CN117281840A - Application of fecal tooth bacillus - Google Patents
Application of fecal tooth bacillus Download PDFInfo
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- CN117281840A CN117281840A CN202311500714.4A CN202311500714A CN117281840A CN 117281840 A CN117281840 A CN 117281840A CN 202311500714 A CN202311500714 A CN 202311500714A CN 117281840 A CN117281840 A CN 117281840A
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Classifications
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
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- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
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- A—HUMAN NECESSITIES
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- A61P39/00—General protective or antinoxious agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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Abstract
The invention relates to the technical field of biomedicine, in particular to application of fecal rodenticide. The invention provides application of shigella dysenteriae in preparing preparations for protecting hematopoietic stem cells, which can be used for preparing preparations for inhibiting radiation-mediated hematopoietic stem cell apoptosis, improving clinical symptoms after irradiation and improving survival rate of irradiated mice; the bacillus dystonia can be used for preparing a preparation for increasing the butyric acid content in animals, wherein the butyric acid does not affect glycolysis of hematopoietic stem cells; the shigella dystonia can also be used for preparing medicines for enhancing the clone forming ability of irradiated hematopoietic stem cells, increasing the FOXO3a transcription level in the hematopoietic stem cells and inhibiting the muscle type pyruvate kinase 2 from entering the hematopoietic stem cell nucleus preparation, and can also be used for preparing medicines for researching and/or treating radioactive diseases, intestinal homeostasis and organ transplantation immunity.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to application of fecal rodenticide.
Background
Radiation damage refers to acute, delayed or chronic damage to body tissue caused by ionizing radiation. Ionizing radiation may damage tissue directly or through secondary reactions. Large doses of radiation can produce visible body effects within days. Small dose-induced DNA changes can cause chronic disease in the irradiated subjects, causing genetic defects in their offspring. Although we have made great progress in the control of radiation damage, how to enhance the tolerance of hematopoietic stem cells (hematopoietic stem cells, HSCs) to ionizing radiation has yet to be studied.
The intestinal flora is referred to as the "second genome" and "second brain" of the human. Intestinal flora and its metabolites are involved in the development and prognosis of a variety of major diseases such as tumors and metabolic system diseases. In addition, intestinal flora affects the targeted differentiation and proliferation of HSCs through its metabolites in the case of infection, aging, etc. Is it possible to use intestinal flora or metabolites thereof to enhance the tolerance of HSCs to IR? Therefore, the discovery of new species, strains or key molecules will provide basis for new therapeutic breakthroughs, and new strategies or targets for developing ideal therapeutic IR-mediated adverse reactions.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides application of the shigella dystonia in preparing a preparation for protecting hematopoietic stem cells, inhibiting radiation-mediated apoptosis of the hematopoietic stem cells, increasing butyric acid content in animals, enhancing cloning capacity of the hematopoietic stem cells, increasing FOXO3a transcription level in the hematopoietic stem cells, inhibiting muscle type pyruvate kinase 2 from entering hematopoietic stem cell nuclei, and application of the shigella dystonia in preparing medicines for researching and/or treating radioactive diseases, intestinal homeostasis and organ transplantation immunity.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides application of shigella dysenteriae in preparation of preparations for protecting hematopoietic stem cells.
The invention provides application of shigella dysenteriae in preparation of a preparation for inhibiting radiation-mediated hematopoietic stem cell apoptosis.
The invention provides application of a bacillus rodenticide in preparing a preparation for increasing butyric acid content in an animal body.
The invention provides application of shigella dysenteriae in preparation of a preparation for enhancing colony forming ability of hematopoietic stem cells.
The invention provides application of a fecal tooth bacillus in preparing a preparation for increasing the FOXO3a transcription level in hematopoietic stem cells.
The invention provides application of shigella dysenteriae in preparing a preparation for inhibiting muscle type pyruvic acid kinase 2 from entering hematopoietic stem cell nuclei.
The invention provides application of shigella dysenteriae in preparing medicines for researching and/or treating radioactive diseases, intestinal homeostasis and organ transplantation immunity.
The invention has the beneficial effects that:
1. the invention provides a new strain for discussing research of intestinal flora participating in regulation of hematopoietic stem cell functions and the like and research and development of biological medicine. The invention has wide prospect and practical value in exploring probiotics to regulate and control the functions of hematopoietic stem cells and protecting the hematopoietic stem cells from irradiation damage.
2. The existing probiotics such as lactobacillus and the like do not obviously improve the damage of irradiation to hematopoietic stem cells. The bacillus dystonia can be used for preparing preparations for protecting hematopoietic stem cells; is used for preparing a preparation for inhibiting radiation-mediated hematopoietic stem cell apoptosis, improving clinical symptoms after irradiation, and improving survival rate of irradiated mice.
3. The bacillus rodenticide can be used for preparing a preparation for increasing the butyric acid content in animals, wherein the increase of the butyric acid content does not affect glycolysis of hematopoietic stem cells.
4. The shigella dystonia can be used for preparing medicines for enhancing the clone forming ability of irradiated hematopoietic stem cells, increasing the FOXO3a transcription level in the hematopoietic stem cells and inhibiting muscle type pyruvate kinase 2 from entering hematopoietic stem cell nuclear preparations, and can also be used for preparing medicines for researching and/or treating radioactive diseases, intestinal homeostasis and organ transplantation immunity.
Drawings
FIG. 1 shows the survival rate of the wild type mice after irradiation and the Western blotting detection result in example 1, wherein A is the survival rate of the wild type mice after irradiation treated with antibiotics, and B is the Western blotting detection result;
FIG. 2 is changes in the abundance of various probiotics in mitochondrial STAT3 conditional knock-in mice after irradiation;
FIG. 3 is changes in the abundance of various probiotics in pre-irradiation mitochondrial STAT3 conditional knock-in mice;
FIG. 4 is the clinical scores of F.rodentia, bifidobacterium pseudolongum B.pseudolarion, L.johnsonii after irradiation given to wild mice;
FIG. 5 is the survival rate of F.rodentia, bifidobacterium pseudolongum B.pseudolarion, L.johnsonii after irradiation given to wild mice;
FIG. 6 is a graph showing the results of short chain fatty acid content measurement, wherein A is the short chain fatty acid content after irradiation of a conditional knockout mouse; b is the same bacterial count (5X 10) 8 ) The short chain fatty acid content in the culture supernatant of F.rodentia, B.pseudolongum, L.johnsonii; c is the short chain fatty acid content given to F.rodenticidum and control mice;
FIG. 7 shows the number of clones of mouse hematopoietic stem cells after irradiation and the mRNA level of the target gene of FOXO3 a;
FIG. 8 is a graph showing the results of glycolysis and glycolytic capacity assays of hematopoietic stem cells;
FIG. 9 shows immunofluorescence staining of irradiated mice CD117 + Changes in muscle type pyruvate kinase 2 in the nucleus, wherein A is immunofluorescent staining to detect CD117 in irradiated mice administered with F.rodenticidum + Muscle type pyruvate kinase 2 change condition in cell nucleus, B is immunofluorescence staining to detect irradiated mouse CD117 given butyric acid + Muscle type pyruvate kinase 2 change in nucleus;
FIG. 10 shows the effect of E.rodent on apoptosis of mouse hematopoietic stem cells after irradiation, wherein A is hematopoietic stem cell apoptosis, B is Western blotting to detect p53, BAX and internal reference beta-actin levels, C is BAX mRNA level, D isImmunofluorescence assay of CD117 in irradiated mice given F.rodenticidum + P53 change in cells, E is immunofluorescence detection of CD117 in irradiated mice fed butyric acid diet + P53 change in cells, F is the survival curve of irradiated mice.
Detailed Description
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
The fecal bacteria used in the examples of the present invention were fecal bacteria f.rodentia purchased from north nanobiology, lactobacillus johnsonii, bifidobacterium pseudolongum b.pseudolongum all purchased from north nanobiology;
mouse Rosa26 LSL-MLS-mSTAT3/LSL-MLS-mSTAT3 Mice are laboratory construction mice (cf. Patent application number: 202010294870. X). Ubc ERT2Cre/+ With Villin Cre/+ Mice were purchased from Shanghai Nannon animal model institute, p53 -/- Giving by the church mountain of university of North Carolina in the United states;
the modified PYG medium is purchased from Shandong Tuo Pu bioengineering Co., ltd.) and comprises the following components: 5.0g of tryptone, 5.0g of peptone, 10.0g of yeast extract, 5.0g of beef extract, 5.0g of glucose, 2.0g of potassium hydrogen phosphate, 1.0mL of tween 80 and CaCl 2 -2H 2 O 0.01g，MgSO 4 -7H 2 0.02g of O, 0.04g of potassium hydrogen phosphate, 0.04g of monopotassium phosphate, 0.4g of sodium bicarbonate, 0.08g of sodium chloride and 1.0mg of resazurin;
MRS medium was purchased from Soy Biotechnology Co., ltd, and its composition was: 10.0g of peptone, 10.0g of beef extract, 4.0g of yeast powder, 20.0g of glucose, 0.2g of magnesium sulfate, 5.0g of sodium acetate, 2.0g of tri-ammonium citrate, 2.0g of dipotassium hydrogen phosphate, 0.04g of manganese sulfate and 1.0mL of tween 80;
the modified MRS medium is purchased from Soy Biotechnology Co., ltd and comprises the following components: 10.0g of peptone, 10.0g of beef extract, 4.0g of yeast powder, 20.0g of glucose, 0.2g of magnesium sulfate, 5.0g of sodium acetate, 2.0g of triammonium citrate, 2.0g of dipotassium hydrogen phosphate, 0.04g of manganese sulfate, 1.0mL of tween 80 and 0.5g of L-cysteine (microphone).
Example 1
1. Isolation of intestinal epithelial cell mitochondria: wild type mice were randomly divided into two groups, one group was given normal drinking water for 3 consecutive weeks, and the other group was given broad-spectrum antibiotic drinking water (penicillin 1g/L, metronidazole 1g/L, neomycin 1g/L, vancomycin 0.25 g/L) for 3 consecutive weeks. After 3 weeks, wild-type mice were given 7.5Gy of systemic radiation. The survival of the mice was observed and the survival rate of the mice was calculated, and the results are shown in FIG. 1A.
After 3 days, 3 mice were randomized through CO from each of the two groups 2 Euthanasia, 75% ethanol soaking for 5min, taking out the mice, placing on a sterile operating table, and allowing the abdomen side to face upwards; cutting a small opening in the middle of the abdomen side of the mouse, tearing the skin and exposing the abdomen wall; the peritoneum was lifted up under the spleen, sheared off, turned up, the intestinal tract exposed, isolated with forceps and ophthalmic scissors and removed, and mitochondria were collected using the pecan tissue mitochondrial isolation kit. The specific operation is as follows: the small intestine tissue with the weight of 100mg is cut, and the tissue is washed once by 1mL of phosphate buffer (0.01M, pH 7.2-7.4, nanjing navigation biotechnology Co., ltd.); subsequently, the tissue was sheared with scissors into very fine tissue fragments and 1000 μl of pre-chilled phosphate buffer (0.01 m, ph 7.2-7.4) was added thereto, and ice-bath for 3min; after 15s of low speed centrifugation (2600 rpm centrifugation), the supernatant was discarded and the precipitated tissue sample was retained. Adding 800 mu L of precooled pancreatin digestion solution into the sediment tissue sample, after ice bath for 20min, centrifuging at a low speed (2600 rpm for 15 s), discarding supernatant, collecting sediment tissue sample and re-suspending the tissue with 200 mu L of mitochondrial separation reagent A; after centrifugation at 2600rpm for 15s, the supernatant was discarded, 800 μl of pre-chilled PMSF-containing mitochondrial separation reagent was added to the precipitated tissue sample, which was then placed in an ice bath and homogenized for 25 times; placing the homogenized tissue sample in a low-temperature centrifuge for centrifugation (4 ℃ C., 3400 rpm) for 5min; after centrifugation, the supernatant was transferred to another centrifuge tube and centrifuged again (4 ℃,6400rpm,10 min). After centrifugation, the supernatant was discarded, and the precipitated tissue pellet was isolated small intestine mitochondria.
2. Western blot detection: the small intestine mitochondria obtained by the separation are subjected to western blotting detection, and the western blotting detection is briefly described as follows: protein lysates containing 1% pmsf, 1% pi and 1% sds were added to the tissue pellet described in step 1 and thoroughly mixed. Taking 12 mu L of protein lysate from each group, adding 3 mu L of 5 Xloading buffer solution (the company of Highway Biotechnology, highway Co., ltd.), vortex mixing, centrifuging briefly, heating at 95deg.C for 3min, centrifuging at low temperature (4deg.C, 13200 rpm) for 15s to ensure that no protein lysate remains on the tube wall; fixing the prefabricated glue (Kirschner Co., ltd.) in electrophoresis tank (Berle life medical products Co., ltd.), pouring sufficient MOPS electrophoresis liquid (Kirschner Co., ltd.), and slightly extracting the glue comb; adding 15 mu L of protein Marker (Norvezan) or protein sample into the gel well, and carrying out electrophoresis at 80V; after electrophoresis, taking out the gel plate, putting the separating gel into membrane transferring liquid, and simultaneously putting a nitrocellulose membrane (Zhenjiang Aibianme biotechnology Co., ltd.) prepared in advance and thick filter paper into the membrane transferring liquid for soaking for 15min; sequentially spreading thick filter paper, NC film, separating gel and another piece of thick filter paper in a semi-dry rotary instrument (Berle life medical products Co., ltd.), and carrying out constant current of 180mA and film rotation for 60min; after the membrane transfer is finished, PBST is used for washing the membrane, after 10min, 5mL of 5% skimmed milk and 2.5 mu L of primary antibody are added into an incubation box filled with a nitrocellulose membrane, and the incubation is carried out by shaking on a shaking table at 4 ℃ overnight; the next day, NC membrane is washed by PBST for 5-10min, and repeated three times; discarding the supernatant, adding 5mL of 5% skimmed milk and 1 μL of secondary antibody into an incubation box with nitrocellulose membrane, and incubating for 2h at room temperature; washing NC membrane with PBST for 10min, repeating for three times; freshly prepared color development solution (new sirame biotechnology, su) was added dropwise, developed using a burle chemiluminescent imager (burle life medicine products, inc.) and the image stored for subsequent analysis. The detection result is shown in FIG. 1B.
As can be seen from fig. 1, antibiotics reduce the survival rate of irradiated mice; western blot detection found: antibiotics are used to promote STAT3 localization to the mitochondria of intestinal epithelial cells.
3. Breeding target mice: breeding mice with 6-8 weeks of age, and irradiating 12 weeks of age;
synthesis of CoxThe 4singal-STAT3 gene is inserted into a CAG-LSL-Cox4singal-STAT3-WPRE-pA expression frame at a fixed point at a Rosa26 gene locus by a homologous recombination mode to construct a homologous recombination vector. Microinjection of Cas9 mRNA, gRNA and homologous recombination vector into fertilized eggs of C57BL/6J mice, followed by transplantation of the injected fertilized eggs into uterus of pseudopregnant female mice to produce chimeric mice; the chimeric mice are mated with C57BL/6J mice to obtain F1 generation Rosa26 LSL-MLS-mSTAT3/+ A mouse; after 3 generations of mating and breeding with C57BL/6J mice, rosa26 LSL-MLS-mSTAT3/+ Mice and Rosa26 LSL-MLS-mSTAT3/+ Mice were mated to obtain Rosa26 LSL-MLS-mSTAT3/LSL-MLS-mSTAT3 And (3) a mouse.
Rosa26 at 6-8 weeks of age LSL-MLS-mSTAT3/LSL-MLS-mSTAT3 Mice and Villin Cre/+ Mice were caged to give offspring mice (Rosa 26) LSL-MLS-mSTAT3/+ ；Villin Cre/+ Mice), then the offspring mice were combined with Rosa26 LSL-MLS-mSTAT3/LSL-MLS-mSTAT3 Mice were caged to give Rosa26 LSL-MLS-mSTAT3/LSL-MLS-mSTAT3w Villin Cre/+ A mouse;
rosa26 LSL-MLS-mSTAT38LSL-MLS-mSTAT3 Mice and Ubc ERT2Cre/+ Mice were caged to give offspring mice (Rosa 26) LSL-MLS-mSTAT3/+ ；Ubc ERT2Cre/+ ) Progeny mice and Rosa26 were then prepared LSL-MLS-mSTAT3/+ ；Ubc ERT2Cre/+ Mice were caged to give Rosa26 LSL-MLS-mSTAT3/LSL-MLS-mSTAT3 ；Ubc ERT2Cre/+ And (3) a mouse.
4. The irradiated model mouse cecal content was collected: administration of Rosa26 at 12 weeks of age LSL-MLS-mSTAT3/LSL-MLS-mSTAT3 ；Villin Cre/+ Mice (with unlimited sex) were irradiated systemically at 7.5 Gy. After 3 days, through CO 2 Euthanasia, 75% ethanol soaking for 5min, taking out the mice, placing on a sterile operating table, and allowing the abdomen side to face upwards; cutting a small opening in the middle of the abdomen side of the mouse, tearing the skin and exposing the abdomen wall; the peritoneum was lifted up under the spleen, sheared off, turned up to expose the intestinal tract, the intestinal tract was isolated with forceps and ophthalmological scissors and removed, the cecal contents were removed, sub-packaged and placed in sterile centrifuge tubes and frozen at-80 ℃.
5. And (5) sending detection and sequencing: the cecal content was sent to the Shanghai Meiji Biotechnology Co.Ltd for macrogene sequencing analysis.
6. Real-time quantitative fluorescent PCR detection of relative DNA expression quantity of strains: the DNA of the cecum content was extracted using a fecal genomic DNA extraction kit (Kangji Biotech Co., ltd.) as follows: placing 100mg of fecal sample into a centrifuge tube, adding 1mL Buffer SW into the centrifuge tube, shaking by vortex for 3min, centrifuging at 12000rpm for 1min, and discarding the supernatant; adding 1mL Buffer SL, and vortex oscillating for 3min; a water bath at 65 ℃ for 20min, and vortex oscillation is carried out for 15s every 5min during the period; centrifuging at 12000rpm for 3min, and transferring the supernatant to a new centrifuge tube; adding 1mL Buffer GL into the supernatant, mixing the supernatant for 25 times by reversing the supernatant, placing the supernatant on ice for 5min, and centrifuging the supernatant at 12000rpm for 5min; adding the supernatant into an adsorption column filled into a collecting pipe, centrifuging at 12000rpm for 1min, pouring out waste liquid in the collecting pipe, and putting the adsorption column back into the collecting pipe; adding 500 mu L Buffer GW1 into the adsorption column, centrifuging at 12000rpm for 1min, pouring out the waste liquid in the collecting pipe, putting the adsorption column back into the collecting pipe, and centrifuging at 12000rpm for 1min; adding 500 μl Buffer GW2 into the adsorption column, centrifuging at 12000rpm for 3min, pouring out the waste liquid in the collecting tube, and standing the adsorption column at room temperature for 5min; placing the adsorption column in a new centrifuge tube, suspending and dripping 60 μl of sterilized water into the middle part of the adsorption column, standing at room temperature for 2-5min, centrifuging at 12000rpm for 1min, collecting DNA solution, and detecting the relative abundance of target strain in the collected DNA solution by real-time quantitative fluorescence PCR (Roche life sciences). The analysis results are shown in fig. 2-3, wherein fig. 2 is the change in the abundance of the various probiotics in the mitochondrial STAT3 conditional knock-in mice after irradiation, and fig. 3 is the change in the abundance of the various probiotics in the mitochondrial STAT3 conditional knock-in mice before irradiation; in the figure, F.rodentum is fecal bacillus, B.pseudoturntable is bifidobacterium pseudolongum, L.johnsonii is lactobacillus johnsonii, P.merdae is bacteroides faecalis, A.muciniti philia is akaman, and butyl tricimonas is vibrio.
As can be seen from fig. 2, after irradiation, the abundance of fecal bacteria f.rodentia, bifidobacterium pseudolongum (bifidobactirium_pseudosolongum, b.pseudosolongum), lactobacillus johnsonii (lactobacilli_johnsonii, l.johnsonii), bacteroides faecalis (Parabacteroides merdae, p.merdae) decreases, the abundance of akamain mucilaginous bacteria (Akkermansia muciniphila, a.mucinphilia) increases; the abundance of the genus vibrio Butyricimonas is also reduced. As can be seen from FIG. 3, before irradiation, the abundance of F.rodenticidum, bifidobacterium pseudolongum (Bifidobacterium pseudolongum, B.pseudolongum) and Lactobacillus johnsonii (Lactobacillus johnsonii, L.johnsonii) decreased, whereas the abundance of Acremonium muciniphilum (Akkermansia muciniphila, A.mucinilla), paramycola (Parabacteroides merdae, P.merdae) and Vibrio butyricum were not significantly different. In summary, the abundance of F.rodenticidum, bifidobacterium pseudolongum (B.pseudolongum), and Lactobacillus johnsonii (L.johnsonii) was reduced before and after irradiation.
EXAMPLE 2 cultivation of Strain and construction of model
(1) Culturing of faecium rodent F: adding 1.0L distilled water, pH7.2, adding 33.6g of modified PYG culture medium, heating and boiling to obtain CO 2 Cooling under environment, adding 5mg of hemin and 0.1mg of vitamin K into every 100mL of culture medium 1 And 0.05g cysteine hydrochloride. Sterilizing at 121deg.C for 15min. Taking the fecal tooth bacillus as anaerobic bacteria and taking OD 600 0.214 F.rodenticide 85. Mu.L was added to 15mL of the medium, and the mixture was cultured in an anaerobic incubator at 37℃for 48 hours.
(2) Culture of lactobacillus johnsonii: 48.3g MRS medium was taken, 1.0L distilled water was added thereto, pH was 5.7, and sterilization was performed at 121℃for 15 minutes. OD is taken 600 About 0.350 L.johnsonii 1206. Mu.L was added with 50mL of the medium and incubated in an anaerobic incubator at 37℃for 48h.
(3) Culture of bifidobacterium pseudolongum b.pseudoolongum: 48.83g of modified MRS medium was taken, 1.0L of distilled water was added thereto, pH was 6.2, and the mixture was sterilized at 121℃for 15 minutes. OD is taken 600 Bifidobacterium pseudolongum B.pseudoturntable 522. Mu.L of 0.253 was added to 50mL of the medium, and the mixture was cultured in an anaerobic incubator at 37℃for 48 hours.
(4) Irradiation model of fecal bacteria transplantation: the mice were given clean control medium (modified PYG medium), F.rodenticidum (1.5X10) to wild type mice 4 days before irradiation, respectively 7 Individual/individual), bifidobacterium pseudolongum b.pseudolongum (5 x 10) 8 Individual/individual), lactobacillus johnsonii (2X 10) 8 4 times total, followed by 7.5Gy irradiation to mice; after irradiation, the corresponding irradiated mice were administered with PYG medium or probiotics every two days.
(5) Conditional knock-in murine irradiation model: under normal-grade feeding conditions, conditional knockin mice Rosa26 were given LSL -MLS-mSTAT3/LSL-MLS-mSTAT3 And Rosa26 LSL-MLS-mSTAT3/LSL-MLS-mSTAT3 ；Villin Cre/+ Mice were irradiated to 7.5Gy systemically.
(6) Conditioned knock-in mice fed with butyrate irradiation model: under normal grade feeding conditions, intraperitoneal injection was given to conditional knock-in mice Rosa26 LSL-MLS-mSTAT3/LSL-MLS-mSTAT3 ；Ubc ERT2Cre/+ Mice 75mg/kg tamoxifen (MedChemExp Biotechnology Co., ltd.) were used once every two days for a total of 7 times. 2 days after the last 1 dose, tamoxifen-induced Rosa26 was administered LSL-MLS-mSTAT3/LSL-MLS-mSTAT3 ；Ubc ERT2Cre/+ Mice were irradiated to 7.5Gy systemically. The irradiated mice were divided into two groups and given a normal diet, diet containing 5% butyric acid, respectively.
Example 3 Effect of faecal bacteria on clinical scoring and survival of mice after irradiation
1. Clinical scoring of mice: the irradiated mice of example 2 (4) were observed for a mice hair (normal: 0; not smooth: 1; spot-fried: 2; very-fried: 3), a posture (normal: 0; bow back: 1; bow back, head lifting not: 4; head lifting not to maintain upright posture: 6), a behavior (normal: 0; slight change in behavior: 1; walking posture and breathing rate significantly changed: 3; only moving under external stimulus: 6), a weight reduction ratio [ (original weight-existing weight)/original weight ]: < 5% >: 0 minutes; 5-10%: 1, dividing; 10 to 14.9 percent: 2, dividing; 15-19.9%: 3 minutes; 20-24.9%: 4, dividing; > 25%:5 minutes), appetite (normal: 0 minutes; appetite reduction: 1, dividing; no food was consumed for the first observation: 2, dividing; the second observation did not eat food: 3 minutes) dynamically monitoring the phenotype score of the irradiated mice of example 2 (4). Mice were euthanized over 12 minutes. The scoring results are shown in fig. 4.
As can be seen from fig. 4, the bacillus dystonia f.rodenticum was able to reduce the clinical score of irradiated mice; but bifidobacterium pseudolongum b.pseudoolongum and lactobacillus johnsonii increased the clinical score of irradiated mice.
2. Survival rate of mice: the irradiated mice of example 2 (4) were kept under normal-grade feeding conditions, and the survival of the mice was observed daily. The survival rate was calculated and the calculation result is shown in fig. 5.
As can be seen from FIG. 5, under normal-grade feeding conditions, F.rodentia can increase the survival rate of irradiated mice; but bifidobacterium pseudolongum b.pseudoolongum and lactobacillus johnsonii did not significantly affect the survival rate of irradiated mice.
Example 4 Effect of faecal bacterium on the butyrate content in mice
CO after 3d irradiation of the irradiated mice of examples 2 (4), (5) 2 Euthanasia, 75% ethanol soaking for 5min, taking out the mice, placing on a sterile operating table, and allowing the abdomen side to face upwards; cutting a small opening in the middle of the abdomen side of the mouse, tearing the skin and exposing the abdomen wall; lifting the peritoneum on the lower side of the spleen, cutting, turning up, exposing the intestinal tract, separating and taking out the intestinal tract by forceps and an ophthalmic scissors, taking out the cecum content, placing the cecum content and culture medium supernatant with the same bacterial count as in examples 2 (1-3) into a sterile centrifuge tube, and carrying out short chain fatty acid content detection on the culture medium supernatant by Shanghai Meiji biological medical science and technology Co., ltd, wherein the detection result is shown in figure 6, and A is the short chain fatty acid content after the conditional knock-in mice of example 2 (5) are irradiated; b is the short chain fatty acid content in the supernatant of the culture medium of Lactobacillus johnsonii, lactobacillus pseudolongum B.pseudolongum, lactobacillus johnsonii, of example 2 (1-3) with the same bacterial count F.rodenium; c is the short chain fatty acid content of mice given F.rodentia and control group of example 2 (4).
As can be seen from fig. 6, the butyric acid content was significantly reduced in irradiated conditional knockout mice (fig. 6A); the butyric acid content in the supernatant of the F.rodentia culture medium was increased (FIG. 6B); faecalis f.rodentia was able to increase butyrate content in irradiated mice (figure 6C).
EXAMPLE 5 Effect of F.rodent on the clonogenic potential of mouse hematopoietic Stem cells, FOXO3a transcriptional level in hematopoietic Stem cells
1. Collection of hematopoietic Stem cell enriched CD117 + Bone marrow cells: CO after 3d irradiation of the irradiated mice of examples 2 (4), (5) 2 Euthanized, 75% ethanol soaked for 5min, the mice were removed on a sterile bench with the ventral side facing up, the tibia and femur of the mice were isolated and removed, and the remaining tissues surrounding the tibia and femur were further isolated and removed, and then the tibia and femur were placed in a cell culture dish containing 2mL RPMI-1640 medium containing green chain diab (Gibco) and 10% fbs (Gibco). The bone ends of the tibia and femur were cut with an ophthalmic scissors, and then the complete medium was aspirated with a 1mL syringe, and the bone marrow cells were rinsed from one end of the bone into a 15mL sterile centrifuge tube. Collecting single cell suspension, centrifuging at low speed (25deg.C, 800 rmp) for 5min to obtain spleen cell precipitate, re-suspending with erythrocyte lysate (Fumais biotechnology Co., ltd.), mixing well, standing for 1min to allow erythrocyte to be fully lysed, adding equal volume of phosphate buffer (0.01M, pH 7.2-7.4, nanjing navigation biotechnology Co., ltd.) to terminate lysis, centrifuging at low speed (25deg.C, 800 rmp) for 5min, and re-suspending with 2mL of phosphate buffer (0.01M, pH 7.2-7.4, nanjing navigation biotechnology Co., ltd.) for use.
2. Antibody labeling: the cell suspension obtained in step 1 was then combined with mouse CD117 + Monoclonal antibody (BD diluted 1:200) was incubated at 4deg.C for 20min, after which cells were washed with 1mL of phosphate buffer (0.01M, pH 7.2-7.4, nanjing navigation Biotechnology Co., ltd.) and the pellet was collected by low-speed centrifugation (25 ℃,1000rpm,5 min), followed by resuspension of the cells with 3mL of phosphate buffer (0.01M, pH 7.2-7.4, nanjing navigation Biotechnology Co., ltd.) for use;
3. flow-sorting cells: the flow cell sorter (FACSAria III, BD), the on-board software (BD FACSDiva Software) and all lasers were turned on, the flow was adjusted and sorting parameters were set (Drop Delay:26.53, left tube rack number 58) for analysis to obtain CD117 + Two populations of cells, sorting and harvesting CD117 + Cells were centrifuged at low speed (25 ℃,800 rpm) for 5min in a 15mL centrifuge tube to collect cell clusters and resuspended in appropriate amount of RPMI-1640 medium;
4. cell cloning: the number of cells was counted using a hemocytometer.
3000 CDs 117 were taken + Cells were resuspended in 2mL methylcellulose semisolid medium (stemcel), thoroughly mixed and transferred to 6-well cell culture plates (n=3/group).
Cell cloning procedure for conditional knock-in murine irradiation model: 6000 CDs 117 were taken + Cells were resuspended in 4mL methylcellulose semisolid medium (stemcel), thoroughly mixed and transferred to 6-well cell culture plates containing equal volumes of RPMI-1640 or butyric acid at a final concentration of 5mM (n=3/group). Phosphate buffer solution (0.01M, pH 7.2-7.4, nanjing navigation biotechnology Co., ltd.) is added into the gaps between the holes to balance humidity, and the mixture is placed at 37 ℃ and 5% CO 2 、20％O 2 Culturing in an incubator for 10 days. Cell clusters were counted under a microscope. The results are shown in FIG. 7A.
As can be seen from FIG. 7A, F.rodentum and its metabolite butyric acid enhance the cloning number of HSCs after irradiation.
PCR detection of the target Gene: after counting cells using a hemocytometer, 5X 10 cells were collected 5 CD117 + Cells were centrifuged at low speed (25 ℃,1000rmp,5 min) to collect cell clusters, using Cells-to-CT TM 1-step Power SYBR TM RNA was extracted using Green kit (Siemens' flywheel), cDNA was synthesized using 5×all-In-One RTMastermix kit (abm), and the target gene mRNA level of FOXO3a was detected using real-time fluorescent quantitative PCR. The detection result is shown in fig. 7B.
As can be seen in FIG. 7B, F.rodenticum and butyric acid increased FOXO3a mRNA levels in HSCs.
Example 6 Effect of butyric acid on glycolysis and glycolytic Capacity of hematopoietic Stem cells
Collection of CD117 in bone marrow of conditional knockin murine irradiation model according to example 5, steps 1-3 + And (3) cells. Cell counts were counted using a hemocytometer, and after counting, cell culture plates were prepared: the 22.4. Mu.s of the mixture was prepared with phosphate buffer (0.01M, pH 7.2-7.4)g/mL of Cell-Tak (Corning), and the prepared Cell-Tak was added to SeaHorseXF Cell culture plate (Agilent), 50. Mu.L/well, and left to stand at room temperature for 20min; and then washed with sterilized distilled water 2 times for standby. Inoculating cells: according to 1X10 5 The/well will CD117 + Inoculating cells into SeaHorseXF cell culture plate, adding culture medium with the same volume into four background holes A1, B4, C3 and D6, and adding CO 2 The incubator was incubated overnight. Instrument preparation: the Seahorse instrument (agilent) and computer were turned on and the Wave software (agilent) was turned on, allowing the instrument to warm to 37 ℃ and preheat overnight. Hydration probe and cell treatment: a1000. Mu.L of SeaHorseXF calibrator was added to Utilityplate (Agilent) and the probe was submerged, and the test plate was returned to Utilityplate and placed at 37℃without CO 2 Overnight in the incubator>12h) Hydrating the probe; preparing detection solution by SeahorseXF Base Medium (Agilent), adding glutamine (Xuzhou Microcomputer obtained bioengineering Co., ltd.), adjusting pH to 7.4 with NaOH, filtering, and placing in a 37 deg.C water bath for 60min; cell plate from CO 2 Taking out from the incubator, after confirming good cell status under microscope, sucking out the growth medium with a pipette, adding 1mL of preheated detection solution to the remaining 50. Mu.L, sucking out 1mL of liquid (repeating twice), adding 450. Mu.L of detection solution to the cell plate, and placing the cell plate in the absence of CO 2 Incubator for 60min; drug required for configuration experiment: glucose (micro-keman bioengineering, inc. Of bradycardia, slow): to 2mL of the test solution, 108mg of Glucose was added; oligomycin (Oligomycin) (Shanghai Ala Biotechnology Co., ltd.): to 2mL of the detection solution, 4. Mu.L of the primary concentration was added at 10 -2 Oligomycin of M; 2-Deoxy-D-glucose (2-Deoxy-D-glucose, 2-DG) (MedChemExpress Biotech Co., ltd., U.S.): to 2mL of the test solution was added 328mg of 2-DG. Adding the medicine: adding the prepared medicines into each hole respectively: glucose:56 μl/well, oligomycin: 62. Mu.L/well, 2-DG: 69. Mu.L/well, and the sample was checked on the machine. After the end of the experiment, the cells were counted using a hemocytometer and experimental data were performed using wave software. The process of calculating from first dose (Glucose) to second dose (Oligomycin) by wave software is that the cells are glycolytic level under basal conditions, and calculating from first dose (Glucose) to second dose (Oligomycin)The third dosing (2-DG) is the whole process of maximum glycolytic capacity of the cell. The analysis results are shown in FIG. 8.
As can be seen from FIG. 8, the metabolic product butyric acid of F.rodent does not significantly affect glycolysis and glycolytic capacity of hematopoietic stem cells.
EXAMPLE 7 Effect of faecal bacterium on muscle type pyruvate kinase 2 entry into the hematopoietic Stem cell nucleus
Immunofluorescent staining: CO 3 days after irradiation of the irradiation model for fecal fungus transplantation in example 2 (4) and the conditional knock-in mice irradiation model fed with butyric acid in (6) 2 Euthanized, 75% ethanol soaked for 5min, the mice were removed and placed on a sterile console with the ventral side facing up, the tibia and femur of the mice were isolated, removed and fixed, and decalcification, dehydrated wax dipping (confirmed), embedding and slicing were commissioned by the wuhanseville biotechnology company limited.
a. Dewaxing: baking the slices in a incubator at 60 ℃ for 60min; followed by washing for 5min each in the order of xylene-absolute ethanol-95% ethanol-70% ethanol-PBS-PBST-PBS.
b. Antigen retrieval: 800mL of 0.01M sodium citrate buffer solution (pH 6.0) is boiled and poured into a repair cup, the repair cup is heated with high fire by a microwave oven for 3min, the dewaxed slide glass is placed into the repair cup, the repair cup is continuously heated with medium fire for 5min and then cooled to 25 ℃, and then the repair cup is washed for 2min respectively according to the sequence of PBS-PBST-PBS.
c. Dyeing: anti-CD 117 is added dropwise + Primary antibody, placing the slide in a wet box and incubating overnight at 4 ℃; then washing for 3min according to the sequence of PBS-PBST-PBS; dripping secondary antibody HRP, and incubating for 10min at 25 ℃ in dark; then washing for 3min according to the sequence of PBS-PBST-PBS; diluting (Baino panorama) dye with TSA signal amplification solution to 1 Xdye working solution, dripping 100 μl into the sample, completely covering the sample, and incubating at 25deg.C in dark place for 10min; then washing for 3min according to the sequence of PBS-PBST-PBS; repeating the antigen retrieval and the dyeing steps to carry out the next round of dyeing; dripping ready-to-use DAPI, and incubating for 5min at room temperature in dark; dripping a stirring medium and sealing; the sample was observed with a fluorescence microscope (model: leica STELLARIS 5confocal microscope). As a result, as shown in FIG. 9, in FIG. A, the green color is CD117 + The red color is muscle acetoneAcid kinase 2 (PKM 2), blue is nuclear DNA (4, 6-diamidine-2-phenylindole, DAPI); in Panel B, the red color is CD117 + Green for muscle pyruvate kinase 2 (PKM 2), blue for nuclear DNA (4, 6-diamidine-2-phenylindole, DAPI);
as can be seen from FIG. 9, F.rodentia and its metabolite butyric acid inhibit the entry of muscle type pyruvate kinase 2 into CD117 + And (3) cell nucleus.
Example 8 Effect of faecalis on apoptosis of hematopoietic Stem cells
1. Collection of CD117 in bone marrow of conditional knockin murine irradiation model according to example 5, steps 1-3 + And (3) cells. After counting cells using a hemocytometer, the number of cells was counted at 1X10 5 The/well will CD117 + Cells were inoculated into 24-cell culture plates containing 900. Mu.L of RPMI-1640 medium and 100. Mu.L of fetal bovine serum (Gibco), and 10. Mu.L of RPMI-1640 medium or 10. Mu.L of butyric acid at a concentration of 500mM were added thereto, and after 24 hours, the number of apoptotic cells was then measured using the (red fluorescence) one-step TUNEL apoptosis test kit (MedChemExp Biotechnology Co., USA) as follows: collecting and washing cells: low-speed centrifugation (25 ℃,1000rpm,5 min) to collect cells, washing with an appropriate amount of PBS 2 times, 5min each time; fixing: adding 4% paraformaldehyde to the collected cell mass for fixation (shaking table at 4 ℃ C. For 30 min); washing the cells: cells were collected by low-speed centrifugation (25 ℃,1000rpm,5 min), followed by addition of 3ml fbs to the pellet and shaking of the sample at 4 ℃ for 5min; low-speed centrifugation (25 ℃,1000rpm,5 min) to collect cells, the procedure was repeated 2 times, PBS was discarded after centrifugation was completed, and excess liquid was aspirated with filter paper; penetration: adding a penetrating fluid into the washed cell mass, and incubating at room temperature for 5min; repeating the step of washing the cells; TUNEL staining: adding prepared 50 mu LTUNEL working solution (5 mu LTdT and 45 mu L Cy 3-dUTPLabaringmix) into the cell mass, and incubating at 37 ℃ for 60min in a dark place; repeating the washing step 3 times; subsequently, 500. Mu.LPBS was added to the sample to suspend the cells thoroughly and apoptotic cells were detected under a fluorescence microscope. The detection results are shown in FIG. 10A.
2. P53, BAX and internal reference β -actin levels were detected using western blot techniques: example(s)5x 10 sorted in step 1-3 5 CD117 + Cells were added with 30. Mu.L of cell lysate containing 1% PMSF (volume ratio), 1% PI (volume ratio) and 1% SDS. Taking 12 mu L of protein lysate from each group, adding 3 mu L of 5 Xloading buffer solution (the company of Highway Biotechnology, highway Co., ltd.), vortex mixing, centrifuging briefly, heating at 95deg.C for 3min, centrifuging at low temperature (4deg.C, 13200 rpm) for 15s to ensure that no protein lysate remains on the tube wall; fixing the prefabricated glue (Kirschner Co., ltd.) in electrophoresis tank (Berle life medical products Co., ltd.), pouring sufficient MOPS electrophoresis liquid (Kirschner Co., ltd.), and slightly extracting the glue comb; adding 15 mu L of protein Marker (Norvezan) or protein sample into the gel well, and carrying out electrophoresis at 80V; after electrophoresis, taking out the gel plate, putting the separating gel into membrane transferring liquid, and simultaneously putting a nitrocellulose membrane (Zhenjiang Aibianme biotechnology Co., ltd.) prepared in advance and thick filter paper into the membrane transferring liquid for soaking for 15min; sequentially spreading thick filter paper, NC film, separating gel and another piece of thick filter paper in a semi-dry rotary instrument (Berle life medical products Co., ltd.), and carrying out constant current of 180mA and film rotation for 60min; after the membrane transfer is finished, PBST is used for washing the membrane, after 10min, 5mL of 5% skimmed milk and 2.5 mu L of primary antibody are added into an incubation box filled with a nitrocellulose membrane, and the incubation is carried out by shaking on a shaking table at 4 ℃ overnight; the next day, NC membrane is washed by PBST for 5-10min, and repeated three times; discarding the supernatant, adding 5mL of 5% skimmed milk and 1 μL of secondary antibody into an incubation box with nitrocellulose membrane, and incubating for 2h at room temperature; washing NC membrane with PBST for 10min, repeating for three times; freshly prepared color development solution (new sirame biotechnology, su) was added dropwise, developed using a burle chemiluminescent imager (burle life medicine products, inc.) and the image stored for subsequent analysis. The detection result is shown in fig. 10B.
3. CD117 detection in bone marrow in a conditional knock-in murine irradiation model according to example 5, steps 1-3 + Apoptosis-related genes such as BAXmRNA levels in cells. The detection result is shown in fig. 10C.
3. Detection of CD117 in bone marrow in an irradiation model of faecal fungus transplantation and conditional knock-in mice fed with butyric acid according to example 7 + P53 levels in cells. The detection results are shown in FIGS. 10D-E, wherein FIG. 10D shows immunofluorescence detectionCD117 in irradiated mice given F.rodenticidum + P53 change in cells, red: CD117, off-white: p53, blue: nuclear DNA (4, 6-diamidine-2-phenylindole, DAPI). FIG. 10E shows immunofluorescence detection of CD117 in irradiated mice fed with butyric acid + P53 change in cells, red: CD117, green: p53, blue: nuclear DNA (4, 6-diamidine-2-phenylindole, DAPI);
4. p53 is to be used -/- Mice and Rosa26 LSL-MLS-mSTAT3/LSL-MLS-mSTAT3 ；Ubc ERT2Cre/+ Mice were caged to give Rosa26 LSL-MLS-mSTAT3/+ ；Ubc ERT2Cre/+ ；p53 +/- Mice, subsequently Rosa26 LSL-MLS-mSTAT3/+ ；Ubc ERT2Cre/+ ；p53 +/- Mice and Rosa26 LSL-MLS-mSTAT3/LSL-MLS-mSTAT3 Mice were caged to give Rosa26 LSL-MLS-mSTAT3/LSL-MLS-mSTAT3 ；Ubc ERT2Cre/+ ；p53 +/- Mice, and administration of Rosa26 LSL-MLS-mSTAT3/LSL-MLS-mSTAT3 ；Ubc ERT2Cre/+ ；p53 +/- Mice were irradiated systemically (7.5 Gy), survival observed, and survival rate calculated, as shown in fig. 10F.
As can be seen from fig. 10, the renieratia f.rodentum inhibits radiation-mediated apoptosis of HSCs by its metabolite butyrate and is p53 dependent.
From the above examples, the present invention provides the use of fecal tooth bacillus for preparing a preparation for protecting hematopoietic stem cells, inhibiting radiation-mediated apoptosis of hematopoietic stem cells, increasing butyric acid content in animals, enhancing clonality of hematopoietic stem cells, increasing FOXO3a transcription level in hematopoietic stem cells, inhibiting entry of muscle type pyruvate kinase 2 into hematopoietic stem cell nuclei, and for research and/or treatment of radiation diseases, intestinal homeostasis and organ transplantation immunity.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (7)
1. Application of faecalis in preparation of hematopoietic stem cell protecting preparation is provided.
2. Application of faecalis in preparation of preparations for inhibiting radiation-mediated hematopoietic stem cell apoptosis is provided.
3. Use of a bacillus rodenticide for the preparation of a formulation for increasing the butyric acid content in an animal.
4. Use of a shigella dystonia in the preparation of a formulation for enhancing the clonogenic capacity of hematopoietic stem cells.
5. Use of a shigella dystonia for the preparation of a formulation for increasing FOXO3a transcript levels in hematopoietic stem cells.
6. Application of faecalis in preparation of preparation for inhibiting muscle type pyruvate kinase 2 from entering hematopoietic stem cell nucleus is provided.
7. Application of shigella dysenteriae in preparing medicine for researching and/or treating radioactive diseases, intestinal homeostasis and organ transplantation immunity 。
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