Construction method and application of a preeclampsia animal model

Abstract

The application provides a construction method and application of a preeclampsia animal model, and belongs to the technical field of construction of animal models. The construction method of the preeclampsia animal model provided by the application comprises the following steps: during pregnancy of a female mouse, phthalic acid or an adenovirus is given to inhibit activity of QPRT, and thus the preeclampsia animal model is obtained. The female mouse is a 8-week-old female C57BL / 6 mouse, the female mouse is given the phthalic acid or the adenovirus at the 10.5th day or the 16.5th day of pregnancy, the dosage of the phthalic acid is 50 mg / kg, the dosage of the adenovirus is 10 9 copies / ml. The construction method of the animal model provided by the application has the advantages of simple operation, the drug can be concentrated in the local maternal-fetal interface to play a role by the drug administration mode of uterine cavity perfusion, and the construction method can eliminate the whole-body symptoms caused by intraperitoneal injection and intravenous injection; and the mouse model can better simulate the clinical symptoms and indexes of patients with preeclampsia, and provides a new mouse model for research on the pathogenesis of preeclampsia.

Description

Technical Field

[0001] This invention belongs to the field of animal model construction technology in biomedicine, specifically relating to a method for constructing and applying a preeclampsia animal model. Background Technology

[0002] Preeclampsia (PE) is a pregnancy-related, systemic, progressive complication affecting multiple organs, seriously threatening the lives and health of both the mother and fetus. The pathogenesis of PE is unclear, and currently the only effective treatment is delivery; therefore, the latest guidelines attribute the development of PE to placental dysfunction.

[0003] Therefore, there is an urgent need for reasonable animal models to study the pathogenesis of PE and to develop corresponding prevention and treatment methods. Currently known animal models include lipopolysaccharide-induced inflammation models, RUPP surgical vascular stenosis models, vascular endothelial injury models, and gene editing-induced PE models such as APOE knockout mice.

[0004] Although there are reports on the construction of animal models of preeclampsia, the methods used are extremely limited, and there is no unified standard for developing new animal models. Therefore, obtaining a new method for constructing an animal model of preeclampsia is quite difficult.

[0005] On the other hand, due to the complexity and strong heterogeneity of the pathogenesis of preeclampsia, existing animal models can only reflect some of the pathogenic factors. The lipopolysaccharide-induced PE model is caused by widespread inflammation resulting from intraperitoneal injection of lipopolysaccharide; the RUPP procedure induces PE by surgically narrowing blood vessels to reduce uteroplacental blood flow, but this procedure is technically challenging and the effects vary significantly between batches; the vascular endothelial injury model induces PE by directly injecting drugs that damage the vascular endothelium intravenously; and gene-edited mice mainly focus on the knockout or knockdown of a single gene, which also differs significantly from clinical practice. Furthermore, the creation of gene-edited mice is complex and costly.

[0006] In summary, how to provide a new method for constructing and applying a preeclampsia animal model that can better simulate the clinical symptoms and indicators of PE patients, and provide a corresponding animal model for the study of the pathogenesis and mechanism of preeclampsia, has become an urgent technical problem to be solved. Summary of the Invention

[0007] The present invention aims to solve the aforementioned technical problems by providing a new method for constructing a preeclampsia animal model and its application. The technical objective of this invention is to provide a corresponding method for constructing more preeclampsia animal models and to achieve a simpler, lower-component animal model that can well simulate the clinical symptoms and indicators of PE patients.

[0008] The present invention first provides a method for constructing a preeclampsia animal model, the method comprising: administering phthalic acid or adenovirus to female mice during pregnancy to inhibit the activity of QPRT, thereby obtaining the preeclampsia animal model.

[0009] The inventors unexpectedly discovered the relationship between QPRT (quinolinate phosphotransferase) and preeclampsia, thus constructing the aforementioned animal model. Through extensive research, the inventors found that the level of NAM (nicotinamide) in extracellular vesicles (EVs) derived from the placenta of PE patients is reduced, and that NAM in placental-derived EVs has a significant inhibitory effect on macrophage and T cell inflammation at the maternal-fetal interface in both cell and animal experiments, playing a crucial role in maintaining pregnancy.

[0010] Since quinolinate phosphobiose transferase (QPRT) is a key rate-limiting enzyme in the production of NAM, the inventors of this invention inhibited QPRT activity by using phthalic acid (PA) or adenovirus to interfere with the expression level of the QPRT gene (Ad Sh-QPRT). They found that this effectively reduced the level of NAM in trophoblasts and extracellular vesicles of trophoblasts, thereby causing pregnant mice to exhibit a series of PE-like symptoms, including increased embryo resorption rate, decreased fetal weight and crown-rump length, and increased systolic blood pressure in the maternal tail artery, urinary UACR value, and peripheral blood PIGF and sFLT-1 concentrations. Therefore, this invention provides a simple and rapid method for constructing an animal model of preeclampsia.

[0011] Existing methods for constructing animal models of preeclampsia (PE) often employ complex modeling techniques and require high costs. This invention provides a novel animal model that induces a series of PE-like symptoms and a significant increase in the degree of inflammation of several major immune cells at the maternal-fetal interface simply by intrauterine perfusion of PA or AdSh-QPRT in pregnant mice. Recovery is achieved by supplementing with NAM-rich placental EVs. This model is simple to operate, and the intrauterine perfusion administration method allows the drug to exert its effect locally at the maternal-fetal interface, avoiding the systemic symptoms caused by intraperitoneal or intravenous injections. This model requires no complex surgical procedures or intervention in the mouse's genetic background, and it effectively simulates the clinical symptoms and indicators of PE patients, providing a novel mouse model for studying the pathogenesis and mechanism of preeclampsia.

[0012] Furthermore, the female mouse includes rats or mice, the rats including SD rats, Wistar rats, or Lewis rats; the mice include C57BL / 6 mice or Balb / c mice.

[0013] Furthermore, the female mouse was an 8-week-old C57BL / 6 mouse.

[0014] Furthermore, the administration of PA or Ad Sh-QPRT is performed on day 10.5 or day 16.5 of gestation.

[0015] Furthermore, the method of administering PA or Ad Sh-QPRT is intrauterine perfusion.

[0016] Furthermore, the dosage of PA administered is 50 mg / kg, and the concentration of AdSh-QPRT administered is 10 mg / kg. 9 copies / ml.

[0017] The second objective of this invention is to provide an application of the preeclampsia animal model constructed by the method described above in the pathogenesis and research mechanism of preeclampsia.

[0018] A third objective of this invention is to provide an application of the preeclampsia animal model constructed by the method described above in the preparation of drugs for the prevention and treatment of preeclampsia, or in drug screening and efficacy evaluation.

[0019] The fourth objective of this invention is to provide the application of the QPRT gene as a target in animal models of preeclampsia.

[0020] The fifth objective of this invention is to provide the application of the QPRT gene as a target in the preparation of reagents or kits for the prevention and treatment of preeclampsia.

[0021] The beneficial effects of this invention are as follows:

[0022] (1) This invention provides a method for constructing an animal model of preeclampsia. It can effectively reduce the level of NAM in trophoblasts and extracellular vesicles by using phthalic acid to inhibit the activity of QPRT or by interfering with the expression level of QPRT gene through adenovirus, thereby causing pregnant mice to exhibit a series of PE-like symptoms. This animal model is simple to operate, and the intrauterine perfusion administration method can concentrate the drug at the maternal-fetal interface to exert its effect locally, avoiding the systemic symptoms caused by intraperitoneal injection and intravenous injection.

[0023] (2) The method for constructing the preeclampsia animal model provided by the present invention does not require complex surgical procedures or intervention in the genetic background of mice, and the mouse model can better simulate the clinical symptoms and indicators of PE patients, providing a novel mouse model for the study of the pathogenesis and mechanism of preeclampsia.

[0024] (3) The preeclampsia animal model of the present invention can be used to help elucidate the pathogenesis and mechanism of preeclampsia, and can also be used to help screen drugs and evaluate efficacy for the prevention and treatment of preeclampsia. Attached Figure Description

[0025] Figure 1 The level of NAM in placental pEVs was detected by ELISA and statistically analyzed (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; NS, no statistical significance). Figure 2 Placental EVs derived from (A) normal pregnancy (NP) mice are denoted as NP-pEVs; placental EVs derived from mice treated with intrauterine perfusion of PA (PA-pEVs) or adenovirus-mediated interference with QPRT expression (Ad-shQPRT-pEVs) are denoted as NAM. low (B) Eight-week-old female C57BL / 6 mice were mated with ten-week-old male BALB / c mice, and the time when vaginal plugging was detected was taken as day 0.5 of gestation; on days 10.5 and 14.5 of gestation, PA or Ad-shQPRT was instilled into the uterine cavity to establish a PE mouse model; on days 12.5 and 16.5 of gestation, NP-pEVs or NAM were reinfused. low pEVs.

[0026] Figure 3 The levels of NAM in NP-pEVs and PA-pEVs were detected by ELISA (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; NS, no statistical significance). Figure 4 The level of NAM in placental EVs after intrauterine perfusion with Ad-sh NC and Ad-sh QPRT was detected by ELISA (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; NS, no statistical significance).

[0027] Figure 5 The images show representative images of the uterus and fetuses of mice in each group: the Ethanol, PA, NP-pEVs and PA-pEVs groups refer to pregnant mice that received intrauterine perfusion of the same concentration of Ethanol (control), PA, and PA and reinfusion of NP-pEVs and PA-pEVs, respectively.

[0028] Figure 6 The embryo resorption rate of mice in each group is statistically significant (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; NS, no statistical significance).

[0029] Figure 7 Statistical chart of fetal crown-rump length of mice in each group (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; NS, no statistical significance).

[0030] Figure 8 The following is a statistical chart of fetal weight of mice in each group (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; NS, no statistical significance).

[0031] Figure 9 Statistical graphs of tail artery systolic blood pressure at 10.5 days and 16.5 days of gestation for each group of mice (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; NS, no statistical significance).

[0032] Figure 10 Statistical graphs of urinary albumin / creatinine ratio (UACR) for each group of mice (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; NS, no statistical significance). Figure 11 Statistical graph of sFLT-1 concentration in peripheral blood serum of mice in each group as detected by ELISA (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; NS, no statistical significance).

[0033] Figure 12 Statistical chart of PIGF concentration in peripheral blood serum of mice in each group as detected by ELISA (**, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; NS, no statistical significance).

[0034] Figure 13 Statistical chart of sFLT-1 / PIGF ratio in peripheral blood serum of mice in each group (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; NS, no statistical significance). Figure 14 The flow cytometry results of uterine macrophages in each group of mice are as follows: (A) flow cytometry gate plot of uterine macrophages; (B) flow cytometry plot of mean fluorescence intensity of CD86 in uterine macrophages; (C) statistical graph of mean fluorescence intensity of CD86 in uterine macrophages; (D) flow cytometry plot of CD206 proportion in uterine macrophages in each group; (E) statistical graph of CD206 expression proportion in uterine macrophages in each group (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; NS, no statistical significance).

[0035] Figure 15The results of flow cytometry analysis of Th cells in the uterus of mice in each group are as follows: (A) Flow cytometry of IFNγ expression in uterine CD4+ T cells (Th1); (B) Flow cytometry of IL-4 expression in uterine CD4+ T cells (Th2); (C) Flow cytometry of IL-17 expression in uterine CD4+ T cells (Th17); (D) Flow cytometry of Foxp3 expression in uterine CD4+ T cells (Treg); (E) Statistical chart of the proportion of Th cells in the uterus of each group (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; NS, no statistical significance).

[0036] Figure 16 Representative images of the uterus and fetuses of mice in each group: The Ad Sh-NC, Ad Sh-QPRT, NP-pEVs and QPRT-pEVs groups refer to pregnant mice that received intrauterine perfusion of the same concentration of Ad Sh-NC (control), Ad Sh-QPRT, and intrauterine perfusion of Ad Sh-QPRT followed by reinfusion of NP-pEVs and QPRT-pEVs, respectively.

[0037] Figure 17 The embryo resorption rate of mice in each group is statistically significant (**, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; NS, no statistical significance).

[0038] Figure 18 Statistical chart of fetal crown-rump length of mice in each group (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; NS, no statistical significance).

[0039] Figure 19 The following is a statistical chart of fetal weight of mice in each group (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; NS, no statistical significance).

[0040] Figure 20 Statistical graphs of tail artery systolic blood pressure at 10.5 days and 16.5 days of gestation for each group of mice (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; NS, no statistical significance).

[0041] Figure 21The flow cytometry results of uterine macrophages in each group of mice are as follows: (A) Flow cytometry plot of mean fluorescence intensity of CD86 in uterine macrophages; (B) Statistical plot of mean fluorescence intensity of CD86 in uterine macrophages; (C) Flow cytometry plot of CD206 proportion in uterine macrophages in each group; (D) Statistical plot of CD206 expression proportion in uterine macrophages in each group (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; NS, no statistical significance).

[0042] Figure 22 The results of flow cytometry analysis of Th cells in the uterus of mice in each group are as follows: (A) Flow cytometry of IFNγ expression in uterine CD4+ T cells (Th1); (B) Flow cytometry of IL-4 expression in uterine CD4+ T cells (Th2); (C) Flow cytometry of IL-17 expression in uterine CD4+ T cells (Th17); (D) Flow cytometry of Foxp3 expression in uterine CD4+ T cells (Treg); (E) Statistical chart of the proportion of Th cells in the uterus of each group (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; NS, no statistical significance). Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is described in detail below with reference to embodiments. It should be noted that the following embodiments are for explanation and illustration only and are not intended to limit the invention. Non-essential improvements and adjustments made by those skilled in the art based on the above description are still within the scope of protection of this invention.

[0044] Example 1

[0045] (I) Experimental Methods:

[0046] Eight-week-old female C57BL / 6 mice were mated with ten-week-old male BALB / c mice, and the time when the vaginal plug was detected was defined as day 0.5 of gestation. Phthalic acid (PA) at 50 mg / kg or 10 mg / kg was administered to the pregnant mice from days 10.5 and 14.5 of gestation. 9 Each of the adenovirus (Ad Sh-QPRT) copies / ml interfering with QPRT gene expression was administered via intrauterine perfusion once.

[0047] Preeclampsia indicators: embryo resorption rate, placental weight, fetal rat weight and crown-rump length, tail artery systolic pressure, urinary UACR value, peripheral blood PLGF and sFLT-1 concentrations.

[0048] (II) Relevant indicators detected, such as Figures 1-22 The relevant experimental data are as follows:

[0049] 1. Comparison of nicotinamide (NAM) content in extracellular vesicles (EVs) of the placenta between the normal control group (NP) and patients with preeclampsia (PE) showed that the PE patient group had significantly lower levels.

[0050] 2. After intrauterine perfusion of PA or Ad Sh-QPRT, the level of NAM in placental EVs (pEVs) decreased;

[0051] 3. After intrauterine perfusion of PA or Ad Sh-QPRT, pregnant mice exhibited a series of PE-like symptoms, including increased embryo resorption rate, decreased fetal weight and crown-rump length, increased systolic blood pressure in the maternal tail artery, increased urinary UACR value, increased peripheral blood sFLT-1 concentration, decreased PIGF concentration, and increased sFLT-1 / PIGF ratio. Significant imbalance of the uterine immune microenvironment also occurred, such as an increase in the proportion of M1 macrophages, a decrease in the proportion of M2 macrophages, an increase in the proportion of Th1 cells, a decrease in the proportion of Th2 cells, an increase in the proportion of Th17 cells, and a decrease in the proportion of Treg cells.

[0052] Nicotinamide (NAM) is a water-soluble member of the B vitamin family and possesses significant anti-inflammatory effects. Studies have shown that NAM can significantly reduce embryo resorption rate and blood pressure in pregnant mice with PE (premature birth) models, prevent preterm birth, and improve fetal growth restriction. Supplementing pregnant mice with PA (polyacrylamide) via intrauterine perfusion with NP-pEVs containing a high concentration of NAM can alleviate PA-induced PE-like symptoms and immune imbalance, while supplementing with PA-pEVs containing low levels of NAM does not alleviate these symptoms.

[0053] The above experimental results of this invention show that inhibiting the activity of quinolinate phosphotransferase (QPRT) with phthalic acid or adenovirus can effectively reduce the level of NAM in trophoblasts and extracellular vesicles of trophoblasts, thereby causing pregnant mice to exhibit a series of PE-like symptoms, including increased embryo resorption rate, decreased fetal weight and crown-rump length, and increased systolic blood pressure of the maternal tail artery, urinary UACR value, and peripheral blood PIGF and sFLT-1 concentrations.

[0054] Therefore, this invention provides a novel animal model that induces a series of PE-like symptoms and a significant increase in the degree of inflammation of several major immune cells at the maternal-fetal interface in pregnant mice simply by intrauterine perfusion of PA or AdSh-QPRT. Recovery is achieved by supplementing with NAM-rich placental EVs. This model is simple to operate, and the intrauterine perfusion method allows the drug to be concentrated locally at the maternal-fetal interface, avoiding the systemic symptoms caused by intraperitoneal or intravenous injections. This model does not require complex surgical procedures or intervention in the mouse's genetic background, and it effectively simulates the clinical symptoms and indicators of PE patients, providing a novel mouse model for studying the pathogenesis and mechanism of preeclampsia.

Claims

1. A method for constructing a pre-eclampsia animal model, characterized by, The construction method comprises: administering phthalic acid or an adenovirus by uterine cavity perfusion to inhibit the activity of QPRT on the 10.5th day or the 16.5th day of pregnancy of a female mouse to obtain the preeclampsia animal model; the female mouse is an 8-week-old C57BL / 6 mouse; the administration amount of the phthalic acid is 50 mg / kg, and the administration amount of the adenovirus is 10 9 copies / ml.

2. The use of the preeclampsia animal model constructed by the method of claim 1 in the study of the pathogenesis of preeclampsia.

3. The use of the preeclampsia animal model constructed by the method of claim 1 in the preparation of a drug for preventing and treating preeclampsia, or in drug screening and efficacy evaluation.

4. The use of QPRT gene as a target in the preeclampsia animal model constructed by the method of claim 1.

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