Application of medicine for knocking out or reducing RBM39 gene expression in preparation of medicine for treating autoimmune diseases

Abstract

The invention discloses application of a medicine for knocking out or reducing RBM39 gene expression in preparation of a medicine for treating autoimmune diseases, the research finds that RBM39 can be used as a target for treating the autoimmune diseases for the first time, and after RBM39 expression is knocked out or reduced, proliferation of T cells can be inhibited, apoptosis of the T cells can be promoted, Treg cell differentiation can be promoted, and Th1 and Th17 differentiation can be inhibited; the purpose of treating autoimmune diseases is achieved; the target treatment cannot cause weight loss of animals, has no obvious toxic and side effects, and can be used for clinically treating autoimmune diseases, especially autoimmune encephalomyelitis and psoriasis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to the application of a drug for knocking out or reducing the expression of RBM39 gene in the preparation of a drug for treating autoimmune diseases. BACKGROUND

[0002] Autoimmune diseases are caused by the dysfunction of immune cells, which directly or indirectly damage the function of normal tissue cells. CD4 + T helper (TH) cells include Th1 cells, Th17 cells and Treg cell subgroups, which play a crucial role in the pathogenesis of various autoimmune diseases. This is due to the fact that different TH cell subgroups recruit different immune cells and coordinate different immune mechanisms by secreting corresponding cytokines and chemokines through different transcription factors. Under physiological conditions, the balance between Th cell subgroups is maintained by promoting and restraining each other. Once the balance is broken, the imbalance in the proportion and number of cell subgroups will lead to the occurrence of autoimmune diseases. Literature reports that Th1 cells and / or Th17 cells exacerbate inflammation and disease progression by secreting pro-inflammatory cytokines such as IFN-γ and IL-17A in various autoimmune diseases such as insulin-dependent type 1 diabetes (IDDM), multiple sclerosis (MS), inflammatory bowel disease (IBD), rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Regulatory T cells (Treg) can regulate central and peripheral immune tolerance, inhibit the immune response of other immune cells, and maintain the immune homeostasis of the body. At present, the main method for controlling the progression of patients is to use broad-spectrum immunosuppressive drugs. These drugs are usually non-specific target drugs, which have strong toxic side effects on normal tissues with high proliferation and metabolism such as the intestinal tract and liver in addition to the inhibition of immune cells. Therefore, it is crucial to find a target that can specifically inhibit the function of immune cells without affecting the function of other normal cells for the targeted treatment of autoimmune diseases and reducing side effects. SUMMARY

[0003] Therefore, the present application aims to provide a drug for knocking out or reducing the expression of RBM39 gene in the preparation of a drug for treating autoimmune diseases.

[0004] To achieve the above-mentioned purpose, the present application provides the following technical solutions: The application of a drug for knocking out or reducing the expression of RBM39 gene in the preparation of a drug for treating autoimmune diseases.

[0005] Preferably, the autoimmune disease is autoimmune encephalomyelitis, psoriasis, insulin-dependent type 1 diabetes, multiple sclerosis, inflammatory bowel disease, rheumatoid arthritis and systemic lupus erythematosus.

[0006] Preferably, the RBM39 gene expression is knocked out by using a drug inducible gene knockout.

[0007] Preferably, the RBM39 gene knockout treats autoimmune diseases by inhibiting the proliferation of T cells and promoting the apoptosis of T cells.

[0008] Preferably, the RBM39 gene expression is reduced to treat autoimmune diseases by promoting the differentiation of Treg cells and inhibiting the differentiation of Th1 and Th17.

[0009] The present application has the beneficial effects that the present application discloses that RBM39 can be used as a target for treating autoimmune diseases, and knocking out or reducing the expression of RBM39 can treat autoimmune diseases, the treatment process is achieved by inhibiting the proliferation of T cells, promoting the apoptosis of T cells, promoting the differentiation of Treg cells, and inhibiting the differentiation of Th1 and Th17, and the use of the drug does not cause the weight loss of animals, and the weight change has no difference with that of normal control mice; the tissue sections of multiple organs show that the drug has no obvious toxic side effects on each organ. The present application provides a new target for the treatment of autoimmune diseases, and has important significance for the clinical treatment of autoimmune diseases. BRIEF DESCRIPTION OF DRAWINGS

[0010] In order to make the purpose, technical scheme and beneficial effects of the present application clearer, the present application is described below with the help of the following drawings: Figure 1 In order to reduce the expression of RBM39 by gene knockout, the proliferation of T cells can be inhibited, and the apoptosis of activated T cells can be promoted (A: flow cytometry of T cell proliferation in wild type and RBM39 knockout groups; B: statistical graph of T cell proliferation in wild type and RBM39 knockout groups; C: flow cytometry of T cell apoptosis in wild type and RBM39 knockout groups; D: statistical graph of T cell apoptosis in wild type and RBM39 knockout groups); Figure 2 In order to reduce the expression of RBM39 by gene knockout, the differentiation of inflammatory effector T cells can be inhibited and the proportion of anti-inflammatory regulatory T cells can be increased (A: Th1 type T cell differentiation in wild type and RBM39 knockout groups; B: Th17 and Treg cell differentiation in wild type and RBM39 knockout groups).

[0011] Figure 3 In order to reduce the expression of RBM39 by gene knockout, the treatment of autoimmune encephalomyelitis in mice (A: disease score graph of wild type and RBM39 knockout groups of mice after EAE induction; B: H&E section graph of spinal cord lesions of wild type and RBM39 knockout groups of mice after EAE disease induction).

[0012] Figure 4After treating the mice with autoimmune encephalomyelitis by gene knockout to reduce the expression of RBM39, the number of lymphocyte infiltrates in the central nervous system and the proportion of regulatory T cells in the peripheral lymphatic organs (A: flow cytometry of lymphocyte infiltrates in the central nervous system of wild-type and RBM39 knockout group mice after induction of EAE disease; B: statistical chart of the number of lymphocyte infiltrates in the central nervous system of wild-type and RBM39 knockout group mice after induction of EAE disease; C: flow cytometry of the proportion of Treg in the peripheral lymphatic organs of wild-type and RBM39 knockout group mice after induction of EAE disease; D: statistical chart of the proportion of Treg in the peripheral lymphatic organs of wild-type and RBM39 knockout group mice after induction of EAE disease). DETAILED DESCRIPTION

[0013] The present application will be further described below in conjunction with the drawings and specific examples, so that those skilled in the art can better understand the present application and implement it, but the examples are not limiting to the present application.

[0014] Obtaining of RBM39 knockout mice: SAIYE BIOTECHNOLOGY INC., No. S-CKO-03525.

[0015] Obtaining of Imiquimod (IMQ): purchased from Sichuan Mingxin Lide Co., Ltd., National Drug Standard Number: H20030129.

[0016] Example 1, Effect of specifically reducing RBM39 expression on T cell proliferation and apoptosis I. Reducing RBM39 level using drug-induced gene knockout mice: (1) Drug-induced gene knockout mice: The drug-induced gene knockout mice involved in this experiment are Tamoxifen-induced gene knockout mice, which are obtained by knocking out Rbm39 fl / fl mice and Cre ERT2 mice to obtain Rbm39 fl / fl Cre ERT2 mice, which normally express the Rbm39 gene. When treated with Tamoxifen, Tamoxifen is metabolized into an estrogen analogue and combined with the Cre-ERT2 outside the nucleus, making it into a cutting-active Cre recombinase that enters the nucleus to excise the target gene. The mice are purchased from SAIYE BIOTECHNOLOGY INC.

[0017] (2) Coating plate: T cell proliferation activation requires corresponding stimulators. In vitro culture, Anti-CD3 antibody is diluted to 5 μg / ml with phosphate buffer saline before plating, added to the well plate, and the liquid completely covers the bottom of the plate (about 130 μl for a 48-well plate and about 75 μl for a 96-well plate). The plate is incubated at 37°C in a 5% CO2 cell incubator for 3h; (3) Mouse lymphoid organ sampling: Rbm39 fl / fl Cre ERT2+ mice of different ages were selected for the experiment, and the whole body was sprayed with alcohol and placed in a clean bench. The mouse abdominal cavity was exposed, and the mouse spleen and peripheral lymph nodes were taken with clean and sterile ophthalmic forceps. After grinding and lysing red blood cells, a single cell suspension was prepared by filtration; (4) Cell proliferation labeling: The single cell suspension obtained in step (2) was centrifuged (500g, 5min), and CTV(CellTrace™ Violet)-BV421 was diluted with 1xPBS at a ratio of 1:1500. The cell pellet was resuspended and incubated on ice for 30 minutes in the dark. After incubation, the cells were washed once with 1xPBS and counted; (5) In vitro expansion culture: Take an appropriate volume of culture medium (RPMI1640+10% FBS+1% P / S+1% β-mercaptoethanol), add Anti-CD28(2 μg / ml) and IL-2(2 μg / ml) and mix well. The experimental group uses culture medium containing 1 μM Tamoixfen, and the control group uses culture medium containing less than 1% DMSO. The required number of cells and the corresponding culture medium were plated and incubated at 37°C in a 5% CO2 cell incubator for 4 days. (6) Flow cytometry staining and machine: ① Harvest and wash cells: Centrifuge the harvested target cells (500g, 5min), and discard the supernatant. Resuspend the cells with an appropriate volume of 0.5% FACS (1xPBS+0.5% FBS) and centrifuge (500g, 5min). Discard the supernatant. ② Cell viability staining: Dilute the cell viability dye with 1xPBS at a ratio of 1:1000. Resuspend the cells at a volume of 100ul per tube, incubate at room temperature for 15 minutes in the dark, and then wash and centrifuge (500g, 5min) with 0.5% FACS Buffer. Discard the supernatant. ③Cell surface staining: Dilute cell surface dye with FACS Buffer at a ratio of 1:400, resuspend cells at a volume of 50ul per tube, incubate on ice for 30min in the dark, after staining, directly add appropriate volume of 0.5% FACS Buffer to wash and centrifuge (500g, 5min), discard the supernatant; ④Apoptosis staining: Dilute apoptosis dye with Annexin V Binding Buffer at a ratio of 1:100, resuspend cells at a volume of 50ul per tube, incubate at room temperature for 15min in the dark, after staining, directly add appropriate volume of Annexin V Binding Buffer to terminate apoptosis staining, transfer liquid to flow tube with pipette and wait for machine; Important note: Cells labeled with CTV need to be taken on the day of labeling and a certain amount of cells are subjected to the above flow staining to confirm the labeling of CTV on the machine, and the CTV voltage is kept consistent with the initial one when staining and machine is used at the end of subsequent culture.

[0018] Results are shown in Figure 1 It is shown that the use of Tamoxifen to induce knock-out of Rbm39 inhibits the proliferation of T cells and promotes the apoptosis of T cells.

[0019] Example 2, Effect of specifically reducing RBM39 expression on Th1, Th17 and Treg cell differentiation Using drug-inducible gene knockout mice to reduce RBM39 levels: (1) Coating culture plates: the operation steps are the same as in Example 2; (2) Mouse lymphoid organ sampling: the operation steps are the same as in Example 2-I; (3) Cell sorting: centrifuge the single cell suspension obtained in (2) (500g, 5min), resuspend the cells with 500ul of 1x PBS containing 2% FBS, transfer to a 5ml flow tube, and sort according to Naive CD4 + T cell sorting kit instructions, obtain the sorted cell suspension and count; (4) In vitro differentiation culture: take an appropriate volume of culture medium (RPMI1640+10% FBS+1% P / S+1% β-mercaptoethanol), add Anti-CD28 (2 μg / ml) and mix well for standby, and prepare the culture medium according to the differentiation conditions in Table 1: Table 1, differentiation condition medium The experimental group used the culture medium containing 1 μM Tamoxifen, and the control group used the culture medium containing less than 1% DMSO, and the required cells were plated with the corresponding medium, and incubated at 37°C, 5% CO2 cell incubator for 4 days; (5) Flow staining and machine: ① Harvest and wash cells: centrifuge the harvested target cells (500g, 5min), discard the supernatant; resuspend the cells with an appropriate volume of 0.5% FACS (1xPBS+0.5%FBS), centrifuge (500g, 5min), discard the supernatant; ② Cell live / dead staining: dilute the cell death and live dye with 1xPBS at a ratio of 1:1000, resuspend the cells at a volume of 100μl per tube, incubate at room temperature for 15min, and directly add an appropriate volume of 0.5% FACS Buffer to wash and centrifuge (500g, 5min), discard the supernatant; ③ Cell surface staining: dilute the cell surface dye with FACS at a ratio of 1:400, resuspend the cells at a volume of 50μl per tube, incubate on ice for 30min, and directly add an appropriate volume of 0.5% FACS Buffer to wash and centrifuge (500g, 5min), discard the supernatant; ④ Cell fixation: Intracellular staining of cells: use BD Cytofix / Cytoperm TM kit, resuspend the cells at a volume of 50μl per tube, incubate on ice for 30min, and directly add an appropriate volume of 1xBD Perm / Wash TM centrifuge (500g, 5min), discard the supernatant; Intracellular staining of cells: use BD Cytofix / Cytoperm TM kit, resuspend the cells at a volume of 50μl per tube, incubate on ice for 30min, and directly add an appropriate volume of 1xBD Perm / Wash TM centrifuge (500g, 5min), discard the supernatant; ⑤ Intracellular / nuclear staining of cells: Intracellular staining of cells: use 1xBD Perm / Wash TMDilute intracellular staining dye, resuspend cells at a volume of 50 μl per tube, incubate on ice for 30 min in the dark, after staining, directly add the appropriate volume of 0.5% FACS Buffer to wash and centrifuge (500g, 5 min), discard the supernatant; Nuclear staining: dilute nuclear staining dye with 1x Permeabilization Buffer at a ratio of 1:200, resuspend cells at a volume of 50ul per tube, incubate on ice for 30 min in the dark, after staining, directly add the appropriate volume of 0.5% FACS Buffer to wash and centrifuge (500g, 5 min), discard the supernatant; ⑥Flow cytometry: resuspend cells with the appropriate volume of 0.5% FACS Buffer according to the amount of cells, use a pipette to transfer the cell suspension to a flow tube, and wait for the machine; Results are shown in Figure 2 It is shown that after Tamoxifen-induced knockout of Rbm39, the differentiation of Th1 and Th17 is inhibited, and the differentiation of Treg is promoted.

[0020] Example 3, Establishment and treatment of autoimmune encephalomyelitis (EAE) model Prepare each group by injecting 30 μl of 10 mg / ml myelin oligodendrocyte glycoprotein (MOG) + 100 μl of 1x PBS + 100 μl of 5 mg / ml complete Freund's adjuvant (CFA) per mouse.

[0021] Emulsion configuration: connect two threaded syringes in a three-way valve, remove the push rod from one of them, add PBS, MOG and CFA in turn, install the syringes, push the syringes through the three-way valve to remove the gas, push back and forth on the ice until the emulsion is completely emulsified, then transfer it to a syringe, and store it on ice (add a drop of emulsion to a beaker, the drop will remain as a lump, and slowly dissipate, indicating that the emulsion is complete).

[0022] Mouse immunization: anesthetize the mouse with isoflurane, and inject 100 μl of emulsion subcutaneously on both sides of the mouse's back with a 2 ml syringe. Then inject 100 μl of pertussis toxin (PTX: 2 μl of 100 mg / ml PTX + 98 μl of 1x PBS) into the mouse's eye vein with an insulin syringe, and repeat the injection 48 hours later.

[0023] Weigh and score: from the day of immunization, weigh the mice every other day, and start weighing and scoring every day after the onset of symptoms (0 points for normal, 1 point for tail weakness, 2 points for hind limb weakness, 3 points for hind limb paralysis, 4 points for front limb paralysis, 5 points for death, and 0.5 points for intermediate states).

[0024] Criteria for successful modeling: Mice start to show signs of disease 7-14 days after sensitization, with symptoms including, but not limited to, ataxia, limb paralysis, and other clinical signs of multiple sclerosis. Disease grade criteria is ranked from 1 to 5. Grade 1: animal has weakness in the tail; Grade 2: weakness in the tail plus weakness in the limbs; Grade 3: mild paralysis of the limbs; Grade 4: severe paralysis of the limbs, unable to right itself after being flipped over; Grade 5: moribund state.

[0025] Knockout of autoimmune encephalomyelitis model mice reduces RBM39 levels. Part of the brain and lumbar spinal cord were taken after 29 days for H&E staining. The remaining brain and spinal cord tissue were subjected to lymphocyte isolation, and flow cytometry was used to analyze the cells.

[0026] The specific steps of flow cytometry (FACS) are as follows: 1. Cell harvesting: ① Lymph node cell acquisition: The mouse was sacrificed by cervical dislocation and placed on a foam board covered with 70% alcohol disinfectant paper towels. The mouse was cut open, and two axillary, axillary, and inguinal lymph nodes were taken. The cells were ground and filtered in 1x PBS buffer, centrifuged (500g, 5min), and resuspended in 1x PBS buffer.

[0027] ② Spleen cell acquisition: The spleen was taken, and the steps were the same as above. After grinding and filtering, centrifugation (500g, 5min) was performed, and 1ml ACK Lysing Buffer was used for resuspension. The red blood cells were lysed at room temperature for 5min, and after centrifugation (500g, 5min), 1x PBS buffer was used for resuspension.

[0028] ③ Brain and spinal cord cell acquisition: The brain and spinal cord were taken, and the steps were the same as above. The brain and spinal cord were cut and ground, centrifuged (500g, 5min), and resuspended in 50% Percoll. Slowly add to the upper layer of 70% Percoll, centrifuge (18°C, 500g, 30min, slow up and slow down), and then collect the middle layer. After centrifugation (500g, 5min), 1x PBS buffer was used for resuspension.

[0029] 2. Cell death staining: Centrifuge the cell suspension (500g, 5min), discard the supernatant, and resuspend each sample in 100μl dead and live staining solution (zombie yellow: 1x PBS buffer 1:1000). Incubate at room temperature for 15min in the dark, centrifuge (500g, 5min), and resuspend in 1x PBS buffer.

[0030] 3. Cell surface staining: Prepare surface antibody premix solution according to surface antibody: FACS Buffer (0.5% FBS-PBS) 1:400, centrifuge the suspension (500g, 5min), discard the supernatant, resuspend each sample with 50μl surface antibody premix solution, incubate on ice for 30min in the dark, centrifuge (500g, 5min) and resuspend with FACS Buffer.

[0031] 4. Cytokine / transcription factor staining: centrifuge the cell suspension (500g, 5min), discard the supernatant, resuspend each sample with 50μl corresponding cytokine / transcription factor fixative, incubate on ice for 30min in the dark; centrifuge (10000g, 1min) and resuspend each sample with 200μl corresponding Wash Buffer, centrifuge (10000g, 1min) and resuspend each sample with 50μl cytokine / transcription factor staining solution (cytokine antibody: cytokine Wash Buffer 1:200, transcription factor antibody: transcription factor Wash Buffer 1:100), incubate on ice for 30min in the dark, centrifuge (10000g, 1min) and resuspend each sample with 200μl corresponding Wash Buffer. (If staining both cytokine and transcription factor, use transcription factor Kit separately) 5. Sample collection: centrifuge the stained suspension (unfixed 500g, 5min, fixed 10000g, 1min), discard the supernatant, resuspend with 200-500μl FACS, filter through nylon mesh into flow cytometry sample tubes, and perform flow cytometry fluorescence detection.

[0032] By reducing RBM39 expression through gene knockout, the mouse autoimmune encephalomyelitis is treated as shown in Figure 3 and Figure 4 The results show that by specifically reducing the RBM39 protein level through gene knockout, the number of inflammatory cells in the central nervous system of the mouse autoimmune encephalomyelitis model is significantly reduced, and the proportion of regulatory T cells (Treg) in the peripheral lymphatic organs is significantly increased. Therefore, by specifically reducing the RBM39 protein level through gene knockout, the occurrence of the disease in the model mouse is significantly reduced, and the weight loss of the mouse is significantly improved, and the pathological changes in the spinal cord tissue are also significantly better than those in the control group.

[0033] The above results show that specifically reducing the RBM39 protein level can significantly reduce the number of inflammatory cells at the lesion site, increase the proportion of regulatory T cells in the peripheral lymphatic organs of the mouse autoimmune encephalomyelitis model, and further reduce the occurrence of the disease in the model mouse, and the pathological changes in the spinal cord tissue are also significantly better than those in the control group.

[0034] The above-described embodiments are merely preferred embodiments of the present application, and the protection scope of the present application is not limited thereto. Any equivalent substitutions or transformations made by those skilled in the art based on the present application are within the protection scope of the present application. The protection scope of the present application is subject to the claims.

Claims

1. Use of a drug for knocking out or reducing the expression of RBM39 gene in the preparation of a drug for treating autoimmune diseases.

2. Use according to claim 1, characterized in that: The autoimmune disease is autoimmune encephalomyelitis, psoriasis, insulin-dependent type 1 diabetes, multiple sclerosis, inflammatory bowel disease, rheumatoid arthritis and systemic lupus erythematosus.

3. Use according to claim 1, characterized in that: The knocking out of the expression of the RBM39 gene adopts a drug-induced gene knockout.

4. Use according to claim 1, characterized in that: The treatment of autoimmune diseases by knocking out the RBM39 gene is achieved by inhibiting the proliferation of T cells and promoting the apoptosis of T cells.

5. The use according to claim 1, characterized in that: The treatment of autoimmune diseases by reducing the expression of the RBM39 gene is achieved by promoting the differentiation of Treg cells and inhibiting the differentiation of Th1 and Th17.

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