A method for constructing an animal model of liver abscess

Abstract

The present invention provides a method for constructing an animal model of liver abscess, comprising: Irp2 The missing animals are given dextran sulfate sodium salt solution as drinking water at intervals, and the mass concentration of the dextran sulfate sodium salt solution is 1.5-3%, so as to obtain the animal model. The method has a high success rate and strong repeatability, and can construct an animal model with liver abscess, providing new tools and intervention directions for clinical intervention strategies of liver abscess.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal models and specifically relates to a method based on Irp2 Method for constructing a liver abscess animal model in knockout mice. Background Art

[0002] Liver abscess is a suppurative lesion of the liver caused by a variety of microorganisms such as bacteria, fungi or histolytica, with bacterial liver abscess (Pyogenic liver abscess, PLA, hereinafter referred to as liver abscess) being the most common. In view of the improvement of medical conditions and the use of antibiotics, the proportion of liver abscesses caused by Klebsiella pneumoniae in clinical practice is currently high. It is generally believed that the main infection routes of liver abscesses are biliary route, portal vein route, direct invasion, hepatic artery route, open trauma and cryptogenic route. At present, experimental animal models of liver abscesses are mostly established by intraperitoneal injection and oral administration of Klebsiella pneumoniae to establish mouse liver abscess models.

[0003] Purpose of the Invention

[0004] Iron homeostasis at the cellular level is mainly regulated by iron regulatory protein 1 and 2 (IRP1 / 2). Both IRP1 / 2 have RNA binding activity, and their functions are compensatory. Irp2 Whole-body knockout caused neurological diseases and microcytic anemia in mice, and also caused iron overload in some tissues such as the intestine and liver. Irp1 and Irp2 Double knockout mice are embryonic lethal, indicating Irps The essentiality of genes and tissue specificity of functions. In the previous study of the function of IRP2, the applicant unexpectedly found that in the dextran sulfate sodium salt (DSS)-induced inflammatory bowel disease (IBD) model, DSS-treated knockout mice ( Irp2 - / - ) can induce bacterial liver abscess and has the same characteristics as clinical bacterial liver abscess.

[0005] Based on the above research findings, the present invention proposes a method for constructing an animal model of liver abscess, which has a high success rate and strong repeatability. It can construct an animal model with liver abscess, and provide new tools and intervention directions for clinical intervention strategies of liver abscess.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] A method for constructing an animal model of liver abscess, comprising:

[0008] right Irp2 Missing ( Irp2 - / - ) The animals are given dextran sulfate sodium salt (DSS) solution as drinking water at intervals, wherein the mass concentration of the dextran sulfate sodium salt solution is 1.5-3%, thereby obtaining the animal model.

[0009] Preferably, the animal is a mouse.

[0010] Preferably, the Irp2 The missing animals were 8-9 weeks old Irp2 Deletion mice.

[0011] Preferably, the molecular weight of the dextran sulfate sodium salt is 36,000-50,000.

[0012] Preferably, the method of administering dextran sulfate sodium salt solution as drinking water at intervals is: administering dextran sulfate sodium salt solution as drinking water for 6 to 8 consecutive days as one period, and Irp2 The animals were given clean water as drinking water for 3 to 5 consecutive days. Irp2 The drinking water of the deprived animals was only DSS solution.

[0013] Preferably, the clean water is sterile double distilled water.

[0014] Preferably, the method of administering the dextran sulfate sodium salt solution as drinking water at intervals comprises administering the dextran sulfate sodium salt solution as drinking water for two periods in total.

[0015] The present invention also provides application of the animal model constructed by the above method in liver abscess drug screening or liver abscess disease development research.

[0016] Preferably, the liver abscess is a liver abscess caused by enteric infection.

[0017] Said Irp2 The deletion animals and methods for obtaining them are prior art, which are described in "Targeted deletion of the gene encoding iron regulatory protein-2 causes misregulation of ironmetabolism and neurodegenerative disease in mice", Nat Genet. 2001 Feb;27(2):209-14.

[0018] The present invention constructs a mouse enterogenic liver abscess model for the first time with a high success rate and strong repeatability, providing an experimental animal model for studying liver abscess diseases caused by clinical enteritis infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is cultivated in Example 1 of the present invention Irp2 - / - Genotyping results of mice.

[0020] Figure 2 In Example 2 of the present invention Irp2 - / - Body weight and survival rate curves of mice after intermittent administration of DSS and drinking water.

[0021] Figure 3 In Example 2 of the present invention Irp2 - / - Liver tissue phenotypes of mice after intermittent administration of DSS in drinking water.

[0022] Figure 4 In Example 2 of the present invention Irp2 - / - H&E staining results of mice given DSS drinking water at intervals.

[0023] Figure 5 In Example 2 of the present invention Irp2 - / - Gram staining results of liver tissues of mice after intermittent administration of DSS in drinking water.

[0024] Figure 6 In Example 2 of the present invention Irp2 - / - Blood plate analysis results of liver, portal vein and peripheral blood of mice given DSS drinking water at intervals.

[0025] Figure 7 In Example 2 of the present invention Irp2 - / - Comparison of liver function indicators of mice after intermittent administration of DSS to drinking water.

[0026] Figure 8 In Example 2 of the present invention Irp2 - / - Phenotype of liver tissues in mice treated with antibiotics followed by DSS in drinking water.

[0027] Fig. 9 In Embodiment 2 of the present invention Irp2 - / - H&E staining results of liver tissues of mice given DSS in drinking water and then antibiotics.

[0028] Fig.10 In Example 2 of the present invention Irp2 - / - Results of blood plate analysis of liver and portal vein of mice treated with antibiotics after intermittent administration of DSS in drinking water. DETAILED DESCRIPTION

[0029] Example 1 Irp2 - / - Mouse breeding and identification

[0030] 1. Breeding of mice:

[0031] Obtained with existing technology Irp2 The mice were kept at a temperature of 22-28°C, relative humidity of 40%-60%, noise <60 dB, a temperature difference of 2°C between day and night, and a day and night alternation of 12 h each. The drinking water was autoclaved double distilled water, and the water, mouse cages and bedding were changed weekly. The wild-type mice (WT) were observed every day. Irp2 Deletion mice ( Irp2 - / - )’s daily status such as eating, drinking and activities, and regularly record the weight and survival time of the mice.

[0032] Genotyping of mice:

[0033] The genotype of mice was detected by polymerase chain reaction (PCR). The tail of a one-week-old mouse was cut, and its genomic DNA was extracted as template DNA. The detection primers were used for polymerase chain reaction. The detection primer sequences are as follows:

[0034] Irp2 -Forward Primer: 5'-AAAGAGTGGTCCACTGTGAG-3' (primer 870)

[0035] Irp2 -Reverse Primer: 5'-GGACGAGGAAGGAATCACACA-3' (Primer 884)

[0036] Irp2 -KO-Forward Primer: 5'-TGGAATACCGGCAATGGTGG-3' (Primer 871)

[0037] Irp2 -KO-Reverse Primer: 5'-AGCTCATTCCTCCACTCATG-3' (Primer 872)

[0038] The total reaction volume is 20 μL: including 2xMaster Mix (10 μL) + Forward Primer (0.5 μL) + Reverse Primer (0.5 μL) + mouse tail DNA template (1 μL) + ddH 2 O (8 μL).

[0039] The PCR reaction program was: 94°C pre-denaturation for 30 s, 54°C annealing for 30 s, 72°C extension for 30 s, 35 cycles, and 72°C final extension for 10 min. After the PCR reaction was completed under the above conditions, 1% agarose gel electrophoresis was used to separate the amplified PCR products.

[0040] like Figure 1 The results of mouse genotype identification are shown. The DNA product amplified by primers 870 and 884 is 500 bp in length, which is characteristic of the wild-type allele. The DNA product amplified by primers 871 and 872 is 380 bp in length, which is characteristic of the wild-type allele. Irp2 Mutant allele characteristics; the length of the amplified PCR product fragment is both 500 bp and 380 bp Irp2 Heterozygous allele characteristics. Figure 1 The genotypes of mice 1 and 3 are shown as Irp2 - / - The genotypes of mice No. 2 and No. 4 are Irp2 + / - .

[0041] Example 2 Construction of liver abscess animal model

[0042] 1. Experimental Grouping:

[0043] Select 8-9 week old male wild type and Irp2 The knockout mice were randomly divided into three groups, namely, control drinking water group, DSS-induced group, and DSS-induced + antibiotic group.

[0044] Experimental methods

[0045] Dissolve DSS with a molecular weight of 36,000 to 50,000 in autoclaved double distilled water to prepare a DSS solution with a mass fraction of 1.5 to 3%. Irp2 Knockout mice ( Irp2 - / - ) were given DSS drinking water at intervals (1.5-3% DSS drinking water for 6-8 days + normal drinking water for 3-5 days + 1.5-3% DSS drinking water for 6-8 days), and the weight changes, survival rate changes, tissue pathological changes, and tissue bacterial load levels of the mice were recorded. After the mice developed liver abscess phenotype, 40 mg / kg ofloxacin and 50 mg / kg metronidazole were intraperitoneally injected once a day for 5 days, and the tissue pathological changes and tissue bacterial load levels were recorded.

[0046] (1) Liver H&E staining method:

[0047] Dewaxing of paraffin sections: immerse the paraffin sections in xylene for 10 min, change the solution and immerse in xylene again for 10 min, immerse in 100% ethanol for 3 min, change the solution and immerse in 95% ethanol for 3 min, change the solution and immerse in 85% ethanol for 3 min, change the solution and immerse in 70% ethanol for 3 min, change the solution and immerse in 50% ethanol for 3 min, and carefully rinse with tap water.

[0048] Hematoxylin staining: Dewaxed sections were immersed in hematoxylin staining solution for 3-5 min, then immersed in ultrapure water for 3 min, then immersed in ultrapure water for 3 min, then immersed in 0.25% ammonia solution for anti-blueing for 30 s, then immersed in ultrapure water for 2 min, then immersed in 50% ethanol for 1 min, then immersed in 70% ethanol for 1 min, then immersed in 85% ethanol for 1 min, then immersed in 95% ethanol for 1 min.

[0049] Eosin staining: After hematoxylin staining, the sections were stained with eosin for 4-5 min, the solution was changed and immersed in 100% ethanol for 4 min, the solution was changed and immersed in 100% ethanol for 4 min, the solution was changed and immersed in 100% ethanol for 4 min, the solution was changed and immersed in 100% ethanol for 4 min, the solution was changed and immersed in xylene for 10 min, and the solution was changed and immersed in xylene again for 10 min.

[0050] The sections stained with eosin were sealed with neutral gum, examined under a microscope, and images were collected and analyzed.

[0051] (2) Gram staining method for liver tissue sections:

[0052] Dewaxing of paraffin sections: immerse the paraffin sections in xylene for 10 min, change the solution and immerse in xylene again for 10 min, immerse in 100% ethanol for 3 min, change the solution and immerse in 95% ethanol for 3 min, change the solution and immerse in 85% ethanol for 3 min, change the solution and immerse in 70% ethanol for 3 min, change the solution and immerse in 50% ethanol for 3 min, and carefully rinse with tap water.

[0053] Gram staining: add ammonium oxalate crystal violet staining solution to the slices for 10 s - 30 s, wash with water, and spin dry; add 1% iodine solution for 1 min, wash with water, and spin dry; use a Pasteur pipette to draw Gram decolorizing solution from one end of the frosted surface of the slide to rinse the tissue for differentiation until the Gram decolorizing solution flowing down the slices is colorless, wash with water, and spin dry; add fuchsin staining solution to the slices for 20 - 60 s, wash with water. Use filter paper to absorb moisture from the edges, examine under a microscope, and bake in an oven at 60°C.

[0054] Dehydration: The dried slices were quickly placed in three cylinders of anhydrous ethanol for rapid dehydration for 1 s, 3 s, and 5 s, respectively.

[0055] Transparent sealing: Put the slices into xylene for 5 min, transparent tissue blocks, and seal with neutral gum.

[0056] Microscope examination, image acquisition and analysis.

[0057] Experimental results:

[0058] Figure 2 The results show that the drinking time of WT and Irp2 - / - The survival rate and body weight changes of mice showed Irp2 - / - The survival rate and body weight of mice (n=13) were significantly lower than those of WT mice (n=13). Irp2 - / - Mice died on day 10, and on day 17 Irp2 - / - The survival rate of mice was reduced to about 33%, and no death occurred in WT mice. By recording the weight of mice, it was found that WT and Irp2 - / - The weight change trend of mice was basically the same, but during the period of giving sterile double-distilled water, Irp2 - / - The weight recovery of mice was slower than that of WT mice. After continuing to give DSS drinking water, Irp2 - / - The weight of mice continued to decrease, and there was no significant change in WT mice. Irp2 - / - There were significant differences in the body weight of the mice.

[0059] Figure 3 , Figure 4 It shows that after giving DSS water at intervals Irp2 - / - Mice developed liver abscesses, and liver H&E showed severe liver damage. During the interval of DSS drinking water, the earliest death was found in the autopsy Irp2 - / - The mice developed liver abscesses, while the WT mice did not develop liver abscesses after the interval administration of DSS drinking water. Irp2 - / - The mice showed obvious hepatocyte degeneration and necrosis, inflammatory cell infiltration, and clear boundaries between necrotic tissue and surrounding tissue.

[0060] Figure 5 It shows that after the interval of DSS drinking Irp2 - / - Mouse liver tissue was infected with Gram-positive bacteria. WT and Irp2 - / - After the mice were given DSS drinking water at intervals, Gram staining of the liver tissues of the mice revealed Irp2 - / -Gram-positive bacteria staining was observed in the liver tissue of mice, but not in the liver tissue of WT mice, indicating that Irp2 - / - The liver tissue of mice was infected with Gram-positive bacteria after intermittent administration of DSS in drinking water.

[0061] Figure 6 It shows that after the interval of DSS drinking Irp2 - / - Bacterial infection was present in the liver tissue and portal vein blood of mice. Irp2 - / - After the mice were given DSS drinking water at intervals, blood plate smears were performed on the liver tissue, portal vein blood, and peripheral blood, and a large number of bacterial infections were found in the portal vein and liver. This phenomenon did not occur in WT mice after the intermittent DSS drinking water was stopped.

[0062] Figure 7 Shows the interval after giving DSS and drinking water Irp2 - / - Liver function damage in mice. By measuring liver function-related indicators such as alanine aminotransferase (ALT), aspartate aminotransferase (AST) and albumin (ALB), it was found that Irp2 - / - After mice were given DSS drinking water at intervals, the liver function damage indicators increased significantly, indicating that liver damage occurred.

[0063] Give DSS after the drinking water is finished Irp2 - / - Antibiotic treatment of mice, Figure 8 show, Irp2 - / - The symptoms of liver abscess in mice were significantly alleviated. Through gross analysis of the liver, it was found that liver abscess appeared after the interval administration of DSS and drinking water ended, that is, after 6-8 days of DSS + 3-5 days of water + 6-8 days of DSS ( Irp2 - / - +DSS, and then the mice were given antibiotics (40 mg / kg / d ofloxacin and 50 mg / kg / d metronidazole) by intraperitoneal injection for 5 days ( Irp2 - / - +DSS+antibiotics, the size of liver abscesses was significantly smaller than that of mice not given antibiotics ( Irp2 - / - +DSS+no antibiotics).

[0064] Give DSS after the drinking water is finished Irp2 - / - Antibiotic treatment of liver abscess in mice Fig. 9 Liver H&E showed that the extent of liver abscesses was reduced. H&E staining results of tissue paraffin sections showed that after the interval of DSS drinking water, Irp2- / - In mice treated with antibiotics, liver abscess, hepatocyte degeneration, necrosis, and inflammatory cell infiltration were significantly reduced compared with those in mice not treated with antibiotics.

[0065] Give DSS after the drinking water is finished Irp2 - / - Antibiotic treatment of liver abscess in mice Fig.10 The results showed that bacterial infection in liver tissue and portal vein blood was significantly reduced. Irp2 - / - Mice with liver abscess were treated with antibiotics, and blood plate smears were performed on liver tissue and portal vein blood. It was found that the amount of bacterial infection in the portal vein and liver was significantly lower than that in mice not treated with antibiotics.

[0066] Based on the above analysis, compared with wild-type mice, Irp2 After the mice were given DSS drinking water at intervals, obvious liver abscesses appeared in their livers. After being treated with liver abscess-related drugs, the symptoms were significantly relieved, but not in wild-type mice. The success rate of this model is almost 100%, indicating that Irp2 The deletion plays an important role in liver abscess caused by intestinal damage. The construction of this model mouse can be used to study the model of liver abscess disease caused by intestinal infection and screen drugs.

Claims

1. A method for constructing an animal model of liver abscess, characterized in that: include: right Irp2 The missing animals are given dextran sulfate sodium salt solution as drinking water at intervals, wherein the mass concentration of the dextran sulfate sodium salt solution is 1.5-3%, thereby obtaining the animal model; The method of administering dextran sulfate sodium salt solution as drinking water at intervals is as follows: administering dextran sulfate sodium salt solution as drinking water for 6 to 8 consecutive days is one period, and Irp2 The missing animals were given clean water as drinking water for 3 to 5 consecutive days.

2. The construction method according to claim 1, characterized in that: The animal is a mouse.

3. The construction method according to claim 2, characterized in that: Said Irp2 The missing animals were 8-9 weeks old Irp2 Deletion mice.

4. The construction method according to claim 1, characterized in that: The molecular weight of the dextran sulfate sodium salt is 36000-50000.

5. The construction method according to claim 1, characterized in that: The method of administering the dextran sulfate sodium salt solution as drinking water at intervals includes administering the dextran sulfate sodium salt solution as drinking water for a total of 2 periods.

6. The construction method according to claim 1, characterized in that: The clean water is sterile double distilled water.

7. Use of the animal model constructed by the construction method according to any one of claims 1 to 6 in liver abscess drug screening or liver abscess disease development research.

8. The use according to claim 7, characterized in that: The liver abscess is caused by enterogenic infection.

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