WO2021047607A1 - 非igf1r结合型的物质在预防和/或治疗炎症性疾病中的应用 - Google Patents

非igf1r结合型的物质在预防和/或治疗炎症性疾病中的应用 Download PDF

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WO2021047607A1
WO2021047607A1 PCT/CN2020/114587 CN2020114587W WO2021047607A1 WO 2021047607 A1 WO2021047607 A1 WO 2021047607A1 CN 2020114587 W CN2020114587 W CN 2020114587W WO 2021047607 A1 WO2021047607 A1 WO 2021047607A1
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igf2
igf1r
igf2r
macrophages
inflammatory
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PCT/CN2020/114587
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French (fr)
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时玉舫
王莹
王雪枫
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中国科学院上海营养与健康研究所
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Priority to EP20863556.5A priority Critical patent/EP4046658A4/en
Priority to CN202080006025.6A priority patent/CN113164606A/zh
Priority to US17/641,539 priority patent/US20220401515A1/en
Priority to AU2020346948A priority patent/AU2020346948A1/en
Publication of WO2021047607A1 publication Critical patent/WO2021047607A1/zh

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Definitions

  • the present invention relates to the field of biomedicine, in particular to the application of non-IGF1R-binding substances in the prevention and/or treatment of inflammatory diseases.
  • IGF2 Insulin like growth factor 2
  • IGF2 receptors include IGF1R and IGF2R, among which IGF2R has the highest affinity, followed by binding to IGF1R, and almost no binding to insulin receptor 2 .
  • IGF2 tyrosine kinase IGF1R open, but with no positive correlation IGF2R and IR 2.
  • IGF2R Intracellular Golgi apparatus
  • IGF2R is not the only receptor of IGF2
  • IGF2 is not the only ligand of IGF2R. Its ligands also include mannose-6-phosphate.
  • the main function of IGF2R is to transport its ligands to lysosomes. 4 complete protein processing or degradation.
  • studies have found that, the IGF2R on the cell membrane can be cut off in a soluble form, into the blood and other liquid environment, effective binding IGF2, 4,5 inhibition IGF2 function.
  • Autoimmune diseases are diseases caused by the immune system's abnormal attacks on one's own organs and tissue cells. According to data released by the US Department of Health and Human Services, there are currently 24 million Americans-7% of the US population suffers from more than 80 types. The torment of autoimmune diseases involves almost every part of the body6,7 . The pathogenic factors of most autoimmune diseases are not yet clear, and may be related to factors such as genetics, infections, drugs, and the environment. The disease course is longer, with repeated remissions and attacks, and more women than men are affected. Most autoimmune diseases involve the participation of phagocytes composed of monocyte precursor cells, monocytes and macrophages.
  • activated macrophages secrete milk fat globule-epidermal growth factor 8 (MFG-E8), the C-terminal domain of MFG-E8 and apoptotic cells phosphatidylserine binding, mediates phagocytosis of apoptotic cells, autoimmune diseases clear attack and death in apoptosis and immune cell debris, to help the body to restore homeostasis 8.
  • MFG-E8 milk fat globule-epidermal growth factor 8
  • apoptotic cells phosphatidylserine binding
  • MFG-E8-mediated macrophage phagocytosis of apoptotic fragments can also inhibit the regulation of lipopolysaccharide and other pro-inflammatory effects on macrophages, including p38, ERK1/2, c-JNK, MAPK and 10 inhibition of p65.
  • Inflammation is the body's defense response to its own and foreign inflammatory factors. Inflammatory factors include biological factors, physical factors, chemical factors, foreign bodies, necrotic tissues, allergic reactions and pathological changes. Inflammatory reactions are affected by inflammatory factors. 11 and the body's own regulation. In inflammatory diseases in both natural immunity to participate, but also to participate in adaptive immunity, in the early innate immune generated inflammation lead to tissue injury site, and determine the impact of late innate and adaptive immune response type 12. When the inflammatory regulation is disordered or obstructed, inflammatory diseases occur, and the ability to resist stimuli and pathogens is lost, which accelerates the aggravation of the disease. According to the speed of the inflammatory response, the inflammatory response includes both acute and chronic inflammation.
  • the first category is steroidal anti-inflammatory drugs, that is, steroid drugs, such as adrenal cortex hormones, male hormones, and estrogen, which have certain anti-inflammatory effects. long-term use will cause endocrine disorders such as multi-system dysfunction 13-15.
  • the second category is non-steroidal anti-inflammatory drugs including aspirin, acetaminophen, indomethacin, naproxen, naproxen, diclofenac, ibuprofen, nimesulide, rofecoxib, celecoxib etc., it is widely used in clinical osteoarthritis, rheumatoid arthritis and other anti-inflammatory treatment 14-16.
  • Nonsteroidal anti-inflammatory drug single action principle primarily anti-inflammatory action by inhibiting the synthesis of prostaglandin 17, and reduce the formation of bradykinin, and reduce platelet aggregation and leukocyte aggregation. Therefore, sterol drugs are limited by side effects, while non-steroid drugs are limited by a single pathway of action, and the combination of drugs has a poor sensitization effect.
  • the purpose of the present invention is to provide a medicine that can treat autoimmune diseases and/or inflammatory diseases more effectively.
  • the first aspect of the present invention provides the use of a non-IGF1R-binding substance for the preparation of a composition or preparation for the prevention and/or treatment of inflammatory diseases.
  • the non-IGF1R-binding substance is selected from the group consisting of IGF2 mutants, vectors expressing IGF2 mutants, antibodies, small molecule compounds, or combinations thereof.
  • the inflammatory disease is selected from the group consisting of peritonitis, inflammatory bowel disease, multiple sclerosis, diabetes, systemic lupus erythematosus, scleroderma, Hashimoto's thyroiditis, autoimmune hepatitis, self Immune uveitis, interstitial lung disease, psoriasis, vitiligo, dermatomyositis, Kawasaki disease, adult ear disease, compulsive spondylitis, sarcoidosis, arthritis associated with start and end inflammation, polyarticular adolescents Idiopathic arthritis, rheumatoid arthritis, graft-versus-host disease, autoimmune pancreatitis, Parkinson's disease, Alzheimer's disease, or a combination thereof.
  • the non-IGF1R-binding substances include substances that have a higher selectivity or affinity for IGF2R than IGF1R, and substances that can efficiently activate IGF2R that do not activate or substantially do not activate IGF1R (such as retinoic acid, etc.).
  • Non-IGF1R ligand Non-IGF1R ligand
  • the vector expressing the IGF2 mutant includes a viral vector.
  • the viral vector is selected from the following group: adenovirus vector, lentivirus vector, or a combination thereof.
  • the non-IGF1R-binding substance also includes one or more substances selected from the group consisting of Plasminogen, Serglycin, and e1a stimulation.
  • Cellular suppressor of E1A-stimulated genes CREG
  • Sgsh sulfamidase
  • phosphorylated ⁇ -glucuronidase Phosphorylated ⁇ -glucuronidase, Gusb
  • the IGF2 mutant has a mutation in the tyrosine corresponding to the 27th position of SEQ ID NO.:1 of the wild-type IGF2 protein.
  • the tyrosine at position 27 is mutated into one or more amino acids selected from the group consisting of leucine, isoleucine, valine, methionine, and alanine. Acid, phenylalanine, serine, proline, threonine, histidine, lysine, tryptophan, arginine, glutamic acid, glycine, aspartic acid, cysteine.
  • the tyrosine at position 27 is mutated to leucine.
  • the IGF2 mutant mutates the tyrosine at position 27 of the wild-type IGF2 protein corresponding to SEQ ID NO.:1 to leucine, and optionally deletes D-domain (SEQ ID NO.:1). ID NO.:1 threonine 62 to glutamic acid 67).
  • the IGF2 mutant has a mutation in the glutamic acid at position 12 of the wild-type IGF2 protein corresponding to SEQ ID NO. 1, and optionally deletes D-domain (SEQ ID NO. 1). :1 threonine at position 62 to glutamic acid at position 67).
  • the glutamic acid at position 12 is mutated to one or more amino acids selected from the group consisting of Asp, Ala, Gln, His, Arg, and Lys.
  • the IGF2 mutant has a mutation in the phenylalanine at position 26 of the wild-type IGF2 protein corresponding to SEQ ID NO. 1, and optionally deletes the D-domain (SEQ ID NO. .:1 threonine 62 to glutamic acid 67).
  • the phenylalanine at position 26 is mutated to one or more amino acids selected from the group consisting of Ser, Asp, Ala, Gln, His, Arg, and Lys.
  • the phenylalanine at position 26 is mutated to serine.
  • the IGF2 mutant has a mutation in the tyrosine at position 27 of the wild-type IGF2 protein corresponding to SEQ ID NO. 1, and optionally deletes D-domain (SEQ ID NO. 1). :1 threonine 62 to glutamic acid 67).
  • the tyrosine at position 27 is mutated to one or more amino acids selected from the group consisting of Leu, Asp, Ala, Gln, His, Arg, Lys.
  • the tyrosine at position 27 is mutated to leucine.
  • the IGF2 mutant has a mutation in the wild-type IGF2 protein corresponding to SEQ ID NO.:1, the 43rd valine, and optionally deletes D-domain (SEQ ID NO. 1). :1 threonine 62 to glutamic acid 67).
  • the valine at position 43 is mutated to one or more amino acids selected from the group consisting of Leu, Asp, Ala, Gln, His, Arg, Lys.
  • valine at position 43 is mutated to leucine.
  • the tyrosine at position 27 is mutated to one or more amino acids selected from the group consisting of Leu, Asp, Ala, Gln, His, Arg, Lys, and the tyrosine at position 43 is Valine is mutated to one or more amino acids selected from the group consisting of Leu, Asp, Ala, Gln, His, Arg, Lys.
  • the tyrosine at position 27 and the valine at position 43 are simultaneously mutated to leucine.
  • the tyrosine at position 27 and the valine at position 43 are mutated to leucine at the same time, and the 62th threon of D-domain (SEQ ID NO.:1) is optionally deleted. Amino acid to glutamic acid at position 67).
  • amino acid sequence of the IGF2 mutant is shown in any one of SEQ ID NO.: 2-58.
  • the IGF2 mutant is a polypeptide having an amino acid sequence shown in any one of SEQ ID NO.: 2-58, an active fragment thereof, or a conservative variant polypeptide thereof.
  • the IGF2 mutant except for the mutations (such as Glu at position 12, Phe at position 26, Tyr at position 27, Val at position 43 and the corresponding mutant sequence for deleting D-domain), the rest of the amino acid sequence It is the same or substantially the same as the sequence shown in SEQ ID NO.:1.
  • the glutamic acid at position 12 is mutated to glutamine.
  • the said substantially identical is at most 50 (preferably 1-20, more preferably 1-10, more preferably 1-5) amino acids are different, wherein, The difference includes the substitution, deletion or addition of amino acids (such as the deletion of T at position 62 to E at position 67), and the IGF2 mutant has the activity of inhibiting inflammation.
  • the homology with the sequence shown in SEQ ID NO.:1 is at least 80%, preferably at least 85% or 90%, more preferably at least 95%, and most preferably at least 98% or 99%.
  • the IGF2 protein is derived from a human or non-human mammal.
  • the IGF2 mutant is selected from the following group:
  • amino acid sequence shown in SEQ ID NO.: 2-58 is formed by the substitution, deletion or addition of one or more (such as 2, 3, 4 or 5) amino acid residues, It also has a polypeptide derived from (a) that inhibits inflammation.
  • the homology between the derived polypeptide and any sequence shown in SEQ ID NO.: 2-58 is at least 60%, preferably at least 70%, and more preferably at least 80%. %, preferably at least 90%, such as 95%, 97%, 99%.
  • the IGF2 mutant is formed by mutation of the wild-type IGF2 protein shown in SEQ ID NO.:1.
  • composition or preparation is also used for one or more purposes selected from the following group:
  • the autoimmune disease is selected from the group consisting of multiple sclerosis, inflammatory bowel disease, autoimmune hepatitis, systemic lupus erythematosus, rheumatoid arthritis, insulin resistance, diabetes, autoimmune Hepatitis, vitiligo, psoriasis, peritonitis, scleroderma, Hashimoto's thyroiditis, graft-versus-host disease, dermatomyositis, Kawasaki disease, adult ear disease, mandatory spondylitis, sarcoidosis, start and stop Spot inflammation-related arthritis, polyarticular juvenile idiopathic arthritis, autoimmune pancreatitis, and some undefined autoimmune diseases, or combinations thereof.
  • the composition includes a pharmaceutical composition.
  • the composition comprises (a) a non-IGF1R-binding substance; and (b) a pharmaceutically acceptable carrier.
  • the composition contains 0.001-99wt%, preferably 0.1-90wt%, more preferably 1-80wt% of non-IGF1R-binding substances, based on the total weight of the composition.
  • the composition is a liquid preparation or a lyophilized preparation.
  • the composition is an injection.
  • the second aspect of the present invention provides a cell preparation including:
  • Phagocytes treated with non-IGF1R-binding substances Phagocytes treated with non-IGF1R-binding substances.
  • the phagocytic cell is selected from the group consisting of monocytes, macrophages, monocyte precursor cells, or a combination thereof.
  • the non-IGF1R-binding substance is selected from the following group: IGF2, IGF2 mutant, vector expressing IGF2 mutant, antibody, small molecule compound, or a combination thereof.
  • the non-IGF1R-binding substance also includes one or more substances selected from the group consisting of Plasminogen, Serglycin, and e1a stimulation.
  • Cellular suppressor of E1A-stimulated genes CREG
  • Sulfamidase Sgsh
  • phosphorylated ⁇ -glucuronidase Phosphorylated ⁇ -glucuronidase, Gusb
  • the cell preparation further includes a non-IGF1R-binding substance.
  • the phagocytic cell has characteristics selected from the following group:
  • IGF1R is not activated or the degree of activation is lower than IGF2R, IGF2R is activated;
  • Phagocytes use oxidative phosphorylation as the main way of obtaining energy; and/or
  • the phagocytic cells are derived from bone marrow, abdominal cavity, peripheral blood, site of inflammation, or a combination thereof.
  • the cell preparation includes a liquid preparation.
  • the cells in the cell preparation basically ( ⁇ 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%) or all of them are passed through (a) Non-IGF1R-binding substance pretreated phagocytic cells and (b) optional non-IGF1R-binding substance constituted.
  • the concentration of the phagocytic cells is 1 ⁇ 10 4 -5 ⁇ 10 7 /ml, preferably 5 ⁇ 10 4 -5 ⁇ 10 6 /ml, more preferably 1 ⁇ 10 5 -1 ⁇ 10 6 /ml.
  • the carrier is selected from the following group: infusion carrier and/or injection carrier, preferably, the carrier is one or more carriers selected from the group: physiological saline, glucose Salt water, or a combination thereof.
  • the cell preparation also includes other drugs for preventing and/or treating inflammatory diseases.
  • the other drugs for preventing and/or treating inflammatory diseases are selected from the group consisting of non-steroidal anti-inflammatory drugs, glucocorticoids, methotrexate, TNF ⁇ neutralizing antibody, TNFR1 antibody, TNFR2 Antibodies, anti-CD20 antibodies, IL-1R antagonists, IL-12 and IL-23p40 neutralizing antibodies, IL-23p19 neutralizing antibodies, IL-17 neutralizing antibodies, IL-17A receptor neutralizing antibodies, or combinations thereof.
  • the other drugs for preventing and/or treating autoimmune diseases are selected from the group consisting of non-steroidal anti-inflammatory drugs, glucocorticoids, methotrexate, TNF ⁇ neutralizing antibody, and TNFR1 antibody , TNFR2 antibody, anti-CD20 antibody, IL-1R antagonist, IL-12 and IL-23p40 neutralizing antibody, IL-23p19 neutralizing antibody, IL-17 neutralizing antibody, IL-17A receptor neutralizing antibody, CTLA- 4 Fusion protein, or a combination thereof.
  • the third aspect of the present invention provides a medicine kit including:
  • the kit further includes:
  • the third container, and the active ingredient contained in the third container (c) other medicines for preventing and/or treating inflammatory diseases, or medicines containing the active ingredient (c).
  • the kit further includes:
  • the fourth container, and the active ingredient contained in the fourth container (d) Other drugs for preventing and/or treating autoimmune diseases, or drugs containing other drugs for preventing and/or treating autoimmune diseases .
  • first container, the second container, the optional third container, and the optional fourth container may be the same or different.
  • the medicine in the first container is a unilateral preparation containing phagocytes treated with a non-IGF1R-binding substance.
  • the medicine in the second container is a unilateral preparation containing non-IGF1R-binding substances.
  • the third container is a single preparation containing other drugs for preventing and/or treating inflammatory diseases.
  • the fourth container is a single preparation containing other drugs for preventing and/or treating autoimmune diseases.
  • the dosage form of the drug is an injection dosage form.
  • the kit also contains instructions, which describe the combined administration of active ingredient (a), active ingredient (b), optional (c) and optional (d) so as to ( i) prevention and/or treatment of inflammatory diseases; and/or (ii) instructions for prevention and/or treatment of autoimmune diseases.
  • the concentration of the non-IGF1R-binding substance is 0.01 ⁇ g-1 mg/kg body weight, preferably 0.1-10 ⁇ g/kg Body weight, more preferably 0.5-5 ⁇ g/kg body weight.
  • the concentration of the phagocytic cells is 0.2*10 4 -1*10 8 cells/kg body weight, which is higher than Best 1*10 5 -5*10 7 /kg body weight, more preferably 1*10 6 -5*10 6 /kg body weight.
  • the concentration of the other drugs for preventing and/or treating inflammatory diseases is 0.0002-40 mg/kg body weight , Preferably 0.001-30 mg/kg body weight, more preferably 0.02-25 mg/kg body weight.
  • the concentration of the other drugs for preventing and/or treating autoimmune diseases is 0.0002 to 0.1 mg /kg body weight, preferably 0.001-0.08 mg/kg body weight, more preferably 0.002-0.01 mg/kg body weight.
  • the fourth aspect of the present invention provides a drug screening method, including the steps:
  • test group In the test group, add a test substance to the cell culture system, and observe the binding activity of the test substance with IGF1R and/or IGF2R in the cells of the test group; in the control group, the same cell culture No test substances are added to the system;
  • the binding activity of the test substance to IGF1R in the cells of the test group decreases; and the binding activity to IGF2R remains unchanged or increases, it indicates that the test substance is a non-IGF1R-binding IGF2 mutant type.
  • the method further includes the steps:
  • step (b) For the non-IGF1R binding type IGF2 mutant obtained in step (a), further determine its binding activity to the IGFBP protein; and/or further test whether its binding to the IGFBP protein remains unchanged or slightly increased.
  • the method further includes the steps:
  • the positive control is IGF2 protein or IGF2 mutant, and its binding activity to IGF1R and/or IGF2R is compared with the non-IGF1R binding type IGF2 mutant obtained in step (a) to IGF1R and / Or the binding activity of IGF2R for comparison.
  • the binding activity of the non-IGF1R-binding IGF2 mutant obtained in step (a) to IGF1R and/or IGF2R is equivalent to the binding activity of the positive control to IGF1R and/or IGF2R.
  • a method for screening candidates for the prevention and/or treatment of inflammatory diseases including:
  • test group In the test group, add a test substance to the cell culture system, and observe the effect of the test substance in the cells of the test group on the expression and/or activity of IGF2R on the cell surface; and/or the test The influence of the substance on the nuclear transport of IGF2R; and/or the influence of the test substance on the redistribution of IGF2R to the nucleus; in the control group, no test substance is added to the culture system of the same cell;
  • test substance of the test group inhibits the expression and/or activity of IGF2R on the cell surface; and/or the test substance increases the nuclear transport of IGF2R; and/or the test substance promotes the redistribution of IGF2R to the nucleus, This indicates that the test substance is a candidate for the prevention and/or treatment of inflammatory diseases.
  • the candidate has IGF2R selectivity; that is, the candidate has a greater selectivity for IGF2R than for IGF1R.
  • the candidate selectively and/or preferentially binds to IGF2R, but does not bind or substantially does not bind to IGF1R.
  • the cell is a cell cultured in vitro.
  • the cell is a phagocytic cell.
  • the phagocytic cell is selected from the group consisting of monocytes, macrophages, monocyte precursor cells, or a combination thereof.
  • the method is an in vitro method.
  • the method is non-diagnostic and non-therapeutic.
  • the fifth aspect of the present invention provides a drug screening method (or a method for screening candidates), including:
  • test group In the test group, add a test substance to the cell culture system, and observe the effect of the test substance on the binding activity of IGF2 and IGF1R and/or IGF2R in the cells of the test group; in the control group, No test substance is added to the same cell culture system;
  • test substance in the cells of the test group inhibits the binding activity of IGF2 and IGF1R; and has no influence or promotion effect on the binding activity of IGF2 and IGF2R, it indicates that the test substance is a non-IGF1R-binding substance ( Or candidate).
  • the non-IGF1R-binding substance is selected from the following group: antibodies, small molecule compounds, or a combination thereof.
  • the method further includes the steps:
  • step (b) For the non-IGF1R-binding substance obtained in step (a), further determine its binding activity to IGFBP protein; and/or further test whether its binding to IGFBP protein remains unchanged or slightly increases.
  • the method further includes the steps:
  • the positive control is IGF2 protein or IGF2 mutant, and its binding activity to IGF1R and/or IGF2R is compared with the non-IGF1R binding substance obtained in step (a) to IGF1R and/or The binding activity of IGF2R was compared.
  • the binding activity of the non-IGF1R-binding substance obtained in step (a) to IGF1R and/or IGF2R is equivalent to the binding activity of the positive control to IGF1R and/or IGF2R.
  • the cell is a cell cultured in vitro.
  • the cell is a phagocytic cell.
  • the phagocytic cell is selected from the group consisting of monocytes, macrophages, monocyte precursor cells, or a combination thereof.
  • the method is an in vitro method.
  • the method is non-diagnostic and non-therapeutic.
  • the sixth aspect of the present invention provides a method for obtaining pro-inflammatory samples and anti-inflammatory samples in vitro, including:
  • the binding activity of IGF2 to IGF2R is further determined.
  • the binding activity of IGF2 to IGF2R is further determined.
  • the seventh aspect of the present invention provides a composition comprising:
  • non-IGF1R-binding IGF2 mutant or candidate obtained by the method of the fourth or fifth aspect of the present invention does not include an IGF2 mutation selected from the following group Type: wild-type IGF2, wild-type IGF2 amino acid fragments 25 to 91, IGF2 mutant with mutation of glutamic acid at position 12 to Asp, Ala, Gln, His, Arg, or Lys, mutation of phenylalanine at position 26 to Ser IGF2 mutant type, tyrosine 27 is mutated to Leu IGF2 mutant, valine 43 is mutated to Leu IGF2 mutant, IGF2 mutant with D-domain deleted, or a combination thereof.
  • the non-IGF1R binding type IGF2 mutant does not include the IGF2 shown in any of SEQ ID NO.: 1-2, 16, 18, 20, 22, 24, 26, 28, 43 Mutant.
  • amino acid sequence of the non-IGF1R-binding IGF2 mutant is shown in SEQ ID NO.: 3-15, 17, 19, 21, 23, 25, 27, 29-42, 44-58 Either shown.
  • the eighth aspect of the present invention provides a method for inhibiting inflammatory activity, including the steps:
  • phagocytic cells are cultured to suppress inflammatory activity.
  • the application concentration of the non-IGF1R-binding substance is 0.0000005-0.0005 mg/ml, preferably, 0.000001-0.0001 mg/ml, more preferably, 0.000005-0.00005 mg/ml.
  • the ninth aspect of the present invention provides a method for preventing and/or treating inflammatory diseases, including:
  • the subject includes non-human mammals and humans.
  • the dosage of the non-IGF1R-binding substance is 0.01 ⁇ g-1 mg/kg body weight, preferably 0.1-10 ⁇ g/kg body weight, more preferably 0.5-5 ⁇ g/kg body weight.
  • the frequency of administration of the non-IGF1R-binding substance is 1 time/day to 2 times/day.
  • the administration includes simultaneous administration or sequential administration.
  • the tenth aspect of the present invention provides a method for preventing and/or treating autoimmune diseases, including:
  • the subject includes non-human mammals and humans.
  • the dosage of the non-IGF1R-binding substance is 0.01 ⁇ g-1 mg/kg body weight, preferably 0.1-10 ⁇ g/kg body weight, more preferably 0.5-5 ⁇ g/kg body weight.
  • the frequency of administration of the non-IGF1R-binding substance is 1 time/day to 2 times/day.
  • the administration includes simultaneous administration or sequential administration.
  • the eleventh aspect of the present invention provides a use of the cell preparation according to the second aspect of the present invention, the kit according to the third aspect of the present invention, or the composition according to the seventh aspect of the present invention, for the preparation of Drugs to prevent and/or treat inflammatory diseases.
  • FIG. 1 shows that low-dose IGF2 inhibits peritonitis.
  • Low-dose IGF2 (L-IGF2, 5-50ng IGF2 per mouse) and high-dose IGF2 (H-IGF2, 1000ng IGF2 per mouse) were used to treat peritonitis mice.
  • FIG. 2 shows that low-dose IGF2 changed the energy metabolism preference of macrophages and the expression trend and potential of inflammatory factors.
  • Low-dose IGF2 L-IGF2, 5-50ng IGF2 per mouse
  • high-dose IGF2 H-IGF2, 1000ng IGF2 per mouse
  • the peritonitis mouse peritoneal macrophages were isolated and analyzed.
  • A-B low-dose IGF2 enhances the oxidative phosphorylation potential of macrophages, while high-dose IGF2 inhibits oxidative phosphorylation.
  • C low-dose IGF2 changed the energy metabolism preference of macrophages and the expression trend and potential of inflammatory factors.
  • low-dose IGF2 reduces the amount of lactic acid produced by macrophages, corresponding to weaker aerobic glycolytic metabolism.
  • D low-dose IGF2 inhibits the expression of LPS-induced pro-inflammatory genes (Cxcl10, IL-18, Tnfa, Cd40),
  • E low-dose IGF2 promotes IL-4 and IL-13-induced anti-inflammatory repair related genes (Retnla, Ym1, Ccl22, Clec7a) expression.
  • the experiment was repeated three times, and the significant difference was completed by student's t test. *P ⁇ 0.05; **P ⁇ 0.01; ***P ⁇ 0.001.
  • FIG. 3 shows that low-dose IGF2 increases the cytoplasmic H + concentration of monocytes (maturing) and macrophages (mature) and reduces the lysosomal H + concentration.
  • Low-dose IGF2 L-IGF2, 5-50ng IGF2 per mouse
  • high-dose IGF2 H-IGF2, 1000ng IGF2 per mouse
  • IGF2 Abdominal monocytes and macrophages of peritonitis mice were separated and analyzed.
  • A Flow cytometric detection of IGF2 regulates the fluorescence intensity of peritoneal monocytes and macrophages JC1, and evaluates the relative mitochondrial membrane potential.
  • B Flow cytometry IGF2 regulates the cytoplasmic pH of peritoneal monocytes and macrophages.
  • C Flow cytometry IGF2 regulates the relative pH value of lysosomes of peritoneal macrophages.
  • D IGF2 regulates the absolute activity of lysosomal type V ATPase of peritoneal macrophages.
  • Figure 4 shows the construction of igf2r gene-deficient mice and igf1r or igf2r conditional knockout mice.
  • AC using CRISPR-Cas9 technology to knock down IGF2R gene expression (complete knockout of IGF2R gene, lethal mouse embryos) by causing frameshift mutations caused by 2bp or 13bp gene deletion, using gene sequencing technology to identify offspring mice, using Flow cytometry to identify knockdown efficiency.
  • DI by crossing IGF1R fl/fl or IGF2R fl/fl mice and Lyz Cre mice to obtain mice that specifically knock out myeloid cells IGF1R or IGF2R. The knockout efficiency was identified based on PCR and flow analysis.
  • lane 1 indicates wild-type mice (124bp)
  • lane 2 indicates Igf1r fl/fl (220bp)
  • lane 3 has no band, indicating that Igf1r fl/fl mice do not have Lyz2 Cre activity
  • Lane 4 (320bp) indicates that Igf1r fl/fl mice possess Lyz2 Cre activity.
  • H Lane 1 indicates wild-type mice (2000 bp)
  • Lane 2 indicates Igf2r fl/fl mice without Lyz2 Cre activity (2200 bp).
  • FIG. 5 shows that low-dose IGF2 inhibits dextran sulfate sodium salt (DSS)-induced inflammatory bowel disease.
  • DSS dextran sulfate sodium salt
  • a mouse model of enteritis was induced by feeding and drinking 4% DSS solution.
  • A-E low-dose IGF2 alleviates DSS-induced inflammatory bowel disease, which is manifested as a significant improvement in body mass index, survival time, stool score and stool bleeding score, and colon length.
  • F-H and IGF2 were used to treat DSS-induced colitis mice, H&E staining, TUNEL and Ki67 immunohistochemical staining of mouse colon tissue. The experiment was repeated three times, and the significant difference was completed by student's t test.
  • Figure 6 shows that macrophages treated with low-dose IGF2 can effectively inhibit inflammatory bowel disease.
  • mice drinking 4% DSS solution to induce a mouse model of inflammatory bowel disease.
  • A-B low-dose or high-dose IGF2-induced peritoneal mononuclear/macrophages (peritoneal macrophages, PM) in the treatment of DSS-induced inflammatory bowel disease, body weight changes and survival curves were recorded. The experiment was repeated three times, and the significant difference was completed by student's t test. **P ⁇ 0.01.
  • FIG. 7 shows that low-dose IGF2 promotes macrophage formation with anti-inflammatory properties.
  • A Low-dose IGF2 improves DSS-induced inflammatory bowel disease and reduces the infiltration of mononuclear cells in colon tissue.
  • BC analysis of the effect of IGF2 on the expression of interleukin-1 ⁇ (IL-1 ⁇ ) by macrophages (CD11b + F4/80 +) infiltrating the colon of mice with inflammatory bowel disease.
  • D Analysis of the effect of IGF2 on the expression of PD-L1 on macrophages (CD11b + F4/80 +) infiltrating the colon of mice with inflammatory bowel disease. The experiment was repeated three times, and the significant difference was completed by the student's t test. ***P ⁇ 0.001.
  • FIG. 8 shows that IGF1 aggravates the progression of inflammatory bowel disease.
  • mice drinking 4% DSS solution to induce a mouse model of inflammatory bowel disease.
  • mice drinking 4% DSS solution to induce a mouse model of inflammatory bowel disease.
  • mice with inflammatory bowel disease were injected with low-dose IGF1 (L-IGF1, 50ng/mouse) and high-dose IGF1 (H-IGF1, 1000ng/mouse) Mouse) or PBS.
  • A observe the survival of mice
  • B measure the changes in colon length
  • C use flow cytometry to detect the proportion of IL-1 ⁇ expressed by macrophages in the colon lamina propria
  • D use flow cytometry to detect the colon lamina
  • Macrophages express the level of PD-L1
  • EG peritonitis mice were injected with L-IGF1, H-IGF1 or PBS, and macrophages were separated from the peritoneal lavage fluid. Under LPS stimulation, IGF1 was analyzed for macrophages The effects of nitric oxide secretion, lactic acid production and glucose consumption.
  • FIG. 9 shows the effect of low-dose IGF2 performed by IGF2R on the anti-inflammatory function of macrophages.
  • Wild-type (WT) mice and IGF2RCKO (IGF2R fl/fl Lyz2 Cre ) mice were given to induce peritonitis, IGF2 (50ng/mouse) or PBS was intraperitoneally injected, and macrophages were separated from the peritoneal lavage fluid 48 hours later.
  • A Flow cytometric analysis of the expression of PD-L1 on macrophages;
  • B Flow cytometric analysis of the expression of IL-1 ⁇ in macrophages. Mice were given to induce peritonitis, low-dose IGF2 or high-dose IGF2 were injected respectively, and IGF2R antibody was given at the same time,
  • C flow cytometry analysis of H + concentration in macrophage cytoplasm.
  • FIG. 10 shows that IGF2R performs the anti-inflammatory task of low-dose IGF2.
  • IGF2RCKO IGF2R fl/fl Lyz2 Cre
  • IGF1RCKO IGF1R fl/fl Lyz2 Cre
  • A weight change of mice
  • B survival of mice
  • C stool score
  • D stool blood score
  • E colonic histopathological analysis
  • F mononuclear cell infiltration in the lamina intestinal of the colon.
  • GL evaluation of high-dose IGF2 on the inflammatory bowel induced in IGF1R fl/fl Lyz2 Cre mice and IGF1R fl/fl mice Influence of disease.
  • L mononuclear cell infiltration in the lamina limbal of the colon.
  • FIG 11 shows that Leu27-IGF2 targeted activation of IGF2R optimally inhibits peritonitis and inflammatory bowel disease.
  • Peritonitis mice were treated with low-dose IGF2 or Leu27-IGF2 (50ng IGF2/Leu27-IGF2 per mouse) and high-dose IGF2 or Leu27-IGF2 (1000ng, or 2000ng IGF2/Leu27-IGF2 per mouse), respectively.
  • Abdominal monocytes and macrophages of peritonitis mice were separated and analyzed.
  • Leu27-IGF2 significantly increases the H + concentration in the cytoplasm of macrophages.
  • HJ stool score, degree of blood in the stool, and colon length evaluated the therapeutic effect of Leu27-IGF2 on DSS-induced inflammatory bowel disease.
  • K Schematic diagram of the effective threshold of wild-type IGF2 and Leu27-IGF2 to relieve Ftg-induced peritonitis and DSS-induced inflammatory bowel disease. The experiment was repeated three times, and the significant difference was completed by the student's t test. *P ⁇ 0.05;**P ⁇ 0.01;***P ⁇ 0.001.
  • Figure 12 shows that Leu27-Des(62-67)-IGF2 inhibits inflammatory bowel disease.
  • Mice were given 4% DSS solution to induce an inflammatory bowel disease model, and low and high doses of Leu27-Des(62-67)-IGF2 were injected on the first day of disease induction.
  • A monitoring the weight change of mice;
  • B survival of mice with inflammatory bowel disease, evaluating the therapeutic effect of Leu27-Des(62-67) IGF2 on inflammatory bowel disease.
  • Significant differences are completed by student’s t test. *P ⁇ 0.05; ***P ⁇ 0.001.
  • FIG 13 shows that activation of IGF2R promotes nuclear distribution and regulates the cytoplasmic pH of macrophages.
  • THP1 cells were treated with different doses of IGF2, AB, and flow cytometry was used to detect the presence of IGF2R on the cell membrane surface.
  • C Western blotting detects the expression of IGF2R at the overall cell level and in the nucleus.
  • the protein nuclear transcription inhibitor-ivermectin (5 ⁇ M) was used to treat bone marrow-derived mononuclear precursor cells treated with different doses of IGF2. After the macrophages matured, D, Western blotting was used to detect the expression of IGF2R at the overall cell level and in the nucleus.
  • THP1 cells were treated with different doses of IGF2 or Leu27-Des(62-67)-IGF2, F, flow cytometry analysis technique was used to detect the expression of IGF2R on the cell membrane surface. Significant differences are completed by student's t test. ***P ⁇ 0.001.
  • non-IGF1R-binding substances can significantly (a) prevent and/or treat inflammatory diseases; and/or (b) prevent and/or treat autoimmune diseases .
  • the inventors completed the present invention.
  • wild-type IGF2 wild-type IGF2 amino acid fragments from positions 25 to 91, glutamic acid at position 12 is mutated to Asp, Ala, Gln, His, Arg, or Lys, IGF2 mutant, phenylalanine at position 26
  • the IGF2 mutant with Ser, the IGF2 mutant with tyrosine 27 mutated to Leu, the IGF2 mutant with valine 43 mutated to Leu, and the IGF2 mutant with D-domain deleted are in SEQ ID NO.:1
  • the truncation, mutation and/or deletion of amino acids are carried out on the basis of.
  • IGF2 protein and its mutants
  • IGF2 Insulin like growth factor 2
  • IGF2 receptors include IGF1R and IGF2R, among which IGF2R has the highest affinity, followed by binding to IGF1R, and almost no binding to insulin receptor 2 .
  • IGF2 and IGF1 have a high degree of homology in amino acid sequence, with 62% overlapping sequences. Therefore, IGF2 can often reflect the biological effects that IGF1 can activate 2 .
  • Current research suggests that the main biological function of IGF2 depends on the activation of tyrosine kinases that activate IGF1R (insulin like growth factor 1 receptor), but has no positive correlation with IGF2R (insulin like growth factor 2 receptor) and IR. From an evolutionary perspective, IGF1R and IR have gene homology, and the signaling pathways activated downstream of IGF1R and IR are very similar. However, the source of IGF2R gene is completely different from that of IGF1R or IR gene.
  • IGF2R is often considered to have the opposite biological function of IGF1R and IR.
  • IGF2R acts as the degradation receptor of IGF2, and inhibits IGF2's effect on IGF1R by internalizing degradation of IGF2. The activation effect.
  • IGF2R Intracellular Golgi apparatus
  • IGF2R is not the only receptor of IGF2
  • IGF2 is not the only ligand of IGF2R. Its ligands also include mannose-6-phosphate.
  • the main function of IGF2R is to transport its ligands to lysosomes. 4 complete protein processing or degradation.
  • the IGF2R on the cell membrane can be cut off in a soluble form, into the blood and other liquid environment, effective binding IGF2, 4,5 inhibition IGF2 function.
  • IGF2 can inhibit animal models of multiple sclerosis and ulcerative colitis, but its specific regulation mechanism is still unclear.
  • the present invention relates to an IGF2 protein and variants thereof.
  • the amino acid sequence of the IGF2 protein is shown in SEQ ID NO.:1.
  • the IGF2 protein or the vector expressing the IGF2 protein of the present invention can (a) prevent and/or treat inflammatory diseases.
  • the IGF2 protein or the vector expressing the IGF2 protein of the present invention can also be used for one or more purposes selected from the following group:
  • the present invention also includes 50% or more of the sequence shown in SEQ ID NO. 1 of the present invention (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more, most preferably 98% or more, such as 99%) homologous polypeptides or proteins with the same or similar functions.
  • SEQ ID NO.: 1 is a human IGF2 protein (AYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPAKSE).
  • the "same or similar function” mainly refers to: "(a) prevention and/or treatment of inflammatory diseases; and/or (i) reduction of pathogenic mononuclear cell infiltration; and/or (ii) inhibition of macrophage Pathogenic phenotype; and/or (iii) reduce the number of macrophages infiltrated; and/or (iv) increase the cytoplasmic H + concentration of macrophages; and/or (v) increase the metabolic propensity of macrophages to oxidize phosphoric acid ; And/or (vi) endowing macrophages with immune memory that significantly inhibits inflammation; and/or (vii) preventing and/or treating autoimmune diseases; and/or (viii) a variety of other macrophages involved Diseases involving inflammation (such as inflammation related to liver fibrosis, renal fibrosis, lung fibrosis and other diseases, inflammation caused by tissue, organ or cell transplantation, and lung cancer, breast cancer, prostate cancer, liver cancer , Pancreatic cancer,
  • the protein of the present invention can be a recombinant protein, a natural protein, or a synthetic protein.
  • the protein of the present invention can be a natural purified product, or a chemically synthesized product, or produced from a prokaryotic or eukaryotic host (for example, bacteria, yeast, higher plant, insect, and mammalian cells) using recombinant technology.
  • a prokaryotic or eukaryotic host for example, bacteria, yeast, higher plant, insect, and mammalian cells
  • the protein of the present invention may be glycosylated or non-glycosylated.
  • the protein of the present invention may also include or not include the initial methionine residue.
  • the present invention also includes IGF2 protein fragments and analogs having IGF2 protein activity.
  • fragment and analogs having IGF2 protein activity refer to a protein that substantially maintains the same biological function or activity as the natural IGF2 protein of the present invention.
  • the mutant of the IGF2 protein of the present invention is mutated in the tyrosine at position 27 of the wild-type IGF2 protein corresponding to SEQ ID NO.:1 (AYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPAKSE), and the IGF2 protein of the present invention is Mutants can significantly (a) prevent and/or treat inflammatory diseases; and/or (b) prevent and/or treat autoimmune diseases.
  • the tyrosine at position 27 is mutated to leucine.
  • the tyrosine at position 27 is mutated to leucine, and D-domain (threonine at position 62 to glutamic acid at position 67 of SEQ ID NO.: 1) is optionally deleted.
  • the IGF2 mutant is a polypeptide having an amino acid sequence shown in any one of SEQ ID NO.: 2-58, an active fragment thereof, or a conservative variant polypeptide thereof.
  • amino acid numbering in the mutant of the present invention is based on SEQ ID NO.:1.
  • amino acid of the mutant may be misaligned with respect to the amino acid numbering of SEQ ID NO.: 1), such as misaligned 1-5 to the N-terminus or C-terminus of the amino acid.
  • the mutein fragment, derivative or analogue of the present invention may be (i) a mutein in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acids
  • the residue may or may not be encoded by the genetic code, or (ii) a mutein with a substitution group in one or more amino acid residues, or (iii) a mature mutein and another compound (such as an extended mutein) Half-life compounds, such as polyethylene glycol) fused to form a mutant protein, or (iv) additional amino acid sequence fused to the mutant protein sequence to form a mutant protein (such as leader sequence or secretory sequence or used to purify the mutant protein)
  • the sequence or proprotein sequence, or the fusion protein formed with the antigen IgG fragment According to the teachings herein, these fragments, derivatives and analogs belong to the scope well known to those skilled in the art.
  • conservatively substituted amino acids are preferably generated by amino acid substitution
  • the present invention also includes that the natural IGF2 protein of the present invention has 50% or more (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more, most preferably 98% or more, such as 99 %) Homologous polypeptides or proteins with the same or similar functions.
  • the protein variant can be obtained by substituting, deleting or adding at least one amino acid by several (usually 1-60, preferably 1-30, more preferably 1-20, most preferably 1-10). Derivative sequences, and adding one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal.
  • the function of the protein is usually not changed, and the addition of one or several amino acids to the C-terminal and/or ⁇ terminal usually does not change the function of the protein.
  • the present invention includes that the difference between the natural IGF2 protein analog and the natural IGF2 protein may be the difference in the amino acid sequence, the difference in the modified form that does not affect the sequence, or both.
  • Analogs of these proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, site-directed mutagenesis or other known biological techniques.
  • Analogs also include analogs having residues different from natural L-amino acids (such as D-amino acids), and analogs having non-naturally occurring or synthetic amino acids (such as ⁇ , ⁇ -amino acids). It should be understood that the protein of the present invention is not limited to the representative proteins exemplified above.
  • Modified (usually not changing the primary structure) forms include: chemically derived forms of proteins in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those that undergo glycosylation modifications during protein synthesis and processing. This modification can be accomplished by exposing the protein to an enzyme that performs glycosylation (such as a mammalian glycosylase or deglycosylase). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, and phosphothreonine). In addition, the mutant protein of the present invention can also be modified.
  • Modified (usually not changing the primary structure) forms include: chemically derived forms of mutein in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those produced by glycosylation modifications during the synthesis and processing of the mutant protein or during further processing steps. This modification can be accomplished by exposing the mutein to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylase. Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, and phosphothreonine). It also includes mutant proteins that have been modified to improve their resistance to proteolysis or optimize their solubility.
  • the present invention also provides a polynucleotide sequence encoding IGF2 protein.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include: DNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • a polynucleotide encoding a mature polypeptide includes: a coding sequence that only encodes the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence (and optional additional coding sequences) of the mature polypeptide and non-coding sequences.
  • polynucleotide encoding a polypeptide may include a polynucleotide encoding the polypeptide, or a polynucleotide that also includes additional coding and/or non-coding sequences.
  • the present invention also relates to variants of the aforementioned polynucleotides, which encode fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the present invention.
  • the variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants.
  • These nucleotide variants include substitution variants, deletion variants and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide. It may be a substitution, deletion or insertion of one or more nucleotides, but it will not substantially change the function of the encoded polypeptide. .
  • the coding nucleic acid sequence of the present invention can be constructed by the method of synthesizing the nucleotide sequence in segments and then performing overlap extension PCR.
  • the present invention also relates to polynucleotides that hybridize with the aforementioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences.
  • the present invention particularly relates to polynucleotides that can hybridize with the polynucleotide of the present invention under stringent conditions (or stringent conditions).
  • stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) adding during hybridization There are denaturants, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90% or more, more Fortunately, hybridization occurs when more than 95%.
  • proteins and polynucleotides of the present invention are preferably provided in an isolated form, and more preferably, are purified to homogeneity.
  • the full-length sequence of the polynucleotide of the present invention can usually be obtained by PCR amplification method, recombination method or artificial synthesis method.
  • primers can be designed according to the relevant nucleotide sequence disclosed in the present invention, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA prepared by a conventional method known to those skilled in the art can be used.
  • the library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.
  • the recombination method can be used to obtain the relevant sequence in large quantities. This is usually done by cloning it into a vector, then transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is short. Usually, by first synthesizing multiple small fragments, and then ligating to obtain fragments with very long sequences.
  • the DNA sequence encoding the protein (or fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequence of the present invention through chemical synthesis.
  • the method of using PCR technology to amplify DNA/RNA is preferably used to obtain the polynucleotide of the present invention.
  • the RACE method RACE-cDNA end rapid amplification method
  • the primers used for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein. And can be synthesized by conventional methods.
  • the amplified DNA/RNA fragments can be separated and purified by conventional methods such as gel electrophoresis.
  • the present invention also relates to a vector containing the polynucleotide of the present invention, a host cell produced by genetic engineering using the vector of the present invention or a mutant protein coding sequence of the present invention, and a method for producing the polypeptide of the present invention through recombinant technology.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant mutein. Generally speaking, there are the following steps:
  • polynucleotide (or variant) of the present invention encoding the mutant protein of the present invention, or use a recombinant expression vector containing the polynucleotide to transform or transduce a suitable host cell;
  • the polynucleotide sequence encoding the mutant protein can be inserted into a recombinant expression vector.
  • recombinant expression vector refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenovirus, retrovirus or other vectors well known in the art. Any plasmid and vector can be used as long as it can replicate and stabilize in the host.
  • An important feature of an expression vector is that it usually contains an origin of replication, a promoter, a marker gene, and translation control elements.
  • Methods well known to those skilled in the art can be used to construct an expression vector containing the DNA sequence encoding the mutein of the present invention and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology.
  • the DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis.
  • promoters are: Escherichia coli lac or trp promoter; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, anti Transcriptional virus LTRs and some other known promoters that can control gene expression in prokaryotic or eukaryotic cells or viruses.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • a vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell so that it can express the protein.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast and plant cells (such as ginseng cells).
  • Enhancers are cis-acting factors of DNA, usually about 10 to 300 base pairs, acting on promoters to enhance gene transcription. Examples include the 100 to 270 base pair SV40 enhancer on the late side of the replication initiation point, the polyoma enhancer on the late side of the replication initiation point, and adenovirus enhancers.
  • Transformation of host cells with recombinant DNA can be performed by conventional techniques well known to those skilled in the art.
  • the host is a prokaryotic organism such as Escherichia coli
  • competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method.
  • the steps used are well known in the art.
  • Another method is to use MgCl2.
  • transformation can also be carried out by electroporation.
  • the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture can be selected from various conventional mediums.
  • the culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • the recombinant polypeptide in the above method can be expressed in the cell or on the cell membrane, or secreted out of the cell. If necessary, the physical, chemical, and other characteristics can be used to separate and purify the recombinant protein through various separation methods. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitation agent (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • non-IGF1R binding type As used herein, the terms “non-IGF1R binding type”, “IGF2R selective type” or “IGF2R selective” are used interchangeably, and refer to the substance that is more selective for IGF2R than for IGF1R, that is, selectively and/ Or preferentially bind to IGF2R, but not or substantially not bind to IGF1R.
  • non-IGF1R-binding substances include substances that have a higher selectivity or affinity for IGF2R than IGF1R, and substances that can efficiently activate IGF2R and do not activate IGF1R (such as non-IGF1R ligands such as retinoic acid).
  • the affinity or selectivity of the non-IGF1R-binding substance for IGF2R is higher than its affinity or selectivity for IGF1R.
  • the non-IGF1R-binding substance is selected from the group consisting of IGF2 mutants, vectors expressing IGF2 mutants, antibodies, small molecule compounds, or combinations thereof.
  • the non-IGF1R-binding substance also includes one or more substances selected from the following group: Plasminogen, Serglycin, e1a stimulator Cellular suppressor of E1A-stimulated genes (CREG), Sulfamidase (Sgsh), phosphorylated ⁇ -glucuronidase (Phosphorylated ⁇ -glucuronidase, Gusb) and other effective anti-inflammatory ligands for IGF2R .
  • Plasminogen Plasminogen, Serglycin, e1a stimulator Cellular suppressor of E1A-stimulated genes (CREG), Sulfamidase (Sgsh), phosphorylated ⁇ -glucuronidase (Phosphorylated ⁇ -glucuronidase, Gusb) and other effective anti-inflammatory ligands for IGF2R .
  • non-IGF1R-binding substances can significantly (a) prevent and/or treat inflammatory diseases; and/or (i) reduce disease-causing mononuclear cell infiltration; and/or (ii) Inhibit the pathogenic phenotype of macrophages; and/or (iii) reduce the number of macrophages infiltrated; and/or (iv) increase the cytoplasmic H + concentration of macrophages; and/or (v) increase macrophage oxidation
  • the metabolic propensity of phosphoric acid; and/or (vi) confers immune memory to macrophages that significantly inhibits inflammation; and/or (vii) prevents and/or treats autoimmune diseases; and/or (viii) is used for macrophages Participate in a variety of other diseases involving inflammation (such as inflammation related to liver fibrosis, renal fibrosis, lung fibrosis and other diseases, inflammation related to tissue, organ or cell transplantation, and lung cancer, breast cancer
  • the present invention provides a compound pharmaceutical composition containing active ingredients (a) phagocytic cells treated with a non-IGF1R-binding substance; (b) optional non-IGF1R-binding substance; and (c) a pharmaceutically acceptable carrier .
  • Such carriers include (but are not limited to): saline, buffer, dextrose, water, glycerol, ethanol, powder, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be made into an injection form, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods.
  • compositions such as injections, solutions, tablets and capsules should be manufactured under sterile conditions.
  • the pharmaceutical combination of the present invention can also be made into powder for inhalation.
  • the dosage form of the pharmaceutical composition of the present invention is an injection.
  • the amount of active ingredient administered is a therapeutically effective amount.
  • the pharmaceutical preparation of the present invention can also be made into a sustained-release preparation.
  • the pharmaceutical composition of the present invention is preferably an injection preparation.
  • the pharmaceutical composition of the present invention can also be used together with other therapeutic agents.
  • the pharmaceutical composition of the present invention may also include additional components selected from the following group: (a) drugs for preventing and/or treating inflammatory diseases; and/or (b) preventing and/ Or a component for the treatment of autoimmune diseases.
  • the effective amount of the active ingredient of the present invention can vary with the mode of administration and the severity of the disease to be treated.
  • the selection of the preferred effective amount can be determined by a person of ordinary skill in the art according to various factors (for example, through clinical trials).
  • the factors include, but are not limited to: the pharmacokinetic parameters of the active ingredients such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's weight, the patient's immune status, and administration The way and so on.
  • the active ingredient of the present invention is about 0.01 ⁇ g-1 mg/kg body weight per day, preferably 0.1-10 ⁇ g/kg body weight, more preferably 0.5-5 ⁇ g/kg body weight. It can be given satisfactory results.
  • the optimal dose will be adjusted according to the course and condition of the disease. For example, according to the urgent requirement of the treatment condition, the dose can be given several times a day, or the dose can be reduced proportionally.
  • the pharmaceutically acceptable carriers of the present invention include (but are not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or Its combination.
  • the choice of carrier should match the mode of administration, which are well known to those of ordinary skill in the art.
  • the present invention also provides a drug that can be used to (a) prevent and/or treat inflammatory diseases; and/or ((i) reduce the infiltration of pathogenic mononuclear cells; and/or (ii) inhibit the infiltration of macrophages Disease phenotype; and/or (iii) reduce the number of macrophages infiltrated; and/or (iv) increase the cytoplasmic H + concentration of macrophages; and/or (v) increase the metabolic propensity of macrophages to oxidize phosphoric acid; (vi) Endow macrophages with immune memory that significantly inhibits inflammation; and/or (vii) prevent and/or treat autoimmune diseases; and/or (viii) use for a variety of other inflammatory reactions involving macrophages Diseases (such as inflammation related to liver fibrosis, renal fibrosis, lung fibrosis and other diseases, inflammation related to tissue, organ or cell transplantation, and lung cancer, breast cancer, prostate cancer, liver cancer, pancreatic cancer, Inflammation
  • the pharmaceutical composition and kit of the present invention are suitable for (a) preventing and/or treating inflammatory diseases; and/or (i) reducing the infiltration of pathogenic mononuclear cells in the colon; and/or (ii) inhibiting macrophage Pathogenic phenotype; and/or (iii) reduce the number of macrophages infiltrated; and/or (iv) increase the cytoplasmic H + concentration of macrophages; and/or (v) increase the metabolic propensity of macrophages to oxidize phosphoric acid ; And/or (vi) endowing macrophages with immune memory that significantly inhibits inflammation; and/or (vii) preventing and/or treating autoimmune diseases; and/or (viii) a variety of other macrophages involved Diseases involving inflammation (such as inflammation related to liver fibrosis, renal fibrosis, lung fibrosis and other diseases, inflammation caused by tissue, organ or cell transplantation, and lung cancer, breast cancer, prostate cancer, liver cancer , Pancreatic cancer,
  • the preparation of the present invention can be taken three times a day to once every ten days, or once every ten days in a sustained-release manner.
  • the preferred way is to take it once a day, because it facilitates the patient's adherence, thereby significantly improving the patient's compliance with medication.
  • the total dose applied daily in most cases should be lower (or equal to or slightly greater than) the daily daily dose of each single drug in most cases.
  • the effective dose of the active ingredient used can vary depending on the mode of administration and the expected dose. The severity of the disease to be treated varies.
  • the present invention also provides the use of the two active ingredients of the present invention or corresponding drugs to (a) prevent and/or treat inflammatory diseases; and/or (i) reduce the infiltration of pathogenic mononuclear cells in the colon; and/or ( ii) inhibit the pathogenic phenotype of macrophages; and/or (iii) reduce the number of macrophages infiltrated; and/or (iv) increase the H + concentration of the cytoplasm of macrophages; and/or (v) increase macrophages
  • the two active ingredients of the present invention can be mixed with one or more pharmaceutically acceptable carriers or excipients, such as solvents, diluents, etc., and can be administered orally in the form of tablets: Pills, pills, capsules, dispersible powders, granules or suspensions (containing, for example, about 0.05-5% suspending agent), syrups (containing, for example, about 10-50% sugar), and elixirs (containing about 20-50% ethanol), Or in the form of a sterile injectable solution or suspension (containing about 0.05-5% suspension in an isotonic medium) for parenteral administration.
  • these pharmaceutical preparations may contain about 0.01-99%, more preferably about 0.1%-90% by weight of the active ingredient mixed with the carrier.
  • the two active ingredients or pharmaceutical compositions of the present invention can be administered by conventional routes, including (but not limited to): intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, oral, intratumoral or topical administration .
  • routes of administration include oral administration, intramuscular administration or intravenous administration.
  • the preferred pharmaceutical composition is a liquid composition, especially an injection.
  • the two active ingredients or drugs of the present invention can also be combined with other (a) prevention and/or treatment of inflammatory diseases; and/or (i) reduction of pathogenic mononuclear cell infiltration; and/or (ii) inhibition of macrophages
  • non-IGF1R-binding substances can significantly (a) prevent and/or treat inflammatory diseases; and/or (i) reduce the infiltration of pathogenic mononuclear cells; and/or (ii) inhibit macrophage The pathogenic phenotype of phages; and/or (iii) reduce the number of macrophages infiltrated; and/or (iv) increase the cytoplasmic H + concentration of macrophages; and/or (v) increase the oxidative phosphoric acid of macrophages Metabolic propensity; and/or (vi) endowed macrophages with immune memory that significantly inhibits inflammation; and/or (vii) prevention and/or treatment of autoimmune diseases; and/or (viii) used for macrophage participation
  • inflammation such as inflammation related to liver fibrosis, renal fibrosis, lung fibrosis and other diseases, inflammation related to tissue, organ or cell transplantation, and lung cancer, breast cancer, prostate Inflammation
  • IGF2 insulin-like growth factor 2
  • IGF2R IGF2 receptor
  • the present invention finds for the first time that low-dose IGF2 can induce non-classical mitochondrial dynamics and give macrophages immune memory against inflammation.
  • High-dose IGF2 can activate IGF1 receptor (IGF1R), inhibit the effect of IGF2R, and promote monotherapy.
  • IGF1R IGF1 receptor
  • Nuclear cells and macrophages use aerobic glycolysis and metabolism to obtain pro-inflammatory capabilities.
  • the present invention finds for the first time that IGF2 mutants with low affinity to IGF1R specifically activate IGF2R, and to the greatest extent alleviate autoimmune diseases and inflammatory diseases such as peritonitis and inflammatory bowel disease. These findings indicate that the targeted activation of IGF2R can determine the long-term immunosuppressive memory of macrophages. IGF2R is an ideal target for the treatment of inflammation-related diseases involving macrophages.
  • the present invention discovered for the first time that the IGF2R, which is directed to activate monocytes, inhibits inflammatory bowel disease.
  • the present invention found for the first time that IGF2 treatment of inflammatory bowel disease relies on macrophages.
  • the present invention discovered for the first time that IGF2 is involved in a variety of other inflammatory diseases in macrophages.
  • Recombinant human insulin-like growth factor-2 (Recombinant human IGF2, 292-G2-250), recombinant mouse-derived macrophage colony stimulating factor (Recombinant mouse M-CSF protein, 416-ML-050) were purchased from R&D Systems.
  • the 27th human source is leucine point mutation IGF2 (Human Leu27-IGF2, TU100) purchased from GroPep Bioreagents.
  • the 27th human source is leucine point mutation IGF2 (Human Leu27-IGF2, TU100). The deletion of the D domain is synthesized in the laboratory.
  • IGF2R +/- mice use the Crisper-Cas9 technology to knock out the 2bp or 13bp base on the 6th exon of the igf2r gene, causing a frameshift mutation. Since homozygous IGF2R deficiency can cause embryonic death, we use IGF2R +/- heterozygous mice to pass down and identify them.
  • the primers used for identification are:
  • Igf2r fl/fl mice insert loxp sequence on both sides of exon 2 of igf2r gene, and the primers used for identification are:
  • Primer 2,5’-CAATTTAGGGCTGAAGCGATGGAT-3’ (SEQ ID NO.: 62)
  • the primers used for Igf1r fl/fl mouse identification are:
  • the primers used for Lyz2 Cre mouse identification are:
  • thioglycolate (Ftg) medium aqueous solution Prepare 5% (mass/volume) thioglycolate (Ftg) medium aqueous solution, sterilize at 121°C under high temperature and high pressure for 30 minutes, and cool to room temperature for later use.
  • Ftg thioglycolate
  • the viscosity should not cause bubbles when shaking.
  • the color of the medium should be changed from blue-green when initially configured to brown when shaking.
  • mice C57BL/6 female mice of the same age in the range of 9-12 weeks were selected for the experiment. Each mouse was slowly injected with 2ml of thioglycolate medium into the abdominal cavity, 24 hours later, IGF2 or other drugs were given, and it was given once at the 24th and 48th hours after the thioglycolate injection, and at the 72nd hour The mice were euthanized and the peritoneal macrophages were isolated.
  • mice that have been euthanized (Ftg) for 72 hours with 75% ethanol for a few minutes, tear off the abdominal fur in the direction perpendicular to the head and tail of the mouse, suck 10ml of pre-cooled phosphate buffer () with a syringe, and remove the phosphoric acid.
  • salt buffer solution into the abdominal cavity of the mouse (to help blow down the adherent cells), clamp the tail of the mouse with the index finger and ring finger of the right hand, gently hold the mouse, shake it up and down three to five times, and slowly use a syringe Aspirate the cell lavage fluid.
  • Peritoneal macrophage lavage fluid needs to be stored at four degrees, and cells are easy to adsorb to the wall of the centrifuge tube at room temperature.
  • DSS dextran sulfate sodium salt
  • the clinical scoring of inflammatory bowel disease was performed according to the following criteria. After feeding the mice with 4% DSS solution for 4 days, “Stool scores” and “Bleeding scores” were evaluated.
  • the "Stool scores” standard is: 0 points, normal stool shape; 1 point, semi-formed stool and no anus attachment; 2 points, semi-formed stool and anal attachment; 3 points, liquid feces attached to the anus.
  • "Bleeding scores” judgment criteria 0 points, fecal occult blood test paper is negative; 1 point, fecal occult blood test paper is positive; 2 points, blood stains can be seen in the cage; 3 points, rectal bleeding, red blood spots on the anus.
  • mice Euthanize the mice induced by 4% DSS for 4-5 days, dissected the mice, and obtained the colon tissue, and placed it in 4°C pre-cooled phosphate buffer solution to wash off the blood stains on the colon surface. Cut the colon longitudinally, and carefully wash away the stool in a four-degree pre-cooled phosphate buffer.
  • Use 1640 culture medium containing 200 ⁇ g/ml DNaseI and 1mg/ml type VIII collagenase add DTT before digestion, shake 200 rpm for 60 minutes, carefully remove the colon basal tissue, cut into small sections, and perform digestion again , Filter with a 70 ⁇ m cell sieve, and centrifuge the filtrate at 400g for 5 minutes to obtain a single cell pellet.
  • Protein staining on cell membrane surface For cells blocked with CD16/32 antibody, dilute the antibody with phosphate buffer at a ratio of 1:100, 100 ⁇ l staining working solution/1 ⁇ 10 7 cells, stain at room temperature for 30 minutes, wash with phosphate buffer, and centrifuge with 200 ⁇ l Resuspend in phosphate buffer, test on the machine or fix with 1% neutral formaldehyde.
  • Intracellular proteins and nuclear proteins need to be performed after the staining of the cell membrane surface proteins is completed.
  • intracellular proteins use the cell fixation and rupture kit (Fixation/Permeabilization Solution and Perm/Wash Buffer) for rupture and staining after fixation; for nuclear proteins, use the cell fixation/cell rupture kit (Foxp3/Transcription Factor) Staining Buffer Set) Stain after fixation and membrane rupture.
  • the oxygen consumption rate measurement experiment and the extracellular acid production rate measurement experiment were performed with or without the following stimulation conditions: 1 ⁇ M oligomycin, 0.75mM FCCP, 100nM rotenone and 1 ⁇ M antimycin, using an extracellular flux analyzer , Agilent) determination.
  • RNA Isolation Kit Use Tiangen Biological Cell/Bacterial RNA Isolation Kit to extract total RNA from cells. You can usually get 300ng/ml RNA in a volume of 30 ⁇ L per sample.
  • the real-time quantitative PCR system is 10 ⁇ L, including 5 ⁇ L SYBR, 4 ⁇ L cDNA, and 1 ⁇ L primers. After spotting the sample in the 384-well plate, centrifuge it at 2500 rpm for 2 minutes at room temperature. In this step, centrifugation at 4°C will form water mist on the membrane.
  • the real-time quantitative PCR conditions are: Step 1, 95°C for 10 minutes, Step 2, 95°C for 15 seconds, Step 3, 60°C for 1 minute, Step 4, 95°C for 15 seconds, Step 5, 60°C for 1 minute , Step 6, 95°C for 15 seconds, of which Step 2 and Step 3 cycle 40 times.
  • the primers involved in the real-time quantitative PCR experiment are as follows:
  • TNFa-F 5’-CCTGTAGCCCACGTCGTAG-3’ (SEQ ID NO.: 69),
  • TNFa-R 5’-GGGAGTAGACAAGGTACAACCC-3’ (SEQ ID NO.: 70);
  • IL-1b-F 5’-GCAACTGTTCCTGAACTCAACT-3’ (SEQ ID NO.: 71),
  • IL-1b-R 5'-ATCTTTTGGGGTCCGTCAACT-3' (SEQ ID NO.: 72);
  • Il18-F 5’-GACTCTTGCGTCAACTTCAAGG-3’ (SEQ ID NO.: 81)
  • Il18-R 5’-CAGGCTGTCTTTTGTCAACGA’ (SEQ ID NO.: 82);
  • Retnla-F 5’-CCAATCCAGCTAACTATCCCTCC-3’ (SEQ ID NO.: 75),
  • Retnla-R 5’-ACCCAGTAGCAGTCATCCCA-3’ (SEQ ID NO.: 76);
  • Dehydration embedding Take the colon tissue at the same position and fix it in 4% paraformaldehyde solution for 24 hours, and then embed it after dehydration treatment in the following steps. 70% ethanol, soak overnight; 80% ethanol, soak for 2 hours; 85% ethanol, soak for 1.5 hours; 90% ethanol, soak for 1 hour; 95% ethanol, soak for 1 hour; 100% ethanol, soak for 30 minutes, repeat the operation once ; Xylene, soak for 5 minutes, repeat the operation once; 60 °C liquid paraffin, soak for 1 hour, repeat the operation once; use the paraffin embedding machine to complete the paraffin embedding.
  • Section staining slice at a thickness of 3 ⁇ m, extend the paraffin tissue section in a 56°C water bath for a few seconds, absorb the paraffin tissue section with a glass slide, bake the slice in an oven at 60°C overnight, and then perform staining according to the following steps.
  • Xylene soak for 5 minutes, repeat the operation once; 100% ethanol, soak for 5 minutes, repeat the operation once; 95% ethanol, soak for 5 minutes, repeat the operation once; 70% ethanol, soak for 5 minutes; distilled water, soak for 5 minutes; Wood essence, dyeing for 5 minutes; tap water and running water for proper time; ammonia water, immersion for proper time; running water for proper time; eosin, dyeing for 1 minute; 95% ethanol, soaking for 1 minute; 100% ethanol, soaking for 1 minute, repeat the operation once.
  • Xylene soak for 1 minute; repeat the operation once; mount the tablet.
  • Antigen-antibody reaction drop the primary anti-Ki67 antibody on the paraffin section, incubate overnight at 4°C; wash four times with pH 7.4 phosphate-Tween buffer for three minutes each time; add horseradish on the paraffin section dropwise Peroxidase-conjugated secondary antibody, incubate at room temperature for 60 minutes; wash 4 times with pH 7.4 phosphate-Tween buffer for 3 minutes each time; after DBA staining for an appropriate time, wash off DBA stain; use hematoxylin Dye counterstain paraffin sections, determine the appropriate time according to the type of tissue; rinse with tap water to remove hematoxylin; use hydrochloric acid alcohol differentiation and ammonia water to reverse blue until hematoxylin staining appears light blue; dehydration mounts.
  • the TUNEL immunohistochemistry experiment was carried out in accordance with the experimental method provided by the In situ cell death detection kit-POD kit (Roche, TUN11684817).
  • Detecting the accumulation of nitric oxide in the supernatant of the culture medium can reflect the activity of inducible nitric oxide synthase (iNOS) of macrophages, and speculate the type of energy metabolism and inflammatory phenotype of macrophages.
  • iNOS inducible nitric oxide synthase
  • the lactate detection kit was used to detect the lactate production capacity of the peritoneal monocytes and macrophages pre-programmed by IGF2. Perform the experiment according to the experimental method provided by Actate Colorimetric Assay Kit II (BioVision, K627-100). Detecting the accumulation of lactic acid in the supernatant of the culture medium can reflect the type of energy metabolism and inflammatory phenotype of macrophages.
  • Glucose detection kit was used to detect the glucose consumption capacity of peritoneal monocytes and macrophages pre-programmed by IGF2.
  • IGF2 insulin growth factor receptor 2
  • Glucose Colorimetric Assay Kit II BioVision, K686-100.
  • the rate of glucose consumption can reflect the type of energy metabolism and inflammatory phenotype of macrophages.
  • the lysosomes of IGF2-treated peritoneal monocytes and macrophages were separated, and the experiment was performed according to the experimental method provided by the Lysosome Isolation Kit (Sigma-Aldrich, LYSISO1-1KT).
  • the separated lysosomes will be used in the V-type ATPase activity detection experiment.
  • Use the ATPase activity detection kit to detect the lysosomal ATPase activity isolated above, which represents the activity of lysosomal type V ATPase.
  • the data are expressed in the form of mean or mean ⁇ S.E.M. or S.D., using Graphpad software for mapping and significant difference analysis.
  • significance of the difference between the two sets of data was analyzed using two-tailed non-parametric Student’s t test. ns means insignificant difference; *P ⁇ 0.05; **P ⁇ 0.01; ***P ⁇ 0.001, P ⁇ 0.05 is considered a significant difference.
  • Example 1 Low-dose IGF2 inhibits peritonitis.
  • L-IGF2 Low-dose IGF2
  • H-IGF2 high-dose IGF2
  • Example 2 Anti-inflammatory macrophages under the control of low-dose IGF2 are dominated by the metabolic preference of oxidative phosphorylation.
  • macrophages Under the control of low-dose IGF2, macrophages exhibit higher levels of OCR:ECAR, indicating that this group of macrophages tend to use oxidative phosphorylation (OXPHOS) as the main way to obtain energy, while the control group and high-dose IGF2 Group macrophages are more inclined to choose aerobic glycolysis to produce energy for the cells.
  • OXPHOS oxidative phosphorylation
  • Lactic acid is an intermediate metabolite of aerobic glycolysis. In the process of aerobic glycolysis, excess lactic acid will be secreted out of the cell by the cells. Therefore, to detect and compare the cumulative amount of lactic acid in the supernatant of the cell culture medium, it is An effective way to measure the level of aerobic glycolysis in cells.
  • LPS Lipopolysaccharide
  • LPS lipopolysaccharide
  • IL-4/113 interleukin-4/13
  • RT-PCR results showed that the expression of inflammatory factors in macrophages classically activated by lipopolysaccharide was inhibited by low-dose IGF2 (L-IGF2), and high-dose IGF2 had no significant regulation on some genes (Figure 2D); the anti-inflammatory factor expression of macrophages activated by interleukin 4/13 is amplified by IGF2 (L-IGF2), and high-dose IGF2 (H-IGF2) has no obvious effect (Figure 2E).
  • L-IGF2 low-dose IGF2
  • H-IGF2 high-dose IGF2
  • Example 3 Low-dose IGF2 inhibits type V ATPase activity, reduces lysosomal H + concentration, promotes cytoplasmic H + concentration, and increases mitochondrial inner membrane potential.
  • Oxidative phosphorylation occurs in the mitochondria, while aerobic glycolysis occurs in the cytoplasm.
  • IGF2 enhances the oxidative phosphorylation capacitation preference of macrophages.
  • peritoneal macrophages mature and mature macrophages treated with low-dose IGF2 (L-IGF2, 50ng per mouse) all showed higher mitochondrial membrane potential ( Figure 3A), suggesting a significant increase The mitochondrial membrane potential provides sufficient H + substrates for the oxidative phosphorylation of macrophages under the control of low-dose IGF2.
  • Example 4 Construction of igf2r gene-deficient mice and igf1r or igf2r conditional knockout mice.
  • IGF2 can bind to IGF1R and IGF2R, and has the greatest affinity with IGF2R. When IGF2 binds to IGF2R, it causes IGF2R to be internalized, and IGF2 will be transported to the lysosome for degradation. Therefore, IGF2R is often considered as a decoy receptor for IGF2. ), and the combination of IGF2 and IGF1R exerts mitogen function and promotes cell proliferation and survival 2 . According to the previous part of the study, we found that high-dose IGF2 reflects the function of mitogen and promotes the proliferation and survival of peritoneal macrophages in peritonitis mice, while low-dose IGF2 has no such function.
  • IGF2 In view of the maximum affinity of IGF2 and IGF2R, we hypothesize that low-dose IGF2 preferentially binds to IGF2R, and induces IGF2R-dependent biological effects, such as the occurrence of non-classical mitochondrial dynamics. High-dose IGF2 “depletes” the outer cell membrane IGF2R. There is still a chance to combine with IGF1R to counteract the biological effects of IGF2R, thereby promoting the proliferation and survival of peritoneal macrophages in peritonitis mice.
  • mice In order to study which receptors IGF2 relies on to achieve bidirectional regulation of monocytes, we constructed genetically engineered mice. In igf2r gene knockout mice, we used Crisper-Cas9 technology to target and knock out the 2bp or 13bp base of exon 6 of igf2 gene, causing frameshift mutation to complete ( Figure 4A). In view of the fact that complete deletion of igf2r gene can cause mouse embryonic death, we finally obtained igf2r knockout heterozygous mice-IGF2R +/- mice for breeding and conservation and experiments, and identification of IGF2R +/- mouse progeny genotypes Rely on sequencing alignment (Figure 4B). We induced mouse peritoneal macrophages, and used flow cytometry to detect the expression of IGF2R in these cells to confirm the knockout efficiency (Figure 4C).
  • IGF1R fl/fl mice and IGF2R fl/fl mice were bred with Lyz2 Cre mice respectively, and Lyz2 was expressed at a high level in myeloid cells 18 until IGF1R fl/fl Lyz2 Cre homozygous mice and IGF2R fl/ fl Lyz2 Cre homozygous mice ( Figure 4D, G).
  • the DNA level and protein level identification results verified the knockout efficiency ( Figure 4E, F, H, I).
  • DSS dextran sulfate sodium salt
  • IGF2 inhibits DSS-induced inflammatory bowel disease
  • the Ki67 positive signal of the low-dose IGF2 group was significantly stronger than the control group and the high-dose IGF2 treatment group, which indicates that the basement membrane is near
  • the monocytes and macrophages played a role in the repair of intestinal villi.
  • the macrophages in the colonic basement membrane of mice treated with low-dose IGF2 could better promote colonic villi repair (Figure 5H).
  • low-dose IGF2 can significantly improve the quality of life and survival time of DSS-induced inflammatory bowel disease mice, while high-dose IGF2 has no therapeutic effect and may even aggravate some symptoms.
  • IGF2 treatment of inflammatory bowel disease relies on macrophages
  • macrophages regulated by low-dose IGF2 can alleviate inflammatory bowel disease, while macrophages regulated by high-dose IGF2 aggravate the progression of inflammatory bowel disease.
  • IGF1 aggravates inflammatory bowel disease
  • IGF2 preferentially binds to IGF2R at low doses, and can bind to IGF1R at high doses. And IGF1 has the ability to bind IGF1R, but not IGF2R. In order to prove the regulatory effect of IGF1R activation on the inflammatory response, the use of IGF1 to treat inflammatory bowel disease was studied.
  • IGF2R performs the function of low-dose IGF2 to induce anti-inflammatory macrophages
  • the study uses myeloid cells to specifically knock out IGF2R mice, establishes a peritonitis model, and analyzes the presence or absence of low-dose IGF2 (L-IGF2, 50ng each (Mice) when injected, the expression levels of PD-L1, IL-1 ⁇ and H + in the cytoplasm of peritoneal macrophages.
  • L-IGF2 low-dose IGF2
  • IGF2R performs the anti-inflammatory task of low-dose IGF2
  • IGF1R fl/fl Lyz2 Cre mice that specifically knock out IGF1R are more resistant to inflammatory bowel disease, no matter in the PBS treatment group or high-dose IGF2(H -IGF2, 1000ng per mouse) in the treatment group, the weight of the mice was significantly higher than the weight of the control group IGF1R fl/fl mice under the corresponding treatment conditions, the survival of the mice was significantly prolonged, the stool score and the degree of blood in the stool were significantly reduced, and the colon Inflammatory infiltration was significantly reduced (Figure 10G-L).
  • IGF2 mutants In order to facilitate the study of the functions of IGF2R and IGF1R, many IGF2 mutants have been designed and reported, including IGF2 mutants that can selectively bind to IGF2R.
  • the researchers mutated the tyrosine at position 27 of the IGF2 peptide to leucine to prepare the Leu27-IGF2 mutant (L27-IGF2).
  • the Leu27-IGF2 mutant has the same affinity for IGF2R and IGFBP protein, and has the same affinity with IGF1R. The affinity is extremely reduced.
  • Leu27-IGF2 can significantly reduce the relative pH of the cytoplasm of peritonitis mouse peritoneal monocytes and peritoneal macrophages. Even at a high dose of 2000ng/ml, we also compared Leu27-IGF2 at a low dose. The effect of changing the pH of the cytoplasm eliminates the possibility of the low titer of Leu27-IGF2 ( Figure 11A). In the study of peritonitis, we found that macrophages after treatment with low-dose Leu27-IGF2 and high-dose Leu27-IGF2 both showed significant production of nitric oxide ( Figure 11B) and lactic acid (Figure 11C) in response to LPS stimulation. decline.
  • IGF2 in this part of the study, we verified the therapeutic effect of IGF2 in a mouse model of DSS-induced inflammatory bowel disease. It was confirmed that low-dose IGF2 effectively inhibited inflammatory bowel disease depends on the same mechanism of action as low-dose IGF2 in the treatment of peritonitis. In both validation models, IGF2R-dependent pathways can give macrophages the immune memory that significantly inhibits inflammation. IGF2 mutants target to activate IGF2R and exert the most stable effect of inhibiting inflammation.
  • Human Leu27-IGF2 is an analogue of IGF-2, which replaces tyrosine with leucine at position 27 of the human IGF2 sequence. Compared with IGF2, the binding ability of Leu27-IGF2 with serum IGF-binding proteins (IGFBPs) remains unchanged, but the binding ability with IGF1 receptor (IGF1R) is reduced by 10-20 times. Therefore, Leu27-IGF2 and IGF2 receptor ( The specificity of IGF2R) binding is enhanced.
  • the IGF2 peptide consists of four domains (Domains) A, B, C, and D. The deletion of the D domain (amino acids 62-67) will significantly enhance its affinity with IGFBP2, IGFBP3, IGFBP4 and IGFBP6, and with IGFBP1, The affinity of IGFBP5 remains unchanged.
  • IGF2R Activation of IGF2R promotes its nuclear transport and regulates the cytoplasmic H + concentration of macrophages
  • IGF2 can reduce the distribution of IGF2R on the cell membrane of THP1 cells, showing a dose-dependent characteristic. Starting from a low dose of 5ng/ml to a high dose of 1000ng ml, the reduction of IGF2R on the cell membrane surface is maximized. Continue to increase the dose of IGF2 on the cell membrane surface. IGF2R no longer continued to decrease ( Figure 13A, B). Using western blotting, it is clear that after treatment of THP1 cells with IGF2, the distribution of IGF2R in the nucleus is significantly increased ( Figure 13C). Blocking the nuclear transport of IGF2R with Ivermectin can inhibit the distribution of IGF2R in the nucleus ( Figure 13D).

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Abstract

提供了非IGF1R结合型的物质在预防和/或治疗炎症性疾病中的应用，具体地，提供了一种非IGF1R结合型的物质的用途，用于制备组合物或制剂，所述组合物或制剂用于预防和/或治疗炎症性疾病。

Description

非IGF1R结合型的物质在预防和/或治疗炎症性疾病中的应用 技术领域
本发明涉及生物医药领域，具体地，涉及非IGF1R结合型的物质在预防和/或治疗炎症性疾病中的应用。
背景技术
胰岛素样生长因子2(insulin like growth factor2，IGF2)属于胰岛素样生长因子家族，与其他家族共同参与调控细胞增殖、分化、代谢、衰老和死亡，影响生物体的发育、生长、健康与疾病 1,2。IGF2受体包括IGF1R、IGF2R，其中与IGF2R具有最高亲和力，其次可以与IGF1R结合，几乎不与胰岛素受体结合 2。
目前研究认为，IGF2的主要生物学功能是依赖于激活IGF1R的酪氨酸激酶开启的，而与IGF2R和IR没有正向关联性 2。
约90％的IGF2R分布于细胞内的高尔基体，剩余10％的IGF2R分布于细胞外膜 3。IGF2R不是IGF2的唯一受体，IGF2也不是IGF2R的唯一配体，其配体还包括甘露糖-6-磷酸，在现有报道中，IGF2R的主要功能是将其配体转运至溶酶体，完成蛋白质加工或者降解 4。此外，有研究发现，细胞膜上的IGF2R可以被剪切下来以一种可溶的形式，进入血液以及其他液体环境，有效结合IGF2，抑制IGF2发挥功能 4,5。
自身免疫性疾病是免疫系统异常攻击自身器官和组织细胞所产生的疾病，美国卫生与人类服务部公布的数据显示，目前有2400万美国人——达到7％的美国人口承受着超过八十种的自身免疫性疾病的折磨，几乎涉及到身体的各个部分 6,7。多数自身免疫性疾病的致病因素尚不清楚，可能与遗传、感染、药物和环境等因素有关，病程较长，出现反复的缓解和发作，发病患者中女性多于男性。多数自身免疫性疾病涉及到了由单核前体细胞、单核细胞和巨噬细胞组成的吞噬细胞的参与。
在多种自身免疫性疾病中，活化的巨噬细胞分泌乳脂肪球-表皮生长因子8(Milk fat globule-epidermal growth factor 8，MFG-E8)，MFG-E8的C端的结构域和凋亡细胞的磷脂酰丝氨酸结合，介导巨噬细胞对凋亡细胞的吞噬，清除自身免疫性疾病中受攻击而凋亡的细胞和死亡的免疫细胞碎片，帮助机体 恢复稳态 8。一些炎症因子如脂多糖，会抑制巨噬细胞表达MFG-E8，抑制凋亡碎片清除而加重炎症反应 9。相反的，MFG-E8介导的巨噬细胞吞噬凋亡碎片的事件，又可以抑制脂多糖等对巨噬细胞的促炎作用的调控，包括对p38、ERK1/2、c-JNK、MAPK以及p65的抑制 10。
炎症是机体对于自身的以及外来的致炎因子产生的防御反应，致炎因子包括生物性因子、物理性因子、化学性因子、异物、坏死组织、变态反应和病理变化，炎症反应受到致炎因子和机体自身的调节 11。在炎症性疾病中既有天然免疫的参与，又有适应性免疫的参与，在炎症产生的早期，天然免疫率先到组织损伤部位，影响和决定了后期天然免疫和适应性免疫的反应类型 12。当炎症调节出现紊乱或障碍时，即发生炎症性疾病，失去抵抗刺激和病原体的能力，加速加重疾病病情。按照炎症反应速度，炎症反应包含了急性和慢性的炎症反应。
目前治疗炎症性疾病和自身免疫性疾病的药物有两大类：第一类是甾体抗炎药，即类固醇类药物，如肾上腺皮质激素、雄性激素、雌性激素，具有一定的抗炎作用，长期使用将引起内分泌紊乱等多系统功能障碍 13-15。第二类是非甾体抗炎药包括阿司匹林、对乙酰氨基酚、吲哚美辛、萘普生、萘普酮、双氯芬酸、布洛芬、尼美舒利、罗非昔布、塞来昔布等，在临床上广泛用于骨关节炎、类风湿性关节炎等抗炎治疗 14-16。非甾体抗炎药作用原理单一，主要通过抑制前列腺素的合成，减少缓激肽的形成，减少白细胞和血小板的聚集和凝集来发挥抗炎作用 17。因此，固醇类药物受限于副作用，非固醇类药物则受限于作用通路单一，联合用药增敏效果不佳。
因此，本领域迫切需要开发一种能更有效治疗自身免疫性疾病和/或炎症性疾病的药物。
发明内容
本发明的目的在于提供一种能更有效治疗自身免疫性疾病和/或炎症性疾病的药物。
本发明第一方面提供了一种非IGF1R结合型的物质的用途，用于制备组合物或制剂，所述组合物或制剂用于预防和/或治疗炎症性疾病。
在另一优选例中，所述非IGF1R结合型的物质选自下组：IGF2突变体、表达 IGF2突变体的载体、抗体、小分子化合物、或其组合。
在另一优选例中，所述炎症性疾病选自下组：腹膜炎、炎症性肠病、多发性硬化、糖尿病、系统性红斑狼疮、硬皮病、桥本甲状腺炎、自身免疫性肝炎、自身免疫葡萄膜炎、间质性肺病、银屑病、白癜风、皮肌炎、川崎病、成人耳氏病、强制性脊柱炎、类肉状瘤病、起止点炎相关的关节炎、多关节青少年特发性关节炎、类风湿性关节炎、移植物抗宿主病、自身免疫性胰腺炎、帕金森病、老年痴呆、或其组合。
在另一优选例中，所述非IGF1R结合型的物质包括对IGF2R的选择性或亲和力高于IGF1R的物质，以及可以高效激活IGF2R的不激活或基本不激活IGF1R的物质(如视黄酸等非IGF1R配体)。
在另一优选例中，所述表达IGF2突变体的载体包括病毒载体。
在另一优选例中，所述病毒载体选自下组：腺病毒载体、慢病毒载体、或其组合。
在另一优选例中，所述非IGF1R结合型的物质还包括选自下组的一种或多种物质：血纤维蛋白溶酶原(Plasminogen)、丝甘蛋白聚糖(Serglycin)、e1a刺激基因的细胞抑制因子(Cellular repressor of E1A-stimulated genes，CREG)、硫酸胺酶(Sulfamidase，Sgsh)、磷酸化的β-葡萄糖醛酸酶(Phosphorylated β-glucuronidase，Gusb)等IGF2R有效配体。
在另一优选例中，所述IGF2突变体在野生型IGF2蛋白的对应于SEQ ID NO.:1的第27位的酪氨酸发生突变。
在另一优选例中，所述第27位的酪氨酸突变为选自下组的一种或多种氨基酸：亮氨酸、异亮氨酸、缬氨酸、甲硫氨酸、丙氨酸、苯丙氨酸、丝氨酸、脯氨酸、苏氨酸、组氨酸、赖氨酸、色氨酸、精氨酸、谷氨酸、甘氨酸、天冬氨酸、半胱氨酸。
在另一优选例中，所述第27位的酪氨酸突变为亮氨酸。
在另一优选例中，所述IGF2突变体在野生型IGF2蛋白的对应于SEQ ID NO.:1的第27位的酪氨酸突变为亮氨酸，并任选的删除D-domain(SEQ ID NO.:1的第62位苏氨酸至第67位谷氨酸)。
在另一优选例中，所述IGF2突变体在野生型IGF2蛋白的对应于SEQ ID NO.:1的第12位的谷氨酸发生突变，并任选的删除D-domain(SEQ ID NO.:1的第62位苏氨酸至第67位谷氨酸)。
在另一优选例中，所述第12位的谷氨酸突变为选自下组的一种或多种氨基酸： Asp、Ala、Gln、His、Arg、Lys。
在另一优选例中，所述IGF2突变体在野生型IGF2蛋白的对应于SEQ ID NO.:1的第26位的苯丙氨酸发生突变，并任选的删除D-domain(SEQ ID NO.:1的62位苏氨酸至67位谷氨酸)。
在另一优选例中，所述第26位的苯丙氨酸突变为选自下组的一种或多种氨基酸：Ser、Asp、Ala、Gln、His、Arg、Lys。
在另一优选例中，所述第26位的苯丙氨酸突变为丝氨酸。
在另一优选例中，所述IGF2突变体在野生型IGF2蛋白的对应于SEQ ID NO.:1的第27位的酪氨酸发生突变，并任选的删除D-domain(SEQ ID NO.:1的62位苏氨酸至67位谷氨酸)。
在另一优选例中，所述第27位的酪氨酸突变为选自下组的一种或多种氨基酸：Leu、Asp、Ala、Gln、His、Arg、Lys。
在另一优选例中，所述第27位的酪氨酸突变为亮氨酸。
在另一优选例中，所述IGF2突变体在野生型IGF2蛋白的对应于SEQ ID NO.:1的第43位的缬氨酸发生突变，并任选的删除D-domain(SEQ ID NO.:1的62位苏氨酸至67位谷氨酸)。
在另一优选例中，所述第43位的缬氨酸突变为选自下组的一种或多种氨基酸：Leu、Asp、Ala、Gln、His、Arg、Lys。
在另一优选例中，所述第43位的缬氨酸突变为亮氨酸。
在另一优选例中，所述第27位的酪氨酸突变为选自下组的一种或多种氨基酸：Leu、Asp、Ala、Gln、His、Arg、Lys，且同时将43位的缬氨酸突变为选自下组的一种或多种氨基酸：Leu、Asp、Ala、Gln、His、Arg、Lys。
在另一优选例中，所述第27位的酪氨酸和第43位的缬氨酸同时突变为亮氨酸。
在另一优选例中，所述第27位的酪氨酸和第43位的缬氨酸同时突变为亮氨酸，并任选的删除D-domain(SEQ ID NO.:1的62位苏氨酸至67位谷氨酸)。
在另一优选例中，所述IGF2突变体的氨基酸序列如SEQ ID NO.:2-58中任一所示。
在另一优选例中，所述的IGF2突变体为具有SEQ ID NO.:2-58任一所示氨基酸序列的多肽、其活性片段、或其保守性变异多肽。
在另一优选例中，所述的IGF2突变体除所述突变(如12位Glu，26位Phe，27位Tyr，43位Val以及对应删除D-domain的突变序列)外，其余的氨基酸序列 与SEQ ID NO.:1所示的序列相同或基本相同。
在另一优选例中，所述第12位的谷氨酸突变为谷氨酰胺。
在另一优选例中，所述的基本相同是至多有50个(较佳地为1-20个，更佳地为1-10个、更佳地1-5个)氨基酸不相同，其中，所述的不相同包括氨基酸的取代、缺失或添加(如第62位T至第67位E的缺失)，且所述的IGF2突变体具有抑制炎症的活性。
在另一优选例中，与SEQ ID NO.:1所示序列的同源性至少为80％，较佳地至少为85％或90％，更佳地至少为95％，最佳地至少为98％或99％。
在另一优选例中，所述IGF2蛋白来源于人或非人哺乳动物。
在另一优选例中，所述IGF2突变体选自下组：
(a)具有SEQ ID NO.:2-58任一所示氨基酸序列的多肽；
(b)将SEQ ID NO.:2-58任一所示氨基酸序列经过一个或多个(如2个、3个、4个或5个)氨基酸残基的取代、缺失或添加而形成的，且具有抑制炎症活性的由(a)衍生的多肽。
在另一优选例中，所述的衍生的多肽与SEQ ID NO.:2-58任一所示序列的同源性至少为60％，较佳地至少为70％，更佳地至少为80％，最佳地至少为90％，如95％、97％、99％。
在另一优选例中，所述IGF2突变体为SEQ ID NO.:1所示的野生型的IGF2蛋白经突变形成的。
在另一优选例中，所述组合物或制剂还用于选自下组的一种或多种用途：
(i)减少致病单个核细胞浸润；
(ii)抑制巨噬细胞的致病表型；
(iii)降低巨噬细胞浸润数目；
(iv)提高巨噬细胞细胞质的H +浓度；
(v)提高巨噬细胞氧化磷酸的代谢倾向性；
(vi)赋予巨噬细胞显著抑制炎症的免疫记忆；
(vii)预防和/或治疗自身免疫性疾病；
(viii)用于巨噬细胞参与的多种其他涉及炎症反应的疾病(如与肝纤维化、肾纤维化、肺纤维化等疾病的发生有关的炎症反应，与组织、器官或细胞移植引起的炎症反应，与肺癌、乳腺癌、前列腺癌、肝癌、胰腺癌、黑色素瘤、淋巴瘤等癌症的发生和转移相关的炎症反应，以及与自身免疫性疾病相关的炎症反应)；
(ix)促进移植组织或细胞的植入；
(x)下调IGF2R在THP1细胞上的表达。
在另一优选例中，所述自身免疫性疾病选自下组：多发性硬化、炎症性肠病、自身免疫性肝炎、系统性红斑狼疮、类风湿性关节炎、胰岛素抵抗、糖尿病、自身免疫性肝炎、白癜风、银屑病、腹膜炎、硬皮病、桥本甲状腺炎、移植物抗宿主病、皮肌炎、川崎病、成人耳氏病、强制性脊柱炎、类肉状瘤病、起止点炎相关的关节炎、多关节青少年特发性关节炎、自身免疫性胰腺炎、以及部分未明确定义的自身免疫性疾病、或其组合。
在另一优选例中，所述组合物包括药物组合物。
在另一优选例中，所述组合物包含(a)非IGF1R结合型的物质；和(b)药学上可接受的载体。
在另一优选例中，所述组合物中含有0.001-99wt％，较佳地0.1-90wt％，更佳地1-80wt％的非IGF1R结合型的物质，按组合物的总重量计。
在另一优选例中，所述的组合物为液态制剂或冻干制剂。
在另一优选例中，所述的组合物为注射剂。
本发明第二方面提供了一种细胞制剂，包括：
经过非IGF1R结合型的物质处理的吞噬细胞。
在另一优选例中，所述吞噬细胞选自下组：单核细胞、巨噬细胞、单核细胞前体细胞、或其组合。
在另一优选例中，所述非IGF1R结合型的物质选自下组：IGF2、IGF2突变体、表达IGF2突变体的载体、抗体、小分子化合物、或其组合。
在另一优选例中，所述非IGF1R结合型的物质还包括选自下组的一种或多种物质：血纤维蛋白溶酶原(Plasminogen)、丝甘蛋白聚糖(Serglycin)、e1a刺激基因的细胞抑制因子(Cellular repressor of E1A-stimulated genes，CREG)、硫酸胺酶(Sulfamidase，Sgsh)、磷酸化的β-葡萄糖醛酸酶(Phosphorylated β-glucuronidase，Gusb)等IGF2R有效抑炎配体。
在另一优选例中，所述细胞制剂还包括非IGF1R结合型的物质。
在另一优选例中，所述吞噬细胞具有选自下组的特征：
(a)IGF1R未激活或激活程度低于IGF2R，IGF2R激活；和/或
(b)吞噬细胞以氧化磷酸化为主要获能方式；和/或
(c)具备选自下组的一个或多个特征：
●低表达IL-1β；
●低表达TNFα；
●低表达CXCL10；
●高表达CCL22；
●高表达CLEC7a；
●高表达PD-L1；
●高表达PD-L2；
●高表达IL-10；
●高表达TGFβ；
●细胞质中的H +浓度增加；
●产生一氧化氮(nitric oxide)，乳酸水平下降。
在另一优选例中，所述吞噬细胞来源于骨髓、腹腔、外周血、炎症发生部位、或其组合。
在另一优选例中，所述细胞制剂包括液体制剂。
在另一优选例中，所述的细胞制剂中的细胞基本上(≥90％、95％、96％、97％、98％、99％、99.5％、99.9％)或全部由(a)经过非IGF1R结合型的物质预处理的吞噬细胞和(b)任选的非IGF1R结合型的物质构成。
在另一优选例中，所述的组合物中，所述吞噬细胞的浓度1×10 4-5×10 7/ml，较佳地5×10 4-5×10 6/ml，更佳地1×10 5-1×10 6/ml。
在另一优选例中，所述的载体选自下组：输液剂载体和/或注射剂载体，较佳地，所述的载体是选自下组的一种或多种载体：生理盐水、葡萄糖盐水、或其组合。
在另一优选例中，所述细胞制剂还包括其他预防和/或治疗炎症性疾病的药物。
在另一优选例中，所述其他预防和/或治疗炎症性疾病的药物选自下组：非甾体抗炎药、糖皮质激素、甲氨蝶呤、TNFα中和抗体、TNFR1抗体、TNFR2抗体、抗CD20抗体、IL-1R拮抗剂、IL-12和IL-23p40中和抗体、IL-23p19中和抗体、IL-17中和抗体、IL-17A受体中和抗体、或其组合。
在另一优选例中，所述其他的预防和/或治疗自身免疫性疾病的药物选自下组：非甾体抗炎药、糖皮质激素、甲氨蝶呤、TNFα中和抗体、TNFR1抗体、TNFR2抗体、抗CD20抗体、IL-1R拮抗剂、IL-12和IL-23p40中和抗体、IL-23p19中和抗体、IL-17中和抗体、IL-17A受体中和抗体、CTLA-4融合蛋白、或其组合。
本发明第三方面提供了一种药盒，包括：
(i)第一容器，以及装于该第一容器中的活性成分(a)经过非IGF1R结合型的物质处理的吞噬细胞，或含有活性成分(a)的药物；
(ii)任选的第二容器，以及装于该第二容器中的活性成分(b)非IGF1R结合型的物质，或含有活性成分(b)的药物；和
(iii)说明书，所述说明书中记载了联合给予活性成分(a)和活性成分(b)从而预防和/或治疗炎症性疾病的说明。
在另一优选例中，所述药盒还包括：
(iv)第三容器，以及装于该第三容器中的活性成分(c)其他预防和/或治疗炎症性疾病的药物，或含有活性成分(c)的药物。
在另一优选例中，所述药盒还包括：
(v)第四容器，以及装于该第四容器中的活性成分(d)其他预防和/或治疗自身免疫性疾病的药物，或含其他预防和/或治疗自身免疫性疾病的药物的药物。
在另一优选例中，所述第一容器、第二容器、任选的第三容器、任选的第四容器可以相同，可以不同。
在另一优选例中，所述的第一容器的药物是含经过非IGF1R结合型的物质处理的吞噬细胞的单方制剂。
在另一优选例中，所述的第二容器的药物是含非IGF1R结合型的物质的单方制剂。
在另一优选例中，所述的第三容器是含其他预防和/或治疗炎症性疾病的药物的单方制剂。
在另一优选例中，所述第四容器是含其他预防和/或治疗自身免疫性疾病的药物的单方制剂。
在另一优选例中，所述药物的剂型为注射剂型。
在另一优选例中，所述试剂盒还含有说明书，所述说明书中记载了联合给予活性成分(a)、活性成分(b)、任选的(c)和任选的(d)从而(i)预防和/或治疗炎症性疾病；和/或(ii)预防和/或治疗自身免疫性疾病的说明。
在另一优选例中，所述含有(b)非IGF1R结合型的物质的制剂中，所述非IGF1R结合型的物质的浓度为0.01μg-1mg/kg体重，较佳地0.1-10μg/kg体重，更佳地0.5-5μg/kg体重。
在另一优选例中，所述含有(a)经过非IGF1R结合型的物质处理的吞噬细胞的制剂中，所述吞噬细胞的浓度为0.2*10 4-1*10 8个/kg体重，较佳地1*10 5-5*10 7 个/kg体重，更佳地1*10 6-5*10 6个/kg体重。
在另一优选例中，所述含有(c)其他预防和/或治疗炎症性疾病的药物的制剂中，所述其他预防和/或治疗炎症性疾病的药物的浓度为0.0002-40mg/kg体重，较佳地0.001-30mg/kg体重，更佳地0.02-25mg/kg体重。
在另一优选例中，所述含有(d)其他预防和/或治疗自身免疫性疾病的药物的制剂中，所述其他预防和/或治疗自身免疫性疾病的药物的浓度为0.0002-0.1mg/kg体重，较佳地0.001-0.08mg/kg体重，更佳地0.002-0.01mg/kg体重。
本发明第四方面提供了一种筛药方法，包括步骤：
(a)测试组中，在细胞的培养体系中添加测试物质，并观察所述测试组的细胞中所述测试物质与IGF1R和/或IGF2R的结合活性；在对照组中，在相同细胞的培养体系中不添加测试物质；
其中，如果测试组的细胞中所述测试物质与IGF1R的结合活性降低；并且与IGF2R的结合活性不变或升高，则表明，所述测试物质为非IGF1R结合型的IGF2突变型。
在另一优选例中，所述方法还包括步骤：
(b)对于步骤(a)所获得的非IGF1R结合型的IGF2突变型，进一步测定其对IGFBP蛋白的结合活性；和/或进一步测试其对IGFBP蛋白的结合保持不变还是略有增加。
在另一优选例中，所述方法还包括步骤：
(c)引入一个阳性对照，所述阳性对照为IGF2蛋白或IGF2突变体，将其对IGF1R和/或IGF2R的结合活性与步骤(a)所获得的非IGF1R结合型的IGF2突变型对IGF1R和/或IGF2R的结合活性进行比较。
在另一优选例中，步骤(a)所获得的非IGF1R结合型的IGF2突变型对IGF1R和/或IGF2R的结合活性与所述阳性对照对IGF1R和/或IGF2R的结合活性相当。
在本发明第四方面，还提供了一种筛选预防和/或治疗炎症性疾病的候选物的方法，包括：
(a)测试组中，在细胞的培养体系中添加测试物质，并观察所述测试组的细胞中所述测试物质对IGF2R在细胞表面的表达和/或活性的影响；和/或所述测试物质对IGF2R核转运的影响；和/或所述测试物质对IGF2R向细胞核的重分布的影响；在对照组中，在相同细胞的培养体系中不添加测试物质；
其中，如果测试组的所述测试物质抑制IGF2R在细胞表面的表达和/或活性；和/或所述测试物质增加IGF2R的核转运；和/或所述测试物质促进IGF2R向细胞核的重分布，则表明，所述测试物质为预防和/或治疗炎症性疾病的候选物。
在另一优选例中，所述的候选物具有IGF2R选择性的候选物；即所述候选物对IGF2R的选择性大于对IGF1R的选择性。
在另一优选例中，所述的候选物选择性地和/或优先结合于IGF2R，而不结合或基本不结合于IGF1R。
在另一优选例中，所述细胞为体外培养的细胞。
在另一优选例中，所述细胞为吞噬细胞。
在另一优选例中，所述吞噬细胞选自下组：单核细胞、巨噬细胞、单核细胞前体细胞、或其组合。
在另一优选例中，所述方法为体外方法。
在另一优选例中，所述的方法是非诊断和非治疗性的。
本发明第五方面提供了一种筛药方法(或筛选候选物的方法)，包括：
(a)测试组中，在细胞的培养体系中添加测试物质，并观察所述测试组的细胞中所述测试物质对IGF2与IGF1R和/或IGF2R的结合活性的影响；在对照组中，在相同细胞的培养体系中不添加测试物质；
其中，如果测试组的细胞中所述测试物质抑制IGF2与IGF1R的结合活性；并且对IGF2与IGF2R的结合活性无影响或有促进作用，则表明，所述测试物质为非IGF1R结合型的物质(或候选物)。
在另一优选例中，所述非IGF1R结合型的物质选自下组：抗体、小分子化合物、或其组合。
在另一优选例中，所述方法还包括步骤：
(b)对于步骤(a)所获得的非IGF1R结合型的物质，进一步测定其对IGFBP蛋白的结合活性；和/或进一步测试其对IGFBP蛋白的结合保持不变还是略有增加。
在另一优选例中，所述方法还包括步骤：
(c)引入一个阳性对照，所述阳性对照为IGF2蛋白或IGF2突变体，将其对IGF1R和/或IGF2R的结合活性与步骤(a)所获得的非IGF1R结合型的物质对IGF1R和/或IGF2R的结合活性进行比较。
在另一优选例中，步骤(a)所获得的非IGF1R结合型的物质对IGF1R和/或 IGF2R的结合活性与所述阳性对照对IGF1R和/或IGF2R的结合活性相当。
在另一优选例中，所述细胞为体外培养的细胞。
在另一优选例中，所述细胞为吞噬细胞。
在另一优选例中，所述吞噬细胞选自下组：单核细胞、巨噬细胞、单核细胞前体细胞、或其组合。
在另一优选例中，所述方法为体外方法。
在另一优选例中，所述的方法是非诊断和非治疗性的。
本发明第六方面提供了一种体外获得促炎样本和抑炎样本的方法，包括：
在第一组实验中，用低剂量的IGF2进行处理，获得抑炎样本；和
在第二组实验中，用高剂量的IGF2进行处理，获得促炎样本。
在另一优选例中，所述促炎样本中，进一步测定IGF2对IGF2R的结合活性。
在另一优选例中，所述抑炎样本中，进一步测定IGF2对IGF2R的结合活性。
本发明第七方面提供了一种组合物，包括：
(a)由本发明第四方面或第五方面所述的方法获得的非IGF1R结合型的IGF2突变型或候选物；其中所述非IGF1R结合型的IGF2突变型不包括选自下组的IGF2突变型：野生型IGF2、野生型IGF2第25～91位氨基酸片段、12位谷氨酸突变为Asp、Ala、Gln、His、Arg、或Lys的IGF2突变型、26位苯丙氨酸突变为Ser的IGF2突变型、27位酪氨酸突变为Leu的IGF2突变型、43位缬氨酸突变为Leu的IGF2突变型、删除D-domain的IGF2突变型、或其组合。
在另一优选例中，所述非IGF1R结合型的IGF2突变型不包括SEQ ID NO.:1-2、16、18、20、22、24、26、28、43中任一所示的IGF2突变型。
在另一优选例中，所述非IGF1R结合型的IGF2突变型的氨基酸序列如SEQ ID NO.:3-15、17、19、21、23、25、27、29-42、44-58中任一所示。
本发明第八方面提供了一种抑制炎症活性的方法，包括步骤：
在非IGF1R结合型的物质(或具有IGF2R选择性的候选物)存在下，培养吞噬细胞，从而抑制炎症活性。
在另一优选例中，所述非IGF1R结合型的物质的施用浓度为0.0000005-0.0005mg/ml，较佳地，0.000001-0.0001mg/ml，更佳地，0.000005-0.00005mg/ml。
本发明第九方面提供了一种预防和/或治疗炎症性疾病的方法，包括：
(a)给需要的对象施用本发明第二方面所述的细胞制剂或本发明第三方面所述的药盒或本发明第七方面所述的组合物。
在另一优选例中，所述的对象包括非人哺乳动物和人。
在另一优选例中，所述非IGF1R结合型的物质的施用剂量为0.01μg-1mg/kg体重，较佳地0.1-10μg/kg体重，更佳地0.5-5μg/kg体重。
在另一优选例中，所述非IGF1R结合型的物质的施用频率为1次/天-2次/天。
在另一优选例中，所述的施用包括同时施用或先后施用。
本发明第十方面提供了一种预防和/或治疗自身免疫性疾病的方法，包括：
(a)给需要的对象施用本发明第二方面所述的细胞制剂或本发明第三方面所述的药盒或本发明第七方面所述的组合物。
在另一优选例中，所述的对象包括非人哺乳动物和人。
在另一优选例中，所述非IGF1R结合型的物质的施用剂量为0.01μg-1mg/kg体重，较佳地0.1-10μg/kg体重，更佳地0.5-5μg/kg体重。
在另一优选例中，所述非IGF1R结合型的物质的施用频率为1次/天-2次/天。
在另一优选例中，所述的施用包括同时施用或先后施用。
本发明第十一方面提供了一种本发明第二方面所述的细胞制剂、本发明第三方面所述的药盒或本发明第七方面所述的组合物的用途，用于制备用于预防和/或治疗炎症性疾病的药物。
应理解，在本发明范围内中，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，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了低剂量IGF2抑制腹膜炎。分别使用低剂量IGF2(L-IGF2，5-50ng IGF2每只小鼠)和高剂量IGF2(H-IGF2，1000ng IGF2每只小鼠)处理腹膜炎小鼠。分离腹膜炎小鼠腹腔单核细胞和巨噬细胞并分析每只小鼠可以分离到的腹腔单核细胞和巨噬细胞的数目。实验重复三次，显著性差异由student’s t检验完成。**P<0.01；***P<0.001。
图2显示了低剂量IGF2改变了巨噬细胞能量代谢偏好和炎症因子表达趋势和潜力。分别使用低剂量IGF2(L-IGF2，5-50ng IGF2每只小鼠)和高剂量IGF2(H-IGF2，1000ng IGF2每只小鼠)处理腹膜炎小鼠。分离腹膜炎小鼠腹腔 巨噬细胞并分析。A-B，低剂量IGF2增强巨噬细胞氧化磷酸化潜能，而高剂量IGF2抑制氧化磷酸化。C，在有无脂多糖(Lipopolysaccharide，LPS)刺激时，低剂量IGF2降低巨噬细胞乳酸产生量，对应较弱的有氧糖酵解代谢。D，低剂量IGF2抑制LPS诱导促炎基因(Cxcl10、IL-18、Tnfa、Cd40)表达，E，低剂量IGF2促进IL-4和IL-13诱导抗炎修复相关基因(Retnla、Ym1、Ccl22、Clec7a)表达。实验重复三次，显著性差异由student’s t检验完成。*P<0.05；**P<0.01；***P<0.001。
图3显示了低剂量IGF2提高单核细胞(maturing)和巨噬细胞(mature)细胞质H +浓度，降低溶酶体H +浓度。分别使用低剂量IGF2(L-IGF2，5-50ng IGF2每只小鼠)和高剂量IGF2(H-IGF2，1000ng IGF2每只小鼠)处理腹膜炎小鼠。分离腹膜炎小鼠腹腔单核细胞和巨噬细胞并分析。A，流式检测IGF2调控腹腔单核细胞和巨噬细胞JC1的荧光强度，评价相对线粒体膜电位。B，流式检测IGF2调控腹腔单核细胞和巨噬细胞细胞质pH值。C，流式检测IGF2调控腹腔巨噬细胞溶酶体相对pH值。D，IGF2调节腹腔巨噬细胞的溶酶体V型ATP酶的绝对活力。
图4显示了构建igf2r基因缺陷小鼠，以及igf1r或igf2r条件性敲除小鼠。A-C，利用CRISPR-Cas9技术，通过造成2bp或者13bp基因缺失引起的移码突变，敲低IGF2R基因表达(IGF2R基因完全敲除，小鼠胚胎致死)，利用基因测序技术鉴定子代小鼠，利用流式细胞术鉴定敲低效率。D-I，通过杂交IGF1R fl/fl或者IGF2R fl/fl小鼠和Lyz Cre小鼠，获得特异性敲除髓系细胞IGF1R或者IGF2R的小鼠。敲除效率根据PCR和流式分析鉴定。E，琼脂糖凝胶电泳结果，泳道1指示野生型小鼠(124bp)，泳道2指示Igf1r fl/fl(220bp)，泳道3无条带，指示Igf1r fl/fl小鼠不具备Lyz2 Cre活力，泳道4(320bp)指示Igf1r fl/fl小鼠具备Lyz2 Cre活力。H，泳道1指示野生型小鼠(2000bp)，泳道2指示Igf2r fl/fl小鼠，不具备Lyz2 Cre活力(2200bp)。
图5显示了低剂量IGF2抑制葡聚糖硫酸钠(dextran sulfate sodium salt，DSS)诱导的炎症性肠病。通过饲饮4％DSS溶液诱导肠炎小鼠模型。A-E，低剂量IGF2缓解DSS诱导的炎症性肠病，表现为体重指数、生存时间、粪便评分和粪便出血评分以及结肠长度的明显改善。F-H，IGF2用于治疗DSS诱导结肠炎小鼠时，小鼠结肠组织的H&E染色、TUNEL和Ki67免疫组织化学染色。实验重复三次，显著性差异由student’s t检验完成。*P<0.05；**P<0.01； ***P<0.001。
图6显示了低剂量IGF2处理的巨噬细胞可以有效抑制炎症性肠病。给予小鼠饮用4％DSS溶液，诱导炎症性肠病小鼠模型。A-B，低剂量或高剂量IGF2诱导的腹腔单核/巨噬细胞(peritoneal macrophages,PM)在DSS诱导的炎症性肠病中的治疗作用，体重改变和生存曲线被记录。实验重复三次，显著性差异由student’s t检验完成。**P<0.01。
图7显示了低剂量IGF2促进抗炎特性巨噬细胞形成。给予小鼠饮用4％DSS溶液，诱导炎症性肠病小鼠模型。A，低剂量IGF2改善DSS诱导的炎症性肠病，减少结肠组织中单个核细胞的浸润。B-C，分析IGF2对炎症性肠病小鼠的结肠中浸润的巨噬细胞(CD11b +F4/80 +)的表达白介素-1β(IL-1β)的影响。D，分析IGF2对炎症性肠病小鼠的结肠中浸润的巨噬细胞(CD11b +F4/80 +)上PD-L1表达水平的影响。实验重复三次，显著性差异由student’s t检验完成。***P<0.001。
图8显示了IGF1加重炎症性肠病进展。给予小鼠饮用4％DSS溶液，诱导炎症性肠病小鼠模型。给予小鼠饮用4％DSS溶液，诱导炎症性肠病小鼠模型。在疾病诱导的第1天、第3天、第5天，给予炎症性肠病小鼠注射低剂量IGF1(L-IGF1，50ng/只小鼠)、高剂量IGF1(H-IGF1，1000ng/只小鼠)或PBS。A，观察小鼠存活；B，测量结肠长度变化；C，利用流式细胞分析技术检测结肠固有层中巨噬细胞表达IL-1β的比例情况；D，利用流式细胞分析技术检测结肠固有层中巨噬细胞表达PD-L1的水平；E-G，给予腹膜炎小鼠注射L-IGF1、H-IGF1或PBS，从腹腔灌洗液中分离巨噬细胞，在LPS刺激下，分析IGF1对巨噬细胞一氧化氮(nitric oxide)分泌、乳酸(lactic acid)产生和葡萄糖消耗的影响。
图9显示了IGF2R执行低剂量IGF2对巨噬细胞抗炎功能的调控作用。给予野生型(WT)小鼠和IGF2RCKO(IGF2R fl/flLyz2 Cre)小鼠诱导腹膜炎，腹腔注射IGF2(50ng/只小鼠)或PBS，48小时后从腹腔灌洗液中分离巨噬细胞。A，流式分析巨噬细胞上PD-L1的表达情况；B，流式分析巨噬细胞中IL-1β的表达情况。给予小鼠诱导腹膜炎，分别注射低剂量IGF2或高剂量IGF2，同时给予IGF2R抗体，C，流式分析巨噬细胞细胞质中H +浓度。
图10显示了IGF2R执行低剂量IGF2的抗炎任务。给予野生型((Lyz2Cre)小鼠、IGF2RCKO(IGF2R fl/flLyz2 Cre)小鼠、IGF1RCKO(IGF1R fl/flLyz2 Cre)小鼠饮用 4％DSS溶液，诱导肠炎小鼠模型。A-F，评价低剂量IGF2对IGF2R fl/flLyz2 Cre小鼠和IGF2R fl/fl小鼠中诱导的炎症性肠病的影响。A，小鼠体重变化；B，小鼠存活；C，粪便评分；D，便血评分；E，结肠组织病理学分析；F，单个核细胞在结肠固有层中的浸润情况。G-L，评价高剂量IGF2对IGF1R fl/flLyz2 Cre小鼠和IGF1R fl/fl小鼠中诱导的炎症性肠病的影响。G，小鼠体重变化；H，小鼠存活；I，粪便评分；J，便血评分；K，结肠组织病理学分析；L，单个核细胞在结肠固有层中的浸润情况。实验重复三次，显著性差异由student’s t检验完成。*P<0.05；**P<0.01；***P<0.001。
图11显示了Leu27-IGF2靶向激活IGF2R最优效抑制腹膜炎和炎症性肠病。分别使用低剂量IGF2或Leu27-IGF2(50ng IGF2/Leu27-IGF2每只小鼠)和高剂量IGF2或Leu27-IGF2(1000ng，或者2000ng IGF2/Leu27-IGF2每只小鼠)处理腹膜炎小鼠。分离腹膜炎小鼠腹腔单核细胞和巨噬细胞并分析。A，Leu27-IGF2明显增加巨噬细胞细胞质中H +浓度。B-C，不同剂量IGF2和Leu27-IGF2处理下腹腔巨噬细胞产生一氧化氮(nitric oxide)和乳酸的能力。D-E，不同剂量IGF2和Leu27-IGF2处理下腹腔巨噬细胞在LPS刺激下，表达IL-1β和PD-L1的水平。给予小鼠饮用4％DSS溶液，诱导炎症性肠病模型。F，体重指数评价Leu27-IGF2对DSS诱导的炎症性肠病的缓解作用。G，小鼠存活情况评价Leu27-IGF2对DSS诱导的炎症性肠病的治疗作用。H-J，粪便评分、便血程度、结肠长度评价Leu27-IGF2对DSS诱导的炎症性肠病的治疗作用。K，野生型IGF2和Leu27-IGF2缓解Ftg诱导的腹膜炎和DSS诱导的炎症性肠病的有效阈值示意图。实验重复三次，显著性差异由student’s t检验完成。*P<0.05；**P<0.01；***P<0.001。
图12显示了Leu27-Des(62-67)-IGF2抑制炎症性肠病。给予小鼠饮用4％DSS溶液，诱导炎症性肠病模型，并在疾病诱导的第1天开始注射低剂量和高剂量Leu27-Des(62-67)-IGF2。A，监测小鼠体重变化；B，炎症性肠病小鼠存活情况，评价Leu27-Des(62-67)IGF2对炎症性肠病的治疗作用。显著性差异由student’s t检验完成。*P<0.05；***P<0.001。
图13显示了激活IGF2R促进核分布，调控巨噬细胞细胞质pH值。利用不同剂量IGF2处理THP1细胞，A-B，流式细胞分析技术检测细胞膜表面IGF2R的存在量。C，Western blotting检测IGF2R在总体细胞水平和细胞核中的表达情况。利用蛋白质核转录抑制剂——ivermectin(5μM)处理不同剂量IGF2 处理的骨髓来源单核前体细胞，在巨噬细胞成熟后，D，Western blotting检测IGF2R在总体细胞水平和细胞核中的表达情况。E，流式细胞分析技术检测巨噬细胞细胞质中H +浓度。利用不同剂量IGF2或Leu27-Des(62-67)-IGF2处理THP1细胞，F，流式细胞分析技术检测细胞膜表面IGF2R的表达量。显著性差异由student’s t检验完成。***P<0.001。
具体实施方式
本发明人经过广泛而深入的研究，首次意外地发现，非IGF1R结合型的物质可显著(a)预防和/或治疗炎症性疾病；和/或(b)预防和/或治疗自身免疫性疾病。在此基础上，本发明人完成了本发明。
如本文所用，野生型IGF2、野生型IGF2第25～91位氨基酸片段、12位谷氨酸突变为Asp、Ala、Gln、His、Arg、或Lys的IGF2突变型、26位苯丙氨酸突变为Ser的IGF2突变型、27位酪氨酸突变为Leu的IGF2突变型、43位缬氨酸突变为Leu的IGF2突变型、删除D-domain的IGF2突变型均为在SEQ ID NO.:1的基础上进行的氨基酸的截取、突变和/或删除。
IGF2蛋白及其突变体
胰岛素样生长因子2(insulin like growth factor2，IGF2)属于胰岛素样生长因子家族，与其他家族共同参与调控细胞增殖、分化、代谢、衰老和死亡，影响生物体的发育、生长、健康与疾病 1,2。IGF2受体包括IGF1R、IGF2R，其中与IGF2R具有最高亲和力，其次可以与IGF1R结合，几乎不与胰岛素受体结合 2。
IGF2与IGF1在氨基酸序列上具有高度的同源性，存在62％的重叠序列，因此，IGF2常常可以体现出IGF1所能激活的生物学效应 2。目前研究认为，IGF2的主要生物学功能是依赖于激活IGF1R(insulin like growth factor 1 receptor)的酪氨酸激酶开启的，而与IGF2R(insulin like growth factor 2 receptor)和IR没有正向关联性。从进化的角度看，IGF1R与IR具有基因的同源性，而且，IGF1R与IR下游激活的信号通路是十分相似的。然而IGF2R的基因则与IGF1R或者IR基因的来源完全不同，因而，IGF2R常常被认为具有与IGF1R、IR相反的生物学功能，IGF2R作为IGF2的降解受体，通过内化降解IGF2 来抑制IGF2对于IGF1R的激活作用。
约90％的IGF2R分布于细胞内的高尔基体，剩余10％的IGF2R分布于细胞外膜 3。IGF2R不是IGF2的唯一受体，IGF2也不是IGF2R的唯一配体，其配体还包括甘露糖-6-磷酸，在现有报道中，IGF2R的主要功能是将其配体转运至溶酶体，完成蛋白质加工或者降解 4。此外，细胞膜上的IGF2R可以被剪切下来以一种可溶的形式，进入血液以及其他液体环境，有效结合IGF2，抑制IGF2发挥功能 4,5。本发明的前期研究发现IGF2可以抑制多发性硬化和溃疡性结肠炎等疾病的动物模型，但是其具体调控机制仍不清楚。
具体地，本发明涉及一种IGF2蛋白及其变体，在本发明的一个优选例中，所述IGF2蛋白的氨基酸序列如SEQ ID NO.:1所示。本发明的IGF2蛋白或表达IGF2蛋白的载体可(a)预防和/或治疗炎症性疾病。
本发明的IGF2蛋白或表达IGF2蛋白的载体还可用于选自下组的一种或多种用途：
(i)减少致病单个核细胞浸润；
(ii)抑制巨噬细胞的致病表型；
(iii)降低巨噬细胞浸润数目；
(iv)提高巨噬细胞细胞质的H +浓度；
(v)提高巨噬细胞氧化磷酸的代谢倾向性；
(vi)赋予巨噬细胞显著抑制炎症的免疫记忆；
(vii)预防和/或治疗自身免疫性疾病；
(viii)用于巨噬细胞参与的多种其他涉及炎症反应的疾病(如与肝纤维化、肾纤维化、肺纤维化等疾病的发生有关的炎症反应，与组织、器官或细胞移植引起的炎症反应，与肺癌、乳腺癌、前列腺癌、肝癌、胰腺癌、黑色素瘤、淋巴瘤等癌症的发生和转移相关的炎症反应，以及与自身免疫性疾病相关的炎症反应)；
(ix)促进移植组织或细胞的植入；
(x)下调IGF2R在THP1细胞上的表达。
本发明还包括与本发明的SEQ ID NO.:1所示序列具有50％或以上(优选60％以上，70％以上，80％以上，更优选90％以上，更优选95％以上，最优选98％以上，如99％)同源性的具有相同或相似功能的多肽或蛋白。
其中，SEQ ID NO.:1为人源IGF2蛋白(AYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPAKSE)。
所述“相同或相似功能”主要是指：“(a)预防和/或治疗炎症性疾病；和/或(i)减少致病单个核细胞浸润；和/或(ii)抑制巨噬细胞的致病表型；和/或(iii)降低巨噬细胞浸润数目；和/或(iv)提高巨噬细胞细胞质的H +浓度；和/或(v)提高巨噬细胞氧化磷酸的代谢倾向性；和/或(vi)赋予巨噬细胞显著抑制炎症的免疫记忆；和/或(vii)预防和/或治疗自身免疫性疾病；和/或(viii)用于巨噬细胞参与的多种其他涉及炎症反应的疾病(如与肝纤维化、肾纤维化、肺纤维化等疾病的发生有关的炎症反应，与组织、器官或细胞移植引起的炎症反应，与肺癌、乳腺癌、前列腺癌、肝癌、胰腺癌、黑色素瘤、淋巴瘤等癌症的发生和转移相关的炎症反应，以及与自身免疫性疾病相关的炎症反应)；和/或(ix)促进移植组织或细胞的植入；和/或(x)下调IGF2R在THP1细胞上的表达”。
本发明的蛋白可以是重组蛋白、天然蛋白、合成蛋白。本发明的蛋白可以是天然纯化的产物，或是化学合成的产物，或使用重组技术从原核或真核宿主(例如，细菌、酵母、高等植物、昆虫和哺乳动物细胞)中产生。根据重组生产方案所用的宿主，本发明的蛋白可以是糖基化的，或可以是非糖基化的。本发明的蛋白还可包括或不包括起始的甲硫氨酸残基。
本发明还包括具有IGF2蛋白活性的IGF2蛋白片段和类似物。如本文所用，术语“片段”和“类似物”是指基本上保持本发明的天然IGF2蛋白相同的生物学功能或活性的蛋白。
在一优选实施方式中，本发明的IGF2蛋白的突变体在野生型IGF2蛋白的对应于SEQ ID NO.:1(AYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPAKSE)的第27位的酪氨酸发生突变，并且本发明的IGF2蛋白的突变体可显著(a)预防和/或治疗炎症性疾病；和/或(b)预防和/或治疗自身免疫性疾病。
优选的，所述第27位的酪氨酸突变为亮氨酸。
优选的，所述第27位的酪氨酸突变为亮氨酸，并任选的删除D-domain(SEQ ID NO.:1的第62位苏氨酸至第67位谷氨酸)。
在一优选实施方式中，所述的IGF2突变体为具有SEQ ID NO.:2-58任一所示氨基酸序列的多肽、其活性片段、或其保守性变异多肽。
应理解，本发明突变体中的氨基酸编号基于SEQ ID NO.:1作出，当某一突变体与SEQ ID NO.:1所示序列的同源性达到80％或以上时，突变体的氨基酸编号可能会有相对于SEQ ID NO.:1)的氨基酸编号的错位，如向氨基酸的N 末端或C末端错位1-5位，而采用本领域常规的序列比对技术，本领域技术人员通常可以理解这样的错位是在合理范围内的，且不应当由于氨基酸编号的错位而使同源性达80％(如90％、95％、98％)的、具有相同或相似的可显著(a)预防和/或治疗炎症性疾病；和/或(i)减少致病单个核细胞浸润；和/或(ii)抑制巨噬细胞的致病表型；和/或(iii)降低巨噬细胞浸润数目；和/或(iv)提高巨噬细胞细胞质的H +浓度；和/或(v)提高巨噬细胞氧化磷酸的代谢倾向性；(vi)赋予巨噬细胞显著抑制炎症的免疫记忆；和/或(vii)预防和/或治疗自身免疫性疾病；和/或(viii)用于巨噬细胞参与的多种其他涉及炎症反应的疾病(如与肝纤维化、肾纤维化、肺纤维化等疾病的发生有关的炎症反应，与组织、器官或细胞移植引起的炎症反应，与肺癌、乳腺癌、前列腺癌、肝癌、胰腺癌、黑色素瘤、淋巴瘤等癌症的发生和转移相关的炎症反应，以及与自身免疫性疾病相关的炎症反应)；和/或(ix)促进移植组织或细胞的植入；和/或(x)下调IGF2R在THP1细胞上的表达的活性不在本发明突变体的范围内。
本发明的突变蛋白片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的突变蛋白，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的突变蛋白，或(iii)成熟突变蛋白与另一个化合物(比如延长突变蛋白半衰期的化合物，例如聚乙二醇)融合所形成的突变蛋白，或(iv)附加的氨基酸序列融合到此突变蛋白序列而形成的突变蛋白(如前导序列或分泌序列或用来纯化此突变蛋白的序列或蛋白原序列，或与抗原IgG片段的形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。本发明中，保守性替换的氨基酸最好根据表I进行氨基酸替换而产生。
表I
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
本发明还包括与本发明的天然IGF2蛋白具有50％或以上(优选60％以上，70％以上，80％以上，更优选90％以上，更优选95％以上，最优选98％以上，如99％)同源性的具有相同或相似功能的多肽或蛋白。在蛋白质变体可以经过若干个(通常为1-60个，较佳地1-30个，更佳地1-20个，最佳地1-10个)取代、缺失或添加至少一个氨基酸所得的衍生序列，以及在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在所述蛋白中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能，在C末端和/或\末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。本发明包括天然IGF2蛋白类似物与天然IGF2蛋白的差别可以是氨基酸序列上的差异，也可以是不影响序列的修饰形式上的差异，或者兼而有之。这些蛋白的类似物包括天然或诱导的遗传变异体。诱导变异体可以通过各种技术得到，如通过辐射或暴露于诱变剂而产生随机诱变，还可通过定点诱变法或其他已知分了生物学的技术。类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物，以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解，本发明的蛋白并不限于上述例举的代表性蛋白。
修饰(通常不改变一级结构)形式包括：体内或体外蛋白的化学衍生形式如 乙酸化或羧基化。修饰还包括糖基化，如那些在蛋白质合成和加工中进行糖基化修饰。这种修饰可以通过将蛋白暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。此外，还可以对本发明突变蛋白进行修饰。修饰(通常不改变一级结构)形式包括：体内或体外的突变蛋白的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化，如那些在突变蛋白的合成和加工中或进一步加工步骤中进行糖基化修饰而产生的突变蛋白。这种修饰可以通过将突变蛋白暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的突变蛋白。
本发明还提供了编码IGF2蛋白的多核苷酸序列。本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括：DNA、基因组DNA或人工合成的DNA，DNA可以是单链的或是双链的。编码成熟多肽的多核苷酸包括：只编码成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。本发明还涉及上述多核苷酸的变异体，其编码与本发明有相同的氨基酸序列的多肽的片段、类似物和衍生物。此多核苷酸的变异体可以是天然发生的等位变异体或非天然发生的变异体。这些核苷酸变异体包括取代变异体、缺失变异体和插入变异体。如本领域所知的，等位变异体是一个多核苷酸的替换形式，它可能是一个或多个核苷酸的取代、缺失或插入，但不会从实质上改变其编码的多肽的功能。
根据本文所述的核苷酸序列，本技术领域人员可方便地用各种已知方法制得本发明的编码核酸。这些方法例如但不限于：PCR，DNA人工合成等，具体的方法可参见J.萨姆布鲁克，《分子克隆实验指南》。作为本发明的一种实施方式，可通过分段合成核苷酸序列再进行重叠延伸PCR的方法来构建本发明的编码核酸序列。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严格条件(或严紧条件)下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC，0.1％SDS，60℃；或(2)杂交时加有变性剂，如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等； 或(3)仅在两条序列之间的相同性至少在90％以上，更好是95％以上时才发生杂交。
本发明的蛋白和多核苷酸优选以分离的形式提供，更佳地，被纯化至均质。
本发明多核苷酸全长序列通常可以通过PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法，可根据本发明所公开的有关核苷酸序列，尤其是开放阅读框序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增而得有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按正确次序拼接在一起。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。
目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
应用PCR技术扩增DNA/RNA的方法被优选用于获得本发明的多核苷酸。特别是很难从文库中得到全长的cDNA时，可优选使用RACE法(RACE-cDNA末端快速扩增法)，用于PCR的引物可根据本文所公开的本发明的序列信息适当地选择，并可用常规方法合成。可用常规方法如通过凝胶电泳分离和纯化扩增的DNA/RNA片段。
表达载体
本发明也涉及包含本发明的多核苷酸的载体，以及用本发明的载体或本发明突变蛋白编码序列经基因工程产生的宿主细胞，以及经重组技术产生本发明所述多肽的方法。
通过常规的重组DNA技术，可利用本发明的多聚核苷酸序列可用来表达或生产重组的突变蛋白。一般来说有以下步骤：
(1).用本发明的编码本发明突变蛋白的多核苷酸(或变异体)，或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；
(2).在合适的培养基中培养的宿主细胞；
(3).从培养基或细胞中分离、纯化蛋白质。
本发明中，编码突变蛋白的多核苷酸序列可插入到重组表达载体中。术语 “重组表达载体”指本领域熟知的细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒或其他载体。只要能在宿主体内复制和稳定，任何质粒和载体都可以用。表达载体的一个重要特征是通常含有复制起点、启动子、标记基因和翻译控制元件。
本领域的技术人员熟知的方法能用于构建含本发明突变蛋白编码DNA序列和合适的转录/翻译控制信号的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。这些启动子的代表性例子有：大肠杆菌的lac或trp启动子；λ噬菌体PL启动子；真核启动子包括CMV立即早期启动子、HSV胸苷激酶启动子、早期和晚期SV40启动子、反转录病毒的LTRs和其他一些已知的可控制基因在原核或真核细胞或其病毒中表达的启动子。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。
此外，表达载体优选地包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状，如真核细胞培养用的二氢叶酸还原酶、新霉素抗性以及绿色荧光蛋白(GFP)，或用于大肠杆菌的四环素或氨苄青霉素抗性。
包含上述的适当DNA序列以及适当启动子或者控制序列的载体，可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞；真菌细胞如酵母、植物细胞(如人参细胞)。
本发明的多核苷酸在高等真核细胞中表达时，如果在载体中插入增强子序列时将会使转录得到增强。增强子是DNA的顺式作用因子，通常大约有10到300个碱基对，作用于启动子以增强基因的转录。可举的例子包括在复制起始点晚期一侧的100到270个碱基对的SV40增强子、在复制起始点晚期一侧的多瘤增强子以及腺病毒增强子等。
本领域一般技术人员都清楚如何选择适当的载体、启动子、增强子和宿主细胞。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的 DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔、脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
非IGF1R结合型的物质
如本文所用，术语“非IGF1R结合型”、“IGF2R选择型”或“IGF2R选择性”可互换使用，指所述物质对IGF2R的选择性大于对IGF1R的选择性，即选择性地和/或优先结合于IGF2R，而不结合或基本不结合于IGF1R。
在本发明中，非IGF1R结合型的物质包括对IGF2R的选择性或亲和力高于IGF1R的物质，以及可以高效激活IGF2R的不激活IGF1R的物质(如视黄酸等非IGF1R配体)。在一优选实施方式中，所述非IGF1R结合型的物质对IGF2R的亲和力或选择性高于其对IGF1R的亲和力或选择性。在一优选实施方式中，所述非IGF1R结合型的物质选自下组：IGF2突变体、表达IGF2突变体的载体、抗体、小分子化合物、或其组合。
在一优选实施方式中，所述非IGF1R结合型的物质还包括选自下组的一种或多种物质：血纤维蛋白溶酶原(Plasminogen)、丝甘蛋白聚糖(Serglycin)、e1a刺激基因的细胞抑制因子(Cellular repressor of E1A-stimulated genes，CREG)、硫酸胺酶(Sulfamidase，Sgsh)、磷酸化的β-葡萄糖醛酸酶(Phosphorylated β-glucuronidase，Gusb)等IGF2R有效抑炎配体。
在本发明中，首次意外的发现，非IGF1R结合型的物质可显著(a)预防和/或治疗炎症性疾病；和/或(i)减少致病单个核细胞浸润；和/或(ii)抑制巨噬细胞的致病表型；和/或(iii)降低巨噬细胞浸润数目；和/或(iv)提高巨噬细胞细胞 质的H +浓度；和/或(v)提高巨噬细胞氧化磷酸的代谢倾向性；和/或(vi)赋予巨噬细胞显著抑制炎症的免疫记忆；和/或(vii)预防和/或治疗自身免疫性疾病；和/或(viii)用于巨噬细胞参与的多种其他涉及炎症反应的疾病(如与肝纤维化、肾纤维化、肺纤维化等疾病的发生有关的炎症反应，与组织、器官或细胞移植引起的炎症反应，与肺癌、乳腺癌、前列腺癌、肝癌、胰腺癌、黑色素瘤、淋巴瘤等癌症的发生和转移相关的炎症反应，以及与自身免疫性疾病相关的炎症反应)；和/或(ix)促进移植组织或细胞的植入；和/或(x)下调IGF2R在THP1细胞上的表达。
复方药物组合物和药盒
本发明提供了含有活性成分(a)经过非IGF1R结合型的物质处理的吞噬细胞；(b)任选的非IGF1R结合型的物质；以及(c)药学上可接受的载体的复方药物组合物。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、粉剂、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。诸如片剂和胶囊之类的药物组合物，可通过常规方法进行制备。药物组合物如针剂、溶液、片剂和胶囊宜在无菌条件下制造。本发明的药物组合也可以被制成粉剂用于雾化吸入。本发明的药物组合物的剂型为注射剂。活性成分的给药量是治疗有效量。本发明的药物制剂还可制成缓释制剂。本发明的药物组合物优选为注射制剂。此外，本发明药物组合物还可与其他治疗剂一起使用。并且，本发明的药物组合物还可以包括额外的组分，所述额外的组分选自下组：(a)预防和/或治疗炎症性疾病的药物；和/或(b)预防和/或治疗自身免疫性疾病的组分。
本发明所述的活性成分的有效量可随给药的模式和待治疗的疾病的严重程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于：所述的活性成分的药代动力学参数例如生物利用率、代谢、半衰期等；患者所要治疗的疾病的严重程度、患者的体重、患者的免疫状况、给药的途径等。通常，当本发明的活性成分每天以约0.01μg-1mg/kg体重，较佳地0.1-10μg/kg体重，更佳地0.5-5μg/kg体重。的剂量给予，能得到令人满意的效果。最佳剂量将根据病程和病情适当调整，例如，由治疗状况的迫切要求，可每天给予若干次分开的 剂量，或将剂量按比例地减少。
本发明所述的药学上可接受的载体包括(但不限于)：水、盐水、脂质体、脂质、蛋白、蛋白-抗体缀合物、肽类物质、纤维素、纳米凝胶、或其组合。载体的选择应与给药方式相匹配，这些都是本领域的普通技术人员所熟知的。
本发明还提供了一种可用于(a)预防和/或治疗炎症性疾病的药物；和/或((i)减少致病单个核细胞浸润；和/或(ii)抑制巨噬细胞的致病表型；和/或(iii)降低巨噬细胞浸润数目；和/或(iv)提高巨噬细胞细胞质的H +浓度；和/或(v)提高巨噬细胞氧化磷酸的代谢倾向性；(vi)赋予巨噬细胞显著抑制炎症的免疫记忆；和/或(vii)预防和/或治疗自身免疫性疾病；和/或(viii)用于巨噬细胞参与的多种其他涉及炎症反应的疾病(如与肝纤维化、肾纤维化、肺纤维化等疾病的发生有关的炎症反应，与组织、器官或细胞移植引起的炎症反应，与肺癌、乳腺癌、前列腺癌、肝癌、胰腺癌、黑色素瘤、淋巴瘤等癌症的发生和转移相关的炎症反应，以及与自身免疫性疾病相关的炎症反应)；和/或(ix)促进移植组织或细胞的植入；和/或(x)下调IGF2R在THP1细胞上的表达的药盒，该药盒含有：
(i)第一容器，以及位于该第一容器中的活性成分(a)经过非IGF1R结合型的物质处理的吞噬细胞，或含有活性成分(a)的药物；和
(ii)任选的第二容器，以及装于该第二容器中的活性成分(b)非IGF1R结合型的物质，或含有活性成分(b)的药物；和
(ii)说明书，所述说明书中记载了给予活性成分(a)从而(a)预防和/或治疗炎症性疾病；和/或(i)减少致病单个核细胞浸润；和/或(ii)抑制巨噬细胞的致病表型；和/或(iii)降低巨噬细胞浸润数目；和/或(iv)提高巨噬细胞细胞质的H +浓度；和/或(v)提高巨噬细胞氧化磷酸的代谢倾向性；和/或(vi)赋予巨噬细胞显著抑制炎症的免疫记忆；和/或(vii)预防和/或治疗自身免疫性疾病；和/或(viii)用于巨噬细胞参与的多种其他涉及炎症反应的疾病(如与肝纤维化、肾纤维化、肺纤维化等疾病的发生有关的炎症反应，与组织、器官或细胞移植引起的炎症反应，与肺癌、乳腺癌、前列腺癌、肝癌、胰腺癌、黑色素瘤、淋巴瘤等癌症的发生和转移相关的炎症反应，以及与自身免疫性疾病相关的炎症反应)；和/或(ix)促进移植组织或细胞的植入；和/或(x)下调IGF2R在THP1细胞上的表达的说明。
本发明的药物组合物和药盒适用于(a)预防和/或治疗炎症性疾病；和/或(i)减少结肠中致病单个核细胞浸润；和/或(ii)抑制巨噬细胞的致病表型；和/或(iii)降低巨噬细胞浸润数目；和/或(iv)提高巨噬细胞细胞质的H +浓度；和/或(v)提 高巨噬细胞氧化磷酸的代谢倾向性；和/或(vi)赋予巨噬细胞显著抑制炎症的免疫记忆；和/或(vii)预防和/或治疗自身免疫性疾病；和/或(viii)用于巨噬细胞参与的多种其他涉及炎症反应的疾病(如与肝纤维化、肾纤维化、肺纤维化等疾病的发生有关的炎症反应，与组织、器官或细胞移植引起的炎症反应，与肺癌、乳腺癌、前列腺癌、肝癌、胰腺癌、黑色素瘤、淋巴瘤等癌症的发生和转移相关的炎症反应，以及与自身免疫性疾病相关的炎症反应)；和/或(ix)促进移植组织或细胞的植入；和/或(x)下调IGF2R在THP1细胞上的表达。
本发明制剂可以每一天服用三次到每十天服用一次，或者以缓释方式每十天服用一次。优选的方式是每天服用一次，因为这样便于病人坚持，从而显著提高病人服药的顺应性。
服用时，极大多数病例一般每天应用的总剂量应低于(或少数病例等于或略大于)各个单药的每天常用剂量，当然，所用的活性成分的有效剂量可随给药的模式和待治疗的疾病的严重程度等而有所变化。
治疗方法
本发明还提供了用本发明的两种活性成分或相应的药物来(a)预防和/或治疗炎症性疾病；和/或(i)减少结肠中致病单个核细胞浸润；和/或(ii)抑制巨噬细胞的致病表型；和/或(iii)降低巨噬细胞浸润数目；和/或(iv)提高巨噬细胞细胞质的H +浓度；和/或(v)提高巨噬细胞氧化磷酸的代谢倾向性；和/或(vi)赋予巨噬细胞显著抑制炎症的免疫记忆；和/或(vii)预防和/或治疗自身免疫性疾病；和/或(viii)用于巨噬细胞参与的多种其他涉及炎症反应的疾病(如与肝纤维化、肾纤维化、肺纤维化等疾病的发生有关的炎症反应，与组织、器官或细胞移植引起的炎症反应，与肺癌、乳腺癌、前列腺癌、肝癌、胰腺癌、黑色素瘤、淋巴瘤等癌症的发生和转移相关的炎症反应，以及与自身免疫性疾病相关的炎症反应)；和/或(ix)促进移植组织或细胞的植入；和/或(x)下调IGF2R在THP1细胞上的表达的方法，它包括给哺乳动物施用有效量的活性成分(a)经过非IGF1R结合型的物质处理的吞噬细胞，或者施用含有所述活性成分(a)的药物组合物；以及(b)非IGF1R结合型的物质，或含有活性成分(b)的药物组合物。
当本发明两种活性成分被用于上述用途时，可与一种或多种药学上可接受的载体或赋形剂混合，如溶剂、稀释剂等，而且可以用如下形式口服给药：片剂、丸剂、胶囊、可分散的粉末、颗粒或悬浮液(含有如约0.05-5％悬浮剂)、 糖浆(含有如约10-50％糖)、和酏剂(含有约20-50％乙醇)，或者以无菌可注射溶液或悬浮液形式(在等渗介质中含有约0.05-5％悬浮剂)进行非肠胃给药。例如，这些药物制剂可含有与载体混合的约0.01-99％，更佳地约为0.1％-90％(重量)的活性成分。
本发明的两种活性成分或药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：肌内、腹膜内、静脉内、皮下、皮内、口服、瘤内或局部给药。优选的给药途径包括口服给药、肌内给药或静脉内给药。
从易于给药的立场看，优选的药物组合物是液态组合物，尤其是注射剂。
此外，本发明的两种活性成分或药物还可与其他(a)预防和/或治疗炎症性疾病；和/或(i)减少致病单个核细胞浸润；和/或(ii)抑制巨噬细胞的致病表型；和/或(iii)降低巨噬细胞浸润数目；和/或(iv)提高巨噬细胞细胞质的H +浓度；和/或(v)提高巨噬细胞氧化磷酸的代谢倾向性；和/或(vi)赋予巨噬细胞显著抑制炎症的免疫记忆；和/或(vii)预防和/或治疗自身免疫性疾病；和/或(viii)用于巨噬细胞参与的多种其他涉及炎症反应的疾病(如与肝纤维化、肾纤维化、肺纤维化等疾病的发生有关的炎症反应，与组织、器官或细胞移植引起的炎症反应，与肺癌、乳腺癌、前列腺癌、肝癌、胰腺癌、黑色素瘤、淋巴瘤等癌症的发生和转移相关的炎症反应，以及与自身免疫性疾病相关的炎症反应)；和/或(ix)促进移植组织或细胞的植入；和/或(x)下调IGF2R在THP1细胞上的表达的组分或药物(非甾体抗炎药、糖皮质激素、甲氨蝶呤、TNFα中和抗体、TNFR1抗体、TNFR2抗体、抗CD20抗体、IL-1R拮抗剂、IL-12和IL-23p40中和抗体、IL-23p19中和抗体、IL-17中和抗体、IL-17A受体中和抗体、CTLA-4融合蛋白)联合使用。
本发明的主要优点包括：
(1)本发明首次发现，非IGF1R结合型的物质可显著(a)预防和/或治疗炎症性疾病；和/或(i)减少致病单个核细胞浸润；和/或(ii)抑制巨噬细胞的致病表型；和/或(iii)降低巨噬细胞浸润数目；和/或(iv)提高巨噬细胞细胞质的H +浓度；和/或(v)提高巨噬细胞氧化磷酸的代谢倾向性；和/或(vi)赋予巨噬细胞显著抑制炎症的免疫记忆；和/或(vii)预防和/或治疗自身免疫性疾病；和/或(viii)用于巨噬细胞参与的多种其他涉及炎症反应的疾病(如与肝纤维化、肾纤维化、肺纤维化等疾病的发生有关的炎症反应，与组织、器官或细胞移植引起的炎症反应，与肺癌、乳腺癌、前列腺癌、肝癌、胰腺癌、黑色素瘤、淋巴瘤等癌症的发 生和转移相关的炎症反应，以及与自身免疫性疾病相关的炎症反应)；和/或(ix)促进移植组织或细胞的植入；和/或(x)下调IGF2R在THP1细胞上的表达。
(2)本发明首次发现，胰岛素样生长因子2(IGF2)赋予诱导巨噬细胞抗炎的免疫记忆。这一特性依赖于IGF2受体(IGF2R)。功能性封闭IGF2R，IGF2不再赋予巨噬细胞抵抗炎症的免疫记忆。
(3)本发明首次发现，低剂量的IGF2可以诱导非经典的线粒体动力学，赋予巨噬细胞抵抗炎症的免疫记忆，高剂量IGF2会激活IGF1受体(IGF1R)，抑制IGF2R的效应，促进单核细胞和巨噬细胞利用有氧糖酵解代谢获得促炎能力。
(4)本发明首次发现，与IGF1R低亲和力的IGF2突变体，特异性激活IGF2R，最大程度缓解腹膜炎和炎症性肠病等自身免疫性疾病和炎症性疾病。这些发现说明，IGF2R靶向性激活能够决定巨噬细胞长期的免疫抑制记忆，IGF2R是治疗巨噬细胞参与的炎症相关疾病的理想靶点
(5)本发明首次发现，定向激活单核细胞的IGF2R抑制炎症性肠病。
(6)本发明首次发现，IGF2治疗炎症性肠病依赖于巨噬细胞。
(7)本发明首次发现，IGF2在巨噬细胞参与的多种其他炎症反应疾病。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
如无特别说明，实施例所用的所有材料和试剂均为市售产品。
材料与方法
1试剂
本研究所涉及的实验试剂较多，为了减少字数，节约版面，将按照试剂的不同品牌依次列出。
重组人源胰岛素样生长因子-2(Recombinant human IGF2，292-G2-250)，重组鼠源巨噬细胞集落刺激因子(Recombinant mouse M-CSF protein，416-ML-050)购买于R&D Systems。
人源第27为亮氨酸点突变IGF2(Human Leu27-IGF2，TU100)购买于GroPep Bioreagents。
人源第27为亮氨酸点突变IGF2(Human Leu27-IGF2，TU100)删除D结构域为实验室合成。
2实验小鼠构建和鉴定
IGF2R +/-小鼠利用Crisper-Cas9技术敲除igf2r基因第6外显子上2bp或13bp碱基，造成移码突变。由于IGF2R缺陷纯合子会胚胎致死，我们以IGF2R +/-杂合子小鼠保种传代和鉴定。鉴定使用引物为：
Primer 1,5’-CGTGAAGTCTGTCTATGGAAGAGACTGGACC-3’(SEQ ID NO.:59)，
Primer 2,5’-CATTACCAAGCTCACACTCCCTTCTCTTCTCTATT-3’(SEQ ID NO.:60),
基因型结果判定方法将体现于实验结果部分。
Igf2r fl/fl小鼠在igf2r基因第2外显子两侧插入loxp序列，鉴定使用引物为：
Primer 1,5’-CCCCGGTGTTGAGGTTGATAGATA-3’(SEQ ID NO.:61)，
Primer 2,5’-CAATTTAGGGCTGAAGCGATGGAT-3’(SEQ ID NO.:62),
基因型结果判定方法将体现于实验结果部分。
Igf1r fl/fl小鼠鉴定使用引物为：
Primer 1,5’-CTTCCCAGCTTGCTACTCTAGG-3’(SEQ ID NO.:63)，
Primer 2,5’-CAGGCTTGCAATGAGACATGGG-3’(SEQ ID NO.:64)，
Primer 3,5’-TGAGACGTAGCGAGATTGCTGTA-3’(SEQ ID NO.:65)
基因型结果判定方法将体现于实验结果部分。
Lyz2 Cre小鼠鉴定使用引物为：
Primer 1,5’-CCCAGAAATGCCAGATTACG-3’(SEQ ID NO.:66)，
Primer 2,5’-CTTGGGCTGCCAGAATTTCTC-3’(SEQ ID NO.:67)，
Primer 3,5’-TTACAGTCGGCCAGGCTGAC-3’(SEQ ID NO.:68)
基因型结果判定方法将体现于实验结果部分。
3腹膜炎小鼠模型
配置5％(质量/体积)的巯基乙酸盐(Ftg)培养基水溶液，在121℃高温高压条件下灭菌30分钟，冷却至室温后备用。使用前检查巯基乙酸盐培养基的物理性状，粘稠度以不至于摇晃时出现气泡为佳，培养基颜色由最初配置时的 蓝绿色，转变为摇晃时呈现棕褐色为宜。
选取9-12周范围内，同周龄的C57BL/6雌性小鼠进行实验。每只小鼠缓慢注射2ml巯基乙酸盐培养基至腹腔，24小时后，给予IGF2或其他药物治疗，在巯基乙酸盐注射后的第24小时和第48小时分别给药一次，第72小时安乐死小鼠，分离腹腔巨噬细胞。
4腹腔巨噬细胞分离
用75％乙醇浸泡已经安乐死的(Ftg)诱导72小时的小鼠数分钟，沿垂直于小鼠头尾的方向撕开腹部毛皮，用注射器吸取10ml预冷的磷酸盐缓冲液()，将磷酸盐缓冲液用力快速注射(有助于将贴壁细胞吹下)进入小鼠腹腔，右手食指无名指夹住小鼠尾巴，轻轻握住小鼠，上下剧烈摇晃三至五次，用注射器缓慢地将细胞灌洗液吸出，这个过程中要避免刺穿肠道而污染细胞，避免吸入脂肪组织而堵塞针头。腹腔巨噬细胞灌洗液需四度保存，常温下细胞容易吸附离心管管壁。
5炎症性肠病模型
预先配置16％(质量/体积)葡聚糖硫酸钠(dextran sulfate sodium salt，DSS)水溶液，用0.45μM滤头过滤除菌后传递进入SPF屏障。以常规饮用水对于16％DSS溶液进行4倍稀释至4％终浓度。选择9-12周龄雌性小鼠喂食4％DSS溶液至不同天数，终止日期根据实验目的不同而定。在最初验证IGF2治疗效果的实验中，4％DSS溶液持续喂养，生存曲线统计完成后终止；在后续实验中通常连续喂养5天的4％DSS溶液，不同批次的实验的具体饮用时间将会在实验结果中体现。
对于炎症性肠病的临床评分，按以下标准进行。在用4％DSS溶液喂养小鼠4天后，评价“Stool scores”和“Bleeding scores”。“Stool scores”的标准为：0分，粪便形态正常；1分，半成型粪便且肛门无附着；2分，半成型粪便且存在肛门附着；3分，液态粪便附着于肛门。“Bleeding scores”的判断标准：0分，粪便潜血试纸阴性；1分，粪便潜血试纸阳性；2分，笼具可见血痕；3分，直肠出血，肛门呈现红色血斑。
6结肠浸润淋巴细胞分离
安乐死4％DSS诱导4-5天的小鼠，解剖小鼠，获取结肠组织，放置于4℃预冷的磷酸盐缓冲液中，洗去结肠表面血污。纵向剪开结肠，在四度预冷的磷酸盐缓冲液中，小心洗去粪便。用含有200μg/ml DNaseI和1mg/ml VIII 型胶原酶的1640培养液，于消化前加入DTT，摇床200转/分钟消化60分钟，小心取出结肠基底层组织，剪成小段后，进行再次消化，以70μm细胞筛网过滤，将滤液以400g转速离心5分钟，获得单细胞沉淀。
配置40％和80％的Percoll分离液，用2ml体积的40％浓度的Percoll重悬单细胞沉淀，向40％浓度的Percoll下层的离心管底部，小心加入80％的Percoll分离液，适当控制对移液枪的压力，防止两层分离液混合浑浊。调节离心机温度至25℃，升降加速梯度为0，2000转/分钟离心25分钟。收集40％和80％的Percoll分离液中间层细胞，用10ml 4℃预冷的磷酸盐缓冲液洗涤至少两次，以完全去除Percoll，最终获得纯净的结肠来源的单个核细胞。
7流式细胞分析
因为单核细胞和巨噬细胞相对高水平表达IgG的受体——CD16/32，对于单核细胞和巨噬细胞抗体染色，需要提前用无荧光素偶联的CD16/32抗体封闭细胞(抗体：磷酸盐缓冲液＝1：50比例稀释抗体，100μl抗体工作液/1×10 7细胞，室温孵育30分钟)。因为巨噬细胞具有易贴壁且不易消化的特性，巨噬细胞染色时应该选择超低吸附的培养板来承放细胞。
细胞膜表面蛋白染色。对于CD16/32抗体封闭过的细胞，按照1：100比例用磷酸盐缓冲液稀释抗体，100μl染色工作液/1×10 7细胞，室温染色30分钟后以磷酸盐缓冲液洗涤，离心后用200μl磷酸盐缓冲液重悬，上机检测或者用1％的中性甲醛固定。
细胞内和细胞核内蛋白染色。细胞内蛋白以及细胞核内蛋白需要在细胞膜表面蛋白染色完成后进行。对于细胞内蛋白，使用细胞固定和破膜试剂盒(Fixation/Permeabilization Solution and Perm/Wash Buffer)进行破膜和固定后染色；对于细胞核蛋白，使用细胞固定/细胞破膜试剂盒(Foxp3/Transcription Factor Staining Buffer Set)固定和破膜后染色。
8细胞外通量分析(海马实验)
将腹腔巨噬细胞或者THP1细胞(人单核细胞)的培养液更换为Seahorse专用培养基(XF medium)，培养基添加了10mM葡萄糖，2mM谷氨酰胺和2mM丙酮酸。氧气消耗率测定实验和胞外产酸率测定实验在添加或者不添加以下刺激条件下：1μM寡霉素，0.75mM FCCP，100nM鱼藤酮和1μM抗霉素，使用细胞外流量分析仪(extracellular flux analyzer，Agilent)测定。
9实时定量PCR
使用天根生物细胞/细菌RNA分离试剂盒提取细胞总RNA，每个样本通常可以获得30μL体积的300ng/ml的RNA。利用Takara反转录试剂盒将全部RNA反转录为cDNA(PCR条件为：37℃持续15分钟，85℃灭活5秒钟，温度下降至4℃保存)。cDNA可在-20℃条件下保存数天，时间延长会发生降解。
在使用cDNA前，用双蒸水对cDNA进行10倍稀释，实时定量PCR体系为10μL，包含5μL SYBR，4μL cDNA，1μL引物。于384孔板点好样本后，2500转/分在室温下离心2分钟，此步骤若以4℃条件离心将会在膜上形成水雾。实时定量PCR条件为：Step 1，95℃持续10分钟，Step 2，95℃持续15秒，Step 3，60℃持续1分钟，Step 4，95℃持续15秒，Step 5，60℃持续1分钟，Step 6，95℃持续15秒，其中Step 2和Step 3循环40次。
实时定量PCR实验所涉及的引物如下：
TNFa-F：5’-CCTGTAGCCCACGTCGTAG-3’(SEQ ID NO.:69)，
TNFa-R：5’-GGGAGTAGACAAGGTACAACCC-3’(SEQ ID NO.:70)；
IL-1b-F：5’-GCAACTGTTCCTGAACTCAACT-3’(SEQ ID NO.:71)，
IL-1b-R：5’-ATCTTTTGGGGTCCGTCAACT-3’(SEQ ID NO.:72)；
Il18-F：5’-GACTCTTGCGTCAACTTCAAGG-3’(SEQ ID NO.:81)
Il18-R：5’-CAGGCTGTCTTTTGTCAACGA’(SEQ ID NO.:82)；
Cxcl10-F：5’-CCAAGTGCTGCCGTCATTTTC-3’(SEQ ID NO.:73)，
Cxcl10-R：5’-GGCTCGCAGGGATGATTTCAA-3’(SEQ ID NO.:74)；
Retnla-F：5’-CCAATCCAGCTAACTATCCCTCC-3’(SEQ ID NO.:75)，
Retnla-R：5’-ACCCAGTAGCAGTCATCCCA-3’(SEQ ID NO.:76)；
CCL22-F：5’-AGGTCCCTATGGTGCCAATGT-3’(SEQ ID NO.:77)，
CCL22-R：5’-CGGCAGGATTTTGAGGTCCA-3’(SEQ ID NO.:78)；
Clec7a-F：5’-GACTTCAGCACTCAAGACATCC-3’(SEQ ID NO.:79)，
Clec7a-R：5’-TTGTGTCGCCAAAATGCTAGG-3’(SEQ ID NO.:80)；
Ym1-F:5’-CAGGTCTGGCAATTCTTCTGAA-3’(SEQ ID NO.:83),
Ym1-R：5’-GTCTTGCTCATGTGTGTAAGTGA-3’(SEQ ID NO.:84)；
10H&E染色
脱水包埋：取相同位置的结肠组织于4％多聚甲醛溶液中固定24小时，依次经过以下步骤脱水处理后包埋。70％乙醇，浸泡过夜；80％乙醇，浸泡2小时；85％乙醇，浸泡1.5小时；90％乙醇，浸泡1小时；95％乙醇，浸泡1 小时；100％乙醇，浸泡30分钟，重复操作一次；二甲苯，浸泡5分钟，重复操作一次；60℃液体石蜡，浸泡1小时，重复操作一次；使用石蜡包埋机完成石蜡包埋。
切片染色：以3μm厚度切片，56℃水浴延展石蜡组织切片数秒钟，用载玻片吸附石蜡组织切片，于60℃烘箱中过夜烘片，然后按如下步骤进行染色。二甲苯，浸泡5分钟，重复操作一次；100％乙醇，浸泡5分钟，重复操作一次；95％乙醇，浸泡5分钟，重复操作一次；70％乙醇，浸泡5分钟；蒸馏水，浸泡5分钟；苏木精，染色5分钟；自来水流水冲洗适当时间；氨水，浸洗适当时间；流水冲洗适当时间；伊红，染色1分钟；95％乙醇，浸泡1分钟；100％乙醇，浸泡1分钟，重复操作一次。二甲苯，浸泡1分钟；重复操作一次；封片剂封片。
11免疫组化
石蜡切片抗原复性：石蜡切片于60℃烘箱中烘片两小时；用pH 7.4的磷酸盐缓冲液浸洗石蜡切片三次，每次三分钟；将脱蜡水化的石蜡切片置于沸腾的pH＝6.0的柠檬酸盐缓冲液中煮沸十分钟；自来水流水自然冷却后，用蒸馏水浸洗两次，每次三分钟；用pH 7.4的磷酸盐缓冲液浸洗两次，每次三分钟。每张石蜡切片滴加一滴3％双氧水，室温孵育十分钟。
抗原抗体反应：于石蜡切片上滴加抗Ki67的一抗，4℃孵育过夜；用pH 7.4的磷酸盐-吐温缓冲液浸洗四次，每次三分钟；于石蜡切片上滴加辣根过氧化物酶偶联的二抗，室温孵育60分钟；用pH 7.4的磷酸盐-吐温缓冲液浸洗4次，每次3分钟；DBA染色适当时间后，洗去DBA染色剂；使用苏木素染料复染石蜡切片，根据组织类型确定合适的时间；自来水冲洗，去除苏木苏；使用盐酸酒精分化和氨水反蓝，至苏木苏染色呈现淡蓝色；脱水封片。
TUNEL免疫组化实验按照In situ cell death detection kit-POD kit(Roche，TUN11684817)所提供的实验方法进行实验。
12一氧化氮检测
吸取脂多糖刺激24小时的巨噬细胞的培养基50μL，加入到96孔平底透光平板中，每个样本设置3个副孔。添加50μL Griess试剂至每孔混匀，室温避光孵育15分钟后，在酶标仪540nm波长下检测吸光值。对于定量检测的实验，可以预先配置100mM NaNO 2标准品母液，梯度稀释为0，5，10，20，30，40，50，100μM的不同浓度的标准品，添加等体积Griess试剂，相同条件孵 育和读板后绘制标准曲线。检测培养基上清中一氧化氮的累积量，可以反映出巨噬细胞诱导型一氧化氮合酶(Inducible nitric oxide synthase，iNOS)的活力，推测巨噬细胞能量代谢类型和炎症表型。
13乳酸检测
使用乳酸检测试剂盒检测IGF2预编程的腹腔单核细胞和巨噬细胞的乳酸产生能力。按照Lactate Colorimetric Assay Kit II(BioVision，K627-100)提供的实验方法进行实验。检测培养基上清中乳酸的累积量，可以反映出巨噬细胞能量代谢类型和炎症表型。
14葡萄糖浓度测定
使用葡萄糖检测试剂盒检测IGF2预编程的腹腔单核细胞和巨噬细胞的葡萄糖消耗能力。按照Glucose Colorimetric Assay Kit II(BioVision，K686-100)提供的实验方法进行。葡萄糖消耗速率可以反映出巨噬细胞的能量代谢类型和炎症表型。
15 JC1染色
JC1染色属于活细胞染色，预先配置JC1染色工作液(JC1储液：JC1染色缓冲液：双蒸水：完全培养基＝1：10：89：100的体积比配置)，100μl染色工作液/1×107细胞，37℃培养箱中染色30分钟后立即上机检测。收集BD calibur流式细胞仪的FL1和FL2通道下的荧光信息。根据JC1活细胞染色特点：当线粒体膜电势较高时，JC1寡聚到线粒体外膜，呈现红色荧光，当线粒体膜电势降低时，JC1以单体形式分布在细胞质，呈现绿色荧光，因此比较红色荧光和绿色荧光强度，可以评价单细胞水平的线粒体膜电势的高低。
16细胞质和溶酶体pH检测
活细胞染色，细胞所处环境的pH和葡萄糖浓度会对细胞内pH产生影响。配置pH染色工作液(pH染色储液：活化液：磷酸盐缓冲液＝1：10：89体积比配置)，100μl染色工作液/1×10 7细胞，37℃培养箱中染色30分钟后立即上机检测。对于细胞质pH检测，使用pHrodo Green AM染料，对于溶酶体pH染色，使用LysoSensor Green DND-189或LysoSensor Yellow/Blue DND-160染料。对于细胞质和细胞器pH定量，则需要结合使用pH检测标准品试剂盒(Intracellular pH Calibration Buffer Kit)。
17溶酶体分离和ATP酶活力检测
分离IGF2处理的腹腔单核细胞和巨噬细胞的溶酶体，按照Lysosome  Isolation Kit(Sigma-Aldrich，LYSISO1-1KT)提供的实验方法进行实验。分离获得的溶酶体将用于V型ATP酶活力检测实验。使用ATP酶活性检测试剂盒检测以上分离获得的溶酶体的ATP酶活性，代表了溶酶体V型ATP酶的活力。按照ATPase Activity Assay Kit(BioVision，K417)提供的实验方法进行实验。
18数据统计分析
数据以均值或均值±S.E.M.或S.D.的形式表示，使用Graphpad软件作图和显著差异分析，本研究中对于两组数据之间的差异显著性使用双尾非参数Student’s t检验进行分析。ns，表示不显著差异；*P<0.05；**P<0.01；***P<0.001，P<0.05被认为存在显著差异。
实施例1低剂量IGF2抑制腹膜炎。
分别使用低剂量IGF2(L-IGF2，5-50ng IGF2每只小鼠)和高剂量IGF2(H-IGF2，1000ng IGF2每只小鼠)处理腹膜炎小鼠。分离腹膜炎小鼠腹腔单核细胞和巨噬细胞并分析。
结果显示，与对照组(PBS)和高剂量IGF2处理组相比，低剂量IGF2可以显著抑制腹膜炎小鼠腹腔中巨噬细胞的浸润(图1)，表明低剂量IGF2可以抑制腹膜炎小鼠炎症情况。
实施例2低剂量IGF2调控下的抗炎巨噬细胞以氧化磷酸化的代谢偏好性为主。
分别使用低剂量IGF2(L-IGF2，5-50ng IGF2每只小鼠)和高剂量IGF2(H-IGF2，1000ng IGF2每只小鼠)处理腹膜炎小鼠，分离腹膜炎小鼠腹腔巨噬细胞并分析细胞的代谢状态。海马实验(Seahorse technology)是研究细胞能量代谢偏好的金标准。我们通过海马实验研究发现，低剂量IGF2调控下腹腔巨噬细胞的最大氧气消耗率(Oxygen consumption rate，OCR)显著高于对照组，来源于高剂量IGF2调控下腹腔巨噬细胞的最大氧气消耗率则低于对照组(图2A)。所以，低剂量IGF2增加了巨噬细胞的氧气消耗潜力，而高剂量IGF2逆转了这种潜力。
此外，我们还利用海马实验测定了这三组细胞的最大胞外酸化率(Extracellular acidification rate，ECAR)，发现，低剂量IGF2调控下巨噬细胞的胞外最大产酸率显著低于PBS对照组和高剂量IGF2组。这些结果被 更好的体现于最大氧气消耗率与胞外最大产酸率(OCR：ECAR)的比对值(图2B)。低剂量IGF2调控下巨噬细胞体现更高水平的OCR：ECAR值，说明这组巨噬细胞倾向于以氧化磷酸化(Oxidative phosphorylation，OXPHOS)为主要方式来获取能量，而对照组和高剂量IGF2组的巨噬细胞则更倾向于选择有氧糖酵解的方式产生能量供细胞所需。
乳酸是有氧糖酵解的中间代谢产物，在有氧糖酵解的过程中，过量的乳酸会被细胞分泌到细胞外，因此，检测和对比细胞培养基上清中乳酸的累积量，是衡量细胞进行有氧糖酵解水平的有效方式。我们利用脂多糖(Lipopolysaccharide，LPS)再次刺激这些分离得到的腹腔巨噬细胞，24小时后检测培养基上清中乳酸含量，结果发现，低剂量IGF2处理组的腹腔巨噬细胞的培养基上清中的乳酸积累量显著低于PBS和高剂量IGF2处理组(图2C)。上述研究表明，低剂量IGF2赋予小鼠腹腔巨噬细胞更强的氧化磷酸化获能潜力，而高剂量的IGF2则逆转了这一特性，甚至增加了细胞的无氧糖酵解获能偏好。以氧化磷酸化为主要获能方式的巨噬细胞，往往具备替代激活巨噬细胞的特征，体现抵抗炎症的表型。为了验证不同剂量IGF2预编程的腹腔巨噬细胞的免疫表型，研究分别用脂多糖(LPS)以及白介素-4/13(IL-4/13)分别处理这些分离出来的巨噬细胞，模拟经典激活途径和替代激活途径刺激巨噬细胞。实时定量PCR(Real-time PCR，RT-PCR)结果显示，脂多糖经典激活的巨噬细胞的炎症因子表达被低剂量IGF2(L-IGF2)抑制，高剂量IGF2对部分基因无显著调控(图2D)；白介素4/13激活的巨噬细胞的抗炎因子表达被IGF2(L-IGF2)放大，高剂量IGF2(H-IGF2)无明显作用(图2E)。
这些结果说明，低剂量IGF2引起的氧化磷酸化能量代谢偏好能够促进巨噬细胞体现较强的抗炎表型，而高剂量IGF2逆转了低剂量IGF2的效应，将能量代谢方式回归为与对照组接近的有氧糖酵解为主的方式，相应地逆转了炎症表型。
实施例3低剂量IGF2抑制V型ATP酶活性，降低溶酶体H +浓度，促进细胞质H +浓度，提高线粒体内膜电势。
氧化磷酸化发生于线粒体，而有氧糖酵解发生于细胞质。为了探索低剂量IGF2如何增强巨噬细胞氧化磷酸化获能偏好，我们对于细胞线粒体进行了研究。研究发现，来源于低剂量IGF2(L-IGF2，50ng每只小鼠)处理的腹腔巨噬 细胞(成熟中和成熟巨噬细胞)均体现更高的线粒体膜电位(图3A)，提示显著增高的线粒体膜电位为低剂量IGF2调控下的巨噬细胞进行氧化磷酸化提供了充足的H +底物。同时，低剂量IGF2处理的腹膜炎小鼠腹腔巨噬细胞均呈现较低的细胞质pH，甚至接近pH 5.5(图3B)。在正常情况下，细胞质H +浓度受细胞外膜的和酸性细胞器的V型ATP酶的严格调控，通常细胞质的pH值会处于7.4左右。为了验证细胞质H +浓度上调是否是因为酸性细胞器功能缺陷引起的，我们检测了最重要的负责H +平衡调节的细胞器——溶酶体(Lysosome)的pH。研究发现，低剂量IGF2调控下巨噬细胞的溶酶体pH被上调至6.5，显著高于对照组和高剂量IGF2组巨噬细胞的溶酶体的pH(大约4.5-5.5)(图3C)。此外，分离出细胞溶酶体，检测溶酶体上V型ATP酶的活力。结果发现，低剂量IGF2显著降低巨噬细胞的V型ATP酶的活力，而高剂量IGF2对于V型ATP酶的活力没有显著影响(图3D)。
实施例4构建igf2r基因缺陷小鼠，以及igf1r或igf2r条件性敲除小鼠。
IGF2可以与IGF1R和IGF2R结合，与IGF2R具有最大亲和力，当IGF2与IGF2R结合后，引起IGF2R内化，IGF2将被运往溶酶体发生降解，因此IGF2R往往被认为是IGF2的诱骗受体(Decoy receptor)，而IGF2与IGF1R结合则发挥丝裂原功能，促进细胞增殖和生存 2。根据前一部分的研究，我们发现高剂量IGF2体现出了丝裂原功能，促进腹膜炎小鼠腹腔巨噬细胞增殖和生存，而低剂量IGF2则没有这样的功能。又鉴于IGF2与IGF2R的最大亲和力，我们假设低剂量IGF2优先结合与IGF2R，诱导IGF2R依赖的生物学效应，比如非经典的线粒体动力学的发生，高剂量IGF2在“耗尽”细胞外膜IGF2R后依然有机会与IGF1R结合，抵消IGF2R的生物学效应，进而促进腹膜炎小鼠腹腔巨噬细胞增殖和生存。
为了研究IGF2分别依赖于何种受体实现对于单核细胞的双向调节功能，我们构建了基因工程小鼠。在igf2r基因全身敲除小鼠中，我们利用Crisper-Cas9技术打靶敲除igf2基因第6外显子2bp或13bp碱基，造成移码突变来完成(图4A)。鉴于igf2r基因完全缺失会引起小鼠胚胎致死，我们最终获得igf2r基因敲除的杂合子小鼠——IGF2R +/-小鼠进行繁殖保种和实验，IGF2R +/-小鼠子代基因型鉴定依赖于测序比对(图4B)。我们诱导了小鼠腹腔巨噬细胞，并用流式检测这些细胞IGF2R的表达，确认了敲除效率(图4C)。
为了构建igf2r条件性敲除小鼠，我们利用ES细胞打靶技术，在igf2r基因第二外显子两端引入了同方向的loxp序列，首先构建出IGF2R fl/fl小鼠。然后将IGF1R fl/fl小鼠以及IGF2R fl/fl小鼠分别与Lyz2 Cre小鼠交配，其中Lyz2在髓系细胞中高水平表达 18，直至获得IGF1R fl/flLyz2 Cre纯合子小鼠和IGF2R fl/flLyz2 Cre纯合子小鼠(图4D，G)。DNA水平和蛋白水平鉴定结果验证了敲除效率(图4E，F，H，I)。
实施例5定向激活单核细胞的IGF2R抑制炎症性肠病。
为了评价IGF2-IGF2R调控的单核细胞和巨噬细胞在其他炎症模型中的作用，我们使用葡聚糖硫酸钠(dextran sulfate sodium salt，DSS)诱导了炎症性肠病模型，这是一种与人类克罗恩病和溃疡性结肠炎症状最为相似的一种实验动物模型 19,20。接下来，我们使用不同剂量的IGF2、IGF2突变体、以及IGF2处理的腹腔巨噬细胞对于DSS诱导的炎症性肠病进行治疗。
5.1低剂量IGF2抑制DSS诱导的炎症性肠病
我们发现，低剂量IGF2(L-IGF2，50ng每只小鼠)可以显著减缓DSS引起的小鼠体重下降，显著延长小鼠生存时间，然而，高剂量IGF2(H-IGF2，1000ng每只小鼠)则加速了小鼠体重下降，不能延长小鼠的生存时间(图5A，B)。此外，我们还对小鼠的其他指标做了记录和评价，比如“Stool Score”和“Bleeding Score”以及结肠的长度，结果发现，低剂量IGF2可以显著降低“Stool Score”和“Bleeding Score”的评分，更好地保持小鼠结肠的长度(图5C-E)。
在组织病理学分析中，我们发现，对照组和高剂量IGF2处理组小鼠的结肠绒毛大部分被破坏，而低剂量IGF2组相对完好(图5F)。在TUNEL免疫组化染色结果中，我们发现，死亡的细胞沿着结肠绒毛向基底层蔓延，对照组和高剂量IGF2处理组的阳性死亡信号明显强于低剂量IGF2处理组(图5G)。在Ki67免疫组化染色结果中，我们发现，Ki67阳性增值信号由基底膜向绒毛方向延伸，低剂量IGF2组的Ki67阳性信号显著强于对照组和高剂量IGF2处理组，这说明了基底膜附近的单核细胞和巨噬细胞对于肠绒毛的修复发挥作用，低剂量IGF2治疗的小鼠的结肠基底膜处的巨噬细胞能够更好地促进结肠绒毛修复(图5H)。
综上所述，低剂量IGF2可以显著改善DSS诱导的炎症性肠病小鼠的生活质量和生存时间，高剂量的IGF2无治疗作用，甚至会加重一些症状。
5.2 IGF2治疗炎症性肠病依赖于巨噬细胞
为了验证IGF2处理的单核细胞和巨噬细胞在炎症性肠病治疗中的关键作用，我们利用PBS、低剂量IGF2(L-IGF2，50ng每只小鼠)，以及高剂量IGF2(H-IGF2，1000ng每只小鼠)处理的巨噬细胞来治疗DSS诱导的炎症性肠病小鼠。结果发现，PBS和高剂量IGF2处理的腹腔巨噬细胞在治疗炎症性肠病小鼠时，均加重小鼠体重下降，缩短小鼠生存时间，而低剂量IGF2处理的腹腔巨噬细胞可以显著改善炎症性肠病小鼠状态，体重指数被显著改善，生存时间得以延长(图6A，B)。
因此，在低剂量IGF2调控的巨噬细胞可以缓解炎症性肠病，而高剂量IGF2调控的巨噬细胞加重的炎症性肠病进展。
5.3低剂量IGF2减少结肠中致病巨噬细胞浸润
鉴于巨噬细胞在DSS诱导的炎症性肠病中发挥的重要作用，我们对于结肠浸润的单个核细胞进行了数目和表型分析。结果表明，低剂量IGF2(L-IGF2，50ng每只小鼠)治疗显著降低了结肠中单个核细胞浸润，而高剂量IGF2(H-IGF2，1000ng每只小鼠)则增加了单个核细胞在结肠组织中的浸润(图7A)。这一现象与低剂量IGF2和高剂量IGF2处理的腹膜炎小鼠的可获得的腹腔巨噬细胞数目相类似。
在检测炎症性肠病小鼠结肠组织中分离到的单个核细胞的表型之前，我们利用脂多糖刺激这些细胞5小时，并加入布雷菲德菌素A(Brefeldin A，BFA)以抑制细胞内因子释放。实验结果表明，相对于对照组的细胞，低剂量IGF2治疗的炎症性肠病小鼠结肠组织的巨噬细胞表达更少的白介素-1β(IL-1β)，而高剂量IGF2治疗的结肠炎小鼠结肠组织的巨噬细胞则显著高表达IL-1β(图7B，C)。同时，我们分析了巨噬细胞中PD-L1的表达水平，研究发现低剂量IGF2处理组巨噬细胞上PD-L1的表达明显增加(图7D)。
这些结果说明，低剂量IGF2抑制了炎症性肠病小鼠结肠组织浸润的巨噬细胞IL-1β水平，上调PD-L1水平，同时降低巨噬细胞浸润数目，而高剂量IGF2治疗则会加重巨噬细胞的这一致病表型，同时增加这一类群细胞的浸润数目。
5.4 IGF1加重炎症性肠病
IGF2在低剂量情况下优先结合IGF2R，高剂量时可以结合IGF1R。而IGF1具备结合IGF1R的能力，但是不结合IGF2R。为了证明IGF1R激活对于炎症反应的调控作用，研究利用IGF1治疗炎症性肠病。研究发现，低剂量IGF1(L-IGF1，50ng每只小鼠)和高剂量IGF1(H-IGF2，1000ng每只小鼠)均可以显著缩短DSS诱导炎症性肠病小鼠生存时间，结肠长度明显缩短，结肠固有层巨噬细胞表达IL-1β水平增加，PD-L1水平下降(图8A-D)。此外，研究还分析了低、高剂量IGF1处理腹膜炎小鼠后，腹腔巨噬细胞在LPS刺激下产生一氧化氮和乳酸以及消耗葡萄糖情况(图8E-G)。研究发现，IGF1明显促进巨噬细胞一氧化氮产生、乳酸分泌以及葡萄糖消耗，提示IGF1通过IGF1R执行促炎功能。
5.5 IGF2R执行低剂量IGF2诱导抗炎巨噬细胞的功能
为了验证低剂量IGF2对巨噬细胞抗炎功能的调控是否依赖于IGF2R，研究利用髓系细胞特异性敲除IGF2R小鼠，建立腹膜炎模型，分析在有无低剂量IGF2(L-IGF2，50ng每只小鼠)注射时，腹腔巨噬细胞PD-L1、IL-1β以及细胞质中H +的表达水平。研究发现，低剂量IGF2可以有效促进巨噬细胞表面PD-L1表达水平，抑制IL-1β表达水平，上调巨噬细胞细胞质中H +浓度。当髓系细胞缺失IGF2R时，低剂量IGF2对于巨噬细胞表达PD-L1、IL-1β以及细胞质中H +浓度的调控作用则被消除(图9A-C)。因此，低剂量IGF2借以IGF2R调控巨噬细胞的抗炎特性。
5.6 IGF2R执行低剂量IGF2的抗炎任务
为了验证IGF2在治疗DSS诱导的炎症性肠病小鼠中的作用机制，是否与我们之前的研究结果相一致，我们给予髓系细胞特异性敲除IGF2R或者IGF1R的小鼠诱导炎症性肠病，并以低剂量IGF2(L-IGF2，50ng每只小鼠)治疗。结果表明，相对于对照组IGF2R fl/fl小鼠，特异性敲除IGF2R的IGF2R fl/flLyz2 Cre小鼠对于炎症性肠病更加敏感，体重下降更为显著，而且低剂量IGF2不再发挥有效的抵抗炎症性肠病的作用，体重持续下降，小鼠存活时间显著缩短，粪便评分和便血程度明显加重，结肠炎症浸润明显增加(图10A-F)。
与之对应的是，相对于对照组IGF1R fl/fl小鼠，特异性敲除IGF1R的IGF1R fl/flLyz2 Cre小鼠对于炎症性肠病更加抵抗，无论在PBS处理组还是高剂量 IGF2(H-IGF2，1000ng每只小鼠)治疗组，小鼠体重都显著高于对照组IGF1R fl/fl小鼠在对应治疗条件下的体重，小鼠存活明显延长，粪便评分和便血程度明显减轻，结肠炎症浸润显著下降(图10G-L)。
以上实验结果表明，在DSS诱导的炎症性肠病小鼠模型中，IGF2的治疗依然依赖于IGF2R来执行抗炎表型的塑造，而IGF1R激活的信号则促进了巨噬细胞在炎症性肠病中的促炎症表型。
5.7 Leu27-IGF2靶向激活IGF2R能够最为有效地抑制腹膜炎和炎症性肠病
为了方便对于IGF2R和IGF1R功能的研究，已经有很多IGF2突变体被设计和报道出来，这其中就包括了可以选择性结合IGF2R的IGF2突变体。研究人员将IGF2肽段的第27位酪氨酸突变为亮氨酸而制备了Leu27-IGF2突变体(L27-IGF2)，Leu27-IGF2突变体与IGF2R以及IGFBP蛋白的亲和力不变，与IGF1R的亲和力极显著降低。
与野生型IGF2相比，Leu27-IGF2可以显著降低腹膜炎小鼠腹腔单核细胞和腹腔巨噬细胞的细胞质相对pH，甚至在2000ng/ml的高剂量下，我们也对比了低剂量下Leu27-IGF2的对于细胞质pH的改变效果，排除了Leu27-IGF2效价低的可能性(图11A)。在腹膜炎的研究中，我们发现，低剂量Leu27-IGF2和高剂量Leu27-IGF2处理后巨噬细胞，在响应LPS刺激时均表现为一氧化氮(图11B)和乳酸(图11C)产生的显著下降。同时，低、高剂量Leu27-IGF2处理巨噬细胞表现为IL-1β水平显著抑制，PD-L1水平明显增加(图11D-E)。这说明，低剂量Leu27-IGF2和高剂量Leu27-IGF2都可以赋予腹膜炎小鼠的腹腔巨噬细胞具备抗炎表型。
同样的，在DSS诱导的炎症性肠病小鼠模型中，低、高剂量Leu27-IGF2都可以显著抑制炎症性肠病小鼠的体重降低，提高小鼠存活(图11F-G)。从这些小鼠的粪便评分、便血情况以及结肠长度也可以反映出低、高剂量Leu27-IGF2对炎症性肠病的治疗效果(图11H-J)。综合上述结果，我们发现Leu27-IGF2有效抑制炎症的阈值相对于野生型IGF2提高了20倍以上(图11K)。
在这一部分研究中，我们验证了IGF2在DSS诱导的炎症性肠病小鼠模型中的治疗作用。确认了低剂量IGF2有效抑制炎症性肠病依赖于与低剂量IGF2治疗腹膜炎相同的作用机制，在这两种验证模型中，IGF2R依赖的途径都可以 赋予巨噬细胞显著地抑制炎症的免疫记忆。IGF2突变体靶向激活IGF2R，发挥最稳定的抑制炎症的效果。
5.8 IGF2突变体对炎症性肠病的治疗作用
Human Leu27-IGF2是IGF-2的类似物，在人类IGF2序列的27位以亮氨酸替代酪氨酸。与IGF2相比，Leu27-IGF2与血清中IGF结合蛋白(IGFBPs)的结合能力不变，但与IGF1受体(IGF1R)的结合能力降低了10～20倍，因此Leu27-IGF2与IGF2受体(IGF2R)结合的特异性增强。IGF2肽段由A、B、C、D四个结构域(Domain)组成，D结构域(62-67位氨基酸)缺失将显著增强其与IGFBP2、IGFBP3、IGFBP4以及IGFBP6的亲和力，而与IGFBP1、IGFBP5的亲和力维持不变。
鉴于IGF2与IGFBP结合可以延长IGF2半衰期，增强IGF2稳定性的特点，我们在Leu27-IGF2基础上删除D结构域，预期获得与IGF1R低亲和力的、IGF2R高选择性的、IGFBP高亲和力的、稳定性更强的IGF2类似物——Leu27-Des[62-67]-IGF2。本发明的研究发现，低剂量(1ng每只小鼠)和高剂量(5ng每只小鼠)Leu27-Des[62-67]-IGF2均可以显著抑制DSS诱导炎症性肠病引起小鼠体重下降，并且明显延长炎症性肠病小鼠存活(图12A，B)。
5.9激活IGF2R促进其核转运，调控巨噬细胞细胞质H +浓度
研究发现，IGF2可以减少THP1细胞细胞膜表面IGF2R的分布，呈现剂量依赖特性，从5ng/ml的低剂量开始，至1000ng ml高剂量为止，细胞膜表面IGF2R减少达到最大化，继续增加IGF2剂量，细胞膜表面IGF2R不再继续减少(图13A，B)。利用western blotting，研究明确IGF2处理THP1细胞后，IGF2R在细胞核中的分布明显增加(图13C)。以Ivermectin阻断IGF2R的核转运，可以抑制IGF2R在细胞核中的分布(图13D)。同时，阻断IGF2R的核转运可以抑制低剂量IGF2诱导的巨噬细胞细胞质中H +浓度的显著增加(图13E)。此外，研究发现，Leu27-Des[62-67]-IGF2可以显著下调IGF2R在THP1细胞上的表达(图13F)。因此，靶向性激活IGF2R可以促进其核转运，促进抗炎巨噬细胞的形成，从而抗炎。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献 被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
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Claims (18)

  1. 一种非IGF1R结合型的物质的用途，其特征在于，用于制备组合物或制剂，所述组合物或制剂用于预防和/或治疗炎症性疾病。
  2. 如权利要求1所述的用途，其特征在于，所述非IGF1R结合型的物质选自下组：IGF2突变体、表达IGF2突变体的载体、抗体、小分子化合物、或其组合。
  3. 如权利要求1所述的用途，其特征在于，所述炎症性疾病选自下组：腹膜炎、炎症性肠病、多发性硬化、糖尿病、系统性红斑狼疮、硬皮病、桥本甲状腺炎、自身免疫性肝炎、自身免疫葡萄膜炎、间质性肺病、银屑病、白癜风、皮肌炎、川崎病、成人耳氏病、强制性脊柱炎、类肉状瘤病、起止点炎相关的关节炎、多关节青少年特发性关节炎、类风湿性关节炎、移植物抗宿主病、自身免疫性胰腺炎、帕金森病、老年痴呆、或其组合。
  4. 如权利要求1所述的用途，其特征在于，所述非IGF1R结合型的物质包括对IGF2R的选择性或亲和力高于IGF1R的物质，以及可以高效激活IGF2R的不激活或基本不激活IGF1R的物质。
  5. 如权利要求2所述的用途，其特征在于，所述IGF2突变体在野生型IGF2蛋白的对应于SEQ ID NO.:1的第27位的酪氨酸发生突变。
  6. 如权利要求2所述的用途，其特征在于，所述IGF2突变体在野生型IGF2蛋白的对应于SEQ ID NO.:1的第27位的酪氨酸突变为亮氨酸，并任选的删除D-domain(SEQ ID NO.:1的第62位苏氨酸至第67位谷氨酸)。
  7. 如权利要求2所述的用途，其特征在于，所述IGF2突变体选自下组：
    (a)具有SEQ ID NO.:2-58任一所示氨基酸序列的多肽；
    (b)将SEQ ID NO.:2-58任一所示氨基酸序列经过一个或多个(如2个、3个、4个或5个)氨基酸残基的取代、缺失或添加而形成的，且具有抑制炎症活性的由(a)衍生的多肽。
  8. 一种细胞制剂，其特征在于，包括：
    经过非IGF1R结合型的物质处理的吞噬细胞。
  9. 如权利要求8所述的细胞制剂，其特征在于，所述吞噬细胞选自下组：单核细胞、巨噬细胞、单核细胞前体细胞、或其组合。
  10. 如权利要求8所述的细胞制剂，其特征在于，所述的组合物中，所述吞噬细胞的浓度1×10 4-5×10 7/ml，较佳地5×10 4-5×10 6/ml，更佳地 1×10 5-1×10 6/ml。
  11. 一种药盒，其特征在于，包括：
    (i)第一容器，以及装于该第一容器中的活性成分(a)经过非IGF1R结合型的物质处理的吞噬细胞，或含有活性成分(a)的药物；
    (ii)任选的第二容器，以及装于该第二容器中的活性成分(b)非IGF1R结合型的物质，或含有活性成分(b)的药物；和
    (iii)说明书，所述说明书中记载了联合给予活性成分(a)和活性成分(b)从而预防和/或治疗炎症性疾病的说明。
  12. 一种筛药方法，其特征在于，包括步骤：
    (a)测试组中，在细胞的培养体系中添加测试物质，并观察所述测试组的细胞中所述测试物质与IGF1R和/或IGF2R的结合活性；在对照组中，在相同细胞的培养体系中不添加测试物质；
    其中，如果测试组的细胞中所述测试物质与IGF1R的结合活性降低；并且与IGF2R的结合活性不变或升高，则表明，所述测试物质为非IGF1R结合型的IGF2突变型。
  13. 一种筛选预防和/或治疗炎症性疾病的候选物的方法，其特征在于，包括：
    (a)测试组中，在细胞的培养体系中添加测试物质，并观察所述测试组的细胞中所述测试物质对IGF2R在细胞表面的表达和/或活性的影响；和/或所述测试物质对IGF2R核转运的影响；和/或所述测试物质对IGF2R向细胞核的重分布的影响；在对照组中，在相同细胞的培养体系中不添加测试物质；
    其中，如果测试组的所述测试物质抑制IGF2R在细胞表面的表达和/或活性；和/或所述测试物质增加IGF2R的核转运；和/或所述测试物质促进IGF2R向细胞核的重分布，则表明，所述测试物质为预防和/或治疗炎症性疾病的候选物。
  14. 一种筛药方法(或筛选候选物的方法)，其特征在于，包括：
    (a)测试组中，在细胞的培养体系中添加测试物质，并观察所述测试组的细胞中所述测试物质对IGF2与IGF1R和/或IGF2R的结合活性的影响；在对照组中，在相同细胞的培养体系中不添加测试物质；
    其中，如果测试组的细胞中所述测试物质抑制IGF2与IGF1R的结合活性；并且对IGF2与IGF2R的结合活性无影响或有促进作用，则表明，所述测试物质为非IGF1R结合型的物质(或候选物)。
  15. 一种体外获得促炎样本和抑炎样本的方法，其特征在于，包括：
    在第一组实验中，用低剂量的IGF2进行处理，获得抑炎样本；和
    在第二组实验中，用高剂量的IGF2进行处理，获得促炎样本。
  16. 一种组合物，其特征在于，包括：
    (a)权利要求12或13或14所述的方法获得的非IGF1R结合型的IGF2突变型或候选物；其中所述非IGF1R结合型的IGF2突变型不包括选自下组的IGF2突变型：野生型IGF2、野生型IGF2第25～91位氨基酸片段、12位谷氨酸突变为Asp、Ala、Gln、His、Arg、或Lys的IGF2突变型、26位苯丙氨酸突变为Ser的IGF2突变型、27位酪氨酸突变为Leu的IGF2突变型、43位缬氨酸突变为Leu的IGF2突变型、删除D-domain的IGF2突变型、或其组合。
  17. 一种抑制炎症活性的方法，其特征在于，包括步骤：
    在非IGF1R结合型的物质(或具有IGF2R选择性的候选物)存在下，培养吞噬细胞，从而抑制炎症活性。
  18. 一种权利要求8所述的细胞制剂、权利要求11所述的药盒或权利要求16所述的组合物的用途，其特征在于，用于制备用于预防和/或治疗炎症性疾病的药物。
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