CN114051974B - Liver cancer model and construction method and application thereof - Google Patents
Liver cancer model and construction method and application thereof Download PDFInfo
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- CN114051974B CN114051974B CN202111323944.9A CN202111323944A CN114051974B CN 114051974 B CN114051974 B CN 114051974B CN 202111323944 A CN202111323944 A CN 202111323944A CN 114051974 B CN114051974 B CN 114051974B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
- A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0331—Animal model for proliferative diseases
Abstract
The invention relates to the technical field of biological models, in particular to a construction method and application of a liver cancer tumor model. Feeding a mouse with a high methionine feed containing 2% of methionine by mass until molding is successful; gavage 2 CCl times per week from week 1 4 Until the molding is successful; starting from week 1, intraperitoneal injection of DEN is performed 1 time per week for 4 weeks; the molding time was 8 weeks. The invention combines the methionine-rich feed with diethyl nitrosamine (DEN) and carbon tetrachloride (CCl) 4 ) The method has the advantages of simple operation, short period (8 weeks), high success rate (the tumor formation rate is 93%) and low mortality rate when constructing the liver cancer tumor model. Has irreplaceable necessity of other models and has wide research and application prospect.
Description
The technical field is as follows:
the invention relates to the technical field of biological models, in particular to a construction method and application of a liver cancer tumor model.
The background art comprises the following steps:
in 2020, primary liver cancer is the sixth most commonly diagnosed cancer worldwide and the third leading cause of cancer death, with about 90.6 new cases and 83 deaths. In most regions, men have 2 to 3 times as much morbidity and mortality as women. Liver cancer ranks fifth in global morbidity and second in male mortality. Most highly developed cancers are observed primarily in transitional countries, such as east asia (mongolia has far more prevalence than any other country), southeast asia (e.g., thailand, cambodia, vietnam), and north and west africa. Liver cancer is the leading cause of cancer death in Mongolia, thailand, cambodia, egypt, critical Mara, and another 18 countries. Primary liver cancer includes hepatocellular carcinoma (HCC) (75% -85%) and intrahepatic bile duct cancer (10% -15%) as well as other rare types. The major risk factors for HCC are chronic infection with Hepatitis B Virus (HBV) or Hepatitis C Virus (HCV), consumption of aflatoxin-contaminated food, heavy drinking, excess body weight, type 2 diabetes, and smoking. Most patients have metastasis when finding the liver cancer, the treatment difficulty of the metastatic liver cancer is high, the prognosis is extremely poor, and further research on the metastatic liver cancer is urgently needed.
Animal models are a well-established tool for understanding the pathogenesis of diseases, determining therapeutic targets and screening effective drugs. Due to the complex etiology and heterogeneity of tumors, developing animal models of HCC that resemble the human disease environment is a challenge. Currently available animal models can be classified as follows: a chemical induction model, (b) a transgenic mouse (GEM) model, and (c) a transplantation model.
Transgenic mouse (GEM) models are expensive, require a large amount of manpower and material resources, are long in cycle and are less in use. The most commonly used tumor cell planting method at present adopts the way of injecting suspension tumor cells through subcutaneous, rat tail vein, abdominal cavity and the like to form an ectopic liver cancer model, but the model has the most fundamental defects: for example, the most studied subcutaneous transplanted tumor model is easy to form envelope, is not easy to transfer and is easy to undergo central necrosis, and needs to be improved to construct a better in-situ model, so that the model can be attached to the tumor growth microenvironment of the liver cancer in the human body to the maximum extent, the biological characteristics of the tumor in the human body can be simulated, and the tumor growth microenvironment can be further studied.
Chemical induction models are relatively common models. Humans are inevitably exposed to toxic compounds and there are several well-known chemically inducible substances that can induce liver damage and are therefore used as promoters in HCC models, such as Diethylnitrosamine (DEN), carbon tetrachloride (CCl 4), thioacetamide (TAA), 5-benzene-5-ethyl barbital (PB). These chemicals mainly cause hepatocyte cell death, steatosis or cholestasis, followed by inflammation and fibrosis. In clinic, we find that the hyperhomocysteinemia has various manifestations, such as fatty liver, hepatic fibrosis and the like, and the hepatic fibrosis further develops into cirrhosis and liver cancer. However, no model for the development of hyperhomocysteinemia into liver cancer exists at present to simulate the pathogenesis.
The invention content is as follows:
in order to solve the technical problems, the invention provides a homomethionine combined with Diethylnitrosamine (DEN) and carbon tetrachloride (CCl) 4 ) The established liver cancer tumor model can better simulate the pathogenesis of hyperhomocysteinemia, and compared with the DEN + CCl commonly used at present, the establishment of the liver cancer model 4 The carcinogenic model (the modeling time is more than 20 weeks), the modeling time is obviously shortened, and the success rate is higher.
The construction method of the liver cancer tumor model comprises the following steps: feeding a mouse with a high methionine feed containing 2% of methionine by mass until molding is successful; per week CCl 2 gavages from week 1 4 Until the molding is successful; injecting DEN intraperitoneally from week 1, 1 time per week, and continuously injecting for 4 weeks;
furthermore, the molding mice are male C57BL/6J mice of 6-8 weeks old;
further, the CCl 4 Adopting corn oil to dilute the mixture to 15 percent (v: v), wherein the intragastric administration dose is 5mL/kg each time;
further, the DEN concentration of the intraperitoneal injection is 10 mug/mL, and the injection dose is 100mg/kg;
further, during molding, the mice freely drink water and eat feed, and the sterilized feed and the sterilized water are periodically replaced;
furthermore, mice were placed in clean EVC mouse cages during molding, and sterilized padding, feed and sterilized water were replaced every 3 days;
further, the molding time is 8 weeks;
further, after 8 weeks of modeling, the mice are killed, the liver is taken out for pathological histochemistry determination of the liver cancer of the mice, the tissue structure, the transfer and the invasion conditions of the liver are observed through HE dyeing and masson pine dyeing, the bump on the surface of the liver of the modeling module is observed by naked eyes, the modeling module is seen by HE dyeing to have tumor giant cells and pathological nuclear division, and the modeling module is seen by masson dyeing to have obvious fibrosis.
The invention also provides a liver cancer tumor model constructed by the method, and application thereof, in particular application in preparing liver disease related medicines, and application in treatment, detection and/or prognosis of liver related diseases.
Has the advantages that:
1. the invention adopts the combination of homomethionine and diethyl nitrosamine (DEN) and carbon tetrachloride (CCl) for the first time 4 ) Constructing a liver cancer tumor model which can be widely applied to basic and clinical researches, (1) being beneficial to monitoring the biological behavior of the hyperhomocysteinemia developing into the liver cancer; (2) is beneficial to monitoring the sensitivity of radiotherapy and chemotherapy and biological target therapy of the liver cancer caused by the etiology and provides the selection of the optimal treatment scheme; (3) the non-human animal model of liver cancer is used for the research of screening the medicine for preventing or treating liver cancer caused by hyperhomocysteinemia.
2. The invention has simple operation, short period (8 weeks), high success rate (the tumor formation rate is 93 percent) and low mortality. Therefore, the establishment of the research platform has the irreplaceable necessity of other models, and the research platform is also bound to be paid more and more attention by scientific and technical workers and has wide research prospect.
Description of the drawings:
FIG. 1 is a molding flow chart.
FIG. 2 is a graph of staining of mouse masson; a is normal group; b: 2% homomethionine + DEN + CCl at week 5 4 And (4) grouping.
FIG. 3 is a graph of staining of mouse masson; a is normal group; c: 2% homomethionine + DEN + CCl at week 8 4 And (4) grouping.
FIG. 4 is a graph of staining of mouse masson; a is normal group; d: DEN + CCl at week 20 4 And (4) grouping.
FIG. 5 mouse HE staining profile; a is normal group; b: 2% homomethionine + DEN + CCl at week 5 4 And (4) grouping.
FIG. 6 graph of HE staining of mice; a is normal group; c: 2% homomethionine + DEN + CCl at week 8 4 And (4) grouping.
FIG. 7 graph of mouse HE staining; a is normal group; d: DEN + CCl at week 20 4 And (4) grouping.
The staining patterns in FIGS. 2-7 are 40 Xmagnification by light microscopy.
The specific implementation mode is as follows:
in order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present patent and are not intended to limit the present invention.
The flow of the molding method provided by the present invention is shown in fig. 1, and the present invention will be further explained with reference to the specific embodiment.
Some of the materials and methods used in the present invention are as follows, and the materials involved in the present invention are commercially available:
CCl 4 (corn oil diluted to 15% concentration), corn oil, DEN (physiological saline diluted to 10 μ g/mL concentration), 2% high methionine feed, normal feed, 4% formalin. Wherein, the feed containing 2% of high methionine content and the normal feed (AIN 93G standard feed) are purchased from Nantong Terofen feed science and technology Co.
Male C57BL/6J mice (purchased from Beijing Wintoda Biotechnology, inc.) 6-8 weeks old weighed 14-16g, randomly grouped, 1 group of 30 mice.
Example 1 construction method of a liver cancer tumor model
Step S1: establishing an in-situ heterogeneous mouse liver cancer model, which specifically comprises the following steps:
step S1a: selecting 120 male C57BL/6J mice with age of 6-8 weeks;
step S1b: control group was set, 2% homomethionine + DEN + CCl 4 Experimental group, DEN + CCl 4 Experimental groups:
control group: 30 mice. Feeding with normal feed, and not treating.
2% homomethionine + DEN + CCl 4 Experimental groups: 60 mice (divided into two groups of 30 mice each). Feeding with 2% high methionine feed and drinking water freely. Placing the mouse in a clean EVC mouse cage, adding a sterilized feed, and replacing sterilized water; EVC cages were delivered to the animal house. Replacing the sterilized padding, the feed and the sterilized water every 3 days, and observing the vitality and the growth state of the mouse;
per week CCl 2 gavages from week 1 4 (diluted with corn oil to 15% concentration), the dosage is 5mL/kg each time until the molding is successful;
DEN (10. Mu.g/mL) was intraperitoneally injected 1 time a week, 100mg/kg each time for 4 weeks from week 1.
DEN+CCl 4 Experimental groups: 30 mice. With 2% homomethionine + DEN + CCl 4 Compared with experimental groups, 2% high-methionine feed is replaced by normal feed without methionine, and the rest experimental settings are the same.
Step S2: the tissue structure and the metastasis and invasion conditions of the tumor are observed through HE staining. The method specifically comprises the following steps:
step S2a: a group of 2% homomethionine + DEN + CCl was sacrificed after 5 and 8 weeks, respectively 4 Mice, DEN + CCl sacrificed after 20 weeks 4 The abdominal cavity and the thoracic cavity were opened in the experimental group and the normal group, the gross morphology, the growth infiltration condition and the distant metastasis condition of the liver tumor tissue were observed, and the liver was fixed in 4% formalin.
Step S2b: formalin-fixed liver tissues are embedded in paraffin, 4-micrometer sections are cut, hematoxylin-eosin (HE) staining is carried out, liver tissue specimens are collected and subjected to routine pathological histological detection, and the condition of liver fibrosis is evaluated by applying masson staining. The results are as follows:
2% homomethionine + DEN + CCl at week 5 4 The experimental group was stained with masson, and the results are shown in fig. 2, with mild fibrosis;
2% homomethionine + DEN + CCl at week 5 4 The results of HE staining in the experimental group are shown in fig. 5, and no abnormal cells were observed.
2% homomethionine + DEN + CCl at week 8 4 The experimental group was stained with masson, and the results are shown in fig. 3, showing that significant fibrosis was seen in the experimental group;
2% homomethionine + DEN + CCl at week 8 4 The results of HE staining in the experimental group are shown in FIG. 6, which shows that the nucleus cytoplasm of the experimental group is increased in proportion, the nucleus is multi-linear, the staining is deep, the nucleolus is increased and irregular, the split image is increased, the tumor is diffused, and the size is about 2-3 mm;
the appearance shows that the liver is provided with gray tumor tissues, the surface is nodular and hard, the gray tumor tissues are independently or dispersedly distributed on the surface and the edge of the liver, and part of the nodules can be fused into larger nodules;
and no tumor metastasis is observed in organs after other parts are dissected.
DEN + CCl at week 20 4 The experimental group was stained with masson, and the results are shown in fig. 4, with mild fibrosis;
DEN + CCl at week 20 4 The results of HE staining in the experimental group are shown in fig. 7, and no abnormal cells are found;
the appearance shows that the whole liver has smooth surface, ruddy color and slightly hard texture.
The normal group was stained with masson at week 20, and as a result, no fibrosis was observed as shown in each panel a;
HE staining in the normal group was performed at week 20, and as a result, no abnormal cells were observed as shown in each panel a;
appearance the whole liver appearance surface is smooth, the color is ruddy, and the texture is soft.
And step S3: the tumor formation rate was calculated and the results are shown in Table 1, and 2% homomethionine + DEN + CCl was obtained by the method of this example 4 Of the 30 experimental mice in the experimental group, 3 died at the end of 8 weeks of molding, and 25 of the remaining 27 survivors had nodules with a mortality rate of 10% and a neoplasia rate of 93%.
TABLE 1
By combining the dyeing results of the masson, HE and the tumorigenicity data, 2 percent homomethionine + DEN + CCl can be determined 4 The experimental group successfully models liver cancer tumors.
Through repeated tests by adopting the method, the content of the 2% homomethionine + DEN + CCl is increased 4 The experimental groups can be successfully molded in 8 weeks, and FIGS. 2-7 are the dyeing pattern of masson pine and HE dyeing of multiple repeated experimentsFigure (a). The result shows that the molding method of the invention is stable, and the tumor forming rate and the survival rate of the molding can be kept above 90%.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not to be understood as limiting the scope of the patent. It should be noted that, for those skilled in the art, various changes, combinations and improvements can be made in the above embodiments without departing from the patent concept, and all of them belong to the protection scope of the patent. Therefore, the protection scope of this patent shall be subject to the claims.
Claims (4)
1. A construction method of a liver cancer model is characterized by comprising the following steps: feeding a mouse with a high methionine feed containing 2% of methionine by mass until molding is successful; per week CCl 2 gavages from week 1 4 Until the molding is successful; starting from week 1, intraperitoneal injection of DEN is performed 1 time per week for 4 weeks;
the molding time is 8 weeks;
the CCl 4 Adopting corn oil to dilute to 15% concentration, wherein the stomach filling dosage is 5mL/kg each time;
the DEN concentration of the intraperitoneal injection is 10 mu g/mL, and the injection dose is 100mg/kg.
2. The method for constructing a liver cancer model according to claim 1, wherein the model-making mouse is a male C57BL/6J mouse of 6-8 weeks old.
3. The method of claim 1, wherein after 8 weeks of modeling, the mice are sacrificed and the liver is removed, and pathological histochemical analysis of the liver cancer is performed by HE staining and Masson staining.
4. Use of the mouse liver cancer model constructed according to any one of claims 1 to 3 in the preparation of a medicament related to liver diseases.
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