Use of myeloid-derived suppressor cells in preparation of a medicament or agent for treating alzheimer's disease

Abstract

The application relates to the field of biomedical technology, and particularly relates to application of bone marrow-derived suppressor cells in preparation of drugs or reagents for treating Alzheimer's disease. The application supplements exogenous mMDSCs in an AD model transgenic mouse for the first time, improves the AD condition aggravated by Pg from two aspects of function and neuropathology, obtains certain curative effect, has clinical application potential, provides a new strategy and research and development direction for preparation of drugs or reagents for promoting Alzheimer's disease caused by Porphyromonas gingivalis, and has guiding significance.

Description

TECHNICAL FIELD

[0001] The application relates to the field of biomedical technology, and particularly relates to application of bone marrow-derived suppressor cells in preparation of drugs or reagents for treating Alzheimer's disease. BACKGROUND

[0002] Periodontitis is a chronic infectious disease caused by multiple factors, and the initial factor of periodontitis is plaque biofilm, and Porphyromonas gingivalis (Pg) is one of the important members of plaque biofilm. Long-term chronic Pg infection exists in the oral cavity of periodontitis patients, which will lead to systemic inflammatory state and is a risk factor for various systemic diseases, including Alzheimer's disease (AD). The correlation between Pg and AD and other cognitive impairment diseases has become a research hotspot in recent years.

[0003] AD is the most common type of senile dementia and has become the fifth leading cause of death in the world. Generally, AD patients can survive for 8-10 years after showing clinical symptoms. With the growth of the aging population, the number of AD patients worldwide is expected to exceed 152 million in 2050. So far, there is no cure for AD except long-term oral medication for symptomatic treatment. These challenges will bring huge economic and spiritual burden to families and society. At the same time, people over 65 years old are also a high-risk group for periodontitis, and AD patients cannot maintain oral hygiene due to cognitive and behavioral decline, so the risk of periodontitis is greatly increased, and the Pg content in the oral cavity is significantly increased. Pg can induce immune inflammatory response of the body and is an important risk factor for AD, so Pg and AD may form a vicious cycle, thereby continuously aggravating local and systemic immune inflammatory response.

[0004] At present, many studies have found that Pg promotes the occurrence and development of AD, but the specific mechanism has not been elucidated. Pg and its virulence factors from the oral cavity can enter the blood circulation, cause systemic inflammatory state, and may promote neuroinflammatory activation, thereby leading to the occurrence and development of AD. We attempt to find the key immune cells or factors of Pg in promoting the occurrence and development of AD from the immune inflammatory mechanism, and preliminarily explore the action mechanism and potential value for treating AD.

[0005] Myeloid-derived suppressor cells (MDSCs) are important peripheral immune cells that play a strong immunosuppressive role under pathological conditions such as infection, and are divided into two subgroups of monocytic MDSCs (mMDSCs) and granular MDSCs (gMDSCs). Clinical studies have reported that MDSCs are closely related to the occurrence and development of AD. Therefore, it is of great significance to study whether Pg infection promotes the occurrence and development of AD by regulating MDSCs and their subgroups. SUMMARY

[0006] The application aims at providing the application of myeloid-derived suppressor cells in the preparation of a drug or reagent for treating Alzheimer's disease.

[0007] The application achieves the above-mentioned purpose by the following technical solutions.

[0008] The application of myeloid-derived suppressor cells in the preparation of a drug or reagent for treating Alzheimer's disease.

[0009] The application of myeloid-derived suppressor cells in the preparation of a drug or reagent for treating periodontitis to promote Alzheimer's disease.

[0010] The periodontitis is caused by Porphyromonas gingivalis infection.

[0011] The myeloid-derived suppressor cells are monocytic myeloid-derived suppressor cells (mMDSCs).

[0012] The application further provides a drug for treating Alzheimer's disease, which comprises monocytic myeloid-derived suppressor cells.

[0013] Further, the drug further comprises a pharmaceutically acceptable excipient.

[0014] The application has the following beneficial effects.

[0015] (1) The application uses flow cytometry to sort and obtain mouse bone marrow-derived mMDSCs, which are injected into 5xFAD mice infected with Pg through the tail vein, proving that the supplement of exogenous mMDSCs increases the proportion and immunosuppressive function of mMDSCs in multiple organs of 5xFAD mice reduced by Pg, improves the immune microenvironment of the periphery and the center, and thus improves the AD condition aggravated by Pg.

[0016] (2) The present invention discovered that mMDSCs are the key target cells for periodontitis-induced AD. In vitro expansion and purification of mMDSCs followed by intravenous injection can effectively alleviate AD symptoms. Furthermore, the present invention's findings provide new strategies and research directions for the preparation of drugs or reagents that promote Alzheimer's disease through Porphyromonas gingivalis, which is of guiding significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Figure 5. Changes in the proportions of MDSCs and their subsets in 5xFAD mice after infection with Pg. 5xFAD mice were divided into a control group (AD) and an experimental group (AD+Pg). A: Flow cytometry plot showing the changes in the proportions of MDSCs in the blood (BL) and spleen (SP) of the two groups of mice; B: Flow cytometry plot showing the changes in the proportions of mMDSCs and gMDSCs in the blood and spleen of the two groups of mice; C: Statistical graph showing the changes in the proportions of MDSCs in the blood and spleen of the two groups of mice; D: Statistical graph showing the changes in the proportions of mMDSCs in the blood and spleen of the two groups of mice; E: Statistical graph showing the changes in the proportions of gMDSCs in the blood and spleen of the two groups of mice. *P < 0.05, **P < 0.01.

[0018] Figure 2 The changes in the proportions of MDSCs and their subpopulations after primary cells of 5xFAD mice were infected with different types of Pg. Primary bone marrow cells of 5xFAD mice were divided into a blank control group (Control), a wild-type Pg strain (33277), a heat-inactivated strain (HI-33277), and a gingipain mutant strain (136). Among them, A: Flow cytometry shows the changes in the proportions of MDSCs in the four groups of cells; B: Flow cytometry shows the changes in the proportions of mMDSCs and gMDSCs in the four groups of cells; C: Statistical graph shows the changes in the proportions of MDSCs in the four groups of cells; D: Statistical graph shows the changes in the proportions of mMDSCs in the four groups of cells; E: Statistical graph shows the changes in the proportions of gMDSCs in the four groups of cells. **P<0.01.

[0019] Figure 3Figure 5 shows behavioral testing results after 5xFAD mice were supplemented with exogenous mMDSCs. 5xFAD mice were divided into control, experimental, and treatment groups. A: Flow cytometric plot showing the gating strategy and sorting efficiency for exogenous mMDSCs sorting by flow cytometry; B: Escape latency of the three groups of mice on the first five days of the MWM experiment; C: Number of times the three groups of mice crossed the original platform on the sixth day of the MWM experiment; D: Time spent in the target quadrant on the sixth day of the MWM experiment; E: Distance traveled in the target quadrant (TQ) on the sixth day of the MWM experiment; F: Average swimming speed of the three groups of mice on the sixth day of the MWM experiment; G: Movement trajectories of the three groups of mice on the sixth day of the MWM experiment. *P < 0.05, **P < 0.01.

[0020] Figure 4 Immunofluorescence staining results of 5xFAD mice supplemented with exogenous mMDSCs. 5xFAD mice were divided into control, experimental, and treatment groups. A: Immunofluorescence images showing Aβ deposition in the cortex and hippocampus of the three groups of mice; B: Immunofluorescence images showing neurons in the cortex and hippocampus of the three groups of mice; C: Immunofluorescence images showing microglia in the cortex and hippocampus of the three groups of mice; D: Immunofluorescence images showing M2 microglia in the cortex and hippocampus of the three groups of mice; E, H: Quantitative statistics (A, D). *P < 0.05, **P < 0.01. Scale bar, 50 μm.

[0021] Figure 5 Figure 5: Changes in the proportions of endogenous MDSC subsets in 5xFAD mice after supplementation with exogenous mMDSCs. 5xFAD mice were divided into control, experimental, and treatment groups. A: Flow cytometry plots showing the proportions of MDSC subsets in peripheral blood (BL), spleen (SP), and bone marrow (BM) of the three groups of mice; B: Statistical graphs showing the proportions of mMDSCs in peripheral blood, spleen, and BM of the three groups of mice; C: Statistical graphs showing the proportions of gMDSCs in peripheral blood, spleen, and BM of the three groups of mice. *P < 0.05, **P < 0.01, ***P < 0.001.

[0022] Figure 6 Figure 5: Changes in the proportion of IL-10-producing cells in endogenous MDSC subsets after supplementation of exogenous mMDSCs in 5xFAD mice. 5xFAD mice were divided into control, experimental, and treatment groups. A: Changes in the proportion of IL-10-producing mMDSCs in the peripheral blood (BL) of the three groups of mice; B: Changes in the proportion of IL-10-producing mMDSCs in the bone marrow (BM) of the three groups of mice; C: Changes in the proportion of IL-10-producing gMDSCs in the spleen (SP) of the three groups of mice. *P < 0.05, **P < 0.01, ***P < 0.001.

[0023] Figure 7 Figure 8 is the functional changes of endogenous MDSCs after 5xFAD mice are supplemented with exogenous mMDSCs, the 5xFAD mice are divided into a control group, an experimental group and a treatment group, wherein A: Arg1 gene expression in the spleen (SP) of the three groups of mice; B: Nos2 gene expression in the spleen of the three groups of mice; C: IL-10 gene expression in the spleen of the three groups of mice; D: Arg1 gene expression in the bone marrow (BM) of the three groups of mice; E: Nos2 gene expression in the bone marrow of the three groups of mice; F: IL-10 gene expression in the bone marrow of the three groups of mice. *P<0.05.

[0024] Figure 8 Figure 9 is the changes of peripheral immune microenvironment after 5xFAD mice are supplemented with exogenous mMDSCs, the 5xFAD mice are divided into a control group, an experimental group and a treatment group, wherein A: the proportion change of IL-6-producing CD8 + cells in the peripheral blood (BL) of the three groups of mice; B: the proportion change of IL-6-producing CD4 + cells in the mesenteric lymph nodes (MLN) of the three groups of mice; C: the proportion change of IL-6-producing CD8 + cells in the mesenteric lymph nodes of the three groups of mice; D: the proportion change of IL-10-producing CD4+ cells in the mesenteric lymph nodes of the three groups of mice; E: the proportion change of IL-10-producing CD4+ cells in the spleen (SP) of the three groups of mice; F: the proportion change of T helper 1 cells in the mesenteric lymph nodes of the three groups of mice; G: the proportion change of regulatory T cells in the mesenteric lymph nodes of the three groups of mice; H: the proportion change of B10 cells in the spleen of the three groups of mice. *P<0.05, **P<0.01, ***P<0.001.

[0025] Figure 9 Figure 10 is the changes of central immune microenvironment after 5xFAD mice are supplemented with exogenous mMDSCs, the 5xFAD mice are divided into a control group, an experimental group and a treatment group, wherein A: the immunofluorescence image shows the number change of IL-10-producing immune cells in the cortex and hippocampus of the three groups of mice; B: the quantitative statistical chart shows the number change of IL-10-producing immune cells in the cortex and hippocampus of the three groups of mice. ***P<0.001. DETAILED DESCRIPTION

[0026] In order to facilitate the understanding of those skilled in the art, the present application will be further described below in conjunction with the examples, and the content mentioned in the embodiments is not a limitation of the present application.

[0027] Example 1

[0028] The present embodiment adopts flow cytometry to detect the proportion changes of MDSCs and their subgroups in the peripheral blood, spleen and bone marrow of 5xFAD mice after in vivo and in vitro infection of Pg; and by detecting the proportion changes of MDSCs and their subgroups after infection of different types of Pg (using Pg wild strain, heat-inactivated strain and gingipain triple mutant strain) in the primary cells of 5xFAD mice, the key components of Pg in regulating MDSCs and their subgroups are further explored.

[0029] The experimental method used in the present embodiment is as follows:

[0030] 1.1 Experimental animals: 3-month-old 5xFAD transgenic mice were used, and littermate negative mice were used as wild-type controls, 12 in each group, with a body weight of 20-30 g. The two kinds of mice were randomly divided into control group and experimental group, 6 in each group.

[0031] 1.2 Pg culture

[0032] (1) Pg W83 strain is used for in vivo and in vitro experiments. The specific experimental steps are as follows:

[0033] Recovery: Take out the Pg frozen bacteria, quickly melt at room temperature, and take 400 μL for inoculation into 5 mL BHI liquid medium, and mix thoroughly. Use anaerobic bag and anaerobic box to build anaerobic environment, recover at 37°C for 2-3 days, until the bacterial solution is turbid.

[0034] Identification: Use a sterile inoculation ring to dip a ring of recovered Pg bacterial solution, and draw a line on the BHI solid medium. Use anaerobic bag and anaerobic box to build anaerobic environment, and culture at 37°C for 5-7 days. The round black colonies with special odor and surrounding hemolysis ring are Pg.

[0035] Subculture: Pick Pg monoclonal colonies on BHI solid medium and inoculate into 5 mL BHI liquid medium. After that, subculture: take 200 μL of Pg bacterial solution from the previous generation and inoculate into 5 mL BHI liquid medium, mix thoroughly. Use anaerobic bag and anaerobic box to build anaerobic environment, and culture at 37°C for 2-3 days until the bacterial solution is turbid. The turbid Pg bacterial solution can be used for subculture and subsequent experiments. After 5 subcultures, discard the tube and take another tube of frozen bacteria to repeat the recovery-identification-subculture steps.

[0036] Freezing: After 2-3 subcultures, the Pg has the best activity and performance, and the turbid bacterial solution can be frozen for preservation. Mix Pg bacterial solution and 50% sterilized glycerol thoroughly according to a volume ratio of 1:1, and then divide into freezing tubes, seal with sealing film, and freeze at -80°C.

[0037] (2) Pg ATCC33277 wild strain, heat-inactivated strain and gingiprotein triple mutant KDP136 (A rgpA A rgpB A kgp) were used for in vitro experiments. The culture method of Pg ATCC33277 wild strain was the same as that of W83 strain. Pg heat-inactivated strain was obtained by incubating the bacterial suspension of Pg ATCC33277 wild strain at 70°C for 1 h, and then inoculating it on BHI solid medium for anaerobic culture for 5 days. Pg KDP136 was stored and recovered under the same conditions as W83 strain. Subculture was performed using an antibiotic selective medium: BHI liquid medium containing 5-10 μg / mL chloramphenicol, 5 μg / mL erythromycin and 1 μg / mL tetracycline.

[0038] 1.3 The specific steps for establishing a Pg-infected mouse model are as follows:

[0039] (1) Preparation of Pg suspension

[0040] The turbid Pg bacterial solution was centrifuged at 4500 rpm for 10 min at 4°C, and the supernatant was discarded. The bacterial solution was washed once with 5 mL of sterile PBS and centrifuged at 4500 rpm for 10 min at 4°C, and the supernatant was discarded. The bacterial solution was resuspended in sterile PBS to form a Pg bacterial suspension.

[0041] The concentration of the Pg bacterial suspension was determined using a NanoDrop ultramicro spectrophotometer: 1 μL of the sample was taken after thorough mixing, and the absorbance at 600 nm was measured for viable cell counting. The average value was obtained after three measurements.

[0042] The Pg bacterial suspension was centrifuged at 4500 g for 10 min at 4°C, and the supernatant was discarded. PBS containing 2% carboxymethyl cellulose was prepared as the solvent, and 1.0 x 10 9 The colony-forming unit Pg was resuspended in 0.1 mL of solvent to form a Pg suspension.

[0043] (2) Intraoral bacterial coating method was used, and Pg suspension was given to the experimental and treatment groups, and solvent was given to the control group, 0.1 mL per mouse. The treatment was performed every two days for 6 weeks.

[0044] 1.4 In vivo experiment of the effect of Pg on the proportion of MDSCs

[0045] (1) The specific steps for establishing a Pg-infected mouse model are as follows:

[0046] (2) Flow cytometry detection

[0047] ① Collection of tissue organs

[0048] Peripheral blood, spleen and femur and tibia of wild type mice and 5xFAD mice were collected.

[0049] Peripheral blood: Mice were anesthetized with isoflurane at a concentration of 3%. Blood was collected by enucleation, and collected in heparin sodium anticoagulation blood collection tubes, and immediately mixed gently to prevent coagulation, and placed at room temperature.

[0050] Spleen: Mice were sacrificed by cervical dislocation, the left upper abdominal skin was cut to expose the abdominal cavity, and the reddish-brown long strip-shaped spleen was removed and immediately placed in a 1.5 mL EP tube containing sterile PBS and placed on ice.

[0051] Femur and tibia: The skin of both lower limbs was stripped, cut off at the hip joint and ankle joint, and the femur and tibia were completely removed, the muscle tissue was stripped, and immediately placed in a 1.5 mL EP tube containing sterile PBS and placed on ice.

[0052] ②Cell preparation

[0053] Preparation of spleen single cell suspension: 4 mL of pre-cooled PBS was added to a 60 mm cell culture dish, the spleen was placed in it, ground, filtered with a 40 μm sterile cell filter, mixed gently by blowing, and collected into a 5 mL flow tube. Centrifuged at 1500 rpm for 8 min at 4°C, the supernatant was discarded, 1 mL of pre-cooled PBS was added for resuspension, and a single cell suspension was formed.

[0054] Preparation of bone marrow single cell suspension: 4 mL of pre-cooled PBS was added to a 60 mm cell culture dish, and a sterile 1 mL syringe was used to extract pre-cooled PBS for standby. The bone ends of the femur and tibia were cut to expose the red bone marrow cavity, and the syringe was inserted into one end of the bone marrow cavity, and PBS was injected to flush the bone marrow tissue out into the culture dish, which was mixed gently by blowing and collected into a 5 mL flow tube. Centrifuged at 1500 rpm for 8 min at 4°C, the supernatant was discarded, 1 mL of pre-cooled PBS was added for resuspension, and a single cell suspension was formed.

[0055] Red blood cell lysis: 3 mL of red blood cell lysis buffer was added to each tube of peripheral blood, spleen and bone marrow single cell suspension, and lysed at room temperature for 5 min, with 2 times of inversion mixing during the period. Centrifuged at 1300 rpm for 5 min at 4°C, and the supernatant was discarded.

[0056] Washing and cell counting: Each sample was resuspended and washed once with 4 mL of pre-cooled PBS. If there is flocculent precipitate, it is sucked out with a gun head. 2 μL is sucked for cell counting. Centrifuged at 1300 rpm for 5 min at 4°C, and the supernatant was discarded.

[0057] Resuspension: According to the cell counting results, an appropriate amount of pre-cooled PBS was added for resuspension, and placed on ice for standby.

[0058] ③Cell staining

[0059] Anti-mouse CD16 / 32 antibody and LIVE / DEAD TM Dead cell stain reagent, incubate on ice for 15 min in the dark. Wash once with pre- chilled PBS, centrifuge at 1300 rpm for 5 min at 4°C, discard the supernatant, and resuspend with 1 mL of pre-chilled PBS. Add cell surface antibody staining, incubate on ice for 20 min in the dark. Wash twice with pre-chilled PBS, centrifuge at 1300 rpm for 5 min at 4°C, discard the supernatant. Resuspend the cells in pre-chilled PBS containing 1.5% paraformaldehyde, and detect the proportion of MDSCs and their subsets on the machine. Use the following antibodies: CD11b APC or eF506, Gr1 PC5 or FITC, Ly6C PE, Ly6G PC7 or SB600. All data were collected on Cytoflex or Fortessa.

[0060] 1.5 Experiment on the influence of Pg on the function of MDSCs

[0061] (1) Similarly, use the bone marrow collection and cell preparation method in 1.4 above, and resuspend the cells in RPMI-1640 complete culture medium for use.

[0062] (2) Flow sorting of MDSC subsets

[0063] Resuspend the cells in pre-chilled PBS, add anti-mouse CD16 / 32 antibody, and block for 15 min on ice. Wash the cells once with pre-chilled PBS, centrifuge at 1300 rpm for 5 min at 4°C, discard the supernatant. Add anti-mouse CD11b APC, Gr1 FITC, Ly6C PE cell surface antibody staining, and incubate on ice for 20 min in the dark. Wash the cells once with pre-chilled PBS, centrifuge at 1300 rpm for 5 min at 4°C, discard the supernatant. Resuspend with pre-chilled PBS containing 2% fetal bovine serum, and place on ice in the dark for use.

[0064] Prepare several 5 mL sterile flow tubes, add 1 mL of RPMI-1640 medium containing 20% fetal bovine serum to each tube as a collection tube. Choose a nozzle with a diameter of 85 mm, and perform four-way sorting on the sample tube. The position of the circle door is: CD11b + Gr1 + Ly6C + The cell population is mMDSCs, CD11b + Gr1 + Ly6C - The cell population is gMDSCs. Collect the tube, tightly cap the tube, invert and mix, and place on ice, and replace the new collection tube in time.

[0065] Collect mMDSCs and gMDSCs from all collection tubes, centrifuge at 1500 rpm for 8 min at 4°C, and discard the supernatant. Resuspend the cells in RPMI-1640 complete medium, and wait for use.

[0066] (3) Pg treatment of bone marrow cells, mMDSCs, and gMDSCs, respectively, 2 ng / mL recombinant mouse granulocyte-macrophage colony-stimulating factor is added to the cells, and the cells are mixed thoroughly. The cells are inoculated into a 24-well plate at a concentration of 2×10 6 7 4 / mL, 2×10 7 4 / mL of Pg is added at the same time, and the cells are cultured in a cell incubator containing 5% carbon dioxide at 37°C for 48 h.

[0067] (4) MDSCs stimulation culture: after Pg treatment of the cells, 0.1 μg / mL lipopolysaccharide and 100 U / mL IFN-γ are directly added to the culture well plate, and the cells are stimulated and cultured at 37°C in a cell incubator containing 5% carbon dioxide overnight.

[0068] (5) Collection of mesenteric lymph nodes and preparation of cells, followed by magnetic separation of CD4+ T cells

[0069] Resuspend the prepared mesenteric lymph node cells in 2 mL of pre-cooled PBS, add 20 μL of anti-mouse CD4 PE antibody, and incubate on ice in the dark for 20 min. Centrifuge at 1300 rpm for 5 min at 4°C, and discard the supernatant. Resuspend by adding 450 μL of MACS buffer, add 50 μL of anti-PE magnetic beads, mix thoroughly, and incubate on ice in the dark for 20 min. Wash by adding 5 mL of MACS buffer, centrifuge at 1300 rpm for 5 min at 4°C, and discard the supernatant. Resuspend by adding 500 μL of MACS buffer, and wait for use.

[0070] Place the magnetic separation column on a magnetic stand. Wash the magnetic separation column with 1 mL of PBS for 3 times. Add the above mesenteric lymph node cell suspension to the magnetic separation column, and let it flow down naturally. Wash the magnetic separation column with 1 mL of MACS buffer for 3 times, and place a container below to collect the waste liquid. Remove the magnetic separation column from the magnetic stand, add 3 mL of MACS buffer to the magnetic separation column, and inject by pressure, repeat 3 times, and collect the target cells into a 15 mL flow tube. Mix thoroughly, count the cells, centrifuge at 1500 rpm for 8 min at 4°C, and discard the supernatant. Wash once with 10 mL of PBS, centrifuge at 1500 rpm for 5 min at 4°C, and discard the supernatant. According to the cell count result and experimental requirements, resuspend with an appropriate amount of pre-cooled PBS or RPMI-1640 medium, and place on ice for use.

[0071] (6) T cell proliferation inhibition experiment

[0072] ①Cell proliferation staining marker

[0073] Add 10 mmol / L cell proliferation dye eFluor 450 to 2 mL room temperature PBS TM 450 4 μL as a staining marker solution, mix well by blowing, and wait for use at room temperature in the dark.

[0074] The above mesenteric lymph node cells or CD4 + T cells were resuspended in 2 mL room temperature PBS. Add 2 mL of staining marker solution for resuspension, and incubate at 37°C in the dark for 10 min. Wash twice with 12 mL of pre-cooled PBS, centrifuge at 1500 rpm for 8 min at 4°C, and discard the supernatant. Resuspend the cells in RPMI-1640 medium, place on ice, and wait for use in the dark.

[0075] ②Cell co-culture

[0076] Plate coating: Dilute anti-mouse CD3 antibody to 5 μg / mL with PBS, coat a 48-well plate, 200 μL / well, and incubate overnight at 4°C.

[0077] Plate washing: Wash the 48-well plate twice with PBS, 500 μL / well, and aspirate the washing solution under negative pressure.

[0078] Co-culture: After cell counting, stimulate the bone marrow cells and mesenteric lymph node cells in a 1:1 ratio, and co-culture the stimulated MDSC subgroups and CD4 + T cells. Resuspend the cells in RPMI-1640 complete medium containing 1 μg / mL anti-mouse CD28 antibody, and incubate at 37°C in a cell culture incubator containing 5% carbon dioxide for 48-72 h.

[0079] ③Flow cytometry detection

[0080] Collect the cell co-culture solution, wash once with pre-cooled PBS, centrifuge at 1300 rpm for 5 min at 4°C, and discard the supernatant. Resuspend the cells in pre-cooled PBS containing 1.5% paraformaldehyde, and detect the dilution degree of dye eFluor 450 to evaluate T cell proliferation. TM 450.

[0081] 1.6 Statistical analysis

[0082] Use FlowJo V10 software to analyze the flow cytometry data. Use GraphPad Prism 9 software for statistical analysis of experimental data. Use t-test statistical method to analyze and compare the experimental data results between each two groups. The experimental data is expressed as mean ± standard deviation. When P<0.05, the difference between groups is statistically significant.

[0083] The embodiment explores the ratio change of MDSCs and its subsets in 5xFAD mice after Pg infection by the in vivo experiment of 1.4 above, and the experimental results are shown in Figure 1 Figure 1 The experimental results show that the ratio of mMDSCs in multiple organs decreases in wild type mice and 5xFAD mice after Pg infection, which indicates that Pg may have an important regulatory effect on mMDSCs.

[0084] In addition, the role of bacterial activity and gingivin in Pg regulating MDSCs and its subsets in 5xFAD mice is explored by the experimental method of 1.5 above, and the experimental results are shown in Figure 2 The results show that compared with the blank control group, the ratio of MDSCs and gMDSCs in the Pg wild strain treatment group of 5xFAD mice increases significantly (P<0.05), and the ratio of mMDSCs decreases significantly (P<0.05, Figure 2 C, D, E). Pg ATCC33277 infection produces similar results to W83 strain, which indicates that Pg strains may regulate MDSCs and its subsets in 5xFAD mice through the same mechanism. Regarding the key active ingredients of Pg regulating MDSCs and its subsets, it is found in adult wild type mice that Pg live bacteria down-regulate mMDSCs in a non- gingivin-dependent manner; while in adult 5xFAD mice, Pg down-regulates mMDSCs through the active ingredient of gingivin.

[0085] Embodiment 2

[0086] This embodiment explores whether the AD condition aggravated by Pg is alleviated by supplementing exogenous mMDSCs in 5xFAD mice, and provides an experimental idea: the Pg infection model of 5xFAD mice is established by intraoral coating method, mMDSCs are obtained from the bone marrow primary cells of wild type mice by flow cytometry, and mMDSCs are injected into 5xFAD mice after Pg infection.

[0087] Specifically, the experimental method used in this embodiment is as follows:

[0088] 2.1 Experimental animals: 19 5xFAD transgenic mice of 3 months old, 20-30 g, were randomly divided into three groups: 5 in the control group, 7 in the experimental group, and 7 in the treatment group.

[0089] 2.2 Pg W83 strain was cultured by the same operation steps as in embodiment 1.

[0090] 2.3 Establishment of Pg infected mouse model:

[0091] (1) Preparation of Pg suspension, the specific operation steps are the same as in embodiment 1.​

[0092] (2) Oral smearing was used. The experimental and treatment groups were given Pg suspension, while the control group was given solvent, 0.1 mL / mouse. Treatment was performed every two days for 6 weeks.

[0093] 2.4 The specific steps for supplementing exogenous mMDSCs are as follows:

[0094] (1) mMDSCs were sorted by flow cytometry, and the specific operation steps were the same as those in Example 1.

[0095] (2) 1×10 7 Resuspend mMDSCs in sterile, pre-chilled PBS at a concentration of 100 μL / mL, mix thoroughly, and place on ice. Mice in the treatment group were injected with 100 μL of the mMDSC suspension via the tail vein, while the other two groups were injected with 100 μL of PBS via the tail vein once a week for 4 weeks.

[0096] Example 3

[0097] This example uses the MWM experiment to conduct behavioral tests on the mice in Example 2 to evaluate their cognitive function. The experimental results are as follows: Figure 3 Statistical analysis of experimental data was performed using GraphPad Prism 9 software. Two-way analysis of variance was used to analyze the escape latency results of the MWM experiment. Student's t-test was used to analyze the experimental data between each other group. Data are expressed as mean ± standard deviation. Differences between groups were considered statistically significant when P < 0.05.

[0098] Depend on Figure 3 The results showed that Pg infection exacerbated spatial cognitive impairment in 5XFAD mice, and supplementation with exogenous mMDSCs reversed the Pg-induced cognitive impairment. On the other hand, Pg infection may impair motor function and endurance in mice, whereas injection of exogenous mMDSCs may lead to a corresponding recovery of motor function. Therefore, Pg infection can further impair spatial learning and memory abilities in 5XFAD mice, exacerbate cognitive impairment, and potentially lead to decreased motor function. However, supplementation with exogenous mMDSCs improved spatial learning and memory abilities, alleviated cognitive impairment, and improved motor function in Pg-infected 5XFAD mice.

[0099] Example 4

[0100] In this example, immunofluorescence staining was performed on frozen sections of the brains of 5xFAD mice after Pg infection and exogenous mMDSCs injection in Example 2 to detect and analyze the amyloid plaques (Aβ) in the cerebral cortex and hippocampus of the three groups of mice. 1–42 ) deposition, neurons (NeuN +) and microglia (Iba-1 + The specific experimental method is as follows: the immunofluorescence staining step is carried out by using a brain tissue freezing microtome, but 0.5% triton X-100 solution prepared by using 0.01 mol / L PBS solution is added dropwise to the sample area of the section after ice water bath and before immunostaining blocking, and the membrane is broken at room temperature for 20 min. The primary antibodies used include: mouse anti-mouse Aβ1-42 antibody (1:200 dilution), mouse anti-mouse NeuN antibody (1:200 dilution), rabbit anti-mouse Iba-1 antibody (1:500 dilution), mouse anti-mouse CD206 antibody (1:200 dilution) and rabbit anti-mouse IL-10 antibody (1:500 dilution). The hippocampus and cortex regions of the brain are observed under a fluorescence microscope, and image data is collected. The fluorescence intensity of Aβ deposition, the number of neurons and activated microglia, and the proportion of M2 type are calculated using ImageJ software.

[0101] The experimental results of this example are shown in Figure 4 The results show that Pg infection aggravates Aβ deposition, promotes the activation and proliferation of microglia and reduces the polarization of microglia to neuroprotective M2 type microglia, and increases the loss of neurons. After supplementing exogenous mMDSCs, the above AD-like neuropathological changes are improved.

[0102] Example 5

[0103] In this example, flow cytometry is used to detect the proportion change of endogenous MDSCs subgroups in peripheral blood, spleen and bone marrow of 5xFAD mice supplemented with exogenous mMDSCs in Example 2, and the results are shown in Figure 5

[0104] ​The experimental method used in this example is as follows: First, collect the peripheral blood, spleen, mesenteric lymph nodes and bone marrow of three groups of mice. The method of collecting tissue organs and preparing cells is the same as that of Example 1. But before blocking CD16 / 32, add 10 μg / mL lipopolysaccharide, 50 ng / mL PMA and 500 ng / mL ionomycin and 2 μmol / L monensin in RPMI-1640 complete medium, and stimulate and culture in a 37°C incubator for 5h. Wash once with pre-cooled PBS, then block, dead and surface stain. Then use Cytofix / Cytoperm Plus fixation / permeabilization solution kit to fix and break the membrane of cells at room temperature for 30 min, and wash twice with pre-cooled perm / wash diluent. Add intracellular antibodies (perm / wash diluent preparation), incubate at room temperature for 1h in the dark, and wash twice with pre-cooled PBS. Finally, resuspend the cells in pre-cooled PBS and detect as soon as possible. The antibodies used in flow cytometry are as follows: CD11b eF506, Gr1 PC5 or FITC, Ly6G SB600, CD4 PE / Dazzle, CD8 AF700, CD19 SB780, IL-6 eF450, IL-10 APC. All data are collected on Fortessa, and analyzed using FlowJo software.

[0105] By Figure 5 As can be seen from the results, the proportion of mMDSCs in the peripheral blood, spleen and bone marrow of the experimental group was significantly lower than that of the control group (P<0.05), and after injection of exogenous mMDSCs, the proportion of mMDSCs in the three kinds of tissue organs was significantly increased compared with the experimental group (P<0.05, Figure 5 B).

[0106] In addition, the proportion of gMDSCs in the peripheral blood, spleen and bone marrow of the experimental group was significantly higher than that of the control group (P<0.05), and after injection of exogenous mMDSCs, the proportion of gMDSCs in the three kinds of tissue organs was significantly decreased compared with the experimental group (P<0.05, Figure 5 C). The above results show that in 5xFAD mice, Pg infection significantly reduces the proportion of mMDSCs and increases the proportion of gMDSCs, and the supplementation of exogenous mMDSCs can partially reverse this change.

[0107] Example 6

[0108] In this example, flow cytometry is used to detect the proportion of IL-10-producing MDSCs and their subgroups in 5xFAD mice infected with Pg in Example 2, and the experimental results are shown in Figure 6 Figure 6 ​The experimental results showed that Pg infection reduced the proportion of IL-10-producing mMDSCs and gMDSCs, which may in turn reduce the secretion level of IL-10, while supplementation of exogenous mMDSCs could partially restore it.

[0109] To further evaluate the immunosuppressive function of MDSCs, qRT-PCR was used to detect the gene expressions of Arg1, Nos2, and IL-10 in the spleen and bone marrow of the three groups of mice. Figure 7 shown. Figure 7 The results showed that compared with the experimental group, the expressions of Arg1, Nos2 and IL-10 in the spleen of the treatment group showed an upward trend, but there was no statistical difference (P>0.05, Figure 7 A~C). Compared with the experimental group, the expression of Arg1 in the treatment group was significantly increased (P<0.05, Figure 7 D), the expression trend of IL-10 was similar but with no statistical difference (P>0.05, Figure 7 F), the expression of Nos2 had no significant change (P>0.05, Figure 7 E).

[0110] The above results generally indicate that in 5xFAD mice, Pg infection significantly reduces the proportion of mMDSCs and immunosuppressive function in multiple tissues and organs, and supplementation of exogenous mMDSCs can partially reverse this change.

[0111] Example 7

[0112] This example uses flow cytometry to detect changes in the ratio of regulatory T cells and regulatory B cells in the peripheral blood, spleen, and mesenteric lymph nodes of 5xFAD mice infected with Pg in Example 2. The experimental results are shown in Figure 2. Figure 8 shown.

[0113] Depend on Figure 8 Results (A), (B), and (C) indicate that Pg infection promotes CD4 + T cells and CD8 + T cells produce the inflammatory cytokine IL-6, and supplementation of exogenous mMDSCs can significantly reduce the proportion of T cells that can produce IL-6. Figure 8 The results of D and E show that Pg infection inhibits CD4 + T cells produce the anti-inflammatory cytokine IL-10, and supplementation with exogenous mMDSCs significantly increased the proportion of T cells that could produce IL-10. This suggests that Pg infection reduces IL-10 expression in spleen and bone marrow cells and reduces the proportion of IL-10-producing mMDSCs and gMDSCs, while supplementation with exogenous mMDSCs can partially restore this.

[0114] In addition to secreting the cytokine IL-10, MDSCs can inhibit the proliferation of effector immune cells. In one aspect, Figure 8 FThe results show that in 5xFAD mice, Pg infection promotes the proliferation of T helper 1 cells, while the supplementation of exogenous mMDSCs can significantly inhibit the proliferation of T helper 1 cells. From Figure 8 GThe results show that in 5xFAD mice, Pg infection has no significant effect on regulatory T cells, while the supplementation of exogenous mMDSCs can promote the proliferation of regulatory T cells. In another aspect, Figure 8 HThe results show that in 5xFAD mice, Pg infection significantly reduces B10 cells, while the supplementation of exogenous mMDSCs can promote the proliferation of B10 cells.

[0115] Example 8

[0116] This embodiment detects the expression level changes of immune suppression related genes Arg1, Nos2 and IL-10 genes of MDSCs by qRT-PCR, and further explores the changes of central immune microenvironment by immunofluorescence staining, and the experimental results are shown in Figure 9

[0117] The experimental method of qRT-PCR detection is as follows:

[0118] (1) Total RNA was extracted by Trizol method

[0119] First, the spleen and bone marrow cells of the three groups of mice were sampled and the cells were treated according to the method of Example 1. The cells were transferred to a sterile 1.5 mL EP tube, centrifuged at 2000 rpm for 5 min at 4°C, and the washing liquid PBS was discarded. 1 mL of Trizol was added to each sample, and it was allowed to stand at room temperature for 5 min. 200 μL of chloroform was added to each tube, and it was mixed thoroughly by shaking for 15 s, and allowed to stand at room temperature for 3 min. Centrifuge at 12000 rpm for 15 min at 4°C, and carefully pipette the uppermost supernatant into a new 1.5 mL EP tube. Add an equal volume of isopropanol to each tube, mix well after shaking, and let stand at room temperature for 10 min. Centrifuge at 12000 rpm for 10 min at 4°C, and discard the supernatant. Add 1 mL of 75% ethanol (prepared with nuclease-free water) to each tube, shake well, and centrifuge at 10000 rpm for 5 min at 4°C. Discard the supernatant. Open the EP tube cap and dry the precipitate at room temperature for 10-15 min. Add 30 μL of nuclease-free water to each tube, heat at 55-60°C for 10 min to completely dissolve the precipitate, and immediately place on ice. The obtained solution is the total RNA.

[0120] (2) Purity and concentration detection of RNA

[0121] ​The purity and concentration of the extracted total RNA were detected using a NanoDrop ultramicro spectrophotometer. After each sample was thoroughly mixed, 1 μL was taken for detection of its concentration and OD values at 230 nm, 260 nm and 280 nm wavelengths, and the average value was taken after 3 measurements. The RNA sample was diluted with nuclease-free water to a concentration of 150-600 ng / μL. At this time, the sample should be subjected to RT-qPCR as soon as possible, or stored at -80°C for later use.

[0122] (3) Removal of residual genomic DNA

[0123] Take 1 μg of total RNA template, 3 μL of 5×gDNA Digester Mix, and then add RNase-Free H2O to prepare a 15 μL reaction system to remove residual genomic DNA. After mixing by blowing, centrifuge immediately, incubate at 42°C for 2 min, and then immediately place on ice.

[0124] (4) Reverse transcription reaction

[0125] Take 15 μL of the above reaction solution, add ⅢSuperMix plus to prepare a 20 μL reverse transcription reaction system. After mixing, centrifuge immediately, and the reverse transcription reaction program is set as follows: 25°C for 5 min, 55°C for 15 min, and 85°C for 5 min to obtain a cDNA template, which is diluted 10 times with nuclease-free water.

[0126] (5) qRT-PCR reaction

[0127] Take 1 μL of the above diluted cDNA template to prepare a 10 μL reaction system (Table 1). The primer sequences used are shown in Table 2. The two-step amplification program is set as follows: 95°C for 5 min for pre-denaturation; 95°C for 10 s for denaturation, 60°C for 30 s for annealing / extension, for a total of 45 cycles. Check the amplification curve and melting curve, and perform quantitative analysis. WT and 5xFAD transgenic mice are used as their own control group, and the 2-△△CT method is used to calculate the relative expression of Arg1, Nos2 and IL-10 genes in the samples.

[0128] Table 1 qRT-PCR reaction system

[0129]

[0130] Table 2 qRT-PCR primer sequences

[0131]

[0132] From Figure 9The results show that in 5xFAD mice, Pg infection significantly reduces IL-10-producing immune cells in the brain, and supplementing exogenous mMDSCs can increase the number of IL-10-producing immune cells, possibly secreting higher levels of IL-10, and reducing inflammation in the brain.

[0133] In summary, according to the experimental results of Example 9 of the present application, timely and sufficient supplement of exogenous mMDSCs after Pg infection in 5xFAD mice can up-regulate the proportion and function of endogenous mMDSCs in multiple tissues and organs, and improve the immune microenvironment of the periphery and the center.

[0134] In the present application, bone marrow-derived mMDSCs of mice are obtained by flow cytometry sorting, and are injected into Pg-infected 5xFAD mice via the tail vein, which proves that supplement of exogenous mMDSCs increases the proportion and immunosuppressive function of mMDSCs reduced by Pg in multiple organs of 5xFAD mice, and improves the immune microenvironment of the periphery and the center, thereby improving the AD condition aggravated by Pg.

[0135] In summary, the present application supplements exogenous mMDSCs in AD model transgenic mice for the first time, improves the AD condition aggravated by Pg from the aspects of function and neuropathology, and obtains certain therapeutic effect, which has clinical application potential. The present application provides a new technical inspiration and has guiding significance for prevention and treatment of cognitive impairment and AD-like neuropathological changes caused by Pg.

[0136] The above specific embodiments are further illustrations of the technical solutions and beneficial effects of the present application, and are not limitations of the embodiments. Any obvious replacement without departing from the concept of the present application is within the protection scope of the present application.

Claims

1. Use of myeloid-derived suppressor cells for the preparation of a medicament for the treatment of Alzheimer's disease, characterized in that: The myeloid-derived suppressor cells are monocyte-like myeloid-derived suppressor cells, and the Alzheimer's disease is a periodontal porphyromonas gingivalis infection-promoted Alzheimer's disease.

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