CN101748097A - Human breast cancer cell line with high lung metastasis - Google Patents
Human breast cancer cell line with high lung metastasis Download PDFInfo
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- CN101748097A CN101748097A CN200910151028A CN200910151028A CN101748097A CN 101748097 A CN101748097 A CN 101748097A CN 200910151028 A CN200910151028 A CN 200910151028A CN 200910151028 A CN200910151028 A CN 200910151028A CN 101748097 A CN101748097 A CN 101748097A
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Abstract
The invention belongs to the field of microbial and animal cell lines, and particularly relates to a human breast cancer cell line MDA-MB-231/08-001 with spontaneous high lung metastasis potentiality and an establishment method thereof. The cells of the cell line are humanized cells that have 92 expression genes different from those of parent cells, higher in-vitro growth capacity, multiplication capacity, anti-invasion capacity, tumor forming property and lung metastasis than the parent cells; and in 4 weeks after the cells are inoculated, the in-situ tumor volume of a scid mouse is 4,224.67+/-1,038.95mm<3>, the weight is 2.09+/-0.51g, the spontaneous lung metastasis is 100 percent, and the number in a metastasis lesion is 41 in each lung. The cell line and an established animal model can simulate the whole process from the growth of the tumor cells at implantation position to the formation of the metastasis lesion at a target organ, which is quite consistent with the clinical practical condition, and can be conveniently used for the research of breast cancer lung metastasis mechanisms and related medicaments.
Description
Technical field
The invention belongs to technical field of microbe cell line, be specifically related to a kind of MCF-7 and establishment method thereof with spontaneous lung high metastatic potential.
Background technology
It is reported that mammary cancer has become the primary malignant tumour of serious harm WomanHealth.The cancer statistical report was pointed out in 2008, if American Women was lived by 85 years old, then will have people's (about 12.7%) of 1/8 to suffer from breast cancer in life.Mammary cancer ranks first in all female tumors, accounts for 26%, considerably beyond the 2nd lung cancer and the 3rd colorectal cancer, more makes other cancers too far behind to catch up.In recent years, along with the change of expanding economy and people life style, the breast cancer incidence of China also is obvious ascendant trend, has occupy the first in the various malignant tumours of big city women.In Shanghai, become the primary malignant tumour of women from mammary cancer in 1989, the thick sickness rate of mammary cancer in 1991 is 36.6/10 ten thousand, 1997 is 46.0/10 ten thousand, 2000 be to rise to 74.2/10 ten thousand in 56.2/10 ten thousand, 2004, increase progressively 3.96% every year.Clinical studies show, distant metastasis are that the patient with breast cancer treats failure and main causes of death always.Autopsy data shows, dies among the patient of mammary cancer, and 60%～80% shifts with lung.Therefore, strengthen necessaryly to the research of breast cancer cell metastasis, setting up reliable lung high-transfer cell system and animal model then is the task of top priority.
Prior art discloses in MCF-7, and the clone that can be used for mice-transplanted tumor model transfer research has MDA-MB-435, MDA-MB-231, MCF-7, MAII and BCML-TA299 etc.Wherein MDA-MB-435 behind nude mice mammary fat pad (mfp) in-situ inoculating 4,8 and 12 when week the lung rate of transform be respectively 30%, 70% and 100%, wherein, subclone cell MDA-MB-435HM lung rate of transform when 4 weeks reaches 100%.MDA-MB-435 once was extensive use of for a long time as typical case's representative of the high metastatic breast cancer clone of lung.But the origin of cell about MDA-MB-435 causes much controversies in recent years, has research to point out that this clone may come from melanoma but not mammary cancer (Nat Genet, 2000); MDA-MB-231 also is a kind of MCF-7 commonly used, have the short advantages such as (15 days) of tumor formation rate height (100%), latent period, but its spontaneous lung rate of transform is not high, generally is no more than 10%.Obviously, directly carrying out lung transfer correlative study with MDA-MB-231 is not that institute is suitable.Fortunately existing people isolates a kind of high transitivity subclone MDA-MB-231LM2 (4175) from MDA-MB-231, only with this quite few cell (2 * 10
3) tail vein injection can make mouse that lung takes place and shift, the bone metastasis model can be successfully set up in the left ventricle injection.At this cell (1 * 10 of 13 mfp in-situ inoculatings
6) the BALB/c nude mice in detected lung's metastasis in respect of 7 during by 38 days, the lung rate of transform is 53.8% (Nature, 2005).In view of the lung that causes through the disposable a large amount of injection cancer cells of tail vein shifts the clinical practice situation that departs to a great extent, and the spontaneous lung rate of transform still lower (about 50%) during this cell of in-situ inoculating and experimental period long (last and surpassed for 5 weeks), therefore thinking that it is necessary isolating the higher subclone clone of the spontaneous lung rate of transform from MDA-MB-231, also is possible.
Summary of the invention
The purpose of this invention is to provide a kind of high-lung-metastasis human breast cancer cell line--MDA-MB-231/08-001H.High-lung-metastasis human breast cancer cell line of the present invention can be used for the research of mammary cancer lung transfer mechanism and related drugs.
The present invention adopts MCF-7 MDA-MB-231 (the ATCC numbering: HTB-26 of American type culture collection (ATCC), get by separating in 51 years old Caucasia women with breast cancer patient's the hydrothorax) be parental cell, utilize scid and BALB/c nu/nu mouse by combination, tail vein injection and mammary fat pad (mfp) cross inoculation, the short metastasis of orthotopic transplantation knurl unloading forms, step sizing in the body, it is tactful that amplification in vitro and going down to posterity waits, separate the MCF-7 MDA-MB-231/08-001H that has obtained having the spontaneous lung high metastatic potential, in preservation on August 25 in 2008, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), the Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, 100101, deposit number: CGMCC No.2635, classification name: MCF-7.
This clone has following biological characteristics: detect through microsatellite DNA, the result shows that MDA-MB-231/08-001H and its parental cell MDA-MB-231 are Humanized cell, but has 92 difference expression genes between the two at least; All be higher than its parental cell at external cell growth, multiplication capacity and invasive ability; It becomes knurl and the equal significance of lung rate of transform ground to surpass parental cell scid mouse and BALB/c Model in Nude Mice.Inoculation MDA-MB-231/08-001H cell 4 week back is 4224.67 ± 1038.95mm3 at its primary tumor volume of scid mouse, and weight is 2.09 ± 0.51g (n=6), and the spontaneous lung rate of transform is 100%, and the metastasis median is 41 a/lung.Relative therewith, the primary tumor volume is 749.63 ± 282.35mm3 behind the inoculation MDA-MB-231 cell, and weight is 0.37 ± 0.13g (n=6), and the spontaneous lung rate of transform is 0%.Also obtained similar result the BALB/c nude mouse.
Clone of the present invention has following characteristics:
1) genetic background is clear, and biological characteristics is stable.This clone comes from widely used MCF-7 MDA-MB-231, belongs to Humanized cell through evaluation, and basic biological characteristics such as its growth, invasive ability, one-tenth knurl is consistent with parental cell, through repeatedly go down to posterity and modify after still keep stablizing.
2) use face width, be suitable for setting up human breast carcinoma transplanted tumor model at the panimmunity deficient animals.This clone both can be set up the transplanted tumor model the scid mouse of T, B cell double defect, also can successfully set up the transplanted tumor model the BALB/c nude mouse that has only the T cell defect.
3) method of setting up model approaches the clinical practice situation, spontaneous lung rate of transform height, and experimental period is short.This clone adopts mammary fat pad (mfp) inoculation, no matter still all can reliablely and stablely set up the transplanted tumor model the BALB/c nude mouse the scid mouse, and the lung rate of transform during 4 weeks can reach 100%, and metastasis is intensive and convergence often arranged.
Clone of the present invention and the animal model of setting up thus can be simulated from the grow into whole process that at target organ form metastasis of tumour cell at implantation site, very identical with the clinical practice situation, can be advantageously used in the research of mammary cancer lung transfer mechanism and related drugs.
Description of drawings
Fig. 1 is the transplanted tumor of human breast carcinoma MDA-MB-231/08-001H clone the scid mouse.
Fig. 2 human breast carcinoma MDA-MB-231/08-001H clone shifts at the lung of scid mouse and BALB/c nude mice, and arrow indication wherein is a metastasis.
Embodiment
The screening of embodiment 1 lung high-transfer cell and build and be
Cell involved in the present invention all is incubated in Leibovitz ' the s L-15 cell culture medium that contains 10% foetal calf serum (FCS).Culture temperature is 37 ℃, and atmosphere surrounding is 5%CO
2/ 95% air, humidity are saturated humidity.When going down to posterity, the trypsin digestion and cell with 0.25% goes down to posterity in 1: 3 ratio.
1. the first run of lung high-transfer cell screening
The MDA-MB-231 cell that 1. will be in the growth logarithmic phase is made single cell suspension with 0.25% tryptic digestion;
2. under the aseptic condition, with the 1ml syringe with tumor cell inoculation in the fat pad (mfp) of the 6 week female nude mouses of BALB/c in age (BALB/c nude mice) left sides, second nipple or be injected directly into tail vein (i.v.), capacity is adjusted into 1 * 10
7Cell/0.1ml/ is (mfp) or 2 * 10 only
6Cell/0.1ml/ is (i.v.) only;
3. conventional raised for 4 weeks after, nude mouse is put to death in the broken end bloodletting, and with 75% alcohol-pickled sterilization 5 minutes;
4. nude mouse is dissected in aseptic technique, complete taking-up bilateral lung, and rinsing is 3 times in physiological saline;
5. under dissecting microscope, use eye scissors clip lung metastasis, after it is shredded, move in 6 orifice plates and cultivate;
6. after treating cell attachment, change fresh cell culture medium;
7. be cleared into fibrocyte with half digestion method and half mechanical process;
The tumour cell of 8. external continuation amplification epicycle screening gained, part is used for the lower whorl screening, and part gives frozen standby.
The step sizing of lung high-transfer cell and build and be
Carry out the 2nd～6 with method and take turns the screening of lung transitional cell, each takes turns cell called after m1～5 (mfp inoculations) respectively, v1～5 (tail vein injection) or [v (n) m (n)] (n=1～5) (tail vein injection+mfp inoculation) of gained.Wherein 2m4 exceeded for 20 generations in external long-term cultivation, became the clone of continuous passage, called after MDA-MB-231/08-001H.
MDA-MB-231/08-001H is behind the genetic modification of Luci (luciferase) and green fluorescent protein (GFP), and it becomes knurl, lung transfer characteristics to keep stable.
The present invention by combination utilize that scid and BALB/c nu/nu mouse, tail vein injection and mammary fat pad (mfp) cross inoculation, the short metastasis of orthotopic transplantation knurl unloading form, step sizing, amplification in vitro and strategy such as go down to posterity in the body, separate the MCF-7 MDA-MB-231/08-001H that has obtained having the spontaneous lung high metastatic potential.This clone vitro culture well-grown went down to posterity once in per 5 days.
MDA-MB-231/08-001H and parent MDA-MB-231 all amplify the product with the human breast carcinoma tissue same clip on the D14S68 microsatellite locus, and the nude mice healthy tissues is not seen amplified fragments, the homology that shows MDA-MB-231/08-001H and human breast carcinoma tissue, and MDA-MB-231/08-001H has genetic stability in continuous passage and the vitro culture process in xenotransplantation, nude mouse.
The present invention adopts the CCK8 method to trace the cell growth curve of MDA-MB-231/08-001H and MDA-MB-231, and the result shows that the growth of MDA-MB-231/08-001H cell, multiplication capacity are higher than its parental cell; The result of flow cytometry analysis shows that in MDA-MB-231/08-001H and MDA-MB-231 cell, the shared ratio of S phase cell is respectively 60.44% and 45.10%.
Embodiment 2 mice-transplanted tumors and lung shift experiment
1.BALB/c nude mouse
1. will be in the MDA-MB-231 of logarithmic phase and MDA-MB-231/08-001H cell with 0.25% tryptic digestion, make single cell suspension respectively;
2. under the aseptic condition, with the fat pad of tumor cell inoculation in nude mouse left side second nipple, inoculum size is 1 * 10 with the 1ml syringe
7Cell/0.1ml/ only;
3. observe the knurl that becomes of the healthy state of nude mouse and tumour cell every other day;
4. after 4 weeks, each group is randomly drawed 6 nude mouses, gets its lung with BouinShi liquid-solid fixed 24 hours, is dipped to the lung tissue color restoration with raw spirit again, and till metastasis is white tubercle, and each lung of observed and recorded shifts nodular quantity under dissecting microscope; Cut the capable routine pathology inspection of part transplanted tumor, redundance is stored in the liquid nitrogen.
2.scid mouse
With 6 the week ages female scid mouse be object, method is tested with the BALB/c nude mouse.The tail vein injection amount is 2 * 10
6Cell/0.1ml/, the mfp inoculum size is 1 * 10
7Cell/0.1ml/ only.
The result shows:
The Transwell result of experiment confirms that the MDA-MB-231/08-001H cell has stronger invasive ability than its parental cell.The cell count that the former penetrates artificial basilar membrane is 182 ± 14/HP, and the cell count that the latter penetrates artificial basilar membrane is 94 ± 6.3/HP (P=0.01).
Inoculation MDA-MB-231/08-001H cell 4 week back is 4224.67 ± 1038.95mm3 at its primary tumor volume of scid mouse, and weight is 2.09 ± 0.51g (n=6), and the spontaneous lung rate of transform is 100%, and the metastasis median is 41 a/lung.Relative therewith, the primary tumor volume is 749.63 ± 282.35mm3 behind the inoculation MDA-MB-231 cell, and weight is 0.37 ± 0.13g (n=6), and the spontaneous lung rate of transform is 0%.Also obtained similar result the BALB/c nude mouse.
Claims (5)
1. high-lung-metastasis human breast cancer cell line, it is characterized in that described cell is high-lung-metastasis human breast cancer cell line MDA-MB-231/08-001H, deposit number: CGMCC No.2635, has following biological characteristics: be Humanized cell, 92 difference expression genes arranged with its parental cell; In-vitro cell growth, multiplication capacity and invasive ability are higher than its parental cell; Become the knurl and the lung rate of transform to surpass parental cell, scid mouse primary tumor volume is 4224.67 ± 1038.95mm3 after 4 weeks of inoculation, and weight is 2.09 ± 0.51g, and the spontaneous lung rate of transform is than the parental cell height, and the metastasis number is many than parental cell.
2. by the described high-lung-metastasis human breast cancer cell line of claim 1, it is characterized in that described parental cell is MDA-MB-231, ATCC numbering: HTB-26.
3. by the described high-lung-metastasis human breast cancer cell line of claim 1, it is characterized in that its biological characteristics of described clone keeps stablizing through repeatedly going down to posterity and modifying the back.
4. by the described high-lung-metastasis human breast cancer cell line of claim 1, it is characterized in that described clone can set up the transplanted tumor model and/or set up the transplanted tumor model the BALB/c nude mouse that has only the T cell defect the scid mouse of T, B cell double defect.
5. by the described high-lung-metastasis human breast cancer cell line of claim 1, it is characterized in that the described clone spontaneous lung rate of transform is 100%, the metastasis median is 41 a/lung.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101775369B (en) * | 2008-11-04 | 2011-10-26 | 复旦大学附属肿瘤医院 | High-lung-metastasis human breast cancer cell line |
| CN114196617A (en) * | 2021-12-22 | 2022-03-18 | 中国医学科学院基础医学研究所 | Organ-specific breast cancer metastasis model and establishment method and application thereof |
-
2009
- 2009-06-26 CN CN200910151028A patent/CN101748097A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101775369B (en) * | 2008-11-04 | 2011-10-26 | 复旦大学附属肿瘤医院 | High-lung-metastasis human breast cancer cell line |
| CN114196617A (en) * | 2021-12-22 | 2022-03-18 | 中国医学科学院基础医学研究所 | Organ-specific breast cancer metastasis model and establishment method and application thereof |
| CN114196617B (en) * | 2021-12-22 | 2023-12-22 | 中国医学科学院基础医学研究所 | Organ-specific breast cancer metastasis model, and establishment method and application thereof |
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Application publication date: 20100623 |