MZ B cell overexpressing Hspa13 gene and application thereof

Abstract

The invention provides the MZ B cell overexpressing the Hspa13 gene and the application of the MZ B cell overexpressing the Hspa13 gene, the MZ B cell overexpressing the Hspa13 gene has the effect of promoting CD4 + T cells to be differentiated into regulatory T cells and also has the effect of treating systemic lupus erythematosus, a foundation is laid for further research on the function of the Hspa13 gene in the future, and a new direction is provided for treating systemic lupus erythematosus.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular, relates to MZ B cells overexpressing Hspa13 gene and applications thereof. Background Art

[0002] Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by T-cell-dependent B lymphocyte hyperfunction, the production of multiple autoantibodies and the formation of immune complexes, leading to multi-system damage. The disease is still difficult to cure because the pathogenesis is not clear. Some patients may still deteriorate even after stable maintenance treatment, and the prognosis is poor. Kidney damage is an important factor affecting the prognosis of systemic lupus erythematosus. End-stage renal failure caused by lupus kidney is one of the main causes of death in SLE patients. Lupus nephritis is the most common secondary glomerulonephritis in China. Patients may show corresponding nephritis or nephrotic syndrome manifestations such as massive proteinuria, increased blood creatinine, hematuria, etc. The most commonly used classification method for lupus nephritis is the WHO classification scheme, including mesangial, local proliferative, membranous nephropathy, and diffuse proliferative nephropathy. The incidence of the first three in lupus nephritis patients is about 15% each, and the latter is 50%. A small number of cases also show sclerotic nephropathy. Literature research shows that most lupus nephritis patients die within 2 years after diagnosis, and the survival curve after 3 years is relatively flat, with 1-year, 5-year, and 10-year overall survival rates of 88.3%, 74.5%, and 60.2%, respectively. Hypertension is a key factor affecting the survival rate of lupus nephritis patients. It can be seen that the average survival of lupus nephritis patients is lower than that of SLE patients. Nowadays, with the continuous maturity of renal replacement therapy such as hormones, immunosuppressants, cyclophosphamide shock therapy, dialysis, kidney transplantation, plasma exchange, etc., rational use of drugs, and active improvement of kidney lesions, the prognosis and survival of lupus nephritis patients are gradually improved.

[0003] The Hspa13 gene is member 13 of the heat shock protein family A (Hsp70), which encodes a member of the heat shock protein 70 family and is associated with microsomes. It is involved in the processing of cytosolic and secretory proteins, and the removal of denatured or misfolded proteins. Studies have shown that the Hspa13 gene is associated with a variety of diseases such as Bartter syndrome type 1 and Hyperprostaglandin E syndrome type 1; however, the relationship between the Hspa13 gene and systemic lupus erythematosus nephritis is still unclear. Summary of the invention

[0004] In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide an MZ B cell overexpressing the Hspa13 gene and its application.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides a method for regulating CD4 + Application in differentiation of T cells into regulatory T cells.

[0007] Furthermore, the MZ B cells overexpressing the Hspa13 gene have the ability to promote CD4 + The role of T cells in differentiating into regulatory T cells.

[0008] Furthermore, the MZ B cells overexpressing the Hspa13 gene have the ability to promote CD4 + The differentiation of T cells into regulatory T cells was confirmed by the following method: MZ B cells overexpressing the Hspa13 gene were combined with CD4 + T cells were co-cultured at a ratio of 1:1; after the culture, the cells were collected and flow stained to analyze the proportion of regulatory T cells.

[0009] Furthermore, the method for constructing MZ B cells overexpressing the Hspa13 gene includes: stimulating MZ B cells with LPS, PMA and lonomycin.

[0010] Furthermore, the MZ B cells were stimulated with LPS and cultured for 72 hours, and PMA and lonomycin were added in the last 5 hours before the end of the 72-hour culture.

[0011] Furthermore, the cell suspension density was adjusted to 1×10 6 Pieces / ml.

[0012] Furthermore, the concentrations of LPS, PMA and lonomycin are 10 ug / ml, 50 ng / ml and 1 ug / ml respectively.

[0013] Furthermore, the culture medium of the MZ B cells includes MEM culture medium, DMEM culture medium, and RPMI-1640 culture medium.

[0014] Preferably, the culture medium of the MZ B cells is RPMI-1640 culture medium.

[0015] In some embodiments, the MZ B cells are a subpopulation of B cells located in the marginal zone at the junction of the red and white pulp of the spleen, accounting for approximately 20% of the human peripheral B cells, and are the first line of defense against blood-borne pathogen infection. MZ B cells are non-T cell dependent and can independently produce humoral immune responses without relying on T cells. This characteristic makes them particularly important in the early stages of infection. In addition, MZ B cells can rapidly differentiate into plasma cells and secrete IgM antibodies, which play a key role in neutralizing a variety of pathogens. In addition to directly producing antibodies, MZ B cells can also activate T cells through antigen presentation, thereby further enhancing the immune response. Studies in recent years have found that MZ B cells also have the ability to "steal" the functions of dendritic cells, obtain the MHC molecules of dendritic cells through cytophagy, and then communicate with T cells to trigger an immune response.

[0016] In some embodiments, the LPS refers to lipopolysaccharide, which is an endotoxin mainly composed of the cell wall of Gram-negative bacteria. In immunological research, LPS is often used as a stimulant to activate immune cells and induce them to produce inflammatory responses.

[0017] In some embodiments, the PMA refers to phorbol ester, which is an organic compound that is carcinogenic and can activate protein kinase C (PKC); in cell biology research, PMA is often used as a stimulator to activate a variety of cell types, including T cells and B cells; PMA can simulate the signal transduction process within the cell, thereby studying the mechanism of cell activation and proliferation.

[0018] In some embodiments, the lonomycin refers to ionomycin, which is a calcium ion carrier that can increase the concentration of intracellular calcium ions; in immunological research, lonomycin is often used as a stimulator in combination with PMA to activate T cells and induce them to produce cytokines.

[0019] In a specific embodiment of the present invention, using LPS+PMA+lonomycin to stimulate MZ B cells can increase the expression level of the Hspa13 gene in the MZ B cells, thereby constructing MZ B cells that overexpress the Hspa13 gene.

[0020] In some embodiments, the MZ B cell culture medium includes, but is not limited to, MEM culture medium, DMEM culture medium, and RPMI-1640 culture medium. MEM culture medium is suitable for a variety of cells grown in monolayers, DMEM culture medium is suitable for high-density suspension cell culture, and RPMI-1640 culture medium is widely used in the culture of suspension cells. In a specific embodiment of the present invention, RPMI-1640 culture medium is selected to culture MZ B cells.

[0021] In some embodiments, CD4 + T cells are effector T cells that differentiate from the initial T cells in peripheral lymphoid tissue after being attacked by antigens. They are mainly involved in cellular immunity. + T cells can be activated by antigen-MHC complexes and differentiate into a variety of specific subtypes, including regulatory T cells (Tregs). These cell subsets play different roles in the body's immune response and immune regulation.

[0022] The regulatory T cells (Treg) are a type of T cell subpopulation with immunosuppressive function, and their main function is to maintain immune homeostasis by suppressing excessive immune responses. Among them, induced regulatory T cells (iTreg) are peripheral CD4 + Differentiated from T cells, they can inhibit the functions of antigen-specific T cells, B cells, and antigen-presenting cells (APCs), thereby regulating immune responses. iTreg cell surface markers are CD4 and CD25, and they mainly express inhibitory cytokines such as IL-10 and TGF-β, exerting immunosuppressive effects by secreting these cytokines.

[0023] In a specific embodiment of the present invention, experiments have shown that MZ B cells stimulated by LPS+PMA+lonomycin, i.e., MZ B cells overexpressing the Hspa13 gene, are related to CD4 + T cell co-culture can significantly increase the proportion of regulatory T cells, proving that MZ B cells overexpressing the Hspa13 gene have the ability to promote CD4 + The role of T cells in differentiating into regulatory T cells.

[0024] In the present invention, the MZ B cells are derived from CD19 cre Hspa13 fl / fl Mouse or Hspa13 fl / fl The Hspa13 fl / fl The mice were used as control mice because loxp sequences were inserted on both sides of the target gene Hspa13, but the expression of Hspa13 was not affected. cre Hspa13 fl / fl The mice carry Cre recombinase, which recognizes the loxp sequence sites on both sides of the Hspa13 gene and cuts and deletes the Hspa13 sequence in the middle of the loxp sites, thereby knocking out the expression of Hspa13 in B cells.

[0025] The second aspect of the present invention provides the use of MZ B cells overexpressing the Hspa13 gene in preventing and / or treating systemic lupus erythematosus or systemic lupus erythematosus-related diseases.

[0026] Furthermore, the systemic lupus erythematosus-related diseases include dermatitis, anemia, lupus nephritis, heart disease, pneumonia, neurological and mental diseases.

[0027] Preferably, the systemic lupus erythematosus-related disease is lupus nephritis.

[0028] Furthermore, the MZ B cells overexpressing the Hspa13 gene have the effect of treating lupus nephritis.

[0029] Furthermore, the effect of overexpressing the Hspa13 gene in treating lupus nephritis was verified by the following method: MZ B cells overexpressing the Hspa13 gene were infused into lupus mice through the tail vein. Two weeks after the infusion, the spleen of the mice was taken to prepare a lymphocyte suspension, which was subjected to flow staining to analyze the proportion of regulatory T cells; a part of the kidney of the mouse was taken for HE staining to analyze the pathological changes of the kidney, and the other part was used to detect the changes of inflammatory factors in the renal tissue using Real-time PCR.

[0030] Furthermore, the method for constructing MZ B cells overexpressing the Hspa13 gene includes: stimulating MZ B cells with LPS, PMA and lonomycin.

[0031] Furthermore, the MZ B cells were stimulated with LPS and cultured for 72 hours, and PMA and lonomycin were added in the last 5 hours before the end of the 72-hour culture.

[0032] Furthermore, the cell suspension density was adjusted to 1×10 6 Pieces / ml.

[0033] Furthermore, the concentrations of LPS, PMA and lonomycin are 10 ug / ml, 50 ng / ml and 1 ug / ml respectively.

[0034] Furthermore, the culture medium of the MZ B cells includes MEM culture medium, DMEM culture medium, and RPMI-1640 culture medium.

[0035] Furthermore, the culture medium of the MZ B cells is RPMI-1640 culture medium.

[0036] In a specific embodiment of the present invention, the diseased lupus mice are selected from MRL / lpr mice, which are important experimental animal models for autoimmune diseases such as systemic lupus erythematosus. They carry apoptosis-related mutations in the Fas gene (lpr gene), which lead to apoptosis defects in T cells, B cells and macrophages, and then induce symptoms such as T cell hyperplasia, systemic lymphadenopathy, arthritis and immune complex glomerulonephritis.

[0037] In a specific embodiment of the present invention, it is experimentally demonstrated that from control mice (Hspa13 fl / fl MZ B cells obtained from the spleen of MRL / lpr mice were stimulated with LPS+PMA+lonomycin, which significantly increased the proportion of protective regulatory T cells in the spleen of MRL / lpr mice and significantly reduced the inflammatory cell infiltration and inflammatory factor levels in the renal arterioles and paravascular stroma of MRL / lpr mice. cre Hspa13 fl / fl MZ B cells sorted from mouse spleen had no significant effect.

[0038] The third aspect of the present invention provides a cell therapy composition for preventing and / or treating systemic lupus erythematosus or systemic lupus erythematosus-related diseases.

[0039] Furthermore, the cell therapy composition includes MZ B cells overexpressing the Hspa13 gene.

[0040] The term "prevention" as used herein means preventing the occurrence of systemic lupus erythematosus in a subject at risk of the disease or the recurrence of a disappeared systemic lupus erythematosus. The term "treatment" means controlling, alleviating or relieving the pathological progression of systemic lupus erythematosus.

[0041] The fourth aspect of the present invention provides an MZ B cell overexpressing the Hspa13 gene or the cell therapy composition according to the third aspect of the present invention for use in the preparation of a medicament for treating systemic lupus erythematosus or a disease related to systemic lupus erythematosus or in the preparation of an in vitro promoter for CD4 + Application of reagents for differentiating T cells into regulatory T cells.

[0042] Furthermore, the systemic lupus erythematosus-related diseases include dermatitis, anemia, lupus nephritis, heart disease, pneumonia, neurological and mental diseases.

[0043] Furthermore, the method for constructing MZ B cells overexpressing the Hspa13 gene includes: stimulating MZ B cells with LPS, PMA and lonomycin.

[0044] Furthermore, the MZ B cells were stimulated with LPS and cultured for 72 hours, and PMA and lonomycin were added in the last 5 hours before the end of the 72-hour culture.

[0045] Furthermore, the cell suspension density was adjusted to 1×10 6 Pieces / ml.

[0046] Furthermore, the concentrations of LPS, PMA and lonomycin are 10ug / ml, 50ng / ml and 1ug / ml respectively.

[0047] Furthermore, the culture medium of the MZ B cells includes MEM culture medium, DMEM culture medium, and RPMI-1640 culture medium.

[0048] Furthermore, the culture medium of the MZ B cells is RPMI-1640 culture medium.

[0049] A fifth aspect of the present invention provides an in vitro non-therapeutic destination promoting CD4 + Methods for differentiation of T cells into regulatory T cells.

[0050] Furthermore, the method comprises: combining MZ B cells overexpressing the Hspa13 gene with CD4 + T cell co-culture.

[0051] Furthermore, the method for constructing MZ B cells overexpressing the Hspa13 gene includes: stimulating MZ B cells with LPS, PMA and lonomycin.

[0052] Furthermore, the MZ B cells were stimulated with LPS and cultured for 72 hours, and PMA and lonomycin were added in the last 5 hours before the end of the 72-hour culture.

[0053] Furthermore, the cell suspension density was adjusted to 1×10 6 Pieces / ml.

[0054] Furthermore, the concentrations of LPS, PMA and lonomycin are 10 ug / ml, 50 ng / ml and 1 ug / ml respectively.

[0055] Furthermore, the culture medium of the MZ B cells includes MEM culture medium, DMEM culture medium, and RPMI-1640 culture medium.

[0056] Furthermore, the culture medium of the MZ B cells is RPMI-1640 culture medium.

[0057] A sixth aspect of the present invention provides use of an agent that promotes Hspa13 in the preparation of a medicament for preventing and / or treating systemic lupus erythematosus or a disease related to systemic lupus erythematosus.

[0058] Furthermore, the agent for promoting Hspa13 includes an agent for promoting the expression of the Hspa13 gene and an agent for promoting the activity of the Hspa13 gene expression product.

[0059] Preferably, the drug further comprises a pharmaceutically acceptable carrier.

[0060] In some embodiments, the Hspa13-promoting agent refers to any substance that can increase the activity of Hspa13 protein, improve the stability of Hspa13 gene or protein, upregulate the expression of Hspa13 protein, increase the effective action time of Hspa13 protein, or promote the transcription and translation of Hspa13 gene, and these substances can be used in the present invention as substances useful for upregulating Hspa13, so as to prevent or treat systemic lupus erythematosus. For example, the Hspa13-promoting agent includes a nucleic acid promoter and a protein promoter. The Hspa13-promoting agent includes but is not limited to a vector that overexpresses Hspa13, an Hspa13 protein or its active peptide.

[0061] In some embodiments, the pharmaceutically acceptable carriers and / or excipients include, but are not limited to, diluents, binders, surfactants, wetting agents, adsorption carriers, lubricants, fillers, and disintegrants.

[0062] In some embodiments, the diluent includes but is not limited to: lactose, sodium chloride, glucose, urea, starch, water, etc. In some embodiments, the binder includes but is not limited to: starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, alginic acid and alginate, xanthan gum, hydroxypropyl cellulose and hydroxypropyl methylcellulose, etc. In some embodiments, the surfactant includes but is not limited to: polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, stearic acid monoglyceride, hexadecanol, etc. In some embodiments, the humectant includes but is not limited to: glycerol, starch, etc. In some embodiments, the adsorption carrier includes but is not limited to: starch, lactose, bentonite, silica gel, kaolin and bentonite, etc. In some embodiments, the lubricant includes but is not limited to: zinc stearate, glyceryl monostearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, monolauric sucrose acid ester, sodium lauryl sulfate, magnesium lauryl sulfate, magnesium dodecyl sulfate, etc. In some embodiments, the filler includes but is not limited to: mannitol (granular or powdered), xylitol, sorbitol, maltose, erythrose, microcrystalline cellulose, polymeric sugar, coupling sugar, glucose, lactose, sucrose, dextrin, starch, sodium alginate, laminarin powder, agar powder, calcium carbonate and sodium bicarbonate, etc. In some embodiments, the disintegrant includes but is not limited to: cross-linked vinyl pyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, cross-linked sodium carboxymethyl cellulose, soybean polysaccharide, etc.

[0063] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0064] The present invention proves through experiments that MZ B cells overexpressing Hspa13 gene are in CD4 + It plays an important regulatory role in the process of T cell differentiation and can significantly promote CD4 + T cells differentiate into regulatory T cells; and it is demonstrated that MZ B cells overexpressing the Hspa13 gene can alleviate the symptoms of mice with systemic lupus erythematosus and can be used to prepare products for the prevention and / or treatment of systemic lupus erythematosus. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 This is a diagram showing the relationship between the Hspa13 gene and T cell differentiation; Figure 1 A is a diagram showing the expression of Hspa13 by B cells after induction and stimulation in vitro; Figure 1 B is a flow cytometry analysis of the expression of Hspa13 in B cell subsets after stimulation; Figure 1 C is CD4 + Figure 1. Results of co-culture of T cells and MZ B cells;

[0066] Figure 2 This is the result of MZ B cell treatment of lupus mice; Figure 2 A is a graph showing the expression of Hspa13 in MRL / lpr mice; Figure 2 B is the effect of MZ B cells from different sources on the proportion of regulatory T cells in lupus mice; Figure 2 C is a picture of kidney pathological staining of lupus mice with MZ B cells from different sources; Figure 2 D is a graph showing the effects of MZ B cells from different sources on inflammatory factors in lupus mice. DETAILED DESCRIPTION

[0067] The present invention is further described below in conjunction with specific embodiments, which are only used to explain the present invention and are not to be construed as limiting the present invention. It will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to these embodiments without departing from the principles and purposes of the present invention, and that the scope of the present invention is defined by the claims and their equivalents. The experimental methods for which specific conditions are not specified in the following examples are usually tested under conventional conditions or under conditions recommended by the manufacturer.

[0068] Example 1 Application of Hspa13-overexpressing MZ B cells in promoting regulatory T cell differentiation

[0069] 1. Experimental methods

[0070] 1) Obtaining spleen lymphocytes from mice (Beijing Biotechnology Co., Ltd., 6-8 weeks old C57BL / 6J mice): After killing the mice, the spleen was taken and ground under sterile conditions to prepare a single cell suspension. The cell suspension was slowly added to a centrifuge tube containing an equal volume of mouse lymphocyte separation fluid (Tianjin Haoyang Biological Products Technology Co., Ltd., LTS1092) and centrifuged at room temperature (2000r / min, 20min). After the centrifugation, the cell suspension of the lymphocyte layer was aspirated from the centrifuge tube using a pipette under sterile conditions. Add 3 times the volume of pre-cooled saline (Wuhan Sewell Biotechnology Co., Ltd., G4702-500ML) to the aspirated cell suspension, mix well and centrifuge at room temperature (1500r / min, 10min). Discard the supernatant, add 6-8ml of pre-cooled saline to the cell pellet to resuspend the cells, repeat the washing and centrifugation once (1500r / min, 10min), and obtain the lymphocyte pellet.

[0071] 2) Mouse B cell isolation: Mouse CD45R (B220) magnetic bead sorting reagent (Miltenyi Biotec, Miltenyi Biotech, Germany, 130-049-501) was used to separate B cells from lymphocytes. The specific steps were as follows: Lymphocyte sediment was resuspended in cell sorting buffer (1*PBS (Wuhan Sewell Biotechnology Co., Ltd., G4202-500ML) + 0.5% BSA (Shanghai Biotechnology Co., Ltd., A500023) + 2nM EDTA (Sigma-Aldrich, E9884)) at 10 μl per 10 μl. 7 cells, resuspend the cells in 90ul buffer). 710ul anti-CD45R (B220) magnetic bead-coupled antibody reagent was added to each cell suspension, mixed, and incubated at 4°C in the dark for 15min. 2ml pre-cooled buffer was added to the cell suspension for washing, mixed, and centrifuged at room temperature (1500r / min, 10min) to obtain a cell precipitate. 500ul sorting buffer was added to the cell precipitate to resuspend the cells. The cell sorting column (Miltenyi Biotec-Miltenyi Biotech Co., Ltd., Germany, 130-042-201) was placed on a magnetic sorting rack (Miltenyi Biotec-Miltenyi Biotech Co., Ltd., Germany, 130-042-303), 1ml sorting buffer was added to the cell sorting column to rinse and balance the sorting column, and 500ul cell suspension was added to the sorting column. In the magnetic field environment, B cells labeled with magnetic bead-coupled antibodies were adsorbed on the sorting column. Add 500ul pre-cooled sorting buffer to the sorting column to elute the remaining unlabeled non-B cells, and repeat the washing three times. Remove the sorting column from the magnetic sorting rack and place it on a sterile centrifuge tube (Nice Life Science Co., Ltd., 601002), add 1ml of pre-cooled sorting buffer to the sorting column, and use the plunger rod of the sorting column to pressurize the B cells adsorbed on the sorting column to the centrifuge tube. If residual B cells are not prevented on the sorting column, the above elution operation can be repeated once. Collect the B cell suspension and centrifuge (1500r / min, 10min) at room temperature to obtain a B cell precipitate.

[0072] 3) In vitro stimulation culture of B cells: The B cell pellet was resuspended in RPMI-1640 medium (Sigma-Aldrich, R8758, containing 10% FBS (Gibco, A5256701) and penicillin-streptomycin double antibody mixture (Wuhan Sewell Biotechnology Co., Ltd., G4003-100ML)), and the cell count was performed to adjust the cell suspension density to 1*10 6 B cells (1*10 6 / ml), and the stimulator LPS (10ug / ml, Sigma-Aldrich, L6529) was added to treat B cells, and cultured in vitro for 72h. PMA (50ng / ml, Sigma-Aldrich, P8139) and Ionomycin (1ug / ml, Sigma-Aldrich, I0634) were added to stimulate the cells in the last 5h before the end of the 72h culture.

[0073] 4) Detection of Hspa13 expression in B cells after stimulation and proliferation: Real-time PCR was used to detect the expression of Hspa13 in B cells after stimulation and proliferation, and then flow cytometry was used to analyze that Hspa13-positive cells were mainly present in the MZ B cell subpopulation. The stimulated B cell suspension was collected and centrifuged at room temperature (1500r / min, 10min) to obtain the B cell precipitate. On the one hand, TRIzol reagent (Sigma-Aldrich, T9424) was added to the B cell precipitate, and RNA was extracted using the classic TRIzol method, followed by reverse transcription to obtain cDNA, and the mRNA level of Hspa13 was amplified using the Real-time PCR method. On the other hand, the B cell precipitate was resuspended in 4% paraformaldehyde (Sigma-Aldrich, P6148), and the cells were fixed for 20min at room temperature in a dark environment. Resuspend the B cell pellet (1-5x10 6 Cells / mL), add an appropriate amount of specific anti-Hspa13 antibody (0.1-10ug / mL, Abcam, ab219172) to the cell suspension. Incubate at 4°C for 30 minutes. Wash the cells twice with pre-cooled FACS washing solution to remove unbound primary antibodies and impurities. During washing, centrifuge at 1500r / min for 10 minutes. Dilute the Alexa Fluor series fluorescent secondary antibody (0.1-10ug / mL, Invitrogen, A11008) with pre-cooled FACS washing solution and resuspend the cells, incubate at 4°C in the dark for 30 minutes. Wash the cells twice again with pre-cooled FACS washing solution to remove unbound secondary antibodies and impurities. Resuspend the B cell precipitate labeled with Hspa13 antibody in precooled FACS washing solution (1*PBS+1%BSA+0.1% sodium azide), add flow cytometry antibodies of B cell subset marker molecules (such as CD21-PE (Biolegend, 123409), CD23-APC (Biolegend, 101629), CD1d (Biolegend, 123507), CD5 (Biolegend, 100615), etc.) (final concentration 0.1-10ug / mL), and incubate at 4°C for 30min. Wash the cells twice with precooled FACS washing solution (centrifuge at 1500r / min for 10min) to remove unbound flow cytometry antibodies. Resuspend the cell precipitate with 4% paraformaldehyde (Sigma-Aldrich, P6148), and analyze the expression of Hspa13 in B cell subsets by flow cytometry.

[0074] 5) Sorting spleen MZ B cells using magnetic beads: MZ B cells were separated from spleen lymphocytes using mouse marginal zone (MZ) and follicular zone (FO) B cell magnetic bead sorting reagent (Miltenyi Biotec-Miltenyi Biotech Co., Ltd., Germany, 130-100-366). The specific steps are as follows: mice were killed to prepare spleen cell suspension, and lymphocytes were obtained using lymphocyte separation solution (Tianjin Haoyang Biological Products Technology Co., Ltd., LTS1092) (the method is as above). Lymphocyte sediments were resuspended in pre-cooled cell sorting buffer (1*PBS, PH7.2+0.5% BSA+2nM EDTA) (according to 10 8 cells, resuspend the cells in 400ul buffer). 8Add 100ul of biotin-labeled antibody reagent (Miltenyi Biotec-Miltenyi Biotech Co., Ltd., Germany, 130-110-366) to each cell suspension, mix well, and incubate at 4°C for 5min. Add 300ul of pre-cooled sorting buffer and 200ul of anti-biotin-labeled magnetic beads (Miltenyi Biotec-Miltenyi Biotech Co., Ltd., Germany, 130-100-366), mix well, and continue incubation at 4°C for 10min. Add 10ml of pre-cooled sorting buffer to the cell suspension, mix well, wash, and centrifuge at room temperature (1500r / min, 10min) to obtain a cell pellet. Add 500ul of sorting buffer to the cell pellet to resuspend the cells. The cell sorting column was placed on a magnetic sorting rack, 3 ml of sorting buffer was added to the cell sorting column to rinse and balance the sorting column, and 500 ul of cell suspension was added to the sorting column. In a magnetic field environment, the unlabeled cells (i.e., MZ B cells and FO B cell suspension) that passed through the sorting column were collected in a centrifuge tube. The cell suspension was centrifuged at room temperature (1500 r / min, 10 min) to collect MZ B cells and FO B cells. The cells were resuspended in 450ul pre-cooled sorting buffer, and 50ul anti-CD23 magnetic bead antibody reagent (Miltenyi Biotec-Germany Miltenyi Biotech Co., Ltd., 130-100-366) was added. After mixing, the cells were incubated at 4°C for 10min. 10ml pre-cooled sorting buffer was added to the cell suspension and mixed, and then washed. The cells were centrifuged at room temperature (1500r / min, 10min) to obtain a cell precipitate. The cells were resuspended in 500ul pre-cooled sorting buffer. The cell sorting column was placed on a magnetic sorting rack, and 3ml of sorting buffer was added to the cell sorting column to rinse and balance the sorting column. Then, 500ul of cell suspension was added to the sorting column. In the magnetic field environment, the FO B cells labeled with the magnetic bead-coupled antibody were adsorbed on the sorting column, and the cells that passed through the sorting column and were not labeled were collected in a centrifuge tube, namely, MZ B cells. Add 500ul of pre-cooled sorting buffer to the sorting column to elute the remaining unlabeled MZ B cells, and repeat the washing three times. Collect the MZ B cell suspension and centrifuge at room temperature (1500r / min, 10min) to obtain the MZ B cell precipitate.

[0075] 6) CD4 + T cell sorting: using mouse CD4 +CD4+T cells were separated from spleen lymphocytes using T cell magnetic bead separation reagent (Miltenyi Biotec, Germany, 130-117-043). The specific steps were as follows: mice were sacrificed to prepare spleen cell suspension, and lymphocytes were obtained using lymphocyte separation solution (the method is as above). Lymphocyte precipitates were resuspended in cell separation buffer (1*PBS, PH7.2+0.5% BSA+2nM EDTA) (at a rate of 10 7 cells, resuspend the cells in 90ul buffer). 7 Add 10ul anti-CD4 magnetic bead sorting reagent to each cell (cells), mix well, and incubate at 4°C for 15min. Add 2ml pre-cooled buffer to the cell suspension for washing, mix well, and centrifuge at room temperature (1500r / min, 10min) to obtain a cell pellet. Add 500ul sorting buffer to the cell pellet to resuspend the cells. Place the cell sorting column on the magnetic sorting rack, add 1ml sorting buffer to the cell sorting column to rinse and balance the sorting column, then add 500ul cell suspension to the sorting column. In the magnetic field environment, CD4 + T cells are adsorbed to the sorting column. 500ul of pre-cooled sorting buffer is added to the sorting column to elute the remaining unlabeled non-CD4 + T cells, repeat the washing three times. Remove the sorting column from the magnetic sorting rack and place it on a sterile centrifuge tube. Add 1 ml of pre-cooled sorting buffer to the sorting column. Use the plunger rod of the sorting column to pressurize the CD4 + T cells were eluted into centrifuge tubes. No residual CD4 + T cells, repeat the above steps once. Collect CD4 + The T cell suspension was centrifuged at room temperature (1500 r / min, 10 min) to obtain CD4 + T cell pellet.

[0076] 7) MZ B cells and CD4 + T cell co-culture: The sorted MZ B cells were resuspended in RPMI-1640 medium (Sigma-Aldrich, R8758, containing 10% FBS and penicillin-streptomycin double antibody mixture) and the cell suspension density was adjusted to 1*10 6 / ml, stimulated in vitro (LPS (10ug / ml, Sigma-Aldrich, L6529) was added and cultured for 72h; PMA (50ng / ml, Sigma-Aldrich, P8139) and Ionomycin (1ug / ml, Sigma-Aldrich, I0634) were added in the last 5h before the end of the 72h culture. The cell suspension was collected and centrifuged at 1500r / min for 10min to collect the induced and stimulated MZ B cells. The sorted MZ B cells (1*10 6 cells / ml) and CD4 + T cells (1*10 6 After being evenly mixed, the cells were inoculated in a 24-well plate for co-culture for 72 hours. After the experiment, the cells were collected and flow cytometry was performed to analyze the proportion of regulatory T cells (Tregs). Specifically, the cell pellet (1x10 6 Cells / mL), add an appropriate amount of specific anti-CD4 antibody (0.1-10ug / mL, BioLegend, 100537) to the cell suspension. Incubate at 4°C for 30min. Wash the cells twice with pre-cooled FACS wash solution to remove unbound primary antibodies and impurities. When washing, centrifuge at 1500r / min for 10min. Resuspend the cell pellet with 4% paraformaldehyde and fix the cells at room temperature in the dark for 20min. Collect the cells by centrifugation at 1500r / min for 10min at room temperature. Resuspend the cells with pre-cooled FACS wash solution (1*PBS+1%BSA+0.1% sodium azide), add the marker intracellular factor foxp3 flow cytometry antibody of regulatory T cells (Tregs) (final concentration 0.1-10ug / mL), and incubate at 4°C in the dark for 30min. The cells were washed twice with pre-cooled FACS washing solution (centrifugation at 1500 r / min for 10 min) to remove unbound flow cytometry antibodies. The cell pellet was resuspended with 4% paraformaldehyde, and the proportion of regulatory T cells (Tregs) was detected and analyzed using flow cytometry.

[0077] 2. Experimental results

[0078] After induction and stimulation (LPS+PMA+Ionomycin) in vitro, mature B cells obtained from spleen sorting were analyzed by Real-time PCR for the expression of Hspa13. Figure 1 As shown in A, the transcriptional expression level of Hspa13 was significantly increased after stimulation. Flow cytometry was further used to analyze the B cell subsets expressing Hspa13, and it was found that Hspa13 was mainly expressed in CD21 +CD23 - MZ B cells (about 93.2%) were enriched and highly expressed ( Figure 1 B) MZ cells obtained by spleen sorting after induction and stimulation in vitro (LPS+PMA+Ionomycin) and CD4 + T cells were co-cultured in vitro, and regulatory T cells were detected and analyzed by flow cytometry. It was found that MZ B cells with Hspa13 gene deletion (from CD19 cre Hspa13 fl / fl The ability of Hspa13-overexpressing MZ B cells (obtained from Hspa13 mice) to promote the differentiation of regulatory T cells fl / fl The mice were sorted) and significantly lower than those Figure 1 C).

[0079] Example 2 Application of Hspa13-overexpressing MZ B cells in improving renal pathology in lupus mice

[0080] 1. Experimental methods

[0081] MZ B cell infusion for lupus mice treatment: The sorted MZ B cells were resuspended in RPMI-1640 medium (Sigma-Aldrich, R8758, containing 10% FBS and double antibody) and the cell suspension density was adjusted to 1*10 6 Cells were resuspended in precooled saline and infused into the tail vein of lupus mice (MRL / lpr mice) for treatment (10 7 cells / each). Two weeks after the infusion, the mice were killed. The spleen and kidney tissues of the mice were taken. The spleen lymphocyte suspension was prepared (the method is the same as in Example 1), and the regulatory T cells (Treg) were detected and analyzed by flow cytometry (the method is the same as in Example 1). A part of the kidney tissue was fixed with a tissue fixative (4% paraformaldehyde) and stained with HE to analyze the pathological changes of the kidney. Another part of the kidney tissue was used to extract RNA, and the changes in inflammatory factors in the kidney tissue were detected by Real-time PCR. + The T cell co-culture method is the same as in Example 1.

[0082] 2. Experimental results

[0083] MZ B cells were isolated from the spleen of MRL / lpr mice, and real-time PCR analysis revealed that the expression level of Hspa13 in MZ B cells of diseased mice (after 12 weeks of age) was significantly reduced compared with that of early non-disease mice (before 8 weeks of age). Figure 2 A). MZ B cells obtained by spleen sorting were stimulated in vitro (LPS+PMA+Ionomycin) and then injected into the tail vein of lupus mice for treatment. Flow cytometry results showed that MZ B cells with Hspa13 gene deletion (from CD19 cre Hspa13 fl / fl After infusion of Hspa13-expressing MZ B cells into diseased MRL / lpr mice, the proportion of protective regulatory T cells in the spleen could not be increased, while infusion of MZ B cells that stimulated high expression of the Hspa13 gene could significantly increase the proportion of regulatory T cells ( Figure 2 B). The results of renal pathological HE staining showed that MZ B cells with Hspa13 gene deletion (from CD19 cre Hspa13 fl / fl However, infusion of MZ B cells that stimulated high expression of the Hspa13 gene significantly reduced the infiltration of inflammatory cells in the renal arterioles and paravascular interstitium ( Figure 2 C) and reduce the level of inflammatory factors in kidney tissue ( Figure 2 D).

[0084] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. MZ B cells overexpressing Hspa13 regulate CD4 + Application in differentiation of T cells into regulatory T cells, It is characterized in that The MZ B cells overexpressing the Hspa13 gene have the ability to promote CD4 + The role of T cells in differentiating into regulatory T cells.

2. The use according to claim 1, It is characterized in that The MZ B cells overexpressing the Hspa13 gene have the ability to promote CD4 + The differentiation of T cells into regulatory T cells was verified by the following method: MZB cells overexpressing the Hspa13 gene were crossed with CD4 + T cells were co-cultured at a ratio of 1:1; after the culture, the cells were collected and flow stained to analyze the proportion of regulatory T cells.

3. Application of MZ B cells overexpressing the Hspa13 gene in the prevention and / or treatment of systemic lupus erythematosus or systemic lupus erythematosus-related diseases.

4. The use according to claim 3, It is characterized in that The systemic lupus erythematosus-related diseases include dermatitis, anemia, lupus nephritis, heart disease, pneumonia, neurological and mental diseases; Preferably, the systemic lupus erythematosus-related disease is lupus nephritis; Preferably, the MZ B cells overexpressing the Hspa13 gene have the effect of treating lupus nephritis; Preferably, the effect of overexpressing the Hspa13 gene in treating lupus nephritis is verified by the following method: MZ B cells overexpressing the Hspa13 gene are infused into lupus mice with disease through the tail vein. Two weeks after the infusion, the spleen of the mice is taken to prepare a lymphocyte suspension, which is subjected to flow staining to analyze the proportion of regulatory T cells; a part of the kidney of the mouse is taken for HE staining to analyze the pathological changes of the kidney, and the other part is used to detect the changes of inflammatory factors in the kidney tissue using Real-time PCR.

5. The use according to claim 1 or claim 3, It is characterized in that The method for constructing the MZB cells overexpressing the Hspa13 gene comprises: stimulating the MZ B cells with LPS, PMA and lonomycin; Preferably, the MZ B cells are cultured for 72 hours after being stimulated with LPS, and PMA and lonomycin are added in the last 5 hours before the end of the 72-hour culture; Preferably, the cell suspension density is adjusted to 1×10 6 Pieces / ml; Preferably, the concentrations of LPS, PMA and lonomycin are 10ug / ml, 50ng / ml and 1ug / ml respectively; Preferably, the culture medium of the MZ B cells includes MEM culture medium, DMEM culture medium, RPMI-1640 culture medium; Preferably, the culture medium of the MZ B cells is RPMI-1640 culture medium.

6. A cell therapy composition for preventing and / or treating systemic lupus erythematosus or systemic lupus erythematosus-related diseases, It is characterized in that The cell therapy composition includes MZ B cells overexpressing the Hspa13 gene.

7. The MZ B cells overexpressing the Hspa13 gene or the cell therapy composition according to claim 6 are used in the preparation of a medicament for treating systemic lupus erythematosus or a disease related to systemic lupus erythematosus or in the preparation of a drug for promoting CD4 + Application in reagents for differentiating T cells into regulatory T cells; Preferably, the systemic lupus erythematosus-related diseases include dermatitis, anemia, lupus nephritis, heart disease, pneumonia, neurological and psychiatric diseases.

8. The cell therapy composition according to claim 6 or the use according to claim 7, It is characterized in that The method for constructing MZ B cells overexpressing the Hspa13 gene comprises: stimulating MZ B cells with LPS, PMA and lonomycin; Preferably, the MZ B cells are cultured for 72 hours after being stimulated with LPS, and PMA and lonomycin are added in the last 5 hours before the end of the 72-hour culture; Preferably, the cell suspension density is adjusted to 1×10 6 Pieces / ml; Preferably, the concentrations of LPS, PMA and lonomycin are 10ug / ml, 50ng / ml and 1ug / ml respectively; Preferably, the culture medium of the MZ B cells includes MEM culture medium, DMEM culture medium, RPMI-1640 culture medium; Preferably, the culture medium of the MZ B cells is RPMI-1640 culture medium.

9. An in vitro non-therapeutic destination to promote CD4 + Methods for differentiation of T cells into regulatory T cells, It is characterized in that The method comprises: combining MZ B cells that overexpress the Hspa13 gene with CD4 + T cell co-culture.

10. Use of an agent promoting Hspa13 in the preparation of a drug for preventing and / or treating systemic lupus erythematosus or systemic lupus erythematosus-related diseases; Preferably, the agent promoting Hspa13 includes an agent promoting the expression of the Hspa13 gene and an agent promoting the activity of the Hspa13 gene expression product.

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