Application of enabling mesenchymal stem cells to improve autism-like behavior and immune regulation

Abstract

The invention relates to application of energized mesenchymal stem cells to preparation of a medicine or a medicine composition for preventing and / or treating autism spectrum disorder, and the energized mesenchymal stem cells are mesenchymal stem cells treated by IFN-gamma and TNF-alpha. Specifically, the endogenous mesenchymal stem cells are used for treating BTBR mice, so that social behavior defects, anxiety states and repeated inscribing behaviors of the mice can be remarkably improved, and no acute toxic reaction is generated.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of empowering mesenchymal stem cells to improve autism-like behavior and immune regulation. Background Art

[0002] Individuals with autism spectrum disorder (ASD) often exhibit significant social and communication impairments, along with repetitive behaviors and a narrow range of interests, all of which severely impact their quality of life and social adaptation. The global prevalence of ASD is increasing, garnering widespread public attention. According to recent statistics released by the US Centers for Disease Control and Prevention (CDC), 1 in 36 children (2.76%) aged 8 years are diagnosed with ASD, with the prevalence in boys being 3.8 times higher than in girls. While significant progress has been made in ASD research, the complexity of its etiology and pathogenesis means that despite intensive research, treatment remains a significant challenge. To date, no medication has been approved to treat the core symptoms of ASD. Risperidone and aripiprazole, approved by the US Food and Drug Administration (FDA), are only effective in alleviating the hyperactive and aggressive behaviors associated with ASD.

[0003] Therefore, exploring effective treatments for the core symptoms of ASD remains of great clinical significance. Summary of the Invention

[0004] The purpose of the present invention is to provide a medicine or a pharmaceutical composition for treating autism spectrum disorder.

[0005] In a first aspect, the present invention provides the use of empowered mesenchymal stem cells for preparing a medicament or pharmaceutical composition for preventing and / or treating autism spectrum disorders;

[0006] Wherein, the empowered mesenchymal stem cells are mesenchymal stem cells treated with IFN-γ and TNF-α.

[0007] In another preferred embodiment, the autism spectrum disorder has characteristics selected from the group consisting of social communication disorder, repetitive stereotyped behavior, compulsive behavior, or a combination thereof.

[0008] In another preferred embodiment, the empowered mesenchymal stem cells are mesenchymal stem cells treated with 1-100 ng / ml IFN-γ and 1-100 ng / ml TNF-α; preferably, treated with 1-50 ng / ml IFN-γ and 1-50 ng / ml TNF-α; more preferably, treated with 5-20 ng / ml IFN-γ and 5-20 ng / ml TNF-α.

[0009] In another preferred embodiment, when the mesenchymal stem cells are treated with IFN-γ and TNF-α for T1, T1 is 12 to 36 hours, preferably T1 is 18 to 30 hours, and more preferably T1 is 20 to 28 hours, thereby obtaining empowered mesenchymal stem cells.

[0010] In another preferred embodiment, the empowered mesenchymal stem cells are mesenchymal stem cells treated with 1-100 ng / ml IFN-γ and 1-100 ng / ml TNF-α.

[0011] In another preferred embodiment, the empowered mesenchymal stem cells are prepared by the following steps:

[0012] The mesenchymal stem cells were treated with 10 ng / ml IFN-γ and 10 ng / ml TNF-α for 24 hours to obtain empowered mesenchymal stem cells.

[0013] In another preferred embodiment, the mesenchymal stem cells are derived from umbilical cord blood, preferably from human umbilical cord blood.

[0014] In another preferred embodiment, the drug or pharmaceutical composition is also used for:

[0015] (1) Promote Treg cell proliferation;

[0016] (2) reduced expression of pro-inflammatory cytokine mRNA in the periphery and cerebral cortex;

[0017] (3) reduce the number of Iba1+ microglia in the cerebral cortex; and / or

[0018] (4) Promote the number of Neun+ neurons.

[0019] In another preferred embodiment, the microglial cells include activated microglial cells.

[0020] In another preferred embodiment, the pro-inflammatory cytokine is selected from the group consisting of IL-1β, TNF-α or a combination thereof.

[0021] In another preferred embodiment, the drug or pharmaceutical composition is administered to a subject selected from the group consisting of a rodent or a primate.

[0022] In another preferred embodiment, the subject is not elderly.

[0023] In another preferred embodiment, the pharmaceutical dosage form is an oral or parenteral dosage form.

[0024] In another preferred embodiment, the oral dosage form is a tablet, powder, granule or capsule, or an emulsion or syrup.

[0025] In another preferred embodiment, the non-oral dosage form is an injection or injection.

[0026] In another preferred embodiment, the pharmaceutical composition is selected from the group consisting of injection, inhalant, tincture, powder, granule, capsule, oral solution, tablet, pill, suspension, emulsion, lozenge, or pill.

[0027] In another preferred embodiment, the pharmaceutical composition is administered orally.

[0028] In another preferred embodiment, the pharmaceutical composition may be a single compound or a mixture of multiple compounds.

[0029] In another preferred embodiment, the subject of administration of the drug or pharmaceutical composition is a human or non-human mammal.

[0030] In another preferred embodiment, the dosage of the drug or pharmaceutical composition (based on empowered mesenchymal stem cells) is 1×10 3 ~1×10 8 / week, preferably 1×10 4 ~1×10 7 / week, preferably 5 × 10 4 ~1×10 6 per week, for example, about 2×10 5 per week.

[0031] In another preferred embodiment, the empowered mesenchymal stem cells have one or more of the following characteristics:

[0032] (c1) has multidirectional differentiation potential;

[0033] (c2) low telomerase activity; and / or

[0034] (c3) No malignant clones are formed.

[0035] In a second aspect, the present invention provides a method for promoting Treg cell proliferation and / or inhibiting Th17 cell proliferation, comprising the steps of:

[0036] Co-culturing empowered mesenchymal stem cells with Treg cells and / or Th17 cells to promote Treg cell proliferation and / or inhibit Th17 cell proliferation;

[0037] Wherein, the empowered mesenchymal stem cells are mesenchymal stem cells treated with IFN-γ and TNF-α.

[0038] In another preferred embodiment, the method is in vitro.

[0039] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0040] In a third aspect, the present invention provides a method for preventing and / or treating autism spectrum disorder, comprising the steps of:

[0041] administering a preventively and / or therapeutically effective amount of empowered mesenchymal stem cells to a subject in need thereof, thereby preventing and / or treating autism spectrum disorder;

[0042] Wherein, the empowered mesenchymal stem cells are mesenchymal stem cells treated with IFN-γ and TNF-α.

[0043] In another preferred embodiment, the autism spectrum disorder has characteristics selected from the group consisting of social communication disorder, repetitive stereotyped behavior, compulsive behavior, or a combination thereof.

[0044] In another preferred embodiment, the empowered mesenchymal stem cells are prepared by the following steps:

[0045] The mesenchymal stem cells were treated with 10 ng / ml IFN-γ and 10 ng / ml TNF-α for 24 hours to obtain empowered mesenchymal stem cells.

[0046] In another preferred embodiment, the mesenchymal stem cells are derived from umbilical cord blood, preferably from human umbilical cord blood.

[0047] In another preferred embodiment, the subject is not elderly.

[0048] In another preferred embodiment, the dosage of the empowered mesenchymal stem cells is 1×10 3 ~1×10 8 / week, preferably 1×10 4 ~1×10 7 / week, preferably 5 × 10 4 ~1×10 6 per week, for example, about 2×10 5 per week.

[0049] In another preferred embodiment, the empowered mesenchymal stem cells are continuously administered for 1-20 weeks, preferably 2-12 weeks, and more preferably 4-8 weeks.

[0050] In another preferred embodiment, the empowered mesenchymal stem cells are administered 1-7 times a week, preferably 1-5 times a week, and more preferably 1-3 times a week.

[0051] In another preferred embodiment, the subject includes a human or a non-human mammal.

[0052] In another preferred embodiment, the non-human mammals include rodents and primates, preferably mice, rats, rabbits, and monkeys.

[0053] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 Shown are the behavioral results of MSCs-treated BTBR mice.

[0055] Figure 2 Shown are the chondrogenic staining images of MSCs-IT and MSCs.

[0056] Figure 3 Telomerase expression levels in MSCs, MSCs-IT and HeLa cells are shown.

[0057] Figure 4 The results of soft agar experiments of MSCs, MSCs-IT and HeLa cells are shown.

[0058] Figure 5 Shown are the changes in mouse body weight and organ mass within 14 days after a single injection of MSCs-IT.

[0059] Figure 6 showed that MSCs-IT treatment improved the social preference of BTBR mice.

[0060] Figure 7 It was shown that MSCs-IT treatment improved the anxiety-like behavior of BTBR mice without affecting the mice's motor ability.

[0061] Figure 8 showed that MSCs-IT treatment improved repetitive stereotypic behaviors in BTBR mice.

[0062] Figure 9 It was shown that MSCs-IT can promote Treg cell proliferation and inhibit Th17 cell proliferation in in vitro experiments.

[0063] Figure 10 It was shown that MSCs-IT treatment increased the proportion of Treg cells in the peripheral blood and spleen of BTBR mice.

[0064] Figure 11 It was shown that MSCs-IT treatment reduced the proportion of Tfh cells in the spleen of BTBR mice.

[0065] Figure 12 It was shown that MSCs-IT treatment decreased the relative expression of proinflammatory factors in peripheral blood.

[0066] Figure 13It was shown that MSCs-IT treatment increased the proportion of Treg cells in the cerebral cortex of BTBR mice.

[0067] Figure 14 showed that MSCs-IT treatment upregulated Iba1 in the cortex of BTBR mice + The number of microglia decreased.

[0068] Figure 15 It was shown that MSCs-IT treatment decreased the proportion of M1 microglia and the M1 / M2 ratio in the cortex of BTBR mice.

[0069] Figure 16 It was shown that MSCs-IT treatment reduced the relative expression of pro-inflammatory factors in the cortex of BTBR mice.

[0070] Figure 17 showed that MSCs-IT treatment has an effect on the NeuN in the cortex of BTBR mice. + It has a protective effect on neurons and reduces their death. DETAILED DESCRIPTION

[0071] After extensive and in-depth research, a large number of experiments and screening, the inventors unexpectedly discovered for the first time that empowered mesenchymal stem cells (MSCs-IT) can be used to prepare drugs or pharmaceutical compositions for preventing and / or treating autism spectrum disorders, wherein the empowered mesenchymal stem cells are mesenchymal stem cells treated with IFN-γ and TNF-α. Experiments have shown that treating BTBR mice with empowered mesenchymal stem cells can significantly improve the mice's social behavior defects, anxiety states, and repetitive stereotyped behaviors without producing acute toxic reactions. MSCs-IT treatment synergistically exerts an immunomodulatory effect by upregulating the proportion of Treg cells, thereby improving peripheral and central inflammation in BTBR mice and thus improving their ASD-like symptoms. The present invention was completed on this basis.

[0072] the term

[0073] In order to make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions, because such methods and conditions can change. It should also be understood that the terms used herein are intended only to describe specific embodiments, and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0074] As used herein, the term “comprise” or variations thereof such as “include” or “comprising”, etc., is understood to include the stated elements or components but does not exclude other elements or components.

[0075] The term "about" can refer to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0076] As used herein, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range should be understood to include the value of any integer within the range and, where appropriate, fractional values ​​thereof (e.g., tenths and hundredths of an integer).

[0077] As used herein, the term "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0078] As used herein, the terms "mesenchymal stem cells" and "MSCs" are used interchangeably.

[0079] Peripheral and central nervous system inflammation in ASD

[0080] Dysregulation of innate immunity plays a significant role in the development of ASD. Compared with typically developing children, patients with ASD exhibit activation and imbalance of innate immunity in the periphery. Peripheral blood monocytes and lymphoblasts from children and adults with ASD produce excessive amounts of proinflammatory cytokines, such as interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), both at baseline and after stimulation with lipopolysaccharide (LPS). Furthermore, patients with ASD often suffer from chronic gastrointestinal dysfunction, and analysis of the intestinal microbiome has shown that the prevalence of Clostridium difficile (a toxin-producing bacterium) is significantly higher in ASD patients than in controls. When the intestinal mucosal barrier is damaged, these bacteria can spread throughout the body through the bloodstream. Furthermore, monocytes from patients with ASD exhibit abnormal innate immune responses when stimulated by LPS or other Toll-like receptor ligands, producing excessive amounts of proinflammatory cytokines. Astrocyte proliferation and microglial activation have been observed in different brain regions of ASD patients, particularly in the cerebral cortex and cerebellum. In these regions and in the cerebrospinal fluid, the expression levels of immune-related genes such as IL-6, TNF-α, monocyte chemoattractant protein-1 (MCP-1), transforming growth factor-β1 (TGF-β1), interferon-γ (IFN-γ), and interleukin-8 (IL-8) are significantly increased.

[0081] Numerous studies have found that ASD patients have obvious abnormalities in the ratio of Teff / Treg cells, especially the imbalance of Treg cell subsets and changes in cytokine expression, which may play an important role in the pathological process of ASD. Treg cells play a key role in regulating immune activation and inhibiting autoimmune responses. Studies have found that insufficient number of Treg cells may be the basis for the association between ASD and immune system disorders. + The levels of Treg cells and their related molecules (such as Foxp3, TGF-β and IL-10) are significantly reduced. Ahmad's team's research shows that compared with normal children, Foxp3 in ASD children is significantly reduced. + Treg cells have systemic functional defects. + CD4 + T cells, T-bet + CD4 + T cells and GATA-3 + CD4 + The proportion of T cells is significantly higher than that of normal children. A 2022 meta-analysis also pointed out that compared with the control group, the level of Treg cells in the blood of ASD patients was significantly decreased, the levels of pro-inflammatory cytokines (such as IL-1β, IL-6, IFN-γ) increased, and the levels of anti-inflammatory cytokines (such as TGF-β, IL-10) decreased. The above studies show that some ASD patients have immune function imbalances, that is, there may be patients with immune subtypes of ASD.

[0082] BTBR mice

[0083] There are currently a variety of autism animal models, including transgenic animal models (specific gene mutations) and idiopathic animal models (inbred or environmentally induced), such as the Fmr1 knockout mouse model, the VPA (valproic acid-induced) mouse model, and the BTBR inbred mouse model.

[0084] BTBR mice are widely regarded as a classic model of ASD. They show obvious behavioral defects consistent with the diagnostic criteria of ASD and are relatively stable. The typical characteristics of BTBR are decreased social skills and increased repetitive or compulsive behaviors. In the three-box social experiment, their low social level is particularly prominent. Compared with typical C57 mice, BTBR mice spend significantly less time participating in social activities in their infancy, and their sniffing and following behaviors are also reduced. The study found that their peripheral circulation showed inflammatory characteristics, the differentiation ability of MI-type macrophages was enhanced, the number of NK cells was reduced, and similar to ASD patients, there was Foxp3 +Treg cell function is defective, Th1 and Th2 cells in the spleen and blood increase, and the number of autoantibodies also increases. The study also found that the inhibition of Th17 immune response in the periphery and brain of BTBR mice (manifested by the reduction of CD4 + The BTBR mouse model, characterized by its distinct ASD-like behavioral manifestations and abnormal immune responses, is an ideal animal model for studying the efficacy of immunotherapy for ASD.

[0085] Empowering mesenchymal stem cells

[0086] The immunomodulatory properties of mesenchymal stem cells (MSCs) are plastic and susceptible to the dynamic influence of the inflammatory microenvironment under pathophysiological conditions. In an inflammatory environment, MSCs may exhibit different immunomodulatory properties, exerting both immunosuppressive effects and inducing immune responses.

[0087] In order to enhance the immunoregulatory ability of MSCs, pretreatment with inflammatory cytokines before treatment can enhance the immunosuppressive effect of MSCs. In particular, pretreatment of MSCs with cytokines such as TNF-α and IFN-γ has been shown to significantly promote the secretion of anti-inflammatory mediators (such as IL-10 and TGF-β), which help induce an immunosuppressive state and regulate the production of Treg cells. In addition, MSCs (MSCs-IT) that have undergone this "empowerment" treatment will express a large amount of immunoregulatory factors (such as IDO, TSG6, PD-L1, IL-6, and PGE2, etc.) and enhance the expression of immunosuppression-related genes (INOS). Through the above mechanism, MSCs-IT can synergize with Treg cells to exert immunosuppressive functions, thereby improving the immune imbalance of ASD patients and reducing neuroinflammation.

[0088] In the present invention, the terms "empowered mesenchymal stem cells" and "MSCs-IT" are used interchangeably, both referring to mesenchymal stem cells treated with IFN-γ and TNF-α.

[0089] Drug or pharmaceutical composition

[0090] The pharmaceutical composition provided by the present invention preferably contains 0.1-99 wt% of the first active ingredient, with the remainder being the second active ingredient, a pharmaceutically acceptable carrier, a diluent, a solution or a saline solution.

[0091] The first active ingredient of the present invention is the energized mesenchymal stem cells. In addition, it can also be used in combination with other therapeutic agents, namely the second active ingredient.

[0092] The second active ingredient may be any pharmaceutical ingredient capable of preventing and / or treating autism spectrum disorder.

[0093] When necessary, one or more pharmaceutically acceptable carriers may be added to the drug of the present invention, including conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants, etc. in the pharmaceutical field.

[0094] The compounds and pharmaceutical compositions provided by the present invention can be in various forms, such as tablets, injections, capsules, powders, syrups, solutions, suspensions and aerosols, and can be present in suitable solid or liquid carriers or diluents and in suitable sterile devices for injection or infusion.

[0095] The various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional preparation methods in the pharmaceutical field. The unit dosage of the formulation generally contains 0.05-1000 mg of the active compound of the present invention, preferably 1 mg-500 mg of the active compound of the present invention.

[0096] The pharmaceutical compositions of the present invention can be used clinically in mammals, including humans and animals, and can be administered via the oral, nasal, dermal, pulmonary, or gastrointestinal routes. Oral administration is most preferred. The most preferred daily dose is 0.01-400 mg / kg body weight taken as a single dose, or 0.01-200 mg / kg body weight taken in divided doses. Regardless of the route of administration, the optimal individual dose will depend on the specific treatment being used. Typically, a low dose is started and gradually increased until the most suitable dose is found.

[0097] The drugs or inhibitors of the present invention can be administered in various ways, for example, by injection, spraying, nasal drops, eye drops, penetration, absorption, physical or chemical mediated methods, such as introduction into the body into muscle, intradermal, subcutaneous, intravenous, or mucosal tissues; or can be mixed or encapsulated in other substances and introduced into the body.

[0098] Typically, the active ingredient of the present invention or the pharmaceutical composition containing the same can be administered in a unit dosage form, and the administration route can be enteral or parenteral, such as oral, intravenous, intramuscular, subcutaneous, nasal, oral mucosa, eyes, lungs and respiratory tract, skin, vagina, rectum, etc.

[0099] The dosage form can be a liquid dosage form, a solid dosage form, or a semisolid dosage form. Liquid dosage forms can be solutions (including true solutions and colloidal solutions), emulsions (including O / W, W / O, and multiple emulsions), suspensions, injections (including aqueous injections, powder injections, and infusions), eye drops, nasal drops, lotions, and liniments; solid dosage forms can be tablets (including ordinary tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, and orally disintegrating tablets), capsules (including hard capsules, soft capsules, and enteric-coated capsules), granules, powders, micropills, dropping pills, suppositories, films, patches, aerosols (powders), and sprays; semisolid dosage forms can be ointments, gels, pastes, and the like.

[0100] The active ingredient of the present invention can be prepared into common preparations, sustained-release preparations, controlled-release preparations, targeted preparations and various microparticle delivery systems.

[0101] In order to prepare the active ingredients of the present invention into tablets, various excipients well known in the art can be widely used, including diluents, binders, wetting agents, disintegrants, lubricants, and glidants. Diluents can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; wetting agents can be water, ethanol, isopropyl alcohol, etc.; adhesives can be starch slurry, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, acacia slurry, gelatin slurry, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinyl pyrrolidone, polyethylene glycol, etc.; disintegrants can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, etc.; lubricants and glidants can be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0102] The tablets can be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets.

[0103] To prepare the dosing unit as a capsule, the active ingredient of the present invention can be mixed with a diluent and a glidant, and the mixture can be directly placed in a hard or soft capsule. Alternatively, the active ingredient can be first prepared into granules or pellets with a diluent, a binder, and a disintegrant, and then placed in a hard or soft capsule. The diluents, binders, wetting agents, disintegrants, and glidants used to prepare the tablets of the present invention can also be used to prepare the capsules of the present invention.

[0104] To prepare the active ingredient of the present invention as an injection, water, ethanol, isopropanol, propylene glycol, or a mixture thereof can be used as the solvent, and appropriate amounts of solubilizers, cosolvents, pH adjusters, and osmotic pressure regulators commonly used in the art can be added. Examples of solubilizers or cosolvents include poloxamer, lecithin, and hydroxypropyl-β-cyclodextrin; pH adjusters include phosphates, acetates, hydrochloric acid, and sodium hydroxide; and osmotic pressure regulators include sodium chloride, mannitol, glucose, phosphates, and acetates. For lyophilized powder injections, mannitol, glucose, and the like can also be added as support agents.

[0105] Furthermore, if necessary, colorants, preservatives, perfumes, flavorings or other additives may be added to the pharmaceutical preparations.

[0106] The active ingredient or composition of the present invention can be taken alone or in combination with other therapeutic drugs or symptomatic drugs.

[0107] When the active ingredient of the present invention has a synergistic effect with other therapeutic drugs, its dosage should be adjusted according to the actual situation.

[0108] The main advantages of the present invention include:

[0109] 1. MSCs-IT have multidirectional differentiation potential and no risk of tumorigenesis, demonstrating their safety. In vitro experimental results showed that MSCs-IT can promote the proliferation of Treg cells.

[0110] 2. MSCs-IT treatment did not produce acute toxic reactions in BTBR mice, and could significantly improve the mice's social behavior defects, anxiety state and repetitive stereotyped behaviors.

[0111] 3. MSCs-IT treatment significantly increased the proportion of Treg cells in the periphery and cerebral cortex of BTBR mice, while decreasing the proportion of peripheral Tfh cells and the mRNA expression of pro-inflammatory cytokines IL-1β and TNF-α in the periphery and cerebral cortex.

[0112] 4. MSCs-IT treatment can also reduce the number of Iba1+ microglia in the cerebral cortex, especially the reduction of activated microglia, and reduce the death of Neun+ neuronal cells, which has a neuroprotective effect.

[0113] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0114] 1. Materials and Methods

[0115] 1.1 Experimental Animals

[0116] Male mice aged 6 to 8 weeks were used as experimental animals. BTBR mice were purchased from the Jackson Laboratory in the United States and bred in the SPF-grade animal room of the Experimental Center of the Second Affiliated Hospital of Kunming Medical University, with a male-to-female ratio of 1:1. The BTBR mice used in the experiment were all produced through inbreeding, and all mice used in the experiment met SPF standards. The housing environment maintained a humidity of 50% and a temperature of 22°C, with a light cycle of 12 hours of light and 12 hours of darkness. All experiments were conducted in strict accordance with the standards of Kunming Medical University, and behavioral testing was conducted between 2:00 PM and 8:00 PM daily. This animal experiment has passed the review standards of the Experimental Animal Welfare and Ethics Review Committee of the Second Affiliated Hospital of Kunming Medical University.

[0117] 1.2 Mesenchymal stem cells

[0118] MSCs-IT were provided by Yan'an Hospital. The cell line was human umbilical cord-derived MSCs (UCMSC20150831 IT) that were pretreated with IFN-γ and TNF-α (when the cell density was >80%, 10 ng / ml IFN-γ and 10 ng / ml TNF-α were added and stimulated for 24 h).

[0119] 1.3 Experimental plan

[0120] Twenty-seven BTBR mice were randomly divided into three different groups: the blank control group mice did not receive any form of intervention, the negative control group mice were injected with 100 μL of normal saline through the tail vein, and the treatment group mice were injected with 2×10 5 The mice were treated with empowered mesenchymal stem cells resuspended in 100 μL of normal saline once a week for a total of four times. One week after the end of the treatment, the mice were behaviorally tested and samples were collected.

[0121] 1.4 Behavioral experiments

[0122] 1.4.1 Three-Box Social Experiment

[0123] The three-box social interaction experiment consisted of a rectangular box measuring 60 cm x 40 cm x 22 cm. Its sides were colorless and transparent, while its bottom was dark and opaque. The center of the box was divided into three equal-sized areas by two partitions with small doors. The experiment was divided into four phases (40 minutes total): the mouse acclimation phase (10 minutes), the unfamiliar mouse phase 1 (10 minutes), the restriction phase (10 minutes), and the unfamiliar mouse phase 2 (10 minutes). Throughout the experiment, a camera was used to record the mouse's exploration path, and the ambient lighting was kept consistent.

[0124] (1) Mouse adaptation stage: Place empty transparent restraint cages on the left and right sides of the three boxes, put the experimental mice into the middle box, and allow them to explore freely for 10 minutes.

[0125] (2) Social approach preference stage: A male mouse of the same strain and of similar age that has never been in contact with the experimental mouse was placed in a transparent restraint cage with one side empty, and recorded as Stranger 1 (S1).

[0126] (3) Before starting the next stage, the experimental mice were confined to the middle box with a small partition for 10 minutes.

[0127] (4) Social novelty preference stage: Place another male mouse of the same strain and of similar age that has never been in contact with the experimental mouse in an empty transparent restraint cage on the other side, record it as Stranger 2 (S2), and record for 10 minutes. This is the end of the experiment.

[0128] Between two experiments, 75% alcohol was used to clean the experimental equipment to ensure that the residual information of the animals in the previous experiment would not affect the mice in the next experiment.

[0129] We then used the following two indicators to evaluate behavior:

[0130] The social approach preference index is calculated as: (time spent sniffing S1 - time spent sniffing the empty cage) / (time spent sniffing S1 + time spent sniffing the empty cage). This index assesses a mouse's social interest in strangers. By comparing the difference in sniffing time between the stranger (S1) and the empty cage, it reflects the mouse's tendency toward social approach behavior.

[0131] The social novelty preference index (SNP) is calculated as: (sniffing time S2 - sniffing time S1) / (sniffing time S2 + sniffing time S1). This index measures a mouse's preference for novel individuals. By comparing the difference in sniffing time between novel individuals (S2) and familiar individuals (S1), it reflects the mouse's social responsiveness to novel stimuli.

[0132] 1.4.2 Grooming experiment

[0133] Select a clean and transparent observation cage and lay an appropriate amount of clean bedding inside it to ensure the hygiene and comfort of the experimental environment. Subsequently, place the experimental mice in the observation cage and let them adapt to the environment for 10 minutes. Next, use a camera to continuously record the activities of the mice for the next 10 minutes. In order to evaluate the repetitive stereotyped behaviors of mice, the focus was on counting the time the mice spent grooming their hair within 10 minutes, including the behavior of using their mouths and forelimbs to comb their head, back, perineum, tail and other parts of the body. The statistics of these behaviors can provide a more comprehensive understanding of the stereotyped behavioral characteristics of mice.

[0134] 1.4.3 Buried Bead Experiment

[0135] In a box measuring 60cm×40cm×20cm, a 5cm thick wood shavings bedding was laid and evenly compacted to provide a stable experimental environment. Subsequently, 20 marble beads with a diameter of 1.6cm were arranged in order to form a 4×5 square array structure. When the experiment started, the mice were placed in a square white box measuring 40cm×40cm×30cm and allowed to explore freely. The exploration time was set to 10 minutes. After the experiment was over, the arrangement of the beads in the box and the degree of burial were carefully observed. If the buried area of ​​the beads exceeded 50% of the surface area of ​​the beads, they were identified as valid buried beads, and the specific number of valid buried beads was accurately recorded. This experiment can effectively evaluate the repetitive stereotyped behavior of mice.

[0136] 1.5 Treg cell detection

[0137] Cell fixation and membrane permeabilization: To quickly revive cells, place the cells in a 37°C constant temperature water bath. After the cell suspension is completely thawed, transfer it to a flow cytometry tube. Centrifuge at 400×g for 5 minutes to remove the cell freezing solution. Next, wash the cells with PBS buffer to remove impurities. Subsequently, adjust the density of the cell suspension to 1×106 / mL. Dilute 4×Fixation / Permeabilization Concentrate with Fixation / Perm Diluent in proportion to prepare a 1× working solution. Add 1mL of 1×Fixation / Permeabilization Concentrate to the cell sample and gently pipette to resuspend the cells evenly in the solution. Mix the cells and solution thoroughly to ensure the uniformity of the system, and then incubate the mixture in a dark place at room temperature for 30 minutes.

[0138] Cell staining: First, dilute a certain amount of 10× Permeabilization Buffer with purified water to 1× Permeabilization Buffer. Reserve this for later use. After incubation, centrifuge the sample at 400×g for 5 minutes. After centrifugation, carefully remove the supernatant. Next, wash the cells with 2 mL of 1× Permeabilization Buffer. Repeat this wash step twice. After two washes, leave approximately 100 μL of liquid in the tube. Gently pipette the solution to disperse the cells into a uniform suspension. Next, pipette 5 μL of antibody dropwise into each tube of cell suspension. Incubate at room temperature in the dark for 45 minutes to allow the antibody to fully bind to the cells. At the end of the incubation, add 2 mL of 1× Permeabilization Buffer directly to the tube and wash the cells with repeated pipetting. After pipetting evenly, place the tube in a centrifuge and centrifuge at 400×g for 5 minutes. Discard the supernatant and repeat the wash cycle with 2 mL of 1× Permeabilization Buffer and centrifuge at 400×g for 5 minutes. Finally, discard the supernatant and add 450 μL of cell staining buffer to the tube. Resuspend the cells by pipetting. The cell sample is ready for analysis.

[0139] 1.6 Detection of Th1, Th2, and Th17 cells

[0140] Cell culture: Remove the supernatant of the cell cryopreservation solution to prevent the cryopreservation solution from adversely affecting subsequent experiments. After removal, resuspend the cells in 1640 culture medium containing 10% FBS to create a new environment rich in nutrients and suitable for survival. Next, adjust the density of the cell suspension. Take a six-well plate and add 2mL of cell suspension with a density of 1×106 / mL, just enough to cover the bottom of the well. After adding the cell suspension, cytokine secretion should be further promoted. Accurately add 2μL of cell activation mixture (Cell Activation Cocktail (with Brefeldin A)) to each 1mL of cell suspension to stimulate cell activity and promote cytokine secretion. After adding, gently shake the plate in the front, back, left and right directions to allow the cells to fully contact the mixture. Finally, place the six-well plate containing the treated cell suspension in a cell culture incubator. The incubator is set to 37°C and contains 5% CO2, and incubate in this environment for 5 hours. During these 5 hours, the cells continue to grow and carry out physiological activities under suitable conditions, preparing for subsequent experimental steps;

[0141] Cell Fixation: After cell culture is complete, the cell suspension must be processed to ensure accuracy in subsequent experiments. First, gently pipette the cell suspension to mix thoroughly and avoid large local concentration differences. Then, transfer the suspension to a flow cytometer and centrifuge at 400×g for 5 minutes. After centrifugation, carefully discard the supernatant, retaining the cell pellet at the bottom of the tube. Next, wash the cells by slowly adding 2mL of PBS buffer to the flow cytometer. Gently pipette to resuspend the cell pellet and wash it, removing any remaining impurities. After washing, centrifuge again at 400×g for 5 minutes. After washing and centrifugation, add 1mL of cold Fixation Buffer to the flow cytometer and thoroughly pipette the cells to mix them with the Fixation Buffer. To break up the cells and prevent aggregation, shake the tube on a shaker for 5 seconds. After shaking, incubate the tube at room temperature for 15 minutes in the dark. After this 15-minute incubation, centrifuge again at 400×g for 5 minutes and carefully discard the supernatant. Finally, the cells were washed with 2 mL of PBS buffer and centrifuged at 400 × g for 5 min to complete the treatment.

[0142] Cell membrane disruption: Dilute 10× Intracellular Staining Perm Wash Buffer into 1× solution in advance and add the cells to it for resuspending. After resuspension, place the tube containing the cell suspension on an oscillator and set the oscillation time to 5 seconds to ensure that the cells are evenly distributed in the suspension. After the oscillation is completed, incubate the tube at room temperature for 20 minutes. At the end of the incubation period, place the tube in a centrifuge, set the speed to 400×g, and the centrifugation time to 5 minutes. After the centrifugation is completed, pour out the supernatant, leaving about 100μL of liquid in the tube, and use a pipette to blow away the cells in it.

[0143] Cell staining: Add 5 μL of antibody to each tube of cell suspension and incubate at room temperature for 45 minutes in the dark. At the end of the incubation, add 2 mL of 1× Intracellular Staining Perm Wash Buffer directly to the tube. Mix by pipetting to wash the cells. Centrifuge at 400 × g for 5 minutes. Repeat the wash step once more, discard the supernatant, and resuspend the cells in 450 μL of cell staining buffer. The cells are then ready for analysis.

[0144] 1.7 Tfh cell detection

[0145] The cells were quickly placed in a 37°C water bath to recover. After the cell suspension was completely dissolved, it was transferred to a flow cytometry tube and centrifuged at 400×g for 5 minutes to remove the cell freezing solution. Afterwards, the cells were resuspended in PBS buffer for washing and the density of the suspension was adjusted to 1×106 / mL. Leave approximately 100μL of liquid in the tube and blow off the cells. Add 5μL of antibody to each cell suspension and incubate at room temperature in the dark for 45 minutes. After incubation, directly add 2mL of PBS buffer for washing, centrifuge at 400×g for 5 minutes, wash the cells again, and discard the supernatant. Finally, resuspend the cells in 450μL of cell staining buffer and load them for detection.

[0146] 1.8 Immunofluorescence detection

[0147] Rewarming: Take the frozen sections out of the -80°C freezer and place them in a humidified chamber. Allow them to return to room temperature after approximately 30 minutes.

[0148] First, prepare a 3‰ PBST solution and dissolve 150μL of Tritonx-100 in 49.8mL of PBS buffer. Once completely dissolved, store in a refrigerator at 4°C.

[0149] Removal of embedding agent: Rinse three times with 3‰ PBST solution, each time for 5 minutes. After each rinse, carefully aspirate the liquid with an aspirator and remove the remaining liquid with filter paper. This step is mainly used to clean the embedding agent; then use a histochemical pen to circle the tissue.

[0150] Blocking: Use 3% goat serum blocking solution (mix 0.3g BSA powder with 1mL goat serum stock solution and dilute to 10mL with PBS buffer) to block the tissue. Add 50μL of blocking solution to each tissue histochemical circle and then place the wet box at room temperature for more than 1 hour;

[0151] Incubate primary antibodies: After the blocking process is completed, aspirate the liquid in the histochemical circle, then add Iba1+ rabbit antibody (dilution factor of 1:4000) and NeuN+ mouse antibody (dilution factor of 1:1000). All primary antibodies are prepared with blocking solution and incubate in a humidified chamber at 4°C overnight.

[0152] Wash away unbound primary antibody: Remove the wet box from the 4°C refrigerator and let it stand at room temperature for 1 hour, then rinse three times with 3‰ PBST to wash away unbound primary antibody, each time for 5 minutes;

[0153] Incubate with secondary antibodies: Prepare goat anti-rabbit (dilution 1:1000) and goat anti-mouse secondary antibodies (dilution 1:1000) in 3% blocking buffer and incubate in a humidified chamber at room temperature for 1 hour. Note that the secondary antibodies are fluorescently labeled and should be avoided from light during operation.

[0154] Wash away unbound secondary antibody: wash three times with 3‰ PBST, each time for 5 minutes;

[0155] During the sealing process, DAPI was used to stain cell nuclei, and bubbles were strictly avoided during the operation to ensure staining quality. After staining, the samples were placed under a fluorescence microscope, and four images were captured from different fields of view for each observation area of ​​each mouse. These images were then processed, and the average value of the relevant indicators in the four images for each area was calculated for subsequent analysis.

[0156] 1.9 Detection of cytokine expression in the cerebral cortex by fluorescence quantitative PCR

[0157] When using the 7500 Real-Time PCR System, follow the steps below and prepare the reaction mixture according to Table 7:

[0158] Table 7 Reaction system

[0159]

[0160] The quantitative PCR reaction setup is as follows: First, perform a pre-denaturation step at 95°C for 1 minute. After pre-denaturation, the reaction cycle begins, with each cycle consisting of a denaturation step at 95°C for 20 seconds and an annealing and extension step at 60°C for 1 minute, repeated 40 times. At the end of the reaction, the product is stored at 4°C. The melting amplification curve is then analyzed, and the Ct value is accurately recorded. The relative expression of the target gene is then calculated, and statistical analysis is performed.

[0161] 1.10 Statistical Analysis

[0162] Graph drawing: GraphPad Prism (v8.0.2.263) was used for scientific drawing; data processing: statistical analysis was completed using SPSS Statistics (version 27.0).

[0163] Continuous variables were expressed as mean ± standard deviation (mean ± SD); error bars uniformly used standard deviation to represent data dispersion.

[0164] Comparison between two groups: independent sample t test (two-sided); comparison between multiple groups: one-way ANOVA with LSD method; significance threshold: set P < 0.05 as statistically significant.

[0165] 2. Results

[0166] 2.1 The behavior of BTBR mice did not improve after initial MSCs treatment.

[0167] like Figure 1As mentioned above, MSCs treatment did not improve the behavior of BTBR mice.

[0168] 2.2 MSCs-IT Security Testing

[0169] 2.2.1MSCs-IT have good chondrogenic differentiation

[0170] The chondrogenesis assay is an in vitro method used to study chondrocyte differentiation and cartilage tissue formation. MSCs or chondrocytes are cultured in a three-dimensional culture medium containing specific induction factors, combined with a scaffold material to simulate the in vivo microenvironment and promote the production of cartilage-specific matrices (such as type II collagen and proteoglycans). This assay assesses chondrogenic differentiation using Alcian blue histological staining, thereby evaluating the potential for chondrogenic differentiation under specific induction conditions and the molecular mechanisms underlying this process.

[0171] like Figure 2 As shown in the results, MSCs-IT can effectively form cartilage, indicating that it has the same multidirectional differentiation potential as MSCs.

[0172] 2.2.2 Low telomerase activity in MSCs-IT

[0173] Telomerase is a ribonucleoproteinase that extends telomeres and plays a key role in maintaining chromosome stability and cell proliferation. Telomerase is not expressed in most normal somatic cells, but is highly active in stem cells, germ cells, and most cancer cells. Therefore, telomerase detection has important applications in cancer diagnosis, stem cell research, and the exploration of aging mechanisms.

[0174] like Figure 3 As shown, the telomerase activity of MSCs-IT was significantly lower than that of the positive control HeLa cells (cervical cancer cells). The low telomerase activity of MSCs-IT reflects normal cell cycle regulation and the lack of malignant proliferation tendency, which meets the safety requirements for clinical application.

[0175] 2.2.3MSCs-IT rarely form malignant clones

[0176] The soft agar assay is a classic in vitro method for examining the anchorage-independent growth of cells, primarily used to assess their transformation potential and tumorigenicity. While normal cells require anchorage on a solid substrate to proliferate, tumor cells or transformed cells are able to form colonies in semi-solid soft agar culture media. The soft agar assay can be used to examine whether MSCs-IT acquire abnormal proliferation properties after long-term culture, thereby assessing their safety for clinical application.

[0177] like Figure 4As shown in the results, MSCs and MSCs-IT cells, compared to HeLa cells, formed almost no colonies in the soft agar assay. This indicates that MSCs-IT cells lack the ability to form colonies, demonstrating that they have normal anchorage-dependent growth characteristics and that their proliferation behavior is strictly regulated, without undergoing malignant transformation or acquiring tumor cell characteristics. This result further validates the safety of MSCs-IT cells.

[0178] 2.2.4 MSCs-IT did not induce acute toxicity in BTBR mice

[0179] As the starting point of toxicity research, acute toxicity test refers to the experiment of exposing organisms to poisons once or multiple times within 24 hours, which can provide key data for the safety evaluation of the test substance.

[0180] To investigate whether MSCs-IT would induce acute toxicity in transplant recipients, normal saline was mixed with MSCs-IT (2×10 5 cells / 100 μL) were injected into mice through the tail vein.

[0181] like Figure 5 As shown, within 14 days after injection of saline and MSCs-IT, there was no statistically significant difference in body weight between the two groups of mice, and no significant behavioral abnormalities were observed. On day 14, the mice were sacrificed and their organs were weighed, revealing no statistically significant difference in organ mass between the two groups. These results indicate that MSCs-IT does not cause acute toxicity in mice.

[0182] 2.3 Detection of the effectiveness of MSCs-IT treatment in BTBR mice

[0183] 2.3.1 The social preference of BTBR mice was significantly improved after MSCs-IT treatment

[0184] The Three-Chamber Social Test is a classic behavioral experimental method for evaluating the social behavior and social memory of mice. It is widely used in the study of neuropsychiatric diseases such as ASD and schizophrenia. The experimental device consists of three connected transparent boxes, and the middle box is connected to the boxes on both sides by a switchable door. The experiment is divided into multiple stages, including an adaptation period, a social preference test, and a social novelty test. By placing unfamiliar mice or objects in the boxes on both sides, the interaction time and frequency of the experimental mice with the unfamiliar mice or objects are observed to assess their social tendencies and their ability to recognize novel social objects. As a naturally spontaneously formed ASD model mouse, BTBR mice exhibit typical ASD-like behavioral characteristics such as social interaction defects and repetitive stereotyped behaviors.

[0185] The inventors' previous research, through systematic behavioral testing (including three-box social experiments, grooming behavior analysis, and bead burying experiments), found that compared with C57 mice, 6- to 8-week-old BTBR mice exhibited stable and prominent autism-like behavioral characteristics. For example, the time BTBR mice spent on social interactions was significantly reduced, while the time spent alone and in a solitary state was significantly increased. They also exhibited typical stereotyped repetitive behaviors, such as a programmed head-body-tail grooming pattern (with a significant increase in grooming time) and a significant increase in the number of bead buryings. The social avoidance and stereotyped behavior characteristics exhibited by BTBR mice are accompanied by abnormal manifestations of the immune system (an imbalance in the Treg / Teff ratio in peripheral blood and spleen, a significant increase in activated microglia in the brain, and an increase in the expression of pro-inflammatory cytokines in the periphery and central nervous system), which are highly consistent with the core symptoms of autism spectrum disorder and are an ideal research model.

[0186] After BTBR mice were treated with MSCs-IT, it was found that the social approach preference index of mice in the MSCs-IT treatment group was significantly higher than that of BTBR mice (P<0.01). The sniffing time of the mice in the treatment group towards the stranger mouse 1 was also significantly increased (P<0.001), indicating that MSCs-IT significantly improved the social behavior defects of BTBR mice ( Figure 6 ).

[0187] 2.3.2 Anxiety status of BTBR mice was significantly improved after MSCs-IT treatment

[0188] As a classic behavioral testing paradigm, the open field test has important application value in neuroscience research, drug development and evaluation, and psychological and behavioral analysis by quantitatively analyzing the autonomous movement, exploratory tendencies, and anxiety-related behaviors of rodents.

[0189] The experimental setup is usually a square or circular open field surrounded by high walls, and the field is divided into a central area and a peripheral area. The experiment places the animal in the center of the field and records its activity trajectory, total movement distance, number of times it enters the central area, and length of stay within a specified time (10 minutes). The open field experiment can reflect the animal's ability to move independently, its desire to explore, and its anxiety level (animals tend to move in the peripheral areas to avoid exposure to the central area). In the open field experiment, by measuring the total distance the mouse moves, its movement state and excitability can be effectively evaluated. At the same time, the length of time the mouse stays in the central area of ​​the open field is an important indicator reflecting its level of anxiety. Generally speaking, the shorter the time the mouse stays in the central area, the higher its anxiety level; conversely, the longer the stay time, the lower the anxiety level.

[0190] like Figure 7As shown in the figure, compared with BTBR mice, the time spent in the central area by mice treated with MSCs-IT was significantly increased (P<0.01), indicating that the mice's anxiety state was alleviated. There was no statistical difference in the total distance traveled by mice in each group, indicating that MSCs-IT treatment did not affect the mice's exercise ability.

[0191] 2.3.3 Repetitive stereotypic behaviors of BTBR mice were significantly improved after MSCs-IT treatment

[0192] The grooming and bead burying tests are commonly used to assess the severity of stereotypic behaviors in mice. The longer the grooming session, the more severe the stereotypic behavior. In the bead burying test, the more beads a mouse buries, the more pronounced the stereotypic behavior. These tests provide data support for the study of stereotypic behaviors in mice.

[0193] like Figure 8 As shown in the data, compared with untreated BTBR mice, the number of beads buried in mice after MSCs-IT treatment was significantly reduced (P<0.01), and the grooming time was also significantly shortened (P<0.001), indicating that MSCs-IT treatment had a significant effect on improving the repetitive stereotyped behavior of BTBR mice.

[0194] The above results indicate that MSCs-IT treatment can improve the social deficits, repetitive stereotyped behaviors and anxiety state of BTBR mice to varying degrees.

[0195] 2.4 Immunological index detection

[0196] 2.4.1 MSCs-IT can promote Treg cell proliferation and inhibit Th17 cell proliferation in vitro

[0197] The proportion of peripheral Treg cells in BTBR mice was lower than that in C57 mice, indicating an immune overreaction.

[0198] In order to observe the effect of MSCs-IT on the proliferation of Treg cells, the proliferation promotion rate of MSCs-IT on Treg cells was detected. The results showed that MSCs-IT could promote the proliferation of Treg cells and inhibit the proliferation of Th17 cells ( Figure 9 ).

[0199] 2.4.2 MSCs-IT treatment can promote the increase of peripheral Treg cell proportion in BTBR mice

[0200] Does the proportion of Treg cells increase after MSCs-IT treatment of BTBR mice? Flow cytometry was used to analyze the Treg (CD4 + FOXP3 + ) cells in peripheral blood and spleen.

[0201] like Figure 10 As shown in the figure, compared with BTBR mice treated with normal saline, the proportion of Treg cells in the peripheral blood and spleen of BTBR mice treated with MSCs-IT was significantly increased (P<0.001).

[0202] 2.4.3 MSCs-IT treatment reduces the proportion of Tfh cells in the spleen of BTBR mice

[0203] Tfh is a follicular helper T cell that plays an important role in helping B cells produce antibody responses by promoting the formation of germinal centers, affinity maturation, and possible immunoglobulin class switching recombination. In various autoimmune diseases, such as systemic lupus erythematosus (SLE), the number of Tfh cells increases. The number of Tfh cells in the spleen of BTBR mice was abnormally increased. After treatment with MSCs-IT, the number of Tfh cells in the spleen of BTBR mice was significantly reduced (P<0.05) ( Figure 11 ).

[0204] 2.4.4 MSCs-IT treatment reduces the expression of peripheral proinflammatory factors in BTBR mice

[0205] The results of Q-PCR showed that the relative mRNA expression of pro-inflammatory cytokines IL-1β and TNF-α in PBMC (peripheral blood mononuclear cells) of BTBR mice decreased significantly after MSCs-IT treatment, further confirming that MSCs-IT treatment has a significant anti-inflammatory effect in the peripheral area (peripheral blood) of BTBR mice ( Figure 12 ).

[0206] 2.5MSCs-IT treatment alleviates cortical inflammation in BTBR mice

[0207] 2.5.1 MSCs-IT treatment promotes the increase of Treg cell proportion in the cortex of BTBR mice

[0208] The previous results showed that MSCs-IT treatment can reduce peripheral inflammation in BTBR mice. So, does it also reduce neuroinflammation in the brain of mice? We further analyzed Treg (CD4 + FOXP3 + ) cells in the cortex. Compared with the NS-treated BTBR mice, the proportion of cortical Treg cells in the MSCs-IT-treated mice was also significantly increased (P<0.001) ( Figure 13 ).

[0209] 2.5.2 MSCs-IT treatment changes the number and phenotype of Iba1+ microglia in the cortex of BTBR mice

[0210] The cerebral cortex is mechanistically thought to be related to a variety of cognitive functions (including social behavior). Microglia, in terms of number and activation status, are closely related to the pathological processes of neuroinflammation, neurodegenerative diseases, and brain injury. Assessing changes in the number of activated microglia is crucial for understanding their mechanism of action in disease. Immunofluorescence technology has become a common method for detecting the number of microglia due to its high specificity and intuitiveness. In this study, immunofluorescence technology was used to quantitatively analyze the changes in the number of microglia after MSCs-IT treatment by labeling the microglia-specific marker Iba1.

[0211] like Figure 14 As shown in the results, the number of microglia was significantly decreased after treatment (P<0.001), and the number of activated microglia was significantly reduced (P<0.001).

[0212] To explore whether MSCs-IT treatment affects the changes of M1 and M2 microglia in BTBR mice, flow cytometry was used to detect the expression of M1 (CD45 + CD11b + CD86 + ) and M2 microglia (CD45 + CD11b + CD206 + ).

[0213] like Figure 15 As shown in the results, compared with NS-treated BTBR mice, the proportion of M1 microglia (P<0.001) and the ratio of M1 / M2 microglia (P<0.001) in the cortex of BTBR mice treated with MSCs-IT were significantly reduced, while the proportion of M2 microglia remained unchanged. This suggests that MSCs-IT treatment promotes the transformation of microglia from the predominantly pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype.

[0214] The above results showed that MSCs-IT treatment significantly reduced the proportion of M1 microglia in the cortex of autistic BTBR mice, indicating that MSCs-IT may directly affect M1 / M2 polarization.

[0215] 2.5.3 MSCs-IT treatment reduces the expression of pro-inflammatory factors in the cortex of BTBR mice

[0216] To further explore the effect of MSCs-IT treatment on neuroinflammation in BTBR mice, inflammatory cytokines in the cortex of BTBR mice were detected using fluorescence quantitative PCR technology.

[0217] Q-PCR test data showed that the relative expression levels of pro-inflammatory cytokines such as IL-1β and TNF-α mRNA in the cortex of BTBR mice treated with MSCs-IT were significantly decreased ( Figure 16 This result further supports the anti-inflammatory effect of MSCs-IT treatment in the cerebral cortex of BTBR mice.

[0218] 2.5.4 MSCs-IT treatment reduces NeuN+ neuronal death in the cortex of BTBR mice

[0219] Previous studies have found that autistic BTBR mice have significantly fewer neurons than normal C57 mice (data not shown). To investigate whether MSCs-IT treatment affects the number of neurons in the cerebral cortex, neurons were stained using immunofluorescence.

[0220] The results are as follows Figure 17 As shown in the figure, compared with the BTBR mice in the NS group, the number of neurons in the cerebral cortex of the BTBR mice in the MSCs-IT treatment group was significantly increased (P<0.001), indicating that MSCs-IT has a neuroprotective effect.

[0221] 3. Conclusion

[0222] 1. MSCs-IT treatment significantly improved the abnormal behavioral patterns of BTBR mice and is expected to become a potential treatment for ASD.

[0223] 2. MSCs-IT treatment may improve ASD-like symptoms in BTBR mice by upregulating the proportion of Treg cells and synergistically exerting immunomodulatory effects, thereby improving peripheral and central inflammation.

[0224] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. Use of empowering mesenchymal stem cells, characterized in that, for preparing a medicament or pharmaceutical composition for preventing and / or treating autism spectrum disorder; Wherein, the empowered mesenchymal stem cells are mesenchymal stem cells treated with IFN-γ and TNF-α.

2. The use according to claim 1, characterized in that The autism spectrum disorder has a feature selected from the group consisting of social communication impairment, repetitive and stereotyped behaviors, compulsive behaviors, or a combination thereof.

3. The use according to claim 1, characterized in that The empowered mesenchymal stem cells are mesenchymal stem cells treated with 1-100 ng / ml IFN-γ and 1-100 ng / ml TNF-α.

4. The use according to claim 1, wherein The mesenchymal stem cells are derived from umbilical cord blood, preferably from human umbilical cord blood.

5. The use according to claim 1, characterized in that The drug or pharmaceutical composition is also used for: (1) Promote Treg cell proliferation; (2) reduced expression of pro-inflammatory cytokine mRNA in the periphery and cerebral cortex; (3) reduce the number of Iba1+ microglia in the cerebral cortex; and / or (4) Reduce NeuN+ neuronal cell death.

6. The use according to claim 1, wherein The medicament or pharmaceutical composition is administered to a subject selected from the group consisting of a rodent or a primate.

7. The use according to claim 1, characterized in that The subject is not elderly.

8. The use according to claim 1, characterized in that The dosage of the drug or pharmaceutical composition (based on the empowered mesenchymal stem cells) is 1×10 3 ~1×10 8 / week, preferably 1×10 4 ~1×10 7 / week, preferably 5 × 10 4 ~1×10 6 / week.

9. The use according to claim 1, characterized in that The empowered mesenchymal stem cells have one or more of the following characteristics: (c1) has multidirectional differentiation potential; (c2) low telomerase activity; and / or (c3) No malignant clones are formed.

10. A method for promoting Treg cell proliferation and / or inhibiting Th17 cell proliferation, characterized in that: Including steps: Co-culturing empowered mesenchymal stem cells with Treg cells and / or Th17 cells to promote Treg cell proliferation and / or inhibit Th17 cell proliferation; Wherein, the empowered mesenchymal stem cells are mesenchymal stem cells treated with IFN-γ and TNF-α.

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