CN102690785B - Establishment and application of hepatocellular carcinoma cell line HCC-LY5 - Google Patents
Establishment and application of hepatocellular carcinoma cell line HCC-LY5 Download PDFInfo
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Abstract
The invention provides establishment and application of a hepatocellular carcinoma cell line HCC-LY5. Specifically, the hepatocellular carcinoma cell line HCC-LY5 provided in the invention has stable traits, can be subcultured stably and repeatedly, and is applicable to established animal models of hepatocellular carcinoma, thus being an ideal cell line for the basic and preclinical phase application study of hepatocellular carcinoma. In addition, a very small part of the hepatocellular carcinoma cell line HCC-LY5 expresses hepatocellular carcinoma stem cell marker CD133. The invention also provides application of the cell line in establishing model animals, screening drugs, and other aspects.
Description
Technical field
The present invention relates to biology and oncology, relate more specifically to a kind of new hepatocellular carcinoma clone HCC-LY5 and establishment method thereof and application.
Background technology
The generation of the various diseases of the mankind and development are very complicated, and pathogeny and the curative effect mechanism thereof of the various disease of the discussion that go deep into can not be tested with it patient.But can Animal Protection Law be followed; by animal, various disease and biological phenomena are studied; and then push away and unravel silk the secret that the mankind explore human life; explore the rule that development occurs human diseases; to control generation, the development of human diseases; cure diseases and alleviate the ill misery of the mankind, improves life quality, extends the life-span of the mankind.
It is found that for a long time, the development promoting medical science using people itself as experimental subjects is difficult, experience accumulated clinically not only also exists limitation over time and space, and many experiments also also exist various restriction in ethics and methodology.
The animal model (Animal model of human diseases) of human diseases be in biomedical sciences research set up the experimentation on animals object and material with the performance of human diseases simulation.Use animal model to be the very important experimental technique of in modern biomedical research one and means, contribute to more convenient, to be more effectively familiar with human diseases generation, the rule of development and research prophylactico-therapeutic measures.
Tumour cell ties up in the fundamental research of tumour has critical role, and cultured tumor cells in vitro is more difficult, especially set up can long term growth, go down to posterity, there is again the human tumor cell line of certain feature.
For liver cancer field, at present, although there are some hepatoma cell line, but there is multiple shortcoming in these hepatoma cell line, comprising: the instability that goes down to posterity, Tumor formation are poor, the characteristic of clone is inconsistent, liver cancer-specific marker expression is uneven, some does not even express, therefore cannot be satisfactory.
Therefore, this area in the urgent need to develop effective, can stablize go down to posterity, Tumor formation is good and be applicable to set up the various tumor cell lines of animal model, especially hepatoma cell line.
Summary of the invention
Object of the present invention be just to provide a kind ofly stable to go down to posterity, Tumor formation is good and be applicable to set up the various tumor cell lines of animal model.
Another object of the present invention is to provide preparation method and the purposes of described hepatoma cell line.
In a first aspect of the present invention, provide a kind of human liver cancer cell, described liver cancer cell is human hepatocellular carcinoma cell HCC-LY5, and it is deposited in China typical culture collection center, and preserving number is CCTCC NO:C201099.
In a second aspect of the present invention, provide the daughter cell of the human liver cancer cell described in first aspect present invention, wherein said daughter cell can cause nude mice to form liver cancer.
In another preference, described daughter cell retains or the characteristic of human hepatocellular carcinoma cell HCC-LYC5 of whole parental generation substantially.
In another preference, described human liver cancer cell HCC-LY5 or its daughter cell have following characteristic:
A () has lower than 0.2% (about 0.02-0.2%, preferably about 0.1%) cell expressing CD133;
B () has higher Intrahepatic metastasis ability;
C () has certain distant metastasis (as Lung metastases) ability.
In a third aspect of the present invention, provide the purposes of the above-mentioned human liver cancer cell of the present invention or its daughter cell, they are used to produce liver cancer in Mammals.
In another preference, described Mammals is selected from rat, mouse, rabbit, sheep, dog, monkey.
In another preference, described Mammals is immune deficiency laboratory animal.
In another preference, described animal is nude mice (T cell defect), NON/SCID mouse (T cell, B cell and NK cell associating defect).
In a fourth aspect of the present invention, provide a kind of method setting up liver cancer animal model, comprise step:
A () is by 1 × 10
4-1 × 10
9the human liver cancer cell that individual the present invention is above-mentioned or its daughter cell are inoculated in Mammals;
B the Mammals 7-100 days of () culturing step (a), obtains liver cancer animal model.
In another preference, described inoculation is inoculated in lower portion: liver, abdominal cavity, tail vein, subcutaneous location or its combination.
In another preference, described Mammals is immune deficiency laboratory animal.
In another preference, in step (b), cultivate Mammals 14-50 days, obtain liver cancer animal model.
In another preference, described Mammals is selected from rat, mouse, rabbit, sheep, dog, monkey.
In another preference, described animal is nude mice or NON/SCID mouse.
In a fifth aspect of the present invention, provide a kind of method of In Culture Hepatoma Cell, comprise step: in the substratum of applicable cultivation, cultivate the above-mentioned human liver cancer cell of the present invention or its daughter cell.
In a sixth aspect of the present invention, provide a kind of method of screening the candidate compound of Hepatoma therapy, comprise step:
A () is by 1 × 10
4-1 × 10
9the human liver cancer cell that individual the present invention is above-mentioned or its daughter cell are inoculated in Mammals;
B the Mammals 7-100 days of () culturing step (a), obtains liver cancer animal model; With
C test compounds is applied to the liver cancer animal model of step (b) by (), and compare with the liver cancer animal model of the step (b) not using test compounds, cause liver cancer symptom to be improved after wherein using or the test compounds of curing is exactly the candidate compound of Hepatoma therapy;
Or described method comprises:
In (a) test group, in the culture system of human liver cancer cell HCC-LY5 of the present invention, add test compounds, and observe quantity and/or the growing state of liver cancer cell HCC-LY5; In control group, in the culture system of human liver cancer cell HCC-LY5, do not add test compounds, and observe quantity and/or the growing state of liver cancer cell HCC-LY5;
Wherein, if the quantity of liver cancer cell HCC-LY5 or the speed of growth are less than control group in test group, just show that this test compounds is the candidate compound growth of liver cancer cell or propagation being had to inhibiting Hepatoma therapy.
In another preference, it is characterized in that, in step (c), the method for application of test compounds is selected from lower group: be locally applied to liver lesion place, intravenously administrable, oral administration.
In a seventh aspect of the present invention, provide the liver cancer model animal of the formation prepared with aforesaid method of the present invention.
Should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and can combining mutually between specifically described each technical characteristic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, tiredly no longer one by one to state at this.
Accompanying drawing explanation
Fig. 1 shows the Tumor formation of clone HCC-LY5 of the present invention.
Fig. 2 shows the cultivation situation of clone HCC-LY5.The figure illustrates cell when semidrying cultivates 7 days and climb out of the situation (100 ×) of tissue block
Fig. 3 shows clone HCC-LY5 cellular form of the present invention.
Fig. 4 shows the luciferase/GFP double-tagging result of clone HCC-LY5 of the present invention.Left figure is light field (200x), right figure is fluorescence (200x).
Fig. 5 shows HCC-LY5 immunocytochemical stain result.
Fig. 6 shows the expression of results of Flow cytometry HCC-LY5 cell liver-cancer stem cell mark.Fig. 6 A ~ E is flow cytometer showed HCC-LY5 cell CD133, EpCAM, CD44, CD24 and CD45 developed by molecule result respectively.
Fig. 7 shows in clone HCC-LY5 of the present invention does not exist SP cell mass.
Fig. 8 shows HCC-LY5 cell CD133+ of the present invention/-cell mass subcutaneous one-tenth knurl result.Be respectively from top to bottom 10/, 100/ with 1000/ become knurl situation.
Fig. 9 shows HCC-LY5 Cell clonality and compares.HCC-Y5 cell CD133+ (on) with CD133-(under) cell mass plate clone forms and test (500 cells/well) result and show, both distinguish without remarkable.
Figure 10 shows the RT-PCR detected result of the CD133+/-cell mass of different passage number HCC-LY5 cellular segregation.
Figure 11 shows the western blot detected result of HCC-LY5 clone of the present invention.
Embodiment
The present inventor, through extensive and deep research, carries out original cuiture to up to a hundred liver cancer primary tumors and metastasis tumor specimen, finally builds up a kind of liver cancer infinite cell line HCC-LY5.Complete the present invention on this basis.
Specifically, the present inventor, by the hepatocarcinoma of up to a hundred patients, is inoculated in immunodeficient mouse respectively, after growth, carries out continuous passage between nude mice.Get the capable vitro culture of tumor tissues again, set up corresponding body outer cell line.
To the Biological Detection that clone is correlated with.Finally screening obtains a kind of purebred Bel7402.This cell line growth stable (stable went down to posterity for 46 generations), and morphological feature under light microscopic is consistent with induced tumor tissue.Immunohistochemical analysis shows, and the tumor marker of cell and oncogene protein product are high expression level.
Experiment of Zoology shows, cause nude mice production tumour after this clone inoculation nude mice, its histopathology form is consistent with the tumor tissues of former liver cancer patient.
Human liver cancer cell of the present invention not only can prepare liver cancer model animal, also can be used for the method for the drug candidate screening Hepatoma therapy.
The method that one preferably screens medicine (or having growth of tumour cell) comprises step:
In (a) test group, in the culture system of human liver cancer cell HCC-LY5, add test compounds, and observe quantity and/or the growing state of liver cancer cell HCC-LY5; In control group, in the culture system of human liver cancer cell HCC-LY5, do not add test compounds, and observe quantity and/or the growing state of liver cancer cell HCC-LY5;
Wherein, if the quantity of liver cancer cell HCC-LY5 or the speed of growth are less than control group (there is difference) in test group, then the growth of this test compounds to tumour cell or propagation is pointed out to have restraining effect;
In addition, if the quantity of liver cancer cell HCC-LY5 or the speed of growth are greater than control group (there is difference) in test group, then the growth of this test compounds to tumour cell or propagation is pointed out to have promoter action.
In another preference, described being less than represents the ratio of test group with the quantity of control group or ratio≤0.5 of the speed of growth, more preferably≤0.25.
In another preference, described being greater than represents the ratio of test group with the quantity of control group or ratio >=2 of the speed of growth, more preferably >=4.
In another preference, described remarkable difference is P < 0.05.
A kind of method of preferred screening medicine comprises step:
A () is by 1 × 10
4-1 × 10
9(preferably 1 × 10
5-1 × 10
8more preferably 1 × 10
6-1 × 10
7) above-mentioned human liver cancer cell HCC-LY5 is inoculated in Mammals;
B the Mammals 7-100 days of () culturing step (a), obtains liver cancer animal model;
C test compounds is applied to the liver cancer animal model of step (b) by (), and compare with the liver cancer animal model of the step (b) not using test compounds, cause liver cancer symptom to be improved after wherein using or the test compounds of curing is exactly the candidate compound of Hepatoma therapy.
Major advantage of the present invention is:
A the proterties of () hepatoma cell line HCC-LY5 of the present invention is stablized, Absorbable organic halogens repeatedly goes down to posterity;
B the Tumor formation of () hepatoma cell line HCC-LY5 of the present invention is good, hereditary property is clear and definite, is the basis of liver cancer and the ideal cell line (efficiency assay) of preclinical phase applied research.
Functional study (as by growth behavior, motion and transfer ability observation etc.) in c external, body that () hepatoma cell line HCC-LY5 of the present invention can be used for liver cancer related gene;
D () hepatoma cell line HCC-LY5 of the present invention can be used for external, the interior screening of body (as by growth behavior, motion and transfer ability observation etc.) of liver cancer sensitive drug.
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition is as people such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise per-cent and number be weight percentage and parts by weight.
Embodiment 1
The foundation of hepatoma cell line HCC-LY5
A () is originated
This clone comes from a Male Hepatocellular Carcinoma Patients, and (patient code: 603320), its clinical information is as shown in the table.
Table 1
(b) method
Get the hepatocarcinoma of this liver cancer patient, be inoculated in immunodeficient mouse, and get into tumor tissue and repeatedly carry out inoculating and going down to posterity, finally obtain the stable clone gone down to posterity.Specifically comprise following process:
In addition, in June, to third generation mouse, the male NOD/SCID mouse of knurl 5 is cut in the operation of subcutaneous transplantation knurl.In July, observe and cut bilateral lymphadenectasis after knurl, see after dissection that lung compressing tablet is transparent, do original cuiture, and get lymphoglandula and pass 4 NOD/SCID mouse, get lung and pass 4 NOD/SCID mouse.In August, again carry out cutting knurl, and get bilateral lymphoglandula and pass 4 NOD/SCID mouse.
No pain puts to death lotus knurl NOD/SCID mouse, and asepsis extracts Subcutaneous tumor.Tumour is placed in the PBS culture dish that precooling is housed, fully cleans out the necroses of tumor capsule and tumour, use eye scissors tumor tissues to be cut into the fritter of about 5 × 5 × 5mm size, add liver cell culture liquid in a small amount and cultivated by semidrying.Liver cell culture liquid composition comprises william ' s E substratum, adds 10% foetal calf serum, 100IU/ml penicillin and 100 μ g/ml Streptomycin sulphates.The tissue block that novel species enters changed liquid after 24 hours, changed liquid next day of in cell cultivation process.Can observe cell around tissue block after semidrying cultivates 48 hours to grow, the Polygons cell observing similar Epithelial morphology after 72 hours climbs out of.By choosing cloning amplification epithelial cell clone, and adopt repeatedly the inoblast in differential attachment method removal culture dish.Primary cell obtains a large amount of form homogeneous epithelioid cell when being passaged to for the 9th generation, called after HCC-LY5.
The one-tenth knurl growth curve of clone HCC-LY5 as shown in Figure 1.Result shows, this clone has good Tumor formation.
Current HCC-LY5 passage is to the 46th generation, and Growth of Cells is stablized, in good condition, is continuing (Fig. 2) in Secondary Culture.
This strain human hepatocellular carcinoma clone HCC-LY5 obtained is deposited in China typical culture collection center (CCTCC) (Wuhan, China) on September 27th, 2010, and preserving number is CCTCC NO:C201099.
Embodiment 2
The biological characteristics of Bel7402 HCC-LY5
In the present embodiment, by ordinary method, the purebred Bel7402 that preserving number is CCTCC NO:C201099 is carried out to the detection of biological characteristics.Result is as follows:
2.1 cellular fories:
Observing HCC-LY5 liver cancer cell under inverted phase contrast microscope is Polygons or circle, and form is homogeneous, goes down to posterity (Fig. 3) every 72 hours.
2.2 enzymes/green fluorescent protein double-tagging
The luciferase/GFP double-tagging result of HCC-LY5 clone, as shown in Fig. 4 (right figure), shows that cell is all marked by green fluorescent protein (GFP).
2.3HCC-LY5 cellular immunization cytochemistry is analyzed
HCC-LY5 liver cancer cell is planted on cell chip, is analyzed the expression of liver cell associated protein by immunocytochemical method.The antibody used and extent of dilution as shown in table 2 below.
Table 2
| Antibody | Company | Lot number | Dilution situation |
| AFP | Beckman | IM1595 | Do not dilute |
| Albumin | DAKO | A0001 | 1∶100 |
| CK-18 | Santa Cruz | SC-8020 | 1∶20 |
| CK-19 | Santa Cruz | SC-6278 | 1∶25 |
| Hap Par1 | DAKO | M7158 | 1∶10 |
| Fibroblast | DAKO | M0877 | 1∶25 |
| E-Cadherin | Santa Cruz | SC-7870 | 1∶25 |
| β-Catenin | CST | #9562 | 1∶10 |
| N-Cadherin | Santa Cruz | SC-7939 | 1∶25 |
| Vimentin | DAKO | M0725 | 1∶25 |
| ABCB1 | Chemicon | MAB4336 | 1∶25 |
| ABCB4 | Santa Cruz | SC-13131 | 1∶10 |
| ABCG2 | Abcam | mab4155 | 1∶25 |
| ABCC1 | Chemicon | MAB4100 | 1∶25 |
| ABCC2 | Santa Cruz | SC-5770 | 1∶25 |
| ABCC3 | Santa Cruz | SC-59612 | 1∶10 |
| Glypican-3 | BioMosaics | B0025 | 1∶25 |
| Oct4 | Santa Cruz | SC-5279 | 1∶10 |
| Notch1 | Beckman | 552466 | 1∶25 |
| CD133 | Santa Cruz | SC-19365 | 1∶25 |
| CD90 | BD | 555593 | 1∶10 |
| DLK1 | Abcam | ab21682 | 1∶25 |
Result shows, HCC-LY5 cell high expression level hepatocyte markers Albumin, CK19, Hap Par1, Glypican-3 albumen and epithelial cell mark E-Cadherin albumen, and having bile duct cell mark CK18 protein expression, this is consistent with its source liver tumor tissue expression situation.HCC-LY5 liver cancer cell expresses mesenchymal cell markers Fibroblast, N-Cadherin and Vimentin albumen equally in addition, and resistance is correlated with ABC gene family ABCB1, ABCB4, ABCG2, ABCC1, ABCC2 and ABCC3 albumen, prompting cell may have stronger aggressive and resistance.We have detected the expression of liver-cancer stem cell associated protein at HCC-LY5 cell simultaneously, find that Oct4, Notch1, CD90, Delta-like 1 homologue have expression at HCC-LY5 cell.
In addition, there is the expression of liver cell sign A FP albumen in HCC-LY5 cell simultaneously, and the expression of its ABCB4, ABCG2, Notch1 albumen is very strong, and we detect the expression of liver-cancer stem cell mark CD133 albumen on HCC-LY5 cell, infer that the differentiation degree of HCC-LY5 liver cancer cell is lower and the heterogeneity of tumour is stronger, this point also confirmed by the tumor tissues HE coloration result of this tumour patient.
2.4 flow cytometry
The expression of (a) HCC-LY5 cell liver-cancer stem cell mark
Tumor stem cell (CSCs) be a small set of be present in tumor cell line or tumor tissues few in number, but due to its low differentiation, high drug-resistance, high resistance radiotherapy characteristic, cause tumor chemoradiotherapy weak effect, be easy to the specific tumor cells of transfer and recurrence.According to reported in literature, CD133, CD90, CD44 and EpCAM can as the marks of hepatic carcinoma stem cell.Therefore, the flow cytometry expression of HCC-LY5 liver cancer cell related neoplasms stem cell markers and CD24, CD45 albumen is also passed through in the present embodiment.
Result is as shown in following table 3 and Fig. 6.
Table 3
It should be noted that in this HCC-LY5 cell have a small amount of cell expressing CD133 albumen (a kind of liver-cancer stem cell mark).
The detection of (b) side population cell
In addition, past has research to confirm, side population cell (Side Population in tumour, SP cell) there is stronger one-tenth knurl ability and resistance, therefore by Hoechst 33342 fluorescent staining, flow cytomery method, have detected SP cell in HCC-LY5 cell and whether exist.
Result as shown in Figure 7, shows there is not SP cell mass in HCC-LY5 cell.
2.5 one-tenth knurl experiments
According to the flow cytometer showed result to HCC-LY5 cell liver-cancer stem cell mark, CD133+/-the cell mass extracted in HCC-LY5 cell divides 10/, 100/, 1000/three groups to carry out the subcutaneous one-tenth knurl experiment of NOD/SCID mouse, it is subcutaneous that cell divides left and right sides to inject same mouse respectively, often organizes cell and inject 7.The subcutaneous one-tenth knurl of HCC-LY5 cell CD133 cell mass is tested and was stopped after 8 weeks.
Result shows, and 10, HCC-LY5 cell can become knurl, and the one-tenth knurl ability of HCC-LY5 CD133-cell mass is better than CD133+ cell mass (Fig. 8 and table 4).
Table 4HCC-LY5 cell CD133+/-cell mass subcutaneous one-tenth knurl result
2.6 colony formation
Be extracted HCC-LY5 cell CD133+/-cell mass, the cell kind of some amount is entered 6 orifice plates, cultured continuously 2 weeks, observe its plate clone formational situation.
Result shows, HCC-LY5 cell CD133+/-cell mass, and the plate clone energy for growth between the positive and negative cells there is no remarkable difference (Fig. 9).
2.7HCC-LY5 cell CD133+/-cell population cycle analysis
By flow cytometer, cell cycle analysis is carried out to the CD133+/-cell mass of HCC-LY5 cell derived.
Result shows, and in the CD133+ cell mass of HCC-LY5 cell, G2+S phase cell is more than CD133-cell mass (table 5).
Table 5
| HCC-LY5 | ％G1 | ％G2 | ％S |
| CD133+ | 43.588 | 18.829 | 37.583 |
| CD133- | 59.141 | 11.289 | 29.570 |
2.8HCC-LY5 cell CD133+/-cell mass RT-PCR detects
By RT-PCR method, the expression of the CD133+ that the HCC-LY5 cell that have detected different algebraically is separated/-cell mass liver cell genes involved, Testing index comprises ALPL, WT1, CK19, Notch1, Foxa2.
Detected result shows, the HCC-LY5 cell of different algebraically be separated the expression basically identical (Figure 10) of the CD133+/-cell mass liver cell genes involved obtained.
2.9HCC-LY5 cell Western Blot detects
Also WesternBlot detection is carried out to CD133+ and the CD133-cell that HCC-LY5 sorting obtains in the present embodiment.Result as shown in figure 11.
Result shows: in CD133+ cell, CD133 protein expression, CD133-cell CD133 albumen are not expressed, but DNMT1, Erk1/2, alpha-tubulin, CXCR4, snail at CD133+ cells at CD133+ and CD133-cell no significant difference.
Embodiment 3
The animal model of liver cancer is set up with Bel7402
The Bel7402 HCC-LY5 (preserving number: CCTCC C201099) obtained in Example 1, after Digestive system (0.25% pancreatin, 0.02%EDTA, pH7.3) digestion, collects and counts, with 1 × 10
6or 1 × 10
7a cell/mouse quantity is the Balb/c system nude mouse of right side of mice wall of the chest subcutaneous vaccination in 20 4-6 all ages.
After inoculation between 14-60 days, all there is tumour in 20 mouse successively.The method identical by embodiment 2 carries out pathology detection, confirms to induce hepatocellular carcinoma on animal model.
Embodiment 4
Screening of medicaments
Get the animal pattern of preparation in 18 embodiments 3, be divided into three groups at random.Adopt double-blind method, to the animal of each group, respectively by the one in three groups of test substances below abdominal injection, what namely (a) was known has the positive compound vincristine sulphate suppressing liver cancer cell growth effect, the negative compound PBS of (b) known unrestraint effect; And (c) blank (only containing injection solvent), and observe into knurl result.
Although do not know to give which kind of material during administration, but become knurl result to conform to the tumor suppression of given tester, namely tumour mean size is: the negative compound of blank (without becoming knurl) < positive compound (cis-platinum) <.This shows, the liver cancer model animal of preparing by clone of the present invention can be used for the drug candidate screening Hepatoma therapy.
Culture presevation
Human hepatocellular carcinoma cell line HCC-LY5 of the present invention, be deposited in China typical culture collection center (CCTCC) (Wuhan, China) on September 27th, 2010, preserving number is CCTCC NO:C201099.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (8)
1. a human liver cancer cell, is characterized in that, described liver cancer cell is human hepatocellular carcinoma cell HCC-LY5, and it is deposited in China typical culture collection center, and preserving number is CCTCC NO:C201099.
2. the daughter cell of human liver cancer cell as claimed in claim 1, is characterized in that, described daughter cell can cause nude mice to form liver cancer.
3. human liver cancer cell as claimed in claim 1 or daughter cell according to claim 2, it is characterized in that, described cell has following characteristic:
A () has lower than 0.2% cell expressing CD133;
B () has Intrahepatic metastasis ability; With
C () has distant metastasis ability.
4. human liver cancer cell as claimed in claim 1 or daughter cell according to claim 2, is characterized in that, described cell expressing mesenchymal cell markers Fibroblast, N-Cadherin and Vimentin albumen.
5. human liver cancer cell as claimed in claim 1 or daughter cell according to claim 2, is characterized in that, described cell expressing resistance is correlated with ABC gene family ABCB1, ABCB4, ABCG2, ABCC1, ABCC2 and ABCC3 albumen.
6. human liver cancer cell as claimed in claim 1 or daughter cell according to claim 2, is characterized in that, described cell expressing liver-cancer stem cell associated protein Oct4, Notch1, CD90 and Delta-like 1 homologue.
7. a method for In Culture Hepatoma Cell, is characterized in that, comprises step: in the substratum of applicable cultivation, cultivates human liver cancer cell according to claim 1 or its daughter cell.
8. screen a method for the candidate compound of Hepatoma therapy, it is characterized in that, described method comprises step:
In test group, in the culture system containing the human liver cancer cell HCC-LY5 described in claim 1, add test compounds, and observe quantity and/or the growing state of liver cancer cell HCC-LY5; In control group, in containing the culture system of human liver cancer cell HCC-LY5, do not add test compounds, and observe quantity and/or the growing state of liver cancer cell HCC-LY5;
Wherein, if the quantity of liver cancer cell HCC-LY5 or the speed of growth are less than control group in test group, just show that this test compounds is the candidate compound growth of liver cancer cell or propagation being had to inhibiting Hepatoma therapy.
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| CN108467854B (en) * | 2017-02-23 | 2021-08-31 | 中国科学院上海营养与健康研究所 | Novel bone-specific metastatic hepatoma cell and preparation thereof |
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