Pre-eclampsia mouse model as well as construction method and application thereof

Abstract

The invention discloses a preeclampsia mouse model. The preeclampsia mouse model is constructed by the following method: (1) combining a female mouse with a male mouse; (2) in the middle stage of pregnancy, intragastric administration of hypoxanthine and oteracil potassium salt is carried out on the pregnant mouse for a period of time; the invention also discloses a construction method and application of the gene. The invention provides a novel preeclampsia modeling mode, hypoxanthine and oteracil potassium salt are combined for use, and the administration dosage and the administration mode of hypoxanthine and oteracil potassium salt are controlled, so that the serum uric acid level of a model mouse is stably increased, preeclampsia change occurs in the model mouse, and the model mouse can be used for treating preeclampsia. The stability, specificity and pathophysiology characteristics of the model are ensured to be similar to those of human preeclampsia, so that the uric acid-induced preeclampsia mouse model is successfully constructed. The uric acid induced preeclampsia mouse model provides a valuable tool for researching the pathogenesis of preeclampsia, evaluating a new treatment method and understanding the effect of serum hyperuricemia in preeclampsia.

Description

Technical Field

[0001] The present invention relates to the technical field of animal models, and in particular to a preeclampsia mouse model and a construction method and application thereof. Background Art

[0002] Animal models of preeclampsia (PE) are important tools for studying the pathogenesis of the disease and developing treatments. The following are some existing animal model technologies for preeclampsia and their shortcomings:

[0003] 1. Transgenic preeclampsia animal model: It provides a better platform to study the pathogenesis of preeclampsia, especially the function of related pathogenic genes. However, there is no ideal transgenic preeclampsia animal model that can fully replicate all the characteristics of human preeclampsia.

[0004] 2. Angiogenic factor inhibitor model: Injection of adenovirus carrying sFLT-1 into the tail vein of rats on the 8th or 9th day of pregnancy leads to preeclampsia-like changes such as increased blood pressure, proteinuria and kidney lesions in the late pregnancy, but no infiltration of trophoblast cells is shown. In addition, some inducers can also cause hypertension, proteinuria, etc. when used in the non-pregnant period, which lacks pregnancy specificity.

[0005] 3. Oxidative stress model: Multiple hypoxia-inducible factor 1α (HIF-1α) overexpression models have been used to study the pathogenesis of oxidative stress in preeclampsia. No abnormalities in trophoblast cell infiltration were found in this model, and the pathological changes and progression of preeclampsia cannot be fully reflected.

[0006] 4. Inflammatory immune overactivation model: The preeclampsia model is constructed by injecting LPS or inflammatory factors TNF-α, IL-6, etc. Each model can only simulate some of the characteristics of preeclampsia. Due to the diversity of pathophysiology and clinical manifestations of preeclampsia, there is currently no single animal model that can effectively simulate all the characteristics of preeclampsia.

[0007] 5. Reduced Uterine Perfusion Pressure (RUPP) Model: Surgery is used to reduce blood flow to the uterus to simulate certain characteristics of preeclampsia. This model does not show insufficient trophoblast infiltration. In addition, surgical artery blockage is not the real cause of preeclampsia.

[0008] In summary, although a variety of animal models of preeclampsia have been established, each model has its limitations, and no model can fully simulate all the characteristics of human preeclampsia. These limitations are one of the important reasons why the cause of preeclampsia is unknown and there is a lack of effective treatment.

[0009] Therefore, we developed a more ideal, simpler and more economical animal model of preeclampsia, which can simulate the clinical manifestations of preeclampsia patients during pregnancy, mainly hypertension, proteinuria, fetal growth restriction, as well as the phenotypes of placental dysfunction and decreased trophoblast cell infiltration. Summary of the invention

[0010] The purpose of the present invention is to provide a preeclampsia mouse model and a construction method and application thereof, so as to solve the deficiencies of the prior art.

[0011] The purpose of the present invention is achieved through the following technical solutions:

[0012] The first aspect of the present invention provides a preeclampsia mouse model, wherein the preeclampsia mouse model is constructed by the following method:

[0013] (1) The female mouse was placed in a cage with the male mouse. The thrombus was checked the next day. If the thrombus was found, it was confirmed that the female mouse was pregnant and the gestational age was set at 0.5 days.

[0014] (2) Orally administer hypoxanthine and potassium salt of oxalic acid to pregnant mice starting from the second trimester for a period of time to obtain the preeclampsia mouse model.

[0015] Furthermore, in step (2), the second trimester is 7.5 days of pregnancy, the dosage of hypoxanthine is 600 mg / kg mouse / d, and the dosage of potassium salt of oxonate is 600 mg / kg mouse / d, and the administration is continued by oral gavage until 16.5 days of pregnancy.

[0016] Furthermore, in step (2), the concentration of hypoxanthine is 24 mg / ml, and the concentration of potassium salt of oxonoic acid is 24 mg / ml.

[0017] Furthermore, the female mice in step (1) are female mice of 6-10 weeks old, and the male mice are male mice of 6-10 weeks old.

[0018] A second aspect of the present invention provides a method for constructing a preeclampsia mouse model, comprising the following steps:

[0019] (1) The female mouse was placed in a cage with the male mouse. The thrombus was checked the next day. If the thrombus was found, it was confirmed that the female mouse was pregnant and the gestational age was set at 0.5 days.

[0020] (2) Orally administer hypoxanthine and potassium salt of oxalic acid to pregnant mice starting from the second trimester for a period of time to obtain the preeclampsia mouse model.

[0021] Furthermore, in step (2), the second trimester is 7.5 days of pregnancy, the dosage of hypoxanthine is 600 mg / kg mouse / d, and the dosage of potassium salt of oxonate is 600 mg / kg mouse / d, and the administration is continued by oral gavage until 16.5 days of pregnancy.

[0022] Furthermore, in step (2), the concentration of hypoxanthine is 24 mg / ml, and the concentration of potassium salt of oxonoic acid is 24 mg / ml.

[0023] Furthermore, the female mice in step (1) are female mice of 6-10 weeks old, and the male mice are male mice of 6-10 weeks old.

[0024] The third aspect of the present invention provides the use of the above-mentioned preeclampsia mouse model in studying the pathogenesis of preeclampsia.

[0025] A fourth aspect of the present invention provides the use of the above-mentioned preeclampsia mouse model in screening and evaluating the efficacy of drugs for preventing and treating preeclampsia.

[0026] Beneficial effects of the present invention:

[0027] The present invention provides a novel preeclampsia modeling method, which combines hypoxanthine and oxonic acid potassium salt, and controls the dosage and administration method of hypoxanthine + oxonic acid potassium salt, so that the serum uric acid level of the model mice is stably increased, and the model mice show preeclampsia changes. The stability, specificity and pathophysiological characteristics of the model are similar to those of human preeclampsia, thereby successfully constructing a uric acid-induced preeclampsia mouse model. The uric acid-induced preeclampsia mouse model of the present invention has the following advantages:

[0028] 1. Simulate the pathological characteristics of human preeclampsia: The uric acid-induced preeclampsia mouse model of the present invention not only has the clinical manifestations of preeclampsia with adverse pregnancy outcomes such as hypertension, proteinuria, and fetal growth restriction that are unique to pregnancy, but can also simulate the phenotypes of placental dysfunction and decreased placental trophoblast cell infiltration in preeclampsia. Compared with other preeclampsia models, the uric acid-induced preeclampsia mouse model of the present invention can fully simulate the clinical characteristics of preeclampsia and can be used for preeclampsia-related research.

[0029] 2. Simple operation and low cost: In the construction of the uric acid-induced preeclampsia mouse model of the present invention, the administration method of hypoxanthine + potassium salt of oxalic acid is oral gavage. The construction method is simple to operate, low in cost, and easy to repeat experiments and large-scale application.

[0030] 3. Provides a stable model: Hypoxanthine is a product of purine metabolism in the body and is converted into uric acid under the action of xanthine oxidase. Oxononic acid potassium salt is a commonly used uricase inhibitor, which blocks the degradation of uric acid by inhibiting the activity of uricase, thereby leading to increased serum uric acid levels. The present invention combines the two to increase uric acid production while reducing uric acid excretion, thereby creating a high uric acid level in the body. This type of modeling method under the influence of multiple factors is more stable than a single drug administration modeling method, and the increase in uric acid levels is more significant.

[0031] 4. Better understanding of the pathogenesis of preeclampsia: The uric acid-induced preeclampsia mouse model of the present invention is of great value in exploring the occurrence and development mechanism of preeclampsia, especially in the study of preeclampsia related to high uric acid levels, its advantages are particularly significant.

[0032] In summary, the uric acid-induced preeclampsia mouse model of the present invention provides a valuable tool for studying the pathogenesis of preeclampsia, evaluating new treatments, and understanding the role of serum hyperuricemia in preeclampsia. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Figure 1 is the appearance of the fetuses and placentas in the control group and the uric acid-induced preeclampsia group. Figure A is the appearance of the fetuses after dissection of the pregnant mice in the control group and the uric acid-induced preeclampsia group. The top is the appearance of the fetuses in the control group, and the bottom is the appearance of the fetuses in the uric acid-induced preeclampsia group. Figure B is the appearance of the placentas and fetuses in the control group and the uric acid-induced preeclampsia group. The first row is the placenta of the control group, the second row is the fetuses in the control group, the third row is the placenta of the uric acid-induced preeclampsia group, and the fourth row is the fetuses in the uric acid-induced preeclampsia group.

[0034] Figure 2 The following are statistical diagrams of fetal and placental growth and development in the control group and the uric acid-induced preeclampsia group. A is the comparison of head-to-rump length of fetal mice between the control group and the uric acid-induced preeclampsia group; B is the comparison of fetal weight between the control group and the uric acid-induced preeclampsia group; C is the comparison of placental weight between the control group and the uric acid-induced preeclampsia group.

[0035] Figure 3 It is a line graph of the changes in blood pressure (systolic pressure) in the control group and the uric acid-induced preeclampsia group.

[0036] Figure 4 Statistical graph of 24-hour urine protein / urine creatinine ratio in the control group and uric acid-induced preeclampsia group.

[0037] Figure 5 Statistical graphs of UA (Figure A), Cr (Figure B), and sFLT1 (Figure C) concentrations in the serum of pregnant mice in the control group and uric acid-induced preeclampsia group.

[0038] Figure 6 HE staining and statistical graphs of placental tissues in the control group and uric acid-induced preeclampsia group. A is the HE staining of placental tissues in the control group and uric acid-induced preeclampsia group; B is the statistical graph of the ratio of the labyrinth layer to the connection area; C is the statistical graph of the ratio of the labyrinth layer to the total placental area.

[0039] Figure 7The immunofluorescence staining and statistical graphs of the placenta tissues in the control group and the uric acid-induced preeclampsia group. A is the immunofluorescence staining of CK7 in the control group and the uric acid-induced preeclampsia group (2X), DAPI is the DAPI fluorescent dye, and Merge is the combined superposition of CK7 and DAPI; B is the infiltration degree of trophoblast cells in the placenta in the control group and the uric acid-induced preeclampsia group.

[0040] Figure 8 Immunofluorescence staining of CK7, SMA, and vWF in the control group and uric acid-induced preeclampsia group (20X), and DAPI is DAPI fluorescent dye.

[0041] In the above figures, * indicates P ≤ 0.05. DETAILED DESCRIPTION

[0042] The present invention is further explained below in conjunction with the examples and drawings. The following examples are only used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0043] Example 1

[0044] 1.1 Construction of uric acid-induced preeclampsia mouse model

[0045] Thirty 6-8 week old C57BL / 6 mice (18-22 g in weight) were ordered, including 20 female mice and 10 male mice, and they were adaptively raised for one week. Afterwards, the female mice were caged with the male mice at a ratio of 2:1 (in the evening), and the thrombus was checked the next morning (the thrombus was found on the morning of the next day after cage closure, indicating that the female mouse was pregnant, and the pregnancy was determined to be 0.5 days). The female mice with thrombus were randomly divided into two groups, 8 in each group, namely the control group and the experimental group (uric acid-induced preeclampsia group). Starting from 7.5 days of pregnancy, the control group was orally gavaged with 1X PBS (diluted from 20XPBS (Sangon Biotech, B548117-0500), pH 7.2-7.6), 1XPBS dosage was 500ul / mouse / d; the experimental group was orally gavaged with hypoxanthine (ABCONE, H03235; concentration was 24mg / ml, prepared with 1XPBS (same as above)) + oxonic acid potassium salt (COOLABER, CO8101; concentration was 24mg / ml, prepared with 1X PBS (same as above)), hypoxanthine dosage was 600mg / kg mouse / d, oxonic acid potassium salt dosage was 600mg / kg mouse / d, and gavage was continued until 16.5 days of pregnancy (gavage was performed at the same time every day). Adaptive feeding was carried out in an SPF-level environment, with normal feeding of feed and water, and subsequent experiments were carried out in a clean-level environment, with normal feeding of feed and water.

[0046] 1.2 Pathophysiological characteristics of the uric acid-induced preeclampsia mouse model

[0047] (1) The blood pressure (systolic blood pressure) of pregnant mice was measured for two consecutive days starting from 5.5 days of pregnancy, and the blood pressure (systolic blood pressure) of pregnant mice was measured every other day starting from 7.5 days of pregnancy (blood pressure was measured before oral gavage on the same day).

[0048] (2) On day 16.5 of gestation, the pregnant mice were placed in metabolic cages (after oral gavage on the same day), and 24-hour urine was collected to detect urine protein and urine creatinine.

[0049] (3) On gestational day 17.5, the eyes of pregnant mice were enucleated and blood was collected for serological tests (uric acid (UA), creatinine (Cr), and soluble fms-like tyrosine kinase-1 (sFLT-1)). The pregnant mice were euthanized, and the fetuses and placentas were dissected. The head-to-rump length, fetal weight, and placental weight of the fetuses were measured, and the placentas were sampled and tested (HE staining and immunofluorescence staining analysis).

[0050] The blood pressure (systolic pressure) of pregnant mice was measured with a sphygmomanometer, the 24-hour urine and serum were tested with a biochemical analyzer, the head-to-rump length of fetuses was measured with a ruler, the fetal weight and placenta weight were measured with a balance, and the placenta tissue was tested with HE staining and immunofluorescence staining.

[0051] Fetuses and placentas in each group Figure 1 The results of head-to-rump length, fetal weight, and placental weight of each group of fetuses are shown in Figure 2 The results showed that the head-to-rump length, fetal weight, and placental weight of the fetuses in the uric acid-induced preeclampsia group were reduced, showing the phenotype of fetal growth restriction in preeclampsia.

[0052] The results of blood pressure (systolic pressure) of pregnant mice in each group are shown in Figure 3 The results showed that the blood pressure of the uric acid-induced preeclampsia group began to rise at 9.5 days of pregnancy, and its blood pressure level showed a stable increase before autopsy, showing the phenotype of preeclampsia hypertension.

[0053] The results of urine protein and urine creatinine in each group are shown in Figure 4 ,The results showed that the urine protein / urine creatinine ratio in the uric acid-induced preeclampsia group increased, showing the phenotype of preeclampsia proteinuria.

[0054] The results of serum UA, Cr and sFLT-1 in each group are shown in Figure 5 The results showed that the concentrations of UA, Cr, and sFLT1 in the serum of the uric acid-induced preeclampsia group increased, among which the increase in sFLT1 could cause extensive vascular endothelial damage, leading to clinical manifestations such as hypertension and proteinuria.

[0055] The results of HE staining analysis of placental tissue in each group are shown in Figure 6The results showed that the degree of placental trophoblast cell infiltration in the uric acid-induced preeclampsia group was reduced, the ratio of the labyrinth layer to the junction area decreased, and the proportion of the labyrinth layer in the total placental area decreased.

[0056] The results of immunofluorescence staining analysis of placental tissue in each group are shown in Figure 7 and Figure 8 The results showed that compared with the control group, the expression of cytokeratin 7 (CK7) was reduced, and the expression of smooth muscle actin (SMA) and von Willebrand factor (vWF) was increased in the uric acid-induced preeclampsia group, indicating that uric acid-induced pregnant mice had poor placental spiral artery remodeling.

Claims

1. A mouse model of preeclampsia, characterized in that: The preeclampsia mouse model was constructed by the following method: (1) The female mouse was placed in a cage with the male mouse. The thrombus was checked the next day. If the thrombus was found, it was confirmed that the female mouse was pregnant and the gestational age was set at 0.5 days. (2) Orally administer hypoxanthine and potassium salt of oxalic acid to pregnant mice starting from the second trimester for a period of time to obtain the preeclampsia mouse model.

2. A mouse model of preeclampsia according to claim 1, characterized in that: Step (2) The second trimester is 7.5 days of pregnancy, and the dosage of hypoxanthine is 600 mg / kg mouse / d, and the dosage of potassium salt of oxonate is 600 mg / kg mouse / d, and the administration is continued by oral gavage until 16.5 days of pregnancy.

3. A mouse model of preeclampsia according to claim 2, characterized in that: In step (2), the concentration of hypoxanthine is 24 mg / ml, and the concentration of potassium salt of oxonoic acid is 24 mg / ml.

4. A mouse model of preeclampsia according to claim 1, 2 or 3, characterized in that: The female mice in step (1) are female mice of 6-10 weeks old, and the male mice are male mice of 6-10 weeks old.

5. A method for constructing a preeclampsia mouse model, characterized in that: The steps include: (1) The female mouse was placed in a cage with the male mouse. The thrombus was checked the next day. If the thrombus was found, it was confirmed that the female mouse was pregnant and the gestational age was set at 0.5 days. (2) Orally administer hypoxanthine and potassium salt of oxalic acid to pregnant mice starting from the second trimester for a period of time to obtain the preeclampsia mouse model.

6. The method for constructing a preeclampsia mouse model according to claim 5, characterized in that: Step (2) The second trimester is 7.5 days of pregnancy, and the dosage of hypoxanthine is 600 mg / kg mouse / d, and the dosage of potassium salt of oxonate is 600 mg / kg mouse / d, and the administration is continued by oral gavage until 16.5 days of pregnancy.

7. The method for constructing a preeclampsia mouse model according to claim 6, characterized in that: In step (2), the concentration of hypoxanthine is 24 mg / ml, and the concentration of potassium salt of oxonoic acid is 24 mg / ml.

8. The method for constructing a mouse model of preeclampsia according to claim 5, 6 or 7, characterized in that: The female mice in step (1) are female mice of 6-10 weeks old, and the male mice are male mice of 6-10 weeks old.

9. Use of the preeclampsia mouse model according to any one of claims 1 to 4 in studying the pathogenesis of preeclampsia.

10. Use of the preeclampsia mouse model according to any one of claims 1 to 4 in screening and evaluating the efficacy of drugs for preventing and treating preeclampsia.

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