CN115005162A - Construction method of mouse model for senile sepsis thrombocytopenia fitting with clinical current situation - Google Patents
Construction method of mouse model for senile sepsis thrombocytopenia fitting with clinical current situation Download PDFInfo
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- CN115005162A CN115005162A CN202210765830.8A CN202210765830A CN115005162A CN 115005162 A CN115005162 A CN 115005162A CN 202210765830 A CN202210765830 A CN 202210765830A CN 115005162 A CN115005162 A CN 115005162A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
- A01K67/027—New breeds of vertebrates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/20—Animals treated with compounds which are neither proteins nor nucleic acids
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/30—Animals modified by surgical methods
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
Abstract
A construction method of a mouse model for senile sepsis thrombocytopenia fitting the clinical situation relates to the technical field of medical detection. The method comprises the following specific steps: selecting a mouse; feeding; single tail vein injection of endotoxin; injecting normal saline with the same volume into tail vein of the control group; observing and detecting; and (6) detecting and analyzing. The invention has the beneficial effects that: a20-month-old C57BL/6J mouse is taken as a model preparation object, an endotoxin tail vein injection method is adopted to construct a model construction method of the mouse model for senile sepsis thrombocytopenia, which conforms to the clinical situation, the model for senile sepsis thrombocytopenia is provided for the research on pathogenesis and drug treatment of sepsis thrombocytopenia, the model for senile sepsis thrombocytopenia more conforms to the clinical practical sepsis thrombocytopenia is provided, the model for senile mouse is more close to the age characteristics of clinical sepsis patients in terms of body age and various functions, the endotoxin is injected by veins, the interference factors are few, and the model stability is strong.
Description
Technical Field
The invention relates to the technical field of medical detection, in particular to a method for constructing a mouse model for senile sepsis thrombocytopenia, which is attached to the current clinical situation.
Background
In order to better study the pathogenesis of sepsis and find a method for prevention and treatment, researchers have developed various methods for constructing sepsis models, such as: cecal ligation and perforation, self-excretory abdominal cavity transplantation, bacterial attack (intraperitoneal, intravenous or tracheal pulmonary injection), endotoxin attack (intraperitoneal and intravenous injection) and the like, so as to duplicate and simulate the pathogenesis process of clinical sepsis patients. The sepsis model prepared by the cecum ligation and perforation method is high in acceptance among a plurality of models, but the method has more influencing factors in the construction process, and uncontrollable factors exist in the cecum ligation position, the perforation size, the force and the like, so that the stability and the repeatability of the model are poor. And the existing animal model preparation mostly adopts the low-age animals, especially uses rodent low-age rats and mice to prepare the sepsis model, and the prepared murine sepsis model has stronger self-healing capability and can not fit clinical practice. In addition, currently prepared sepsis models are less involved in the simulation of sepsis thrombocytopenia. Therefore, there is a need to construct animal models that better fit clinical practice to reproduce the clinical features and pathogenesis of sepsis thrombocytopenia.
C57BL/6J is the most widely used inbred line mouse and the first mouse which is completely sequenced by genome, the age comparison study of the mouse and the human shows that the age of the 18-24 month old mouse is equivalent to the age of 56-69 years of human, and the old C57BL/6J mouse is applied to related studies such as immunology, cancer, long life intervention, biomarkers and the like.
In the prior art, all the molding mice are young healthy mice, the resistance to molding induction factors such as endotoxin is strong, the self-healing capability is strong, the morbidity process of sepsis cannot be well simulated, the interference factors of cecal ligation and perforation are more, and the preparation stability and the repeatability of the model are poor.
Disclosure of Invention
The invention aims to provide a method for constructing a mouse model for reducing senile sepsis platelets, which conforms to the clinical situation and aims at overcoming the defects and defects in the prior art, a 20-month-old C57BL/6J mouse is taken as a model preparation object, an endotoxin tail vein injection method is adopted to construct the mouse model for reducing senile sepsis platelets, so that the mouse model for reducing the sepsis platelets, which is more fit for clinical practice, is provided for research on pathogenesis and medicament treatment of the sepsis platelets, is closer to the age characteristics of clinical sepsis patients in terms of body age and various functions, and the endotoxin is injected by veins, so that few interference factors are caused, and the model is strong in stability.
In order to achieve the purpose, the invention adopts the following technical scheme: the construction method of the mouse model for senile sepsis thrombocytopenia according to the current clinical situation comprises the following specific steps: selecting a mouse: adopting a C57BL/6J mouse with the age of 20 months and the weight of 30-35 g; feeding: after the selection, the breeding should be carried out adaptively for 1 week; single tail vein injection of endotoxin: weighing the weight of the mouse, injecting endotoxin into the tail vein of the mouse of the model group according to the dose of 15mg/kg, and injecting equal volume of normal saline into the mouse of the control group according to the weight; the tail vein of the control group was injected with physiological saline: the amount of saline injected should be equal to the amount of endotoxin injected; and (3) observation and detection: blood is taken from inner canthus before injection, 12h, 24h, 48h and 72h after injection to detect the thrombopenia count of the mouse to evaluate the thrombocytopenia, the survival state of the mouse (whether the hair of the mouse is glossy, whether the activity is normal, the reaction speed to external stimulation, drinking water and defecation conditions, whether congestion, adhesion, ulceration and other conditions exist in eyes and the death condition of the mouse) is continuously observed for 72h, blood is taken after 72h to separate serum, the lung tissue of the mouse is separated after the mouse is treated, and the normal saline is washed clean and put into 4 percent paraformaldehyde for fixed storage; detection and analysis: detecting the peripheral blood platelet count of mice, detecting the expression of TNF-alpha, IL-1 beta and IL-18 inflammatory factors in serum by an enzyme-linked immunosorbent assay, analyzing and observing the lung injury condition by lung tissue pathological analysis, and observing the detaining condition of blood platelets in lung tissue by an immunohistochemical method.
After the technical scheme is adopted, the invention has the beneficial effects that: a20-month-old C57BL/6J mouse is used as a model preparation object, an endotoxin tail vein injection method is adopted to construct an aged sepsis thrombocytopenia mouse model, the sepsis thrombocytopenia model which is more suitable for clinical practice is provided for pathogenesis and drug therapy research of sepsis thrombocytopenia, the aged mouse model is closer to the age characteristic of clinical sepsis patients in terms of organism age and various functions, intravenous endotoxin is used, interference factors are few, and the model stability is strong.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a graph showing the platelet count at each time point in the model group mice after endotoxin injection in the present invention.
FIG. 2 is a graph showing the survival of mice in the model group and the control group in the present invention for 72 h.
FIG. 3 is a graph showing the expression of peripheral blood inflammatory factors in mice of the model group and the control group in the present invention.
Detailed Description
Referring to fig. 1 to fig. 3, the technical solution adopted by the present embodiment is: the method comprises the following specific steps: selecting a mouse: adopting a C57BL/6J mouse with the age of 20 months and the weight of 30-35 g; feeding: after the selection, the breeding should be carried out adaptively for 1 week; single tail vein injection of endotoxin: weighing the weight of the mouse, injecting endotoxin into the tail vein of the mouse of the model group according to the dose of 15mg/kg, and injecting equal volume of normal saline into the mouse of the control group according to the weight; tail vein injection of control group with physiological saline: the amount of saline injected should be equal to the amount of endotoxin injected; and (3) observation and detection: blood is taken from inner canthus before injection, 12h, 24h, 48h and 72h after injection to detect the thrombopenia count of the mouse to evaluate the thrombocytopenia, the survival state of the mouse (whether the hair of the mouse is glossy, whether the activity is normal, the reaction speed to external stimulation, drinking water and defecation conditions, whether congestion, adhesion, ulceration and other conditions exist in eyes and the death condition of the mouse) is continuously observed for 72h, blood is taken after 72h to separate serum, the lung tissue of the mouse is separated after the mouse is treated, and the normal saline is washed clean and put into 4 percent paraformaldehyde for fixed storage; detection and analysis: detecting the peripheral blood platelet count of mice, detecting the expression of TNF-alpha, IL-1 beta and IL-18 inflammatory factors in serum by an enzyme-linked immunosorbent assay, analyzing and observing the lung injury condition by lung tissue pathological analysis, and observing the detaining condition of blood platelets in lung tissue by an immunohistochemical method.
The above description is only for the purpose of illustrating the technical solutions of the present invention and not for the purpose of limiting the same, and other modifications or equivalent substitutions made by those skilled in the art to the technical solutions of the present invention should be covered within the scope of the claims of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Note: specific values and conclusions:
1. the platelet count of the model group mice is reduced when endotoxin is injected for 12h, the platelet count is lowest at 24h, and the platelet count is relieved at 48h and 72h but does not reach the platelet count value before injection, and the reference is made to figure 1;
2. after the model group mice are molded, the mice gradually have dark and upright hair, reduced activity, slow response or no response to external stimulation, reduced times of drinking water and diet, diarrhea, conjunctival congestion, increased secretion, even adhesion and fester; the control mice had normal hair, regular activity, normal response to external stimuli, normal drinking water, normal defecation and normal eyes.
3. The mice in the model group die at the 16 th hour of the model making, and the survival rate of the mice is 39.57% until 72 hours; the control group mice did not die during the observation period, see fig. 2, where LPS is endotoxin group and Sham is control group;
4. detecting the peripheral blood serum inflammatory factor expression condition of each group of mice by an enzyme-linked immunosorbent assay:
the inflammatory factor is detected by adopting a commercial enzyme-linked immunosorbent assay kit of the corresponding inflammatory factor, and the specific method comprises the following steps: taking out the kit, and balancing to room temperature; diluting the concentrated washing liquid and the standard substance; sample adding: and a blank hole and a sample hole to be detected are respectively arranged. Adding standard substance and universal diluent for the sample into the blank hole, adding the sample or standard substances with different concentrations into the other corresponding holes, diluting appropriately according to the condition, sealing the plate by using a sealing plate film, and then incubating for 90 minutes at 37 ℃; washing: carefully uncovering the sealing plate film, discarding liquid, spin-drying, filling washing liquid into each hole, standing for 30 seconds, then discarding, repeating the steps for 5 times, and patting dry; adding biotin antibody: taking out the biological plasticizing antibody working solution 20min before use for dilution, adding the biotinylation antibody diluent into blank holes, adding the biotinylation antibody working solution (100 mu l/hole) into the rest holes, sealing the plate by a sealing plate film, and then incubating for 60 minutes at 37 ℃; adding enzyme: taking out the working solution of the enzyme conjugate 20min before use for dilution, respectively adding the diluent of the enzyme conjugate into blank holes, and adding the working solution of the enzyme conjugate (100 mu l/hole) into the rest holes; sixthly, incubation: sealing the plate with sealing plate film, incubating at 37 deg.C for 30min, and keeping away from light; seventhly, washing the plate for 5 times; adding a color developing substrate (100 mu l/hole), incubating for 15 minutes at 37 ℃, and keeping out of the sun; ninthly, adding a terminating solution (100 mu l/hole), and uniformly mixing to measure the OD450 value.
As a result: the expression levels of TNF-alpha, IL-1 beta and IL-18 inflammatory factors of mice in the model group are all higher than those of the control group, and the difference has statistical significance, see figure 3, in which LPS is an endotoxin group and Sham is the control group;
5. taking lung tissues of all groups of mice to carry out fixation and histopathological analysis, and the specific method comprises the following steps:
the lung and spleen were removed, washed clean and placed in a bottle containing 5ml of 4% paraformaldehyde reagent, and the specimen was dehydrated, embedded in paraffin, tabletted (4 μm thick), HE-stained, observed with an optical microscope and analyzed by photography.
As a result: the lung tissue is an important organ of the body and is also the organ in which sepsis most often accumulates first. Lung pathological analysis shows that inflammatory cells infiltrate into lung tissues of the model group, and inflammatory cells can be seen in blood vessels in the lung; whereas no inflammatory cell infiltration was seen in the control group.
6. The immunohistochemistry method for observing the entrapment condition of the platelets in the lung tissue comprises the following specific steps:
(1) paraffin embedded section: the fixed lung tissue was removed and dehydrated in an automatic dehydration apparatus. The dehydration sequence is as follows. 70% ethanol I, 70% ethanol II, 80% ethanol, 90% ethanol, 95% ethanol I, 95% ethanol II, anhydrous ethanol I, anhydrous ethanol II, xylene I, xylene II, paraffin I, and paraffin II. After dehydration, paraffin embedding was performed and the paraffin tissue blocks were stored in a-20 ℃ refrigerator. Carefully cutting a paraffin tissue block by using a blade until the paraffin tissue block exposes lung tissues, then cutting the lung tissues with the thickness of 4 mu m, paving the cut tissues in warm water at 40-45 ℃ by using forceps to fully extend the tissues, then using an anti-falling glass slide to take the tissues out, adjusting the temperature to 65 ℃ on a baking machine, baking for 0.5h, and dewaxing the baked slices, wherein the dewaxing sequence is as follows: xylene I for 10 minutes, xylene II for 10 minutes, absolute ethanol I for 5 minutes, absolute ethanol II for 5 minutes, 95% ethanol II for 10 minutes, and 80% ethanol for 10 minutes. Then washed 3 times with PBST buffer for 5 minutes each;
(2) antigen retrieval: the antigen retrieval solution was heated for 5 minutes. The tissue sections were then repaired in a 95 ℃ water bath for 20 minutes. Taking out the tissue section and standing to room temperature. Washed 3 times with PBST buffer for 5 minutes each;
(3) blocking endogenous peroxidase: placing the slices into 3% hydrogen peroxide solution (hydrogen peroxide: pure water: 1:9), incubating at room temperature in dark for 10min, placing the slides in PBS (PH7.4), and washing on a decolorizing shaker for 3 times, each time for 5 min;
(4) BSA or serum blocking: after the section is slightly dried, a circle is drawn around the tissue by a grouping pen (the antibody is prevented from flowing away), the tissue is uniformly covered by 3 percent BSA in the circle in a dropwise manner, and the section is sealed for 30min at room temperature;
(5) plus primary antibody (anti-mouse CD 41): gently removing the confining liquid, dripping PBS (phosphate buffer solution) on the slices to prepare primary antibodies according to a certain proportion, and flatly placing the slices in a wet box for incubation at 4 ℃ overnight. (small amounts of water added in wet boxes to prevent evaporation of antibody);
(6) adding a secondary antibody: slides were washed 3 times in PBS (pH7.4) with shaking on a destaining shaker for 5min each time. After the section is slightly dried, dripping secondary antibody (HRP mark) of the corresponding species of the primary antibody into the ring to cover the tissue, and incubating for 50min at room temperature;
(7) DAB color development: DAB color was added dropwise to the tissue. Specific staining was viewed under a mirror. Use of
Stopping the color development by flowing water;
(8) counterstaining cell nuclei: performing Harris hematoxylin counterstaining for about 3min, and washing for 15min with running water;
(9) dewatering and sealing: placing the slices in 80% alcohol for 6 min-95% alcohol for 6 min-anhydrous alcohol I for 6 min-anhydrous alcohol II for 6 min-xylene I for 5min, dehydrating, removing the slices from xylene, air drying, and sealing with neutral gum;
(10) microscopic examination and image acquisition and analysis;
as a result: CD41 is an expression protein on the surface of platelets, and platelet expression can be assessed by detecting expression of CD41 protein. The result of immunohistochemical analysis shows that the positive expression of CD41 in lung tissue of the mouse in the model group is obviously increased compared with that in the control group. The mice in the model group are prompted to have the platelet sequestration phenomenon in the lungs.
Claims (6)
1. The construction method of the mouse model for senile sepsis thrombocytopenia fitting the clinical status is characterized in that: the method comprises the following specific steps:
1) selecting a mouse;
2) feeding;
3) single tail vein injection of endotoxin;
4) normal saline was injected into the tail vein of the control group;
5) observing and detecting;
6) and (6) detecting and analyzing.
2. The method for constructing a mouse model for senile sepsis thrombocytopenia according to claim 1, wherein: the selected mice should be 20-month-old C57BL/6J mice with the weight of 30-35 g.
3. The method for constructing a mouse model for senile sepsis thrombocytopenia according to claim 1, wherein: the plants should be acclimatized for 1 week after selection.
4. The method for constructing a mouse model for senile sepsis thrombocytopenia according to claim 1, wherein: the mice were weighed, and administered with endotoxin at a dose of 15mg/kg to the tail vein of the mice in the model group, and the mice in the control group were injected with an equal volume of physiological saline according to the body weight.
5. The method for constructing a mouse model for senile sepsis thrombocytopenia according to claim 1, wherein: the observation and detection in the step 5 are specifically as follows: blood is taken from inner canthus before injection, 12h, 24h, 48h and 72h after injection to detect the thrombopenia of the mice for evaluating the thrombopenia, the survival state of the mice (whether the hair of the mice is glossy, whether the activity is normal, the reaction speed to external stimulation, drinking water and defecation conditions, the situations of congestion, adhesion, ulceration and the like of eyes and the death situation of the mice) is continuously observed for 72h, blood is taken after 72h to separate serum, the lung tissues of the mice are separated after the mice are treated, and the normal saline is washed clean and put into 4 percent paraformaldehyde for fixed storage.
6. The method for constructing a mouse model for senile sepsis thrombocytopenia according to claim 1, wherein: the detection and analysis in the step 6 specifically comprises the following steps: detecting the peripheral blood platelet count of mice, detecting the expression of TNF-alpha, IL-1 beta and IL-18 inflammatory factors in serum by an enzyme-linked immunosorbent assay, analyzing and observing the lung injury condition by lung tissue pathological analysis, and observing the detaining condition of blood platelets in lung tissue by an immunohistochemical method.
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| WO2021078246A1 (en) * | 2019-10-24 | 2021-04-29 | 深圳市脉唐生物科技有限公司 | Pharmaceutical composition for preventing or treating sepsis, kit, use thereof and treatment method thereof |
| CN112402442A (en) * | 2020-11-27 | 2021-02-26 | 云南省第二人民医院 | Method for constructing sepsis rat model with multiple organ dysfunction syndrome |
| CN114304063A (en) * | 2021-12-13 | 2022-04-12 | 中国人民解放军联勤保障部队第九二〇医院 | Method for constructing mouse sepsis model under simulated real battlefield environment |
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