Composition for enhancing treatment efficiency of mesenchymal stem cells and application

Abstract

The invention relates to a composition for enhancing the treatment efficiency of mesenchymal stem cells and application, and belongs to the technical field of cell drug treatment. The composition for enhancing the treatment efficiency of the mesenchymal stem cells comprises a PARP inhibitor and an inflammatory factor. Mesenchymal stem cells are pretreated by the composition to obtain the mesenchymal stem cells with enhanced treatment efficiency, and the mesenchymal stem cells are used for preparing drugs for treating immune diseases characterized by abnormal inflammatory reaction. The PARP inhibitor is combined with the inflammatory factor to treat the mesenchymal stem cells, so that the intracellular signal transduction and transcriptional activator protein 1 (STAT1) phosphorylation level can be improved, and the expression of immunosuppressive factor indoleamine dioxygenase 1 (IDO1) and programmed cell death ligand 1 (PD-L1) can be increased, thereby enhancing the immune regulation function of the stem cells; and a gain effect of treating immune diseases with abnormal inflammatory reaction as a main characteristic is realized.

Description

Technical Field

[0001] The present invention relates to the technical field of cell drug therapy, and in particular to a composition and application thereof for enhancing the therapeutic efficacy of mesenchymal stem cells. Background Art

[0002] Mesenchymal stem cells (MSCs) are a type of adult stem cell that is widely present in a variety of tissues and organs. They are closely related to individual growth and development, maintenance of tissue homeostasis, and organ damage repair. In addition to having strong self-renewal capabilities and multiple differentiation potentials, MSCs also have immune regulation capabilities. When the body is damaged, both 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, and promoting injury repair and tissue regeneration. Therefore, MSCs have become an important research object in the field of life medicine, bringing hope to the treatment of a variety of refractory and intractable diseases.

[0003] Due to its convenient source, easy access and low immunogenicity, MSCs are expected to become one of the cell therapy preparations for various inflammatory diseases. Compared with first-line immunosuppressive drugs, MSCs do not systematically reduce the function of the immune system, but instead reduce the body's inflammatory damage and promote tissue regeneration and repair by targeting the immune response under high inflammation.

[0004] In actual clinical trials, it was found that when using MSCs to treat inflammatory diseases, if the amount of cells injected is too low, the expected therapeutic effect will not be achieved; on the contrary, if the amount of stem cell infusion is increased, the risk of thrombosis will increase significantly, causing serious adverse reactions. Therefore, it is particularly important in clinical practice to improve the immune function characteristics of the infused cells at the original dose. Summary of the invention

[0005] In order to solve the many pain points of the above-mentioned treatments, the present invention provides a composition and application for enhancing the therapeutic efficacy of mesenchymal stem cells. The improved mesenchymal stem cells described in the present invention are obtained by treating with a clinical drug PARP inhibitor in combination with inflammatory factors. The present invention uses PARP inhibitors in combination with inflammatory factors to pre-treat mesenchymal stem cells in vitro to promote the phosphorylation level of intracellular signal transducer and activator of transcription 1 (STAT1), increase the expression of immunosuppressive factors indoleamine dioxygenase 1 (IDO1) and programmed cell death ligand 1 (PD-L1), thereby enhancing the immune regulation function of stem cells and enhancing the efficacy of mesenchymal stem cells in treating immune diseases characterized by abnormal inflammatory responses, such as colitis and hepatitis.

[0006] The present invention is achieved through the following technical solutions:

[0007] The first object of the present invention is to provide a composition for enhancing the therapeutic efficacy of mesenchymal stem cells, wherein the composition comprises a PARP inhibitor and an inflammatory factor.

[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-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 are multipotent stem cells with self-renewal and multidirectional differentiation potential. Umbilical cord-derived mesenchymal stem cells (UC-MSCs) are one of the main types of MSCs used in current clinical trials, and can differentiate into a variety of interstitial tissues, such as bone, cartilage, fat, and bone marrow hematopoietic tissue. The cell use examples listed in the present invention are implemented in accordance with the standards of the Ethics Committee of Soochow University.

[0014] It is particularly pointed out that the human pluripotent stem cells used in the present invention cannot develop into a complete individual and have been approved by the Ethics Committee of Soochow University for use in scientific research.

[0015] The second object of the present invention is to provide a cell medicine for enhancing the therapeutic efficacy of mesenchymal stem cells, wherein the cell medicine is obtained by culturing mesenchymal stem cells using the composition.

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

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

[0018] The third object of the present invention is to provide a drug for treating immune diseases characterized by inflammatory response, comprising the cell drug.

[0019] In some embodiments of the present invention, the inflammatory response includes an immune disease characterized by an abnormal inflammatory response; the immune disease characterized by an abnormal inflammatory response includes colitis, hepatitis, graft-versus-host disease, and the like.

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

[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 comprises a pharmaceutically acceptable carrier.

[0023] In some embodiments of the present invention, the carrier is selected from one or more of a disintegrant, a diluent, a lubricant, a binder, a wetting agent, a flavoring agent, a suspending agent, a surfactant and a preservative.

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

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

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

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

[0028] The present invention uses PARP inhibitors to pre-treat mesenchymal stem cells in vitro, which can show a more efficient therapeutic effect compared to the same number of mesenchymal stem cells that have not been pre-treated, thereby providing a novel enhancement strategy for the treatment of immune diseases (such as colitis and hepatitis) characterized by abnormal inflammatory responses.

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

[0030] The present invention provides a composition and 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 therapeutic enhancement effect on immune diseases characterized by abnormal inflammatory reactions, such as enteritis and hepatitis, is achieved. 5 ) MSCs pretreated with DMSO and inflammatory factors failed to significantly alleviate the disease signs in mice. However, the infusion of the same number of MSCs pretreated with PARP inhibitors and inflammatory factors significantly improved the level of transaminase in the serum of hepatitis mice and inhibited liver tissue necrosis. It also alleviated the disease symptoms of enteritis mice, such as weight loss, diarrhea, blood in the stool, intestinal tissue thickening and edema, structural damage, and immune cell infiltration. Therefore, pretreatment of MSCs with PARP inhibitors can enhance the immune regulation function of stem cells and achieve a gain effect in stem cell disease treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

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

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

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

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

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

[0037] (1) Use trypsin to digest and passage the MSCs in the culture dish, resuspend and centrifuge them, and then inoculate them into a new culture dish for expansion and culture;

[0038] (2) Fresh culture medium was replaced every two days until the cell confluence reached 80% to 90%. Only MSCs within 10 generations were used in this experimental study.

[0039] 2. Hepatitis model / colitis animal experiment

[0040] 2.1.1 Experimental animals

[0041] The 8-10-week-old male C57BL / 6J mice weighing about 25 g were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. and were raised in strict accordance with the SPF barrier system standards. The animal experiments involved in the present invention were approved by the Animal Experiment Ethics Committee of Soochow University.

[0042] 2.1.2 Establishment of hepatitis model

[0043] On the day of the experiment, C57BL / 6J mice were randomly divided into four groups, and concanavalin A solution (ConA, molecular weight: 102000Da) was injected into the tail vein at a dose of 15 mg / kg to induce an acute liver injury model. The control mice were injected with the same volume of PBS as the isotype control of the experiment. ConA activates T lymphocytes in the body and causes them to overproliferate. The excessive inflammatory response leads to acute necrosis of mouse liver cells, which eventually induces the occurrence of hepatitis in the body.

[0044] 2.1.3 Treatment of hepatitis model

[0045] After 30 min of induction of acute liver injury model in mice, 2×10 5 The mice were given a tail vein infusion of MSCs pretreated with PARP inhibitor Rucaparib and inflammatory factors IFN-γ and TNF-α (10 ng / mL) for 24 hours to treat the disease. The model control mice were given a tail vein infusion of the same volume of PBS. After 12 hours, the peripheral blood and liver tissues of the mice were collected for detection.

[0046] 2.1.4 Serum aspartate aminotransferase concentration detection

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

[0048] (2) The matrix solution was preheated in a 37°C water bath and then added to a 96-well plate at 20 μL / well. Then, 5 μL / well of serum was added to the test wells.

[0049] (3) Mix the serum sample and matrix solution in the well plate repeatedly with a pipette, taking care to avoid generating bubbles;

[0050] (4) Place in a 37°C incubator in an air bath for 30 min;

[0051] (5) Add chromogenic solution to the 96-well plate at 20 μL / well and add 5 μL of deionized water to the control wells;

[0052] (6) Mix the serum, matrix solution, and color development solution in the well plate repeatedly with a pipette, taking care to avoid generating bubbles;

[0053] (7) Place in a 37°C incubator in an air bath for 20 min;

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

[0055] (9) Read the absorbance value of the sample at a wavelength of 510 nm using an ELISA 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 colitis animal model

[0058] On the 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), and the control mice drank normal water. They were fed for a total of 7 days to induce colitis.

[0059] 2.2.2 Treatment of colitis model

[0060] On the second day of induction of colitis in mice, the mice were infused with 2×10 cells / mL pretreated with PARP inhibitor Rucaparib (10 μM) and inflammatory factors IFN-γ and TNF-α (10 ng / mL) for 24 h. 5 The same volume of MSCs was used to treat the disease in model control mice, and the same volume of PBS was infused into the tail vein of the mice.

[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) Diarrhea severity (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) Body 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 mucosa and submucosa;

[0084] 3 points: Penetration of 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: loss of the entire crypt and epithelial cells.

[0090] (3) Inflammatory cell infiltration (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 Kit to extract total cellular RNA

[0096] (1) Remove the culture medium with a suction pump 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 lysate into a cannula and centrifuge at 12000 rpm for 1 min;

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

[0100] (5) Centrifuge again at 12000 rpm for 1 min;

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

[0102] (7) Discard the supernatant, centrifuge again for 1 min without adding washing solution;

[0103] (8) Transfer the tube and add Elution Buffer for elution. Add 25 μL to 50 μL Elution Buffer according to the amount of cells extracted and centrifuge at 12,000 rpm for 1 min.

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

[0105] 3.2 Reverse transcription of total RNA into cDNA

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

[0107] Table 1

[0108]

[0109] Perform reverse transcription in a common PCR instrument. After the reaction is complete, store the sample in a -20°C refrigerator or proceed directly to subsequent experiments.

[0110] 3.3 Fluorescence real-time quantitative PCR

[0111] (1) All target gene primers tested were 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 into the 384-well plate using a dispenser;

[0116] (4) Perform gene quantitative detection in a real-time fluorescence quantitative PCR instrument, and the operating procedure is as follows:

[0117] Table 3

[0118]

[0119] (5) The expression level of the gene is 2 -ΔCt The calculation means, ΔCt(treated)=[Ct(target gene)-Ct(β-ACTIN)].

[0120] (6) Specific real-time fluorescence quantitative PCR primer sequence list:

[0121] Table 4

[0122]

[0123] 4. Western Blotting

[0124] 4.1 Reagent configuration:

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

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

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

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

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

[0130] 4.2 Total cell protein extraction

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

[0132] 4.3 Protein content determination

[0133] The protein concentration was determined using a BCA assay kit.

[0134] 4.4 Protein denaturation

[0135] Ensure that the concentration of the loaded protein is consistent, add the corresponding volume of 5× bromophenol blue solution and mix well, use a metal bath to denature the protein at 99°C for 8min to 10min, and use the sample immediately after denaturation or store it at -80°C.

[0136] 4.5 Electrophoresis

[0137] (1) Prepare separation gel (5 mL / piece): Mix 1.5 mol / L Tris-HCl (pH = 8.8), 30% polyacrylamide, TEMED, 10% APS and 10% SDS according to the recipe, carefully inject into the separation gel, cover the top layer with anhydrous ethanol, and let stand for at least 30 min;

[0138] (2) Preparation of stacking gel (2 mL / piece): 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 upper end of the separation gel;

[0139] (3) Sample loading: The protein sample loading volume is 10 μg, and an appropriate amount of loading buffer is added to fill 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, and the blank wells are filled with loading buffer;

[0140] (4) Electrophoresis: The voltage is set to a constant voltage of 80 V to 90 V. When the sample reaches the separation gel, the voltage is increased to 100 V to 120 V. The electrophoresis is stopped at an appropriate time 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 make a "sandwich structure": follow the following sequence: fiber pad - filter paper - PVDF membrane - gel - filter paper - fiber pad (the transfer buffer was pre-cooled at 4 °C before use), transfer conditions were 260 mA, 1.5 h;

[0142] (6) Membrane blocking: After transfer, soak 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, and add blocking solution to block it at room temperature for 1 h.

[0143] (7) Incubate with primary antibody: Recover the blocking solution and wash the membrane three times with 0.1% TBST (a volume larger than the blocking solution), 10 min / time. Add an appropriate amount of diluted antibody and shake gently at 4°C overnight;

[0144] (8) Incubation with secondary antibody: Wash the membrane three times with 0.1% TBST for at least 10 min each time to remove the remaining primary antibody as much 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, and finally soak it in the membrane washing solution. Prepare ECL developer and observe and detect the protein content using an ultra-sensitive fully automatic imaging analysis system.

[0146] 5. Flow Cytometry Analysis

[0147] (1) Digest the cells to be tested with trypsin to prepare a single cell suspension;

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

[0149] (3) Incubate at 4°C for 20–30 min.

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

[0151] (5) Remove the supernatant with a suction pump, resuspend the cell pellet with 150 μL of PBS containing 1% FBS, and detect the expression of cell surface proteins by flow cytometry.

[0152] 6. Histopathological Analysis

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

[0154] After the experiment, the left lateral lobe of the mouse liver / colorectum was taken and placed in a tissue embedding frame, fixed in 4% PFA at room temperature for 24 h to 48 h, and then the corresponding pathological examination was performed.

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

[0156] The liver tissue / colorectal tissue fixed with 4% paraformaldehyde solution was dehydrated, transparentized, wax-impregnated and embedded, and then made into tissue wax blocks to prepare for the next step of H&E staining.

[0157] The process of paraffin-embedded tissue preparation is as follows:

[0158] (1) Gradient alcohol dehydration:

[0159] 70% alcohol, overnight;

[0160] 80% alcohol, 2h;

[0161] 85% alcohol, 1.5h;

[0162] 90% alcohol, 40min;

[0163] 95% alcohol, 20min;

[0164] 100% alcohol 1, 15min;

[0165] 100% alcohol 2, 15min.

[0166] (2) Replacement of alcohol:

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

[0168] Xylene 1, 10min;

[0169] Xylene 2, 10min.

[0170] (3) Wax dipping:

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

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

[0173] (4) Embedding: Embed the tissue on an embedding machine and place the embedded tissue on a freezing table to allow it to solidify quickly to obtain a wax block.

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

[0175] 6.3 H&E staining

[0176] (1) Slice baking: Bake the slices in an oven at 60°C for 45 min to 1 h to melt all the wax on the slices.

[0177] (2) Dewaxing: Dewax the sections in xylene three times, each time for 10 min.

[0178] (3) Rehydration: Rehydrate the dewaxed sections in 100% alcohol, 95% alcohol, 85% alcohol, and 75% alcohol in turn, soaking for 4 min each time.

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

[0180] (5) Staining: Liver nuclei are insensitive. Stain the slides in hematoxylin for 4 min, and colorectal tissues in hematoxylin for 2 min. Wash off the floating color, drain the water, and then stain in eosin for 20 s. Wash off the floating color, drain the water, and dry in a fume hood.

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

[0182] 7. Statistical Analysis

[0183] Graphpad Prism 8 software was used for data processing and result presentation. All data are mean ± standard deviation, and the data were tested using ANOVA. * represents P < 0.05; ** represents P < 0.01; *** represents P < 0.001; **** represents P < 0.0001.

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

[0185] Figure 1In the experiment (AG), MSCs were treated with IFN-γ (10 ng / mL) and TNF-α (10 ng / mL) in combination with PARP inhibitors (10 μM) for 24 hours, and then the expression levels of intracellular immunosuppressive factors and the phosphorylation level of STAT1 were detected. The results showed that the use of the inflammatory factor combination IFN-γ and TNF-α as a means to empower the immune regulation function of MSCs and the detection of intracellular STAT1 phosphorylation levels by WB showed that the activation of inflammatory factors would lead to a significant increase in the phosphorylation level of intracellular STAT1 in MSCs, thereby activating the gene transcription and protein expression of downstream immunosuppressive factors IDO1 and PD-L1. The use of PARP inhibitors can further increase the phosphorylation level of STAT1 and promote the further production of immunosuppressive factors IDO1 and PD-L1.

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

[0187] The present invention further clarifies that PARP inhibitor pretreatment enhances the therapeutic effect of MSCs in two disease models of hepatitis and colitis. The present invention is verified in two diseases of hepatitis and colitis. The therapeutic effect of MSCs with different pretreatments on hepatitis and colitis is as follows Figure 2 As shown. A: 30 minutes after ConA-induced hepatitis, MSCs under different pretreatment conditions were infused into mice with ConA-induced acute liver injury to evaluate the enhancing effect of PARP inhibitors on MSCs disease treatment, among which mice treated with only PBS served as isotype controls for disease treatment. B: Serum aspartate aminotransferase levels in mice were analyzed using enzyme-linked immunosorbent assay. C: H&E staining was used to analyze liver necrosis in mice. D: Statistical analysis of liver necrosis in mice. E: MSCs with different treatments were intraperitoneally administered to mice on the second day of DSS-induced colitis, among which mice treated with only PBS served as isotype controls for disease treatment. F: Weight change curves of mice in each group during the disease period. G: The disease activity index of the body was calculated based on the weight loss, diarrhea, rectal bleeding and body activity of colitis mice. H: Colon tissues of mice in each group were dissected and separated after euthanasia. I: Colon length of mice in each group. J: H&E staining was used to analyze intestinal wall thickening, crypt damage and inflammatory cell infiltration. K: Comprehensive evaluation of the histological scores of colon tissues in each group. Scale bar: 250 μm.

[0188] The results of in vivo animal experiments showed that PARP inhibitors combined with inflammatory factors IFN-γ and TNF-α pretreatment can enhance the therapeutic effect of MSCs on acute liver injury and colitis in mice. Specifically, the serum transaminase levels of mice treated with MSCs with the combined treatment were significantly reduced, and the area of ​​central lobular necrosis was significantly reduced. In addition, the combined treatment of MSCs can further improve the weight loss, diarrhea and bloody stool symptoms caused by colitis, the activity of mice is improved, the disease activity index is reduced, and the shortening of the colon is also significantly improved ( Figure 2 ). In terms of pathology, intestinal wall thickening, crypt damage and inflammatory cell infiltration were also improved, greatly reducing the comprehensive histological score of colitis mice. These experimental results show that mesenchymal stem cells treated with PARP inhibitors and inflammatory factors have stronger immunosuppressive ability, can significantly improve hepatitis, an immune disease characterized by abnormal inflammatory response, and can be used as a novel disease stem cell drug treatment preparation.

[0189] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A composition for enhancing the therapeutic efficacy of mesenchymal stem cells, characterized in that: The composition includes a PARP inhibitor and an inflammatory factor.

2. The composition according to claim 1, characterized in that The inflammatory factors include IFN-γ and TNF-α.

3. The composition according to claim 1, characterized in that The concentration of the inflammatory factor is 1 ng / mL-20 ng / mL.

4. The composition according to claim 1, characterized in that The PARP inhibitor is selected from one or more of rucaparib, olaparib and niraparib.

5. The composition according to claim 1, characterized in that The concentration of the PARP inhibitor is 1 μM-20 μM.

6. A cell medicine for enhancing the therapeutic efficacy of mesenchymal stem cells, characterized in that: The cell medicine is obtained by culturing mesenchymal stem cells using the composition according to any one of claims 1 to 5.

7. The cell medicine according to claim 6, characterized in that The culture time is 24h-48h.

8. The cell medicine according to claim 6, characterized in that: The mesenchymal stem cells are derived from bone marrow, fat or umbilical cord.

9. A drug for treating an immune disease characterized by an inflammatory response, characterized in that: A cell medicine comprising any one of claims 6 to 8.

10. The immune disease drug according to claim 9, characterized in that: The inflammatory response includes immune diseases characterized by abnormal inflammatory response; the immune diseases characterized by abnormal inflammatory response are colitis, acute hepatitis and graft-versus-host disease.

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