CN115211405A - Method for establishing experimental mouse mastitis model - Google Patents
Method for establishing experimental mouse mastitis model Download PDFInfo
- Publication number
- CN115211405A CN115211405A CN202210903343.3A CN202210903343A CN115211405A CN 115211405 A CN115211405 A CN 115211405A CN 202210903343 A CN202210903343 A CN 202210903343A CN 115211405 A CN115211405 A CN 115211405A
- Authority
- CN
- China
- Prior art keywords
- mouse
- mice
- liquid
- staphylococcus aureus
- flat
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 208000004396 mastitis Diseases 0.000 title claims abstract description 35
- 238000000034 method Methods 0.000 title claims abstract description 18
- 241000699666 Mus <mouse, genus> Species 0.000 claims abstract description 92
- 241000699670 Mus sp. Species 0.000 claims abstract description 54
- 210000005075 mammary gland Anatomy 0.000 claims abstract description 54
- 239000007788 liquid Substances 0.000 claims abstract description 47
- 210000002445 nipple Anatomy 0.000 claims abstract description 37
- 241000191967 Staphylococcus aureus Species 0.000 claims abstract description 35
- 241000755266 Kathetostoma giganteum Species 0.000 claims abstract description 29
- 241000588724 Escherichia coli Species 0.000 claims abstract description 19
- 230000001580 bacterial effect Effects 0.000 claims abstract description 19
- 230000001575 pathological effect Effects 0.000 claims abstract description 16
- 239000008267 milk Substances 0.000 claims abstract description 15
- 210000004080 milk Anatomy 0.000 claims abstract description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 12
- 238000005520 cutting process Methods 0.000 claims abstract description 12
- 230000003444 anaesthetic effect Effects 0.000 claims abstract description 9
- 235000009161 Espostoa lanata Nutrition 0.000 claims abstract description 5
- 240000001624 Espostoa lanata Species 0.000 claims abstract description 5
- 210000000683 abdominal cavity Anatomy 0.000 claims abstract description 5
- 210000005252 bulbus oculi Anatomy 0.000 claims abstract description 5
- 230000035800 maturation Effects 0.000 claims abstract description 5
- 230000006651 lactation Effects 0.000 claims description 12
- 241000894006 Bacteria Species 0.000 claims description 11
- 239000000243 solution Substances 0.000 claims description 9
- RNFNDJAIBTYOQL-UHFFFAOYSA-N chloral hydrate Chemical compound OC(O)C(Cl)(Cl)Cl RNFNDJAIBTYOQL-UHFFFAOYSA-N 0.000 claims description 6
- 229960002327 chloral hydrate Drugs 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- 238000003860 storage Methods 0.000 claims description 6
- 238000007865 diluting Methods 0.000 claims description 5
- 238000002474 experimental method Methods 0.000 claims description 5
- 229930040373 Paraformaldehyde Natural products 0.000 claims description 4
- 210000001015 abdomen Anatomy 0.000 claims description 4
- 210000004369 blood Anatomy 0.000 claims description 4
- 239000008280 blood Substances 0.000 claims description 4
- 230000008014 freezing Effects 0.000 claims description 4
- 238000007710 freezing Methods 0.000 claims description 4
- 239000007924 injection Substances 0.000 claims description 4
- 238000002347 injection Methods 0.000 claims description 4
- 239000012452 mother liquor Substances 0.000 claims description 4
- 229920002866 paraformaldehyde Polymers 0.000 claims description 4
- 239000000725 suspension Substances 0.000 claims description 4
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 claims description 3
- 238000009631 Broth culture Methods 0.000 claims description 3
- 230000003321 amplification Effects 0.000 claims description 3
- 239000001963 growth medium Substances 0.000 claims description 3
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 3
- 238000012449 Kunming mouse Methods 0.000 claims description 2
- 230000001954 sterilising effect Effects 0.000 claims description 2
- 239000010413 mother solution Substances 0.000 claims 1
- 238000010276 construction Methods 0.000 abstract description 4
- 238000000465 moulding Methods 0.000 abstract description 4
- 238000010241 blood sampling Methods 0.000 abstract 1
- 210000001519 tissue Anatomy 0.000 description 17
- 239000002158 endotoxin Substances 0.000 description 15
- 229920006008 lipopolysaccharide Polymers 0.000 description 15
- 102000003896 Myeloperoxidases Human genes 0.000 description 11
- 108090000235 Myeloperoxidases Proteins 0.000 description 11
- 230000000694 effects Effects 0.000 description 8
- 210000000481 breast Anatomy 0.000 description 6
- 210000000440 neutrophil Anatomy 0.000 description 6
- 239000008055 phosphate buffer solution Substances 0.000 description 6
- 206010002091 Anaesthesia Diseases 0.000 description 4
- 230000037005 anaesthesia Effects 0.000 description 4
- 235000013365 dairy product Nutrition 0.000 description 4
- 230000008595 infiltration Effects 0.000 description 4
- 238000001764 infiltration Methods 0.000 description 4
- 230000002757 inflammatory effect Effects 0.000 description 4
- 230000036285 pathological change Effects 0.000 description 4
- 231100000915 pathological change Toxicity 0.000 description 4
- FFRBMBIXVSCUFS-UHFFFAOYSA-N 2,4-dinitro-1-naphthol Chemical compound C1=CC=C2C(O)=C([N+]([O-])=O)C=C([N+]([O-])=O)C2=C1 FFRBMBIXVSCUFS-UHFFFAOYSA-N 0.000 description 3
- 241000283690 Bos taurus Species 0.000 description 3
- 206010061218 Inflammation Diseases 0.000 description 3
- 241000191940 Staphylococcus Species 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 230000004054 inflammatory process Effects 0.000 description 3
- 210000002540 macrophage Anatomy 0.000 description 3
- 210000004303 peritoneum Anatomy 0.000 description 3
- 239000002504 physiological saline solution Substances 0.000 description 3
- 230000035935 pregnancy Effects 0.000 description 3
- 230000000638 stimulation Effects 0.000 description 3
- 238000011725 BALB/c mouse Methods 0.000 description 2
- 230000000740 bleeding effect Effects 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 230000000249 desinfective effect Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 210000001508 eye Anatomy 0.000 description 2
- 210000001703 glandular epithelial cell Anatomy 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000001338 necrotic effect Effects 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 230000008961 swelling Effects 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 230000000451 tissue damage Effects 0.000 description 2
- 231100000827 tissue damage Toxicity 0.000 description 2
- OTLLEIBWKHEHGU-UHFFFAOYSA-N 2-[5-[[5-(6-aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy]-3,4-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-3,5-dihydroxy-4-phosphonooxyhexanedioic acid Chemical compound C1=NC=2C(N)=NC=NC=2N1C(C(C1O)O)OC1COC1C(CO)OC(OC(C(O)C(OP(O)(O)=O)C(O)C(O)=O)C(O)=O)C(O)C1O OTLLEIBWKHEHGU-UHFFFAOYSA-N 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 206010006298 Breast pain Diseases 0.000 description 1
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 208000006662 Mastodynia Diseases 0.000 description 1
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 208000030270 breast disease Diseases 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 239000002095 exotoxin Substances 0.000 description 1
- 231100000776 exotoxin Toxicity 0.000 description 1
- 239000008098 formaldehyde solution Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 1
- 230000036732 histological change Effects 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 230000004660 morphological change Effects 0.000 description 1
- 208000013435 necrotic lesion Diseases 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 208000026777 severe mastitis Diseases 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000008719 thickening Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000001993 wax Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000008096 xylene Substances 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/02—Breeding vertebrates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61D—VETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS
- A61D7/00—Devices or methods for introducing solid, liquid, or gaseous remedies or other materials into or onto the bodies of animals
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/44—Staphylococcus
- C12R2001/445—Staphylococcus aureus
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Environmental Sciences (AREA)
- Genetics & Genomics (AREA)
- Animal Behavior & Ethology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Public Health (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Biomedical Technology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a method for establishing a mastitis model of an experimental mouse, which comprises the following steps: preparing escherichia coli liquid and staphylococcus aureus liquid; mice in a selective maturation stage; injecting an anesthetic into the abdominal cavity of the mouse, and wiping the mammary gland of the mouse and the skin around the mammary gland by using an alcohol cotton ball; clamping the fourth pair of nipples of the mouse by using surgical forceps, and cutting off the nipple tips by using surgical scissors, wherein the cut part is 1mm long; sucking 20 mu L of bacterial liquid by using a flat-head microinjector, extending the flat-head microinjector into the milk duct of the fourth pair of nipples, wherein the extension depth is 4-5mm, the flat-head microinjector vertically enters the milk duct, slowly injecting the bacterial liquid into mammary glands, and then slowly rotating the flat-head microinjector to leave the milk duct; killing the mouse after blood sampling of eyeballs, and preparing pathological sections; the invention effectively improves the successful construction probability of the mastitis model, and leads the molding rate to reach 95 percent.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for establishing a mastitis model of an experimental mouse.
Background
Mastitis is considered to be one of the most common breast diseases in the world, and is an inflammation of the breast tissue, including redness, swelling, heat, breast pain, and gland tissue damage; and poses a serious threat to humans and mammals, especially to dairy cows. For more severe mastitis, acinar secretion is impaired, resulting in loss of lactation function at the site of infection. Mastitis is also a major source of economic losses for dairy farmers. Mastitis typically has clinical symptoms that are subject to varying degrees of infection and inflammatory changes. It can be caused by a variety of pathogens, and in general, occurs primarily in bacterial infections, with the most common pathogens being escherichia coli and staphylococcus aureus. Coli elicits a strong immune response in the mammary gland through Lipopolysaccharide (LPS) on the bacterial cell wall, causing mastitis. Staphylococcus aureus can repeatedly infect mammary tissue, induce mammary inflammatory reaction, apoptosis, and released exotoxin can cause systemic symptoms, resulting in death of dairy cows. Therefore, the search for safe treatment for mastitis is urgent, and before searching for treatment drugs, researchers need to construct mastitis models for experiments, but in the process of establishing mastitis models, mice are easy to die, the model construction process is affected, and the molding rate is low.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provides a method for establishing a mastitis model of an experimental mouse, effectively improves the successful construction probability of the mastitis model, and makes the modeling rate reach 95%.
The purpose of the invention is realized by the following technical scheme: a method for establishing a mastitis model of an experimental mouse, comprising the following steps:
s1, preparation of experimental bacteria liquid
The bacterial liquid comprises escherichia coli bacterial liquid and staphylococcus aureus bacterial liquid;
diluting the escherichia coli mother liquor to obtain escherichia coli liquid, wherein the concentration of the escherichia coli liquid is 0.2mg/ml;
performing shake culture on Staphylococcus aureus strains, and after the Staphylococcus aureus is amplified to logarithmic phase, resuspending to 2 × 10 8 CFU/mL to obtain staphylococcus aureus liquid;
s2, selecting mice
Mice with a selective maturation period of 8-10 weeks, of which mice the mother mouse: the proportion of the male mice is 2:1, the male mice are raised in the same cage, the pure mice are not easy to be matched in the gestation period of 21 days and the lactation period of 21 days, and if the purchased mice cannot be matched and pregnant for a long time, the breeding of experimental mice needs to be carried out in a laboratory;
s3, treatment of experimental mice
Anesthetizing the mice:
injecting an anesthetic into the abdominal cavity of the mouse, after the mouse is completely anesthetized, keeping the mouse in a supine position with the front side facing upwards, unhairing, exposing the nipple of the mouse, wiping the mammary gland of the mouse and the skin around the mammary gland with an alcohol cotton ball, fully sterilizing, and keeping the mammary gland and the skin around the mammary gland clean;
and (3) mammary gland treatment: clamping the fourth pair of nipples of the mouse by using a flat-head surgical forceps with the left hand, and cutting off the nipple tips by using a surgical scissors with the right hand to expose the section of the nipples, so that the bacteria liquid can be conveniently injected at the later stage, wherein the cut part is 1mm long;
injecting bacterial liquid:
0.2mg/mL LPS 50. Mu.L or 2X 10 was aspirated using a flat-head microinjector 8 20 mu L of CFU/mL golden yellow staphylococcus liquid, extending the flat-head micro-injector into the milk duct of the fourth pair of nipples, wherein the extension depth is 4-5mm, the flat-head micro-injector vertically enters the milk duct, slowly injecting the liquid in the flat-head micro-injector into the mammary gland to finish stimulation, then slowly rotating the flat-head micro-injector to leave the milk duct, and lightly massaging the nipples and the skin around the mammary gland by hands to enable the liquid to be better absorbed, performing the same treatment on the left nipple and the right nipple of the fourth pair of nipples of the mouse, then placing the mouse in a mouse cage, and waiting for anesthesia and awakening;
and S4, after a period of time, carrying out eyeball blood collection on the mouse, and then killing the mouse to prepare pathological sections.
Further, the mice in step S2 are all in the seventh day of lactation, the mice are isolated from the newborn mice for at least 1h, and the lactation is stopped and fasted.
Furthermore, the mice can be any of BALB/C mice, kunming mice and C57 mice.
Further, the anesthetic is 10% chloral hydrate in step S3, and the injection amount of the chloral hydrate is 0.1mL, and the dosage is for a mouse with a body weight of 50 g.
Further, in step S4, the preparation method of the pathological section includes:
cutting skin (not cutting peritoneum) along the ventral midline of the lower abdomen of the mouse, separating to two sides, exposing the fourth pair of mammary glands, peeling off the fourth pair of mammary glands, putting the mammary glands on one side into 4% paraformaldehyde solution, making pathological sections, and freezing the rest mammary gland tissues in a 4 ℃ refrigerator for short-term storage or a-80 ℃ refrigerator for later-stage experiment.
Further, the experimental bacteria liquid is prepared by the following specific steps:
1mg/mL of E.coli 055: b5, diluting the mother liquor to 0.2mg/mL concentration by using PBS (phosphate buffer solution), thus obtaining Escherichia coli (LPS) bacterial liquid;
inoculating the staphylococcus aureus ATCC 35556 strain into a broth culture medium, carrying out amplification culture in a shaking table environment with the temperature of 37 ℃ and the pH value of 7.4, after the staphylococcus aureus is amplified to a logarithmic phase, using an autoclaved PBS solution for carrying out heavy suspension until the concentration of the staphylococcus aureus is 2 x 10 8 And (3) CFU/mL, and then placing the sample at 4 ℃ for storage to obtain the staphylococcus aureus liquid, so that the establishment of the model is ensured.
The beneficial effects of the invention are: the invention uses a micro flat-head injector to inject bacteria such as LPS or staphylococcus aureus suspension into the mammary tissue of the treated experimental mouse through a mammary duct, and the flat-head injector injects vertically to ensure that the bacteria can completely enter the mammary tissue, and the result of pathological change, pathological histological change and MPO activity is observed by naked eyes to show that the mastitis model established by the experimental mouse is successfully established, and the molding rate is as high as 95 percent; the method can be widely applied to the research of prevention and treatment of mastitis induced by escherichia coli and staphylococcus aureus, screening, research and development of related drugs and the like, and provides a good experimental animal model for researching the prevention and treatment of mastitis of escherichia coli and staphylococcus aureus of people and dairy cows and the mechanism of blood-milk barrier damage.
Drawings
FIG. 1 is a healthy mouse nipple before modeling;
FIG. 2 is a mouse nipple with the tip removed;
FIG. 3 is a mouse after modeling;
FIG. 4 is mouse mammary tissue after modeling;
FIG. 5 is a graph of morphological changes of mouse mammary tissue after modeling;
FIG. 6 is a graph of pathological changes after modeling of mouse mammary gland tissues;
FIG. 7 is a graph of MPO changes after modeling of mouse mammary tissue.
Detailed Description
The technical solutions of the present invention are further described in detail below with reference to the accompanying drawings, but the scope of the present invention is not limited to the following descriptions.
Examples
A method for establishing a mastitis model of an experimental mouse, comprising the following steps:
s1, preparation of experimental bacteria liquid
The bacterial liquid comprises escherichia coli bacterial liquid and staphylococcus aureus bacterial liquid;
1mg/mL of E.coli 055: b5, diluting the mother liquor to 0.2mg/mL concentration by using PBS (phosphate buffer solution), thus obtaining Escherichia coli (LPS) bacterial liquid;
inoculating the staphylococcus aureus ATCC 35556 strain into a broth culture medium, carrying out amplification culture in a shaking table environment with the temperature of 37 ℃ and the pH value of 7.4, after the staphylococcus aureus is amplified to a logarithmic phase, using an autoclaved PBS solution for carrying out heavy suspension until the concentration of the staphylococcus aureus is 2 x 10 8 CFU/mL, and then placing at 4 ℃ for storage to obtain staphylococcus aureus liquid;
s2, selecting a mouse
Mice with a selective maturation period of 8-10 weeks, of which mice the mother mouse: the proportion of the male mice is 2:1, the male mice are raised in the same cage, the pregnancy period is 21 days, the lactation period is 21 days, the mice are all on the seventh day of the lactation period, the mice and the newborn mice are isolated for at least 1h, and the lactation is stopped and fasted; the mouse is a BALB/c mouse;
s3, treatment of experimental mice
Anesthetizing the mice:
injecting an anesthetic into the abdominal cavity of the mouse, after the mouse is completely anesthetized, keeping the mouse in a state of facing upwards and lying on the back, unhairing, exposing the nipple of the mouse, wiping the mammary gland of the mouse and the skin around the mammary gland by using an alcohol cotton ball, fully disinfecting, and keeping the mammary gland and the skin around the mammary gland clean; the anesthetic is 10% chloral hydrate, and the injection amount of a mouse with the weight of 50g is 0.1mL;
and (3) mammary gland treatment: clamping the fourth pair of nipples of the mouse by using a flat-head surgical forceps with the left hand, and cutting off the nipple tips by using a surgical scissors with the right hand to expose the section of the nipples, so that the bacteria liquid can be conveniently injected at the later stage, wherein the cut part is 1mm long;
injecting bacterial liquid:
0.2mg/mL LPS 50. Mu.L or 2X 10 was aspirated using a flat-head microinjector 8 20 mu L of CFU/mL golden yellow staphylococcus liquid, extending the flat-head micro-injector into the milk duct of the fourth pair of nipples, wherein the extension depth is 4-5mm, the flat-head micro-injector vertically enters the milk duct, slowly injecting the liquid in the flat-head micro-injector into the mammary gland to finish stimulation, then slowly rotating the flat-head micro-injector to leave the milk duct, and lightly massaging the nipples and the skin around the mammary gland by hands to enable the liquid to be better absorbed, performing the same treatment on the left nipple and the right nipple of the fourth pair of nipples of the mouse, then placing the mouse in a mouse cage, and waiting for anesthesia and awakening;
s4, after a period of time, carrying out eyeball blood collection on the mouse, and then killing the mouse to prepare a pathological section, wherein the preparation method of the pathological section comprises the following steps:
cutting skin (not cutting peritoneum) along the ventral midline of the lower abdomen of the mouse, separating to two sides, exposing the fourth pair of mammary glands, peeling off the fourth pair of mammary glands, putting the mammary glands on one side into 4% paraformaldehyde solution, making pathological sections, and freezing the rest mammary gland tissues in a 4 ℃ refrigerator for short-term storage or a-80 ℃ refrigerator for later-stage experiment.
Comparative example:
a method for establishing a mastitis model of an experimental mouse, comprising the following steps:
s1, selecting a mouse
Mice with a selective maturation phase of 8-10 weeks, of which mice the mother mouse: the proportion of the male mice is 2:1, the male mice are raised in the same cage, the pregnancy period is 21 days, the lactation period is 21 days, the mice are all on the seventh day of the lactation period, the mice and the newborn mice are isolated for at least 1h, and the lactation is stopped and fasted; the mouse is a BALB/c mouse;
s2, treatment of laboratory mice
Anesthetizing the mice:
injecting an anesthetic into the abdominal cavity of the mouse, after the mouse is completely anesthetized, keeping the mouse in a state of facing upwards and lying on the back, unhairing, exposing the nipple of the mouse, wiping the mammary gland of the mouse and the skin around the mammary gland by using an alcohol cotton ball, fully disinfecting, and keeping the mammary gland and the skin around the mammary gland clean; the anesthetic is 10% chloral hydrate, and the injection amount of a mouse with the weight of 50g is 0.1mL;
and (3) mammary gland treatment: clamping the fourth pair of nipples of the mouse by using a flat-head surgical forceps with the left hand, and cutting off the nipple tips by using a surgical scissors with the right hand to expose the section of the nipples, so that the bacteria liquid can be conveniently injected at the later stage, wherein the cut part is 1mm long;
and (3) injecting physiological saline:
a flat head micro-injector is used for sucking 20 mu L of physiological saline, the flat head micro-injector is extended into a breast duct of the fourth pair of nipples, the extension depth is 4-5mm, the flat head micro-injector vertically enters the breast duct, the physiological saline in the flat head micro-injector is slowly injected into mammary glands to finish stimulation, then the flat head micro-injector is slowly rotated to leave the breast duct, the nipples and the skin around the mammary glands can be lightened by hands, bacteria liquid can be better absorbed, the left nipple and the right nipple of the fourth pair of nipples of the mouse are treated in the same way, and then the mouse is placed in a mouse cage to wait for anesthesia and awakening;
s3, after a period of time, carrying out eyeball blood collection on the mouse, and then killing the mouse to prepare a pathological section, wherein the preparation method of the pathological section comprises the following steps:
cutting skin (not cutting peritoneum) along the ventral midline of the lower abdomen of the mouse, separating to two sides, exposing the fourth pair of mammary glands, peeling off the fourth pair of mammary glands, putting the mammary glands on one side into 4% paraformaldehyde solution, making pathological sections, and freezing the rest mammary gland tissues in a 4 ℃ refrigerator for short-term storage or a-80 ℃ refrigerator for later-stage experiment.
Evaluation was performed on the mice of the example group and the mice of the comparative example group:
evaluation of mouse mastitis model:
the success of the mastitis construction of the mouse can be determined from the following aspects:
(1) Pathological changes of the eye: after the milk duct is injected with LPS (or golden yellow staphylococcus liquid) for 24h, the shape and color change of the fourth pair of nipples of the mouse can be observed, whether congestion and swelling exist or not, whether bleeding spots, necrotic lesions exist or not and the like.
(2) Histopathological changes: mice stimulated with LPS (or staphylococcus aureus) have a marked thickening of the acinar wall of the mammary gland, and the acinar cavity is filled with a large number of neutrophils and macrophages, with congestion and edema.
(3) Myeloperoxidase (MPO) activity: MPO activity is an important index of infiltration degree of neutrophils and macrophages, and the MPO activity of mouse mammary tissue stimulated by LPS (or staphylococcus aureus) is obviously increased.
(II) pathological test of mammary gland tissue
After sacrifice, the 4 th pair of mammary gland tissues were collected, cut into 0.5cm x 0.5cm pieces, loaded into an embedding box and labeled, fixed in 10% formaldehyde solution for 24h → alcohol gradient dehydration (75% alcohol 24h → 85% alcohol 12h → 95% I alcohol 12h → 95% II alcohol 12h → 100% I alcohol 0.5h → 100% II alcohol 0.5 h) → xylene transparency 2min → wax immersion, 4 total jars, 0.5 h/jar → paraffin embedded mammary gland tissues → sections 5mm in water at 40 ℃ to spread the tissues in a spreading manner → dragging the sections with a glass slide and labeling the tissues → 70 ℃ baking 4h → HE conventional staining → gelatin sealing → observation of the pathological changes of the mammary gland tissues of the mice under a microscope.
And (III) weighing, homogenizing and centrifuging mouse mammary tissue, collecting supernatant, adding an indicating reagent according to the operation of MPO kit instructions, and measuring the MPO activity.
MPO activity = (ODtest-ODcontrol)/[ 11.3 weight (g) ].
And (IV) morphological observation shows that the mammary tissue of the control group is soft and flat, has boundaries, is milky white in color, and can clearly see blood vessels and milk contained in the mammary tissue (figure 5A). Mammary tissue induced by LPS or Staphylococcus aureus liquid has visible divergence of tissue lees, is bright red, contains many necrotic foci, and has massive bleeding points (LPS model group: FIG. 5B, and Staphylococcus aureus model group: 5C).
(V) the result of the mouse mammary gland histopathology section is shown in the figure, the structure of the mammary lobules of the control group is clear, acinus is complete and uniform, and mammary epithelial cells are flat (figure 6A); LPS-induced breast tissue: most acinar structures are destroyed, a large number of inflammatory mediators such as neutrophils are infiltrated in the cavity, and necrotic and exfoliated glandular epithelial cells are remarkably increased (fig. 6B); staphylococcus aureus-induced breast tissue damage was severe, with increased inflammatory secretions within the acinar cavity, massive neutrophil infiltration, and exfoliated glandular epithelial cells (fig. 6C). HE staining results show that a mouse mastitis model is successfully established by using LPS and staphylococcus aureus induction.
(VI) the MPO activity value mainly refers to the degree of infiltration of neutrophils and macrophages into tissues, and can be used as an important index for evaluating the severity of inflammation. As shown in FIG. 7, after mastitis is induced by LPS and Staphylococcus aureus, infiltration of inflammatory mediators such as neutrophils is obvious, and the MPO activity value is 3.96 +/-0.48U/g and 3.38 +/-0.40U/g, which are sharply increased and remarkably different (p is less than 0.01) compared with that of the control group of 0.20 +/-0.11U/g.
Seventhly, mastitis models of 60 BALB/c experimental mice are established, and 1 mouse of the 60 mice dies accidentally after anesthesia; 1 mouse died after mastitis was established, the cause was unknown, and no model was established when sampling; in addition, one mouse is sampled after a mastitis model is established for 24 hours, and the pathological result shows that mastitis is not established; after evaluation of 57 mice, mastitis is found to be established, and the molding rate reaches 95%.
The foregoing is illustrative of the preferred embodiments of this invention, and it is to be understood that the invention is not limited to the precise form disclosed herein and that various other combinations, modifications, and environments may be resorted to, falling within the scope of the concept as disclosed herein, either as described above or as apparent to those skilled in the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (6)
1. A method for establishing a mastitis model of an experimental mouse is characterized by comprising the following steps:
s1, preparation of experimental bacteria liquid
The bacterial liquid comprises escherichia coli bacterial liquid and staphylococcus aureus bacterial liquid;
diluting the escherichia coli mother liquor to obtain escherichia coli liquid, wherein the concentration of the escherichia coli liquid is 0.2mg/ml;
performing shake culture on Staphylococcus aureus strains, and after the Staphylococcus aureus is amplified to logarithmic phase, resuspending to 2 × 10 8 CFU/mL to obtain staphylococcus aureus liquid;
s2, selecting a mouse
Mice with a selective maturation period of 8-10 weeks, of which mice the mother mouse: the proportion of the male mouse is 2:1;
s3, treatment of laboratory mice
Anesthetizing the mice:
injecting an anesthetic into the abdominal cavity of the mouse, after the mouse is completely anesthetized, keeping the mouse in a supine position with the front side facing upwards, unhairing, exposing the nipple of the mouse, wiping the mammary gland of the mouse and the skin around the mammary gland with an alcohol cotton ball, fully sterilizing, and keeping the mammary gland and the skin around the mammary gland clean;
and (3) mammary gland treatment: clamping the fourth pair of nipples of the mouse by using surgical forceps, and cutting off the nipple tips by using surgical scissors, wherein the cut-off part is 1mm long;
injecting bacterial liquid:
0.2mg/mL LPS 50. Mu.L or 2X 10 was aspirated using a flat-head microinjector 8 20 mu L of CFU/mL golden staphylococcus aureus bacterial liquid, extending the flat-head microinjector into the milk duct of the fourth pair of nipples to the extension depth of 4-5mm, enabling the flat-head microinjector to vertically enter the milk duct, slowly injecting the bacterial liquid in the flat-head microinjector into mammary gland, and then slowly rotating the flat-head microinjector to leave the milk duct;
and S4, after a period of time, carrying out eyeball blood collection on the mouse, and then killing the mouse to prepare pathological sections.
2. A method of establishing a model for experimental mouse mastitis according to claim 1, wherein: the mice are all in the seventh day of lactation in the step S2, the mice are isolated from the newborn mice for at least 1h, and the lactation is stopped and fasted.
3. A method of establishing a model for mastitis in laboratory mice according to claim 2, wherein the method comprises the steps of: the mice can be any of BALB/C mice, kunming mice and C57 mice.
4. A method of establishing a model for experimental mouse mastitis according to claim 1, wherein: in the step S3, the anesthetic is 10% chloral hydrate, and the injection amount of the chloral hydrate is 0.1mL.
5. The method for establishing a mastitis model of an experimental mouse as claimed in claim 1, wherein in the step S4, the pathological section is prepared by:
cutting the skin along the ventral midline of the lower abdomen of the mouse, separating to two sides, exposing the fourth pair of mammary glands, peeling off the fourth pair of mammary glands, putting the mammary glands on one side into 4% paraformaldehyde solution, making pathological sections, and freezing the rest mammary gland tissues in a refrigerator at 4 deg.C for short-term storage or at-80 deg.C for later-stage experiment.
6. A method of establishing a model for experimental mouse mastitis according to claim 1, wherein: the experimental bacteria liquid is prepared by the following specific steps:
1mg/mL of E.coli 055: diluting the B5 mother solution to 0.2mg/mL concentration by PBS solution to obtain Escherichia coli liquid;
inoculating the staphylococcus aureus ATCC 35556 strain into a broth culture medium, carrying out amplification culture in a shaking table environment with the temperature of 37 ℃ and the pH value of 7.4, after the staphylococcus aureus is amplified to a logarithmic phase, using an autoclaved PBS solution for carrying out heavy suspension until the concentration of the staphylococcus aureus is 2 x 10 8 And (5) CFU/mL, and then storing at 4 ℃ to obtain the staphylococcus aureus liquid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210903343.3A CN115211405A (en) | 2022-07-26 | 2022-07-26 | Method for establishing experimental mouse mastitis model |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210903343.3A CN115211405A (en) | 2022-07-26 | 2022-07-26 | Method for establishing experimental mouse mastitis model |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115211405A true CN115211405A (en) | 2022-10-21 |
Family
ID=83613716
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210903343.3A Pending CN115211405A (en) | 2022-07-26 | 2022-07-26 | Method for establishing experimental mouse mastitis model |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115211405A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106923929A (en) * | 2017-02-24 | 2017-07-07 | 西北农林科技大学 | A kind of mammary gland of mouse injection needle and its application |
| CN111387133A (en) * | 2020-02-25 | 2020-07-10 | 中国农业大学 | Method for establishing model for inhibiting mouse mastitis by using Klebsiella pneumoniae bacteriophage |
| CN111990330A (en) * | 2020-07-28 | 2020-11-27 | 扬州大学 | Method for constructing mouse staphylococcus aureus type mastitis model |
-
2022
- 2022-07-26 CN CN202210903343.3A patent/CN115211405A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106923929A (en) * | 2017-02-24 | 2017-07-07 | 西北农林科技大学 | A kind of mammary gland of mouse injection needle and its application |
| CN111387133A (en) * | 2020-02-25 | 2020-07-10 | 中国农业大学 | Method for establishing model for inhibiting mouse mastitis by using Klebsiella pneumoniae bacteriophage |
| CN111990330A (en) * | 2020-07-28 | 2020-11-27 | 扬州大学 | Method for constructing mouse staphylococcus aureus type mastitis model |
Non-Patent Citations (1)
| Title |
|---|
| 高瑞娟等: "金黄色葡萄球菌、大肠杆菌联合诱发小鼠乳腺炎病理模型", 中国兽医学报, vol. 38, no. 5, pages 1029 - 1034 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Joe et al. | Basidiobolus ranarum as a cause of subcutaneous mycosis in Indonesia | |
| CN105769916B (en) | Application of the source for mesenchymal stem cells excretion body in preparation treatment preeclampsia drug or preparation | |
| CN111387133A (en) | Method for establishing model for inhibiting mouse mastitis by using Klebsiella pneumoniae bacteriophage | |
| CN109674819A (en) | Placenta mesenchyma stem cell preparation and its purposes for treating hardening illness | |
| Adlam et al. | Natural and experimental staphylococcal mastitis in rabbits | |
| CN111990330B (en) | Method for constructing mouse staphylococcus aureus type mastitis model | |
| Udeh et al. | Effects of type of extender and storage conditions on the motility of goat spermatozoa | |
| CN103642800A (en) | siRNA molecule composition, and applications thereof in treatment of hypertrophic scars | |
| CN115211405A (en) | Method for establishing experimental mouse mastitis model | |
| CN103952377A (en) | Cell line used for separation culture and multiplication of Orf virus (ORFV) as well as preparation method and application of cell line | |
| CN103800900B (en) | Staphylococcus aureus and the mammitis of cow vaccine that its deactivation is obtained | |
| CN107174657A (en) | Prepare the method for the antigen composition of targeting glioma and glioma stem cells and the vaccine containing the antigen composition | |
| Sayid | Advances in bovine follicular aspiration technique | |
| CN107427555A (en) | Healthy reproduction and suppress the composition and method of hyperketonemia using IL 8 to increase the output of milk and improve in mammal | |
| CN109517772A (en) | The building of Lactococcus lactis MG1363 a kind of and its application in treatment puerpera's cracked nipple | |
| CN105230607A (en) | Honeybee artificial insemination buffer solution, and preparation method thereof | |
| Tatsuo | Acid-fast bacilli detected in umbilical codes and skins of human at cases of surgical operation. | |
| CN105941308B (en) | A method of cleaning grade is cultivated by drug purification and screening method and tests tree shrew | |
| CN103800901A (en) | Streptococcus and dairy cow mastitis vaccine obtained by inactivating streptococcus | |
| Abou-Eisha et al. | Dermatophytes in animals and their zoonotic importance in Suez canal area | |
| CN103800899A (en) | Milk cattle mastitis vaccine | |
| CN103948639A (en) | Method for constructing trichophyton mentagrophytes infected rabbit model | |
| Foshay | Induction of a Virulence in Pasteurella tularensis | |
| CN103756942B (en) | One strain coli strain and the mammitis of cow vaccine that bacterial strain inactivation is obtained | |
| Panjaitan et al. | Case Study of Bovine Papilloma Virus in Aceh Cattle in Lhoknga Aceh Besar |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |