CN101831404B

CN101831404B - Lymph gland targeted metastatic human hepatoma cell strain and establishing method thereof

Info

Publication number: CN101831404B
Application number: CN 201010153268
Authority: CN
Inventors: 吴伟忠; 陶中华; 李薇薇; 孙惠川; 汤钊猷; 樊嘉; 钦伦秀; 王鲁
Original assignee: Zhongshan Hospital Fudan University
Current assignee: Zhongshan Hospital Fudan University
Priority date: 2010-04-22
Filing date: 2010-04-22
Publication date: 2012-05-23
Anticipated expiration: 2030-04-22
Also published as: CN101831404A

Abstract

The invention provides a lymph gland targeted metastatic human hepatoma cell strain. The mother cell is a red fluorescent protein-expressed hepatoma cell HCCLM3-R. The human hepatoma cell strain is characterized in that the cell is named as lymph gland targeted metastatic human hepatoma cell strain HCCLM3-R-LnM1 according to the classified nomenclature with a preservation number of 3645, and has the following biological characteristics: the cell is polygonal epithelioid and is in adherent growth and loss of contact growth inhibition, is of a hypo-triploid karyotype, and has 50-58 of chromosomes each of which secretes AFP proteins. The establishing method comprises the following steps: using subcutaneous and liver orthotopic transplantation tumor models of a nude mouse to carry out spontaneous lymphatic metastasis screening, and then carrying out in-vitro cloning, amplification and purification on the lymphatic metastasis human hepatoma cell. The invention has wide application prospects and potential social and economic values in the basic research on hepatoma and the pre-clinic research on medicament.

Description

Lymph gland targeted metastatic human hepatoma cell strain and establishment method thereof

Technical field

The present invention relates to lymph gland targeted metastatic human hepatoma cell strain and establishment method thereof; Be specifically related to and can stably express red fluorescent protein (RFP), the human hepatoma cell strain HCCLM3-R-LnM1 and the establishment method thereof of the lymph gland targeted transfer ability of tool, belong to mikrobe 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 like 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.

Lymphoglandula and lungs transfer often appear in liver cancer patient clinically.Lack corresponding research object at present both at home and abroad, the organ target property transfer of molecules mechanism of liver cancer cell is known little about it, 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) (Li Y, 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; With ClinCancer Res.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).All do not see report in the domestic and foreign literature about the lymph gland targeted metastatic human hepatoma cell strain of ability stably express fluorescence.

Summary of the invention

The lymph gland targeted 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 chromosomal integration type, Bel7402 stably express red fluorescent protein encoding sox, the lymph gland targeted transfer ability of tool.

It is parent cell that the present invention expresses liver cancer cell HCCLM3-R with red fluorescent protein; Adopt the subcutaneous and spontaneous nodus lymphoideus transferring rate screening of liver orthotopic transplantation knurl model of nude mice; The nodus lymphoideus transferring rate human liver cancer cell carries out the method for body outer clone amplification purification again, has set up a kind of lymph gland targeted metastatic human hepatoma cell strain (HCCLM3-R-LnM1).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: 3645, classification name: lymph gland targeted transfer human liver cancer cell strain (HCCLM3-R-LnM1).

Said 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: express red fluorescent protein human hepatoma cell strain (HCCLM3-R).

Subcutaneous and the liver orthotopic transplantation knurl model of said 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 appearance, 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 HS: the red fluorescent protein encoding sox 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; Increasing luciferase expression intensity, is that 560nm, emission wavelength are under the condition of 585nm in excitation wavelength, 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 characteristic, one of which has stronger transfer ability than parent cell; Its two, comprise the high heterogeneity that different targets shift subclones with parent cell and compare, this cell heterogeneity is lower; Have lymph gland targeted transfer clonal selection advantage, nodus lymphoideus transferring rate at first appears in the 2nd week after the orthotopic transplantation of nude mice liver, and the rate of transform is about 71.4%; Lymphonodi coeliaci occurring to the 4th week 100% shifts; And lung shifts in that to begin to occur about 14.3%, the 4 week the 3rd week about 28.6%, but late period, whole body shifted.

The establishment method of described lymph gland targeted 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 the abdominal cavity, take out nodus lymphoideus transferring rate kitchen range tissue, with nodus lymphoideus transferring rate kitchen range tissue mill, the centrifugal cell precipitation that obtains, through cultivating and clone step by step, sorting acquisition lymph gland targeted metastatic human hepatoma cell strain.

The present invention is maternal cell with high transfer characteristics hepatoma cell strain HCCLM3R, and the nodus lymphoideus transferring rate kitchen range liver cancer cell in the employing nude mice liver TIS transplantation model (referring to applying for a patent 200710045675.8) obtains through cultured and amplified in vitro as cloned object; Lymph gland targeted transfer human liver cancer cell strain HCCLM3-R-LnM1 confirms at first to take place nodus lymphoideus transferring rate through nude mice liver TIS transplantation model; The liver cancer cell that obtains have identical genetic background with maternal liver cancer cell HCCLM3R; But have characteristics such as the red fluorescence of the HS of expression, stabilization characteristics of genetics continuous passage, lymph gland targeted transfer clonal selection advantage.

The present invention obtains the lymph gland targeted metastatic human hepatoma cell HCCLM3-R-LnM1 that red fluorescence is expressed, and 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 subcutaneous foundation with 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 lymphoglandula 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.

Embodiment 1

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, supply 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 nodus lymphoideus transferring rate property liver cancer cell: the orthotopic transplantation knurl grows to the 42nd day; Put to death nude mice; Open the abdominal cavity, launch mesentery, observe the size and number (the Leica fluor stereomicroscope of stomach wall and mesenteric lymph nodes MET; IPP software), get 4-6 nodus lymphoideus transferring rate kitchen range tissue through the fluorescence location.

The clone and the amplification of 4, nodus lymphoideus transferring rate property liver cancer cell: with 200 purpose filter screens, sex organization is milled into cell suspension with nodus lymphoideus transferring rate, centrifugal, and (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 subsequent use.

The Function Identification of 5, nodus lymphoideus transferring rate property luciferase expression liver cancer cell: the subcutaneous and liver orthotopic transplantation knurl of nude mice of preparation nodus lymphoideus transferring rate property HCCLM3-R-LnM1 liver cancer cell.Put to death the nude mice of liver cancer orthotopic transplantation knurl week about, observe 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 lymph gland targeted metastatic human hepatoma cell strain HCCLM3-R-LnM1 that the present invention obtained confirms nodus lymphoideus transferring rate at first takes place through nude mice liver TIS transplantation model, occurs abdominal cavity, abdominal lymph nodes,parietal and whole body subsequently successively and shifts; The lymph gland targeted metastatic liver cancer cell has identical genetic background with maternal liver cancer cell HCCLM3R; But and have characteristics such as expressing the red fluorescence of HS, stabilization characteristics of genetics continuous passage, lymph gland targeted transfer clonal selection advantage.Find that through external long-term cultured continuously the red fluorescence expression intensity is high, fluorescence is 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 to imitate to treat 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 human organs targeted 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

1. lymph gland targeted metastatic human hepatoma cell strain, parent cell are that red fluorescent protein is expressed liver cancer cell HCCLM3-R, it is characterized in that said cell divide called after: lymph gland targeted transfer human liver cancer cell strain HCCLM3-R-LnM1; Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number: CGMCC3645 has following biological characteristics: cell is Polygons epithelium appearance; Adherent growth, the forfeiture of contact growth-inhibiting, hypo-triploid caryogram; Chromosome number 50-58 bar is all secreted AFP albumen, but the red fluorescence of stably express HS; Stabilization characteristics of genetics, can have lymph gland targeted transfer clonal selection advantage in continuous passage under the normal condition, nodus lymphoideus transferring rate at first appears in the 2nd week after the orthotopic transplantation of nude mice liver; The rate of transform is 71.4%, reaches 100% to the 4th all rates of transform, and lung shifts beginning in the 3rd week and occurs; Be that 14.3%, the 4 week was 28.6%, but late period, whole body shifted.