CN102690785A - 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 and application.
Background technology
The generation of human various diseases and development are very complicated, and the pathogeny and the curative effect mechanism thereof of the discussion various diseases that go deep into can not experimentize the patient on one's body.But can follow Animal Protection Law; Through animal various diseases and biological phenomena are studied, and then pushed away and unravel silk the secret that the mankind explore the human life, explore the rule of human diseases incidence and development; Generation, development with the control human diseases; Cure diseases with alleviate human ill misery, improve life quality, prolong the human life-span.
It is found that for a long time; Is difficult with people itself as the development that experimental subjects promotes medical science; The experience that is accumulated clinically not only exists limitation on time and space, and many experiments also exist various restrictions on ethics and methodology.
The animal model of human diseases (Animal model of human diseases) is to set up experimentation on animals object and the material with the performance of human diseases simulation in the biomedical sciences research.Using animal model is very important experimental technique and means in the modern biomedical research, help more convenient, more effectively be familiar with generation, the rule of development and the research prophylactico-therapeutic measures of human diseases.
Tumour cell ties up in the fundamental research of tumour has critical role, and cultured tumor cells in vitro is than difficulty, and especially setting up can long term growth, go down to posterity, and has the human tumor cell line of certain characteristic again.
For the liver cancer field; At present; Though some SMMC-7721s are arranged; But there is multiple shortcoming in these SMMC-7721s, comprising: the instability that goes down to posterity, become that knurl property characteristic relatively poor, clone is inconsistent, the liver cancer-specific marker expression is uneven, some is not even expressed etc., and therefore can't be satisfactory.
Therefore, this area press for exploitation effectively, can stablize and go down to posterity, become knurl property good and be applicable to the various tumor cell lines of setting up animal model, especially SMMC-7721.
Summary of the invention
The object of the invention just provides and a kind ofly stable goes down to posterity, becomes knurl property good and be applicable to the various tumor cell lines of setting up animal model.
Another object of the present invention provides the preparation method and the purposes of described SMMC-7721.
In first aspect of the present invention, a kind of human liver cancer cell is provided, said liver cancer cell is human liver cell liver cancer cell HCC-LY5, and it is deposited in Chinese typical culture collection center, and preserving number is CCTCC NO:C201099.
In second aspect of the present invention, the daughter cell of the described human liver cancer cell of first aspect present invention is provided, wherein said daughter cell can cause nude mice to form liver cancer.
In another preference, described daughter cell keeps or the characteristic of the human liver cell liver cancer cell HCC-LYC5 of whole parental generation basically.
In another preference, described human liver cancer cell HCC-LY5 or its daughter cell have following characteristic:
(a) have and be lower than 0.2% (about 0.02-0.2%, preferably about 0.1%) cell expressing CD133;
(b) transfer ability in the higher liver is arranged;
(c) certain metastasis (shifting like lung) ability is arranged.
In the third aspect of the invention, the purposes of the above-mentioned human liver cancer cell of the present invention or its daughter cell is provided, they are used in Mammals, produce liver cancer.
In another preference, described Mammals is selected from rat, mouse, rabbit, sheep, dog, monkey.
In another preference, described Mammals is the immunodeficient laboratory animal.
In another preference, described animal is nude mice (a T cell defect), NON/SCID mouse (T cell, B cell and NK cell associating defective).
In fourth aspect of the present invention, a kind of method of setting up liver cancer animal model is provided, comprise step:
(a) with 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 of culturing step (a) days, obtain liver cancer animal model.
In another preference, described inoculation is to be inoculated in following position: liver, abdominal cavity, tail vein, subcutaneous location or its combination.
In another preference, described Mammals is the immunodeficient laboratory animal.
In another preference, cultivated Mammals 14-50 days in the step (b), 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.
Aspect the of the present invention the 5th, a kind of method of vitro culture liver cancer cell is provided, comprise step: in being fit to the substratum of cultivating, cultivate above-mentioned human liver cancer cell or its daughter cell of the present invention.
Aspect the of the present invention the 6th, a kind of method of screening the candidate compound of treatment liver cancer is provided, comprise step:
(a) with 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 of culturing step (a) days, obtain liver cancer animal model; With
(c) test compounds is applied to the liver cancer animal model of step (b); And compare with the liver cancer animal model of the step of not using test compounds (b), the test compounds that causes liver cancer doing well,improving or healing after wherein using is exactly the candidate compound of treatment liver cancer;
Perhaps said method comprises:
(a) in the 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 the test group, just show that this test compounds is the candidate compound that growth or propagation to liver cancer cell have inhibiting treatment liver cancer.
In another preference, it is characterized in that, in step (c), the method for application of test compounds is selected from down group: be locally applied to liver lesion place, intravenously administrable, oral administration.
Aspect the of the present invention the 7th, the liver cancer model animal with the formation of method for preparing of the present invention is provided.
In should be understood that within the scope of the present invention, above-mentioned each technical characterictic of the present invention and hereinafter can mutual combination between specifically described each technical characterictic in (like embodiment), thus constitute new or optimized technical scheme.As space is limited, this tired no longer one by one stating.
Description of drawings
Fig. 1 has shown the one-tenth knurl property of clone HCC-LY5 of the present invention.
Fig. 2 has shown the cultivation situation of clone HCC-LY5.Cell climbs out of the situation (100 *) of tissue block when the figure illustrates semidrying and cultivating 7 days
Fig. 3 has shown clone HCC-LY5 cellular form of the present invention.
Fig. 4 has shown the luciferase/GFP double-tagging result of clone HCC-LY5 of the present invention.Left side figure is light field (200x), and right figure is fluorescence (200x).
Fig. 5 has shown HCC-LY5 immunocytochemical stain result.
Fig. 6 has shown the expression of results of Flow cytometry HCC-LY5 cell liver-cancer stem cell mark.Fig. 6 A～E is respectively flow cytometer showed HCC-LY5 cell CD133, EpCAM, CD44, CD24 and CD45 developed by molecule result.
Fig. 7 has shown and has not had the SP cell mass among the clone HCC-LY5 of the present invention.
Fig. 8 shown HCC-LY5 cell CD133+ of the present invention/-the subcutaneous one-tenth knurl of cell mass result.Be respectively from top to bottom 10/, 100/ with 1000/ become the knurl situation.
Fig. 9 has shown that HCC-LY5 cell clone formation ability relatively.HCC-Y5 cell CD133+ (on) show that with CD133-(descending) cell mass plate clone formation experiment (500 cells/well) result both do not have remarkable difference.
Figure 10 shown the CD133+ of different passage number HCC-LY5 cellular segregation/-the RT-PCR detected result of cell mass.
Figure 11 has shown the Western trace detected result of HCC-LY5 clone of the present invention.
Embodiment
The inventor is through extensive and deep research, and up to a hundred liver cancer primary tumors and MET tumor specimen are carried out formerly being commissioned to train fosterly, builds up a kind of liver cancer infinite cell line HCC-LY5 at last.Accomplished the present invention on this basis.
Particularly, the inventor is inoculated in immunodeficient mouse respectively with up to a hundred patients' primary hepatocarcinoma tissue, after growth, carries out continuous passage between nude mice.Get the capable vitro culture of tumor tissues again, set up corresponding in vitro clone.
The Biological Detection that pair cell system is correlated with.Finally screening obtains a kind of purebred Bel7402.This cell line growth stable (stable going down to posterity for 46 generations), and the morphological feature under light microscopic is consistent with the induced tumor tissue.Immunohistochemical analysis shows that the tumor marker and the oncogene protein product of cell are high expression level.
Experiment of Zoology shows, causes nude mice production tumour behind this clone inoculation nude mice, and 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 the liver cancer model animal, also can be used for screening the method for the drug candidate of treating liver cancer.
The method of a kind of preferred screening medicine (or have growth of tumour cell) comprises step:
(a) in the 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 (having the significance difference) in the test group, then point out growth or the propagation of this test compounds that restraining effect is arranged to tumour cell;
In addition, if the quantity of liver cancer cell HCC-LY5 or the speed of growth are greater than control group (having the significance difference) in the test group, then point out growth or the propagation of this test compounds that promoter action is arranged to tumour cell.
In another preference, described less than expression test group and the ratio of the quantity of control group or ratio≤0.5 of the speed of growth, more preferably≤0.25.
In another preference, described greater than expression test group and the ratio of 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) with 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 of culturing step (a) days, obtain liver cancer animal model;
(c) test compounds is applied to the liver cancer animal model of step (b); And compare with the liver cancer animal model of the step of not using test compounds (b), the test compounds that causes liver cancer doing well,improving or healing after wherein using is exactly the candidate compound of treatment liver cancer.
Major advantage of the present invention is:
(a) proterties of SMMC-7721 HCC-LY5 of the present invention is stable, can stablize repeatedly and go down to posterity;
(b) the one-tenth knurl property of SMMC-7721 HCC-LY5 of the present invention is good, and hereditary property is clear and definite, is the basis of liver cancer and the desirable clone (efficiency assay) of preclinical phase applied research.
(c) SMMC-7721 HCC-LY5 of the present invention can be used for external, the interior functional study of body (as through growth behavior, motion and transfer ability observation etc.) of liver cancer related gene;
(d) SMMC-7721 HCC-LY5 of the present invention can be used for external, the interior screening of body (as through growth behavior, motion and transfer ability observation etc.) of liver cancer sensitive drug.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example; Usually according to people such as normal condition such as Sambrook; Molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press; 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber be weight percentage and parts by weight.
The foundation of SMMC-7721 HCC-LY5
(a) source
This clone comes from male sex's liver cancer patient, and (patient's numbering: 603320), its clinical information is as shown in the table.
Table 1
(b) method
Get the primary hepatocarcinoma tissue of this liver cancer patient, be inoculated in immunodeficient mouse, and get into tumor tissue and inoculate repeatedly and go down to posterity, finally obtain the stable clone that goes down to posterity.Specifically comprise following process:
In addition, in June, to third generation mouse, 5 male NOD/SCID mouse of knurl are cut in the operation of subcutaneous transplantation knurl.In July, observe and cut bilateral lymphadenectasis after the knurl, see after the dissection that the lung compressing tablet is transparent, do and formerly be commissioned to train fosterly, and get lymphoglandula and pass 4 NOD/SCID mouse, get lung and pass 4 NOD/SCID mouse.In August, cut knurl once more, and get 4 NOD/SCID mouse of bilateral lymphoglandula biography.
No pain is put to death lotus knurl NOD/SCID mouse, and asepsis is extracted Subcutaneous tumor.Tumour is placed the PBS petridish that precooling is housed, fully clean out the downright bad part of tumour coating and tumour, use eye scissors that tumor tissues is cut into the big or small fritter of about 5 * 5 * 5mm, add a small amount of liver cell culture liquid and cultivate through semidrying.Liver cell culture liquid composition comprises william ' s E substratum, adds 10% foetal calf serum, 100IU/ml penicillium mould and 100 μ g/ml Streptomycin sulphates.The tissue block that novel species is gone into was changed liquid after 24 hours, changed liquid next day of in the cell cultivation process.The semidrying cultivation can have been observed cell after 48 hours and grow around tissue block, the Polygons cell of observing similar epithelium form after 72 hours climbs out of.Clone through choosing PCR cloning PCR amplification epithelial cell, and adopt repeatedly the differential adherent method to remove the inoblast in the petridish.Primary cell obtains the epithelioid cell of a large amount of form homogeneous, called after HCC-LY5 when being passaged to for the 9th generation.
The one-tenth knurl growth curve of clone HCC-LY5 is as shown in Figure 1.The result shows that this clone has good one-tenth knurl property.
At present 46 generations of HCC-LY5 passage to the, the cell growth is stable, in good condition, continuing to go down to posterity cultivate in (Fig. 2).
This strain human liver cell SMMC-7721 HCC-LY5 that obtains is deposited in Chinese 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, use ordinary method preserving number to be carried out the detection of biological characteristics as the purebred Bel7402 of CCTCC NO:C201099.The result is following:
2.1 cellular form:
It is Polygons or circle that inverted phase contrast microscope is observed the HCC-LY5 liver cancer cell down, form homogeneous, every went down to posterity at a distance from 72 hours (Fig. 3).
2.2 enzyme/green fluorescent protein double-tagging
The luciferase/GFP double-tagging result of HCC-LY5 clone shows that cell is all by green fluorescent protein (GFP) mark shown in Fig. 4 (right figure).
2.3HCC-LY5 cellular immunization cytochemistry method is analyzed
The HCC-LY5 liver cancer cell is planted on the cell chip, analyzes the expression of liver cell GAP-associated protein GAP through immunocytochemical method.Employed antibody and extent of dilution are as shown in table 2 below.
Table 2
| Antibody | Company | Lot number | The dilution situation | |
| AFP | Beckman | IM1595 | Do not dilute | |
| | DAKO | A0001 | 1∶100 | |
| CK-18 | Santa?Cruz | SC-8020 | 1∶20 | |
| CK-19 | Santa?Cruz | SC-6278 | 1∶25 | |
| Hap? | DAKO | M7158 | 1∶10 | |
| | 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 | |
| | DAKO | M0725 | 1∶25 | |
| | Chemicon | MAB4336 | 1∶25 |
[0098]?
| ABCB4 | Santa?Cruz | SC-13131 | 1∶10 | |
| ABCG2 | Abcam | mab4155 | 1∶25 | |
| | Chemicon | MAB4100 | 1∶25 | |
| ABCC2 | Santa?Cruz | SC-5770 | 1∶25 | |
| ABCC3 | Santa?Cruz | SC-59612 | 1∶10 | |
| Glypican-3 | | 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 |
The result shows; HCC-LY5 cell high expression level liver cell mark Albumin, CK19, Hap Par1, Glypican-3 albumen and epithelial cell mark E-Cadherin albumen; And bile duct cell mark CK18 protein expression is arranged, this is consistent with its source liver tumor tissue expression situation.The HCC-LY5 liver cancer cell is expressed mesenchymal cell mark Fibroblast, N-Cadherin and Vimentin albumen equally in addition; And the relevant ABC gene family ABCB1 of resistance, ABCB4, ABCG2, ABCC1, ABCC2 and ABCC3 albumen, the prompting cell possibly have stronger aggressive and resistance.We have detected the expression of liver-cancer stem cell GAP-associated protein GAP at the HCC-LY5 cell simultaneously, find that Oct4, Notch1, CD90, DLK1 albumen all have expression at the HCC-LY5 cell.
In addition; There is the proteic expression of liver cell sign A FP simultaneously in the HCC-LY5 cell; And its ABCB4, ABCG2, the proteic expression of Notch1 are very strong; And we detect the proteic expression of liver-cancer stem cell mark CD133 on the HCC-LY5 cell, and the differentiation degree of supposition HCC-LY5 liver cancer cell is lower and heterogeneity tumour is stronger, and this point is also confirmed by the tumor tissues HE coloration result of this tumour patient.
2.4 flow cytometry
(a) expression of HCC-LY5 cell liver-cancer stem cell mark
Tumor stem cell (CSCs) is a small set ofly to be present in tumor cell line or the tumor tissues few in numberly, but because its low differentiation, high drug-resistance, high anti-radiotherapy characteristic, causes the tumor chemoradiotherapy weak effect, is easy to the specific tumor cells that shifts and recur.According to reported in literature, CD133, CD90, CD44 and EpCAM can both be as the marks of liver cancer tumor stem cell.Therefore, in the present embodiment also through flow cytometry HCC-LY5 liver cancer cell related neoplasms stem cell markers and CD24, the proteic expression of CD45.
Result such as following table 3 are with shown in Figure 6.
Table 3
It should be noted that has small amounts of cells to express CD133 albumen (a kind of liver-cancer stem cell mark) in this HCC-LY5 cell.
(b) detection of side group cell
In addition; Past has research to confirm that the side group cell in the tumour (Side Population, SP cell) has stronger one-tenth knurl ability and resistance; Therefore through Hoechst 33342 fluorescent stainings, flow cytometer detection method, detected whether the SP cell exists in the HCC-LY5 cell.
The result is as shown in Figure 7, shows not have the SP cell mass in the HCC-LY5 cell.
2.5 become the knurl experiment
According to flow cytometer showed result to HCC-LY5 cell liver-cancer stem cell mark; Extract CD133+ in the HCC-LY5 cell/-cell mass divide 10/only, 100/only, 1000/only three groups 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, and every group of cell injected 7.The subcutaneous one-tenth knurl of HCC-LY5 cell CD133 cell mass is tested in the back termination of 8 weeks.
The result shows that 10 in 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+/-the subcutaneous one-tenth knurl of cell mass result
2.6 the clone forms experiment
Extracted HCC-LY5 cell CD133+/-cell mass, the cell kind of some amount is gone into 6 orifice plates, in 2 weeks of cultured continuously, observe its plate clone and form situation.
Result's demonstration, HCC-LY5 cell CD133+/-cell mass, the plate clone energy for growth between the positive and the negative cells does not have remarkable difference (Fig. 9).
2.7HCC-LY5 cell CD133+/-the cell mass cell cycle analysis
Through flow cytometer, to the CD133+ in HCC-LY5 cell source/-cell mass carried out cell cycle analysis.
The result shows that G2+S phase cell is more than CD133-cell mass (table 5) in the CD133+ cell mass of HCC-LY5 cell.
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 detection
Through the RT-PCR method, detected the isolating CD133+ of HCC-LY5 cell of different algebraically/-cell mass liver cell Expression of Related Genes situation, detect index and comprise ALPL, WT1, CK19, Notch1, Foxa2.
Detected result shows, the HCC-LY5 cell of different algebraically separate the CD133+ that obtains/-cell mass liver cell Expression of Related Genes basically identical (Figure 10).
2.9HCC-LY5 cell Western Blot detects
The CD133+ and the CD133-cell that also the HCC-LY5 sorting are obtained in the present embodiment have carried out the WesternBlot detection.The result is shown in figure 11.
The result shows: CD133 protein expression, CD133-cell CD133 albumen are not expressed in the CD133+ cell, but DNMT1, Erk1/2, alpha-tubulin, CXCR4, snail are expressed in CD133+ and CD133-cell no significant difference in the CD133+ cell.
Embodiment 3
Set up the animal model of liver cancer with the Bel7402
(preserving number: CCTCC C201099), (0.25% pancreatin, 0.02%EDTA pH7.3) after the digestion, collect and counting, with 1 * 10 through Digestive system to get the Bel7402 HCC-LY5 that obtains among the embodiment 1
6Or 1 * 10
7A cell/mouse quantity is nude mouse in the subcutaneous vaccination of the right side of mice wall of the chest at the Balb/c in 20 4-6 age in week.
Between inoculation back 14-60 days, tumour all appears in 20 mouse successively.Carry out pathology with embodiment 2 identical methods and detect, confirm on animal model, to have brought out hepatocellular carcinoma.
Embodiment 4
Screening of medicaments
Get the animal pattern of preparation among 18 embodiment 3, be divided into three groups at random.Adopt double-blind method; Animal to each group; A kind of through in three groups of test substances below the abdominal injection respectively, promptly (a) is known has the positive compound vincristine sulphate that suppresses the liver cancer cell growth effect, (b) negative Compound P BS of known unrestraint effect; And (c) blank (only containing injection solvent), and observe into the knurl result.
Though during administration and do not know to give which kind of material, become knurl result and the tester that is given but knurl property conforms to, promptly the tumour mean size is: blank (not having into knurl)＜positive compound (cis-platinum)＜negative compound.This shows that the liver cancer model animal for preparing with clone of the present invention can be used for screening the drug candidate of treatment liver cancer.
Culture presevation
Human hepatocellular carcinoma cell line HCC-LY5 of the present invention is deposited in Chinese typical culture collection center (CCTCC) (Wuhan, China) on September 27th, 2010, and preserving number is CCTCC NO:C201099.
All documents in that the present invention mentions are all quoted as a reference in this application, are just quoted such as a reference separately as each piece document.Should be understood that in addition after having read above-mentioned teachings of the present invention, those skilled in the art can do various changes or modification to the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (10)
1. a human liver cancer cell is characterized in that, said liver cancer cell is human liver cell liver cancer cell HCC-LY5, and it is deposited in Chinese 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, said daughter cell can cause nude mice to form liver cancer.
3. human liver cancer cell as claimed in claim 1 or the described daughter cell of claim 2 is characterized in that said cell has following characteristic:
(a) have and be lower than 0.2% (about 0.02-0.2%) cell expressing CD133;
(b) has transfer ability in the liver; With
(c) has metastasis (shifting) ability like lung.
4. the purposes of human liver cancer cell as claimed in claim 1 or its daughter cell is characterized in that, is used for producing liver cancer Mammals.
5. purposes as claimed in claim 4 is characterized in that described Mammals is selected from rat, mouse, rabbit, sheep, dog, monkey.
6. a method of setting up liver cancer animal model is characterized in that, comprises step:
(a) with 1 * 10
4-1 * 10
9The described human liver cancer cell of individual claim 1 or its daughter cell are inoculated in Mammals;
(b) the Mammals 7-100 of culturing step (a) days, obtain liver cancer animal model.
7. method as claimed in claim 6 is characterized in that described Mammals is selected from rat, mouse, rabbit, sheep, dog, monkey.
8. the method for a vitro culture liver cancer cell is characterized in that, comprises step: in being fit to the substratum of cultivating, cultivate the described human liver cancer cell of claim 1 or its daughter cell.
9. method of candidate compound of screening treatment liver cancer is characterized in that said method comprises step:
(a) with 1 * 10
4-1 * 10
9The described human liver cancer cell of individual claim 1 or its daughter cell are inoculated in Mammals;
(b) the Mammals 7-100 of culturing step (a) days, obtain liver cancer animal model; With
(c) test compounds is applied to the liver cancer animal model of step (b); And compare with the liver cancer animal model of the step of not using test compounds (b), the test compounds that causes liver cancer doing well,improving or healing after wherein using is exactly the candidate compound of treatment liver cancer;
Perhaps said method comprises:
(a) in the test group, in the culture system of the described human liver cancer cell HCC-LY5 of claim 1, 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 the test group, just show that this test compounds is the candidate compound that growth or propagation to liver cancer cell have inhibiting treatment liver cancer.
10. method as claimed in claim 9 is characterized in that, in step (c), the method for application of test compounds is selected from down group: be locally applied to liver lesion place, intravenously administrable, oral administration.
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| CN103305467A (en) * | 2013-05-31 | 2013-09-18 | 湖南大学 | Human liver cancer cell line HLCZ02 and application thereof |
| CN108467855A (en) * | 2017-02-23 | 2018-08-31 | 中国科学院上海生命科学研究院 | New lung specificity transfer liver cancer cells and its preparation |
| CN108467854A (en) * | 2017-02-23 | 2018-08-31 | 中国科学院上海生命科学研究院 | New bone transspecific liver cancer cells and its preparation |
| CN114891747A (en) * | 2022-04-19 | 2022-08-12 | 济南微生态生物医学省实验室 | Human hepatocellular carcinoma cell strain and application thereof |
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| CN103305467A (en) * | 2013-05-31 | 2013-09-18 | 湖南大学 | Human liver cancer cell line HLCZ02 and application thereof |
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| CN108467855A (en) * | 2017-02-23 | 2018-08-31 | 中国科学院上海生命科学研究院 | New lung specificity transfer liver cancer cells and its preparation |
| CN108467854A (en) * | 2017-02-23 | 2018-08-31 | 中国科学院上海生命科学研究院 | New bone transspecific liver cancer cells and its preparation |
| 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 |
| CN108467855B (en) * | 2017-02-23 | 2021-09-07 | 中国科学院上海营养与健康研究所 | Novel lung-specific metastatic hepatoma cell and preparation thereof |
| CN114891747A (en) * | 2022-04-19 | 2022-08-12 | 济南微生态生物医学省实验室 | Human hepatocellular carcinoma cell strain and application thereof |
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