A mouse model, its construction method and application

Abstract

This invention provides a mouse model of hypothyroidism combined with pulmonary hypertension, its construction method, and its application, belonging to the field of animal experimental model construction technology. In this invention, a hypothyroid mouse model was obtained by intragastric administration of methimazole and feeding mice under normoxic conditions. The hypothyroid mouse model was then further modified by intragastric administration of methimazole, followed by continuous subcutaneous injection of the vascular endothelial growth factor inhibitor Su5416, and feeding the mice under hypoxic conditions to obtain a mouse model of hypothyroidism combined with pulmonary hypertension. This invention successfully established a stable mouse model of hypothyroidism combined with pulmonary hypertension through a combination of drug intervention and hypoxic feeding.

Description

Technical Field

[0001] This invention belongs to the field of animal experimental model construction technology, and in particular relates to a mouse model of hypothyroidism combined with pulmonary hypertension, its construction method and application. Background Technology

[0002] Hypothyroidism, commonly known as underactive thyroid, is a systemic hypometabolic syndrome caused by various factors that weaken the production or function of thyroid hormones. The most common causes are autoimmune diseases, medications, and surgery, leading to insufficient thyroid hormone production or impaired utilization. Hypothyroidism often has an insidious onset and a long course, with many patients lacking specific symptoms for a considerable period. As the condition progresses, it can progressively worsen, leading to systemic symptoms such as chills and fatigue. When the heart is involved, pericardial effusion and heart failure can occur; severe cases can result in myxedema coma.

[0003] Pulmonary hypertension (PH) refers to a hemodynamic and pathophysiological state in which pulmonary artery pressure rises above a certain threshold. It can lead to right heart failure and can be an independent disease, a complication, or a syndrome. Pulmonary hypertension can be classified into five main categories: ① arterial pulmonary hypertension; ② pulmonary hypertension caused by left ventricular disease; ③ pulmonary hypertension caused by hypoxia and / or lung disease; ④ chronic thromboembolic pulmonary hypertension; and ⑤ pulmonary hypertension caused by multiple mechanisms and / or unknown mechanisms. Common symptoms of pulmonary hypertension include dyspnea, fatigue, weakness, decreased exercise tolerance, syncope, chest pain, and leg and ankle edema.

[0004] Observational studies estimate that the prevalence of hypothyroidism in patients with pulmonary hypertension ranges from 10% to 24%, a significantly higher rate than in the general population. Furthermore, research indicates that pulmonary artery pressure and pulmonary vascular resistance are higher in patients with hypothyroidism than in those with normal thyroid function. More notably, patients with pulmonary hypertension and hypothyroidism tend to have poorer cardiac function and a poorer prognosis, and high TSH levels are associated with increased mortality in pulmonary hypertension patients. The occurrence of hypothyroidism and pulmonary hypertension complications has attracted widespread attention in the medical community. Recent studies have gradually revealed a close link between hypothyroidism and pulmonary hypertension (PH), but the specific mechanisms by which hypothyroidism affects PH are poorly understood. Therefore, establishing reliable animal models is crucial for research on hypothyroidism complicated by pulmonary hypertension. Currently, existing animal models only address hypothyroidism and pulmonary hypertension individually; animal models of hypothyroidism complicated by pulmonary hypertension are lacking. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a mouse model of hypothyroidism combined with pulmonary hypertension, its construction method and application.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for constructing a mouse model of hypothyroidism complicated with pulmonary hypertension, comprising the following steps:

[0008] 1) A hypothyroid mouse model was obtained by intragastric administration of methimazole and feeding the mice under normoxic conditions;

[0009] 2) The hypothyroid mouse model was continued to be administered methimazole by gavage and the vascular endothelial growth factor inhibitor Su5416 was continuously injected subcutaneously. The hypothyroid mouse model with pulmonary hypertension was obtained by feeding the mouse under hypoxic conditions.

[0010] Preferably, the mouse is a C57BL / 6 mouse.

[0011] Preferably, the amount of methimazole administered by gavage in step 1) is 30-50 mg / kg / day; and the duration of gavage administration of methimazole is 3-5 weeks.

[0012] Preferably, in step 2), the amount of methimazole administered by gavage is 30-50 mg / kg / day; and the duration of gavage administration of methimazole is 3-4 weeks.

[0013] Preferably, the injection dose of the vascular endothelial growth factor inhibitor Su5416 in step 2) is 20 mg / kg / w.

[0014] Preferably, the vascular endothelial growth factor inhibitor Su5416 is injected over a period of 3 to 4 weeks.

[0015] Preferably, the oxygen concentration of the low-oxygen environment in step 2) is 9-11%.

[0016] This invention also provides the application of the hypothyroidism combined with pulmonary hypertension mouse model constructed by the above construction method in pathological studies of hypothyroidism combined with pulmonary hypertension or in vitro drug testing and screening.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] This invention successfully established a hypothyroidism model in C57BL / 6 mice by continuous gavage administration of methimazole followed by tail-dissection and blood sampling to assess thyroid function. Subsequent subcutaneous injection of the vascular endothelial growth factor inhibitor Su5416 and subsequent hypoxic rearing resulted in the establishment of a pulmonary hypertension model. Right ventricular systolic pressure was measured via right heart catheterization, and pulmonary blood flow acceleration time and ejection time were detected by echocardiography. In summary, this invention successfully established a stable mouse model of hypothyroidism combined with pulmonary hypertension through a combination of drug intervention and hypoxic rearing. Attached Figure Description

[0019] Figure 1 This is a simplified flowchart of the mouse modeling process for hypothyroidism complicated with pulmonary hypertension;

[0020] Figure 2 These are the right ventricular waveform curves and right ventricular systolic pressure measurements of mice in different treatment groups (where A is the right ventricular pressure curve of each group of mice; B is a comparison of right ventricular systolic pressure (RVSP) of each group of mice, compared with the CON group). P <0.01; compared with the SuHx group, ## P <0.01; Compared with the SuHx+MMI group, & P <0.05.

[0021] Figure 3 The values ​​of PTA and PAT / PET measured by echocardiography in mice of different treatment groups are as follows: (A) Pulmonary valve Doppler signal was recorded by pulsed Doppler ultrasound at the aortic level in the short-axis view of the left ventricle, and pulmonary blood flow acceleration time (PAT) and pulmonary blood flow ejection time (PET) were measured; (B) Comparison of PAT values ​​among mice in each group; (C) Comparison of PAT / PET values ​​among mice in each group, compared with the CON group. P <0.01; compared with the SuHx group, # P <0.05, ## P <0.01; compared with the SuHx+MMI group, & P <0.05.

[0022] Figure 4 HE staining was used to observe pulmonary vascular remodeling in mice under different treatment groups: pulmonary vessels of mice in each group under a microscope (200×, scale bar = 50 μm).

[0023] Figure 5 Masson staining was used to observe pulmonary vascular fibrosis in mice of each group (200×, scale bar = 50μm). Detailed Implementation

[0024] This invention provides a method for constructing a mouse model of hypothyroidism complicated with pulmonary hypertension, comprising the following steps:

[0025] 1) A hypothyroid mouse model was obtained by intragastric administration of methimazole and feeding the mice under normoxic conditions;

[0026] 2) The hypothyroid mouse model was continued to be administered methimazole by gavage and the vascular endothelial growth factor inhibitor Su5416 was continuously injected subcutaneously. The hypothyroid mouse model with pulmonary hypertension was obtained by feeding the mouse under hypoxic conditions.

[0027] In this invention, a hypothyroid mouse model is obtained by administering methimazole via gavage to mice and raising them under normoxic conditions. The mice are preferably C57BL / 6 mice; the preferred gavage dose of methimazole is 30-50 mg / kg / day, more preferably 40 mg / kg / day; the preferred gavage duration of methimazole is 3-5 weeks, more preferably 4 weeks.

[0028] In this invention, a hypothyroid mouse model is continuously administered methimazole by gavage and subcutaneously injected with the vascular endothelial growth factor inhibitor Su5416. The preferred gavage dose of methimazole is 30-50 mg / kg / day, more preferably 40 mg / kg / day; the preferred gavage duration of methimazole is 3-4 weeks; the preferred injection dose of the vascular endothelial growth factor inhibitor Su5416 is 20 mg / kg / week; the preferred injection duration of the vascular endothelial growth factor inhibitor Su5416 is 3-4 weeks; and a hypothyroid mouse model with pulmonary hypertension is obtained by raising the mouse in a hypoxic environment; the preferred oxygen concentration of the hypoxic environment is 9-11%, more preferably 10%.

[0029] This invention also provides the application of the hypothyroidism combined with pulmonary hypertension mouse model constructed by the above construction method in pathological studies of hypothyroidism combined with pulmonary hypertension or in vitro drug testing and screening.

[0030] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0031] C57BL / 6 mice were purchased from the Experimental Animal Center of Gansu University of Traditional Chinese Medicine and were housed at the SPF-grade Animal Experimental Center of Gansu University of Traditional Chinese Medicine.

[0032] Mouse TSH enzyme-linked immunosorbent assay kit, catalog number: E-EL-M1153C, purchased from Wuhan Yilairuit Biotechnology Co., Ltd., China;

[0033] Mouse FT4 enzyme-linked immunosorbent assay kit, catalog number: MM-202305, purchased from Jiangsu Enzyme Immunoassay Co., Ltd., China.

[0034] Example 1

[0035] I. Construction of a model of hypothyroidism complicated with pulmonary hypertension

[0036] Eight-week-old male SPF-grade C57BL / 6 mice were administered methimazole (MMI) 40 mg / kg / day by gavage for four consecutive weeks. Blood was collected from the tail to assess thyroid function (serum TSH and FT4 levels). After successful establishment of the hypothyroidism model, the mice were subcutaneously injected with the vascular endothelial growth factor inhibitor Su5416 (20 mg / kg / w) for three consecutive weeks. During this period, the mice were placed in a normobaric hypoxic chamber with an oxygen concentration of 10%. A simplified flowchart of the modeling process is shown below. Figure 1 As shown.

[0037] The experiment was divided into 5 groups: CON, control group; SuHx, pulmonary hypertension group; MMI, hypothyroidism group; SuHx+MMI, pulmonary hypertension combined with hypothyroidism group; and SuHx+MMI+L-T4, L-T4 intervention group.

[0038] II. Signs of Successful Model Construction

[0039] Hypothyroidism model: Serum TSH and FT4 levels were measured. TSH levels were significantly higher than those in the control group, while FT4 levels were significantly lower, indicating that the hypothyroidism model was successfully established.

[0040] A mouse model of hypothyroidism complicated with pulmonary hypertension was established. Right ventricular systolic pressure (RVSP) measured by right heart catheterization was higher than that in the control group (gold standard). Pulmonary artery acceleration time (PAT) and pulmonary ejection time (PET) were measured by echocardiography, and the calculated PAT / PET ratio (negatively correlated with pulmonary artery pressure) was lower in the mouse model compared to the control group. After model establishment, mice were sacrificed, and further pathological examination of lung tissue sections using hematoxylin-eosin (HE) and Masson staining revealed thickened pulmonary vessel walls, narrowed lumens, and increased collagen deposition in the adventitia compared to the control group. Blood samples were collected from the eyeballs to measure serum TSH and FT4 levels. TSH was significantly higher than in the control group, while FT4 was significantly lower. This established the mouse model of hypothyroidism complicated with pulmonary hypertension.

[0041] III. Thyroid Function Testing

[0042] After the modeling was completed, the mice were anesthetized, and their eyeballs were removed to collect blood. The blood was collected in a sterile tube and allowed to stand at room temperature for about 15-30 minutes to allow the blood to clot naturally. The clotted blood was then centrifuged at 4°C for 15 minutes (2000 rpm) to separate the serum and clot. The upper layer of serum was carefully aspirated into a sterile centrifuge tube using a pipette, taking care not to disturb the lower layer of red blood cells or clots. The tube was then stored at -80°C for later use.

[0043] 1. Detection of serum TSH concentration by enzyme-linked immunosorbent assay (ELISA)

[0044] (1) Take the kit and serum sample out of the refrigerator 20 minutes before the test and place them at room temperature (18-25°C); dilute the concentrated washing buffer (1:24) with double distilled water; centrifuge the standard at 10000×g for 1 minute, add 1 mL of standard & sample diluent, cap the tube and let it stand for 10 minutes, then mix thoroughly until the standard solution is dissolved to prepare a standard working solution of 80 ng / mL. Continue to prepare standard solutions of the following concentrations according to the instructions: 40, 20, 10, 5, 2.5, 1.25, 0 ng / mL; centrifuge the biotinylated antibody concentrate at 800×g for 1 minute, and then dilute it 100 times to the working concentration with the biotinylated antibody diluent; centrifuge the HRP enzyme conjugate concentrate at 800×g for 1 minute, and then dilute it 100 times to the working concentration with the diluent.

[0045] (2) Operating steps: Set up standard wells, blank wells, and sample wells respectively. Add 100µL of standard solution to the standard wells, 100µL of standard solution and sample diluent to the blank wells, and 100µL of sample diluent to the remaining wells; then cover the plate and incubate at 37°C for 90 minutes; after incubation, discard the liquid in the wells without washing, then add 100µL of biotinylated antibody working solution to each well and continue incubation at 37°C for 1 hour; after incubation, discard the liquid and pat dry on absorbent paper, fill each well with washing solution, soak for 1 minute, then discard and pat dry, repeat this washing process 3 times; then add 100µL of HRP enzyme conjugate working solution to each well, cover the plate and incubate at 37°C for 30 minutes, repeat the washing 5 times; finally add to each well Add 90 µL of substrate solution (TMB), cover with the plate membrane, and incubate at 37°C in the dark for 15 minutes. Preheat the microplate reader 15 minutes beforehand. When a clear gradient appears in the first four standard wells, add 50 µL of stop solution to each well. After the reaction is complete, quickly measure the OD value of each well at 450 nm using the microplate reader. Subsequently, based on the known concentrations of the standards and their corresponding OD values, plot a standard curve and calculate the linear regression equation. Substitute the OD value of the sample to be tested into this equation to calculate the sample concentration. Finally, after appropriate dilution conversion, the exact concentration of TSH in the sample can be obtained.

[0046] 2. Detection of serum FT4 concentration by enzyme-linked immunosorbent assay (ELISA)

[0047] (1) Remove the serum sample and ELISA kit from the refrigerator and leave them at room temperature for half an hour; dilute the concentrated washing solution 30 times with distilled water.

[0048] (2) Prepare the following concentrations of standard according to the kit instructions: 12, 6, 3, 1.5, 0.75, 0 pmol / L.

[0049] (3) Blank wells, standard wells, and sample wells were set up separately. No sample or enzyme-labeled reagent was added to the blank wells, and the rest of the operation was the same as the other wells. For the standard wells, 50 µL of standard solution was added. In the sample wells, 40 µL of sample diluent was added first, followed by 10 µL of sample to ensure that the final dilution factor of the sample reached 5 times.

[0050] (4) Seal the plate with a sealing film and incubate at 37°C for 30 minutes.

[0051] (5) Remove the sealing film, shake off the liquid, fill each hole with detergent, let it stand for 30 seconds, and then discard the liquid. Repeat this process 5 times, and then pat dry.

[0052] (6) Add 50 μL of enzyme-labeled reagent to each well, except for the blank wells; repeat step ⑤.

[0053] (7) First add 50 μL of colorimetric reagent A to each well, then add 50 μL of colorimetric reagent B. Gently shake to mix evenly, and incubate at 37°C in the dark for 10 minutes.

[0054] (8) The OD value of each well was measured at a wavelength of 450 nm using an ELISA reader. A linear regression equation was established by the concentration of the standard and the OD value. The concentration was calculated by substituting the OD value of the sample into the equation, and then multiplied by the dilution factor to obtain the FT4 concentration of the sample.

[0055] Experimental results are shown in Table 1.

[0056] Table 1 Statistical Analysis of FT4 and TSH Levels

[0057]

[0058] Experimental Results: Compared with the control group, mice administered MMI via gavage showed a 25.1% decrease in serum FT4 and a 31.7% increase in TSH, both statistically significant, indicating a successful establishment of the hypothyroidism model. Compared with the CON group, the MMI group, SuHx+MMI group, and SuHx+MMI+L-T4 group all showed decreased serum FT4 levels and increased TSH levels, with statistically significant differences. P <0.05); Compared with the SuHx+MMI group, the serum FT4 level of mice in the SuHx+MMI+L-T4 group was increased and the TSH level was decreased, and the results showed statistical differences ( P <0.05); There was no statistically significant difference in serum FT4 and TSH levels between the SuHx group and the CON group ( P >0.05).

[0059] IV. Pulmonary hypertension detection

[0060] 1. Right ventricular systolic pressure measurement

[0061] After ultrasound measurement, the mouse body weight (BW) was measured and recorded. The mouse was anesthetized by intraperitoneal injection of chloral hydrate, fixed in a supine position on a mouse board, and the hair on the right side of the neck was shaved. The skin was incised along the right side of the neck, and the right common jugular vein was bluntly dissected. After ligating the distal end of the vein with cotton thread, the common jugular vein was lifted, and a "V" shaped incision was made towards the ventricle. A micro-tip pressure catheter was inserted into the right common jugular vein and connected to the Powerlab physiological recorder through a pressure sensor. The catheter was advanced to the right ventricle, and a large-amplitude ventricular wave could be seen on the Powerlab physiological recorder screen. After stabilization for 1 minute, the right ventricular systolic pressure (RVSP) was measured by the displayed waveform curve and pressure value.

[0062] Experimental results: such as Figure 2 As shown in the figure, the right ventricular systolic pressure (RVSP) measured by right heart catheterization in mice with pulmonary hypertension and hypothyroidism was higher than that in the control group. Compared with the CON group, the RVSP increase in the MMI group was not significant and showed no statistical difference, while the RVSP increase in the SuHx group was significant and the results showed a statistical difference. P <0.01); Compared with the SuHx group, the RVSP in the SuHx+MMI group was significantly increased, and the results showed a statistically significant difference ( P <0.01); Compared with the SuHx+MMI group, the RVSP of the SuHx+MMI+L-T4 group was lower, and the results showed a statistically significant difference ( P <0.05).

[0063] 2. PAT and PAT / PET testing

[0064] Echocardiography of mice was recorded using a high-resolution small animal ultrasound imaging system (Vevo 3100LT Visualsonics) with a probe frequency of 30 MHz. Pulmonary hemodynamics and right ventricular function were assessed according to the references ([1] Zhu Z., Godana D., Li A., et al. Echocardiographic assessment of right ventricular function in experimental pulmonary hypertension. Pulmonary circulation. 2019, 9(2):2045894019841987. [2] Kohut A., Patel N., Singh H. Comprehensive Echocardiographic Assessment of the Right Ventricle in Murine Models. J Cardiovasc Ultrasound. 2016, 24(3):229-38.). Hair was removed from the chest of mice using depilatory cream. After successful anesthesia with 3% isoflurane, the mice were fixed on a preheated 37°C imaging platform. Electrogel was applied to the limbs and connected to the electrocardiogram leads. Echocardiography was performed when the heart rate stabilized at 400-500 beats / min, and the heart rate (HR) of each mouse was recorded. Pulsed Doppler ultrasound was used to record the Doppler signal of pulmonary valve blood flow in the short-axis section of the left ventricle, and the pulmonary artery acceleration time (PAT) and pulmonary ejection time (PET) were measured.

[0065] Experimental results: such as Figure 3 As shown in the figure, the PAT / PET ratio in mice with pulmonary hypertension and hypothyroidism was lower than that in the control group. Compared with CON, the PAT and PAT / PET values ​​in the SuHx group were both lower, and the results showed statistical differences. P <0.01); compared with the SuHx group, the PAT of the SuHx+MMI group ( P <0.01), PAT / PET ( P The <0.05) value decreased; compared with the SuHx+MMI group, the PAT and PAT / PET values ​​of the SuHx+MMI+L-T4 group increased, and the results showed statistical differences. P <0.01). There was no statistically significant difference between the MMI group and the CON group in the above indicators.

[0066] 3. HE staining

[0067] (1) Experimental preparation

[0068] ① Reagent preparation: Prepare hematoxylin staining solution, eosin staining solution, graded alcohols, xylene, neutral resin, etc.

[0069] ② Section preparation: The previously embedded paraffin blocks of mouse lung tissue are sectioned (the largest section of one lobe of lung is cut to include as many blood vessels as possible), with a thickness of 4~6μm.

[0070] (2) HE staining steps

[0071] ① Dewaxing and hydration: The sections were dewaxed in xylene and then hydrated by passing them through a gradient of alcohols.

[0072] ② Hematoxylin staining: Place the section in hematoxylin staining solution and allow it to stain thoroughly for 10 minutes.

[0073] ③ Rinsing and Differentiation: The sections were rinsed with tap water to remove excess staining solution, and then differentiated with 1% hydrochloric acid alcohol.

[0074] ④ Blueing: Place the slice in a weakly alkaline aqueous solution to blue the cell nuclei, making them appear blue.

[0075] ⑤ Eosin staining: Immerse the slide in eosin staining solution for 3 minutes.

[0076] ⑥ Dehydration and clearing: Dehydrate again with a gradient of alcohols, then clear with xylene.

[0077] ⑦ Mounting: Add a drop of neutral resin to the slide and cover it with a coverslip to seal it.

[0078] (3) Morphological observation

[0079] Locate pulmonary arterioles near alveolar tissue under an optical microscope and acquire images (outer diameter 50-200 μm). Use Image J image analysis software to measure the lumen area (LA) and total vascular area (TA), and calculate the pulmonary vascular wall area index (WA%), WA% = (TA-LA) / TA×100%.

[0080] Experimental results: such as Figure 4As shown in the figure. HE staining results of lung tissue showed that, compared with the CON group, the MMI group of mice did not show obvious pathological abnormalities in lung tissue, while the other groups all showed varying degrees of pulmonary vascular wall thickening and lumen narrowing, which is consistent with the typical pathological features of pulmonary vascular remodeling. Compared with the SuHx group, the SuHx+MMI group of mice showed more prominent pulmonary vascular wall thickening and aggravated lumen narrowing, some even approaching occlusion. Compared with the SuHx+MMI group, the pathological manifestations of pulmonary vascular wall thickening and lumen narrowing in the SuHx+MMI+L-T4 group of mice were significantly alleviated.

[0081] 4. Masson staining

[0082] (1) Experimental preparation

[0083] ① Reagent preparation: Prepare the relevant reagents for Masson staining, including Weigert iron hematoxylin staining solution, acid fuchsin staining solution, phosphomolybdic acid solution, aniline blue staining solution, etc.

[0084] ②Slice preparation: Slice the paraffin block of mouse lung tissue (the largest section of one lobe of lung, cutting as many blood vessels as possible), and the slice thickness is usually 4~6μm.

[0085] (2) Masson staining procedure

[0086] ① Dewaxing and hydration: The sections were dewaxed in xylene and then hydrated by passing them through a gradient of alcohols.

[0087] ②Weigert iron hematoxylin staining: Immerse the sections in Weigert iron hematoxylin staining solution for 1 minute.

[0088] ③ Rinsing and Differentiation: Rinse the sections with tap water, and then differentiate them with 1% hydrochloric acid alcohol.

[0089] ⑤ Acid fuchsin staining: Immerse the sections in acid fuchsin staining solution for staining.

[0090] ⑥ Phosphomolybdic acid differentiation: Use phosphomolybdic acid solution to differentiate and remove excess acidic fuchsin.

[0091] ⑦ Aniline blue staining: Immerse the section in 2.5% aniline blue solution for 15 seconds.

[0092] ⑧ Dehydration and clearing: Dehydrate again with a gradient of alcohols, then clear with xylene.

[0093] ⑨ Mounting: Add a drop of neutral resin to the slide and cover it with a coverslip to seal it.

[0094] (3) Morphological observation

[0095] Using an optical microscope, locate and image pulmonary arterioles near the alveolar tissue (outer diameter 50-200 μm) to observe the distribution and proliferation of collagen fibers around the pulmonary vessels. Assess the degree of pulmonary vascular fibrosis based on the color and density of the collagen fibers. More collagen fiber proliferation and a darker color indicate more severe fibrosis. Simultaneously observe the morphology and structure of the vessels to see if there are any structural changes caused by fibrosis.

[0096] Experimental Results: In the CON group, collagen fibers were evenly distributed in the lung tissue of mice, with lighter staining and fewer fibers, and normal pulmonary vessel thickness. Compared with the CON group, mice in the SuHx group had many disordered proliferating collagen fibers in the adventitia and surrounding tissues of pulmonary vessels, with an expanded and darker blue-stained area, along with thickened pulmonary vessel walls and narrowed lumens. Compared with the SuHx group, mice in the SuHx+MMI group had more collagen fiber deposition, increased vessel wall thickness, and even narrower lumens. Compared with the SuHx+MMI group, mice in the SuHx+MMI+L-T4 group showed significantly improved collagen fiber deposition, and some improvement in vessel wall thickening and lumens narrowing. These observations indicate that mice in the SuHx group exhibited pulmonary vascular remodeling, which was exacerbated by hypothyroidism, while L-T4 intervention improved the pathological manifestations of pulmonary vessels.

[0097] As can be seen from the above embodiments, the present invention has successfully established a stable mouse model of hypothyroidism combined with pulmonary hypertension by combining drug intervention and hypoxia feeding.

[0098] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing a mouse model of hypothyroidism combined with pulmonary arterial hypertension, characterized in that, Comprise the following steps: 1) obtain hypothyroid mouse model by intragastric administration of methimazole in mice and feeding in normoxic environment; The intragastric amount of methimazole is 30-50 mg / kg / d; the intragastric administration time of methimazole is 3-5 weeks; 2) obtain hypothyroid combined with pulmonary arterial hypertension mouse model by continuing intragastric administration of methimazole, continuous subcutaneous injection of vascular endothelial growth factor inhibitor Su5416 and feeding in hypoxic environment; The injection amount of vascular endothelial growth factor inhibitor Su5416 is 20 mg / kg / w; The injection time of vascular endothelial growth factor inhibitor Su5416 is 3-4 weeks; The oxygen concentration of the hypoxic environment is 9-11%.

2. The construction method of claim 1, wherein, The mouse is C57BL / 6 mouse.

3. The construction method of claim 1, wherein, The intragastric amount of methimazole in step 2) is 30-50 mg / kg / d; the intragastric administration time of methimazole is 3-4 weeks.

4. The application of hypothyroid combined with pulmonary arterial hypertension mouse model constructed by the construction method of any one of claims 1-3 in drug testing and screening.

Images