CN111317747A

CN111317747A - Composition of intestinal flora regulator and mesenchymal stem cells and application thereof

Info

Publication number: CN111317747A
Application number: CN202010212014.5A
Authority: CN
Inventors: 张益�; 肖锷; 吕婉琪
Original assignee: Peking University School of Stomatology
Current assignee: Peking University School of Stomatology
Priority date: 2020-03-24
Filing date: 2020-03-24
Publication date: 2020-06-23
Also published as: WO2021189558A1

Abstract

The composition disclosed by the invention can be suitable for treating diabetes, effectively enhances the treatment effect of the mesenchymal stem cells on treating the diabetes, particularly autoimmune type I diabetes, reduces the inflammation of pancreatic islets, enables more pancreatic islet β cells to be stored, provides support for clinical transformation and application of the mesenchymal stem cells, and has a good application prospect.

Description

Composition of intestinal flora regulator and mesenchymal stem cells and application thereof

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to a composition of an intestinal flora regulator and mesenchymal stem cells and application thereof.

Background

There are a number of active microbial communities within the gastrointestinal tract of mammals, including bacteria, archaea, eukaryotes, viruses, and the like, known as "gut microbiota" in quantities of about 10¹⁴The total number of genes encoded by these microorganisms is about 100 times that of the human body's own genes. These microorganisms constitute the intestinal flora with the largest number of bacteria. They have been co-evolved with the host for a long time, form a complex and mutual relationship, and are also considered as "new organs" of the host. The intestinal flora plays an important role in maintaining the steady state of the organism, including digestion and absorption of food, stimulation of the maturation of the immune system, regulation of metabolism, resistance to colonization and proliferation of pathogenic microorganisms in the intestinal tract, and the like. However, when the ecological balance of the intestinal flora is disrupted and the composition of the intestinal microorganisms is changed, the above-mentioned function is also affected, which is called imbalance of the intestinal flora. In some cases, such as over-use of antibiotics and improper dietary structure, which can lead to disturbance of intestinal flora structure, the disease can be promoted and the disease can be accelerated, and the regulation of disturbed intestinal flora is beneficial for the treatment of the disease.

Although the cause of T1DM is still clear, the prevailing opinion is that the cause of T1DM is a multigenic disease affected by environmental factors, studies of related scholars show that genetic risk factors are essential for the disease but insufficient, many environmental impact factors including viral infection, diet, use of e.g. pregnancy infections and antibiotics, etc., have been proposed as candidates for T1 gestational infections and use of antibiotics, and studies of intestinal tract flora of mice show that the proportion of T1 enteropathogens is increased, and studies of obesity-related bacteria show that the proportion of T1 diabetes mellitus is increased, and studies of intestinal tract flora of mice show that the proportion of diabetes mellitus is increased.

In recent years, Mesenchymal Stem Cells (MSCs) are widely used in the research of the treatment of various diseases, and the good immunoregulatory capacity of the MSCs has been considered to have a wide application prospect in the treatment of autoimmune diseases.

Disclosure of Invention

One of the purposes of the present invention is to overcome the defects in the prior art, and thus provide a composition of an intestinal flora regulator and mesenchymal stem cells, which is suitable for treating diabetes, effectively enhances the stability of the mesenchymal stem cells in treating T1DM, especially autoimmune type I diabetes, can reduce inflammation of pancreatic islets, allows more pancreatic islet β cells to be preserved, and provides support for clinical transformation and application of the mesenchymal stem cells.

In order to achieve the above purpose, the invention provides the following technical scheme:

a composition comprising a first agent consisting of mesenchymal stem cells; and the combination of (a) and (b),

a second agent consisting of an intestinal flora modulator.

In some embodiments, the gut flora modulator is a gut probiotic and/or a broad spectrum antibiotic.

Intestinal probiotics generally refer to bacteria or fungi that grow in the intestines and stomach of the human body and play a positive role in human health. Antibiotics (antibiotics) are secondary metabolites with anti-pathogen or other activities generated by microorganisms (including bacteria, fungi and actinomycetes) or higher animals and plants in the life process, and chemical substances capable of interfering with other life cell development functions, are mainly used for treating various bacterial infections or disease-causing microbial infection diseases, and generally do not have serious side effects on hosts of the antibiotics; broad-spectrum antibiotics refer to drugs having a relatively broad antimicrobial spectrum, and are simply drugs that are capable of resisting most bacteria, such as chloramphenicol, aureomycin, oxytetracycline, tetracycline, thiamphenicol, and the like. Not only can strongly inhibit most gram-negative bacteria and gram-positive bacteria, but also can inhibit rickettsia, spirochete and certain protozoa.

In some embodiments, the intestinal probiotic is selected from one or more of bifidobacteria, lactic acid bacteria, streptococci, bacilli;

preferably: the Bifidobacterium is selected from one or more of Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium odonta, Bifidobacterium animalis, etc., Bifidobacterium lactis, Bifidobacterium adolescentis, and Bifidobacterium bifidum;

preferably: the lactobacillus is selected from one or more of acidophilus, lactobacillus reuteri, lactobacillus rhamnosus, lactobacillus johnsonii, bacillus coagulans and lactobacillus bulgaricus;

preferably: the streptococcus is faecalis streptococcus;

preferably: the bacillus is selected from one or more of bacillus clausii, bacillus coagulans and bacillus subtilis;

in some embodiments, the broad-spectrum antibiotic is one or more of polypeptide broad-spectrum antibiotic, macrolide broad-spectrum antibiotic, 8-phosphopolysaccharide broad-spectrum antibiotic, polyether broad-spectrum antibiotic, aminoglycoside broad-spectrum antibiotic, and chemically synthesized broad-spectrum antibiotic.

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

In some embodiments, the mesenchymal stem cell is selected from one or more of adipose mesenchymal stem cell, bone marrow mesenchymal stem cell, umbilical cord mesenchymal stem cell, placental mesenchymal stem cell, dental pulp mesenchymal stem cell, skin mesenchymal stem cell, urine mesenchymal stem cell, periosteal mesenchymal stem cell, preferably adipose mesenchymal stem cell.

In some embodiments, the gut flora modulator in the composition is a broad spectrum antibiotic with a ratio of solutes to the mesenchymal stem cells in the broad spectrum antibiotic of (80-1000) mg (0.5-5) × 10⁶And (4) respectively.

In some embodiments, the composition has a mass of broad spectrum antibiotic solute of 130mg and a number of mesenchymal stem cells of 1 × 10⁶And (4) respectively.

In some embodiments, the intestinal flora modulator in the composition is a bifidobacterium with an optical density value of 0.8 or more, the ratio of the bifidobacterium to the mesenchymal stem cells is 1 × 10^9-12CFU：(0.5-5)× 10⁶And (4) respectively.

In some embodiments of the present invention, the,the composition comprises 1 × 10⁶Mesenchymal stem cell and 1 × 10⁹CFU bifidobacterium.

When the composition is applied, the mesenchymal stem cells may be optionally administered by a route selected from the group consisting of intravenous injection, intramuscular injection, subcutaneous injection, intrathecal injection or infusion, and intraorgan infusion. For example, for intravenous injection, systemic administration may be intravenous or tail vein injection. Intra-organ infusion includes, among other things, infusion into anatomical spaces, such as the gallbladder, gastrointestinal lumen, esophagus, pulmonary system (by inhalation), and/or bladder. The broad spectrum antibiotics are administered orally or intravenously, and the probiotics are administered orally through the digestive tract.

In some embodiments, both agents in the composition are provided in the form of a single pharmaceutical composition, and in some embodiments, a kit or combined dispenser package containing each of the two agents is contemplated. It is to be understood that the present disclosure encompasses co-administration of either agent to a subject, whether such administration is in a single formulation or in a combination of separate formulations, and whether such administration is simultaneous or staggered.

In some embodiments, the mesenchymal stem cells are taken from excess adipose tissue of a liposuction patient.

In some embodiments, the number of passages of the mesenchymal stem cell is 3-6.

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

In some embodiments, the composition further comprises a pharmaceutically acceptable carrier; the dosage form of the composition is preferably selected from lyophilized powder injection, tablet or capsule.

In some embodiments, the dosage form is selected from an injection or a tablet.

In some embodiments, there is also provided the method for extracting mesenchymal stem cells, comprising the steps of:

(1) adding enzyme into the adipose tissue for digestion, and then carrying out centrifugal filtration and heavy suspension;

(2) culturing the resuspended cells in vitro, adding trypsin to digest the cells when the cell confluence is more than 80%, and then inoculating the cells into a new culture dish for subculture;

(3) when subculture is carried out to the third generation, washing with PBS for 2-5 times, and digesting with 0.2-0.3 wt% of trypsin to obtain mesenchymal stem cells to be identified;

(4) and sequentially fixing and sealing the mesenchymal stem cells to be identified, incubating the surface marker antibody at 4-6 ℃ in a dark place, taking CD90-FITC, CD44-PE, CD73-APC and CD105-CY5.5 as isotype control substances to make corresponding isotype control, and identifying the mesenchymal stem cells when the surface marker antibodies are CD90, CD44, CD73 and CD105 and are expressed at more than 95%.

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 dispase and collagenase type I in a mass ratio of 1.8-2.2: 1.

In some embodiments, the digestion in step (1) is performed at a temperature of 35-38 ℃ and shaking at a speed of 400-500rpm/min for more than 30min, and the centrifugal filtration comprises first filtering under sterile conditions using a 70nm cell sieve, and then centrifuging the suspension at a speed of 2400-2600rpm/min for 6-8min, the resuspension being performed in α -MEM medium containing 10-12 wt% fetal bovine serum.

In some embodiments, the in vitro culture in step (2) is to transfer the resuspended cells into a new culture dish for culture until the cells grow into fusiform adherent growth, wherein the temperature of the culture is 35-38 ℃, and the volume fraction of carbon dioxide is 4-6%.

In some embodiments, the fixing in step (4) is performed by fixing the cells with 3-5 wt% paraformaldehyde for 10-20min, and the blocking is performed by blocking with 0.8-12 wt% bovine serum albumin for 20-30 min.

On one hand, the invention also provides application of the intestinal flora regulator in preparing concomitant medicaments of mesenchymal stem cell oral products.

The term "mesenchymal stem cell article of clothing" in the embodiments of the present invention includes, but is not limited to, a product or method that enables mesenchymal stem cells to enter an animal. As an illustrative example, the product capable of allowing the mesenchymal stem cell medicine to enter the animal body includes a freeze-dried powder injection, an injection, a tablet or a capsule. As illustrative examples, methods for allowing a stem cell drug to enter an animal include oral administration and injection.

"concomitant medication" in embodiments of the invention includes medications that come into the body with one or more other medications. Concomitant medications may be introduced into the body at the same time or at approximately the same time. As an illustrative example, the nearly simultaneous entries may be one after the other; or into the body at different times on the same day. For example, multiple agents or active ingredients thereof may be formulated into one formulation, or two or more formulations may be introduced into the body at least substantially simultaneously. E.g., within about 1 hour of each other.

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

In some embodiments, the mesenchymal stem cell oral product is an adipose mesenchymal stem cell for ingestion into an animal; preferably, the animal is a mammal; preferably, the mammal is a human.

In one aspect, the present invention also provides a stem cell administration system, comprising: mesenchymal stem cell oral products, and, concomitant products; the concomitant product is selected from broad-spectrum antibiotics and/or bifidobacteria; the mesenchymal stem cell medicine comprises adipose 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 previously.

In one aspect, the invention also provides a method of promoting the prevention and treatment of type I diabetes in a mesenchymal stem cell oral product, the method comprising administering an intestinal flora modulator simultaneously or substantially simultaneously during the administration of the mesenchymal stem cell oral product.

Drawings

FIG. 1A is a graph showing the proportion of CD 44-positive cells in the flow cytometry assay of example 2;

FIG. 1B is a graph of the proportion of CD73 positive cells in the flow cytometry assay of example 2;

FIG. 1C is the proportion of CD90 positive cells in the flow cytometry assay of example 2;

FIG. 1D is the proportion of CD105 positive cells in the flow cytometry assay of example 2;

FIG. 2A is a schematic flow chart of the staged treatment of NOD/Ltj mice with streptozotocin, ADSCs and broad spectrum antibiotics of examples 1-4;

FIG. 2B is a graph showing a comparison of blood glucose changes in four groups of mice, i.e., PBS group, ADSCs + Abx group, and Co-household group, as described in example 3;

FIG. 2C is a graph comparing the pancreatic HE staining results and corresponding islet inflammation scores of four groups of mice, PBS group, ADSCs + Abx group, and Co-used group, as described in example 3;

FIG. 2D is a graph showing the results of pancreatic insulin immunohistochemical staining and β cell staining score quantification for four mice of PBS group, ADSCs + Abx group, and Co-used group described in example 3;

FIG. 2E is a graph showing Elisa results of serum insulin detection of four groups of mice, i.e., PBS group, ADSCs + Abx group, and Co-household group, as described in example 3;

FIG. 3A is a graph showing the comparison of the blood glucose changes of the ADSCs, ADSCs + ampicillin + vancomycin, ADSCs + neomycin and ADSCs + metronidazole in example 4;

FIG. 4A is a graph showing a comparison of blood glucose levels in PBS, ADSCs + Abx and Abx groups in example 5;

FIG. 5A is a graph showing the quantitative results of fecal bacteria in four groups of mice, i.e., PBS group, ADSCs + Abx group, and Co-household group, as described in example 6;

FIG. 5B is a graph showing the diversity analysis of α in four groups of mouse fecal bacteria, PBS group, ADSCs + Abx group, and Co-household group, as described in example 6;

FIG. 5C is a graph showing the diversity analysis of β in four groups of mouse fecal bacteria, PBS, ADSCs + Abx, and Co-household groups as described in example 6;

FIG. 5D is a graph showing the comparison of the abundance of different bacteria (Bifidobacterium) among the ADSCs, ADSCs + Abx and Co-used groups in example 6;

FIG. 6A is a graph showing the results of intestinal colonization by Bifidobacterium (B.breve) in the ADSCs, ADSCs + Abx, Co-owned and ADSCs + B.breve groups in example 7;

FIG. 6B is a graph showing the comparison of the blood glucose changes of the ADSCs, ADSCs + Abx and ADSCs + B. breve groups in example 7;

FIG. 6C is a graph comparing the results of HE staining of pancreas of ADSCs, ADSCs + Abx and ADSCs + B. breve groups and corresponding islet inflammation scores in example 7

Fig. 6D is a graph of pancreatic insulin immunohistochemical staining results and β cell staining score quantification for the ADSCs group, ADSCs + Abx group, and ADSCs + b.breve group described in example 7;

FIG. 7A is a graph showing the comparison of the concentration of FITC-dextran in the serum of the ADSCs, ADSCs + Abx, ADSCs + B. breve group in example 8;

FIG. 7B is a graph comparing the serum endotoxin concentrations in the ADSCs, ADSCs + Abx and ADSCs + B. breve groups of example 8;

FIG. 7C is a comparison graph of the thickness of mucus layer and scanogram obtained by staining colon tissue with Alisin blue in the ADSCs group, the ADSCs + Abx group and the ADSCs + B.breve group in example 8;

FIG. 8A is a graph comparing the bacterial load of pancreatic tissue in the ADSCs, ADSCs + Abx and ADSCs + B. breve groups as described in example 9;

FIG. 8B is a graph showing in situ hybridization staining of EUB338 probe for pancreatic tissue in the ADSCs, ADSCs + Abx and ADSCs + B. breve groups in example 9, and comparison of the numbers of bacteria observed in a high power field;

fig. 8C is a scan obtained by MafA immunohistochemical staining of pancreatic tissues in the ADSCs group, the ADSCs + Abx group, and the ADSCs + b. breve group in example 9, and a comparison graph of percentage content of positive cells.

Detailed Description

The technical solutions of the present invention are further illustrated by the following specific examples, which do not represent limitations to the scope of the present invention. Insubstantial modifications and adaptations of the present invention by others of the concepts fall within the scope of the invention.

The Mesenchymal Stem Cells (MSC) in the embodiment of the invention are derived from mesoderm at the early development stage, belong to pluripotent stem cells, can be differentiated into various tissue cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelium and the like under specific induction conditions in vivo or in vitro, still have multidirectional differentiation potential after continuous subculture and cryopreservation, can be used as ideal seed cells for repairing tissue organ injury caused by aging and pathological changes, ADSCs are Adipose Derived Stem Cells (ADSCs), the adipose mesenchymal stem cells are pluripotent stem cells derived from adipose tissues, have self-renewal and multidirectional differentiation capacity, can secrete various bioactive factors, and have wide application prospects in tissue injury and repair.

T1DM in the present invention is type 1diabetes mellitis (T1 DM).

Breve in the invention is bifidobacterium breve.

All animal experiments in the examples of the invention were approved by the ethical committee of the department of medicine of Beijing university (ethical number: LA 2019190).

The PBS in the present invention is Phosphate Buffered Saline (PBS), which generally acts as a solvent to solubilize the protective agent. It is a buffer solution which is most widely used in biochemical research, and the main component of the buffer solution is Na₂HPO₄、KH₂PO₄NaCl and KCl.

The ampicillin of the invention is a broad-spectrum semisynthetic penicillin, which is also called ampicillin, ampelin, savericin, methicillin, ampicillin, dipyridamole and AB-PC, is an β -lactam antibiotic with extremely low toxicity.

Metronidazole is nitroimidazole derivative, and can inhibit oxidation-reduction reaction of amoeba protozoon, so that nitrogen chain of the protozoon is broken. The antibacterial spectrum includes bacteroides fragilis and other bacilli, clostridium, bacillus aerogenes, eubacterium, peptococcus and peptostreptococcus, etc. The bactericidal concentration is slightly higher than the bacteriostatic concentration. Metronidazole kills cells and anaerobic microorganisms growing in the absence of oxygen, and its metabolites, which are produced upon reduction in the human body, also have an anti-anaerobic effect, but have no effect on aerobic and facultative anaerobic bacteria.

Neomycin is produced by streptomyces fradiae and belongs to the class of aminoglycoside antibiotics. The antibacterial spectrum of the neomycin is similar to that of other aminoglycoside drugs, and the neomycin has good antibacterial activity on gram-negative bacteria and partial antibacterial action on gram-positive bacteria. Neomycin has complete cross-resistance to kanamycin and partial cross-resistance to streptavidin.

Vancomycin is limited to systemic infection with methicillin-resistant staphylococcus aureus (MRSA) and intestinal and systemic infections with clostridium difficile. Has good antibacterial effect on methicillin-sensitive and drug-resistant strains in staphylococcus including staphylococcus aureus and coagulase-negative staphylococcus, various streptococcus, streptococcus pneumoniae, enterococcus and other gram-positive bacteria, and also has good effect on bacillus proteus, bacillus anthracis, diphtheria bacillus and the like which are difficult to distinguish.

"comprising" or "including" is intended to mean that the compositions (e.g., media) and methods include the recited elements, but not excluding others. When used in defining compositions and methods, "consisting essentially of … …" is meant to exclude other elements having any significance to the combination of the stated objects. Thus, a composition consisting essentially of the elements defined herein does not exclude other materials or steps that do not materially affect the basic and novel characteristics of the claimed invention. "consisting of … …" refers to trace elements and substantial process steps excluding other components. Embodiments defined by each of these transition terms are within the scope of the present invention.

Example 1 construction of a diabetic mouse model:

7-8 week-old female NOD/Ltj mice (non-obese diabetic NOD mice, non-obesity diabetes mellitus, NOD mice) were selected, purchased from the center of the department of medicine, Beijing university, and raised in an environment free of specific pathogenic microorganisms, kept at constant temperature and humidity, and maintained in a circadian (12:12) rhythm. One week after mouse adaptation, 4 consecutive days were injected with streptozotocin (STZ; 40 mg/kg; Sigma-Aldrich, USA) to accelerate the progression of diabetes. Blood glucose levels were measured by tail vein blood sampling 7 days after injection and mice with blood glucose levels >13.9mmol/L for 3 consecutive days were diagnosed with diabetes.

Example 2 extraction and identification of Adipose Derived Stem Cells (ADSCs):

a) washing redundant adipose tissues taken from a liposuction patient with physiological saline for 3 times in a super clean bench, adding enzyme (0.4mg/ml dispase: 0.2mg/ml collagenase I1: 1) with three times of the adipose volume for digestion, carrying out filtration by using a 70nm cell sieve under the aseptic condition at 37 ℃ and 450rpm, digesting for 30min, centrifuging the filtered suspension at 2500rpm/min for 5min, discarding the supernatant, adding α -MEM culture medium containing 10% fetal calf serum for re-suspension, transferring the re-suspended cells into a new 10cm culture dish, placing the culture dish in a cell incubator at 37 ℃ for culture, carrying out carbon dioxide volume fraction of 5%, digesting the cells into a long fusiform after adherent growth of Cs, digesting the cells by using 0.25% trypsin when the cells converge to 80%, and inoculating the cells into a new 10cm culture dish at the inoculation ratio of 1:3 for subculture.

b) To identify the ADSCs, the ADSCs subcultured to the third generation were washed 3 times with PBS, digested with 0.25% trypsin, and cells were collected. Fixing the cells with 4% paraformaldehyde for 10-20min, blocking with 1% BSA for 20-30min, incubating the surface marker antibody and the corresponding isotype control at 4 ℃ in the dark: CD90-FITC, CD44-PE, CD73-APC, CD105-CY 5.5. All antibodies were purchased from BD Biosciences. Detection was performed using an Accuri C6 flow cytometer (BDBiosciences). Flow cytometry identification results show that the expression of mesenchymal stem cell surface markers CD44, CD73, CD90 and CD105 is more than 95% (figure 1A, figure 1B, figure 1C and figure 1D), and the mesenchymal stem cell surface markers are identified as ADSCs.

Example 3 broad spectrum antibiotic cocktail treatment can enhance the effect of ADSCs on T1 DM:

a) grouping setting: on the basis of example 1, diabetic mice with onset were selected for random grouping:

PBS group: PBS was injected into the tail vein of the group of mice as a negative control group, which was referred to as PBS group;

ADSCs group, the mice of the group are injected with 1.0 × 10 content through tail vein after onset⁶Injecting PBS suspension of ADSCs for 100 μ l twice at an interval of one week, wherein the group is called ADSCs group;

ADSCs + Abx group: in order to regulate the intestinal flora, while ADSCs are injected for the first time, a broad-spectrum antibiotic mixture (Abx, ampicillin (1mg/ml), metronidazole (1mg/ml), neomycin (1mg/ml) and vancomycin (0.5mg/ml)) is added into drinking water as an intestinal flora regulator, and the mixture is treated for 1 week and is called an ADSCs + Abx group;

co-used group: in order to research the effect of the intestinal flora in the treatment of ADSCs, mice of ADSCs + Abx groups which are treated by antibiotics and mice of ADSCs groups which are not treated by antibiotics are subjected to cage-mixing feeding, the intestinal flora of the mice of the ADSCs groups is re-planted into the intestinal tracts of the mice of the ADSCs + Abx groups by utilizing the fecal feeding property of the mice, and the mice are reformed into co-housed groups.

Referring to FIG. 2A, a schematic of a staged treatment procedure for NOD/Ltj mice with streptozotocin, ADSCs, and broad spectrum antibiotics (including single components, generic antibacterials, and cocktail) in examples 1-4 is shown.

b) Evaluation of therapeutic effect: the blood sugar detection result shows that the ADSCs treatment can effectively reduceThe blood sugar of mice with low T1DM is further lower than that of mice treated by the ADSCs alone, and the blood sugar of mice with co-used group is temporarily reduced during the period of antibiotic feeding and gradually increased after being mixed with cages to approach the blood sugar of mice with ADSCs (figure 2B); HE staining and islet inflammation score (scoring standard reference)

E, et al, nat Immunol 2017.PMID 28346408) results showed that islet inflammation was lighter in mice in the ADSCs + Abx group than in the other three groups (fig. 2C), insulin immunohistochemical staining results showed that islet β cells were also more strongly stained in mice in the ADSCs + Abx group than in the other three groups (fig. 2D), and serum insulin Elisa assay results also showed that the serum insulin level was higher in mice in the ADSCs + Abx group than in the other groups (fig. 2E).

Example 4 broad spectrum antibiotics, either alone or in combination with the same type of antibacterium, did not substantially enhance the effect of ADSCs on the treatment of T1 DM:

in order to explore the influence of each component in the broad-spectrum antibiotic mixture on the T1DM treatment effect of ADSCs, the four components are divided into three groups according to the bacterial types of the action of four antibiotics, wherein one group of ampicillin and vancomycin mainly aims at gram-positive bacteria; neomycin group, mainly against gram-negative bacteria; the metronidazole group is mainly aimed at anaerobes. The antibiotic solution is respectively combined with ADSCs, and the blood sugar level change of the mice after the combination is observed. The specific embodiment is as follows:

a) grouping setting: the diabetic mice with the onset of disease were randomly selected and divided into the following 4 groups:

ADSCs + ampicillin + vancomycin group: in order to regulate the intestinal flora, while ADSCs are injected for the first time, ampicillin (1mg/ml) and vancomycin (0.5mg/ml) are added into drinking water as intestinal flora regulators, and the mixture is treated for 1 week and is called an ADSCs + ampicillin + vancomycin group;

ADSCs + metronidazole group: in order to regulate the intestinal flora, metronidazole (1mg/ml) is added into drinking water as an intestinal flora regulator while the ADSCs are injected for the first time, and the mixture is treated for 1 week and is called an ADSCs + ampicillin + vancomycin group;

ADSCs + neomycin group: in order to regulate the intestinal flora, while ADSCs are injected for the first time, neomycin (1mg/ml) is added into drinking water as an intestinal flora regulator, and the mixture is treated for 1 week and is called ADSCs + ampicillin + vancomycin group.

b) Evaluation of therapeutic effect: the blood glucose change results showed that when ampicillin + vancomycin, neomycin, or metronidazole were used in combination with ADSCs, the blood glucose in mice was similar to the ADSCs group and did not further decrease (fig. 3A). Combining the results of example 3, it is demonstrated that only a mixture of ampicillin, vancomycin, neomycin and metronidazole can further enhance the therapeutic effect of ADSCs.

Example 5 the effect of the broad spectrum antibiotic cocktail alone on treatment of T1DM was not significant:

to exclude the effect of antibiotic cocktail on blood sugar of T1DM mice, we designed the Abx group without ADSCs and with broad-spectrum antibiotic cocktail alone, and observed blood sugar changes of mice, as follows:

a) grouping setting: selecting diabetic mice with onset for random grouping:

PBS group: PBS is injected into the tail vein of the group of mice to serve as a negative control group, which is called a PBS group;

ADSCs + Abx group: in order to regulate the intestinal flora, while ADSCs are injected for the first time, a broad-spectrum antibiotic mixture (Abx, ampicillin (1mg/ml), metronidazole (1mg/ml), neomycin (1mg/ml) and vancomycin (0.5mg/ml) are added into drinking water as an intestinal flora regulator, and the mixture is treated for 1 week and is called an ADSCs + Abx group;

group Abx: the group of mice did not receive the treatment of ADSCs, only the broad-spectrum antibiotic cocktail (Abx, ampicillin (1mg/ml), metronidazole (1mg/ml), neomycin (1mg/ml) and vancomycin (0.5mg/ml)) was added to the drinking water as the intestinal flora regulator, and treated for 1 week, which was called the Abx group;

b) evaluation of therapeutic effect: the blood glucose change results showed that the blood glucose of mice treated with Abx alone without ADSCs did not differ significantly from the PBS group, indicating that Abx alone did not have a blood glucose lowering effect (fig. 4A). The results of examples 3 and 4 show that the ADSCs can be used in combination with Abx to achieve better therapeutic effect.

Example 6 treatment with broad-spectrum antibiotic cocktail changes intestinal flora:

to investigate how the gut flora changes after Abx use and that bacterial changes may lead to enhanced therapeutic effects after ADSCs + Abx combination, we collected four groups of mouse stools at 1 and two week time points and extracted fecal DNA for 16s rRNA Real-time PCR quantitative detection and 16s rRNA sequencing Real-time results showing that bacterial counts in stools were significantly reduced after antibiotic treatment for 1w, while bacterial counts gradually recovered after antibiotic water withdrawal for the second week (2w), with no statistical difference from the other groups (fig. 5A), 16s rRNA sequencing results, α and β diversity at 2w showing that the flora structure was not significantly altered by treatment with cs alone, while the ads cs + Abx treatment formed a unique flora (fig. 5B and 5C) differential bacterial analysis showing that the bifidobacteria in the mouse stools of the cs + Abx group were significantly higher than the adscus-cs group and the adshoco-5D (fig. 5B and 5C).

Example 7 bifidobacterium breve treatment can enhance the stem cell therapeutic effect:

based on the above results, we intend to treat mice with Bifidobacterium breve (B.breve) and ADSCs simultaneously and observe the blood glucose changes of the mice, the mice treated in the manner are named ADSCs + B.breve group, wherein B.breve (1.3001) is purchased from China general microbiological culture Collection center, cultured in MRS medium (containing 0.05% cysteine) at 37 ℃ under anaerobic environment, and 1.0 × 10⁹Breve mice after the first ADSCs treatment were gavaged for 1 week. Collecting mouse feces at 2-week time point, extracting feces DNA, and performing real-time PCR and horizontal gel electrophoresis detection confirmed successful colonization of the mouse gut by b.breve (fig. 6A).

The results show that the blood sugar of mice in ADSCs + B.breve group is obviously lower than that of mice in ADSCs group, the blood sugar is similar to that of ADSCs + ABx group (figure 6B). The pancreas tissue HE staining and islet inflammation scoring results show that the islet proportion of mice treated by ADSCs + B.breve and having islet inflammation scoring of > 75% is reduced compared with that of ADSCs group (figure 6C), while the insulin staining results also show that islet β cells are stained more strongly after the ABx/B.breve treatment compared with that of ADSCs group (figure 6D). the result shows that the increase of bifidobacterium proportion after the ADSCs + Abx treatment is the reason for enhancing the treatment effect of ADSCs, and the treatment effect of treating T1DM by ADSCs can be further enhanced through B.breve gastric lavage treatment.

Example 8 broad spectrum antibiotic cocktail/b.breve treatment can reduce intestinal permeability:

t1DM patients and animal models are often associated with increased intestinal permeability. Intestinal permeability was measured by gavage with fluorescein isothiocyanate labeled dextran (FITC-dextran, 4kd), and the results showed that the serum FITC-dextran fluorescence intensity was lowest in ADSCs + Abx group mice and ADSCs + b.breve group, indicating decreased intestinal permeability compared to ADSCs + Abx/b.breve treatment in mice treated with ADSCs alone (fig. 7A). The serum endotoxin test showed the same result, i.e., the serum endotoxin level of mice in the ADSCs + Abx/B.breve group was lower than that in the ADSCs group (FIG. 7B). Again, ADSCs + Abx/b. breve treatment decreased intestinal permeability in mice. The results of aliskiren blue staining also showed thickening of the intestinal mucus layer of mice after ADSCs + Abx/b. Breve treatment decreased mouse intestinal permeability by thickening the colonic mucus layer as described above.

Example 9 broad spectrum antibiotic cocktail/b.breve treatment can reduce ectopic colonization of the pancreas by intestinal flora, increase insulin transcription:

in order to detect the change of ectopic colonization of pancreas by intestinal flora, Real-time PCR quantitative detection and bacterial universal probe EUB338 staining are carried out, the Real-time PCR quantitative detection result (figure 8A) and the bacterial universal probe EUB338 staining result (figure 8B) both show that the number of bacteria in the pancreas is reduced compared with that in the ADSCs group, MafA is an important transcription factor of β cell insulin gene, and the MafA immunohistochemical staining result shows that the number of mouse positive cells in the ADSCs + Abx/B. breve group is more than that in the ADSCs group (figure 8C), which is consistent with the insulin staining result in figure 6D.

In conclusion, the broad-spectrum antibiotic mixture or the probiotic B.breve treatment can enhance the treatment effects of the ADSCs on reducing blood sugar and insulin inflammation when the ADSCs are treated simultaneously. Breve treatment reduces ectopic colonization of intestinal flora on pancreas by reducing intestinal permeability, thereby enhancing transcription and expression of insulin, and finally further enhancing the effect of treating T1DM by ADSCs.

Therefore, the invention aims at the problem that the mesenchymal stem cells have poor treatment effect in clinical application of treating type I diabetes, and the effect of treating type I diabetes by using the human adipose derived mesenchymal stem cells is enhanced by using broad-spectrum antibiotics or bifidobacteria to adjust the intestinal flora. The invention lays a foundation for emphasizing the important function of the intestinal flora in the process of treating the type I diabetes by the mesenchymal stem cells and also for the clinical transformation application of the mesenchymal stem cells in treating the type I diabetes.

In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the appended claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

1. A composition, comprising:

comprising a first agent consisting of mesenchymal stem cells; and the combination of (a) and (b),

a second agent consisting of an intestinal flora modulator.

2. The composition of claim 1, wherein the gut flora modulator is a gut probiotic and/or a broad spectrum antibiotic;

preferably, the intestinal probiotics are selected from one or more of bifidobacteria, lactic acid bacteria, streptococcus and bacillus;

preferably: the streptococcus is faecalis streptococcus;

preferably, the broad-spectrum antibiotics are one or more of polypeptide broad-spectrum antibiotics, macrolide broad-spectrum antibiotics, 8-phosphopolysaccharide broad-spectrum antibiotics, polyether broad-spectrum antibiotics, aminoglycoside broad-spectrum antibiotics and chemically synthesized broad-spectrum antibiotics;

preferably, the broad spectrum antibiotic is selected from a mixture consisting of ampicillin solution, metronidazole solution, neomycin solution and vancomycin solution;

preferably, the mesenchymal stem cells are selected from one or more of adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, placenta mesenchymal stem cells, dental pulp mesenchymal stem cells, skin mesenchymal stem cells, urine mesenchymal stem cells and periosteum mesenchymal stem cells, preferably adipose mesenchymal stem cells.

3. The composition of claim 2, wherein the concentration ratio of the ampicillin solution, the metronidazole solution, the neomycin solution and the vancomycin solution in the mixture is 1:1:1:0.4-0.6 in sequence.

4. The composition of claim 3, wherein the ratio of solutes in the broad spectrum antibiotic to the mesenchymal stem cells is (80-1000) mg (0.5-5) × 10⁶A plurality of;

preferably, the mass of the broad spectrum antibiotic solute in the composition is 130mg, and the number of mesenchymal stem cells is 1 × 10⁶And (4) respectively.

5. The composition of claim 2, wherein the gut flora modulator is a bifidobacterium with an optical density value of 0.8 or greater, and the ratio of the bifidobacterium to the mesenchymal stem cells is 1 × 10^9-12CFU：(0.5-5)×10⁶A plurality of;

preferably, the composition comprises 1 × 10⁶Mesenchymal stem cell and 1 × 10⁹CFU bifidobacterium.

6. Use of a composition according to claim 1 for the preparation of a medicament for the treatment of type I diabetes.

7. The composition of claim 1, further comprising a pharmaceutically acceptable carrier;

preferably, the dosage form of the composition is preferably selected from freeze-dried powder injection, tablet or capsule;

preferably, the dosage form is selected from an injection or a tablet.

8. The application of the intestinal flora regulator in preparing concomitant medicaments of mesenchymal stem cell oral products;

preferably, the mesenchymal stem cell oral product is selected from adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells or dental pulp mesenchymal stem cells;

preferably, the mesenchymal stem cell oral administration product is adipose mesenchymal stem cells for taking in animal bodies;

preferably, the animal is a mammal; preferably, the mammal is a human;

preferably, the gut flora modulator is a gut probiotic and/or a broad spectrum antibiotic;

preferably, the intestinal probiotic is a bifidobacterium.

9. A stem cell administration system, comprising:

the mesenchymal stem cell oral product of claim 9, and,

a companion product; the concomitant product is selected from broad-spectrum antibiotics and/or bifidobacteria;

the mesenchymal stem cell medicine comprises adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells or dental pulp mesenchymal stem cells.