Compositions of intestinal flora modulators in combination with mesenchymal stem cells and uses thereof

Abstract

This invention belongs to the field of biomedicine and relates to a composition of intestinal flora regulators and mesenchymal stem cells and its application. The composition of this invention is applicable to the treatment of diabetes, effectively enhancing the therapeutic effect of mesenchymal stem cells in diabetes treatment, especially autoimmune type 1 diabetes, reducing pancreatic islet inflammation, and preserving more pancreatic β cells. It provides support for the clinical translation and application of mesenchymal stem cells and has promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and particularly relates to the composition of intestinal flora regulators and mesenchymal stem cells and their applications. Background of the Invention

[0002] The gastrointestinal tract of mammals contains a diverse and active microbial community, including bacteria, archaea, eukaryotes, and viruses, collectively known as the "gut microbiota," which comprises approximately 10... 14 These microorganisms encode approximately 100 times more genes than the human body itself. Among these microorganisms, bacteria are the most numerous, forming the gut microbiota. They have co-evolved with the host over a long period, forming a complex and mutually beneficial relationship, and are considered a "new organ" of the host. The gut microbiota plays a crucial role in maintaining homeostasis, including food digestion and absorption, stimulating the maturation of the immune system, regulating metabolism, and resisting the colonization and proliferation of pathogenic microorganisms in the gut. However, when the ecological balance of the gut microbiota is disrupted and the composition of the gut microbiota changes, these functions are also affected; this is known as gut microbiota dysbiosis. In some cases, such as excessive use of antibiotics or an inappropriate diet leading to gut microbiota imbalance, the occurrence and progression of diseases can be promoted, while regulating the dysbiosis can be beneficial for disease treatment.

[0003] Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia. Symptoms of diabetes include the "three highs and one low" (polyuria, polydipsia, polyphagia, and weight loss), and are most common in type 1 diabetes. Type 2 diabetes often presents with less obvious symptoms or only some symptoms. Hyperglycemia is caused by defects in insulin secretion or impaired biological action, or both. Type 1 diabetes mellitus (T1DM) is an autoimmune disease characterized by the infiltration and attack of pancreatic β-cells by T lymphocytes or other immune cells, leading to the destruction and reduction of β-cell numbers, ultimately resulting in an absolute deficiency of insulin. Although the etiology of T1DM remains unclear, the mainstream view considers it a polygenic disease influenced by environmental factors. Studies have shown that genetic risk factors are necessary but not sufficient for the disease, and many environmental factors, including viral infections, diet, pregnancy-related infections, and antibiotic use, have been proposed as candidate causes of T1DM. Furthermore, studies have shown that the gut microbiota structure of patients with type 1 diabetes mellitus (T1DM) differs significantly from that of healthy individuals, exhibiting an increased proportion of Bacteroidetes and a decreased proportion of Firmicutes, along with lower gut microbiota diversity and poorer stability. Non-obese diabetic mice (NOD mice), a type of spontaneously diabetic mouse, have shown that antibiotic treatment that disrupts their healthy gut microbiota before the onset of T1DM can accelerate the progression of T1DM and increase its incidence. These studies all demonstrate a significant link between gut microbiota and T1DM.

[0004] Currently, insulin therapy remains the most common treatment for type 1 diabetes mellitus (T1DM), which aims to restore blood glucose levels to near normal while preventing hypoglycemia. However, although exogenous insulin can improve blood glucose levels, it cannot address fluctuating blood glucose levels because it does not improve the function of pancreatic β-cells in secreting insulin. While islet and pancreas transplantation can improve insulin secretion, its widespread availability is hampered by donor scarcity and surgical risks. In recent years, mesenchymal stem cells (MSCs) have been widely used in research for the treatment of various diseases, and their excellent immunomodulatory capabilities are considered to have broad application prospects in the treatment of autoimmune diseases. In a streptozotocin (STZ)-induced T1DM mouse model, MSC transplantation can reduce pancreatic inflammation, increase serum insulin levels and intrapancreatic insulin content, thereby alleviating hyperglycemia symptoms. However, to date, due to unsatisfactory clinical trial results, there are still no approved stem cell drugs worldwide for the treatment of T1DM. Enhancing the therapeutic efficacy of MSCs is a pressing issue that needs to be addressed. Summary of the Invention

[0005] One of the objectives of this invention is to overcome the deficiencies described in the prior art, thereby providing a composition of an intestinal flora regulator and mesenchymal stem cells. This composition is suitable for treating diabetes, effectively enhancing the stability of mesenchymal stem cell therapy for T1DM, especially autoimmune type 1 diabetes, reducing pancreatic islet inflammation, and preserving more pancreatic β cells, thus providing support for the clinical translation and application of mesenchymal stem cells.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A composition comprising a first agent composed of mesenchymal stem cells; and,

[0008] The second dose consists of gut microbiota regulators.

[0009] In some implementations, the gut microbiota regulator is a gut probiotic and / or a broad-spectrum antibiotic.

[0010] Probiotics generally refer to bacteria or fungi that grow in the human gut and have a positive effect on human health. Antibiotics are secondary metabolites produced by microorganisms (including bacteria, fungi, and actinomycetes) or higher plants and animals during their life processes. They are chemical substances that can interfere with the developmental functions of other living cells and are mainly used to treat various bacterial infections or diseases caused by pathogenic microorganisms. Generally, they do not produce serious side effects on their hosts. Broad-spectrum antibiotics refer to drugs with a relatively broad antibacterial spectrum; simply put, they are drugs that can resist most bacteria, such as chloramphenicol, chlortetracycline, oxytetracycline, tetracycline, and thiamphenicol. They not only strongly inhibit most Gram-negative and Gram-positive bacteria but also inhibit rickettsiae, spirochetes, and some protozoa.

[0011] In some embodiments, the intestinal probiotics are selected from one or more of Bifidobacterium, Lactobacillus, Streptococcus, and Bacillus.

[0012] Preferably, the Bifidobacterium is selected from one or more of Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium dentalis, Bifidobacterium animalis, Bifidobacterium lactis, Bifidobacterium adolescentis, and Bifidobacterium bifidum.

[0013] Preferably, the lactic acid bacteria are selected from one or more of the following: Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus johnsonii, Bacillus coagulans, and Lactobacillus bulgaricus.

[0014] Preferably, the streptococcus is *Streptococcus faecalis*.

[0015] Preferably, the Bacillus is selected from one or more of Bacillus claurifolia, Bacillus coagulans, and Bacillus subtilis;

[0016] In some embodiments, the broad-spectrum antibiotic is one or more of the following: polypeptide broad-spectrum antibiotics, macrolide broad-spectrum antibiotics, phosphate-containing polysaccharide broad-spectrum antibiotics, polyether broad-spectrum antibiotics, aminoglycoside broad-spectrum antibiotics, and chemically synthesized broad-spectrum antibiotics.

[0017] In some embodiments, the broad-spectrum antibiotic is selected from a mixture of ampicillin solution, metronidazole solution, neomycin solution, and vancomycin solution; the concentration ratio of ampicillin solution, metronidazole solution, neomycin solution, and vancomycin solution in the mixture is 1:1:1:0.4-0.6 respectively.

[0018] In some embodiments, the mesenchymal stem cells are selected from one or more of adipose-derived mesenchymal stem cells, bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, dental pulp mesenchymal stem cells, skin mesenchymal stem cells, urine mesenchymal stem cells, and periosteal mesenchymal stem cells, with adipose-derived mesenchymal stem cells being preferred.

[0019] In some embodiments, the gut microbiota regulator in the composition is a broad-spectrum antibiotic, and the ratio of the solute in the broad-spectrum antibiotic to the mesenchymal stem cells is (80-1000) mg : (0.5-5) × 10⁻⁶. 6 indivual.

[0020] In some embodiments, the composition contains 130 mg of broad-spectrum antibiotic solute and 1 × 103 mesenchymal stem cells. 6 indivual.

[0021] In some embodiments, the gut microbiota regulator in the composition is Bifidobacterium with an optical density value of 0.8 or higher, and the ratio of Bifidobacterium to mesenchymal stem cells is 1 × 10⁻⁶. 9-12 CFU: (0.5-5)×10 6 indivual.

[0022] In some embodiments, the composition contains 1×10 6 One mesenchymal stem cell and 1×10 9 CFU (bifidobacterium acnes)

[0023] When applying the composition, the mesenchymal stem cells can be administered optionally via a route comprising intravenous injection, intramuscular injection, subcutaneous injection, intrathecal injection or infusion, and intra-organ infusion. For example, for intravenous injection, it can be administered via systemic intravenous administration or tail vein injection. Intra-organ infusion includes infusion into anatomical spaces, such as the gallbladder, gastrointestinal lumen, esophagus, pulmonary system (by inhalation), and / or bladder. Broad-spectrum antibiotics are administered orally or intravenously, and probiotics are administered orally via the digestive tract.

[0024] In some embodiments, the two reagents in the composition are provided as a single pharmaceutical composition, and in other embodiments, a kit or dispenser package containing each of the two reagents may be contemplated. It should be understood that the scope of this invention covers the co-administration of either of the two reagents to a subject, whether such administration is as a single formulation or as a separate combination of formulations, and whether such administration is simultaneous or staggered.

[0025] In some embodiments, the mesenchymal stem cells are taken from excess adipose tissue from liposuction patients.

[0026] In some embodiments, the mesenchymal stem cells are of the 3rd to 6th generation.

[0027] In some embodiments, the use of the composition in the preparation of a medicament for treating type 1 diabetes is also provided.

[0028] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier; the dosage form of the composition is preferably selected from lyophilized powder for injection, injection, tablets, or capsules.

[0029] In some embodiments, the dosage form is selected from injections or tablets.

[0030] In some embodiments, a method for extracting the mesenchymal stem cells is also provided, comprising the following steps:

[0031] (1) Add enzymes to the adipose tissue for digestion, then centrifuge, filter and resuspend;

[0032] (2) After resuspending the cells, culture them in vitro. When the cells are more than 80% confluent, add trypsin to digest the cells and then inoculate them into new culture dishes for passage culture.

[0033] (3) When the culture reaches the third generation, wash with PBS 2-5 times and digest with 0.2-0.3wt% trypsin to obtain the mesenchymal stem cells to be identified;

[0034] (4) The mesenchymal stem cells to be identified were fixed and blocked sequentially, and surface marker antibodies were incubated at 4-6℃ in the dark. At the same time, CD90-FITC, CD44-PE, CD73-APC, and CD105-CY5.5 were used as isotype controls. When the surface marker antibodies were CD90, CD44, CD73, and CD105, and the expression was above 95%, the mesenchymal stem cells were identified.

[0035] In some embodiments, the volume of the enzyme in step (1) is more than three times that of the adipose tissue, and the enzyme comprises a dispersant enzyme and a type I collagenase in a mass ratio of 1.8-2.2:1.

[0036] In some embodiments, the digestion in step (1) is performed by shaking at 400-500 rpm / min for more than 30 minutes at a temperature of 35-38°C, the centrifugation filtration includes first filtering with a 70 nm cell sieve under sterile conditions, and then centrifuging the suspension at 2400-2600 rpm / min for 6-8 minutes, and the resuspension is performed in α-MEM medium containing 10-12 wt% fetal bovine serum.

[0037] In some embodiments, the in vitro culture in step (2) involves transferring the resuspended cells into a new culture dish for culture until the cells adhere to the wall and grow into a spindle shape, wherein the culture temperature is 35-38°C and the volume fraction of carbon dioxide is 4-6%.

[0038] In some embodiments, the fixation in step (4) is to fix cells with 3-5 wt% paraformaldehyde for 10-20 min, and the blocking is to block with 0.8-12 wt% bovine serum albumin for 20-30 min.

[0039] On the one hand, the present invention also provides the use of gut microbiota regulators as a companion drug in the preparation of mesenchymal stem cell oral products.

[0040] The "mesenchymal stem cell oral products" in this invention include, but are not limited to, products or methods that enable mesenchymal stem cells to enter an animal body. As exemplary examples, products enabling mesenchymal stem cell oral products to enter an animal body include lyophilized powder for injection, injections, tablets, or capsules. As exemplary examples, methods enabling stem cell oral products to enter an animal body include oral administration and injection.

[0041] In this invention, "concomitant medication" includes medications that enter the body together with one or more other medications. Concomitant medications may enter the body simultaneously or nearly simultaneously. As an exemplary example, nearly simultaneous entry can mean entering one after another; or entering the body at different times on the same day. For example, multiple agents or their active ingredients may be formulated into a single preparation, or two or more preparations may enter the body at least substantially simultaneously. For example, mutually within about 1 hour.

[0042] In some embodiments, the mesenchymal stem cell oral product is selected from adipose-derived mesenchymal stem cells, bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, or dental pulp mesenchymal stem cells.

[0043] In some embodiments, the mesenchymal stem cell product is adipose-derived mesenchymal stem cells for ingestion by an animal; preferably, the animal is a mammal; preferably, the mammal is a human.

[0044] On one hand, the present invention also provides a stem cell administration system, comprising: a mesenchymal stem cell product, and a companion product; the companion product is selected from broad-spectrum antibiotics and / or bifidobacteria; the mesenchymal stem cell product comprises adipose-derived mesenchymal stem cells, bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, or dental pulp mesenchymal stem cells; the broad-spectrum antibiotic or bifidobacteria is as described above.

[0045] On the one hand, the present invention also provides a method for promoting the prevention and treatment of type 1 diabetes with mesenchymal stem cell products, the method comprising taking an intestinal flora regulator simultaneously or almost simultaneously with taking the mesenchymal stem cell products. Attached Figure Description

[0046] Figure 1A The percentage of CD44-positive cells in flow cytometry analysis in Example 2;

[0047] Figure 1B The percentage of CD73-positive cells in flow cytometry analysis in Example 2;

[0048] Figure 1C The percentage of CD90-positive cells in flow cytometry analysis in Example 2;

[0049] Figure 1D The percentage of CD105-positive cells in flow cytometry analysis in Example 2;

[0050] Figure 2A This is a schematic diagram of the process of treating NOD / Ltj mice in stages using streptozotocin, ADSCs and broad-spectrum antibiotics in Examples 1-4.

[0051] Figure 2B This is a comparison chart of blood glucose changes in the four groups of mice described in Example 3: PBS group, ADSCs group, ADSCs+Abx group, and Co-housed group.

[0052] Figure 2C The results of HE staining of the pancreas of mice in the four groups of mice described in Example 3 (PBS group, ADSCs group, ADSCs+Abx group, and Co-housed group) and the corresponding pancreatic islet inflammation score comparison chart.

[0053] Figure 2D The results of pancreatic insulin immunohistochemical staining and quantitative β-cell staining scores of the four groups of mice described in Example 3: PBS group, ADSCs group, ADSCs+Abx group, and Co-housed group.

[0054] Figure 2E This is a comparison chart of serum insulin ELISA results for the four groups of mice described in Example 3: PBS group, ADSCs group, ADSCs+Abx group, and Co-housed group.

[0055] Figure 3A This is a comparison chart of blood glucose changes in the ADSCs group, ADSCs+ampicillin+vancomycin group, ADSCs+neomycin group and ADSCs+metronidazole group in Example 4;

[0056] Figure 4A This is a comparison chart of blood glucose changes in the PBS group, ADSCs group, ADSCs+Abx group, and Abx group in Example 5;

[0057] Figure 5A This is a comparison chart of the fecal bacterial quantification results of the four groups of mice described in Example 6: PBS group, ADSCs group, ADSCs+Abx group, and Co-housed group.

[0058] Figure 5B This is a comparison chart showing the fecal bacterial α diversity analysis of the four groups of mice described in Example 6: PBS group, ADSCs group, ADSCs+Abx group, and Co-housed group.

[0059] Figure 5C This is a comparison chart of fecal bacterial β diversity analysis in the four groups of mice described in Example 6: PBS group, ADSCs group, ADSCs+Abx group, and Co-housed group.

[0060] Figure 5D This is a comparison chart of the differential bacterial (Bifidobacterium) abundance in the ADSCs group, ADSCs+Abx group and Co-housed group in Example 6;

[0061] Figure 6A The image shows the intestinal colonization results of Bifidobacterium (B. breve) in the ADSCs group, ADSCs+Abx group, Co-housed group, and ADSCs+B. breve group in Example 7.

[0062] Figure 6B This is a comparison chart of blood glucose changes in the ADSCs group, ADSCs+Abx group, and ADSCs+B.breve group described in Example 7;

[0063] Figure 6C The image shows the HE staining results of the pancreas in the ADSCs group, ADSCs+Abx group, and ADSCs+B.breve group described in Example 7, along with a comparison of the corresponding pancreatic islet inflammation scores.

[0064] Figure 6D The results of pancreatic insulin immunohistochemical staining and quantitative β-cell staining scores in the ADSCs group, ADSCs+Abx group and ADSCs+B.breve group described in Example 7 are shown.

[0065] Figure 7A This is a comparison chart of serum FITC-dextran concentrations in the ADSCs group, ADSCs+Abx group, and ADSCs+B.breve group described in Example 8;

[0066] Figure 7B This is a comparison chart of serum endotoxin concentrations in the ADSCs group, ADSCs+Abx group, and ADSCs+B.breve group described in Example 8;

[0067] Figure 7C The images show a comparison of the colon tissue obtained by alcinocyanine blue staining and the thickness of the mucus layer in the ADSCs group, ADSCs+Abx group and ADSCs+B.breve group described in Example 8.

[0068] Figure 8A Example 9 shows a comparison of bacterial load in pancreatic tissue among the ADSCs group, the ADSCs+Abx group, and the ADSCs+B.breve group;

[0069] Figure 8B This is a comparison of the EUB338 probe in situ hybridization staining images of pancreatic tissue in the ADSCs group, ADSCs+Abx group and ADSCs+B.breve group described in Example 9, and the number of bacteria observed under high magnification.

[0070] Figure 8C The images shown are scans of pancreatic tissue obtained by MafA immunohistochemical staining in the ADSCs group, ADSCs+Abx group and ADSCs+B.breve group described in Example 9, and a comparison of the percentage of positive cells. Detailed Implementation

[0071] The following specific embodiments further illustrate the technical solution of the present invention. These specific embodiments do not represent a limitation on the scope of protection of the present invention. Non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the scope of protection of the present invention.

[0072] The mesenchymal stem cells (MSCs) in this invention are derived from the mesoderm in early development and are pluripotent stem cells. Under specific induction conditions in vivo or in vitro, MSCs can differentiate into various tissue cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, myocardium, and endothelium. Even after continuous passage culture and cryopreservation, they retain multi-lineage differentiation potential and can serve as ideal seed cells for repairing tissue and organ damage caused by aging and disease. ADSCs are adipose-derived stem cells (ADSCs). ADSCs are pluripotent stem cells derived from adipose tissue, possessing self-renewal and multi-lineage differentiation capabilities, and can secrete various bioactive factors, showing broad application prospects in tissue damage and repair. In the following embodiments of this invention, ADSCs are obtained from excess or discarded adipose tissue of patients undergoing fat grafting (ethics number: PKUSSIRB-201948106), and the extracted ADSCs are cultured in α-MEM (minimum essential medium).

[0073] In this invention, T1DM refers to type 1 diabetes mellitus (T1DM).

[0074] In this invention, B. breve is a short-type Bifidobacterium.

[0075] All animal experiments in this invention embodiment were approved by the Ethics Committee of Peking University School of Medicine (Ethics No.: LA2019190).

[0076] The PBS used in this invention is phosphate buffer saline, which is generally used as a solvent to dissolve and protect reagents. It is one of the most widely used buffer solutions in biochemical research, and its main components are Na₂HPO₄, KH₂PO₄, NaCl, and KCl.

[0077] The ampicillin of this invention is a broad-spectrum semi-synthetic penicillin, also known as ampicillin, savicillin, thiamethoxam, ampicillin, pamoatein, and AB-PC. It is a β-lactam antibiotic with extremely low toxicity. Its antibacterial spectrum is similar to that of penicillin. It was the first penicillin to have "broad-spectrum" antibacterial activity against Gram-positive bacteria, including Streptococcus pneumoniae, Streptococcus pyogenes, some Staphylococcus aureus isolates (but not penicillin- or methicillin-resistant strains), Eubacterium, and some Enterococci. It is one of the few antibiotics effective against multidrug-resistant Enterococcus faecalis and Enterobacter faecalis. It also has antibacterial activity against some Gram-negative bacteria such as Neisseria meningitidis, some Haemophilus influenzae, and some Enterobacteriaceae.

[0078] Metronidazole is a nitroimidazole derivative that inhibits the redox reaction of amoebae, causing the nitrogen chain of the protozoa to break. Its antibacterial spectrum includes Bacteroides fragilis and other Bacteroides species, Fusobacterium, Aerobacterium, Eubacterium, Peptococcus, and Peptostreptococcus. Its bactericidal concentration is slightly higher than its bacteriostatic concentration. Metronidazole has a killing effect on cells and anaerobic microorganisms growing under hypoxic conditions. Its metabolites generated during reduction in the human body also have anti-anaerobic activity, but it has no effect on aerobic or facultative anaerobic bacteria.

[0079] Neomycin is produced by *Streptomyces flexneri* and belongs to the aminoglycoside class of antibiotics. Its antibacterial spectrum is similar to other aminoglycoside drugs. Neomycin has good antibacterial activity against Gram-negative bacteria and partial antibacterial activity against Gram-positive bacteria. Neomycin exhibits complete cross-resistance with kanamycin and partial cross-resistance with streptomycin.

[0080] Vancomycin is limited to the treatment of systemic infections caused by methicillin-resistant Staphylococcus aureus (MRSA) and intestinal and systemic infections caused by Clostridium difficile. It exhibits good antibacterial activity against most Gram-positive Staphylococcus species, including Staphylococcus aureus and methicillin-sensitive and methicillin-resistant coagulase-negative staphylococci, various streptococci, Streptococcus pneumoniae, and Enterococcus spp. It also shows good activity against Clostridium difficile, Bacillus anthracis, and Corynebacterium diphtheriae.

[0081] The terms "comprising" or "including" are intended to indicate that a composition (e.g., a medium) and a method include the listed elements, but do not exclude other elements. When used to define compositions and methods, "consistently of" means excluding other elements that are of any significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements defined herein does not exclude other materials or steps that do not substantially affect the essential and novel features of the claimed invention. "Constitutes of" means excluding trace elements and substantial method steps that are other components. Embodiments defined by each of these transitional terms are within the scope of this invention.

[0082] Example 1: Construction of a diabetic mouse model:

[0083] Female NOD / Ltj mice (non-obese diabetic mice, NOD mice) aged 7-8 weeks were purchased from the Experimental Animal Center of Peking University School of Medicine. They were housed in a specific pathogen-free environment with constant temperature and humidity, maintaining a 12:12 circadian rhythm. After one week of acclimatization, the mice were injected intraperitoneally with streptozotocin (STZ; 40 mg / kg; Sigma-Aldrich, USA) for four consecutive days to accelerate the progression of diabetes. Seven days after injection, blood glucose levels were measured via tail vein sampling. Mice with blood glucose levels >13.9 mmol / L for three consecutive days were diagnosed with diabetes.

[0084] Example 2: Extraction and identification of adipose-derived stem cells (ADSCs):

[0085] a) Excess adipose tissue from liposuction patients was washed three times with physiological saline in a laminar flow hood. Then, an enzyme solution (0.4 mg / ml dispersin: 0.2 mg / ml type I collagenase = 1:1) three times the volume of the fat was added for digestion. Digestion was performed at 37°C and 450 rpm for 30 min using a constant temperature shaker. The cells were filtered through a 70 nm cell sieve under aseptic conditions. The suspension was centrifuged at 2500 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in α-MEM medium containing 10% fetal bovine serum. The resuspended cells were transferred to new 10 cm culture dishes and incubated at 37°C with 5% CO2. After ADSCs adhered and grew into spindle-shaped cells, when the cell confluence reached approximately 80%, the cells were digested with 0.25% trypsin and seeded at a 1:3 ratio into new 10 cm culture dishes for passage.

[0086] b) To identify ADSCs, passaged ADSCs to the third generation were washed three times with PBS, digested with 0.25% trypsin, and the cells were collected. Cells were fixed with 4% paraformaldehyde for 10-20 min, then blocked with 1% BSA for 20-30 min, and incubated at 4°C in the dark with surface marker antibodies and corresponding isotype controls: CD90-FITC, CD44-PE, CD73-APC, and CD105-CY5.5. All antibodies were purchased from BD Biosciences. Detection was performed using an Accuri C6 flow cytometer (BD Biosciences). Flow cytometry results showed that the expression of mesenchymal stem cell surface markers CD44, CD73, CD90, and CD105 was above 95%. Figure 1A , Figure 1B , Figure 1C , Figure 1D ), identified as ADSCs.

[0087] Example 3: Treatment with a broad-spectrum antibiotic mixture can enhance the efficacy of ADSCs in treating T1DM:

[0088] a) Grouping: Based on Example 1, diabetic mice with pre-existing disease were randomly grouped:

[0089] PBS group: Mice in this group were injected with PBS via the tail vein as a negative control group; this group is called the PBS group.

[0090] ADSCs group: Mice in this group were injected via tail vein with a solution containing 1.0 × 10⁻⁶ mg / L after the onset of the disease. 6 100 μl of PBS suspension was injected twice, with a one-week interval between injections. This group was called the ADSCs group.

[0091] ADSCs+Abx group: In order to regulate the intestinal flora, at the same time as the first injection of ADSCs, a broad-spectrum antibiotic mixture (Abx, ampicillin (1 mg / ml), metronidazole (1 mg / ml), neomycin (1 mg / ml) and vancomycin (0.5 mg / ml)) was added to the drinking water as an intestinal flora regulator and treated for 1 week. This group is called ADSCs+Abx group.

[0092] co-housed group: In order to study the role of gut microbiota in ADSCs treatment, we mixed ADSCs+Abx group mice that received antibiotic treatment with ADSCs group mice that did not receive antibiotic treatment. Taking advantage of the fact that mice eat feces, we recolonized the gut microbiota of ADSCs group mice into the intestines of ADSCs+Abx group mice and reorganized them into co-housed group.

[0093] Please refer to the flowcharts illustrating the phased treatment of NOD / Ltj mice using streptozotocin, ADSCs, and broad-spectrum antibiotics (including single-component, similar antimicrobial spores, and mixtures) in Examples 1-4. Figure 2A .

[0094] b) Evaluation of treatment efficacy: Blood glucose test results showed that ADSCs treatment could effectively reduce blood glucose in T1DM mice. Furthermore, the blood glucose levels in the ADSCs+Abx group were further lower than those in the ADSCs-only group. In contrast, the blood glucose levels in the co-housed group showed a brief decrease during antibiotic feeding, but gradually increased after mixing with other mice, approaching the levels in the ADSCs group. Figure 2B HE staining and pancreatic islet inflammation score (scoring criteria refer to...) E, et al. NatImmunol 2017. PMID 28346408) The results showed that the pancreatic islet inflammation in the ADSCs+Abx group was less than that in the other three groups. Figure 2CImmunohistochemical staining results for insulin showed that the staining of pancreatic β cells in the ADSCs+Abx group was also stronger than that in the other three groups. Figure 2D Serum insulin ELISA results also showed that the serum insulin level in the ADSCs+Abx group was higher than that in other groups. Figure 2E ).

[0095] Example 4: Broad-spectrum antibiotics, either single-component or in combination with similar antimicrobial species, did not significantly enhance the efficacy of ADSCs in treating T1DM.

[0096] To investigate the effects of each component in a broad-spectrum antibiotic mixture on the efficacy of ADSCs in treating type 1 diabetes mellitus (T1DM), we divided the four antibiotics into three groups based on the bacterial types they target: ampicillin + vancomycin (primarily targeting Gram-positive bacteria); neomycin (primarily targeting Gram-negative bacteria); and metronidazole (primarily targeting anaerobic bacteria). The above antibiotic solutions were then used in combination with ADSCs, and changes in blood glucose levels in mice after combined administration were observed. The specific implementation plan is as follows:

[0097] a) Grouping: Diabetic mice with pre-existing disease were randomly divided into the following 4 groups:

[0098] ADSCs group: Mice in this group were injected via tail vein with a solution containing 1.0 × 10⁻⁶ mg / L after the onset of the disease. 6 100 μl of PBS suspension was injected twice, with a one-week interval between injections. This group was called the ADSCs group.

[0099] ADSCs+Ampicillin+Vancomycin group: In order to regulate the intestinal flora, at the same time as the first injection of ADSCs, ampicillin (1 mg / ml) and vancomycin (0.5 mg / ml) were added to the drinking water as intestinal flora regulators and treated for 1 week. This group is called ADSCs+Ampicillin+Vancomycin group.

[0100] ADSCs + Metronidazole group: In order to regulate the intestinal flora, metronidazole (1 mg / ml) was added to the drinking water as an intestinal flora regulator at the same time as the first injection of ADSCs and treated for 1 week. This group is called ADSCs + Ampicillin + Vancomycin group.

[0101] ADSCs + Neomycin group: In order to regulate the intestinal flora, neomycin (1 mg / ml) was added to the drinking water as an intestinal flora regulator at the same time as the first injection of ADSCs and treated for 1 week. This group is called ADSCs + Ampicillin + Vancomycin group.

[0102] b) Evaluation of treatment efficacy: Blood glucose changes showed that after ampicillin + vancomycin, neomycin, or metronidazole were used in combination with ADSCs, the blood glucose levels in mice were similar to those in the ADSCs group, with no further decrease. Figure 3A Based on the results of Example 3, it is demonstrated that only a mixture of ampicillin, vancomycin, neomycin, and metronidazole can further enhance the therapeutic effect on ADSCs.

[0103] Example 5: The use of broad-spectrum antibiotic mixtures alone was not effective in treating T1DM.

[0104] To eliminate the influence of the antibiotic mixture itself on blood glucose in T1DM mice, we designed an Abx group that did not use ADSCs and used the broad-spectrum antibiotic mixture alone, and observed changes in blood glucose in the mice. The specific implementation plan is as follows:

[0105] a) Grouping: Selected diabetic mice with pre-existing disease were randomly assigned to groups:

[0106] PBS group: Mice in this group were injected with PBS via the tail vein as a negative control group, and were referred to as the PBS group.

[0107] ADSCs group: Mice in this group were injected via tail vein with a solution containing 1.0 × 10⁻⁶ mg / L after the onset of the disease. 6 100 μl of PBS suspension was injected twice, with a one-week interval between injections. This group was called the ADSCs group.

[0108] ADSCs+Abx group: In order to regulate the intestinal flora, at the same time as the first injection of ADSCs, a broad-spectrum antibiotic mixture (Abx, ampicillin (1 mg / ml), metronidazole (1 mg / ml), neomycin (1 mg / ml) and vancomycin (0.5 mg / ml)) was added to the drinking water as an intestinal flora regulator and treated for 1 week. This group is called ADSCs+Abx group.

[0109] Abx group: Mice in this group did not receive ADSCs treatment, but only had a broad-spectrum antibiotic mixture (Abx, ampicillin (1 mg / ml), metronidazole (1 mg / ml), neomycin (1 mg / ml) and vancomycin (0.5 mg / ml)) added to their drinking water as an intestinal flora regulator for 1 week. This group was called Abx group.

[0110] b) Evaluation of treatment efficacy: Blood glucose changes showed that there was no significant difference in blood glucose levels between mice treated with Abx alone and those treated with ADSCs without ADSCs and the PBS group, indicating that Abx treatment alone did not have a blood glucose-lowering effect. Figure 4A Based on the results of Examples 3 and 4, it is demonstrated that combining ADSCs with Abx can achieve better therapeutic effects.

[0111] Example 6: Treatment with a broad-spectrum antibiotic mixture alters the gut microbiota.

[0112] Abx use alters the structure and abundance of the gut microbiota. To investigate how the gut microbiota changes after Abx use and which bacterial alterations might contribute to the enhanced therapeutic effect of combined ADSCs and Abx administration, we collected fecal samples from four groups of mice at 1-week and 2-week time points. Fecal DNA was extracted for 16S rRNA real-time PCR quantification and 16S rRNA sequencing. Real-time results showed a significant decrease in fecal bacterial count after 1 week of antibiotic treatment, which gradually recovered 1 week after antibiotic withdrawal, i.e., at the second week (2 weeks), with no statistically significant difference compared to the other groups. Figure 5A 16S rRNA sequencing results at 2 weeks showed that α and β diversity did not significantly alter the bacterial community structure when ADSCs were treated alone, while ADSCs+Abx treatment resulted in a unique bacterial community. Figure 5B and 5C Differential bacterial analysis showed that the abundance of Bifidobacteria in the feces of mice in the ADSCs+Abx group was significantly higher than that in the ADSCs group and the co-housed group. Figure 5D ).

[0113] Example 7: Treatment with Bifidobacterium breve can enhance the efficacy of stem cell therapy.

[0114] Based on the above research results, we plan to treat mice simultaneously with Bifidobacterium breve (B. breve) and ADSCs and observe the changes in blood glucose levels. Mice treated in this manner were named the ADSCs+B. breve group. B. breve (1.3001) was purchased from the China General Microbiological Culture Collection Center and cultured in MRS medium (containing 0.05% cysteine) under anaerobic conditions at 37°C. Using 1.0 × 10⁻⁶... 9 Mice were administered B. breve via gavage for one week after their first ADSC treatment. Fecal samples were collected at two weeks, and fecal DNA was extracted. Real-time PCR and horizontal gel electrophoresis confirmed successful colonization of B. breve in the mouse intestine. Figure 6A ).

[0115] The results showed that the blood glucose level in the ADSCs+B. breve group was significantly lower than that in the ADSCs group, and similar to that in the ADSCs+Abx group. Figure 6B HE staining and islet inflammation scoring of pancreatic tissue showed that the proportion of islets with an inflammation score >75% in mice treated with ADSCs+B. breve was reduced compared to the ADSCs group. Figure 6C Insulin staining results also showed that the staining of pancreatic β cells was stronger after Abx / B. breve treatment than that of the ADSCs group. Figure 6DThis result indicates that the increased proportion of Bifidobacteria after ADSCs+Abx treatment is the reason for the enhanced therapeutic effect of ADSCs, and that B. breve gavage treatment can further enhance the therapeutic effect of ADSCs in treating T1DM.

[0116] Example 8: Treatment with a broad-spectrum antibiotic mixture / B. breve can reduce intestinal permeability:

[0117] Increased intestinal permeability is a common symptom in patients with type 1 diabetes mellitus (T1DM) and in animal models. Intestinal permeability was assessed by gavage administration of fluorescein isothiocyanate-labeled dextran (FITC-dextran, 4 kDa). Results showed that the serum FITC-dextran fluorescence intensity was lowest in mice treated with ADSCs+Abx and ADSCs+B. breve, indicating that ADSCs+Abx / B. breve treatment reduced intestinal permeability compared to ADSCs-only treatment. Figure 7A Serum endotoxin assays showed the same result: serum endotoxin levels were lower in the ADSCs+Abx / B. breve group than in the ADSCs group. Figure 7B This further demonstrates that ADSCs+Abx / B. breve treatment reduced intestinal permeability in mice. Alixin blue staining results also showed that ADSCs+Abx / B. breve treatment resulted in thickening of the intestinal mucus layer in mice. Figure 7C The above indicates that ADSCs+Abx / B.breve treatment reduces intestinal permeability in mice by thickening the colonic mucus layer.

[0118] Example 9: Treatment with a broad-spectrum antibiotic mixture / B. breve can reduce ectopic colonization of gut microbiota in the pancreas and increase insulin transcription.

[0119] Increased intestinal permeability allows gut bacteria to cross the intestinal barrier and invade distal organs, including the pancreas. Therefore, changes in intestinal permeability are closely related to distal organ function. To detect changes in ectopic colonization of the gut microbiota in the pancreas, we performed real-time PCR quantitative detection and staining with the universal bacterial probe EUB338. Real-time PCR quantitative detection results ( Figure 8A ) and staining results of the universal bacterial probe EUB338 ( Figure 8B Both studies showed that the number of bacteria in the pancreas of ADSCs+Abx / B. breve mice was reduced compared to the ADSCs group. MafA is an important transcription factor of the β-cell insulin gene, and MafA immunohistochemical staining results showed that the number of positive cells in the ADSCs+Abx / B. breve group was higher than that in the ADSCs group. Figure 8C This is related to Figure 6D The staining results for insulin were consistent.

[0120] In conclusion, concurrent administration of broad-spectrum antibiotic mixtures or probiotic B. breve treatment with ADSCs can enhance the therapeutic effects of ADSCs in lowering blood glucose and alleviating pancreatic inflammation. Abx / B. breve treatment, by reducing intestinal permeability and decreasing ectopic colonization of gut microbiota in the pancreas, enhances insulin transcription and expression, ultimately further strengthening the efficacy of ADSCs in treating T1DM.

[0121] This invention addresses the issue of poor therapeutic efficacy of mesenchymal stem cells (MSCs) in the clinical application of type 1 diabetes treatment. By using broad-spectrum antibiotics or Bifidobacteria to regulate the gut microbiota, it enhances the efficacy of human adipose-derived MSCs in treating type 1 diabetes. This invention emphasizes the crucial role of gut microbiota in MSC therapy for type 1 diabetes and lays the foundation for its clinical translation.

[0122] Given the many possible embodiments in which the principles of the disclosed invention can be applied, it should be understood that the illustrated embodiments are merely preferred examples of the invention and should not be considered as limiting the scope of the invention. Rather, the scope of the invention is defined by the appended claims. Therefore, we claim protection for all inventions falling within the scope and spirit of these claims.

Claims

1. A composition characterized in that, include: The first dose, composed of mesenchymal stem cells; and, The second dose, composed of gut microbiota regulators; The intestinal flora regulator is either an intestinal probiotic or a broad-spectrum antibiotic. The broad-spectrum antibiotic is a mixture of ampicillin solution, metronidazole solution, neomycin solution, and vancomycin solution; The intestinal probiotics are selected from Bifidobacteria; This composition is suitable for the treatment of type 1 diabetes.

2. The composition according to claim 1, characterized in that, The mesenchymal stem cells are selected from one or more of adipose-derived mesenchymal stem cells, bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, dental pulp mesenchymal stem cells, skin mesenchymal stem cells, urine mesenchymal stem cells, and periosteal mesenchymal stem cells.

3. The composition of claim 1, wherein The mesenchymal stem cells are selected from adipose-derived mesenchymal stem cells.

4. The composition of claim 1, wherein The Bifidobacterium is selected from Bifidobacterium longum (Bifidobacterium longum). Bifidobacterium longum ), Bifidobacterium breve Bifidobacterium breve Bifidobacterium densiflorum ( Bifidobacterium dentium Bifidobacterium animalis ( Bifidobacterium animalis Bifidobacterium adolescentis ( ) Bifidobacterium adolescentis Bifidobacterium bifidum ( Bifidobacterium bifidum One or more of them.

5. The composition of claim 4, wherein The Bifidobacterium longum ( Bifidobacterium longum ) Selected from Bifidobacterium infantis ( Bifidobacterium longum subsp. infantis ).

6. The composition of claim 4, wherein The animal bifidobacteria ( Bifidobacterium animalis ) Selected from Bifidobacterium lactis ( Bifi dobacterium animalis subsp. lactis ).

7. The composition of claim 1, wherein The concentration ratios of ampicillin solution, metronidazole solution, neomycin solution, and vancomycin solution in the mixture are 1:1:1:0.4-0.6, respectively.

8. The composition according to claim 7, characterized in that, The ratio of the solute in the broad-spectrum antibiotic to the mesenchymal stem cells is (80-1000) mg : (0.5-5) × 10⁻⁶ mg. 6 indivual.

9. The composition of claim 8, wherein, The composition contains 130 mg of broad-spectrum antibiotic solute and 1 × 103 mesenchymal stem cells. 6 indivual.

10. The composition of claim 1, wherein The intestinal flora regulator in the composition is Bifidobacterium with an optical density value of 8 or higher, and the ratio of Bifidobacterium to mesenchymal stem cells is 1 × 10⁻⁶. 9-12 CFU: (0.5-5)×10 6 indivual.

11. The composition of claim 10, wherein The composition contains 1×10 6 One mesenchymal stem cell and 1×10 9 CFU (bifidobacterium acnes) 12. Use of the composition according to claim 1 in the preparation of a medicament for treating type 1 diabetes.

13. The composition of claim 1, wherein The composition also contains a pharmaceutically acceptable carrier; The dosage form of the composition is selected from lyophilized powder for injection, injection, tablets or capsules.

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