Construction method and application of mouse amniotic embolism model

Abstract

The application discloses a kind of construction method and application of mouse amniotic embolism model, which comprises the following steps: S1, anesthetizing processing to late pregnant mouse, obtain the amniotic fluid filtrate and placental filtrate of the late pregnant mouse;S2, the placental filtrate is mixed with the amniotic fluid filtrate at 1:0.5~4 volume ratio, to obtain amniotic fluid-placenta mixed solution;S3, the amniotic fluid-placenta mixed solution is injected into healthy female mouse using tail vein injection method, i.e.get.The construction method of the application selects normal female mouse as modeling object for the first time, which helps to reduce modeling cost, and using the construction method of the application can effectively simulate the occurrence of amniotic embolism, reduce acute mortality, improve modeling efficiency, and provide disease model basis for subsequent research on the pathophysiological mechanism of amniotic embolism.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for constructing and applying a mouse amniotic fluid embolism model. Background Technology

[0002] Amniotic fluid embolism (AFE) is a serious labor complication caused by the sudden entry of amniotic fluid into the maternal bloodstream during delivery, resulting in acute pulmonary embolism, anaphylactic shock, disseminated intravascular coagulation (DIC), renal failure, or sudden death. Currently, AFE is believed to be caused by the abnormal release of trophoblasts or other substances into the maternal circulation. Although there are no definitive risk factors for AFE, potential factors are generally considered to include: ① Maternal factors, such as advanced maternal age, race, pre-pregnancy hypertension, asthma, illicit drug use, and a history of multiple pregnancies; ② Pregnancy factors, such as placenta accreta (PAS), placental abruption, uterine rupture, polyhydramnios, chorioamnionitis, preeclampsia, fetal growth restriction, and stillbirth; ③ Delivery factors, such as gestational age <34 weeks, cervical ripening, cesarean section, assisted vaginal delivery, and manual removal of the placenta. Among these factors, PAS has the strongest correlation with AFE and is positively correlated with its severity.

[0003] Currently, the mechanism of amniotic fluid embolism is unclear. While the mechanical embolism of pulmonary vessels by formed elements of amniotic fluid can lead to respiratory and circulatory failure and ultimately multiple organ dysfunction syndrome (MODS), research has found that the cause of pulmonary circulatory problems is not solely due to mechanical embolism caused by formed elements in the amniotic fluid. The release of vasoactive substances after amniotic fluid enters the bloodstream is a crucial factor. The interaction between the coagulation storm and immune storm caused by amniotic fluid entering the bloodstream creates a vicious cycle, ultimately leading to MODS. Due to the unclear mechanism of amniotic fluid embolism, there is a lack of definitive diagnostic methods in clinical practice, and diagnosis is mainly based on exclusion. Amniotic fluid embolism primarily manifests as sudden cardiovascular failure, severe respiratory distress, hypoxia, or seizures. Treatment mainly involves respiratory and circulatory support, anticoagulation, and other symptomatic supportive therapies. However, due to its rapid progression, there is currently no effective causal treatment for amniotic fluid embolism, resulting in an extremely low cure rate and making it a very serious obstetric complication. Therefore, establishing effective animal models of amniotic fluid embolism and elucidating the mechanisms of its occurrence are of great significance for developing diagnostic, preventive, and treatment strategies for amniotic fluid embolism. In related techniques, to obtain effective animal models of amniotic fluid embolism, a mixture of amniotic fluid and / or meconium is typically injected. This requires a high injection volume to induce typical symptoms; however, high doses can easily lead to acute death during injection (i.e., death within 5-10 minutes), with a mortality rate exceeding 60%. Conversely, using relatively low injection volumes fails to induce typical symptoms, resulting in low model establishment efficiency.

[0004] Therefore, there is an urgent need to find an animal model of amniotic fluid embolism that can reduce acute mortality and has high modeling efficiency. Summary of the Invention

[0005] This invention aims to address at least one of the technical problems existing in the prior art. To this end, this invention proposes a method for constructing a mouse amniotic fluid embolism disease model and its application. This method selects normal female mice as the modeling subjects, reducing the use of pregnant mice and lowering modeling costs. Simultaneously, this construction method can effectively simulate the occurrence of amniotic fluid embolism, reduce acute mortality, and improve modeling efficiency, providing a disease model basis for subsequent research on the pathophysiological mechanisms of amniotic fluid embolism.

[0006] In a first aspect, the present invention provides a method for constructing a mouse model of amniotic fluid embolism, comprising the following steps: S1. Anesthetize mice in late pregnancy and obtain amniotic fluid filtrate and placental filtrate from the mice in late pregnancy. S2. The placental filtrate and the amniotic fluid filtrate are mixed at a volume ratio of 1:0.5~4 to obtain an amniotic fluid-placental mixture; S3. The amniotic fluid-placental mixture is injected into a healthy female mouse via tail vein injection to obtain the final product.

[0007] The construction method according to embodiments of the present invention has at least the following beneficial effects: The present invention uses normal female mice as the model subjects to construct the amniotic fluid embolism disease model, which reduces the use of pregnant mice and lowers the modeling cost. At the same time, the construction method can effectively simulate the occurrence of amniotic fluid embolism, reduce acute mortality, and improve modeling efficiency, providing a disease model basis for subsequent research on the pathophysiological mechanism of amniotic fluid embolism.

[0008] In some embodiments of the present invention, the anesthesia treatment includes intraperitoneal injection of 0.8-1.2% sodium pentobarbital into the late-pregnancy mice.

[0009] In some embodiments of the present invention, the method for obtaining the amniotic fluid filtrate includes: collecting an amniotic fluid mixture from the isolated uterus of the late-pregnancy mouse, and then filtering it through a filter membrane with a pore size of 30-50 μm.

[0010] In some embodiments of the present invention, the method for collecting the amniotic fluid mixture includes: puncturing the uterine wall and fetal membranes of the isolated uterus with a syringe needle, collecting the amniotic fluid that flows out, and washing it with a ratio of 80-150 μL of physiological saline per fetal mouse to obtain a washing solution, and combining the amniotic fluid and the washing solution to obtain the amniotic fluid mixture.

[0011] It is understood that the fetal mouse in question is a fetal mouse in the womb during late pregnancy.

[0012] In some embodiments of the present invention, the pore size of the filter membrane can be 30 μm, 35 μm, 40 μm, 45 μm or 50 μm. Preferably, it is 40 μm.

[0013] In some embodiments of the present invention, the method for obtaining the placental filtrate includes: separating the placenta of the late-pregnancy mouse, grinding it and mixing it with physiological saline, and then filtering it through a filter membrane with a pore size of 30-50 μm.

[0014] In some embodiments of the present invention, the amount of physiological saline added is 80~150 μL.

[0015] In some embodiments of the present invention, the late-pregnancy mice have a gestational age of 16 to 20 days.

[0016] In some embodiments of the present invention, the late-pregnancy mice are C57 mice.

[0017] In some embodiments of the present invention, in step S2, the volume ratio of the amniotic fluid filtrate to the placental filtrate is 1:0.5~4.

[0018] In some preferred embodiments of the present invention, the volume ratio of the amniotic fluid filtrate to the placental filtrate is 1:0.5~1.5; more preferably, it is 1:0.8~1.2.

[0019] In some embodiments of the present invention, in step S2, the healthy female mouse is a female mouse that is 8-10 weeks old and weighs 20-30g.

[0020] In some embodiments of the present invention, in step S2, the female mouse is a C57 mouse.

[0021] In some embodiments of the present invention, in step S3, the injection volume of the amniotic fluid-placental mixture is 80~120 μL / animal.

[0022] In a second aspect, the invention provides the application of the method for constructing a mouse amniotic fluid embolism disease model as described in the first aspect of the invention in obtaining an animal model of amniotic fluid embolism disease.

[0023] A third aspect of the present invention provides the application of the method for constructing a mouse amniotic fluid embolism disease model as described in the first aspect of the present invention in screening drugs for alleviating and / or treating amniotic fluid embolism.

[0024] Other features and advantages of the present invention will be set forth in the following description. Attached Figure Description

[0025] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 The figures show the statistical results of coagulation and liver function indicators in model mice injected with different proportions of amniotic fluid-placental mixture according to the present invention. Among them, a is the statistical result of PAI-1 concentration in plasma, b is the statistical result of platelet concentration, c is the statistical result of alanine aminotransferase concentration, and d is the statistical result of aspartate aminotransferase concentration.

[0026] Figure 2 This is an observation of the organs of a mouse model after intravenous injection of amniotic fluid-placental mixture according to the present invention.

[0027] Figure 3 HE staining results of organs in model mice after injection of amniotic fluid-placental mixture or physiological saline according to the present invention.

[0028] Figure 4 The figures show the statistical results of biochemical indicators in model mice after injection of amniotic fluid-placental mixture or saline according to the present invention. In the figures, a represents the statistical results of creatine kinase content, b represents the statistical results of lactate dehydrogenase content, and c represents the statistical results of potassium ion content. * p<0.05, **** p <0.0001. Detailed Implementation

[0029] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0030] The terms "preferred," "more preferably," etc., used in this invention refer to embodiments of the invention that provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of this invention.

[0031] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0032] In the description of this invention, the reference term "and / or" includes all and any combination of one or more of the associated listed items.

[0033] In the description of this invention, the terms "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0034] In this embodiment of the invention, both C57BL / 6 pregnant mice and ordinary female mice were purchased from Hunan Slack Jingda Animal Co., Ltd. The physiological saline used was a 0.9% sodium chloride solution.

[0035] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0036] Example 1: A method for constructing a mouse model of amniotic fluid embolism. This embodiment provides a method for constructing a mouse model of amniotic fluid embolism through allogeneic injection, including the following steps: (1) Obtaining amniotic fluid filtrate: C57 mice in late pregnancy (gestational age 19-21 days) were obtained. Due to poor activity levels, pregnant mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg) at the onset of labor. A midline incision was made in the lower abdomen, and a subtotal hysterectomy was performed. The abdomen was closed, fluid was administered, and the mice were resuscitated using a constant-temperature blanket. The isolated uterus was placed in a sterile dish, and the uterine wall and fetal membranes were punctured at an avascular site using a syringe needle. The flowing amniotic fluid was collected and washed with 100 μL of physiological saline per fetal mouse. The amniotic fluid and washing solution were combined and filtered through a 40 μm filter to obtain the amniotic fluid filtrate, which was then used for reprocessing.

[0037] (2) Obtaining placental filtrate: The excised uterus was rinsed with physiological saline and the placenta was separated. The placenta was then thoroughly ground with a 1 mL grinder. The placental grinding fluid was mixed with physiological saline at a ratio of 1:100 μL and then filtered through a 40 μm filter to obtain placental filtrate. The filtrate was then placed on ice for later use.

[0038] (3) Preparation of amniotic fluid-placental mixture: The collected and filtered placental filtrate and amniotic fluid filtrate were thoroughly vortexed at a volume ratio of 2:1 to obtain an amniotic fluid-placental mixture, which was then placed on ice for later use.

[0039] (4) Tail vein injection: Eight-week-old, healthy C57 female mice weighing 20-30 g were used. The amniotic fluid-placental mixture prepared above was injected into each mouse via the tail vein at a dose of 100 μL. After injection, gauze was applied to stop bleeding, and the condition of the mice was observed and recorded.

[0040] Example 2: A method for constructing a mouse model of amniotic fluid embolism. This embodiment provides a method for constructing a mouse model of amniotic fluid embolism through allogeneic injection, including the following steps: (1) Obtaining amniotic fluid filtrate: C57 mice in late pregnancy (gestational age 19-21 days) were obtained. Due to poor activity levels, pregnant mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg) at the onset of labor. A midline incision was made in the lower abdomen, and a subtotal hysterectomy was performed. The abdomen was closed, fluid was administered, and the mice were resuscitated using a constant-temperature blanket. The isolated uterus was placed in a sterile dish, and the uterine wall and fetal membranes were punctured at an avascular site using a syringe needle. The flowing amniotic fluid was collected and washed with 100 μL of physiological saline per fetal mouse. The amniotic fluid and washing solution were combined and filtered through a 40 μm filter to obtain the amniotic fluid filtrate, which was then used for reprocessing.

[0041] (2) Obtaining placental filtrate: The excised uterus was rinsed with physiological saline and the placenta was separated. The placenta was then thoroughly ground with a 1 mL grinder. The placental grinding fluid was mixed with physiological saline at a ratio of 1:100 μL and then filtered through a 40 μm filter to obtain placental filtrate. The filtrate was then placed on ice for later use.

[0042] (3) Preparation of amniotic fluid-placental mixture: The collected and filtered placental filtrate and amniotic fluid filtrate were thoroughly vortexed at a volume ratio of 1:1 to obtain an amniotic fluid-placental mixture, which was then placed on ice for later use.

[0043] (4) Tail vein injection: Eight-week-old, healthy C57 female mice weighing 20-30 g were used. The amniotic fluid-placental mixture prepared above was injected into each mouse via the tail vein at a dose of 100 μL. After injection, gauze was applied to stop bleeding, and the condition of the mice was observed and recorded.

[0044] Example 3: A method for constructing a mouse model of amniotic fluid embolism. This embodiment provides a method for constructing a mouse model of amniotic fluid embolism through allogeneic injection, including the following steps: (1) Obtaining amniotic fluid filtrate: C57 mice in late pregnancy (gestational age 19-21 days) were obtained. Due to poor activity levels, pregnant mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg) at the onset of labor. A midline incision was made in the lower abdomen, and a subtotal hysterectomy was performed. The abdomen was closed, fluid was administered, and the mice were resuscitated using a constant-temperature blanket. The isolated uterus was placed in a sterile dish, and the uterine wall and fetal membranes were punctured at an avascular site using a syringe needle. The flowing amniotic fluid was collected and washed with 100 μL of physiological saline per fetal mouse. The amniotic fluid and washing solution were combined and filtered through a 40 μm filter to obtain the amniotic fluid filtrate, which was then used for reprocessing.

[0045] (2) Obtaining placental filtrate: The excised uterus was rinsed with physiological saline and the placenta was separated. The placenta was then thoroughly ground with a 1 mL grinder. The placental grinding fluid was mixed with physiological saline at a ratio of 1:100 μL and then filtered through a 40 μm filter to obtain placental filtrate. The filtrate was then placed on ice for later use.

[0046] (3) Preparation of amniotic fluid-placental mixture: The collected and filtered placental filtrate and amniotic fluid filtrate were thoroughly vortexed at a volume ratio of 1:2 to obtain an amniotic fluid-placental mixture, which was then placed on ice for later use.

[0047] (4) Tail vein injection: Eight-week-old, healthy C57 female mice weighing 20-30 g were used. The amniotic fluid-placental mixture prepared above was injected into each mouse via the tail vein at a dose of 100 μL. After injection, gauze was applied to stop bleeding, and the condition of the mice was observed and recorded.

[0048] Example 4: A method for constructing a mouse model of amniotic fluid embolism. This embodiment provides a method for constructing a mouse model of amniotic fluid embolism through allogeneic injection, including the following steps: (1) Obtaining amniotic fluid filtrate: C57 mice in late pregnancy (gestational age 19-21 days) were obtained. Due to poor activity levels, pregnant mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg) at the onset of labor. A midline incision was made in the lower abdomen, and a subtotal hysterectomy was performed. The abdomen was closed, fluid was administered, and the mice were resuscitated using a constant-temperature blanket. The isolated uterus was placed in a sterile dish, and the uterine wall and fetal membranes were punctured at an avascular site using a syringe needle. The flowing amniotic fluid was collected and washed with 100 μL of physiological saline per fetal mouse. The amniotic fluid and washing solution were combined and filtered through a 40 μm filter to obtain the amniotic fluid filtrate, which was then used for reprocessing.

[0049] (2) Obtaining placental filtrate: The excised uterus was rinsed with physiological saline and the placenta was separated. The placenta was then thoroughly ground with a 1 mL grinder. The placental grinding fluid was mixed with physiological saline at a ratio of 1:100 μL and then filtered through a 40 μm filter to obtain placental filtrate. The filtrate was then placed on ice for later use.

[0050] (3) Preparation of amniotic fluid-placental mixture: The collected and filtered placental filtrate and amniotic fluid filtrate were thoroughly vortexed at a volume ratio of 1:4 to obtain an amniotic fluid-placental mixture, which was then placed on ice for later use.

[0051] (4) Tail vein injection: Eight-week-old, healthy C57 female mice weighing 20-30 g were used. The amniotic fluid-placental mixture prepared above was injected into each mouse via tail vein at a dose of 80-120 μL. After injection, gauze was applied to stop bleeding, and the condition of the mice was observed and recorded.

[0052] Comparative Example 1 This comparative example provides a method for constructing a mouse model of amniotic fluid embolism by allogeneic injection. The only difference between this method and Example 2 is that in step (4), physiological saline is injected via tail vein injection. The remaining steps are the same.

[0053] Comparative Example 2 This comparative example provides a method for constructing a mouse model of amniotic fluid embolism by allogeneic injection. The only difference between this method and Example 2 is that in step (4), amniotic fluid is injected via tail vein injection. The remaining steps are the same.

[0054] Comparative Example 3 This comparative example provides a method for constructing a mouse model of amniotic fluid embolism by allogeneic injection. The only difference between this method and Example 2 is that in step (4), placental filtrate is injected via tail vein injection. The remaining steps are the same.

[0055] Test Example 1: Coagulation parameters and liver function tests This test example examined coagulation and liver function indicators in the amniotic fluid embolism mouse models constructed in Examples 1-4 and Comparative Examples 1-3. The specific methods are as follows: 1. Cardiac blood sampling: Eight hours after the injection of amniotic fluid-placental mixture, the model mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg). Once the mice were fully anesthetized and there were no corneal or pain reflexes, they were fixed in place. Alcohol was sprayed onto the mouse's abdomen to disinfect and moisten the skin. Then, the skin, muscles, and peritoneum were cut along the rib margins with scissors and forceps. The diaphragm was cut along the costal margin and the xiphoid process was fixed upwards with a needle to fully expose the heart. The pericardium was then carefully bluntly dissected with forceps, and the heart was gently clamped and fixed with curved forceps. A 1 mL syringe moistened with anticoagulant was carefully inserted into the right ventricle from the apex of the heart along the long axis of the heart. After the syringe was inserted into the heart and the needle tip was inserted into the ventricle, blood was slowly collected.

[0056] After blood collection, gently withdraw the needle tip and remove the needle. Transfer 100-200 µL of blood from the syringe into a 1.5 mL EP tube containing EDTA powder. Gently invert to mix and ensure the anticoagulant is evenly distributed for effective anticoagulation. This sample will be used for platelet counting. Inject the remaining blood from the syringe into another clean 1.5 mL EP tube, gently invert to mix and ensure the anticoagulant is evenly distributed for effective anticoagulation, and then centrifuge at 3000×g, 4℃, for 15 min. After centrifugation, transfer the supernatant plasma to another clean EP tube moistened with anticoagulant for the detection of relevant organ parameters.

[0057] 2. Whole blood platelet count test: Take 100 µL of the fresh whole blood collected above and anticoagulated with EDTA, then analyze it using a fully automated veterinary blood cell analyzer, and count the platelet (PLT) concentration of each model mouse. Each group should have at least three replicates.

[0058] 3. Detection of plasma PAI-1 concentration: Plasma PAI-1 concentration was detected using an ELISA kit (purchased from R&D Systems, catalog number DY3828-05). The specific operating steps are as follows: (1) Reagent preparation: Prepare 1×Reagent Dilute, 1×CaptureAntibody, 1×Wash Buffer, 1×Detection Antibody, 1×HRP and standards of different concentration gradients according to the reagent instructions; (2) Preparation of ELISA plates: Calculate the required antibody based on the sample volume, add 50 µL of 1× CaptureAntibody to each well, cover with the adhesion strip, and incubate overnight at room temperature. Then discard the liquid, wash 3 times with Wash Buffer, blot dry, and add 150 µL of 1× Reagent Dilute to each well for blocking at room temperature for 1 hour. Discard the liquid, wash 3 times with Wash Buffer, and blot dry. (3) ELISA detection: First, add 50 µL of diluted sample and standard to each well of the prepared microplate, cover with the adhesion strip, and incubate at room temperature for 2 hours. Discard the liquid, wash 3 times with Wash Buffer, and blot dry. Then, add 50 µL of 1×Detection Antibody to each well, cover with the adhesion strip, and incubate at room temperature for 2 hours. Discard the liquid, wash 3 times with Wash Buffer, and blot dry. Next, add 50 µL of 1×HRP to each well, cover with the adhesion strip, and incubate at room temperature for 20 minutes. Discard the liquid, wash 3 times with Wash Buffer, and blot dry.

[0059] Add 50 µL of Substrate Solution (Color Reagent A and Color Reagent B mixed in a 1:1 ratio) to each well and incubate in the dark at room temperature for 5–20 min. When the color of the reaction from the previous step is clearly visible, add 25 µL of Stop Solution to each well to terminate the reaction. Gently shake the plate to ensure thorough mixing of the liquid. Measure the absorbance at 450 nm, derive the regression equation from the standard curve, calculate the sample concentration, and multiply it by the dilution factor to obtain the actual concentration.

[0060] The concentration of PAI-1 in the plasma of each group of model mice was statistically analyzed using the above method, with three replicates for each group.

[0061] 4. Organ indicator testing: Take 200 µL of the fresh serum collected after centrifugation and determine the concentrations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) using a fully automated veterinary biochemical analyzer.

[0062] Test results as follows Figure 1 As shown in the figure, where a represents the statistical results of PAI-1 concentration in plasma, b represents the statistical results of platelet concentration, c represents the statistical results of alanine aminotransferase (ALT) concentration, and d represents the statistical results of aspartate aminotransferase (AST) concentration, the results show that compared with the saline group, female mice injected with amniotic fluid and placental mixture exhibited significant changes in PAI-1, platelet count, and liver function. The changes in PAI-1 and platelet count were most pronounced when pure placental filtrate was injected. Furthermore, coagulation parameters changed with decreasing placental ratio. When the amniotic fluid-placental mixture ratio was reduced to 1:1, effective changes in PAI-1 and PLT were achieved, and the placental concentration in the mixture was reduced. Therefore, a 1:1 ratio of amniotic fluid filtrate to placental filtrate resulted in a mouse amniotic fluid embolism model that better simulated real amniotic fluid embolism.

[0063] In addition, it is worth noting that when the ratio of amniotic fluid filtrate to placental filtrate was 1:1, the mortality rate of mice after amniotic fluid embolism modeling was about 45%. After amniotic fluid injection, mice quickly developed symptoms such as respiratory distress and heart failure, and most deaths occurred within 30 minutes. Among the female mice that did not die, a considerable number of them showed convulsions, restlessness, jumping, and weakened movement.

[0064] Example 2: Organ pathology examination This test example describes the organ pathology examination of the mouse model of amniotic fluid embolism constructed in Example 2 above. The specific methods are as follows: 1. Organ bleeding: Five to ten minutes after the injection of a 1:1 amniotic fluid-placenta mixture, the female mouse was dissected to expose the thoracic and abdominal organs, and signs of organ bleeding were recorded with a camera.

[0065] 2. HE staining of tissue: (1) Paraformaldehyde fixation: Eight hours after female mice were injected with an amniotic fluid-placental mixture (1:1 ratio), the right atrial appendage was severed, and the blood circulation of all organs was perfused through the apex of the heart using a syringe or fine needle. First, 5 mL of pre-cooled PBS solution was infused, followed by 3 mL of pre-cooled 4% paraformaldehyde. After perfusion, organs such as the liver, lungs, and kidneys were dissected and placed in centrifuge tubes containing 4% paraformaldehyde for further fixation and later use.

[0066] (2) Preparation of slices: ① Dehydration: The tissue samples fixed with paraformaldehyde were dehydrated in a gradient using 75%, 85%, 90%, 95% ethanol, anhydrous ethanol, and anhydrous ethanol for 4 h, 2 h, 1 h, 1 h, 30 min, and 30 min respectively. Then, they were dehydrated with a 1:1 mixture of ethanol and isobutanol for 10 min, and then dehydrated twice with xylene for 10 min each time.

[0067] ②Embedding: The dehydrated tissue is impregnated with paraffin, and then embedded using an embedding machine.

[0068] ③ Sectioning: Cut the wax block into 3 μm pieces and place the sections onto a glass slide using a 40-degree slide spreader.

[0069] ④ Drying the slides: Place the glass slides in a 60 ℃ oven and dry them.

[0070] (3) HE staining: ①Dewaxing: Place the dried slices in the dewaxing solution twice, 20 minutes each time.

[0071] ②Hydration: The dewaxed sections were hydrated with anhydrous ethanol, anhydrous ethanol and 75% ethanol for 5 min in sequence, repeated twice, and finally rinsed with ddH2O.

[0072] ③ Hematoxylin staining: Immerse the slides in hematoxylin staining solution for 3-5 minutes, wash with tap water, and then use a blueing solution to restore blue color.

[0073] ④ Eosin staining: Immerse the slides in eosin solution for 5 min, then wash with tap water.

[0074] ⑤ Dehydration and mounting: Dry the sections and then incubate them with anhydrous ethanol for 5 min × 3 times, followed by xylene for 5 min × 2 times. Then mount the sections and examine them under a microscope.

[0075] Organ status after intravenous injection of amniotic fluid-placental mixture Figure 2As shown. After female mice were injected with a 1:1 mixture of amniotic fluid and placenta via the tail vein, they coughed up bright red blood from their mouth and nose within 5-10 minutes and died. Timely and thorough dissection exposed their thoracic and abdominal cavities, revealing obvious hemorrhages on the lung surface and congestion in the liver.

[0076] The results of HE staining of the tissue are as follows Figure 3 As shown, after the model was established, the tissue was stained with HE. Under the microscope, the lung tissue showed varying degrees of congestion and edema, thrombus formation in the pulmonary small vessels, partial neutrophil infiltration, congestion and dilation of the alveolar walls, and partial thrombus formation in the liver and kidney vessels.

[0077] The above results show that mice obtained using the amniotic fluid thrombosis model construction method of the present invention exhibited symptoms similar to those of amniotic fluid thrombosis, which is consistent with clinical findings.

[0078] Example 3: Biochemical indicator detection This test example describes the detection of biochemical indicators in the mouse models of amniotic fluid embolism constructed in Example 2 and Comparative Example 1, including the detection of creatine kinase (CK), lactate dehydrogenase (LDH), and potassium ion concentrations in the blood. The specific methods are as follows: Referring to the above test example 1, a cardiac puncture was performed to collect blood. Then, 200 µL of the collected fresh serum was used to detect creatine kinase (CK), lactate dehydrogenase (LDH), and potassium ion levels using a fully automated veterinary biochemical analyzer.

[0079] The test results are shown in Figure 4, where a represents the statistical results of creatine kinase content, b represents the statistical results of lactate dehydrogenase content, and c represents the statistical results of potassium ion content. * p <0.05, **** p <0.0001 indicates that after female mice were injected with amniotic fluid-placental mixture via the tail vein, their biochemical indicators changed significantly, which is consistent with clinical findings.

[0080] In summary, this invention provides a method for constructing and applying a mouse amniotic fluid embolism model. The invention first extracts amniotic fluid and placental filtrate from late-pregnancy C57BL / 6J female mice. Then, the amniotic fluid and placental filtrate are mixed in different proportions and injected into the bloodstream of normal 8-10 week pregnant C57BL / 6J female mice via the tail vein to induce amniotic fluid embolism. After model establishment, samples are collected to detect biochemical indicators and assess organ function. Experimental results show that the mortality rate of mice after amniotic fluid embolism modeling is approximately 45%. Mice rapidly develop symptoms such as respiratory distress and heart failure after amniotic fluid injection, with most deaths occurring within 30 minutes. Among the female mice that do not die, similar phenomena to amniotic fluid embolism are observed, such as convulsions, restlessness, jumping, and weakened movement. In the biochemical index detection, after modeling, mice showed elevated PAL-1 and decreased PLT, changes in coagulation indicators, elevated liver function indicators AST and ALT, and impaired heart and kidney function, exhibiting symptoms similar to clinical amniotic fluid embolism and eventually dying. This model effectively simulates the occurrence of amniotic fluid embolism in mice and has good application prospects in the screening of therapeutic drugs or the study of related mechanisms.

[0081] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A method for constructing a mouse model of amniotic fluid embolism, characterized in that, Includes the following steps: S1. Anesthetize mice in late pregnancy and obtain amniotic fluid filtrate and placental filtrate from the mice in late pregnancy. S2. The placental filtrate and the amniotic fluid filtrate are mixed at a volume ratio of 1:0.5~4 to obtain an amniotic fluid-placental mixture; S3. The amniotic fluid-placental mixture is injected into a healthy female mouse via tail vein injection to obtain the final product. The method for obtaining the amniotic fluid filtrate includes: collecting an amniotic fluid mixture from the isolated uterus of the late-pregnancy mouse, and then filtering it through a filter membrane with a pore size of 30-50 μm. The method for obtaining the placental filtrate includes: separating the placenta from the late-pregnancy mice, grinding it, mixing it with physiological saline, and then filtering it through a filter membrane with a pore size of 30-50 μm.

2. The construction method according to claim 1, characterized in that, The anesthesia treatment included intraperitoneal injection of 0.8-1.2% sodium pentobarbital into the late-pregnancy mice.

3. The construction method according to claim 1, characterized in that, The amount of physiological saline added is 80~150 μL.

4. The construction method according to claim 1, characterized in that, The gestational age of the mice in late pregnancy was 16-20 days.

5. The construction method according to claim 4, characterized in that, The mice in late pregnancy were of the C57 breed.

6. The construction method according to claim 1, characterized in that, The healthy female mice are 8-10 weeks old and weigh 20-30 g.

7. The construction method according to claim 6, characterized in that, The female mouse was a C57 mouse.

8. The construction method according to any one of claims 1 to 7, characterized in that, In step S3, the injection volume of the amniotic fluid-placental mixture is 80~120μL / animal.

9. The application of the method for constructing a mouse amniotic fluid embolism disease model as described in any one of claims 1 to 8 in obtaining an animal model of amniotic fluid embolism disease.

10. The application of the method for constructing a mouse amniotic fluid embolism disease model as described in any one of claims 1 to 8 in screening drugs for alleviating and / or treating amniotic fluid embolism.

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