A method for constructing a spontaneous endometrial cancer mouse model and its application

Abstract

The present invention belongs to the technical field of animal model construction, and particularly relates to a method for constructing a spontaneous endometrial cancer mouse model and its application. The construction method includes constructing a mouse model with the genotype Cre; PTEN<supgt;flox / ‑< / supgt>; STK11<supgt;flox / flox< / supgt> through the Cre / loxP system. After activating the expression of Cre recombinase in uterine epithelial cells of this mouse model, the PTEN gene and the STK11 gene are selectively knocked out, thereby spontaneously inducing the occurrence of endometrial cancer. By selectively deleting the PTEN gene and the STK11 gene in uterine epithelial cells, the present invention rapidly changes the uterine pathology, and has the advantages of short tumorigenesis time and high tumorigenesis rate. Moreover, due to the use of in-situ tumorigenesis technology, the mouse model of the present invention can simulate the growth environment of human endometrial cancer, and the histological characteristics of the tumor are extremely close to those of human endometrial cancer, meeting the biological characteristics of human endometrial cancer.

Description

Technical Field

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

[0002] Endometrium cancer (EC) is a group of epithelial malignant tumors that occur in the endometrium. It is common in women before and after menopause and is one of the most common malignant tumors of the female reproductive system. Due to the aging of the human population and the increase in the number of obese people, its incidence rate has been increasing year by year worldwide. It is currently second only to cervical cancer and ranks second among malignant tumors of the female reproductive system. Endometrial cancer is most common in the form of adenocarcinoma originating from endometrial glands. Its pathogenic mechanism is related to the abnormal expression of oncogenes, tumor suppressor genes, and DNA damage repair genes. Animal models are essential for the treatment and mechanism research of endometrial cancer. Establishing an ideal animal model of endometrial cancer is a prerequisite for exploring its occurrence mechanism and screening effective treatment methods.

[0003] Compared with the diversity and high heterogeneity of endometrial cancer phenotypes, the animal models that have been established are still very limited, and the corresponding endometrial cancer models that are actually important research tools are even rarer. At present, the establishment of endometrial cancer transplanted tumor animal models mainly includes two methods: tissue block transplantation and endometrial cancer cell line inoculation. This method is simple to operate, but it is a non-in situ tumor and cannot provide an in situ microenvironment of endometrial tissue, which may lead to the loss of relevant biological characteristics of human tumors during the experiment and cannot simulate the situation in the human body. With the continuous deepening of research on the construction of animal models by genetic engineering methods, there are literatures that study the relationship between MTS1 / p16 gene deletion or p53 / LKBl gene deletion and the occurrence and development of endometrial cancer, and construct corresponding endometrial cancer mouse models. In recent years, new gene markers have continued to emerge, such as PTEN, and mice with conditional intrauterine PTEN deletion (Pgr Cre / + ;PTEN flox / flox ) has a high prevalence of well-differentiated endometrial adenocarcinoma within 90 days. Therefore, it is very necessary and of practical significance to establish a new animal cell model of endometrial cancer to enrich and complete the endometrial cancer model library and provide a suitable in vitro model for studying tumor heterogeneity to promote basic and clinical research on endometrial cancer. Summary of the invention

[0004] In view of the problems existing in the prior art, the present invention provides a method for constructing a spontaneous endometrial cancer mouse model and its application, aiming to solve some of the problems in the prior art or at least alleviate some of the problems in the prior art.

[0005] The present invention is specifically implemented through the following technical solutions:

[0006] The present invention provides a method for constructing a spontaneous endometrial cancer mouse model, and uses the Cre / loxP system to construct a genotype of Cre; PTEN flox / - ;STK11 flox / flox A mouse model; after activating the expression of Cre recombinase in uterine epithelial cells, the mouse model selectively knocks out the PTEN gene and the STK11 gene, spontaneously inducing the occurrence of endometrial cancer.

[0007] Furthermore, the genotype was constructed as Ltf-Cre; PTEN through the Ltf-Cre / loxP system. flox / - ;STK11 flox / flox Mouse model.

[0008] Furthermore, the method for constructing the spontaneous endometrial cancer mouse model comprises the following steps:

[0009] S1. PTEN flox / flox Genotype of mice with STK11 flox / flox Mating of genotyped mice and screening for PTEN flox / flox ;STK11 flox / flox Genotype of F1 mice;

[0010] S2. PTEN flox / flox ;STK11 flox / flox The F1 mice of the same genotype were mated with Ltf-Cre mice, and LTF-Cre; PTEN flox / - ;STK11 flox / - Genotype of F2 mice;

[0011] S3, Ltf-Cre; PTEN flox / - ;STK11 flox / - Genotype of F2 mice and PTEN flox / flox ;STK11 flox / flox The F1 generation mice of the same genotype were mated and screened for Ltf-Cre; PTEN flox / - ;STK11 flox / flox Genotypes of F3 mice.

[0012] Further, in step S1, the PTEN is selected flox / flox The genotype of the mice was the male parent, the STK11 flox / flox Mice of the same genotype were mated with the mother; or the PTEN flox / flox Genotype is maternal, STK11 flox / floxThe genotype mice are mated in the same cage with the father; wherein the ratio of the number of the father to the number of the mother is preferably 1:2.

[0013] Further, in step S2, PTEN is selected flox / flox ;STK11 flox / flox The F1 female mice of the same genotype were mated with LTF-Cre male mice in the same cage, with the ratio of sire to dam being 1:2.

[0014] Further, in step S3, Ltf-Cre; PTEN flox / - ;STK11 flox / - Genotype of F2 male mice and PTEN flox / flox ;STK11 flox / flox The F1 generation female mice of the same genotype were mated in the same cage and screened for Ltf-Cre; PTEN flox / - ;STK11 flox / flox For the F3 generation female mice of the same genotype, the ratio of the number of paternal and maternal parents is 1:2.

[0015] Furthermore, when Ltf-Cre; PTEN flox / - ;STK11 flox / - Genotype of F2 mice and PTEN flox / flox ;STK11 flox / flox After mating with the same F1 generation mice, Ltf-Cre; PTEN flox / - ;STK11 flox / flox Before genotyping the F3 mice, a PCR verification step was also included.

[0016] Furthermore, the primer sequences for PCR verification are shown in SEQ ID NO.1-6.

[0017] In addition, the present invention also provides the method for constructing the spontaneous endometrial cancer mouse model as described above, and the use of the spontaneous endometrial cancer mouse model constructed in studying the occurrence mechanism and drug resistance mechanism of endometrial cancer or screening drugs for treating endometrial cancer.

[0018] The advantages and positive effects of the present invention are:

[0019] The present invention uses the Cre / loxP system to construct a genotype of Cre; PTEN flox / - ;STK11 flox / flox Mouse model, when Cre recombinase is activated and expressed, Cre; PTEN flox / flox ;STK11 flox / floxThe genotype mice will selectively lack the PTEN gene and STK11 gene of uterine epithelial cells, causing rapid changes in uterine pathology and spontaneously inducing the occurrence of endometrial cancer. It has the advantages of short tumor formation time and high tumor formation rate. Moreover, due to the use of in situ tumor formation technology, the mouse model of the present invention can simulate the growth environment of human endometrial cancer. The tumor histological characteristics are very close to those of human endometrial cancer and are consistent with the biological characteristics of human endometrial cancer. It provides valuable experimental tools and suitable animal models for exploring the cause, pathogenesis, objective evaluation and diagnosis, treatment effects and preventive measures of endometrial tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0021] Figure 1 The electrophoresis detection result of the STK11 gene in the embodiment of the present invention;

[0022] Figure 2 This is a graph showing the electrophoresis detection result of the Cre gene according to an embodiment of the present invention;

[0023] Figure 3 This is a diagram showing the electrophoresis detection result of the PTEN gene of an embodiment of the present invention;

[0024] Figure 4 A general diagram of the uterine anatomy of the mouse model of an embodiment of the present invention;

[0025] Figure 5 PTEN is the control group of the present invention flox / - ;STK11 flox / flox Uterine morphology of mice of different genotypes;

[0026] Figure 6 LTF-Cre; PTEN flox / - ;STK11 flox / flox Uterine morphology of genotype mouse models;

[0027] Figure 7 PTEN is the control group of the present invention flox / - ;STK11 flox / flox Uterine pathophysiology diagrams at different resolutions of genotype mice;

[0028] Figure 8 LTF-Cre; PTEN flox / - ;STK11 flox / floxDiagram of uterine pathophysiology at different resolutions in genotype-specific mouse models. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with the examples. The equipment and reagents used in each embodiment and test example can be obtained from commercial sources unless otherwise specified. The specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0030] Based on the information contained in this application, it is easy for those skilled in the art to make various changes to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the defined process, properties or components, because these embodiments and other descriptions are only for illustrating specific aspects of the present invention. In fact, various changes that the technicians in this field or related fields can obviously make to the embodiments of the present invention are all covered within the scope of the appended claims.

[0031] In order to better understand the present invention but not to limit the scope of the present invention, all the numbers used in this application to express the amount, percentage, and other numerical values ​​should be understood as modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may be changed according to the different ideal properties attempted to be obtained. Each numerical parameter should at least be regarded as obtained based on the reported significant figures and by conventional rounding methods.

[0032] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0033] The embodiment of the present invention provides a method for constructing a spontaneous endometrial cancer mouse model, which uses the Cre / loxP system to construct a genotype of Cre; PTEN flox / - ;STK11 flox / flox A mouse model; after activating the expression of Cre recombinase in uterine epithelial cells, the mouse model selectively knocks out the PTEN gene and the STK11 gene, spontaneously inducing the occurrence of endometrial cancer.

[0034] In the context of the present invention, PTEN (Phosphatase and tensin homolog) refers to phosphatase and tensin homolog, which is a protein encoded by the PTEN gene in the human body. The PTEN gene is a tumor suppressor gene that mutates in a variety of human cancers, including endometrial adenocarcinoma. PTEN inhibits the activity of the phosphoinositide 3-kinase (PI3K)-AKT signaling pathway by dephosphorylating phosphatidylinositol-triphosphate (PIP3) involved in AKT activation. The PI3K-AKT signaling pathway regulates a variety of cellular functions, including but not limited to growth, survival and metabolism, and its abnormal activation is associated with cancer development. The loss of PTEN function caused by PTEN gene mutation and expression loss is an early molecular event in endometrial cancer and plays an important role in the development of endometrial hyperplasia to endometrial precancerous lesions. Heterozygous PTEN mice can also develop tumors in multiple organs.

[0035] In the context of the present invention, STK11 (serine / threonine kinase 11) refers to serine / threonine kinase. The STK11 gene encodes serine / threonine kinase and is a tumor suppressor gene. Studies have found that the loss of the STK11 gene is an important cause of endometrial cancer and its late transformation.

[0036] It should be noted that Cre; PTEN flox / - ;STK11 flox / flox Genotype mice refer to: for the PTEN gene, one of the two chromosomes of the mouse carries the PTEN gene and does not carry the LoxP site (sequence), and the other chromosome carries PTEN flox Allele, that is, the other chromosome (PTEN flox Alleles) carry the PTEN gene and carry LoxP sites (sequences) at both ends of the PTEN gene (i.e., upstream and downstream of the gene); for the STK11 gene, both chromosomes of the mouse carry STK11 flox / flox Alleles, that is, both chromosomes carry the PTEN gene and carry LoxP site sequences at both ends of the PTEN gene. At the same time, the mouse contains the Cre gene, and Cre is expressed under control. When the Cre recombinase is highly expressed, the Cre recombinase will mediate the specific recombination between the two LoxP sites, so that the gene sequence between the LoxP sites is deleted, thereby achieving double knockout of the PTEN gene and the STK11 gene.

[0037] In addition, it should be noted that gene knockout refers to a technique of inactivating or deleting a specific gene in an organism by certain means. In the context of the present invention, knockout and deletion have the same meaning.

[0038] In the present invention, the PTEN gene and STK11 gene of uterine epithelial cells are selectively deleted, while the expression of related genes in other tissues is not affected, thereby constructing mice with double deletion of PTEN and STK11 genes in uterine epithelial cells. In the experiment, it was found that after the Cre recombinase was activated and expressed, Cre; PTEN flox / flox ;STK11 flox / flox Genotype mice died rapidly when the aforementioned gene double deletion was achieved. Correspondingly, Cre; PTEN flox / - ;STK11 flox / flox The genotype mice have a stable life cycle after achieving the aforementioned double deletion of the genes, and after the PTEN gene and the STK11 gene are knocked out, the pathology of the epithelial cells changes rapidly, spontaneously inducing the occurrence of endometrial cancer, with a short tumor formation time and a high tumor formation rate. Within one month, obvious changes in the gross appearance of the uterus can be seen with the naked eye. Moreover, due to the use of in situ tumor formation technology, the mouse model of the present invention can simulate the growth environment of human endometrial cancer, and the tumor histological characteristics are extremely close to those of human endometrial cancer, which is consistent with the biological characteristics of human endometrial cancer, providing valuable experimental tools and suitable animal models for exploring the cause, pathogenesis, objective evaluation and diagnosis, treatment effects and preventive measures of endometrial tumors.

[0039] In order to better achieve uterine epithelial cell-specific gene knockout, the selective knockout of PTEN and STK11 genes described above is specifically achieved through the Ltf-Cre / loxP system, that is, the expression of Cre recombinase is regulated by the lactoferrin (Ltf) promoter, and the genotype obtained is Ltf-Cre; PTEN flox / - ;STK11 flox / flox Mouse model.

[0040] In this embodiment, the Cre / loxP recombinase system is used to control the expression period and expression site of the Cre recombinase in combination with the lactoferrin (Ltf) promoter to achieve selective knockout of the PTEN gene and STK11 gene of mouse uterine epithelial cells in a specific spatiotemporal context. The uterus is a complex organ with three main tissue compartments: myometrium, stroma and epithelium, in which the coordination of estrogen and progesterone regulates the proliferation and differentiation of these uterine cell types. The instability of this synergistic effect can cause various gynecological diseases, including but not limited to endometriosis, endometrial cancer, etc. There are four conventional Cre mice that are used for conditional knockout of endometrial cancer genes. The present invention selects lactoferrin (Ltf) promoter as the uterine epithelium-specific Cre gene transcription promoter. This Ltf-Cre strain mouse is highly sensitive to estrogen in the uterus, and the Ltf promoter is not expressed in the immature mouse uterus, but is strongly expressed only in the uterine epithelium of adult mice and causes the Cre recombinase to be activated. Therefore, the specific expression of the Cre recombinase in the uterine epithelium can be achieved. When mice grow to about 2 months old, the Ltf promoter is activated. At the same time, a large amount of Cre recombinase is produced, knocking out the PTEN gene and STK11 gene in the endometrial epithelium, and in situ mouse endometrial adenocarcinoma can be induced at 3 months of age. The gene recombination efficiency of Ltf-Cre mice in uterine epithelial cells is close to 100%, starting in adulthood at 1-2 months old, which is more in line with the period of occurrence and development of human endometrial cancer. In addition, mice with constitutive deletion of Ltf are viable and fertile, and the Ltf-Cre strain of mice can be used for subsequent breeding.

[0041] Screening the target genotype mice by hybridization is helpful to obtain a mouse model with stable traits. Preferably, the present invention adopts PTEN gene flox homozygous mice (i.e. PTEN flox / flox genotype mice) and STK11 gene flox homozygous mice (i.e. STK11 flox / flox genotype mice) and screened for PTEN flox / flox ;STK11 flox / flox Genotype mice were then mated with LTF-Cre mice to screen for the target genotype mice (Ltf-Cre; PTEN flox / - ;STK11 flox / flox ). Specifically, the following steps are included:

[0042] S1. PTEN flox / flox Genotype of mice with STK11 flox / flox Mating of genotyped mice and screening for PTEN flox / flox ;STK11 flox / flox Genotype of F1 mice;

[0043] S2. PTEN flox / flox ;STK11 flox / flox The F1 mice of the same genotype were mated with Ltf-Cre mice, and LTF-Cre; PTEN flox / - ;STK11 flox / - Genotype of F2 mice;

[0044] S3, Ltf-Cre; PTEN flox / - ;STK11 flox / - Genotype of F2 mice and PTEN flox / flox ;STK11 flox / flox The F1 generation mice of the same genotype were mated and screened for Ltf-Cre; PTEN flox / - ;STK11 flox / flox The genotype of the F3 generation mice is the spontaneous endometrial cancer mouse model of the present invention.

[0045] In step S1, the PTEN is selected flox / flox The genotype of the mice was the male parent, the STK11 flox / flox Mice of the same genotype were mated with the mother; or the PTEN flox / flox Genotype is maternal, STK11 flox / flox The genotype mice are mated in the same cage with the father, wherein the ratio of the father to the mother is preferably 1:2. flox / flox ;STK11 flox / flox Genotype of F1 mice can be obtained LTF-Cre; PTEN flox / - ;STK11 flox / - Genotype mice with shortened reproductive cycle.

[0046] Preferably, in step S2, PTEN is selected flox / flox ;STK11 flox / flox The F1 female mice of the same genotype were mated with LTF-Cre male mice in the same cage, with the ratio of sire to dam being 1:2.

[0047] Like most Cre lines, Ltf-Cre may cause the loss of target genes in other unwanted tissues, which may lead to unexpected phenotypes. In the breeding process, the present invention first uses resuscitated Ltf-Cre male mice as a breeding scheme. Frozen and ice-cut tissue chemistry experiments found that variable but usually low Ltf-Cre activity levels were also observed in other epithelial tissues of the spleen, liver, kidney, esophagus, lung, intestine and bladder, but it did not affect its reproductive capacity. Among the various tissues and organs of the offspring, the Ltf-Cre activity in the uterine epithelium was the strongest and most consistent. However, when Ltf-Cre female mice were used for breeding, the off-target rate of Ltf in its offspring was particularly high, which was suspected to be caused by the genetic information carried by the oocytes. Therefore, the breeding scheme of the present invention mainly uses Ltf-Cre males for breeding to reduce the unexpected results caused by the double deletion of PTEN gene and STK11 gene caused by Ltf-Cre off-target on other organs.

[0048] Preferably, in step S3, Ltf-Cre; PTEN flox / - ;STK11 flox / - Genotype of F2 male mice and PTEN flox / flox ;STK11 flox / flox The F1 generation female mice of the same genotype were mated in the same cage and screened for Ltf-Cre; PTEN flox / - ;STK11 flox / flox The genotype of the F3 generation female mice is the spontaneous endometrial cancer mouse model of the present invention.

[0049] Optionally, in the case of Ltf-Cre; PTEN flox / - ;STK11 flox / - Genotype of F2 mice and PTEN flox / flox ;STK11 flox / flox After mating with the same F1 generation mice, Ltf-Cre; PTEN flox / - ;STK11 flox / flox Before genotyping the F3 mice, a PCR verification step is also included. The primer sequences for the PCR verification are shown in SEQ ID NO.1-6.

[0050] Based on the same inventive concept, another embodiment of the present invention provides an application of the spontaneous endometrial cancer mouse model constructed by the method for constructing the spontaneous endometrial cancer mouse model as described above, specifically including application in studying the occurrence mechanism and drug resistance mechanism of endometrial cancer or screening drugs for treating endometrial cancer.

[0051] The application of the spontaneous endometrial cancer mouse model and the method for constructing the spontaneous endometrial cancer mouse model as described above have the same advantages over the prior art, which will not be described in detail here.

[0052] The present invention is further described below in conjunction with specific examples. The experimental methods in the following examples without specifying specific conditions are generally carried out according to the conditions recommended by the manufacturer.

[0053] 1. PTEN flox / flox Genotype mouse recovery

[0054] PTEN purchased from Shanghai Nanmo Biotechnology Co., Ltd. flox / - Female and male mice of the genotype (catalog number: NM-CKO-18004) were housed separately for one week to familiarize themselves with the environment, and then the female and male mice were housed in the same cage at a ratio of 2:1. The pups were numbered after birth, and their toes were clipped for genomic DNA extraction. Proteinase K was inactivated at high temperature, and the supernatant was collected by centrifugation as a PCR template. PCR amplification and electrophoresis were then performed, and the experimental results were recorded. This pup was the F1.

[0055] The F1 mice were mated in the same cage, and genomic DNA was extracted, PCR amplified and electrophoresed. The experimental results were recorded and the young mice were recorded as F2. PTEN was screened. flox / flox Genotype of mice.

[0056] 2. STK11 flox / flox Genotype mouse recovery

[0057] STK11 purchased from Shanghai Nanmo Biotechnology Co., Ltd. flox / - Female and male mice of the genotype (catalog number: NM-CKO-18014) were housed separately for one week to familiarize themselves with the environment, and then the female and male mice were housed in the same cage at a ratio of 2:1. The pups were numbered after birth, and their toes were clipped for genomic DNA extraction. Proteinase K was inactivated at high temperature, and the supernatant was collected by centrifugation as a PCR template. PCR amplification and electrophoresis were then performed, and the experimental results were recorded. This pup was the F1.

[0058] The F1 mice were mated in the same cage, and genomic DNA was extracted, PCR amplified and electrophoresed. The experimental results were recorded and the young mice were recorded as F2. STK11 was screened. flox / flox Genotype of mice.

[0059] 3. PTEN flox / flox ;STK11 flox / flox Genotype mouse construction

[0060] The revived PTEN flox / flox Genotype of mice with STK11 flox / floxThe genotype mice were housed in the same cage at a ratio of 2:1 and mated. The pups were numbered after birth, and their toes were clipped for genomic DNA extraction. Proteinase K was inactivated at high temperature, and the supernatant was collected by centrifugation as a PCR template. PCR amplification and electrophoresis were then performed, and the experimental results were recorded to screen for PTEN. flox / flox ;STK11 flox / flox Genotype of mice.

[0061] 4. LTF-Cre; PTEN flox / - ;STK11 flox / - Genotype mouse construction

[0062] Subsequent studies were conducted using LTF-Cre mice purchased from Shanghai Model Biotechnology Co., Ltd. to express PTEN flox / flox ;STK11 flox / flox Female mice of the genotype were housed with LTF-Cre male mice at a ratio of 2:1 and mated. The pups were numbered after birth, and their toes were clipped for genomic DNA extraction. Proteinase K was inactivated at high temperature, and the supernatant was collected by centrifugation as a PCR template. Then, PCR amplification and electrophoresis were performed, and the experimental results were recorded to screen for LTF-Cre; PTEN flox / - ;STK11 flox / - Genotype of female mice.

[0063] 5. Ltf-Cre; PTEN flox / - ;STK11 flox / flox Genotype mouse construction

[0064] Ltf-Cre;PTEN flox / - ;STK11 flox / - Genotype of male mice and PTEN flox / flox ;STK11 flox / flox Female mice of different genotypes were housed in the same cage at a ratio of 2:1 and mated. The pups were numbered after birth, and the toes of the mice were clipped for genomic DNA extraction. Proteinase K was inactivated at high temperature, and the supernatant was collected by centrifugation as a PCR template. Then, PCR amplification was performed. The PCR program was set as follows: 95°C pre-denaturation for 3 min; 95°C denaturation for 30 s, 60°C annealing for 15 s, 72°C extension for 30 s, 35 cycles; 72°C extension for 5 min, 4°C maintenance. PCR primers are shown in the following table:

[0065]

[0066] After PCR amplification, 2% agarose gel electrophoresis was used for detection. 10 μL of sample was loaded into each well and electrophoresis was continued at a constant voltage of 110 V for 40 min in 1×TAE electrophoresis buffer. The sample was detected under ultraviolet light and photographed and recorded. Figure 1-3 ), and screen the mice containing the target band according to the electrophoresis results to determine whether the corresponding mice are heterozygous or homozygous.

[0067] Figure 1-3 The electrophoresis detection results of STK11 gene, Cre gene and PTEN gene are shown respectively, wherein lanes 1-9 represent the genomic DNA samples of mouse toes, and lane M represents the marker. Figure 1 As can be seen from the results, lanes 1-8 obtained STK11 gene flox homozygous mice; Figure 2 As can be seen, lanes 1-3 are Cre positive, and mice with the Cre gene were obtained; Figure 3 As can be seen from the figure, lanes 2-6 obtained PTEN gene flox heterozygous mice. Figure 1-3 The genotype of the genomic DNA samples in lanes 2-3 is LTF-Cre; PTEN flox / - ;STK11 flox / flox .

[0068] 6. Tumor observation

[0069] Continue to culture Ltf-Cre;PTEN flox / - ;STK11 flox / flox Female mice with the same genotype were observed weekly until they reached sexual maturity and had a fully developed uterus and spontaneously developed endometrial cancer. flox / - ;STK11 flox / flox The genotype of female mice was analyzed, and the abdomen was palpated to understand whether tumors occurred. After 2 months of LTF-Cre activation, the mouse uterus showed rapid pathological changes. At 3 months of age, obvious macroscopic changes in the uterus were visible. The tumor formation time was short and the tumor formation rate was high.

[0070] When the tumor was palpable in the mouse abdomen, the mouse was anesthetized, weighed, and dissected to observe the morphology of the uterus and the tumor. Figure 4-6 At 4 months of age, the mice were killed by cervical dislocation, dissected and sampled, fixed with 4% neutral formaldehyde, dehydrated, embedded in paraffin, sliced, and stained with HE to observe the pathological changes of tissue morphology under a microscope. The results are shown in Figure 7-8 .

[0071] Figure 4 This is a general diagram of the anatomy of the model mouse uterus. Figure 5 PTEN flox / - ;STK11 flox / flox Uterine morphology of genotype mice. Figure 6 The present invention LTF-Cre; PTEN flox / - ;STK11 flox / flox Uterine morphology of the genotype mouse model, in which the control PTEN can be observedflox / - ;STK11 flox / flox The uterus of the genotype mice was normal in shape and size, while the uterus of the mouse model of the present invention had obvious tumors growing in it.

[0072] Figure 7 PTEN flox / - ;STK11 flox / flox Uterine pathophysiology images of genotype mice at different resolutions. Figure 8 The present invention LTF-Cre; PTEN flox / - ;STK11 flox / flox Uterine pathophysiology diagrams at different resolutions of genotype mouse models. It can be seen that the uterine pathology of the mouse model of the present invention is characterized by irregular glands, crowded and disordered glandular ducts, increased atypical cells, and visible nuclear division, which are consistent with the characteristics of endometrial adenocarcinoma. The mouse endometrial cancer model constructed by the method of the present invention can be used in the research of developing or screening endometrial cancer drugs.

[0073] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for constructing a spontaneous endometrial cancer mouse model, characterized in that: Through the Cre / loxP system, the genotype was constructed as Cre; PTEN flox / - ;STK11 flox / flox A mouse model; after activating the expression of Cre recombinase in uterine epithelial cells, the mouse model selectively knocks out the PTEN gene and the STK11 gene, spontaneously inducing the occurrence of endometrial cancer.

2. The method for constructing a spontaneous endometrial cancer mouse model according to claim 1, characterized in that: The genotype was constructed by Ltf-Cre / loxP system; PTEN flox / - ;STK11 flox / flox Mouse model.

3. The method for constructing a spontaneous endometrial cancer mouse model according to claim 2, characterized in that: The following steps are involved: S1. PTEN flox / flox Genotype of mice with STK11 flox / flox Mating of genotyped mice and screening for PTEN flox / flox ;STK11 flox / flox Genotype of F1 mice; S2. PTEN flox / flox ;STK11 flox / flox The F1 mice of the same genotype were mated with Ltf-Cre mice, and LTF-Cre; PTEN flox / - ;STK11 flox / - Genotype of F2 mice; S3, Ltf-Cre; PTEN flox / - ;STK11 flox / - Genotype of F2 mice and PTEN flox / flox ;STK11 flox / flox The F1 generation mice of the same genotype were mated and screened for Ltf-Cre; PTEN flox / - ;STK11 flox / flox Genotypes of F3 mice.

4. The method for constructing a spontaneous endometrial cancer mouse model according to claim 3, characterized in that: In step S1, the PTEN is selected flox / flox The genotype of the mice was the male parent, the STK11 flox / flox Mice of the same genotype were mated with the mother; or the PTEN flox / flox Genotype is maternal, STK11 flox / flox Mice of the same genotype were mated in the same cage as the male parent.

5. The method for constructing a spontaneous endometrial cancer mouse model according to claim 3, characterized in that: In step S2, select PTEN flox / flox ;STK11 flox / flox F1 female mice of the same genotype were mated with LTF-Cre male mice in the same cage.

6. The method for constructing a spontaneous endometrial cancer mouse model according to claim 3, characterized in that: In step S3, select Ltf-Cre; PTEN flox / - ;STK11 flox / - Genotype of F2 male mice and PTEN flox / flox ;STK11 flox / flox The F1 generation female mice of the same genotype were mated in the same cage and screened for Ltf-Cre; PTEN flox / -； STK11 flox / flox Genotype of F3 female mice.

7. The method for constructing a spontaneous endometrial cancer mouse model according to any one of claims 4 to 6, characterized in that: The ratio of male to female parent is 1:

2.

8. The method for constructing a spontaneous endometrial cancer mouse model according to claim 3, characterized in that: In the Ltf-Cre;PTEN flox / - ;STK11 flox / - Genotype of F2 mice and PTEN flox / flox ;STK11 flox / flox After mating with the same F1 generation mice, Ltf-Cre; PTEN flox / - ;STK11 flox / flox Before genotyping the F3 mice, a PCR verification step was also included.

9. The method for constructing a spontaneous endometrial cancer mouse model according to claim 8, characterized in that: The primer sequences for PCR verification are shown in SEQ ID NO. 1-6.

10. Use of the spontaneous endometrial cancer mouse model constructed by the method for constructing a spontaneous endometrial cancer mouse model according to any one of claims 1 to 9 in screening drugs for treating endometrial cancer.

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