CN108467854B - Novel bone-specific metastatic hepatoma cell and preparation thereof - Google Patents
Novel bone-specific metastatic hepatoma cell and preparation thereof Download PDFInfo
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Abstract
The invention relates to a bone-specific metastatic hepatoma cell 3B-BM, in particular to a cell 3B-BM which is preserved with a preservation number of CCTCC NO: the HEP-3B organ-specific metastatic cell line BM of C2016174.
Description
Technical Field
The invention belongs to the field of biology and oncology, and particularly relates to a bone-specific metastatic hepatoma cell.
Background
Hepatocellular carcinoma is the second leading cause of tumor-related death worldwide, and the high metastasis of liver cancer leads to poor prognosis after drug treatment in liver cancer patients, and clinically common liver cancer metastasis is intrahepatic metastasis, lung metastasis and bone metastasis.
The metastasis of liver cancer involves many processes, including the shedding of tumor cells in situ in the liver, invasion of surrounding tissues, entry into the blood system, survival in the blood system and egress of blood vessels to form clones in distant tissue organs, each process involving the change of tumor cells themselves and the interaction between the tumor and the microenvironment, and the specific mechanism of action is not completely understood.
The tumor cell line has an important position in the basic research of tumors, the in vitro culture of tumor cells is difficult, and particularly, the establishment of a human tumor cell line which can grow and passage for a long time and has certain characteristics usually encounters the conditions of unstable passage characteristics, poor tumorigenicity, inconsistent genetic background, large expression difference of specific markers and the like. In the case of liver cancer cells, the in vitro culture conditions are usually very harsh, while randomly obtained cells (especially metastatic cells) usually have no in vitro culture stability.
Therefore, there is an urgent need in the art to develop a liver cancer cell line suitable for the research of modern liver cancer cells and stably applicable to the establishment of animal models, especially a common metastatic liver cancer cell line.
Disclosure of Invention
The invention provides a bone-specific metastatic hepatoma carcinoma cell, which has extremely strong tumorigenic capacity and bone metastasis specificity and stable passage and hereditary characteristics.
The invention provides a bone-specific metastatic hepatoma cell 3B-BM in a first aspect, wherein the cell 3B-BM is a cell with a preservation number of CCTCC NO: the HEP-3B organ-specific metastatic cell line BM of C2016174.
In a second aspect of the present invention, there is also provided progeny of the bone-specific metastatic liver cancer cell of the first aspect of the present invention, wherein the progeny is capable of causing formation of bone-specific metastatic liver cancer in nude mice and/or humans.
In another preferred embodiment, the progeny cells substantially retain (95%,. gtoreq.96%,. gtoreq.97%,. gtoreq.98%,. gtoreq.99%) or all the structure and characteristics of the parent bone-specific metastatic hepatoma carcinoma cell 3B-BM.
In another preferred embodiment, the progeny cells are progeny cells of 3B-BM cells that have been passaged within 15 passages, preferably within 10 passages, within 5 passages, and more preferably within 3 passages.
In another preferred embodiment, the progeny cells retain substantially or all of the characteristics of the parent bone-specific metastatic hepatoma carcinoma cell 3B-BM.
In another preferred embodiment, the cell according to the first aspect of the present invention (or progeny of the second aspect of the present invention), the bone-specific metastatic liver cancer cell (or progeny) has one or more of the following characteristics:
(a) the cells have a specific miRNA expression profile, and the miRNA expression profile comprises the following characteristics
(b) The bone transfer rate of the cells is more than or equal to 70 percent; and/or
(c) The transfer rate of the cells to organs other than bone is less than 30%.
In a third aspect of the present invention, there is provided a use of the bone-specific metastatic liver cancer cell of the first aspect of the present invention (or the progeny cell of the second aspect of the present invention) for preparing a bone-specific metastatic liver cancer model of a non-human mammal or for screening a candidate compound for treating bone-specific metastatic liver cancer.
Characterized in that the mammal is selected from rat, mouse, rabbit, sheep, dog and monkey.
In another preferred embodiment, the mammal is an immunodeficient laboratory animal.
In another preferred embodiment, the animal is a nude mouse, preferably a T cell deficient nude mouse.
In a fourth aspect of the present invention, a method for establishing an animal model of bone-specific metastatic hepatoma carcinoma cells is provided, comprising the steps of:
(i) will be 5X 105-1×106Inoculating a nude mouse with the bone metastasis hepatoma cell (or progeny cells thereof) of the first aspect of the invention;
(ii) and (3) culturing the nude mouse in the step (i) for 21-42 days, taking out hind limbs of the nude mouse, shearing joints at two ends of a bone, sucking serum-free DMEM by a 1ml injection needle, eluting from one end of the bone, repeating the steps, culturing in the obtained mixed cell DMEM medium, and after the cells adhere to the wall, carrying out flow cytometry to sort GFP positive cells so as to obtain the bone metastasis hepatoma cell animal model.
In another preferred embodiment, the mammal is an immunodeficient mouse, such as a nude mouse, cultured for 21-42 days.
In another preferred embodiment, the cells grow faster, reach a density of 80% effect at 4 days by 10cm dish 1, and carry GFP and an m-cherry selection marker.
In another preferred embodiment, the inoculation is carried out at the following sites: tail vein, abdominal cavity, tail vein, subcutaneous site, liver, or combinations thereof.
In a fifth aspect of the present invention, there is provided a method for screening a candidate compound for treating bone-specific metastatic liver cancer, comprising the steps of:
(a) will be 5X 105—1×106Inoculating a mammal with the bone-specific metastatic hepatoma cell (or progeny cells thereof) according to the first aspect of the invention;
(b) culturing the mammal of the step (a) for 21-42 days to obtain a bone-specific metastatic liver cancer animal model; and
(c) administering a test compound to the bone-specific metastatic liver cancer animal model of step (b) and comparing to the bone-specific metastatic liver cancer animal model of step (b) without administration of the test compound, wherein the test compound that results in an improvement or cure in the shape of the bone-specific metastatic liver cancer after administration is a candidate compound for treating bone-specific metastatic liver cancer;
or the method comprises:
(a1) in the test group, a test compound is added into the culture system of the bone-specific metastatic hepatoma carcinoma cells of the first aspect of the invention, and the number and/or growth of the bone-specific metastatic hepatoma carcinoma cells are observed; in the control group, no test compound is added into the culture system of the bone-specific metastatic hepatoma cells, and the number and/or growth condition of the bone-specific metastatic hepatoma cells are observed;
wherein, if the number or growth rate of the bone-specific metastatic liver cancer cells in the test group is less than that of the control group, the test compound is a candidate compound for treating the bone-specific metastatic liver cancer, which has an inhibitory effect on the growth or proliferation of the bone-specific metastatic liver cancer cells.
In a sixth aspect of the present invention, a method for screening a potential liver cancer lung metastasis related gene is provided, which comprises the steps of:
comparing the gene expressed in the bone-specific metastatic hepatoma carcinoma cell (or progeny cells thereof) of the first aspect of the invention with the gene expressed in normal hepatocytes, and screening out the gene with statistically up-regulated or down-regulated expression in the bone-specific metastatic hepatoma carcinoma cell of the first aspect of the invention, wherein the gene is a potential gene related to bone-specific metastatic hepatoma carcinoma.
In another preferred example, the method further comprises:
the obtained potential bone-specific metastatic liver cancer related gene is further subjected to cell experiments and/or animal experiments to select genes which play a definite role in liver cancer lung metastasis.
In a seventh aspect of the present invention, there is provided a method for in vitro culture of a bone-specific metastatic hepatoma cell line, comprising the steps of: culturing the bone-specific metastatic hepatoma cell or progeny cells thereof according to the first aspect of the invention in a suitable culture medium.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows that the first and second generation liver cancer cell lines screened still have the property of random multi-organ metastasis.
FIG. 2, in which Panel A shows that the 3B-BM cell line of the present invention has an extremely strong specific bone metastasis ability after screening; and the B picture shows that the 3B-BM cell line of the invention still has extremely strong specific bone transfer capability when being passaged for the 15 th generation.
FIG. 3 shows that the proliferation potency of the cell line of the present invention is significantly enhanced compared to its parental cells.
Detailed Description
The inventor conducts extensive and intensive research, carries out a large amount of screening and culture on ten cell lines of metastatic liver cancer, finally establishes a new bone specificity metastatic liver cancer cell which has good tumorigenicity, extremely high bone metastasis rate and stable passage characteristic, and can be used for establishing an animal model and treating refractory liver cancer (or drug-resistant liver cancer). More importantly, the invention also compares the characteristics of each specific metastatic cell, finds out the key factor of the metastasis of the liver cancer after entering the blood, and further clarifies the mechanism of the liver cancer metastasis in the process. On the basis of this, the present invention has been completed.
Term(s) for
As used herein, the terms "bone-specific metastatic hepatoma cells", "HEP-3B organ-specific metastatic cell line BM", "human bone-specific metastatic hepatoma cells", "inventive cells", "3B-BM" are used interchangeably and all refer to the bone-specific metastatic hepatoma cell line 3B-BM of the present invention, which was deposited in the chinese typical culture collection (CCTCC) (wuhan, china) at 2016, 10, 18 days, with the deposition number CCTCC NO: C2016174.
cell line screening method and its characteristics
For liver cancer cells, the culture conditions are usually harsh, for example, after the research of the inventor, the in vitro culture conditions of hep3B cells adopted by the invention are usually 10% FBS + DMEM + 1% double antibody, and the cell growth density is 70-80% for about 2 days of passage time. When the liver cancer cells are cultured in this manner, the digestion conditions are required, 0.25% of pancreatin is used, the whole process temperature is maintained at about 37 ℃, and the inoculation of the transferred cells should be performed immediately after the cells are mixed to about 70%.
However, even if the cell culture operation is performed in compliance with a careful and severe culture condition, the present inventors found that the transfer probability of hep-3B cells is extremely low and that there are many transfer sites. The inventor conducts 4 × 7 (4-generation screening, 7 times of repeated generations) batch of metastatic cell culture, and can locate the position of tumor cells in a living level and accurately separate the tumor cells in an isolated state through multiple screening by double markers (GFP and luciferase), so as to obtain the hep-3B bone/lung metastatic cell line, and finds that the hep-3B bone/lung metastatic cell line has an abnormally stable in-vitro passage characteristic, and the cell line still has a strong bone/lung directional metastasis characteristic after passage for 15-20 generations.
A preferred screening method comprises the steps of: screening a liver cancer specific bone metastasis cell line in a two-step continuous mode, firstly, utilizing luciferase catalytic substrate luminescence carried by cells to locate a metastasis of a specific organ at a living body level, taking out the metastasis from a mouse body, and confirming the metastasis as a marked liver cancer cell line again under a fluorescence microscope; shearing two ends of the leg bone, sucking serum-free DMEM by using a 1ml syringe to repeatedly wash the leg bone to obtain a single cell suspension, culturing until the cells adhere to the wall, growing to about 70% density, screening the tumor cells with GFP by using a flow cytometer, and repeating for dozens of batches until the liver cancer cell line which is stably transferred is obtained.
The screening operation was as follows: shearing two ends of the leg bone, sucking serum-free DMEM by using a 1ml syringe to repeatedly wash the leg bone to obtain a single cell suspension, adding erythrocyte lysate, removing erythrocytes, washing for 3 times by pbs, culturing in a DMEM culture medium for 2-3 days until the cell density reaches about 80%, gently digesting cells, setting a GFP (green fluorescent protein) sorting threshold value by taking common mouse organ cells as a control, sorting GFP positive cells to obtain a primary screening result, and performing directional repeated screening to finally obtain the stably transferred liver cancer cell line.
The bone-specific metastatic hepatoma carcinoma cell of the invention has one or more of the following characteristics:
(a) the cells have a specific miRNA expression profile comprising the following characteristics:
(b) the bone metastasis rate of the cells is more than or equal to 70 percent, preferably 75 percent; and/or
(d) The transfer rate of the cells to organs other than bone is less than 30%.
The 3B-BM cell line of the invention can be passaged by those skilled in the art to obtain progeny cells of 3B-BM, according to methods conventional in cell subculture in the art. Of course, in order to maintain the identity of the genetic characteristics of 3B-BM, it is preferable to use a cell line within 15 generations (more preferably within 10 generations, within 5 generations, and within 3 generations) of 3B-BM as progeny cells of 3B-BM, and when the progeny cells are passaged to more than 15 generations, it is determined by sequencing to maintain the homology of the progeny cells. Typically, progeny cells having greater than 95% homology to the parent cell line will be selected in the art and must retain, or substantially retain, the biological properties of the parent cell.
Experiments prove that after passage of 15-20 generations, the liver cancer cells with bone specific metastasis still retain 70% or more, such as 75%, 80%, 85%, 90%, 95%, 98% or 100% of bone metastasis rate.
Applications of
The bone-specific metastatic hepatoma cell line can be used for preparing bone-specific metastatic hepatoma animal models and screening candidate drugs for treating bone-specific metastatic hepatoma.
A preferable method for establishing a bone-specific metastatic liver cancer animal model comprises the following steps:
(i) will be 5X 105-1×106Inoculating a non-human mammal with a bone-specific metastatic hepatoma cell (or progeny cells thereof) according to the first aspect of the invention;
(ii) (ii) culturing the mammal of (i) for 21-42 days to obtain the bone-specific metastatic hepatoma cell animal model.
Wherein, the inoculation is to inoculate the following parts: liver, abdominal cavity, tail vein, subcutaneous site, or combinations thereof; the mammal is an immunodeficient experimental animal.
A preferred method of screening a candidate compound for the treatment of bone-specific metastatic liver cancer, comprising the steps of:
(a) will be 5X 105—1×106The bone-specific metastatic hepatoma cell (or progeny cells thereof) of the first aspect of the invention) Inoculating the mammal;
(b) culturing the mammal of step (a), such as a nude mouse, to obtain a bone-specific metastatic liver cancer animal model; and
(c) administering a test compound to the bone-specific metastatic liver cancer animal model of step (b) and comparing to the bone-specific metastatic liver cancer animal model of step (b) without administration of the test compound, wherein the test compound that results in an improvement or cure in the shape of the bone-specific metastatic liver cancer after administration is a candidate compound for treating bone-specific metastatic liver cancer;
or the method comprises:
(a1) in the test group, a test compound is added into the culture system of the bone-specific metastatic hepatoma carcinoma cells of the first aspect of the invention, and the number and/or growth of the bone-specific metastatic hepatoma carcinoma cells are observed; in the control group, no test compound is added into the culture system of the bone-specific metastatic hepatoma cells, and the number and/or growth condition of the bone-specific metastatic hepatoma cells are observed;
wherein, if the number or growth rate of the bone-specific metastatic liver cancer cells in the test group is less than that of the control group, the test compound is a candidate compound for treating the bone-specific metastatic liver cancer, which has an inhibitory effect on the growth or proliferation of the bone-specific metastatic liver cancer cells.
Preferably, the mammal is an immunodeficient mouse (nude mouse) and the culturing period is 35 days.
In addition, the bone-specific metastatic hepatoma cell line can also be used in a matching way with a liver-specific metastatic hepatoma cell line, and is used for screening genes of potential metastatic hepatoma and screening drugs for treating metastatic hepatoma at different parts.
A preferred method for screening potential bone-specific metastatic liver cancer related genes comprises the steps of:
comparing the gene expressed in the bone specific metastatic liver cancer cell or the filial cell thereof with the gene expressed in the conventional liver cancer cell, and selecting the gene with up-regulation or down-regulation expression in the bone specific metastatic liver cancer cell, wherein the gene is the potential bone specific metastatic liver cancer related gene.
According to the determination of the mRNA and miRNA spectrums of the bone-specific metastatic liver cancer and the data analysis, the method is as follows:
the invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.
Example 1 screening and identification of bone-specific metastatic hepatoma cell lines
The cell is firstly marked with GFP so as to separate bone metastasis cells, secondly, the cell is transformed to stably express luciferase enzyme, can carry out mouse living body imaging detection cell position and quantitative analysis, and simultaneously, the cell is marked with m-chery when constructing luciferase enzyme and is used as a screening marker of luciferase positive cells. The migration capacity of the cell is obviously enhanced compared with that of the original hep-3B cell. Specifically, hep-3B cells marked with luciferase are injected into nude mice via tail vein, and randomly transferred liver cancer foci appear in the mice within 20-40 days, i.e. primary hep-3B cells do not have transferred organ specificity. Selecting a mouse with a target organ transferred from randomly transferred mice, shearing two ends of a leg bone, sucking serum-free DMEM (DMEM) by using a 1ml syringe to repeatedly wash the leg bone to obtain a single cell suspension, culturing until cells adhere to the wall and grow to about 70% of density, sorting tumor cells with GFP by flow cytometry, and displaying a result that only a few tumor cells are contained in the obtained single cell suspension; subsequently, the selected GFP cells were expanded and secondary sorting was continued using the m-cherry carried by the cells, ensuring that tumor cells, but not mouse stromal cells, were obtained. The cells obtained this time were the first generation bone metastasis cell line, and using the cells, the second generation cell line was obtained in the same manner. The first and second generation bone metastasis cell lines (14 batches in total) do not have stable bone metastasis capacity, the bone metastasis rates are about 30% and 40% respectively, random metastasis is still found in the bodies of the mice transfused back, and more bone metastasis occurs outside the bodies, as shown in fig. 1.
Through living body imaging technology and morphological observation, the steps are repeated again, cells with strong bone transfer capacity are screened out, and third and fourth generation bone transfer cell lines (total 14 batches) obtained by re-infusing mice gradually show strong transfer capacity, but the proliferation capacity of most cell lines is gradually weakened after passage. As a result, a strain was obtained from the fourth generation bone metastasis, which had a strong tendency to bone metastasis, and the bone metastasis rate was about 75% or more, and the results of bone metastasis of this cell line after 2 generations (i.e., the cell line of the present invention) are shown in graph A in FIG. 2, while the ability to metastasize outside the bone was decreased. The strain cell still has the characteristics after stable passage for 15 times, namely stable bone transfer characteristics.
Example 2 establishment and identification of mouse model of bone-specific metastatic hepatoma cells
2.1 mixing 5X 105-1×106Inoculating bone-specific metastatic hepatocarcinoma cell (or its progeny cell) to nude mouse to obtainObtaining the bone-specific transfer hepatoma cell mouse. Taking out mouse bones after 21-42 days, shearing two ends of the leg bones, sucking serum-free DMEM by using a 1ml syringe to repeatedly wash the leg bones to obtain a single cell suspension, culturing until cells adhere to the wall, growing to about 70% density, and carrying out flow cytometry to sort GFP positive cells so as to obtain bone specific metastatic hepatoma cells.
The morphology of the obtained transfer specific cell is identified, and the result proves that the morphological characteristics of the transfer specific cell are not obviously different from those of the original liver cancer cell, the transfer specific cell is determined to be the liver cancer cell, but the proliferation capacity of the cell is obviously enhanced, as shown in figure 3.
2.2 passage Activity identification
Through identification, the cell passage activity is better, and the stable specific bone metastasis can still be realized after passage 15-20 generations, as shown in a B picture in figure 2.
Example 3 Generation of Gene expression Profile identification of bone-specific metastatic hepatoma cells
Culturing hep-3B cells and bone transfer specific cells, after 2 passages, part of the cells are delivered to storage, performing genetic characteristic identification on part of the cells in parallel passages, including gene sequencing, miRNA spectrum and mRNA spectrum difference detection, cell adherence, pancreatin digestion when 50-70% of the cells in a culture dish, centrifugally collecting the cells, cracking the cells by a trizol method, RNA extraction, reverse transcription, mRNA expression chip (Bohao biotechnology, Inc.) detection and gene sequencing. As a result, the deposited cells and the cells of each progeny (generations 2-15) were found to have the same genetic characteristics.
Example 4 identification of planting tumor formation Rate
The tumor formation rate of the bone metastasis specific cells is measured by a conventional method, and the result shows that the tumor formation rate of the tail vein is 100%, wherein the bone metastasis specificity is over 75%.
Cell line preservation
The HEP-3B organ specific transfer cell line BM of the present invention is preserved in 2016.10.18 in China Center for Type Culture Collection (CCTCC) (Wuhan, China), with a preservation number of CCTCC NO: C2016174.
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.
Claims (9)
1. The bone-specific metastatic hepatoma carcinoma cell 3B-BM is characterized in that the cell 3B-BM is a cell with a preservation number of CCTCC NO: c2016174 and bone metastasis rate of the cell is 75% or more; and/or the rate of metastasis of organs other than bone of said cells is < 30%.
2. The bone-specific metastatic hepatoma cell 3B-BM of claim 1, wherein said bone-specific metastatic hepatoma cell 3B-BM or progeny cells thereof has one or more of the following characteristics:
(a) the cells have a specific miRNA expression profile, and the miRNA expression profile comprises the following characteristics
(b) The gene expression of the cell has the following characteristics:
wherein the progeny cells are progeny cells of the 3B-BM cells that have been passaged within 15 passages.
3. The use of the bone-specific metastatic liver cancer cell 3B-BM or progeny cells thereof of claim 1, for preparing a bone-specific metastatic liver cancer model of a non-human mammal or for screening candidate compounds for treating bone-specific metastatic liver cancer, wherein the progeny cells are progeny cells of the 3B-BM cells passaged within 15 generations.
4. The use of claim 3, wherein said mammal is selected from the group consisting of rat, mouse, rabbit, sheep, dog, monkey.
5. The use of claim 3, wherein the mammal is an immunodeficient laboratory animal.
6. A method of screening a candidate compound for the treatment of bone-specific metastatic liver cancer comprising the steps of:
(a1) in the test group, adding a test compound into the culture system of the bone-specific metastatic hepatoma carcinoma cells 3B-BM or progeny cells thereof of claim 1, and observing the number and/or growth of the bone-specific metastatic hepatoma carcinoma cells; in the control group, no test compound is added into the culture system of the bone-specific metastatic hepatoma cells, and the number and/or growth condition of the bone-specific metastatic hepatoma cells are observed; wherein the progeny cells are progeny cells of the 3B-BM cells that have been passaged within 15 passages,
wherein, if the number or growth rate of the bone-specific metastatic liver cancer cells in the test group is less than that of the control group, the test compound is a candidate compound for treating the bone-specific metastatic liver cancer, which has an inhibitory effect on the growth or proliferation of the bone-specific metastatic liver cancer cells.
7. A method for screening a potential liver cancer lung metastasis related gene is characterized by comprising the following steps:
comparing the gene expressed in the bone-specific metastatic hepatoma carcinoma cell 3B-BM of claim 1 or progeny cells thereof with the gene expressed in normal hepatocytes, and screening the gene statistically up-regulated or down-regulated in the bone-specific metastatic hepatoma carcinoma cell 3B-BM of claim 1, which is a potential bone-specific metastatic hepatoma carcinoma-associated gene, wherein the progeny cells are progeny cells of the 3B-BM cells that have been passaged within 15 generations.
8. The method of claim 7, wherein the method further comprises:
the obtained potential bone-specific metastatic liver cancer related gene is further subjected to cell experiments and/or animal experiments to select genes which play a definite role in liver cancer lung metastasis.
9. A method for in vitro culture of a bone-specific metastatic hepatoma cell line, comprising the steps of: culturing the bone-specific metastatic hepatoma carcinoma cell 3B-BM or progeny cells thereof of claim 1 in a suitable medium, wherein the progeny cells are progeny cells of the 3B-BM cell that have been passaged within 15 passages.
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