Compositions for enhancing mesenchymal stem cell therapeutic efficacy and uses thereof

Abstract

The present application relates to a composition and application for enhancing mesenchymal stem cell treatment efficiency, and belongs to the technical field of cell drug treatment. The composition for enhancing mesenchymal stem cell treatment efficiency comprises a PARP inhibitor and an inflammatory factor. The mesenchymal stem cells are pretreated by the composition, and the mesenchymal stem cells with enhanced treatment efficiency are obtained, which are used for preparing a therapeutic drug for immune diseases characterized by abnormal inflammatory response. The mesenchymal stem cells are treated by the PARP inhibitor combined with the inflammatory factor in the present application, which can improve the intracellular signal transduction and the phosphorylation level of STAT1, increase the expression of the immune suppressor IDO1 and PD-L1, thereby enhancing the immune regulation function of the stem cells, and realizing the gain effect on the treatment of immune diseases mainly characterized by abnormal inflammatory response.

Description

Technical Field

[0001] This invention relates to the field of cell therapy technology, and more particularly to a composition and its application for enhancing the therapeutic efficacy of mesenchymal stem cells. Background Technology

[0002] Mesenchymal stem cells (MSCs) are adult stem cells widely distributed in various tissues and organs, closely related to individual growth and development, tissue homeostasis, and organ damage repair. Besides possessing strong self-renewal capacity and multiple differentiation potentials, MSCs also have immunomodulatory capabilities. When the body is injured, exogenously infused MSCs and endogenous tissue MSCs can be recruited to the site of injury, coordinating the functions of in situ stromal cells and tissue-specific stem cells through cell replacement or cell empowerment, thereby promoting damage repair and tissue regeneration. Therefore, MSCs have become an important research subject in the field of biomedicine, bringing hope for the treatment of many refractory and intractable diseases.

[0003] Due to their convenient source, easy availability, and low immunogenicity, MSCs hold promise as a cell therapy agent for various inflammatory diseases. Compared to first-line immunosuppressive drugs, MSCs do not systemically reduce the function of the immune system, but rather reduce inflammatory damage and promote tissue regeneration and repair by targeting the immune response in a state of high inflammation.

[0004] In actual clinical trials, it has been found that when using MSCs to treat inflammatory diseases, if the amount of injected cells is too low, the expected therapeutic effect cannot be achieved; conversely, if the amount of stem cells infused is increased, the risk of thrombosis will be significantly increased, leading to serious adverse reactions. Therefore, enhancing the immune function characteristics of the infused cells at the original dose is particularly important in clinical practice. Summary of the Invention

[0005] To address the numerous pain points of the aforementioned treatments, this invention provides a composition and application for enhancing the therapeutic efficacy of mesenchymal stem cells (MSCs). The modified MSCs described in this invention are obtained through treatment with a clinically proven PARP inhibitor combined with inflammatory factors. This invention uses PARP inhibitors combined with inflammatory factors to pretreat MSCs in vitro, thereby promoting the phosphorylation level of intracellular signal transducer and activator of transcription 1 (STAT1), increasing the expression of immunosuppressive factors indoleamine 2,3-dioxygenase 1 (IDO1) and programmed cell death ligand 1 (PD-L1), and thus enhancing the immune regulatory function of stem cells. This enhances the therapeutic efficacy of MSCs in treating immune diseases characterized by abnormal inflammatory responses, such as colitis and hepatitis.

[0006] This invention is achieved through the following technical solution:

[0007] The first objective of this invention is to provide a composition for enhancing the therapeutic efficacy of mesenchymal stem cells, the composition comprising a PARP inhibitor and inflammatory factors.

[0008] In some embodiments of the present invention, the inflammatory factors include IFN-γ (interferon-γ) and TNF-α (tumor necrosis factor-α).

[0009] In some embodiments of the present invention, the concentration of the inflammatory factor is 1 ng / mL to 20 ng / mL, preferably 10 ng / mL.

[0010] In some embodiments of the present invention, the PARP inhibitor is selected from one or more of rucaparib, olaparib, and niraparib, preferably rucaparib.

[0011] In some embodiments of the present invention, the concentration of the PARP inhibitor is 1 μM-20 μM, preferably 10 μM.

[0012] In some embodiments of the present invention, the mesenchymal stem cells are derived from bone marrow, fat, or umbilical cord.

[0013] The mesenchymal stem cells described are pluripotent stem cells with self-renewal and multi-lineage differentiation potential. Umbilical cord-derived mesenchymal stem cells (UC-MSCs) are one of the main types of MSCs used in current clinical trials, and they can differentiate into various mesenchymal tissues, such as bone, cartilage, adipose tissue, and bone marrow hematopoietic tissue. All cell usage examples listed in this invention were carried out in accordance with the ethics committee guidelines of Soochow University.

[0014] It should be noted that the human pluripotent stem cells used in this invention cannot develop into complete individuals, and have all been reviewed and approved by the Ethics Committee of Soochow University for use in scientific research.

[0015] A second objective of this invention is to provide a cell drug for enhancing the therapeutic efficacy of mesenchymal stem cells, said cell drug being obtained by culturing mesenchymal stem cells using the composition described above.

[0016] In some embodiments of the present invention, the processing temperature is 37°C and the time is 24h-48h, preferably 24h.

[0017] In some embodiments of the present invention, the mesenchymal stem cells are selected from mesenchymal stem cells of 10 generations or less.

[0018] A third object of the present invention is to provide a medicament for treating immune diseases characterized primarily by inflammatory responses, comprising the cellular drug.

[0019] In some embodiments of the present invention, the inflammatory response includes immune diseases characterized primarily by abnormal inflammatory responses; the immune diseases characterized primarily by abnormal inflammatory responses include colitis, hepatitis, graft-versus-host disease, etc.

[0020] In some embodiments of the present invention, the drug promotes the expression of immunosuppressive factors in mesenchymal stem cells.

[0021] In some embodiments of the present invention, the dosage form of the drug is an injection.

[0022] In some embodiments of the present invention, the medicament further includes a pharmaceutically acceptable carrier.

[0023] In some embodiments of the present invention, the carrier is selected from one or more of disintegrants, diluents, lubricants, adhesives, humectants, flavoring agents, suspending agents, surfactants, and preservatives.

[0024] In some embodiments of the present invention, the dosage of the cell drug is 4 × 10⁻⁶. 6 Mesenchymal stem cells per kg.

[0025] Of course, those skilled in the art will know that the drug dosage can be increased as needed, i.e., the dosage of the cell drug is ≥4×10⁻⁶. 6 Mesenchymal stem cells per kg.

[0026] In some embodiments of the present invention, the drug enhances the immune regulatory function of stem cells by increasing the phosphorylation level of STAT1 and increasing the expression of immunosuppressive factors.

[0027] In some embodiments of the present invention, immunosuppressive factors IDO1 and PD-L1 are used.

[0028] This invention provides a novel, enhanced strategy for the treatment of immune diseases characterized by abnormal inflammatory responses, such as colitis and hepatitis, by using PARP inhibitors to pretreat mesenchymal stem cells in vitro, which exhibit a more efficient therapeutic effect compared to the same number of untreated mesenchymal stem cells.

[0029] The technical solution of the present invention has the following advantages compared with the prior art:

[0030] This invention provides a composition and its application for enhancing the therapeutic efficacy of mesenchymal stem cells. By using PARP inhibitors and inflammatory factors to pretreat mesenchymal stem cells in vitro, a synergistic effect is achieved in the treatment of immune diseases characterized primarily by abnormal inflammatory responses, such as enteritis and hepatitis. This invention utilizes a single infusion of a low dose (2×10⁻⁶). 5 Pretreatment of MSCs with DMSO and inflammatory factors failed to significantly alleviate disease symptoms in mice. However, infusion of the same amount of MSCs pretreated with PARP inhibitors and inflammatory factors significantly improved serum transaminase levels in hepatitis mice and inhibited liver tissue necrosis. Furthermore, it alleviated symptoms such as weight loss, diarrhea, bloody stools, intestinal thickening and edema, structural damage, and immune cell infiltration in enteritis mice. Therefore, pretreatment of MSCs with PARP inhibitors can enhance the immunomodulatory function of stem cells, achieving a synergistic effect in stem cell disease treatment. Attached Figure Description

[0031] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0032] Figure 1 The expression levels of various intracellular immunosuppressive factors and the degree of STAT1 phosphorylation in MSCs treated with PARP inhibitors in combination with inflammatory factors in Example 1 of this invention;

[0033] Figure 2 This is a characterization of the enhanced therapeutic effect of MSCs on hepatitis and colitis after 24 hours of treatment with PARP inhibitors and inflammatory factors in Example 2 of the present invention. Detailed Implementation

[0034] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0035] The materials and methods used in the following embodiments of the present invention are as follows:

[0036] 1. In vitro expansion and culture of MSCs

[0037] (1) Use trypsin to digest and passage MSCs in culture dishes, resuspend and centrifuge them, and then inoculate them into new culture dishes for amplification culture.

[0038] (2) Change the culture medium every two days until the cell confluence reaches 80% to 90%. Only MSCs within the first 10 generations were selected for this study.

[0039] 2. Animal experiments with hepatitis models / colitis

[0040] 2.1.1 Laboratory Animals

[0041] Male C57BL / 6J mice, aged 8–10 weeks and weighing approximately 25g, used in the experiment were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and were raised strictly according to SPF-grade barrier system standards. All animal experimental procedures involved in this invention were approved by the Animal Experiment Ethics Committee of Soochow University.

[0042] 2.1.2 Establishment of a hepatitis model

[0043] On the day the experiment began, C57BL / 6J mice were randomly divided into four groups. An acute liver injury model was induced by intravenous injection of concanavalin A (ConA, molecular weight: 102000 Da) at a dose of 15 mg / kg. Control mice were injected with the same volume of PBS as isotype controls. ConA activated T lymphocytes in the body, causing them to proliferate excessively. The overreaction of inflammation led to acute necrosis of hepatocytes in the mice, ultimately inducing hepatitis.

[0044] 2.1.3 Treatment of Hepatitis Models

[0045] Thirty minutes after inducing an acute liver injury model in mice, 2×10 5 Mice were treated for the disease by tail vein infusion of MSCs pretreated with the PARP inhibitor Rucaparib and inflammatory factors IFN-γ and TNF-α (10 ng / mL) for 24 h. Control mice were given the same volume of PBS via tail vein infusion. Peripheral blood and liver tissue were collected from the mice 12 h later for analysis.

[0046] 2.1.4 Serum Aspartate Aminotransferase (AST) Concentration Detection

[0047] (1) Collect peripheral blood from mice. Centrifuge at 3000 rpm for 30 min at room temperature, and collect the serum for later use;

[0048] (2) Heat the matrix solution in a 37°C water bath beforehand, then add 20 μL / well to the 96-well plate, and then add 5 μL / well of serum to the test wells.

[0049] (3) Repeatedly pipette the serum sample and matrix solution in the well plate to mix them, taking care to avoid generating air bubbles;

[0050] (4) Incubate in a 37℃ incubator for 30 min;

[0051] (5) Add 20 μL of colorimetric solution to each well in a 96-well plate, and add 5 μL of deionized water to each control well.

[0052] (6) Repeatedly pipette the serum, matrix solution and colorimetric solution in the well plate to mix them well, taking care to avoid generating air bubbles;

[0053] (7) Incubate in a 37℃ incubator for 20 min;

[0054] (8) Add 200 μL of the stop solution to each well and gently shake the 96-well plate. Let it stand at room temperature for 15 min.

[0055] (9) Read the absorbance of the sample at a wavelength of 510 nm using the microplate reader;

[0056] (10) Substitute the obtained OD value into the standard curve to calculate the serum aspartate aminotransferase concentration.

[0057] 2.2.1 Establishment of an animal model of colitis

[0058] On the first day of the experiment, C57BL / 6J mice were randomly divided into four groups. The model group mice drank water containing 4% dextran sulfate (DSS, molecular weight: 36000-5000Da), while the control mice drank normal water. The mice were fed for a total of 7 days to induce colitis.

[0059] 2.2.2 Treatment of colitis model

[0060] On the second day after inducing colitis in mice, mice were given a tail vein infusion of 2×10⁻⁶ nitric acid, which had been pretreated for 24 h with the PARP inhibitor Rucaparib (10 μM) and the inflammatory factors IFN-γ and TNF-α (10 ng / mL). 5 One MSC was used to treat the disease, while the model control mice were given the same volume of PBS infused via the tail vein.

[0061] 2.2.3 Colitis Disease Activity Index

[0062] (1) Weight loss (0-4 points)

[0063] 1 point: Loss of less than 10% of initial body weight;

[0064] 2 points: Loss of 10% to 15% of initial body weight;

[0065] 3 points: Loss of 15% to 20% of initial body weight;

[0066] 4 points: Loss of more than 20% of initial body weight.

[0067] (2) Severity of diarrhea (0-2 points)

[0068] 0 points: No diarrhea

[0069] 1 point: Mild diarrhea

[0070] 2 points: Moderate to severe diarrhea.

[0071] (3) Degree of rectal bleeding (0-2 points)

[0072] 0 points: No bleeding

[0073] 1 point: Mild bleeding;

[0074] 2 points: Moderate to severe bleeding.

[0075] (4) Organism activity (0-2 points)

[0076] 0 points: Normal

[0077] 1 point: Mild depression;

[0078] 2 points: Moderate to severe depression.

[0079] 2.2.4 Histological scoring of colitis

[0080] (1) Degree of intestinal wall thickening (0-3 points)

[0081] 0 points: No thickening;

[0082] 1 point: Mucosal thickening;

[0083] 2 points: Thickening of the mucosa and submucosa;

[0084] 3 points: Penetrating the intestinal wall.

[0085] (2) Degree of crypt damage (0-3 points)

[0086] 0 points: No damage;

[0087] 1 point: Goblet cell loss;

[0088] 2 points: Only the surface epithelial cells are intact;

[0089] 3 points: The entire crypt and epithelial cells are lost.

[0090] (3) Inflammatory cell infiltration status (0-2 points)

[0091] 0 points: No inflammatory cell infiltration;

[0092] 1 point: Mild to moderate infiltration;

[0093] 2 points: Severe infiltration

[0094] 3. RNA extraction and gene expression detection

[0095] 3.1 Total RNA extracted from cells using the kit

[0096] (1) Use a suction pump to remove the culture medium, and wash the cells once with PBS;

[0097] (2) Add 300 μL to 500 μL of cell lysis buffer and lyse the cells at room temperature for 1 min to 2 min;

[0098] (3) Transfer the lysis buffer into the casing and centrifuge at 12000 r.pm for 1 min;

[0099] (4) Add 500 μL of Wash Buffer (containing 75% anhydrous ethanol);

[0100] (5) Centrifuge again at 12000 r.pm for 1 min;

[0101] (6) Discard the lower layer of liquid in the tube, wash again with Wash Buffer, and centrifuge for 1 min;

[0102] (7) Discard the supernatant, do not add washing solution, and centrifuge again for 1 min;

[0103] (8) Transfer to a tube, add Elution Buffer for elution, add 25 μL to 50 μL of Elution Buffer according to the amount of cells extracted, and centrifuge at 12000 r.pm for 1 min;

[0104] (9) After measuring the RNA concentration of each sample with a spectrophotometer, store it in a -80℃ freezer or proceed directly to subsequent experiments.

[0105] 3.2 Total RNA was reverse transcribed into cDNA

[0106] Using Prime Script TM RT Master Mix kit (brand: TAKARA) reverse transcription, the reaction system is shown below, taking a 20μL system as an example, see Table 1 below for details:

[0107] Table 1

[0108]

[0109] Reverse transcription was performed in a standard PCR instrument. After the reaction, the samples were stored at -20°C or directly used for subsequent experiments.

[0110] 3.3 Real-time quantitative PCR

[0111] (1) The primers for the target genes were all synthesized by Shanghai Jima Pharmaceutical Technology Co., Ltd.

[0112] (2) The specific reaction system is as follows:

[0113] Table 2

[0114]

[0115] (3) Add the pre-prepared reaction system to the 384-well plate using a comb gun;

[0116] (4) Gene quantification is performed using a real-time quantitative PCR instrument. The procedure is as follows:

[0117] Table 3

[0118]

[0119] (5) Gene expression levels are expressed in terms of 2 -ΔCt The calculation is expressed as ΔCt(treated) = [Ct(target gene) - Ct(β-ACTIN)].

[0120] (6) Primer sequence listing for specific real-time quantitative PCR:

[0121] Table 4

[0122]

[0123] 4. Immunoblotting assay

[0124] 4.1 Reagent Preparation:

[0125] (1) Electrophoresis buffer: Dissolve 30.2g Tris-base, 188g glycine and 5g SDS in 900mL deionized water, then bring the volume up to 1L to prepare a 10× stock solution and store at room temperature. When using, dilute the stock solution with deionized water to prepare a 1× working solution.

[0126] (2) Transfer buffer: Dissolve 149g glycine and 30g Tris-base in 900mL deionized water and bring the volume to 1L to obtain the 10× transfer buffer stock solution. When using, mix 100mL of the stock solution with 200mL of methanol and 700mL of double-distilled water to prepare the transfer buffer working solution, and pre-cool at 4℃.

[0127] (3) Preparation of TBS buffer: Weigh 4.2g Tris-base and 80g NaCl and dissolve them in 900mL of deionized water. Adjust the pH of the solution to 7.2-7.4 with hydrochloric acid and bring the volume to 1L to prepare a 10×TBS stock solution. When using, dilute with deionized water to 1×TBS and add Tween-20 at a ratio of 0.1% to prepare 1×TBST.

[0128] (4) Preparation of sealing solution: Add 1g of skim milk powder to 20mL of TBST, vortex until the milk powder is completely dissolved to obtain 5% skim milk sealing solution;

[0129] (5) Cell lysis buffer: RIPA lysis buffer + phosphatase inhibitor + PMSF (protease inhibitor), ready to use immediately.

[0130] 4.2 Extraction of total cell protein

[0131] Discard the culture medium supernatant, wash once with PBS, add 100 μL of cell lysis buffer, shake well, and lyse at 4°C for 30 min. After lysis, scrape the cell lysate onto one side of the culture dish with a clean scraper and transfer it to a 1.5 mL centrifuge tube using a pipette.

[0132] 4.3 Protein content determination

[0133] Protein concentration was detected using a BCA assay kit.

[0134] 4.4 Protein denaturation

[0135] Ensure consistent protein concentration during sample loading, add the corresponding volume of 5× bromophenol blue solution and mix well. Denature the protein using a metal bath at 99℃ for 8-10 minutes. After denaturation, use the sample immediately or store it at -80℃.

[0136] 4.5 Electrophoresis

[0137] (1) Preparation of separating gel (5 mL / block): Mix 1.5 mol / L Tris-HCl (pH = 8.8), 30% polyacrylamide, TEMED, 10% APS and 10% SDS according to the formula, carefully pour into the separating gel, cover the top layer with anhydrous ethanol, and let stand for at least 30 min.

[0138] (2) Preparation of stacking gel (2 mL / block): Discard the covering ethanol and inject a mixture containing deionized water, 1.5 mol / L Tris-HCl (pH = 6.8), 30% polyacrylamide, TEMED, 10% APS and 10% SDS into the top of the separating gel.

[0139] (3) Sample loading: The protein sample loading amount is 10 μg. Add an appropriate amount of loading buffer to make up the volume (so that the total volume is the same as the sample well). Marker wells are set on the left and right sides of the sample. Blank wells are filled with loading buffer.

[0140] (4) Electrophoresis: Set the voltage to a constant voltage of 80V to 90V. When the sample reaches the separating gel, increase the voltage to 100V to 120V. Stop electrophoresis in a timely manner according to the size of the target protein.

[0141] (5) Protein transfer: Mark the upper left corner of the PVDF membrane and soak the membrane in methanol to create a "sandwich structure": follow the order as follows: fiber pad -- filter paper -- PVDF membrane -- gel -- filter paper -- fiber pad (the transfer buffer should be pre-cooled at 4°C before use), and the transfer conditions are 260mA and 1.5h.

[0142] (6) Membrane blocking: After the transfer is completed, immerse the membrane in 1×TBST at room temperature for 2 min to 5 min, cut the membrane according to the size of the target protein, place it in the antibody incubation box, add blocking solution and block at room temperature for 1 h;

[0143] (7) Incubation with primary antibody: Recover the blocking buffer, wash the membrane three times with 0.1% TBST (volume greater than the blocking buffer volume), 10 min each time. Add an appropriate amount of diluted antibody and incubate overnight at 4°C with gentle shaking.

[0144] (8) Incubation with secondary antibody: Wash the membrane 3 times with 0.1% TBST, at least 10 min each time, to wash away as much residual primary antibody as possible, add the prepared secondary antibody, and incubate for 1 h;

[0145] (9) Development: Wash the membrane three times with an appropriate volume of 0.1% TBST, for at least 10 min each time. Finally, soak the membrane in the washing solution and prepare ECL developing solution. Use an ultrasensitive fully automated imaging analysis system to observe and detect the protein content.

[0146] 5. Flow cytometry analysis

[0147] (1) Use trypsin to digest the cells to be tested and prepare a single-cell suspension;

[0148] (2) Add the detection antibody at a staining ratio of 1:300, and then use a pipette to mix it up and down to stain it thoroughly;

[0149] (3) Incubate at 4℃ for 20 min to 30 min;

[0150] (4) Neutralize with 150 μL of PBS containing 1% FBS and centrifuge at 800 r.pm for 5 min;

[0151] (5) The supernatant was aspirated by a suction pump, and the cell pellet was resuspended in 150 μL of PBS containing 1% FBS. The expression of cell surface proteins was detected by flow cytometry.

[0152] 6. Histopathological analysis

[0153] 6.1 Preparation of liver tissue samples from mice with acute liver injury / colonic tissue samples from mice with colitis

[0154] After the experiment, the left lateral lobe of the mouse liver / colorectum was placed in a tissue embedding frame and fixed in 4% PFA at room temperature for 24-48 hours, followed by corresponding pathological examinations.

[0155] 6.2 Preparation of paraffin sections of liver / colon tissue

[0156] After being fixed with 4% paraformaldehyde solution, liver / colorectal tissue was dehydrated, cleared, impregnated with paraffin, and embedded to prepare tissue blocks for the next step of H&E staining.

[0157] The process for preparing paraffin-embedded tissues is as follows:

[0158] (1) Gradient alcohol dehydration:

[0159] 70% alcohol, overnight;

[0160] 80% alcohol, 2 hours;

[0161] 85% alcohol, 1.5 hours;

[0162] 90% alcohol, 40 minutes;

[0163] 95% alcohol, 20 minutes;

[0164] 100% alcohol, 1, 15 min;

[0165] 100% alcohol, 2, 15 min.

[0166] (2) Displacement of alcohol:

[0167] Xylene:ethanol = 1:1, 10 min;

[0168] Xylene 1, 10 min;

[0169] Xylene 2, 10 min.

[0170] (3) Wax impregnation:

[0171] Wax tank 1, 45 min to 1 h;

[0172] Wax tank 2, 45 min to 1 h.

[0173] (4) Embedding: Tissue is embedded on an embedding machine and the embedded tissue is placed on a freezing stage to rapidly solidify and obtain a wax block.

[0174] (5) Slide preparation: Prepare 5μm thick tissue paraffin sections, spread and dry them in a 60℃ oven for 20min, and store them at room temperature.

[0175] 6.3 H&E staining

[0176] (1) Baking the slices: Baking the slices in a 60℃ oven for 45 min to 1 h to melt all the wax on the slices.

[0177] (2) Dewaxing: Place the slices in xylene three times in sequence for 10 minutes each time.

[0178] (3) Rehydration: The dewaxed slices were placed in 100% alcohol, 95% alcohol, 85% alcohol and 75% alcohol in sequence for 4 minutes each time.

[0179] (4) Rinse: Gently rinse the film with a small stream of tap water for 2 minutes.

[0180] (5) Staining: The liver nuclei are not sensitive. The slides are stained in hematoxylin for 4 min and the colorectal tissue is stained in hematoxylin for 2 min. The excess stain is washed away, the water is drained, and then eosin is stained for 20 s. The excess stain is washed away, the water is drained, and the slides are dried in a fume hood.

[0181] (6) Sealing: Add an appropriate amount of neutral resin to the slide, cover it with a coverslip to seal it, and avoid the formation of air bubbles.

[0182] 7. Statistical Analysis

[0183] Data processing and results visualization were performed using Graphpad Prism 8 software. All data are presented as mean ± standard deviation. ANOVA was used to analyze the data. * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001; **** indicates P < 0.0001.

[0184] Example 1: PARP inhibitors enhance the immunosuppressive function of MSCs by promoting STAT1 phosphorylation.

[0185] Figure 1In this study (AG), MSCs were treated with a combination of IFN-γ (10 ng / mL) and TNF-α (10 ng / mL) in conjunction with a PARP inhibitor (10 μM) for 24 h. The expression levels of various intracellular immunosuppressive factors and the phosphorylation level of STAT1 were then examined. Results showed that using the combination of inflammatory factors IFN-γ and TNF-α as a means to enhance the immunomodulatory function of MSCs, and through Western blotting to detect intracellular STAT1 phosphorylation levels, revealed that activation of inflammatory factors led to a significant increase in intracellular STAT1 phosphorylation levels in MSCs, thereby activating the gene transcription and protein expression of downstream immunosuppressive factors IDO1 and PD-L1. The use of a PARP inhibitor further increased STAT1 phosphorylation levels, promoting the further production of immunosuppressive factors IDO1 and PD-L1.

[0186] Example 2: PARP inhibitor pretreatment enhances the disease treatment benefits of MSCs.

[0187] This invention aims to further clarify the therapeutic efficacy of PARP inhibitor pretreatment enhancing MSCs in two disease models: hepatitis and colitis. The invention was validated in both hepatitis and colitis. The therapeutic effects of different pretreatments on MSCs in hepatitis and colitis are as follows: Figure 2 As shown in the figures: A: MSCs under different pretreatment conditions were infused into ConA-induced acute liver injury mice 30 minutes after ConA induction of hepatitis to evaluate the synergistic effect of PARP inhibitors on MSC disease treatment. Mice receiving only PBS treatment served as isotype controls. B: Serum aspartate aminotransferase (AST) levels in mice were analyzed using enzyme-linked immunosorbent assay (ELISA). C: Hepatic necrosis in mice was analyzed using H&E staining. D: Statistical analysis of hepatic necrosis in mice was performed. E: MSCs with different treatments were administered intraperitoneally to mice on day 2 of DSS-induced colitis. Mice receiving only PBS treatment served as isotype controls. F: Body weight change curves for each group of mice during the disease period. G: Disease activity index was calculated based on weight loss, diarrhea severity, rectal bleeding severity, and overall physical activity in colitis mice. H: Colon tissues from each group of mice were dissected after euthanasia. I: Colon length in each group of mice. J: Intestinal wall thickening, crypt damage, and inflammatory cell infiltration were analyzed using H&E staining. K: The histological score of the colon tissue in each group was calculated comprehensively. Scale bar: 250μm.

[0188] In vivo animal experiments showed that pretreatment with PARP inhibitors combined with inflammatory factors IFN-γ and TNF-α enhanced the therapeutic efficacy of MSCs in treating acute liver injury and colitis in mice. Specifically, mice treated with the combined MSCs showed a significant decrease in serum transaminase levels and a marked reduction in centrilobular necrosis. Furthermore, the combined MSCs further improved weight loss, diarrhea, and bloody stools induced by colitis, increased activity levels, reduced disease activity index, and significantly improved colonic shortening in mice. Figure 2 In terms of pathology, intestinal wall thickening, crypt damage, and inflammatory cell infiltration were all improved, significantly reducing the overall histological score in colitis mice. These experimental results demonstrate that mesenchymal stem cells treated with PARP inhibitors in combination with inflammatory factors possess stronger immunosuppressive capabilities and can significantly improve hepatitis, an autoimmune disease characterized by abnormal inflammatory responses, potentially serving as a novel stem cell therapy for disease.

[0189] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A composition for enhancing the therapeutic efficacy of mesenchymal stem cells, characterized in that, The composition includes a PARP inhibitor and inflammatory factors; The inflammatory factors are IFN-γ and TNF-α; The concentration of the inflammatory factors is 1 ng / mL to 20 ng / mL; The PARP inhibitor is rucapranib. The concentration of the PARP inhibitor is 1 μM-20 μM.

2. A cell therapy drug for enhancing the therapeutic efficacy of mesenchymal stem cells, characterized in that, The cell drug is obtained by culturing mesenchymal stem cells using the composition of claim 1.

3. The cell-based drug according to claim 2, characterized in that, The incubation period is 24 h to 48 h.

4. The cell-based drug according to claim 2, characterized in that, The mesenchymal stem cells are derived from bone marrow, fat, or umbilical cord.

5. A drug for treating immune diseases characterized by inflammatory responses, characterized in that, It includes the cell-based drug according to any one of claims 2-4.

6. The immunological disease drug according to claim 5, characterized in that, The inflammatory response includes immune diseases characterized by abnormal inflammatory responses; the immune diseases characterized by abnormal inflammatory responses include colitis and acute hepatitis.

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