，给药体系2-5％DMSO+30-40％PEG400+2-5％Tween80+生理盐水)分别采取腹腔注射给药，不含药物的DMSO作为对照组(标记为Vehicle)。对药物处理过的小鼠创伤耳廓进行鉴定。具体实验结果如图6D～6G所示。
其中，图6D为耳洞损伤后21天时处理组Pancratistatin给药对促进耳洞愈合的效果，显示耳洞完全闭合。图6E为Pancratistatin处理21-28天，观察耳洞闭合情况。图6F(HE染色)、6G(Masson染色)的结果显示，小鼠耳洞创口完全闭合，且鉴定为再生事件，染色数据较好地显示了软骨多个发生中心(黑色长箭头)、毛囊(星号)、腺体/皮脂腺(三角形箭头)、肌肉(虚线框选区域)等结构的再生结构。
以上结果显示，Pancratistatin小分子以促进耳洞闭合，并促进再生。
实施例七：干扰素激活ISG促进耳洞再生。
ISG作为干扰素刺激基因，为验证干扰素的促再生效果，我们选取干扰素(IFN)γ作为代表，首先在细胞模型中通过定量qPCR实验验证干扰素(IFN)γ(10ng/ml)能显著促进ISG基因表达，结果如图9A、9B所示，其中图7A显示成纤维细胞ISG基因表达，图7B显示巨噬细胞ISG基因表达。进一步通过小鼠耳洞模型验证其促再生效果，腹腔注射IFNγ(50μg/kg)，每两天注射一次。损伤后第35天检测耳洞再生情况。结果如图7C所示，显示损伤后干扰素γ著促进耳洞愈合。
实验结果显示，细胞因子干扰素激活ISG可以促进耳洞再生。
实施例八：S100A8/A9激活ISG促进耳洞再生。
警报素是机体损伤后增强表达的一种诱导分子，为验证警报素的促再生效果，我们首先在细胞模型中通过定量qPCR实验验证S100A8/A9(5ng/ml)能显著促进ISG基因表达，结果如图8A、8B所示，其中图8A显示成纤维细胞ISG基因表达，图8B显示巨噬细胞ISG基因表达。进一步通过小鼠耳洞模型验证其促再生效果，腹腔注射分别PBS(Vehicle组)，S100A8/A9异源二聚体(Biolegend，765502)，小鼠白蛋白(mALB，对照)每两天注射一 次，注射剂量分别为25、12.5μg/kg。损伤后第35天检测耳洞再生效果。结果如图8C所示，损伤后第35天S100A8/A9(12.5μg/kg)显著促进耳洞愈合，其中一只完全闭合。进一步HE染色和Masson染色鉴定各组织结构再生，如表皮、真皮、毛囊、腺体、软骨等，如图8D。
实验结果显示，细胞因子S100A8/A9激活ISG可以促进耳洞再生。
实施例九：抑制MAPK13(p38)激活ISG促进耳洞再生。
分别干扰应激相关激酶P38、ERK5、JNK，验证其是否具有促进耳洞再生的作用。实验方法为：分别以如下试剂给药于耳洞创伤小鼠模型，检测创伤后21天的耳洞愈合情况。
MAPK13-IN-1(MCE，HY-12839，5mg/kg)选择性P38δ抑制剂；Doramapimod(MCE，HY-10320，5mg/kg)，P38抑制剂；SB203580(MCE，HY-10256，5mg/kg)，选择性P38α/β抑制剂；BIX02189(MCE，HY-12839，5mg/kg)，选择性MEK5、ERK5抑制剂；SP600125(MCE，HY-12041，5mg/kg)选择性JNK抑制剂。n≥5，*p<0.05，***p<0.001，ns：无显著性差异，t检验。实验结果如图9A所示。进一步通过定量qPCR实验验证P38δ抑制剂MAPK13-IN-1能促进ISG基因表达(图9B)。进一步HE染色图显示，耳洞创伤小鼠经MAPK13-IN-1(20mg/kg)处理后，小鼠耳洞创伤部分的表皮、真皮、软骨及其他结缔组织等多种组织结构向前生长，生长连接在一起，创伤已经接近完全愈合(图9C)。同样地，Masson染色较为清晰显示了经MAPK13-IN-1药物处理21天的愈合情况示。MAPK13-IN-1处理后，可以看到实验组经MAPK13-IN-1处理后胶原纤维整体排列更为规律整齐，与野生型更为接近，成束排列，并且最显著的差异发生在肌纤维的再生，实验组观察到明显的肌纤维束的再生(虚线框选区域)，伴随软骨(箭头)附近，且观察到血管包绕周围，但是对照组并没有发现类似现象。总体来说，再生区域组织类型与野生型一致，这些指标很好指示了组织再生事件的发生(图9D)。
实验结果显示，抑制P38及P38δ激活ISG可以促进耳洞再生。
实施例十：抑制视黄酸受体相关孤儿受体激活ISG促进耳洞再生。
通过激活与反向激活干扰黄酸受体相关孤儿受体α(retinoic acid  receptor-related orphan receptorα，RORα)，验证其在耳洞再生中的作用，实验方法为：分别以如下试剂给药于耳洞创伤小鼠模型，检测创伤后21天的耳洞愈合情况，结果显示反向激活RORα促进耳洞，而激活RORα则明显会抑制再生，如图10A所示。进一步通过定量qPCR实验验证Rora反向激动剂SR3335能促进ISG基因表达(图10B显示成纤维细胞ISG基因表达，图10C显示巨噬细胞ISG基因表达)。进一步HE染色结果显示，经SR3335处理后，耳洞创伤小鼠耳洞部分的皮肤，结缔组织等多种组织结构生长连接在一起，创伤已经接近完全愈合，可以观察到表皮、真皮的再生，以及毛囊结构、皮脂腺的出现(图10D)，Masson染色较为清晰显示了经SR3335药物处理21天的愈合情况示。Masson切片染色数据也很好的表征了软骨、毛囊、皮脂腺(箭头)及肌肉(虚线框选区域)等结构的再生结构(图10E)。SR3335(MCE，HY-14413，10mg/kg)，选择性的RORα反向激动剂；SR1078(MCE，HY-10320，1mg/kg)，RORα激动剂；n≥7，**p<0.01，***p<0.001，ns：无显著性差异，t检验。
利用Rora敲除小鼠(从上海南方模式动物中心引进)进一步验证纯合敲除Rora能显著促进小鼠耳洞闭合，并促进各组织再生(图10F)。
实验结果显示，抑制Rora激活ISG可以促进耳洞再生。
实施例十一：组合CRB(C:蛋白质合成抑制剂CHX；R:RARs激活剂全反式视黄酸(All-trans retinoic acid)；B:BMP激活剂BMP(signaling agonist sb4))促进小鼠4mm耳洞闭合及割处再生。
以7周龄小鼠为例，使用5％水合氯醛对小鼠实施麻醉，腹腔注射10mL/kg，按体重进行麻醉。将麻醉好的小鼠进行绑定，使用75％的乙醇对小鼠耳朵和器械进行消毒。以小鼠的耳廓中心，使用直径为4mm的耳洞打孔器对小鼠左右耳廓分别打孔。对于创伤后的小鼠，采用腹腔注射DMSO/CRB(CRB给药量为CHX 20mg/kg、全ATRA 20mg/kg，BMP signaling agonist sb4 10-20mg/kg)的方式每2天给药一次，并且每7天对小鼠进行麻醉，使用游标卡尺对小鼠耳洞的近-远轴(proximal–distal，DPD)和前后轴(anterior–posterior，DAP)进行测量，并计算小鼠被割除耳洞的面积，面积的计算公式为S＝Πx DPD x DAP/4。对药物处理过的小鼠创伤耳廓进行进一步鉴定。
图11A显示分别经DMSO/CRB处理后，小鼠耳洞的闭合情况。结果显示CRB对于直径4mm的耳洞闭合都有促进作用，在给药处理30天后，小鼠耳洞完创口全闭合。
图11B显示小鼠4mm耳洞经药物处理30天后的闭合情况。可以看出，经药物处理后的小鼠，耳洞创伤已经闭合。
图11C显示经药物处理7天后，小鼠耳廓组织HE染色结果和表皮厚度统计。比例尺为200um。经药物处理后的小鼠耳廓有芽基形成，表皮厚度明显降低。n≥3，t检验。
图11D显示经药物处理7天后，小鼠耳廓组织α-SMA免疫荧光染色结果。比例尺为100um。创伤7天后，对照组与药物处理组均出现大量的α-SMA表达，与对照组相比，药物处理组的α-SMA表达呈线性有序排列，而对照组为无序堆积，表达模式与疤痕形成方式类似。
图11E显示经药物(CRB)诱导大于90天后各个组织再生情况，显示表皮、真皮、腺体、毛囊、肌肉、软骨、脂肪、肌肉的再生。“e”指示表皮再生；“d”指示再生的真皮；“g”指示再生的腺体；“ad”指示再生的脂肪组织；“hf”指示再生的毛囊；“c”指示再生的软骨；“m”指示再生的肌肉。
图11F免疫荧光染色血管标记物CD31进一步鉴定血管再生，三角形指示再生的血管。
实施例十二：环己酰亚胺CHX(C)和全反式视黄酸RA(R)优化组合促进小鼠肢端隆起结构(Outgrowth)再生修复
基于耳洞的再生效果，接下来进行了更复杂的损伤表型的再生诱导实验，选用肢端切除作为损伤模型。对ICR小鼠进行肢端造模，并联合使用适当剂量的CHX(100mg/kg)与RARs激活剂全反式视黄酸(RA，20mg/kg)，腹腔连续隔天给药8周，观察再生表型。
实验方法：以8周龄ICR小鼠为模型，使用5％水合氯醛对小鼠实施麻醉，腹腔注射10mL/kg，按体重进行麻醉。将麻醉好的小鼠进行绑定，使用75％的乙醇对小鼠左上肢端和手术器械进行消毒。这里为了准确定义再生的发生，测量肘关节至桡尺骨的距离，保留10mm，其余至手掌、手指等节段进行切除造模(尺骨和桡骨属于前臂的两根骨头，最简单的区别的方法为拇 指一侧为桡骨、小指一侧为尺骨)。腹腔连续隔天给药8周，CHX(100mg/kg,溶于DMSO或生理盐水)、RA(20mg/kg)采取腹腔注射给药，DMSO作为对照组，持续观察表型。不同天数后，对药物处理过的小鼠肢端创伤进行鉴定。具体实验结果如图12A-G所示。图12A为肢端造模的模式图。图12B显示经药物处理20、40、120天后的肢端诱导再生现象。可以看出，经药物处理后的小鼠截肢部位出现outgrowth的生长。比例尺为2mm。图12C为肢端outgrowth长度的统计结果，可以看到CR给药组很好诱导肢端再生，长度与对照组存在极显著差异。n≥3，***p<0.001，t检验。图12D显示经药物CR处理20天和40天后，利用电子计算机断层扫描(CT)获取骨组织重建的过程高清图，CR处理组出现很好的肢端骨组织诱导生长过程，CR处理20天后即可观察到骨组织的伸长重建碎片(右图，三角箭头指示)，而对照组呈现出钝化状态，显示为骨组织的增生积累，未呈现出向前的生长趋势(左图，三角箭头指示)，是一种类似瘢痕修复的发生。图12E为骨再生的更直接关键证据，药物CR处理30后，取出完整桡尺骨进行观察，与CT扫描结果观察一致，可以明显看到骨组织的再生趋势。此外，HE切片染色发现该组合可以促进截肢肢端多组织类型的再生，如皮肤、毛囊、毛细血管、新生骨等组织(图12F，“Ep”指示表皮再生；“CT”指示结缔组织再生；“CV”指示毛细血管再生；“HF”指示毛囊再生；“OT”指示骨组织再生。)。图12G显示经药物处理21、50、120天后，小鼠肢端再生组织Masson染色结果，对照组可以观察到骨组织的钝化形态，与CT结果相符，药物处理组的骨组织呈现生长趋势，重要的是实验组在120天染色观察到肌肉组织的再生，而对照组为无序堆积，表达模式与疤痕形成方式类似。比例尺为2mm，n≥3，t检验。
以上结果显示，环己酰亚胺CHX和全反式视黄酸RA促进肢端切除后隆起结构的再生修复，包括骨、肌肉、皮肤、毛囊、毛细血管、结蹄组织、新生骨组织等再生。
上述实施例结果显示，多种激活ISG基因表达的不同策略均可以促进哺乳动物再生，提升损伤后再生修复。图13示出了激活ISG基因表达促进哺乳动物再生能力的示意图。
前面仅仅示出了本发明的原理，应理解，本发明的范围不预期限制在本文所述的示例性方面，而应包括所有当前已知的和未来开发的等同物。另外，应当指出，在不脱离本发明技术原理的前提下，还可以作出若干改进和修改，这些改进和修改也应被视为本发明的范围。

Claims (21)

  1. 一种能够上调ISG基因表达的物质在促进哺乳动物组织或复杂结构或器官再生修复能力中的应用。
  2. 一种能够上调ISG基因表达的物质在制备用于促进哺乳动物组织或复杂结构或器官再生修复能力的药物或试剂中的应用。
  3. 一种能够上调ISG基因表达的物质在制备用于治疗与哺乳动物组织或复杂结构或器官的再生修复相关的疾病的药物或试剂中的应用。
  4. 根据权利要求1～3中任一项所述的应用，其特征在于，所述能够上调ISG基因表达的物质选自MAPK抑制剂、视黄酸受体相关孤儿受体抑制剂、蛋白质合成抑制剂、干扰素(IFNγ，β，λ)或警报素(S100A8/A9)蛋白中的一种或两种以上。
  5. 根据权利要求1-4中任一项所述的应用，其特征在于，所述促进哺乳动物组织或复杂结构或器官再生修复能力是通过诱导TBK1-IRF3通路激活实现的，优选的，通过对蛋白合成抑制实现的。
  6. 根据权利要求1-4中任一项所述的应用，其特征在于，所述促进哺乳动物组织或复杂结构或器官再生修复能力是通过对p38 MAPK通路的抑制实现的，优选的，通过对P38δ的抑制实现。
  7. 根据权利要求1-4中任一项所述的应用，其特征在于，所述促进哺乳动物组织或复杂结构或器官再生修复能力是通过对视黄酸受体相关孤儿受体α(RORα)的反向激活实现的。
  8. 根据权利要求1-7中任一项所述的应用，其特征在于，所述再生修复为促进组织或器官切除或损伤后的组织或复杂结构或器官的再生。
  9. 根据权利要求1-8中任一项所述的应用，其特征在于，
    所述组织为皮肤、脂肪、肌肉、骨骼、毛囊、血管或神经，
    所述复杂结构为至少包括皮肤、毛囊、腺体、软骨、肌肉、脂肪、血管、神经或与肢体中的两种以上的机体结构，
    所述器官为肺、肝、心、胰岛或肾。
  10. 根据权利要求1～9中任一项所述的应用，其特征在于，所述复杂结构为耳朵、肢体、手指、眼或鼻。
  11. 根据权利要求1-9中任一项所述的应用，其特征在于，所述再生修复为促进皮肤损伤后的再生修复，脱发后的毛发再生，软骨肌肉损伤再生修复，肺、肝、皮肤、心、肾、肌肉纤维化的再生以及血管、神经与肢体损伤后的再生。
  12. 根据权利要求3～11中任一项所述的应用，其特征在于，所述疾病为皮肤烫伤、皮肤创伤、皮肤烧伤、脱发、软骨肌肉损伤、肝纤维化、肺纤维化或肢体损伤。
  13. 根据权利要求4～12中任一项所述的应用，其特征在于，所述MAPK抑制剂为P38抑制剂、选择性P38δ抑制剂的一种或两种以上，优选的，所述P38抑制剂为达马莫德(Doramapimod)，所述选择性P38δ抑制剂为MAPK13-IN-1。
  14. 根据权利要求4～12中任一项所述的应用，其特征在于，所述视黄酸受体相关孤儿受体抑制剂为选择性的RORα反向激动剂，优选的，为SR3335。
  15. 根据权利要求4～12中任一项所述的应用，其特征在于，所述蛋白质合成抑制剂选自环己酰亚胺(Cycloheximide(CHX))、茴香霉素(Anisomycin(Ani))、膜海鞘素B(Didemnin B(DIDB))、波凡霉素(Bouvardin(BVD))、水仙环素(Narciclasine)或水鬼蕉碱(Pancratistatin)中的一种或两种以上。
  16. 一种组合物，其特征在于，包括蛋白质合成抑制剂、全反式视黄酸和BMP激活剂，优选的，所述蛋白质合成抑制剂为环己酰亚胺(Cycloheximide(CHX))，所述BMP激活剂为BMP signaling agonist sb4。
  17. 根据权利要求16所述的组合物，其特征在于，以所述组合物中的环己酰亚胺为1重量份计，全反式视黄酸为0.25～8重量份，BMP signaling agonist sb4为0.25～4重量份。
  18. 根据权利要求16或17任一项所述的组合物，其特征在于，所述组合物的给药方式为腹腔注射、静脉注射、灌胃、口服或皮肤涂抹。
  19. 如权利要求16～18任一项所述的组合物在促进哺乳动物组织或复杂结构或器官再生修复能力或制备用于促进哺乳动物组织或复杂结构或器官再生修复能力的药物或试剂或制备用于治疗与哺乳动物组织或复杂结构或器官的再生修复相关的疾病的药物或试剂中的应用。
  20. 一种促进哺乳动物组织或复杂结构或器官再生修复的方法，其包括向有需要的受试者施用能够上调ISG基因表达的物质或包含能够上调ISG基因表达的物质的组合物。
  21. 根据权利要求20所述的方法，其中，所述能够上调ISG基因表达的物质或包含能够上调ISG基因表达的物质的组合物为权利要求1～15中所涉及的能够上调ISG基因表达的物质或权利要求16～18中任一项所述的组合物。
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