Chimeric cytokine receptor, product and application thereof, macrophage and application of macrophage in inflammation suppression and tissue repair

Abstract

The invention provides a chimeric cytokine receptor, a product and application thereof, a macrophage and application of the macrophage in inflammation suppression and tissue repair, and relates to the technical field of biology. According to the chimeric cytokine receptor provided by the invention, a structural domain combined with rapamycin is fused with cytokine receptors IL4Ra and IL13Ra, so that macrophages expressing the chimeric cytokine receptor can be polarized towards an M2 sample under the action of rapamycin, and the chimeric cytokine receptor plays roles in inhibiting immune response, reducing the inflammation level and promoting the repair of organs in a damaged group; therefore, the purpose of treating autoimmune diseases is achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a chimeric cytokine receptor and its product and application, macrophages and their application in anti-inflammatory and tissue repair. BACKGROUND

[0002] Autoimmune diseases are a class of diseases in which the immune system mistakenly attacks the body's own tissues, affecting a wide range of and complex, involving multiple organ systems. At present, the etiology of most autoimmune diseases has not been fully elucidated, and current treatment drugs such as non-steroidal anti-inflammatory drugs, glucocorticoids and immunosuppressants have greater side effects in use, and have no effect in some populations, so autoimmune diseases still urgently need to explore more effective and feasible treatment options. Macrophages are an important part of the innate immune system, and exist in almost all tissues and organs of the body. Macrophages play an important role in the progression of autoimmune diseases, and abnormal activation of macrophages is one of the pathogenic factors of certain autoimmune diseases. Therefore, editing macrophages for the treatment of autoimmune diseases has great potential.

[0003] In view of this, the present application is proposed. SUMMARY

[0004] The first object of the present application is to provide a chimeric cytokine receptor to achieve artificial controllable bias of M2-like polarization of macrophages.

[0005] The second object of the present application is to provide a nucleic acid.

[0006] The third object of the present application is to provide a vector.

[0007] The fourth object of the present application is to provide the use of the above-mentioned chimeric cytokine receptor, or nucleic acid, or vector in the preparation of controllable M2-like polarization of macrophages.

[0008] The fifth object of the present application is to provide a macrophage to solve the above technical problems.

[0009] The sixth object of the present application is to provide the use of the above-mentioned macrophage.

[0010] In order to achieve the above objects, the following technical solutions are adopted: In a first aspect, the present application provides a chimeric cytokine receptor, comprising a first chimeric receptor and a second chimeric receptor; The first chimeric receptor comprises an extracellular domain, a transmembrane region and an intracellular domain; wherein the extracellular domain comprises an FKBP protein, the transmembrane region is a transmembrane region of CD28 or a transmembrane region of a cytokine receptor IL-4Ra, and the intracellular domain is an intracellular domain of a cytokine receptor IL-4Ra; The second chimeric receptor comprises an extracellular domain, a transmembrane region and an intracellular domain; wherein the extracellular domain comprises the FRB protein, the transmembrane region is the transmembrane region of CD28 or the transmembrane region of the cytokine receptor IL-13Ra, and the intracellular domain is the intracellular domain of the cytokine receptor IL-13Ra.

[0011] As a further technical solution, the amino acid sequence of the FKBP protein is shown as SEQ ID NO. 1: GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO. 1).

[0012] The amino acid sequence of the transmembrane region of the CD28 is shown as SEQ ID NO. 2: FWALVVVAGVLFCYGLLVTVALCVIWT (SEQ ID NO. 2).

[0013] The amino acid sequence of the transmembrane region of the cytokine receptor IL-4Ra is shown as SEQ ID NO. 3: LPLGVTISCLCIPLFCLFCYFSIT (SEQ ID NO. 3).

[0014] The amino acid sequence of the intracellular domain of the cytokine receptor IL-4Ra is shown as SEQ ID NO. 4: (SEQ ID NO.4).

[0015] The amino acid sequence of the FRB protein is shown in SEQ ID NO.5: EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ (SEQ ID NO. 5).

[0016] The transmembrane amino acid sequence of the cytokine receptor IL-13Ra is shown in SEQ ID NO. 6: FYTTMLLTIPVFVAVAVIILLFYL (SEQ ID NO. 6).

[0017] The intracellular amino acid sequence of the cytokine receptor IL-13Ra is shown in SEQ ID NO.7: KRLKIIIFPPIPDPGKIFKEMFGDQNDDTLHWKKYDIYEKQSKEETDSVVLIENLKKAAP (SEQ ID NO. 7).

[0018] As a further technical solution, the N-terminal of the first chimeric receptor further comprises a signal peptide, and the amino acid sequence of the signal peptide is as shown in SEQ ID NO. 8. The N-terminal of the second chimeric receptor further comprises a signal peptide, and the amino acid sequence of the signal peptide is as shown in SEQ ID NO. 8. METDTLLLWVLLLWVPGSTGD (SEQ ID NO. 8).

[0019] As a further technical solution, from N-terminal to C-terminal, the extracellular domain of the first chimeric receptor comprises FKBP protein and fibronectin type III domain in sequence; the amino acid sequences of the FKBP protein and the fibronectin type III domain are as shown in SEQ ID NO. 9: APDNLTLHTNVSDEWLLTWNNLYPSNNLLYKDLISMVNISREDNPAEFIVYNVTYKEPRLSFPINILMSGVYYTARVRVRSQILTGTWSEWSPSITWYN (SEQ ID NO. 9).

[0020] From N-terminal to C-terminal, the extracellular domain of the second chimeric receptor comprises FRB protein and fibronectin type III domain in sequence; the amino acid sequences of the FRB protein and the fibronectin type III domain are as shown in SEQ ID NO. 10: KPDPPHIKHLLLKNGALLVQWKNPQNFRSRCLTYEVEVNNTQTDRHNILEVEEDKCQNSESDRNMEGTSCFQLPGVLADAVYTVRVRVKTNKLCFDDNKLWSDWSEAQSIGKE (SEQ ID NO. 10).

[0021] In a second aspect, the present application provides a nucleic acid encoding the chimeric cytokine receptor.

[0022] As a further technical solution, the sequence of the nucleic acid is as shown in SEQ ID NO. 11, SEQ ID NO. 12 or SEQ ID NO. 13.

[0023] In a third aspect, the present application provides a vector carrying the nucleic acid.

[0024] In a fourth aspect, the present application provides the use of the chimeric cytokine receptor, or the nucleic acid, or the vector in the preparation of a controllable M2-like polarized macrophage.

[0025] In a fifth aspect, the present application provides a macrophage, which carries the nucleic acid, or contains the vector, or expresses the chimeric cytokine receptor.

[0026] In a sixth aspect, the present application provides the use of the macrophage in any one of a-c; a. preparation of a product for reducing inflammation; b. preparation of a product for promoting repair of damaged tissue; c. preparation of a product for treating autoimmune diseases.

[0027] Compared with the prior art, the present application has the following beneficial effects: The chimeric cytokine receptor provided by the present application fuses the domain combined with rapamycin with the cytokine receptors IL4Ra and IL13Ra, so that the macrophage expressing the chimeric cytokine receptor can be biased towards M2-like polarization under the action of rapamycin, thereby playing a role in inhibiting immune response, reducing inflammation level, and promoting repair of damaged tissue, so as to achieve the purpose of treating autoimmune diseases. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed in the specific embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0029] Figure 1 : Schematic diagram of rapamycin-activated cytokine receptor R413 vector and mechanism; A. Schematic diagram of R413 vector; B. Schematic diagram of natural IL-4 / IL-13 receptor activation and R413 activation; Figure 2 : R413 receptor structure screening; A. Schematic diagram of each version of R413 vector; B. and C. Detection of the activation of STAT6 phosphorylation and CD206 of each version of vector after the addition of rapamycin; Figure 3 : M2-like polarization of macrophages overexpressing R413 under the action of rapamycin; A. Stat6 phosphorylation of macrophages overexpressing R413 under the action of rapamycin; B. Stat6 phosphorylation statistics; C. Activation of M2 polarization-related genes of macrophages overexpressing R413 under the action of rapamycin; Figure 4 Adoptive transfer of R413-expressing macrophages for treatment of autoimmune hepatitis; A. Experimental flow chart; B. Survival rate statistics of experimental mice; C. Detection of serum glutamic-oxalacetic transaminase (AST) and glutamic-pyruvic transaminase (ALT) levels of experimental mice; D. Detection of inflammatory factors IL-6, IFN-γ and TNFα levels in serum of experimental mice; E. Detection of liver tissue cell apoptosis of experimental mice; Figure 5 Adoptive transfer of R413-expressing macrophages for treatment of inflammatory bowel disease; A. Experimental flow chart; B. Statistics of body weight changes of experimental mice; C. Statistics of disease index of experimental mice; Figure 6 Detection of colon length and permeability of experimental mice; A. Representative pictures of colon of experimental mice; B. Statistics of colon length of experimental mice; C. Detection of intestinal permeability of experimental mice; Figure 7 Detection of physiological structure of colon of experimental mice by HE; Figure 8 Detection of tissue regeneration ability of colon of experimental mice after treatment by immunofluorescence staining; Figure 9 Detection of expression levels of inflammatory factors in colon tissue of experimental mice; Figure 10 Adoptive transfer of R413-expressing macrophages for treatment of SLE; Figure 11 Detection of urine protein creatinine ratio (A) and anti-double-stranded DNA antibody levels in serum (B) of experimental mice; Figure 12 Detection of IgG, IgM and C3 deposition levels in kidney of experimental mice; Figure 13 Detection of glomerular cell structure in kidney of experimental mice; Figure 14 Detection of glomerular cell ultrastructure in kidney of experimental mice by electron microscopy. DETAILED DESCRIPTION

[0030] The embodiments of the present application will be described in detail below with reference to the embodiments and examples, but those skilled in the art will understand that the following embodiments and examples are only used to illustrate the present application, and should not be regarded as limiting the scope of the present application. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without making creative efforts shall fall within the scope of protection of the present application. If the specific conditions are not specified, the conventional conditions or the conditions recommended by the manufacturer are used. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased on the market.

[0031] Terminology: Macrophages, abbreviated as Mø, are a type of white blood cell found in tissues that develop from monocytes, which in turn develop from precursor cells in the bone marrow. Both macrophages and monocytes are phagocytes and participate in both nonspecific (innate) and specific (cell-mediated) immunity in vertebrates. Their main function is to engulf and digest cellular debris and pathogens, acting in either a fixed or free state, and to activate lymphocytes and other immune cells to respond to pathogens.

[0032] Macrophage polarization: Macrophages are an important part of the innate immune system and play a central role in inflammation and host defense. In response to various environmental factors (e.g., microbial products, damaged cells, activated lymphocytes) or under different pathophysiological conditions, macrophages transform into different functional phenotypes, i.e., classically activated macrophages (M1) and alternatively activated macrophages (M2). Mature macrophages undergo phenotypic and morphological differentiation under various factors, i.e., the polarization of macrophages. According to the different responses to environmental stimuli, macrophages are mainly activated into two phenotypes, M1 and M2. M1 is activated by signals such as IFN-γ and LPS, mainly has the effects of anti-tumor and enhancing immunity, can secrete inflammatory factors, chemotactic factors, effector molecules and TNF-α, etc., among which membrane molecules CD80, surface markers CD64, etc. are representative. M2 is activated by factors such as IL-4 and IL-13, mainly has the potential of inhibiting immune response, promoting angiogenesis, tissue repair and promoting tumor growth, more secretes IL-10, TGF-β, VEGF, etc. Factors, with relatively high expression of ARG1, CD163 and CD206.

[0033] Autoimmune disease: Autoimmune diseases are a class of diseases caused by the abnormal attack of the immune system on normal tissues and organs of the body. Including autoimmune liver disease, inflammatory bowel disease and systemic lupus erythematosus (SLE), etc.

[0034] Rapamycin: A macrolide compound, it was first isolated from Streptomyces in the soil of Easter Island, and was found to have antifungal effects, and later found to have immunosuppressive function. And in 1999, it was approved by FDA as an immunosuppressant for use in reducing the anti-rejection reaction after kidney transplantation.

[0035] In a first aspect, the present application provides a chimeric cytokine receptor comprising a first chimeric receptor and a second chimeric receptor; The first chimeric receptor comprises an extracellular domain, a transmembrane region and an intracellular domain; wherein the extracellular domain comprises an FKBP protein, the transmembrane region is a transmembrane region of CD28 or a transmembrane region of a cytokine receptor IL-4Ra, and the intracellular domain is an intracellular domain of the cytokine receptor IL-4Ra. The second chimeric receptor comprises an extracellular domain, a transmembrane region and an intracellular domain; wherein the extracellular domain comprises an FRB protein, the transmembrane region is a transmembrane region of CD28 or a transmembrane region of a cytokine receptor IL-13Ra, and the intracellular domain is an intracellular domain of the cytokine receptor IL-13Ra.

[0036] The chimeric cytokine receptor provided by the application fuses the domain combined with rapamycin with the cytokine receptors IL4Ra and IL13Ra, so that the macrophages expressing the chimeric cytokine receptor can be biased to M2-like polarization under the action of rapamycin, thereby playing a role in inhibiting immune response, reducing inflammation level, and promoting repair of damaged organs, so as to achieve the purpose of treating autoimmune diseases.

[0037] In some optional embodiments, the amino acid sequence of the FKBP protein is as shown in SEQ ID NO. 1. The amino acid sequence of the transmembrane region of the CD28 is as shown in SEQ ID NO. 2. The amino acid sequence of the transmembrane region of the cytokine receptor IL-4Ra is as shown in SEQ ID NO. 3. The amino acid sequence of the intracellular domain of the cytokine receptor IL-4Ra is as shown in SEQ ID NO. 4. The amino acid sequence of the FRB protein is as shown in SEQ ID NO. 5. The amino acid sequence of the transmembrane region of the cytokine receptor IL-13Ra is as shown in SEQ ID NO. 6. The amino acid sequence of the intracellular domain of the cytokine receptor IL-13Ra is as shown in SEQ ID NO. 7.

[0038] In some optional embodiments, the N-terminal of the first chimeric receptor further comprises a signal peptide, and the amino acid sequence of the signal peptide is as shown in SEQ ID NO. 8. The N-terminal of the second chimeric receptor further comprises a signal peptide, and the amino acid sequence of the signal peptide is as shown in SEQ ID NO. 8.

[0039] In some optional embodiments, from N-terminal to C-terminal, the extracellular domain of the first chimeric receptor comprises an FKBP protein and a fibronectin type III domain in sequence; and the amino acid sequences of the FKBP protein and the fibronectin type III domain are as shown in SEQ ID NO. 9. The extracellular domain of the second chimeric receptor comprises, in order from N-terminus to C-terminus, an FRB protein and a fibronectin type III domain; the amino acid sequences of the FRB protein and the fibronectin type III domain are shown in SEQ ID NO. 10.

[0040] The inventors have found that macrophages containing the chimeric cytokine receptor with the fibronectin type III domain have higher STAT6 phosphorylation levels and CD206 expression levels after rapamycin activation, and have better effects.

[0041] In a second aspect, the present application provides a nucleic acid encoding the chimeric cytokine receptor.

[0042] In some alternative embodiments, the sequence of the nucleic acid is shown in SEQ ID NO. 11, SEQ ID NO. 12 or SEQ ID NO. 13.

[0043] In a third aspect, the present application provides a vector carrying the nucleic acid.

[0044] The vector in the present application refers to a vector capable of carrying nucleic acid, which can be a plasmid, for example.

[0045] In a fourth aspect, the present application provides the use of the above-mentioned chimeric cytokine receptor, or nucleic acid, or vector in the preparation of controllable M2-like polarized macrophages.

[0046] In a fifth aspect, the present application provides a macrophage carrying the nucleic acid, or containing the vector, or expressing the chimeric cytokine receptor.

[0047] The macrophage can be biased towards M2-like polarization under the action of rapamycin, and can play a role in inhibiting immune response, reducing inflammation level, and promoting repair of damaged organs, thereby achieving the purpose of treating autoimmune diseases.

[0048] In a sixth aspect, the present application provides the use of the above-mentioned macrophage in any one of a-c; a. preparation of a product for reducing inflammation; b. preparation of a product for promoting repair of damaged tissue; c. preparation of a product for treating autoimmune diseases.

[0049] The present application will be further described below through specific examples, but it should be understood that these examples are only for more detailed description and should not be understood as limiting the present application in any form.

[0050] Example 1 I. Design principle A cytokine receptor (named R413) for directed M2-like polarization in macrophages was constructed, with the specific structure shown below. Figure 1 The diagram shows a combination of two chimeric receptors: one (FKBP-IL4Ra) has an extracellular domain of the rapamycin-binding protein FKBP and an intracellular domain of the cytokine receptor IL-4Ra; the other (FRB-IL13Ra) has an extracellular domain of the rapamycin-binding protein FRB and an intracellular domain of the cytokine receptor IL-13Ra. Under the influence of rapamycin, they form a heterodimer, mimicking the interaction of the natural cytokines IL-4 / IL-13 with their receptors, activating Stat6 phosphorylation and activating macrophage M2-like polarization-related genes.

[0051] II: Screening of R413 receptor structure To screen for structures capable of controlled polarization, we constructed three versions of the R413 vector, named R413V1, R413V2, and R413V3, as follows: Figure 2 As shown. The transmembrane region of R413 V1 is the transmembrane region of CD28, while the transmembrane regions of R413 V2 and R413 V3 are the transmembrane regions of IL4Ra and IL13Ra, respectively. Compared with R413 V2, R413 V3 has an additional FN3 domain (fibronectin type III domain) in its extracellular region. To test the effectiveness of the three vectors, the experiment was conducted according to the following steps: Vector sequence structure: R413-V1 (sequence number SEQ ID NO.11): Sequence information: Bases 1-63: Signal peptide sequence (igK leader); Bases 64-384: FKBP sequence; 508-588: CD28 transmembrane region; 592-2250: IL4Ra intracellular region sequence; 2260-2313: T2A peptide sequence; 2314-2376: Signal peptide sequence (igK leader); 2377-2646: FRB sequence; 2770-2850: CD28 transmembrane region; 2854-3033: IL13Ra intracellular region.

[0052] R413-V2 (sequence number SEQ ID NO.12): Sequence information: Bases 1-63: Signal peptide sequence (igK leader); Bases 94-414: FKBP sequence; 430-501: Transmembrane region of IL4Ra; 502-2610: Intracellular region sequence of IL4Ra; 2170-2223: T2A peptide sequence; 2224-2286: signal peptide sequence (igK leader); 2287-2556: FRB sequence; 2572-2643: IL13Ra transmembrane region; 2644-2823: IL13Ra intracellular region.

[0053] R413-V3 (sequence is SEQ ID NO. 13): Sequence information: Base 1-63: signal peptide sequence (igK leader); 94-414: FKBP sequence; 430-762: FN3 domain; 754-825: IL4Ra transmembrane region; 826-2484: IL4Ra intracellular region sequence; 2494-2547: T2A peptide sequence; 2548-2610: signal peptide sequence (igK leader); 2611-2880: FRB sequence; 2896-3234: FN3 domain; 3247-3318: IL13Ra transmembrane region; 3319-3498: IL13Ra intracellular region.

[0054] 1. Molecular cloning constructs a lentiviral vector expressing each structure, and prepares a lentivirus; 2. The lentivirus infects macrophages, and after screening to obtain cells expressing each structure, 50 nM rapamycin is added to the culture medium of the macrophages for 30 minutes, and then the degree of STAT6 phosphorylation is detected by flow cytometry; 48 hours after the addition of 50 nM rapamycin, the expression of CD206 is detected by flow cytometry. The STAT6 phosphorylation level and the CD206 expression level are compared, and the results are shown in Figure 2 .

[0055] The results show that R413 V2 and R413 V3 have higher STAT6 phosphorylation levels and CD206 expression levels, and R413 V3 is more effective, so R413 V3 is used for subsequent experiments.

[0056] Three: in vitro functional verification: after the macrophages overexpress R413, they can be polarized to M2-like under the action of rapamycin.

[0057] In order to verify that R413 makes macrophages M2 polarize under the action of rapamycin, macrophages expressing R413 and wild-type macrophages are selected as controls, rapamycin is added, STAT6 phosphorylation and CD206 expression are detected by flow cytometry, and the expression of M2-related marker genes Arg1, Mrc1, and Pparg1 is detected by QPCR, and the results are shown in Figure 3 .

[0058] Conclusion: After overexpression of R413 in macrophages, STAT6 phosphorylation is induced by rapamycin, and downstream genes are activated, leading to M2-like polarization.

[0059] Four: R413 overexpressing macrophages have therapeutic effects on autoimmune hepatitis.

[0060] Eight to ten-week-old male C57 / B6 mice were selected and injected with ConA at a dose of 15 mg / kg via the tail vein to construct an autoimmune hepatitis model. Two hours after modeling, R413-expressing macrophages (R413-Ms+RAPA) were injected via the tail vein. Other groups were as follows: unmodeled group, modeled mice injected with PBS (PBS), modeled mice injected with only rapamycin (RAPA), modeled mice injected with only wild-type macrophages (WT-Ms), and modeled mice injected with wild-type cells and rapamycin (WT-Ms+RAPA). Five mice were included in each group, and blood was collected 14 hours after modeling. Blood routine tests were performed to detect the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), which are indicative of liver damage. The levels of inflammatory factors in the serum were detected by Elisa. Mice died during the experiment, and 24 hours was the endpoint. Liver tissue sections were stained by immunofluorescence TUNEL to detect tissue cell apoptosis and necrosis. The results are shown in Figure 4

[0061] Conclusion: Adoptive transfer of R413 macrophages and injection of rapamycin can significantly improve the survival rate of mice with autoimmune hepatitis, reduce liver damage, reduce the levels of inflammatory factors, and reduce tissue necrosis.

[0062] Five: R413 overexpressing macrophages have therapeutic effects on inflammatory bowel disease Eight to ten-week-old male C57 / B6 mice were selected and injected with 3% DSS (sodium salt of dextran sulfate) added to water for 7 days. Cells and rapamycin were injected intraperitoneally on the third day of modeling. The experiment was divided into three groups: unmodeled, modeled and injected with PBS, and modeled and injected with macrophages and rapamycin treatment (WT-Ms+RAPA), with 10 mice in each group. During the experiment, indicators such as body weight, soft stool, and the degree of blood in stool were continuously monitored, and the disease index was calculated. On the tenth day, the mice were sacrificed. Four mice were given FITC-labeled dextran orally before being sacrificed to detect intestinal permeability, and BrdU was injected intraperitoneally to detect intestinal cell proliferation. After the mice were sacrificed, the colon tissue was removed to measure the length, and HE staining was performed on the sections to detect the structural integrity. Immunofluorescence staining was performed to detect the proportion of Ki67 and BrdU positive cells, which is indicative of tissue repair capacity. The colon tissue was removed, and RNA was extracted for qPCR to detect the expression levels of inflammation-related factors in the tissue.

[0063] The results are shown in Figures 5-9 ​​

[0064] Conclusion: As shown in Figure 5 Figure 6, adoptive transfer of R413 macrophages and injection of rapamycin significantly reduced the disease index of mice with intestinal inflammation.

[0065] As shown in Figure 6 Figure 7, mice treated with adoptive transfer of R413 macrophages had longer colons and lower permeability, indicating that the colon structure was more complete.

[0066] As shown in Figure 7 Figure 8, mice treated with adoptive transfer of R413 macrophages had more complete colon structure.

[0067] As shown in Figure 8 Figure 9, mice treated with adoptive transfer of R413 macrophages had higher proportion of Ki67 and BrdU double positive cells in the colon crypt, indicating that cell therapy promoted tissue repair ability.

[0068] As shown in Figure 9 Figure 10, mice treated with adoptive transfer of R413 macrophages had lower inflammation level in colon tissue.

[0069] Six: R413 overexpressing macrophages have therapeutic effect on SLE MRL / Lpr mice were selected as model mice (which spontaneously develop lupus phenotype), and the experiment was divided into 4 groups: MRL / MPJ control group (healthy control), model mice without treatment, CD20 antibody B cell deletion group (i.e. model mice were injected with anti-CD20 antibody through the tail vein, with an injection amount of 250 μg per mouse, to deplete B cells by antibody), and CD20 antibody B cell deletion and injection of R413 expressing macrophages and rapamycin (i.e. model mice were injected with anti-CD20 antibody through the tail vein, with an injection amount of 250 μg per mouse, and injected with R413 expressing macrophages and rapamycin). Antibodies were injected at 12 weeks, and cells and rapamycin were injected at 13 weeks. The survival of mice was observed during the period. At 19 weeks of age, the experiment was terminated, and urine was collected to detect urine protein creatinine ratio, blood was collected to detect anti-double stranded DNA antibody level in serum, kidney tissue was collected for immunofluorescence staining to detect IgG, IgM, C3 deposition level in kidney, and expression of vimentin and F-actin was detected, and electron microscopy was used to detect the ultrastructure of glomerular cells in the kidney of the experimental mice. The improvement of the pathological structure of the kidney of the mice was detected.

[0070] The results are shown in Figures 10-14 Figure 11.

[0071] Conclusion: As shown in Figure 10 Figure 12, adoptive transfer of R413 macrophages treatment improved the survival of mice.

[0072] As shown in Figure 11As shown, the level of kidney injury after adoptive transfer of R413 macrophages was reduced, and the level of anti-double stranded DNA antibody in serum was reduced.

[0073] As shown, the level of IgG, IgM, C3 deposition in kidney tissue of mice after cell therapy was reduced. Figure 12 As shown, the structure of kidney tissue of mice after cell therapy was more complete.

[0074] As shown, the structure of kidney tissue of mice after cell therapy was more complete. Figures 13-14 As shown, the structure of kidney tissue of mice after cell therapy was more complete.

[0075] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, rather than limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A chimeric cytokine receptor, characterized in that, Including the first chimeric receptor and the second chimeric receptor; The first chimeric receptor includes an extracellular domain, a transmembrane domain, and an intracellular domain; wherein the extracellular domain includes the FKBP protein, the transmembrane domain is the transmembrane domain of CD28 or the transmembrane domain of the cytokine receptor IL-4Ra, and the intracellular domain is the intracellular domain of the cytokine receptor IL-4Ra. The second chimeric receptor includes an extracellular domain, a transmembrane region, and an intracellular domain; wherein the extracellular domain includes the FRB protein, the transmembrane region is the transmembrane region of CD28 or the transmembrane region of the cytokine receptor IL-13Ra, and the intracellular domain is the intracellular domain of the cytokine receptor IL-13Ra.

2. The chimeric cytokine receptor according to claim 1, characterized in that, The amino acid sequence of the FKBP protein is shown in SEQ ID NO.1; The amino acid sequence of the transmembrane region of CD28 is shown in SEQ ID NO.2; The amino acid sequence of the transmembrane region of the cytokine receptor IL-4Ra is shown in SEQ ID NO.3; The amino acid sequence of the intracellular domain of the cytokine receptor IL-4Ra is shown in SEQ ID NO.4; The amino acid sequence of the FRB protein is shown in SEQ ID NO.5; The transmembrane amino acid sequence of the cytokine receptor IL-13Ra is shown in SEQ ID NO. 6; The intracellular domain amino acid sequence of the cytokine receptor IL-13Ra is shown in SEQ ID NO.

7.

3. The chimeric cytokine receptor according to claim 1, characterized in that, The N-terminus of the first chimeric receptor also includes a signal peptide, the amino acid sequence of which is shown in SEQ ID NO.8; The N-terminus of the second chimeric receptor also includes a signal peptide, the amino acid sequence of which is shown in SEQ ID NO.

8.

4. The chimeric cytokine receptor according to claim 1, characterized in that, From the N-terminus to the C-terminus, the extracellular domain of the first chimeric receptor sequentially includes an FKBP protein and a fibronectin type III domain; the amino acid sequences of the FKBP protein and the fibronectin type III domain are shown in SEQ ID NO.9; From the N-terminus to the C-terminus, the extracellular domain of the second chimeric receptor sequentially includes an FRB protein and a fibronectin type III domain; the amino acid sequences of the FRB protein and the fibronectin type III domain are shown in SEQ ID NO.

10.

5. A nucleic acid, characterized in that, The nucleic acid encodes the chimeric cytokine receptor as described in any one of claims 1-4.

6. The nucleic acid according to claim 5, characterized in that, The sequence of the nucleic acid is shown in SEQ ID NO.11, SEQ ID NO.12 or SEQ ID NO.

13.

7. A carrier, characterized in that, The vector carries the nucleic acid as described in claim 5 or 6.

8. The use of the chimeric cytokine receptor according to any one of claims 1-4, or the nucleic acid according to claim 5 or 6, or the vector according to claim 7 in the preparation of controllable M2-like polarized macrophages.

9. A macrophage, characterized in that, The macrophage carries the nucleic acid as described in claim 5 or 6, or contains the vector as described in claim 7, or expresses the chimeric cytokine receptor as described in any one of claims 1-4.

10. The use of the macrophages of claim 9 in any of the following a c; a. Prepare products that reduce inflammation; b. Prepare products that promote the repair of damaged tissues; c. Prepare products for the treatment of autoimmune diseases.

Images