Construction method and application of mouse gastric cancer spontaneous tumor model

Abstract

The application provides a construction method and application of a mouse spontaneous tumor model of gastric cancer, and the construction method comprises the following steps: reducing or inhibiting the expression amount of a TP53 gene and / or the activity of a P53 protein in a mouse, and then chemically inducing the mouse by using a drug to obtain a mouse spontaneous tumor model of gastric cancer; wherein the drug comprises sodium nitrite and ethyl sarcosine, and / or the drug comprises an N-nitroso compound. The application first proposes to use NESS to construct a spontaneous gastric cancer model of a C57 mouse, and the construction method of the mouse spontaneous tumor model of gastric cancer provided by the application has a tumor formation rate of 75% or more; the tumor formation cycle is short, about 3-4 months, and the construction cost is low.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of animal model construction, in particular to a construction method and application of a mouse gastric cancer spontaneous tumor model. BACKGROUND

[0002] Gastric cancer has the highest morbidity and mortality in the world, and is characterized by late stage and large tumor burden. The specific pathogenesis of gastric cancer has not been elucidated, and there is a lack of effective treatment methods. The 5-year survival rate of advanced gastric cancer is only 30%.

[0003] Gastric cancer mouse models are the basis for the study of the etiology of gastric cancer. At present, the mouse models in the field of gastric cancer are divided into spontaneous tumor and transplanted tumor models. The transplanted tumor model adopts subcutaneous or orthotopic transplantation of human or mouse tumor cells or tissue blocks. Mouse cell lines have the advantages of high tumorigenicity and wide selection of tumor-bearing mice. However, due to the lack of mature and stable mouse gastric cancer cell lines, the commonly used mouse gastric cancer cell line is only the MFC cell line established in 1985, which is derived from the foregastric squamous carcinoma of 615 mice and can only be used for tumor-bearing in 615 mice. The feasibility and applicability are limited. Therefore, at present, the mouse gastric cancer transplanted tumor model mostly uses immunodeficient mice to plant human gastric cancer cells or tissues. However, more and more studies have shown that the tumor immune microenvironment plays an important role in the occurrence and development of tumors, and is closely related to the development and application of tumor treatment, especially for gastric cancer, which is one of the most heterogeneous tumors. Since the stomach is a digestive organ with complex tissue structure and diverse physiological functions, there is an interaction between the in vivo and in vitro environments. The occurrence and development of gastric cancer are attributed to the combined action of many environmental and genetic factors. For example, Helicobacter pylori infection is one of the most important risk factors for gastric cancer. Therefore, the tumor immune microenvironment of gastric cancer is also one of the focuses of the current research in the field of gastric cancer. The gastric cancer transplanted tumor model is not a primary tumor and cannot reflect the process of tumor formation in vivo, and cannot simulate the tumor microenvironment. Immunodeficient mice also cannot simulate the immune system of the body, making it difficult to carry out research on the tumor microenvironment and immune-related research of gastric cancer. The use of this model for the etiology of gastric cancer is also greatly limited and controversial.

[0004] The spontaneous tumor model of mice can simulate the development process of gastric cancer in the human body, has a tumor microenvironment, and has an immune system of the body, and is widely recognized as a reliable material for the etiology of gastric cancer. According to different pathogenic mechanisms, the mouse model of gastric cancer is roughly divided into two categories: one is a transgenic mouse model constructed according to genetic factors gene mutation, and the other is an orthotopic induction mouse model constructed according to environmental stimulus factors. However, the current spontaneous tumor model of mouse gastric cancer also faces many challenges. The genetically edited transgenic mouse model has high specificity and unique advantages in the study of molecular mechanisms of signal pathways related to the occurrence and development of gastric cancer, but the production cost is high and the tumor formation period is long. The orthotopic induction mouse model constructed according to environmental stimulus factors has a low tumor formation rate and a long tumor formation period. Therefore, it is necessary to develop a new method for establishing a mouse spontaneous gastric cancer model. SUMMARY

[0005] The purpose of the present application is to provide a construction method and application of a mouse spontaneous gastric cancer tumor model, so as to improve the tumor formation rate in the construction process of the mouse spontaneous gastric cancer tumor model, shorten the tumor formation period, and reduce the construction cost. The specific technical solutions are as follows:

[0006] The first aspect of the present application provides a construction method of a mouse spontaneous gastric cancer tumor model, comprising the steps of: reducing or inhibiting the expression amount of TP53 gene and / or the activity of P53 protein in the mouse, and then chemically inducing the mouse with a drug to obtain a mouse spontaneous gastric cancer tumor model; wherein the drug comprises sodium nitrite and ethyl sarcosine, and / or the drug comprises an N-nitroso compound.

[0007] In some embodiments of the present application, the reduction or inhibition of the expression amount of TP53 gene and / or the activity of P53 protein in the mouse is to specifically knockout the TP53 gene in the mouse, to obtain a TP53 gene knockout mouse; preferably, the TP53 gene knockout mouse is a TP53 gene knockout mouse with heterozygous deletion of TP53 gene in the whole body.

[0008] In some embodiments of the present application, the TP53 gene specifically knocked out in the mouse is at least one of the 2nd exon, the 3rd exon, the 4th exon, the 5th exon and the 6th exon in the TP53 gene in the genome of the specific knockout mouse.

[0009] In some embodiments of the present application, the TP53 gene specifically knocked out in the mouse includes specifically knocking out the TP53 gene in the recipient mouse by using a CRISPR-Cas9 system.

[0010] In some embodiments of the present application, the N-nitroso compound comprises N-nitroso sarcosine ethyl ester, the concentration of the N-nitroso sarcosine ethyl ester is 4 g / 100 mL-7 g / 100 mL; the concentration of the sodium nitrite is 2 g / 100 mL-3 g / 100 mL, and the concentration of the sarcosine ethyl ester is 5 g / 100 mL-10 g / 100 mL.

[0011] In some embodiments of the present application, the drug is administered by gavage, and the amount of the drug is 5 mL / kg body weight-10 mL / kg body weight.

[0012] In some embodiments of the present application, the drug is administered once a week-three times a week, the administration period of the drug is 4 weeks-8 weeks, the mice are 5 weeks old-7 weeks old when the drug is administered for the first time, the body weight of the mice is 20 g-25 g when the drug is administered for the first time, and the mice are C57 mice.

[0013] In some embodiments of the present application, the administration of the drug to the mice specifically comprises the following steps: the drug is administered after the mice are fasted for 2 hours-4 hours; normal diet and water are provided during the interval between administrations; and the mice are normally bred for 8 weeks-24 weeks after the administration of the drug is completed.

[0014] The second aspect of the present application provides a mouse gastric cancer spontaneous tumor model constructed by the method of the first aspect of the present application.

[0015] The third aspect of the present application provides the use of the method of the first aspect of the present application or the mouse gastric cancer spontaneous tumor model of the second aspect of the present application in screening drugs for preventing and / or treating gastric cancer.

[0016] The method for constructing the mouse gastric cancer spontaneous tumor model provided by the present application has the following advantages:

[0017] (1) The induction process is convenient. Compared with the traditional MNU (N-methyl-N-nitrosourea) drinking water, the method is administered by gavage once a week-three times a week, and the operation process is more convenient.

[0018] (2) The induction process is stable, and the difference between groups is small. Compared with drinking water administration, the difference in administration time and the difference in the amount used are smaller.

[0019] (3) The tumor formation rate is high. The tumor formation rate of the traditional MNU compound drinking water is about 30%, while the tumor formation rate of the method is as high as 75% or more.

[0020] (4) The tumor formation period is short. The tumor formation period of the traditional method is about 7-12 months, and the tumor formation period of the method is about 3-4 months.

[0021] (5) The method is easy to carry out. The method does not require light protection treatment, does not require light protection water bottles and other equipment, and is simple to operate.

[0022] Of course, practicing any of the products or methods of the present application does not necessarily require achieving all of the above-mentioned advantages simultaneously. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other embodiments can also be obtained by those skilled in the art based on these drawings.

[0024] Figure 1 FIG. 1 is a schematic diagram and an effect diagram of the construction of a mouse gastric cancer spontaneous tumor model in Example 1 of the present application; wherein: A) is a schematic diagram of the construction of a mouse gastric cancer spontaneous tumor model, B) is an appearance effect diagram of the mouse gastric cancer spontaneous tumor model constructed, C) is a pathological H&E staining result diagram of the primary gastric cancer tissue of the mouse gastric cancer spontaneous tumor model constructed;

[0025] Figure 2 FIG. 2 is a pathological H&E staining result diagram of the primary gastric cancer tissue of one WT mouse without gene modification in Comparative Example 2 of the present application;

[0026] Figure 3 FIG. 3 is a nuclear magnetic detection result diagram of the mouse gastric tissue block;

[0027] Figure 4 FIG. 4 is an effect diagram of transplanting the primary gastric cancer tissue fragments of the mouse gastric cancer spontaneous tumor model obtained in Example 1 of the present application to the subcutaneous tissue of a C57BL / 6 mouse. DETAILED DESCRIPTION

[0028] The technical solutions in the present application will be described clearly and completely below in combination with the embodiments of the present application and the drawings. Obviously, the described embodiments are only some of the embodiments of the present application, not all the embodiments. All other embodiments obtained by those skilled in the art based on the present application are within the scope of protection of the present application.

[0029] In the establishment of a mouse spontaneous gastric cancer model, the tumor formation rate of a single gene mutant mouse model can reach 100%, such as the INS-GAS transgenic mouse, but the tumor formation period is longer, about 20 months; the tumor formation rate of multiple gene mutations is high, and the period is shorter, but the technical requirements are higher. The in situ induction model has a nitroso compound or a Helicobacter pylori induced tumor, and MNU induction is currently being studied and shows a relatively stable tumor formation rate, but the traditional MNU induction method will most likely obtain a pre-gastric squamous carcinoma, and the tumor formation rate in C57BL / 6 mice is about 30%, and the tumor formation period is about 7-12 months; long-term infection of Helicobacter pylori usually induces severe gastritis, and the tumor type is mostly adenocarcinoma, but the tumor formation rate to cancer is low, and the tumor formation rate in C57BL / 6 mice is reported to be about 20%, and the tumor formation period is more than 18 months. The inventors of the present application surprisingly found that the C57 mouse spontaneous gastric cancer model based on TP53 gene knockout has a high tumor formation rate and a short tumor formation time, which can effectively solve the research problem of the lack of TP53 knockout / mutation C57 mouse gastric cancer model.

[0030] The first aspect of the present application provides a method for constructing a mouse gastric cancer spontaneous tumor model, comprising the steps of: reducing or inhibiting the expression amount of TP53 gene and / or the activity of P53 protein in the mouse in vivo, and then giving the mouse a drug for chemical induction to obtain a mouse gastric cancer spontaneous tumor model; wherein the drug comprises sodium nitrite and ethyl sarcosine; and / or the drug comprises an N-nitroso compound.

[0031] The mouse TP53 gene sequence described in the present application is the nucleotide sequence from position 69471174 to 69482699 of GenBank Accession No. NC_000077.7 (Update Date 2024-02), which includes 12 exons.

[0032] The P53 protein encoded by the mouse TP53 gene described in the present application is the protein represented by the amino acid sequence of GenBank Accession No. NP_035770.2 (Update Date 2024-02).

[0033] The drug described in the present application can also contain water, preferably double distilled water.

[0034] The present application first proposes the use of NESS to construct a C57 mouse spontaneous gastric cancer model. The method for constructing a mouse gastric cancer spontaneous tumor model provided by the present application has a high tumor formation rate, the tumor formation rate of the traditional MNU compound drinking water induction is about 30%, while the tumor formation rate of the method can reach 75% or more; the tumor formation period is short, the tumor formation period of the traditional method is about 7-12 months, while the tumor formation period of the method is about 3-4 months, and the construction cost is low.

[0035] In some embodiments of the present application, the reducing or inhibiting the expression level of the TP53 gene and / or the activity of the P53 protein in the mouse is to specifically knock out the TP53 gene in the mouse to obtain a TP53 knockout mouse; preferably, the TP53 knockout mouse is a TP53 knockout mouse with systemic heterozygous deletion of the TP53 gene.

[0036] The gene knockout mice described in this application refer to the phenomenon of inactivating a specific target gene by homologous recombination. Gene knockout is the inactivation of a specific target gene by changing the DNA sequence. This application can utilize gene homologous recombination of mouse embryonic stem cells to heterozygously knock out the TP53 gene on the mouse genome to obtain a TP53 gene knockout mouse with systemic TP53 gene heterozygosity loss. This application is not particularly limited to the specific operating steps for obtaining TP53 gene knockout mice, and the operating methods commonly used in the art can be used.

[0037] In some embodiments of the present application, the TP53 gene in the specific knockout mouse is at least one of exon 2, exon 3, exon 4, exon 5 and exon 6 in the TP53 gene in the specific knockout mouse genome.

[0038] In some embodiments of the present application, the specific knockout of the TP53 gene in the mouse includes using the CRISPR-Cas9 system to specifically knock out the TP53 gene in the recipient mouse.

[0039] In some embodiments of the present application, the N-nitroso compound comprises N-nitrososarcosine ethyl ester (NSEE), and the concentration of the N-nitrososarcosine ethyl ester is 4 g / 100 mL to 7 g / 100 mL. In the present application, the concentration of N-nitrososarcosine ethyl ester can be 4 g / 100 mL, 4.5 g / 100 mL, 5 g / 100 mL, 5.5 g / 100 mL, 6 g / 100 mL, 6.5 g / 100 mL, 7 g / 100 mL, or a range consisting of any two values ​​therebetween. The N-nitrososarcosine ethyl ester of the present application can be prepared from sodium nitrite (NaNO2) and sarcosine ethyl ester.

[0040] In some embodiments of the present application, the concentration of sodium nitrite is 2 g / 100 mL-3 g / 100 mL, and the concentration of ethyl sarcosinate is 5 g / 100 mL-10 g / 100 mL. In the present application, the concentration of sodium nitrite can be 2 g / 100 mL, 2.5 g / 100 mL, 3 g / 100 mL, or a range between any two of the above values; the concentration of ethyl sarcosinate can be 5 g / 100 mL, 6 g / 100 mL, 7 g / 100 mL, 8 g / 100 mL, 9 g / 100 mL, 10 g / 100 mL, or a range between any two of the above values.

[0041] In some embodiments of the present application, the drug is administered by gavage, and the amount of the drug administered is 5 mL / kg body weight-10 mL / kg body weight (for mice). In the present application, the amount of the drug administered can be 5 mL / kg body weight, 6 mL / kg body weight, 7 mL / kg body weight, 8 mL / kg body weight, 9 mL / kg body weight, 10 mL / kg body weight, or a range between any two of the above values. In the present application, the amount of the drug administered is about 200 µL / mouse-250 µL / mouse.

[0042] In some embodiments of the present application, the drug is administered once-3 times per week, the administration period of the drug is 4 weeks-8 weeks, the mice are 5 weeks old-7 weeks old at the time of initial administration of the drug, the mice weigh 20 g-25 g at the time of initial administration of the drug, and the mice are C57 mice. The C57 mice of the present application can be C57BL / 6 mice. In the present application, the drug can be administered once, twice, or three times per week, the administration period of the drug can be 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks, the mice can be 5 weeks old, 5.5 weeks old, 6 weeks old, 6.5 weeks old, or 7 weeks old at the time of initial administration of the drug, and the mice can weigh 20 g, 21 g, 22 g, 23 g, 24 g, 25 g, or a range between any two of the above values at the time of initial administration of the drug.

[0043] In some embodiments of the present application, the administration of the drug to the mice specifically includes the step of administering the drug after the mice have fasted for 2 hours-4 hours, administering normal diet and water during the administration interval, and routinely feeding the mice for 8 weeks-24 weeks after the administration is complete. In the present application, the mice can fast for 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, or a range between any two of the above values, and the mice can be routinely fed for 8 weeks, 10 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24 weeks, or a range between any two of the above values after the administration is complete.

[0044] The application provides a method for constructing a mouse gastric cancer spontaneous tumor model, which is convenient to induce, has less gavage frequency per week compared to traditional MNU drinking water long-term administration under light protection, and is more convenient to operate; the induction process is stable, and the difference between groups is small; compared with drinking water administration, the difference in administration time and the difference in the amount used are smaller; it is easy to carry out, and the method does not require light protection treatment, does not require light protection water bottle and other equipment, and is simple to operate.

[0045] The second aspect of the application provides a mouse gastric cancer spontaneous tumor model constructed by the method of the first aspect of the application.

[0046] The third aspect of the application provides application of the method of the first aspect of the application or the mouse gastric cancer spontaneous tumor model of the second aspect of the application in screening of drugs for preventing and / or treating gastric cancer.

[0047] EMBODIMENT

[0048] Hereinafter, the embodiments of the application are described more specifically by citing examples and comparative examples. Various tests and evaluations are performed according to the following methods. In addition, unless otherwise specified, "parts" and "%" are mass-based.

[0049] Example 1

[0050] (1) The CRISPR-Cas9 system was used to knock out the 2nd, 3rd, 4th, 5th and 6th exons of the TP53 gene in the genome of C57BL / 6 mice, and PCR identification was performed to obtain TP53 gene knockout C57BL / 6 mice with systemic TP53 gene heterozygous deletion (P53 - / + ).

[0051] (2) 30 6-week-old TP53 gene knockout C57BL / 6 mice with systemic TP53 gene heterozygous deletion and weighing 20-25 g were taken, respectively, and after fasting for 3 hours, the C57BL / 6 mice were gavaged with drugs, including 3 g / 100 mL of sodium nitrite and 10 g / 100 mL of sarcosine ethyl ester, and the solvent was double distilled water; the amount of drug was 10 mL / kg of body weight, the administration frequency was twice a week, normal diet and water were given during the interval, and the total administration time was 6 weeks; after the administration was completed, the mice were normally fed for 16 weeks.

[0052] Comparative Example 1

[0053] (1) The CRISPR-Cas9 system was used to knock out the 2nd, 3rd, 4th, 5th and 6th exons of the TP53 gene in the genome of C57BL / 6 mice, and PCR identification was performed to obtain TP53 gene knockout C57BL / 6 mice with systemic TP53 gene heterozygous deletion (P53- / + ).

[0054] (2) Take 6 six-week-old TP53 gene knockout C57BL / 6 mice with a body weight of 20-25 g, which are systemic TP53 gene heterozygous deletion, and fast for 3 hours. Then, the C57BL / 6 mice are given PBS solution by gavage, the drug administration frequency is twice a week, and the normal diet and water are given during the drug administration interval. The total drug administration time is 6 weeks. After the drug administration is completed, the mice are normally bred for 16 weeks.

[0055] Comparative Example 2

[0056] Take 15 six-week-old C57BL / 6 mice (WT) without genetic modification, which have a body weight of 20-25 g, and fast for 3 hours. Then, the C57BL / 6 mice are given drugs by gavage, the drugs include sodium nitrite with a concentration of 3 g / 100 mL and sarcosine ethyl ester with a concentration of 10 g / 100 mL, and the solvent is double distilled water. The drug dosage is 10 mL / kg of body weight, the drug administration frequency is twice a week, and the normal diet and water are given during the drug administration interval. The total drug administration time is 6 weeks. After the drug administration is completed, the mice are normally bred for 16 weeks.

[0057] The mice finally obtained in the above examples and comparative examples are subjected to tissue sampling and histopathological (H&E staining) identification, and the specific results are shown in Table 1 and Figures 1-3 .

[0058] Table 1

[0059]

[0060] From the above results, it can be seen that 23 mice gastric cancer spontaneous tumor models are obtained in Example 1, the tumor formation rate is 76.6% (23 / 30), and the tumor formation period is about 3 months after gavage. In Comparative Example 2, only 6.7% (1 / 15) of the mice gastric cancer spontaneous tumor models are obtained, and the mice gastric cancer spontaneous tumor models are not successfully obtained in Comparative Example 1.

[0061] The pathological results of the mouse gastric cancer spontaneous tumor models obtained in Example 1 of the present application show that the pathological types of the occurred gastric cancers are gastric adenocarcinoma and gastric squamous carcinoma. Figure 1 FIG. 1 is a schematic diagram and effect diagram for the construction of the mouse gastric cancer spontaneous tumor model in Example 1 of the present application; wherein: A) is a schematic diagram for the construction of the mouse gastric cancer spontaneous tumor model, B) is an appearance effect diagram of the mouse gastric cancer spontaneous tumor model constructed, and C) is a pathological H&E staining result diagram of the primary gastric cancer tissue of the mouse gastric cancer spontaneous tumor model constructed.

[0062] Figure 2 FIG. 2 is a pathological H&E staining result diagram of the primary gastric cancer tissue of the WT mouse without genetic modification in Comparative Example 2 of the present application.

[0063] Figure 3The figure is the result of nuclear magnetic detection of the stomach tissue of the mouse; wherein: A is the result of nuclear magnetic detection of the stomach tissue of the WT mouse (not suffering from gastric cancer) in the comparative example 2, B is the result of nuclear magnetic detection of the stomach tissue of the mouse gastric cancer spontaneous tumor model in the example 1; from Figure 3 It can be seen that the stomach wall of the stomach tissue of the mouse gastric cancer spontaneous tumor model in the example 1 is thickened, and the thickness is significantly greater than that of the stomach tissue of the WT mouse (not suffering from gastric cancer) in the comparative example 2.

[0064] The primary gastric cancer tissue fragments of the mouse gastric cancer spontaneous tumor model in the example 1 are transplanted into the subcutaneous of the C57BL / 6 mouse, and the allogeneic transplantation model can be constructed. Figure 4 The figure is the effect of transplanting the primary gastric cancer tissue fragments of the mouse gastric cancer spontaneous tumor model in the example 1 into the subcutaneous of the C57BL / 6 mouse, from Figure 4 It can be seen from the figure that the allogeneic transplantation model is successfully constructed.

[0065] The above is only the preferred embodiment of the present application, and is not used to limit the present application, any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1.A method for constructing a mouse spontaneous tumor model of gastric cancer, comprising the steps of: obtaining TP53 knockout mice with heterozygous deletion of TP53 gene throughout the body by using a CRISPR-Cas9 system to specifically knock out exon 2 in the TP53 gene in the mouse genome; and then chemically inducing the mice by administering a drug to obtain a mouse spontaneous tumor model of gastric cancer. wherein The drug comprises sodium nitrite and ethyl sarcosine, and / or the drug comprises N-nitrosoethyl sarcosine; the concentration of sodium nitrite is 2 g / 100 mL to 3 g / 100 mL, the concentration of ethyl sarcosine is 5 g / 100 mL to 10 g / 100 mL; and the concentration of N-nitrosoethyl sarcosine is 4 g / 100 mL to 7 g / 100 mL. The administering of the drug to the mice specifically comprises the steps of: administering the drug after the mice are fasted for 2 hours to 4 hours; administering normal diet and water during the interval between administrations; and routinely feeding the mice for 8 weeks to 24 weeks after the administration of the drug is completed; the drug is administered by gavage; the amount of the drug is 5 mL / kg body weight to 10 mL / kg body weight; the frequency of administration of the drug is once to three times per week; the administration cycle of the drug is 4 weeks to 8 weeks; the mice are 5 weeks old to 7 weeks old at the time of the initial administration of the drug; the body weight of the mice is 20 g to 25 g at the time of the initial administration of the drug; and the mice are C57 mice. 2.Use of the method of claim 1 in screening drugs for preventing and / or treating gastric cancer.

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