CN107058228B - Establishment and application of MMHRL3 transgenic mouse liver tumor cell line - Google Patents

Abstract

The invention provides H-ras12V transgenic male miceEstablishment and application of a liver tumor cell line MMHRL 3. In particular, the H-rasThe 12V transgenic male mouse liver tumor cell line MMHRL3 has stable character, can be stably passed for multiple times, is suitable for establishing liver tumor animal models, and is an ideal cell line for basic and preclinical application research of liver cancer. In addition, the H-ras12V transgenic male mouse liver tumor cell line MMHRL3 expresses H-rasMutant oncogenes and signaling pathways with highly activated Ras/MAPK and PI 3K/AKT/mTOR. The invention also provides the application of the cell line in establishing model animals, screening drugs and the like.

Description

Establishment and application of MMHRL3 transgenic mouse liver tumor cell line

Technical Field

The invention relates to the field of biology and oncology, in particular to a novel H-ras12V transgenic male mouse liver tumor cell line MMHRL3, an establishing method and application.

Background

The occurrence and progression of human disease is a very complex process. The intensive research on the pathogenesis of various diseases and the clinical drug treatment effect aims to prevent and cure various diseases, and is a constantly struggle target for human beings. However, the method of directly conducting the experiment on the patient cannot be adopted based on humanity. By following the animal protection method, the rules of occurrence and development of various diseases and life phenomena can be deeply researched through animal experiments, so that the purposes of controlling the occurrence and development processes of human diseases, relieving the pain of patients and curing the diseases are achieved.

In the long-term exploration practice of human beings, the clinical disease research finds that the healthy human body and the diseased human body are taken as research objects to hardly promote the rapid and deep development of medicine, and has a plurality of problems of humanity, ethics, morality and the like. In addition, clinical disease research has the limitations of large sample accumulation and long-term observation.

Animal models of human diseases (Animal models of human diseases) refer to experimental animals and materials established in biomedical research with human disease manifestations. The research on the occurrence and development rules of human diseases and therapeutic measures through human disease animal models is a vital experimental method and means in modern biomedical research. Among them, cell culture has been in history for hundreds of years, and is an important method for in vitro basic medical research.

With the change of life style of people, the social industrialization process and the prolonging of life, the incidence of tumor is also increased, and the tumor becomes one of the main factors threatening the life and health of human beings. The liver cancer called "king of cancer" is the key disease prevention and treatment in our country, but its causes are many, the process is complex, the cure rate is poor, the mechanism is still unclear and still needs to be deeply elucidated.

At present, dozens of human liver cancer cell lines are established, but because the source background of each liver cancer cell line is complex, the molecular mechanism is different, and the accurate analysis of the etiology is not facilitated. In addition, the cell lines have the defects of limited tumorigenic capacity, unstable passage and the like, and are not favorable for basic research of liver tumors. Mice are a typical animal model of human diseases, and the etiology of liver cancer can be deeply profiled by establishing a primary liver tumor animal model induced by a single etiology. However, because the in vivo experiment of the mouse is high in cost, time-consuming and labor-consuming, and the influence factors are complex, the establishment of a corresponding mouse liver tumor cell line is urgently needed. However, in contrast, the establishment of mouse hepatoma cell lines is difficult. Therefore, the number of mouse liver tumor cell lines which can be used at present is very large.

Therefore, the research field urgently needs to research and develop a mouse liver tumor cell line which has definite etiology, stable passage and high tumorigenicity and is suitable for establishing various animal models.

Disclosure of Invention

The invention aims to provide a liver tumor cell line for basic research and clinical treatment of liver tumors, and the cell line has the advantages of stable passage, good tumorigenicity and suitability for establishing various liver tumor animal models.

The invention also aims to provide a preparation method and application of the H-ras12V transgenic male mouse liver tumor cell line.

In the first aspect of the invention, the invention provides a H-ras12V transgenic male mouse liver tumor cell, the H-ras12V transgenic male mouse liver tumor cell

The liver tumor cell is H-ras12V transgenic male mouse liver tumor cell MMHRL3 which is preserved in China center for type culture Collection with the preservation number of CCTCC NO: C2016192.

In a second aspect of the invention, there is provided progeny cells of the H-ras12V transgenic male mouse hepatoma cells described in the first aspect of the invention, said progeny cells being capable of causing liver tumor formation in nude mice.

In another preferred embodiment, the described progeny cells substantially retain or completely retain the characteristics of the parental H-ras12V transgenic male mouse hepatoma cell MMHRL 3.

In another preferred example, the H-ras12V transgenic male mouse liver tumor cell MMHRL3 or progeny cells thereof have the following characteristics:

(a) 100% of the cells expressed the H-ras12V transgene;

(b) has the capability of subcutaneous tumor formation of immunodeficient mice.

In the third aspect of the invention, the invention provides the use of the H-ras12V transgenic male mouse liver tumor cell MMHR1 or progeny cells thereof, which can be applied to the generation of liver tumors in nude mice.

In a fourth aspect of the present invention, there is provided a method of establishing an animal model of liver tumor, comprising the steps of:

(a) 2 x 10 to⁶-1×10⁸The H-ras12V transgenic male mouse liver tumor cell MMHRL3 or the progeny cell thereof is inoculated in a nude mouse;

(b) feeding the nude mice of the step (a) for 15-100 days to obtain the liver tumor animal model.

In another preferred embodiment, the inoculation is carried out at the following sites: liver, abdominal cavity, tail vein, subcutaneous site, or combinations thereof.

In another preferred embodiment, the nude mice are cultured in step (b) for 7-100 days to obtain an animal model of liver tumor.

In a fifth aspect of the present invention, there is provided a method for culturing liver tumor cells in vitro, comprising the steps of: the H-ras12V transgenic male mouse liver tumor cell MMHRL3 or progeny cells thereof of the invention are cultured in a suitable medium.

In a sixth aspect of the present invention, there is provided a method for screening a candidate drug for treating liver tumor, comprising the steps of:

(a) 2 x 10 to⁶-1×10⁸The H-ras12V transgenic male mouse liver tumor cell MMHRL3 or the progeny cell thereof is inoculated to a nude mouse;

(b) feeding the nude mice of the step (a) for 15-100 days to obtain a liver tumor animal model;

(c) applying a test drug to the liver tumor animal model obtained in step (b) and a step without applying a drug

(b) The liver tumor animal models obtained in the above steps are compared, wherein the test drug which causes the improvement or cure of the symptoms of the liver tumor after application is the candidate drug for treating the liver tumor.

In another preferred embodiment, in step (c), the test compound is administered in the following manner: local administration to the lesion of liver tumor, intravenous administration, and oral administration.

In a seventh aspect of the invention, there is provided a developed liver tumor model animal prepared by the above-described method of the invention.

It is understood that within the scope of the present invention, the above-mentioned technical features of the present invention and those described in detail below (e.g., examples) can be combined with each other to form new or preferred technical solutions, which are not described herein, but are not described herein.

Drawings

FIG. 1 shows the culture of the H-ras12V transgenic male mouse cell line MMHRL3 of the present invention. FIG. 1A shows the cell climbing out of a liver tumor tissue mass to form a tumor cell mass (200X) when cultured for 8 days in a semidry method; FIG. 1B shows the growth morphology (200X) of the H-ras12V transgenic male mouse cell line MMHRL3 cells cultured to passage 25.

FIG. 2 shows the cell morphology and proliferation of the H-ras12V transgenic male mouse cell line MMHRL3 of the present invention. FIG. 2A shows the cellular morphology (400X) of the H-ras12V transgenic male mouse cell line MMHRL3 of the present invention; FIG. 2B shows the proliferation of the H-ras12V transgenic male mouse cell line MMHRL3 of the present invention.

FIG. 3 shows the genotyping and expression level testing of the H-ras12V transgene of the H-ras12V transgenic male mouse cell line MMHRL3 of the present invention.

FIG. 4 shows the Western Blot assay of the level of activation of the signaling pathway of the H-ras12V transgenic male mouse cell line MMHRL3 of the present invention.

FIG. 5 shows the results of experiments on the H-ras12V transgenic male mouse cell line MMHRL3 subcutaneous inoculation of nude mouse for tumor formation.

Detailed Description

The inventor carries out extensive and intensive research, primary culture is carried out on a liver tumor sample of an H-ras12V transgenic male mouse, and finally an H-ras12V transgenic male mouse cell line MMHRL3 with unlimited proliferation capacity is established. The present invention has been completed based on this finding.

Specifically, the inventor will be a H-ras12V transgenic male mouse liver tumor tissue in vitro culture, establish the corresponding in vitro cell line.

And carrying out related biological detection and identification on the cell line. Screening to obtain a pure H-ras12V transgenic male mouse cell line MMHRL 3. The cell line grew stably (had been stably passaged to 75 passages) and the cell line was morphologically uniform under light microscopy.

Zoology experiments show that the H-ras12V transgenic male mouse cell line MMHRL3 can cause tumors of nude mice after being inoculated to the nude mice, and the histopathological morphology of the tumor is consistent with that of liver tumor tissues of H-ras12V transgenic male mice.

The H-ras12V transgenic male mouse cell line MMHRL3 not only can be used for preparing a liver tumor animal model, but also can be used for screening candidate drugs for treating liver tumors.

A preferred method of screening a candidate agent for treatment of a liver tumor (or liver tumor cell growth) comprising the steps of:

(a) in the test group, a test compound is added into a culture system of the H-ras12V transgenic male mouse liver tumor cell MMHRL3, and the cell number and/or growth condition of the H-ras12V transgenic male mouse liver tumor cell MMHRL3 are observed; in the control group, no test compound is added in a culture system of the H-ras12V transgenic male mouse liver tumor cell MMHRL3, and the cell number and/or growth condition of the H-ras12V transgenic male mouse liver tumor cell MMHRL3 are observed;

wherein, if the number or the growth rate of the H-ras12V transgenic male mouse cells MMHRL3 in the test group exceeds that of the control group (a significant difference exists), the test candidate drug is suggested to have a promoting effect on the growth or proliferation of the liver tumor cells.

In another preferred embodiment, less than that described means that the ratio of the number of the test group to the control group or the ratio of the growth rate is 0.8 or less, or more preferably 0.4 or less.

In another preferred embodiment, the ratio described as greater than or equal to 2.5, or more preferably greater than or equal to 0.5, represents the ratio of the number of test groups to control groups or the ratio of the growth rate.

In another preferred example, the significant difference described is P <0.05.

A preferred method of screening a candidate drug for treating liver tumors comprises the steps of:

(a) 2 x 10 to⁶-1×10⁸(preferably 2X 10)⁶-1×10⁷More preferably 1X 10⁷-1×10⁸) The Hras12V transgenic male mouse liver tumor cell MMHRL3 is inoculated to a nude mouse;

(b) feeding the nude mice of the step (a) for 14-90 days to obtain a liver tumor animal model;

(c) administering a test drug to the liver tumor animal model of step (b) in combination with a step in which no test drug is administered

(b) Wherein a test drug which can improve or cure the symptoms of the liver tumor animal model after administration can be considered as a candidate drug for treating the liver tumor.

The main advantages of the invention are:

(a) the H-ras12V transgenic male mouse hepatoma cell MMHRL3 is from an H-ras12V transgenic male mouse;

(b) the H-ras12V transgenic male mouse hepatoma cell MMHRL3 has stable properties and can be stably subjected to multiple passages;

(c) the H-ras12V transgenic male mouse liver tumor cell MMHRL3 has good tumorigenicity, has clear liver tumor pathogenesis and genetic characteristics, and is an ideal cell line for researching liver tumor mechanism and clinical early-stage trial;

(d) the H-ras12V transgenic male mouse liver tumor cell MMHRL3 can be used for in vitro research of liver tumor related genes;

(e) the H-ras12V transgenic male mouse liver tumor cell MMHRL3 can be used for in vitro screening of liver tumor sensitive drugs.

The invention is further illustrated by the following examples. It should be understood that the following examples are only illustrative of the present invention and are not intended to limit the scope of the present invention. In the following examples, the experiment was carried out according to the conventional experimental conditions or the experimental conditions recommended by the manufacturer without specifying the experimental conditions. Unless otherwise noted, percentages and parts are percentages by weight and parts by weight.

Example 1

Establishment of H-ras12V transgenic male mouse hepatoma cell MMHRL3

(a) Origin of origin

H-ras12V transgenic male mouse hepatoma cell line was derived from H-ras12V transgenic male mice

The information is shown in the following table.

TABLE 1

Obtaining the primary liver tumor tissue of the H-ras12V transgenic male mouse, and directly culturing the liver tumor tissue in vitro

And culturing, repeatedly digesting and passaging to finally obtain the stably passaged H-ras12V transgenic male mouse liver tumor cell line. The method specifically comprises the following steps:

the H-ras12V transgenic male mice were sacrificed painlessly and liver tumors were excised by aseptic technique. Placing liver tumor in 10cm culture dish containing normal temperature sterilized normal saline, cleaning tumor envelope, cutting the liver tumor tissue remained after the above operation to 1 × 1 × 1mm with scalpel³Adding liver tumor tissue culture solution into the tissue blocks with the size, and culturing by a semidry method. The liver tumor tissue culture solution comprises DMEM high glucose culture medium, 10% heat inactivated fetal calf serum, 100IU/ml penicillin-streptomycin and 50mg/ml gentamycin. And (3) replacing the newly inoculated liver tumor tissue block after 24H, and replacing the solution at 72H in the process of culturing the H-ras12V transgenic male mouse liver tumor cells. After 8 days of semi-dry culture, cell climbing out around the liver tumor tissue mass can be observed. At the moment, the liver tumor tissue is subjected to conventional cell culture digestion and plating, and fibroblasts in the original liver tumor tissue culture are removed in the processes of multiple times of digestion and plating. The primary cells can obtain a large amount of epithelial-like cells with uniform morphology from the first passage to the 10 th passage, and are named as MMHRL 3.

At present, the H-ras12V transgenic male mouse hepatoma cell MMHRL3 has been passaged to the 75 th generation, the cell growth is stable, the state is good, and the cell growth is also continued to be passaged and cultured (FIG. 1B and FIG. 2A).

The obtained H-ras12V transgenic male mouse hepatoma cell MMHRL3 is preserved in 2016 at 11 months and 08 days, and is preserved in China Center for Type Culture Collection (CCTCC) (Wuhan & China), with the preservation number of CCTCC NO: c2016192, deposit address: wuhan university school in Wuhan City of Hubei province.

Example 2

Biological characteristics of H-ras12V transgenic male mouse hepatoma cell MMHRL3

In this example, the pure H-ras12V transgenic male mouse hepatoma cell MMHRL3 with the preservation number of CCTCC NO: C2016192 was examined for biological characteristics by the conventional method, and the results are as follows:

2.1 cell morphology

When the liver tumor cells MMHRL3 of the H-ras12V transgenic male mice were observed under a common light microscope, most of the liver tumor cells were circular or polygonal and the cell morphology was uniform (FIG. 2A). The Cell Counting Kit-8(CCK8 Kit) was used to determine the proliferation status of the Cell line, and the procedure was performed exactly according to the Kit instructions (FIG. 2B). The cells proliferated stably.

2.2 genotyping of the H-ras12V transgene

The H-RAS12V transgenic male mouse liver tumor cell MMHR L3 (accession number: C2016192) obtained in example 1 was digested with a digestive juice (0.25% pancreatin, 0.02% EDTA, pH7.3), and then genomic DNA and total RNA were collected and extracted for PCR qualitative identification of RAS transgenic genotype and RT-PCR detection of RAS gene expression.

(a) RAS transgenic PCR qualitative identification method:

h-ras12V transgenic male mouse cell line MMHRL3 genomic DNA was extracted and subjected to general PCR, the loading amounts are shown in Table 2 below, and the results are shown in FIG. 3A

TABLE 2

(b) RAS transgenic RT-PCR quantitative detection method:

extracting MMHRL3 mRNA from H-ras12V transgenic male mouse cell line, synthesizing cDNA according to the method in (a), performing RT-PCR, and loading the sample as shown in Table 3 below, the results are shown in FIG. 3B

TABLE 3

Western Blot assay of hepatoma cells MMHRL3 cells of 3H-ras 12V transgenic male mice

In this example, WesternBlot assay was performed on H-ras12V transgenic male mouse hepatoma cells MMHRL3 cells. The results are shown in FIG. 4.

The results show that: the MEK/ERK and AKT/mTOR signaling pathways of the liver tumor cell MMHRL3 cell of the H-ras12V transgenic male mouse are obviously activated.

Example 3

Establishment of liver tumor animal model by using H-ras12V transgenic male mouse liver tumor cell MMHR1

The H-ra s12V transgenic male mouse liver tumor cell MMHR L3 (accession number: C2016192) obtained in example 1 was digested with a digestive juice (0.25% pancreatin, 0.02% EDTA, pH7.3), collected and counted, and the number was determined to be 2X 10⁶Or 1X 10⁸Cell number/mouse was inoculated to the subcutaneous site of the left and right underarm of the mouse, 5 nude mice aged 4 weeks.

Tumors developed in 5 mice between 15-40 days after inoculation. Biological examination was carried out in the same manner as in example 2, and it was confirmed that hepatocellular liver tumor was induced in the animal model.

Cell line preservation

The H-ras12V transgenic male mouse hepatoma cell MMHRL3 is preserved in the China center for type culture Collection (Wuhan, China) at 2016, 11, 08 days, with the preservation number of CCTCC NO: C2016192.

all documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

SEQUENCE LISTING

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Claims (5)

1. A transgenic male mouse liver tumor cell is characterized in that the liver tumor cell is H-ras12V transgenic male mouse liver tumor cell MMHRL3 which is preserved in China center for type culture Collection with the preservation number of CCTCC NO: C2016192;

the preparation method of the H-ras12V transgenic male mouse hepatoma cell MMHRL3 specifically comprises the following steps: the H-ras12V transgenic male mice are killed painlessly, and liver tumors are cut off by aseptic technique; placing liver tumor in 10cm culture dish containing normal temperature sterilized normal saline, cleaning tumor envelope, cutting the liver tumor tissue remained after the above operation to 1 × 1 × 1mm with scalpel³Adding liver tumor tissue culture solution into the tissue blocks with the sizes, and culturing by a semi-dry method; the liver tumor tissue culture solution comprises DMEM high glucose culture medium, 10% heat inactivated fetal calf serum, 100IU/ml penicillin-streptomycin and 50mg/ml gentamycin are added; changing the liquid after the newly inoculated liver tumor tissue block is cultured for 24 hours, and changing the liquid for 72 hours in the process of culturing the H-ras12V transgenic male mouse liver tumor cells; after 8 days of semi-dry culture, cell climbing-out can be observed around the liver tumor tissue block; at the moment, conventional cell culture digestion and plate paving are carried out on the liver tumor tissue, and fibroblasts in the original liver tumor tissue culture are removed in the processes of multiple times of digestion and plate paving; the primary cells can obtain a large amount of epithelial-like cells with uniform morphology from the first passage to the 10 th passage, and are named as MMHRL 3.

2. The progeny of the H-ras12V transgenic male mouse hepatoma cells of claim 1, wherein said progeny are capable of causing liver cancer in nude mice.

3. The H-ras12V transgenic male mouse hepatoma cell of claim 1 or progeny cell of claim 2 having the following characteristics:

(a) 100% of the cells expressed the H-ras12V transgene;

(b) has the capability of subcutaneous tumor formation of immunodeficient mice.

4. The use of the H-ras12V transgenic male mouse hepatoma cell of claim 1 or the progeny cell of claim 2 for producing hepatoma in nude mice.

5. A method of establishing an animal model of liver tumor comprising the steps of:

(a) 2 x 10 to⁶-1×10⁸The H-ras12V transgenic male mouse hepatoma cells of claim 1 or progeny cells of claim 2 inoculated into nude mice;

(b) feeding the nude mice of the step (a) for 15-100 days to obtain the liver tumor animal model.

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