CN101831405A - Lung-targeting metastatic human hepatoma cell strain and establishing method thereof - Google Patents
Lung-targeting metastatic human hepatoma cell strain and establishing method thereof Download PDFInfo
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Abstract
The invention provides a lung-targeting metastatic human hepatoma cell strain and an establishing method thereof. The lung-targeting metastatic human hepatoma cell strain is characterized in that the mother cell is a red fluorescent protein expression hepatoma cell HCCLM3-R; the cell is named as a lung-targeting metastatic human hepatoma cell strain HCCLM3-R-LM1 by classifying, has the preservation number of 3644, and has the following biological characteristics that the cell is in polygonal epithelium shape, grows clinging to the wall, loses contact growth inhibition, has a hypo-triploid caryogram with the chromosome number of 50-58, secretes AFP protein, and has lung-targeting metastatic clonal selection advantage. The establishing method comprises the following steps of: sieving by adopting nude mouse subcutis and liver orthotopic implantation tumour model autogenetic lung metastasis, and then carrying out in-vitro cloning, purifying and propagating on the lung-targeting metastatic human hepatoma cell. The invention has wide application potential and potential societal and economic benefits in basic research of hepatoma and clinic previous research of medicines.
Description
Technical field
The present invention relates to lung-targeting metastatic human hepatoma cell strain and establishment method thereof, be specifically related to the human hepatoma cell strain HCCLM3-R-LM1 and the establishment method thereof of energy stably express red fluorescent protein (RFP), tool lung target transfer ability, belong to microorganism animal cell line technical field.
Background technology
Primary hepatocellular carcinoma is one of the malignant tumour occurred frequently in the China and even the whole world, and its M ﹠ M all ranks second of malignant tumour.Several different methods treatment liver cancer is arranged clinically, and as alinjection, RF therapy and radiotherapy etc. in excision, TACE, the knurl, but long-term efficacy is limited.9,800 many cases operation of liver cancer patients' clinical long term follow-up statistics are found that the liver cancer patient back 5 years survival rate of performing the operation still is lower than 50% according to Fudan University liver cancer research institute, its major causes of death is the recurrence and the transfer of liver cancer.
Liver perilymph knot and lungs transfer often appear in liver cancer patient clinically, and lack corresponding research object at present both at home and abroad, and the organ target transfer of molecules mechanism of liver cancer cell is known little about it, and also lack the necessary intervention means.For this reason, Fudan University's liver cancer research take the lead in having set up nude mice people hepatoma Metastasis model (LCI-D20) (Sun FX, et al.Int J Cancer.1996 in the world with independent intellectual property right and national patent; 66:239-243.), be cloned into Bel7402 (MHCC97) (Tian J, et al.Br J Cancer.1999 with high metastatic potential; 81:814-821.), filtered out have serial hepatoma cell strains identical genetic background, different metastatic potentials (MHCC97-H, MHCC97-L) (LiY, et al.World J Gastroenterol.2001; 7:630-636. patent No. WO 2003/087766 A3, ZL2003 1 0122611.5) and the human hepatocellular carcinoma cell line HCCLM3 and HCCLM6 (the J Cancer ResClin Oncol.2004 of high transfer characteristics; 130:187-196; J Cancer Res Clin Oncol.2004; 130:460-468; Chinese Medical Journal .2004; 84:675-679; Cancer Genet Cytogenet.2005; 158:180-183; Clin CancerRes.2006; 12:7140-7148), and RFP and the fluorescently-labeled liver cancer cell (HCCLM3RFP/GFP of GFP have been further developed, HCCLM6RFP/GFP) and corresponding fluorescent visual hepatoma Metastasis model (Yang BW, et al.EurJ Gastroenterol Hepatol.2008; 20-1077-1084. number of patent application: 200710045676.2; With 200710045675.8).
There is no report in the domestic and foreign literature about the lung-targeting metastatic human hepatoma cell strain of energy stably express fluorescence.
Summary of the invention
The lung-targeting metastatic human hepatoma cell strain and the establishment method thereof that the purpose of this invention is to provide stably express fluorescence are more particularly set up Bel7402 chromosomal integration type, stably express red fluorescent protein encoding gene, that have lung target transfer ability.
It is parent cell that the present invention expresses liver cancer cell HCCLM3-R with red fluorescent protein, adopt the subcutaneous and liver orthotopic transplantation knurl model spontaneous lung transfer screening of nude mice, lung transfer human liver cancer cell carries out the method for body outer clone amplification purification again, has set up a kind of lung-targeting metastatic human hepatoma cell strain (HCCLM3-R-LM1).In preservation on March 3 in 2010, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center, the address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, deposit number: 3644, classification name: lung target transfer human liver cancer cell strain (HCCLM3-R-LM1).
Described red fluorescent protein is expressed liver cancer cell HCCLM3-R referring to Chinese patent 200710045676.2.In preservation on March 3 in 2010, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center, the address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, deposit number: 3643, classification name: the human hepatoma cell strain (HCCLM3-R) of expressing red fluorescent protein.
Subcutaneous and the liver orthotopic transplantation knurl model of described nude mice is referring to Chinese patent 200710045675.8.
This cell strain has following biological characteristics:
1, have identical genetic background with parent cell, cell is Polygons epithelium sample, adherent growth, and the forfeiture of contact growth-inhibiting, the hypo-triploid caryogram, chromosome number 50-58 bar is all secreted AFP albumen;
But the red fluorescence of 2 stably express high strength: the red fluorescent protein encoding gene of transfection is identical with the red fluorescent protein RFP that has sequence 1 described in the Chinese patent 200710045676.2, through genetic modification, can effectively delay the fluorescent quenching time, increase luciferase expression intensity, in excitation wavelength is that 560nm, emission wavelength are under the condition of 585nm, and available fluorescent microscope is observed;
3, stabilization characteristics of genetics, can be in continuous passage under the normal condition.
4, compare with parent cell, except having the biological characteristics that parent cell has, also show two other feature, one has stronger transfer ability than parent cell; They are two years old, the high heterogeneity that comprises different targets transfer subclones with parent cell is compared, and this cell heterogeneity is lower, has the lung target and shifts the clonal selection advantage, the 2nd week at first occurring lung after the orthotopic transplantation of nude mice liver shifts, the rate of transform is about and 42.9%, the 3 week was about 85.7%, shifts to the 4th week 100% occurring lung, and the lymphonodi coeliaci transfer is beginning appearance the 3rd week, about 57.1%, the 4 week is about 71.5%, but late period, whole body shifted.
The establishment method of described lung-targeting metastatic human hepatoma cell strain is characterized in that, concrete steps are: expressing liver cancer cell HCCLM3-R with red fluorescent protein is parent cell, sets up nude mice subcutaneous transplantation knurl model, obtains the Subcutaneous tumor fritter; Set up nude mice liver transplantation knurl model, the Subcutaneous tumor fritter is implanted the liver surface of nude mice; After 42 days, put to death nude mice, open chest and take out complete two lungs, with lung metastasis tissue mill, the centrifugal cell precipitation that obtains, through cultivating and clone step by step sorting acquisition lung-targeting metastatic human hepatoma cell strain.
The present invention is maternal cell with high transfer characteristics hepatoma cell strain HCCLM3R, and the lung metastasis liver cancer cell in the employing nude mice liver tumor in situ transplantation model (referring to applying for a patent 200710045675.8) obtains through cultured and amplified in vitro as cloned object; Lung target transfer human liver cancer cell HCCLM3-R-LM1 confirms at first to take place lung through nude mice liver tumor in situ transplantation model and shifts; The liver cancer cell HCCLM3-R-LM1 that obtains have identical genetic background with maternal liver cancer cell HCCLM3R; But have characteristics such as the red fluorescence of the high strength of expression, stabilization characteristics of genetics continuous passage, lung target transfer clonal selection advantage.
The present invention obtains lung-targeting metastatic human hepatoma cell HCCLM3-R-LM1 that red fluorescence expresses well-grown in the two dimension of external routine and three-dimensional cell culture system, can be used for the especially research of organ target transfer of molecules mechanism of hepatoma Metastasis; Or the effector cell who differentiates as anti-hepatoma Metastasis efficacy of new drug.In addition, also can be used as the liver cancer cell of nude mice subcutaneous vaccination, be used for the foundation of the subcutaneous and liver original position fluorescent tracing liver cancer model of nude mice, for the variation of finding biological behaviour such as people's liver cancer organ target transfer in the nude mouse as early as possible provides reference, and facilitate for recruitment evaluation at body anti metastasis new drug or other therapeutic interventions.
The present invention has broad application prospects in liver cancer fundamental research and clinical drug early-stage Study and the potential society and economy is worth.
Description of drawings
Fig. 1 is the liver cancer cell transfer case synoptic diagram in the different time node nude mice lung;
Fig. 2 is the liver cancer cell transfer case synoptic diagram in the different time node nude mice abdominal cavity lymphoglandula;
Fig. 3 is that the liver cancer cell in different time node nude mice lung and the lymphonodi coeliaci shifts the quantity synoptic diagram;
Fig. 4 is the liver cancer cell fluorescence area synoptic diagram in different time node nude mice lung and the lymphonodi coeliaci.
Embodiment
Specify the present invention below in conjunction with embodiment.
1, set up HCCLM3R nude mice subcutaneous transplantation knurl model: BALB/c nu/nu male nude mouse, in age in 5-6 week, body weight 15-18 gram is raised in specified-pathogens free my institute nude mice chamber.The high fluorocyte HCCLM3R that shifts of the liver cancer of back left side subcutaneous injection vitro culture, dosage is 2X10
7Cell/only, the aseptic Subcutaneous tumor of getting in growth 4 week back is cut into 1mm
3About the tumour fritter, for subsequent experimental.
2, set up HCCLM3R nude mice liver transplantation knurl model: location nude mice liver, sterilization liver outer skin, aseptic incision skin expose mouse liver, separate Glisson's capsule and scratch an osculum with tweezers, the tumour fritter is implanted liver surface, sew up Glisson's capsule and skin.
3, obtaining of lung secondary liver cancer cell: the orthotopic transplantation knurl grows to the 42nd day, puts to death nude mice, opens chest and takes out complete two lungs, observes lung metastasis size and number (Leica fluor stereomicroscope, IPP software), gets 5-10 2mm through the fluorescence location
3Lung metastasis tissue.
4, the clone of lung secondary liver cancer cell and amplification: with 200 purpose filter screens, lung metastasis tissue is milled into cell suspension, centrifugal (1500rpm * 5min) gets cell precipitation.After 1 * PBS liquid washing 3 times, the secondary liver cancer cell is resuspended in the DMEM complete culture solution that contains 10%FBS, puts 37 ℃, 5%CO
2Cultivate in the incubator and clone step by step, obtain the mono-clonal liver cancer cell until sorting, conventional liquid nitrogen is preserved standby.
5, the Function Identification of lung-targeting metastatic human hepatoma cell HCCLM3-R-LM1: the subcutaneous and liver orthotopic transplantation knurl of preparation lung transitivity HCCLM3-R-LM1 liver cancer cell nude mice.Put to death the nude mice of liver cancer orthotopic transplantation knurl week about, observe the hepatoma Metastasis situation in different time node nude mice lung and the lymphoglandula.Fig. 1 and Fig. 2 are respectively the liver cancer cell transfer case synoptic diagram in different time node nude mice lung and the lymphonodi coeliaci; Fig. 3 is that the liver cancer cell in different time node nude mice lung and the lymphonodi coeliaci shifts the quantity synoptic diagram; Fig. 4 is the liver cancer cell fluorescence area synoptic diagram in different time node nude mice lung and the lymphonodi coeliaci.
The result shows that the lung target transfer human liver cancer cell HCCLM3-R-LM1 that the present invention obtained confirms at first to take place lung through nude mice liver tumor in situ transplantation model and shifts, and occurs other position of whole body subsequently successively and shifts; It has identical genetic background with maternal liver cancer cell HCCLM3R; But have characteristics such as the red fluorescence of the high strength of expression, stabilization characteristics of genetics continuous passage, lung target transfer clonal selection advantage.Find that through external long-term cultured continuously red fluorescence expression intensity height, fluorescence are difficult for by cancellation, luciferase expression is stable, is comparatively ideal fluorescent tracing, organ targeted metastatic human hepatoma cell.Can be used for the molecular mechanism research of hepatoma Metastasis as fluorescently-labeled experimental cell in external two dimension and three-dimensional cell culture system; Also can be used as curative effect and differentiate the observation of curative effect that cell is used for anti-hepatoma Metastasis new drug; Also can be used as the fluorescent mark liver cancer cell of nude mice subcutaneous vaccination, be used for the foundation of subcutaneous model of fluorescent visual people lung-targeting metastatic human hepatoma nude mice and the former bit model of liver, be used for the supervision of the early stage biological behaviour of hepatoma Metastasis, and in the assessment of body medicine or other therapeutic intervention effects.
Claims (3)
1. lung-targeting metastatic human hepatoma cell strain, parent cell is that red fluorescent protein is expressed liver cancer cell HCCLM3-R, it is characterized in that, described cell is the classification name: lung target transfer human liver cancer cell strain HCCLM3-R-LM1, deposit number: 3644, have following biological characteristics: cell is Polygons epithelium sample, adherent growth, the forfeiture of contact growth-inhibiting, hypo-triploid caryogram, chromosome number 50-58 bar, all secrete AFP albumen, but the red fluorescence of stably express high strength, stabilization characteristics of genetics, can be in continuous passage under the normal condition, the lung target shifts the clonal selection advantage, the 2nd week 42.9% occurring lung after the orthotopic transplantation of nude mice liver shifts, had 85.7% the lung transfer to occur in the 3rd week, the 4th week reached 100%, and lymphonodi coeliaci shifts the 3rd week 57.1% of incidence, the 4th week was 71.5%, but late period, whole body shifted.
2. lung-targeting metastatic human hepatoma cell strain as claimed in claim 1 is characterized in that, the fluorescence microscope condition of described lung-targeting metastatic human hepatoma cell HCCLM3-R-LM1 is: excitation wavelength 560nm, emission wavelength 585nm.
3. the establishment method of the described lung-targeting metastatic human hepatoma cell strain of claim 1 is characterized in that, concrete steps are: expressing liver cancer cell HCCLM3-R with red fluorescent protein is parent cell, sets up nude mice subcutaneous transplantation knurl model, obtains the Subcutaneous tumor fritter; Set up nude mice liver transplantation knurl model, the Subcutaneous tumor fritter is implanted the liver surface of nude mice; After 42 days, put to death nude mice, open chest and take out complete two lungs, with lung metastasis tissue mill, the centrifugal cell precipitation that obtains, through cultivating and clone step by step sorting acquisition lung-targeting metastatic human hepatoma cell strain.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102115730A (en) * | 2010-09-29 | 2011-07-06 | 复旦大学附属中山医院 | High metastatic potential hepatoma cell line capable of steady autophagy indication, and establishment method and application method thereof |
| 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 |
| 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 |
| 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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| WO2006031688A2 (en) * | 2004-09-13 | 2006-03-23 | Cohen Stefan A | Method of separating tumor cells with and without lymphotropic metastatic potential in a human carcinoma |
| CN101381706A (en) * | 2007-09-06 | 2009-03-11 | 复旦大学附属中山医院 | Human liver cancer high-transfer cell strain with stable expression of fluorescent protein and construction method thereof |
| CN101380478A (en) * | 2007-09-06 | 2009-03-11 | 复旦大学附属中山医院 | Installation method of fluorescent visual high-transfer human liver cancer nude mouse model |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2006031688A2 (en) * | 2004-09-13 | 2006-03-23 | Cohen Stefan A | Method of separating tumor cells with and without lymphotropic metastatic potential in a human carcinoma |
| CN101381706A (en) * | 2007-09-06 | 2009-03-11 | 复旦大学附属中山医院 | Human liver cancer high-transfer cell strain with stable expression of fluorescent protein and construction method thereof |
| CN101380478A (en) * | 2007-09-06 | 2009-03-11 | 复旦大学附属中山医院 | Installation method of fluorescent visual high-transfer human liver cancer nude mouse model |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102115730A (en) * | 2010-09-29 | 2011-07-06 | 复旦大学附属中山医院 | High metastatic potential hepatoma cell line capable of steady autophagy indication, and establishment method and application method thereof |
| 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 |
| 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 |
| 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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