A method for constructing a mouse model of abortion and its application

Abstract

The present invention provides a method for constructing an abortion mouse model, the method comprising constructing an abortion mouse model by specifically knocking out the Klk8 gene of mouse liver cells. The mouse model constructed by the present invention has phenotypes such as embryo absorption, abortion, and reduced embryo number, which are consistent with the symptoms and pathological process of human recurrent abortion, and has good controllability and stability. It can be used to study the occurrence and development mechanism and prevention and treatment measures of recurrent abortion, as well as for the development and research of new drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a method for constructing an abortion mouse model and an application thereof. Background Art

[0002] Pregnancy loss is defined as the spontaneous end of pregnancy before fetal survival, including all miscarriages from conception to 20-24 weeks of gestation. In contrast, the term "miscarriage" refers to the loss of an intrauterine pregnancy confirmed by ultrasound or histology within 20-24 weeks of gestation. It is challenging to determine the etiology of pregnancy loss, among which genetics, maternal age at conception, environment, and lifestyle factors may be associated with the risk of miscarriage. Population-based cohort studies have found that women who have miscarried are more likely to have a family history of miscarriage, and the frequency of miscarriage in siblings of women with recurrent miscarriage is twice that of the general population. Many studies have shown the impact of maternal age at conception on miscarriage rates, and age-related miscarriages are mainly caused by chromosomal errors (aneuploidy) in conception. These errors prevent further development and disruption of the maternal-fetal interface, both of which can cause bleeding, cramps, and miscarriage. Air pollution and endocrine-disrupting chemicals (including pollutants such as bisphenol A and phthalates that interfere with nuclear receptor signaling) may increase the risk of miscarriage. Smoking and drinking are also possible risk factors for miscarriage, and alcohol has been confirmed to be a risk factor for fetal developmental abnormalities. Other risk factors include structural abnormalities of the mother's uterus and autoimmune diseases, such as antiphospholipid syndrome (APS) and autoimmune liver disease (ALD). These autoimmune diseases increase the risk of pregnancy loss, but why they affect some but not all pregnancies is unclear.

[0003] When a foreign tissue is given to the host, the host will immediately induce an effective immune response to eliminate allogeneic antigens. In special cases, during pregnancy, the fetus is a semi-allogeneic transplant that is different from the maternal host, and the semi-allogeneic fetus enjoys permission to grow in the maternal uterus. In fact, the maternal immune system actively tolerates the presence of the fetus when it is aware of the presence of the fetus, so immune tolerance is essential for a successful pregnancy. The formation of the maternal-fetal interface is a common strategy used by mammals to maintain this immune tolerance, protect the fetus and promote its growth. The maternal-fetal interface refers to the maternal myometrium / decidua and the fetal placenta. First, a successful pregnancy begins with the implantation of a qualified embryo into the endometrium of the recipient pregnancy. In preparation for implantation, the endometrium undergoes remodeling, during which the endometrial luminal epithelium becomes permissive for the adhesion of the embryonic trophectoderm. This includes the spontaneous differentiation of stromal fibroblasts into more anti-inflammatory and stress-resistant decidual cells, as well as the accumulation of innate immune cells in the endometrium. After successful implantation, the placenta with a deep vascular network choroid begins to form, and the extravillous trophoblast cells migrate to the decidua and myometrium to reshape the maternal spiral arteries, thereby forming low-resistance, high-flow blood vessels required for placental blood supply to meet the increasing demand for placental perfusion in late pregnancy. This process is basically completed by the end of early pregnancy. Because the endometrium is the first site of direct contact between maternal immune cells and embryonic cells, there is close contact between trophoblast and fetal-derived cells and maternal cells during pregnancy. In addition to stromal cells, a large part of these cells (about 40%) are maternal immune cells, which may produce immune responses, especially rejection of foreign tissues. Therefore, it is very important to regulate the accumulation of immune cells in the uterine decidua before and after embryo implantation to ensure maternal tolerance to the embryo. If maternal tolerance decreases, it may lead to various pregnancy complications such as miscarriage and preeclampsia.

[0004] The liver is the largest solid organ in the human body. It is a key organ responsible for clearing toxins, producing proteins, and maintaining metabolic homeostasis. Interestingly, the liver is also highly immune active. Behind each functional unit lobule of the liver, there is a heterogeneous, complex, and carefully coordinated system. Although parenchymal cells are usually associated with the main functions of the liver, the liver is rich in innate and adaptive immune cells, and almost all types of immune cells can be found in liver tissue. Therefore, the immune microenvironment of the liver plays a central role in maintaining local and systemic immune homeostasis by propagating inflammation and coordinating anti-inflammation. Inflammatory processes and liver homeostasis are regulated in the liver by a large and diverse population of resident immune cells, as well as acute phase proteins, complement components, cytokines, and chemokines. The liver is mainly affected by circulatory and hormonal changes. During pregnancy, normal pregnancy causes changes in circulation, endocrine, immunity, and metabolism in healthy women. The circulating blood volume increases by 40% during pregnancy, and changes in hyperdynamic circulation are the basis for liver decompensation and deterioration. Therefore, it is not uncommon for liver diseases to occur during pregnancy. Liver lesions during pregnancy may be the result of pregnancy or may be unrelated to pregnancy. However, studies have found that the presence of severe liver disease during pregnancy is often associated with adverse pregnancy outcomes such as miscarriage, eclampsia, premature birth, and fetal growth restriction.

[0005] Due to the medical ethics restrictions on human experiments, animal models have become an important tool for studying the occurrence and development of miscarriage and exploring preventive measures. However, there is currently no animal model that can well reflect the severity of recurrent miscarriage. After successful modeling, the key phenotypes of human recurrent miscarriage may appear partially or completely. This miscarriage animal model provides a guarantee for the disease characteristics, pathogenesis, disease outcomes, research and development and screening of intervention strategies and new therapeutic drugs for recurrent miscarriage.

[0006] Patent application CN201010217681.9 relates to a method for constructing an animal model, specifically an animal experimental research model for the study of the pathogenesis, diagnosis and treatment of autoimmune recurrent spontaneous abortion and antiphospholipid antibody syndrome. An autoimmune recurrent spontaneous abortion mouse model was established by injecting human β2GP-1 into the uterine cavity of BALB / c mice. The method for constructing the animal model of the invention is safe, reliable, simple and economical, reduces the modeling time of the autoimmune RSA mouse model, improves the success rate and efficiency of modeling, and has good application prospects. However, the modeling method is not ideal in terms of safety and modeling efficiency.

[0007] Patent CN202311208218.1 previously applied by the applicant of the present invention provides a method for constructing an abortion mouse model, the method comprising constructing an abortion mouse model by specifically knocking out the Furin gene of mouse endometrial cells. The mouse model constructed by the invention has phenotypes such as embryo implantation failure, embryo absorption, abortion, and reduced embryo number, which are consistent with the symptoms and pathological processes of human recurrent abortion, and have good controllability and stability. It can be used to study the occurrence and development mechanism and prevention and control measures of recurrent abortion and embryo implantation failure, as well as for the development and research of new drugs. However, the patent knocks out genes in the reproductive system of mice, but the cause of many patients with recurrent abortion clinically is that there are diseases in their non-reproductive systems such as liver or kidneys, rather than diseases in the reproductive system. The specific pathogenesis of recurrent abortion caused by these liver and kidney diseases is not clear. However, in the prior art, there are only abortion mouse models related to "knockout genes of the mouse reproductive system", and there is no abortion mouse model related to "knockout genes of the non-reproductive system of mice". Therefore, there is a need in the art for a method for constructing a miscarriage mouse model related to "knockout of genes in the non-reproductive system of mice" for studying recurrent miscarriage associated with non-reproductive system diseases such as autoimmune liver disease, autoimmune kidney disease, other autoimmune diseases, and antiphospholipid antibody syndrome in patients, as well as for the development and research of corresponding new drugs.

[0008] Therefore, there is a need in the art for a new method for constructing a mouse model of abortion and its application. Summary of the invention

[0009] The present invention first provides a method for constructing an abortion mouse model. The method comprises constructing an abortion mouse model by specifically knocking out the Klk8 gene of mouse liver cells.

[0010] In a specific embodiment, loxP sequences are inserted into both ends of the mouse Klk8 gene to mark and effectively knock out the Klk8 gene. The resulting mice with loxP sequences inserted into both ends of the Klk8 gene are named loxP-flox-Klk8 mice, or Klk8 f / f Then, loxP-flox-Klk8 mice were mated with tool mice carrying Alb-Cre enzyme and Klk8 f / f The abortion mouse model, i.e., Klk8 f / f / Alb-Cre mice; the Alb is albumin, which is only expressed in the liver; the tool mice carrying the Alb-Cre enzyme can effectively and specifically knock out the target gene in the liver tissue.

[0011] In a specific embodiment, after obtaining the abortion mouse model, the model mouse Klk8 f / f / Alb-Cre female mice and model mice Klk8 f / f / Alb-Cre mice to breed more model mice Klk8 f / f Klk8 / Alb-Cre mice and control mice f / f Mouse.

[0012] In a specific embodiment, the male-female cage ratio is 2:1 or more.

[0013] In a specific embodiment, female Klk8 is initially selected. f / f The mice carry the flox sequence, while the male mice are tool mice carrying the Alb-Cre enzyme. f / f Compared with mice carrying the flox sequence and female mice carrying the Alb-Cre enzyme, this can make the mice more reproductively efficient.

[0014] In a specific embodiment, the mouse is an SPF grade C57BL / 6 mouse.

[0015] The present invention also provides an application of an abortion mouse model, that is, the abortion mouse model prepared by the above construction method is used for the development of drugs for treating and / or preventing recurrent abortion in animals and humans.

[0016] The present invention also provides a method for screening drugs for treating and / or preventing recurrent spontaneous abortion in animals and humans, the method comprising the following steps: 1) administering the drug to be tested to the mouse model constructed by the above construction method; 2) analyzing and evaluating the therapeutic effect of the drug to be tested, and selecting the test drug that can significantly improve the pathological characteristics of the model mice.

[0017] The present invention has at least the following beneficial effects:

[0018] 1. The present invention uses a mouse model to simulate the natural state of the human body to conduct experiments to study the pathogenesis and treatment, which reduces the necessity of conducting experiments on humans and avoids the ethical disputes of human experiments.

[0019] 2. The present invention constructs an abortion mouse model by specifically knocking out the Klk8 gene in mouse liver cells. The mouse survival rate is high and the knockout efficiency is high. After successful construction, the phenotype is stable, and the symptoms will not be alleviated or disappear with the increase of time and mating number.

[0020] 3. The pathological manifestations of the abortion model in the invention include embryo absorption, abortion, reduced embryo number, and enlarged spleen, etc. It is consistent with the pathological changes of human recurrent abortion, can better simulate the natural disease state of the human body, and has good representativeness.

[0021] 4. The time of abortion in the mouse model of the present invention occurs in the second trimester of pregnancy, which is conducive to shortening the observation and experimental cycle.

[0022] 5. The present invention can verify and observe gene knockout from multiple angles such as genes, proteins, tissues and organs, and the experimental results are real, credible and convincing.

[0023] 6. The abortion mouse model of the present invention specifically knocks out the KLK8 gene in the liver. The present invention does not directly knock out the gene of the mouse reproductive system, but knocks out the gene of its liver. The abortion mouse model obtained by the construction method of the present invention is suitable for studying recurrent abortion related to non-reproductive system diseases such as autoimmune liver disease, autoimmune kidney disease, other autoimmune diseases and antiphospholipid antibody syndrome in patients, as well as for the development and research of corresponding new drugs.

[0024] In general, the present invention constructs a mouse model of abortion by conditionally knocking out the Klk8 gene of mouse liver cells. The mouse model has phenotypes such as embryo absorption, abortion, reduced embryo number, and enlarged spleen, which are consistent with the symptoms and pathological process of human recurrent abortion, and has good controllability and stability. It can be used to study the occurrence and development mechanism and prevention and treatment measures of recurrent abortion, as well as for the development and research of new drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The figure is a schematic diagram of a method for constructing a miscarriage mouse model.

[0026] Figure 2 Schematic diagram of inserting loxP sequences into both ends of the mouse Klk8 gene for marking and effectively knocking out the Klk8 gene.

[0027] Figure 3 This is a validation diagram of the Klk8 gene in the constructed mouse model.

[0028] Figure 4 This is the Alb-Cre gene validation diagram of the constructed mouse model.

[0029] Figure 5 Protein electrophoresis of Klk8 and internal reference in liver tissue of model mice and control mice.

[0030] Figure 6 Schematic diagram of the protein expression ratio of Klk8 to the internal reference in liver tissue of control mice and model mice.

[0031] Figure 7 Klk8 immunohistochemical staining of liver tissues of control mice and model mice.

[0032] Figure 8 Photos of the reproductive systems of model mice and control mice.

[0033] Fig. 9 The figure is a comparison of the number of embryos absorbed by control mice and model mice.

[0034] Fig.10 A comparison chart of the litter sizes of control mice and model mice.

[0035] Fig.11 Photos of spleens of control mice and model mice.

[0036] Fig.12 The figure is a comparison of spleen weights between control mice and model mice. DETAILED DESCRIPTION

[0037] In the present invention, loxP sequence and flox sequence represent the same meaning.

[0038] Kallikrein-related peptidases (KLKs) are a subclass of the serine protease family, mainly divided into two categories: plasma kallikrein and tissue kallikrein. Tissue kallikrein-related peptidases are a class of compounds with similar properties, which can be synthesized by the liver, pancreas, cranial nerves, etc. The currently recognized tissue kallikrein genes are composed of at least 15 members (KLK1 to KLK15). The 15 genes are arranged in sequence and do not interfere with each other. The gene sequence, protein level, and tertiary structure of each member are very similar. And these 15 tissue kallikrein-related peptidase genes are widely expressed in different tissues. The protein encoded by the tissue kallikrein gene is first expressed to generate a precursor complete protein. After the precursor protein is cleaved and processed by proteases in the body, it becomes a mature protein with biological activity and participates in a series of physiological and pathological processes in the body. One of the kallikrein-related peptidases, Klk8, was originally cloned as a neuroprotease in the mouse brain. During development, Klk8 mRNA is expressed in large quantities in the developing heart, lung, pituitary, choroid plexus, epithelial lining of the skin and central nervous system. Studies have found that Klk8 can cleave the extracellular domain of endothelial cadherin, thereby promoting the nuclear translocation of plasmoglobin. This leads to cardiac interstitial and perivascular cardiac fibrosis, endothelial damage and endothelial-to-mesenchymal transformation. It aggravates diabetic cardiac fibrosis and impairs cardiac function in diabetic mice. Klk8 is also widely distributed in the liver. As a secreted serine protease, Klk8 has been shown to cleave a variety of substrates. Klk8 induces the pathogenesis of schizophrenia, mood and anxiety disorders, Alzheimer's disease and other diseases by cleaving and activating synaptic adhesion molecule L1 (L1CAM), ephedrine-type B receptor 2 (EPHB2), neuregulin 1 (NRG1), etc. Klk8 cleaves urokinase-type plasminogen activator receptor (uPAR), protease-activated receptor (PARS), metalloproteinases, etc., and participates in processes such as cell migration, cell cycle regulation, and cell adhesion, thereby participating in a variety of disease processes such as tumors and inflammation. Klk8 cleaves the chemokine CCL14, which acts as a chemoattractant and activator of different leukocytes and is therefore involved in the development of many inflammatory diseases.

[0039] Studies on the pregnancy process of mice have found that after successful mating, implantation and decidualization begin at about 4.5 days of pregnancy, placenta formation begins at 8.5 days, and the placenta is fully formed after 12.5 days. This series of processes is regulated by multiple factors. Therefore, there are many reasons for pregnancy failure, which may be implantation failure or loss during pregnancy after implantation. There are many causes of pregnancy loss, including implantation and decidualization, placenta formation failure, immune tolerance imbalance, hormone imbalance, genetic factors, abnormal uterine structure of the mother itself, and autoimmune diseases. But the mechanism is still unclear. One of the most common views is the imbalance of immune tolerance at the maternal-fetal interface. During pregnancy, leukocyte subsets change, and leukocytes infiltrating in the pregnancy tissue form an inflammatory microenvironment, affecting specific effects, and participating in the coordinated sequence of reproductive events in a coordinated manner, ultimately ending with the maintenance of pregnancy, the start of delivery, fetal delivery, and uterine degeneration. Liver-specific Klk8 knockout mice were constructed using loxp-CRE technology to explore the effects of abnormal liver Klk8 expression at the overall level. The study found that liver-specific Klk8 knockout mice and control groups did not show differences under normal circumstances. However, the study found that the number of pups in female mice with liver Klk8 knockout was less than that in controls. Further research found that the phenotypes of this genetically modified pregnant mouse at 13.5 days of pregnancy were embryo resorption, miscarriage, reduced embryo number and enlarged spleen, but there were no abnormalities in its delivery time, fetal placental weight, absorption point, liver function indicators and liver morphology. And conversely, this phenomenon did not occur when the genetically modified male mice mated with C57BL / 6 female mice.

[0040] The present invention aims to provide a method for constructing a mouse model of abortion by specifically knocking out the Klk8 gene of mouse liver cells, which can better restore the pathological manifestations of the disease, is consistent with the symptoms and pathological process of human recurrent abortion, has good controllability and stability, and can be used to study the occurrence and development mechanism of recurrent abortion, preventive measures, and new drug research.

[0041] The present invention provides a method for constructing an abortion mouse model, wherein loxP sequences are inserted into both ends of the mouse Klk8 gene to mark and effectively knock out the Klk8 gene, and the obtained mice having loxP sequences inserted into both ends of the Klk8 gene are named loxP-flox-Klk8 mice, or Klk8 f / f Then, loxP-flox-Klk8 mice were mated with tool mice carrying Alb-Cre enzyme and backcrossed to purify, thereby obtaining the abortion mouse model, namely Klk8 f / f / Alb-Cre mice. The genotype of mice is usually determined by clipping the toes or tails of mice at 4 weeks of age. f / fThe expression levels of Klk8 protein and mRNA in the liver tissue of / Alb-Cre mice were measured to verify the knockout of Klk8 gene in mouse liver cells and to further determine whether the abortion mouse model was successfully constructed. f / f Female mice with Klk8 f / f / Alb-Cre mice are caged together to breed more model mice. Specifically: male mice of model mice are selected to mate with female mice to give birth to more model mice and control mice. The genotype of the offspring mice can be verified at about 4 weeks after birth, and the Klk8 gene is identified as wild type (neither of the two paired DNA chains has a flox sequence inserted at both ends of its Klk8 gene), heterozygous (only one of the two paired DNA chains has a flox sequence inserted at both ends of its Klk8 gene), or f / f type (both paired DNA chains have a flox sequence inserted at both ends of its Klk8 gene), and f / f type mice are selected to analyze their Alb-Cre genotype to identify whether they are model mice or control mice. The Klk8 gene of the control mouse is f / f type, and the Alb-Cre genotype is negative, so the Klk8 gene is not knocked out in this mouse. Controlling the male-female cage ratio of 2:4 can maximize the mating success rate, thereby ensuring the orderly progress of the model construction experiment and avoiding the problem of prolonged experimental cycle caused by long-term mating failure during the experimental process.

[0042] Albumin (ALB) is a serum protein that is only expressed in the liver and accounts for 5% to 10% of the newly synthesized proteins in the liver. This strain of mice can effectively knock out the target gene specifically in liver tissue. f / f The / Alb-Cre mouse model can specifically knock out the Klk8 gene in mouse hepatocytes, thereby providing a reliable model for clarifying the mechanism of the Klk8 gene in the occurrence and development of recurrent spontaneous abortion.

[0043] The present invention is further described in conjunction with the accompanying drawings and the following embodiments, but the protection scope of the present invention is not limited to the following embodiments.

[0044] Example 1

[0045] Figure 1 and Figure 2 The flowchart of the construction of the mouse model of liver cell-specific Klk8 gene knockout in the present invention is as follows. SPF-grade C57BL / 6 background mice were selected, loxP sequences were first inserted at both ends of the Klk8 gene, and the target gene with loxP fragments was integrated into the original genome to mark and knock out the Klk8 gene, thereby obtaining loxP-flox-Klk8 mice, namely Klk8 f / fThen, loxP-flox-Klk8 mice were mated with tool mice carrying Alb-Cre enzyme and backcrossed to purify, thus obtaining a mouse model with liver cell-specific knockout of Klk8 gene, namely Klk8 f / f / Alb-Cre mice.

[0046] Figure 3 and Figure 4 This is a genetic validation diagram of the constructed mouse model. Figure 3 This is the Klk8 gene verification diagram. Figure 4 This is the Alb-Cre gene validation diagram. Figure 3 The display shows the nucleic acid gel electrophoresis to verify the genotype of the mouse Klk8 gene. The rightmost column is the bright band of the standard reagent, which is used to indicate the size of the nucleic acid. The 1st-2nd column from the right is the blank control; the 3rd-4th column is the wild type, that is, no loxP sequence is inserted at both ends of the Klk8 gene of the two DNA chains, and the nucleic acid size of the two chains is 446bp; the 5th-9th column is the homozygous f / f, that is, loxP sequence is inserted at both ends of the Klk8 gene of the two DNA chains, and the nucleic acid size of the two chains is 595bp. The homozygous Klk8 gene was selected, and the genotype of its Alb-Cre gene was verified by nucleic acid electrophoresis. The verification results are shown in Figure 4 . Figure 4 In the figure, the rightmost column is the bright band of the standard reagent, which is used to indicate the size of the nucleic acid. The first column from the right is the blank control; the second column is the wild type; the third to sixth columns are mice with negative Alb-Cre genotype; the seventh to eleventh columns are Alb-Cre genotype positive abortion mouse models, and the nucleic acid size is 390bp. These mice with homozygous Klk8 gene (i.e. f / f type) and negative Alb-Cre genotype (the Klk8 gene on liver cells has not been knocked out) can all be used as control mice for the mouse model of the present invention. Figure 3 and Figure 4 It can be seen that the mouse model Klk8 obtained by the present invention f / f The genotype of / Alb-Cre mice is homozygous for the Klk8 gene and carries the Alb-Cre gene.

[0047] Figure 5 Model mouse Klk8 f / f Klk8 / Alb-Cre mice and control mice f / f Protein electrophoresis of Klk8 and internal reference in mouse liver tissue, the first row is a schematic diagram of Klk8 protein, its molecular weight is about 28kDa; the second row is a schematic diagram of its β-actin, the internal reference protein, its molecular weight is about 42kDa. The six bands on the right side of the first row correspond to the six mice, all of which are control mice with homozygous Klk8 gene (i.e. f / f type) and negative Alb-Cre genotype (i.e. Klk8 f / fThe protein electrophoresis of mice shows that the protein expression level is high; the six bands on the left in the first row correspond to the six mice, all of which are model mice with homozygous Klk8 gene (i.e. f / f type) and positive Alb-Cre genotype (i.e. Klk8 f / f The protein electrophoresis of WT / Alb-Cre mice shows that the expression of Klk8 protein in the liver tissue of these mice is reduced or even disappeared.

[0048] Figure 6 Schematic diagram of the protein expression ratio of Klk8 in liver tissue of control mice and model mice to that of the internal reference. Figure 6 for Figure 5 The data are shown in Figure 1. Each data point in the figure represents a mouse. Figure 6 It can be seen that the protein expression level in the liver tissue of the control mice was significantly higher than that in the model mice, and there was a statistical difference between the two, and p<0.01 (** represents p<0.01).

[0049] Figure 7 The left side shows the Klk8 immunohistochemical staining of the liver tissues of control mice and model mice. f / f Mouse) 200 times magnified liver tissue Klk8 immunohistochemical staining, the right side is the model mouse (i.e. Klk8 f / f Klk8 immunohistochemical staining of liver tissue of WT / Alb-Cre mice (200 times magnified). Figure 7 As shown, with Klk8 f / f Compared with mice, Klk8 f / f Klk8 expression was significantly decreased in / Alb-Cre mice.

[0050] from Figures 5 to 7 It can be seen that protein immunoblotting and immunohistochemical staining showed that compared with control mice, conditional knockout mice, namely Klk8 f / f The expression level of Klk8 in liver tissue of / Alb-Cre mice was significantly decreased.

[0051] Figure 8-10 The following are the general diagrams of the reproductive system of the control mouse and the model mouse at day 13.5 of pregnancy and the corresponding data diagrams. Figure 8 The above is a model mouse (Klk8 f / f / Alb-Cre mice). Arrows indicate resorbed embryos; Figure 8 The following are control mice (i.e., Klk8 f / f Mouse) reproductive system. Figure 9-10 for Figure 8 The data reflects Fig. 9 It is the data of the number of embryos absorbed (i.e. the number of aborted litters). Fig.10The data of litter size is shown in Figure 1. Each data point in the figure represents one mouse. Figures 8 to 10 As shown, compared with control mice, Klk8 f / f / Alb-Cre mice showed phenotypic changes of embryo resorption and abortion. Fig. 9 and Fig.10 Each square represents a control mouse (i.e., Klk8 f / f mice), each dot represents a model mouse (i.e., Klk8 f / f / Alb-Cre mice). Fig. 9 It can be seen that among the 12 control mice, 8 had zero absorption points, 2 had 1 absorption point, and 2 had 2 absorption points, with an average of 0.5 absorption points. The absorption points of the 13 model mice ranged from 0 to 4, with an average of 1.85 absorption points. There was a statistically significant difference between the two, and p<0.01 (** represents p<0.01). Fig.10 As can be seen, the litter size of the 12 control mice ranged from 6 to 10, with an average of 8.17 pups; while the litter size of the 13 model mice ranged from 5 to 9, with an average of 5.62 pups; there was a statistically significant difference between the two, and p<0.01 (** represents p<0.01).

[0052] Figure 11-12 The following are the gross images and corresponding data of the spleen of the control mice and model mice at 13.5 days of pregnancy. Fig.11 The right side shows the model mouse (Klk8 f / f / Alb-Cre mice) spleen; Fig.11 The left side shows the control mice (i.e., Klk8 f / f Mouse) spleen. Fig.12 for Fig.11 The data show that each data point in the figure represents a mouse; there is a statistical difference between the two, and p<0.05 (* represents p<0.05). Figures 11-12 As shown, compared with control mice, Klk8 f / f / Alb-Cre mice showed phenotypic changes of spleen enlargement.

[0053] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions and substitutions can be made without departing from the concept of the present invention, which should be regarded as belonging to the protection scope of the present invention.

Claims

1. A method for constructing an abortion mouse model, characterized in that: The method comprises knocking out mouse liver cells by specific Klk8 gene, constructing a mouse model of abortion; and specifically comprising inserting loxP sequence into mouse Klk8 Both ends of the gene are used to mark and effectively knock out Klk8 Gene, obtained in Klk8 Mice with loxP sequences inserted at both ends of the gene are named loxP-flox- Klk8 Mouse, or Klk8 f / f mice; then, loxP-flox- Klk8 Mice and carriers Alb -Cre enzyme-expressing mice were mated and Klk8 f / f The mice were backcrossed and purified to obtain the abortion mouse model, i.e. Klk8 f / f / Alb -Cre mice; Alb That is albumin, which is expressed only in the liver; Alb ‐Cre enzyme tool mice can effectively knock out target genes specifically in liver tissue.

2. The method for constructing an abortion mouse model according to claim 1, characterized in that: After obtaining the abortion mouse model, the model mice Klk8 f / f / Alb -Cre female mice and model mice Klk8 f / f / Alb -Cre mice to breed more model mice Klk8 f / f / Alb -Cre mice.

3. The method for constructing an abortion mouse model according to claim 2, characterized in that: The ratio of male to female in the cage should be 2:1 or above.

4. The method for constructing an abortion mouse model according to any one of claims 1 to 3, characterized in that: Select females at the beginning Klk8 f / f Mice carry the flox sequence, while male mice carry Alb -Cre enzyme tool mice.

5. The method for constructing an abortion mouse model according to any one of claims 1 to 3, characterized in that: The mice are SPF grade C57BL / 6 mice.

6. An application of an abortion mouse model, characterized in that: The abortion mouse model prepared by the construction method according to any one of claims 1 to 5 is used for screening drugs for treating and / or preventing recurrent abortion in animals and humans.

7. A method for screening drugs for treating and / or preventing recurrent spontaneous abortion in animals and humans, characterized in that: The method comprises the following steps: 1) administering the drug to be tested to the mouse model constructed by the construction method according to any one of claims 1 to 5; 2) Analyze and evaluate the therapeutic effects of the drugs to be tested, and select the test drugs that can significantly improve the pathological characteristics of the model mice.

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