CN102071168A - High metastasis human breast carcinoma cell subline and establishing method thereof - Google Patents
High metastasis human breast carcinoma cell subline and establishing method thereof Download PDFInfo
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Abstract
The invention relates to a high metastasis human breast carcinoma cell subline MDA-MB-231sci, which has biological and pathological features and special metastasis phenotypes. The biological features are as follows: the MDA-MB-231sci cells are typical polygonal epithelioid cells, have rich cytoplasm and big and round karyon, grow adherent to the wall with almost uniform size and are closely arranged with clear boundary. The pathological features are as follows: a tumor cell of a nude mouse in vivo metastasis part has the similar structure with the subcutaneous tumor cell, short metastasis time and high metastasis rate, wherein the metastasis time is shortened to 26-42 days; a lung metastasis rate is 100%, meanwhile, a part of axillary lymph node metastasis of an animal is performed. An establishing method for the high metastasis human breast carcinoma cell line comprises the following steps of: (1) establishing a human breast carcinoma orthotopic transplantation tumor model; (2) sieving an orthotopic transplantation lung targeted metastasis model; and (3) establishing and passaging a human breast and lung carcinomas targeted high metastasis cell line. In the invention, the success rate is improved by sieving surgical excision tumor of each generation in vivo and corresponding breast.
Description
Technical field
The present invention relates to cancerous cell line, be specifically related to human breast carcinoma high-transfer cell subbreed and establishment method thereof, belong to the technical field of animal cell line.
Background technology
One of biological characteristics that the invasion and attack of tumour malignant tumour when shifting is the most basic, it is comparatively rare that primary tumor causes death, 90% above tumour patient die from tumour cell when the intravital transfer of people.
Mammary cancer is one of able-bodied principal disease of harm women, becomes the modal malignant tumour of women.In the U.S., there are every year 4.5 ten thousand patient with breast cancers dead because of the tumour distant metastasis.
Yet cutter system is still not fully aware of really for metastases at present, is the focus of oncology studies about metastases always.In relevant mammary cancer infiltration, transfer research, MDA-MB-435 is human breast cancer cell strain comparatively commonly used.Bibliographical information, human breast cancer in nude mice fat pad were that the rate of transform of mouse lung is respectively 30%, 70% and 100% when inoculating these 4 weeks of cell, 8 weeks and 12 weeks originally.This shows,, need long experimental period and higher experiment input though this cell strain finally can have the higher lung rate of transform.So bigger limitation in the research of this area has restricted the progress to the Metastasis in Breast Cancer Study on Mechanism.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of high-transfer human breast cancer cell line, to solve the many weak points of existing in prior technology, as the long and higher defective of experiment input human breast cancer cell strain MDA-MB-435 experimental period.
The technical problem that will solve required for the present invention can be achieved through the following technical solutions:
As a first aspect of the present invention, a kind of human breast carcinoma high-transfer cell subbreed is MDA-MB-231sci, has biology and pathological characteristics, also possesses special metastatic phenotype.
Described biological property is,
The MDA-MB-231sci cell is typical Polygons epithelioid cell;
Endochylema is abundant, and karyon is justified greatly, has kept the morphology characteristics of MDA-MB-231 clone;
Adherent growth, size are consistent, arrange closely, and the boundary line is clear.
Described pathological characteristics is that nude mice is used in described transfer, and the metastasis site tumour cell is similar to the cellularstructure of Subcutaneous tumor in the described nude mouse, and shorten transfer time, rate of transform height.
Foreshorten to 26-42 days described transfer time.
The described nude mice rate of transform is 100%.
Human breast carcinoma high-transfer cell subbreed MDA-MB-231sci contains in China typical culture collection center.Preservation address: the Chinese Wuhan City, Hubei Province wuchang, wuhan Luo Jiashan of life science institute of Wuhan University, postcode 430072.Preservation date on July 29th, 2009.Deposit number CCTCC NO:C200955.
As a second aspect of the present invention, the establishment method of human breast carcinoma high-transfer cell subbreed (MDA-MB-231sci) is characterized in that,
(1) foundation of human breast carcinoma orthotopic transplantation knurl model: get 5 of nude mouses, the outer vigorous continuous passage human breast cancer cell strain MDA-MB-231 of incubation growth of collection body, adjusting cell concn is every milliliter 1 * 10
7Individual, every mouse is got 0.2ml, and to be inoculated in the nearly armpit of the right dorsal part of nude mouse subcutaneous;
When transplanted tumor grows to the diameter 15mm left and right sides, the cervical vertebra dislocation method is put to death mouse, strip out transplanted tumor, remove periphery clot and reticular tissue, with the normal saline flushing that contains antibiotic, cut the tumor tissue of well-grown, incarnadine, flesh of fish shape, it is cut into the fritter of the about 1.5mm of diameter, migrate to other nude mouse forward right side mammary fat pad position with No. 20 trochars, set up mammary cancer orthotopic transplantation knurl model;
(2) screening of orthotopic transplantation lung target metastasis model: after setting up orthotopic transplantation knurl model, ocal resection and corresponding mammary gland when treating that knurl is directly grown to 1.5～2.0cm, treat to sacrifice when animal is in moribund condition mouse and carry out system's dissection, get the obvious visible lung of naked eyes metastasis and migrate to other nude mouse mammary fat pad and carry out orthotopic transplantation, carry out high transfer cycle screening by screening mode in the body of mammary gland orthotopic transplantation, ocal resection and corresponding mammary gland, lung metastasis, mammary gland orthotopic transplantation;
Former generation 10 nude mouses, other per generations 4～6 nude mouses, Cycle Screening to lung shifts and to reach 100%, get tumor tissues and set up corresponding high-transfer cell subbreed this moment;
(3) foundation of human breast carcinoma lung target high-transfer cell system and going down to posterity: lotus knurl nude mouse cervical vertebra dislocation method is put to death, and aseptic condition takes out the subcutaneous transplantation knurl down, rejects necrotic tissue, fibrous capsule and blood vessel;
Wash 2 times with containing two anti-PBS, drip 1-2 to tissue and drip the DMEM nutrient solution that contains 10% calf serum, it is cut into small pieces as far as possible with micro-scissors and microforceps;
To shear good tissue block with microforceps and on the 6cm culture dish, evenly place, about each fritter spacing 0.5cm;
In culture dish, inject an amount of nutrient solution then, be placed on 37 ℃, contain 5%CO
2Incubator in.The primary cell of being set up later carries out the routine cultivation with the DMEM nutrient solution that contains 10% calf serum and goes down to posterity.
Cycle Screening to the described in the described step (2) during four generations lung shift and to reach 100%, accompany part animal axillary lymph to carry down simultaneously and move.
Beneficial effect of the present invention:
1, the present invention adopts screening ocal resection of per generation and corresponding mammary gland in the body, improves success ratio.
2, rate of transform raising of the present invention reaches 100%, accompanies part animal axillary lymph to carry down simultaneously and moves.
Description of drawings
Further specify the present invention below in conjunction with the drawings and specific embodiments.
Fig. 1 is the photo of mammary cancer orthotopic transplantation knurl model.
Fig. 2 is the form photo of MDA-MB-231sci cell.
Human breast carcinoma high-transfer cell subbreed MDA-MB-231sci.
Preservation date on July 29th, 2009.
Depositary institution: Chinese typical culture collection center, be called for short CCTCC.
Deposit number CCTCC NO:C200955.
Embodiment
In order to make technique means of the present invention, creation characteristic, to reach purpose and effect is easy to understand,, further set forth the present invention below in conjunction with concrete diagram.
One, material:
1. tumour cell
Human breast carcinoma high-transfer cell MDA-MB-231 is so kind as to give by medical company limited of Hutchison China Trade Holdings (Shanghai) that (primary source is in the American Type Culture Collection of ATCC American Type Culture Collecti numbering ATCC
HTB-26
TM), subcutaneous transplantation knurl MDA-MB-231 is preserved by the Shanghai Inst. of Tumor and goes down to posterity.
2. cell culture reagent
Dulbecco MEM (DMEM) substratum is available from SIGMA-ALDRICH company; Foetal calf serum is available from PAA laboratories GmbH company; Trypsinase is available from GIBCO BRL company; Physiological saline, gentamicin, 10% neutral formalin solution, PBS damping fluid etc. is import packing or homemade analytical reagent.
3. instrument and equipment
Bechtop is produced by Purifying Equipment Co., Ltd., Suzhou, and model is SW-CJ-2FD; CO
2Incubator is the Heraeus product, and model is BB16UV, and Thermon company product, and model is cell150; Thermostat water bath is the grand experimental installation of a last Nereid company limited product, and model is DK-S22; Inverted microscope is Chongqing Optical ﹠ Electrical Instrument Co., Ltd.'s product, and the instrument model is XDS-1B; Digital camera is the NIKON product; The Ultralow Temperature Freezer model is Jouan, and France produces; Liquid-transfering gun is a GILSON company product; The liquid nitrogen container model is MVE CRYSTEM 4000; Culture dish and culturing bottle are Greiner company product; Small electrical thermovacuum Sterilizers, model are XG1.UXD-0.361B.
4. laboratory animal:
SPF level BALB/c-nu/nu nude mouse, mouse 6～7 weeks of age, body weight 18-22g, female.Experiment mice is provided by the Shanghai Inst. of Tumor, and production licence number is SCXK (Shanghai) 2007-0001, and occupancy permit number is SYXK (Shanghai) 2007-0001.Used bedding and padding, drinking-water, full-valence pellet feed and other article that contact with animal are all handled through autoclaving.Experiment and raising condition are in strict accordance with the code requirement of SPF animal.
Two, method:
1. the foundation of human breast carcinoma orthotopic transplantation knurl model
Get 5 of nude mouses, the outer vigorous continuous passage cell of incubation growth of collection body, adjusting cell concn is every milliliter 1 * 10
7Individual, every mouse is got 0.2ml, and to be inoculated in the nearly armpit of the right dorsal part of nude mouse subcutaneous.When transplanted tumor grows to the diameter 15mm left and right sides, the cervical vertebra dislocation method is put to death mouse, strip out transplanted tumor, remove periphery clot and reticular tissue, with the normal saline flushing that contains antibiotic, cut the tumor tissue of well-grown, incarnadine, flesh of fish shape, it is cut into the fritter of the about 1.5mm of diameter, migrate to other nude mouse forward right side mammary fat pad position with No. 20 trochars, set up mammary cancer orthotopic transplantation knurl model (Fig. 1).
2. the screening of orthotopic transplantation lung target metastasis model
After setting up orthotopic transplantation knurl model, ocal resection and corresponding mammary gland when treating that knurl is directly grown to 1.5～2.0cm, treat to sacrifice when animal is in moribund condition mouse and carry out system's dissection, get the obvious visible lung of naked eyes metastasis and migrate to other nude mouse mammary fat pad and carry out orthotopic transplantation, carry out high transfer cycle screening by screening mode in the body of mammary gland orthotopic transplantation, ocal resection and corresponding mammary gland, lung metastasis, mammary gland orthotopic transplantation.Former generation 10 nude mouses, other per generations 4～6 nude mouses, Cycle Screening to the during four generations lung shift and to reach 100%, get tumor tissues and set up corresponding high-transfer cell subbreed this moment.
3. the foundation of human breast carcinoma lung target high-transfer cell system and going down to posterity
Lotus knurl nude mouse cervical vertebra dislocation method is put to death, and aseptic condition takes out the subcutaneous transplantation knurl down, rejects necrotic tissue, fibrous capsule and blood vessel.Wash 2 times with containing two anti-PBS, drip 1-2 to tissue and drip the DMEM nutrient solution that contains 10% calf serum, it is cut into small pieces as far as possible with micro-scissors and microforceps.To shear good tissue block with microforceps and on the 6cm culture dish, evenly place, about each fritter spacing 0.5cm.In culture dish, inject an amount of nutrient solution then, be placed on 37 ℃, contain 5%CO
2Incubator in.The primary cell of being set up later carries out the routine cultivation with the DMEM nutrient solution that contains 10% calf serum and goes down to posterity.
Three, result
1. The selection result in the body
Along with the increase of screening algebraically, shorten transfer time, and rate of transform raising reaches 100%, accompanies part animal axillary lymph to carry down simultaneously and moves.
2. cell cultures is built and is
Table 1 cell cultures is built and is
As shown in table 1, will leave standstill cultivation after the tumor tissue taking-up after 4 screenings in the body.It is adherent that the 2nd day part cell begins, and has a little cell outwards to swim out of around the small tissue blocks, and cell was firmly adherent in the 4th day, changes nutrient solution.After this, go down to posterity, along with the increase inoblast of passage number can be removed fully according to the cell growing state.After this went down to posterity once cell well-grown, called after MDA-MB-231sci in every 3-4 days.Reached for the 20th generation at present.Human breast carcinoma high-transfer cell subbreed MDA-MB-231sci contains in China typical culture collection center, deposit number CCTCC NO:C200955.
2. morphocytology
The MDA-MB-231sci cell of adherent growth is typical Polygons epithelioid cell, and size is consistent, arranges closely, and the boundary line is clear.Endochylema is abundant, and karyon is justified greatly, has kept morphology characteristics (Fig. 2) the HE dyeing of MDA-MB-231 clone basically, 100 times.
The present invention adopts screening ocal resection of per generation and corresponding mammary gland in the body, improves success ratio; Rate of transform raising reaches 100%, accompanies part animal axillary lymph to carry down simultaneously and moves.A lot of biological characteristicses want next step perfect.
More than show and described ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; that describes in the foregoing description and the specification sheets just illustrates principle of the present invention; the present invention also has various changes and modifications without departing from the spirit and scope of the present invention, and these changes and improvements all fall in the claimed scope of the invention.The claimed scope of the present invention is defined by appending claims and equivalent thereof.
Claims (7)
1. a human breast carcinoma high-transfer cell subbreed is MDA-MB-231sci, has biology and pathological characteristics, also possesses special metastatic phenotype.
2. human breast carcinoma high-transfer cell subbreed according to claim 1 is characterized in that, described biological property is,
The MDA-MB-231sci cell is typical Polygons epithelioid cell;
Endochylema is abundant, and karyon is justified greatly, has kept the morphology characteristics of MDA-MB-231 clone;
Adherent growth, size are consistent, arrange closely, and the boundary line is clear.
3. human breast carcinoma high-transfer cell subbreed according to claim 1, it is characterized in that described pathological characteristics is that nude mice is used in described transfer, the metastasis site tumour cell is similar to the cellularstructure of Subcutaneous tumor in the described nude mouse, shorten transfer time, rate of transform height.
4. human breast carcinoma high-transfer cell subbreed according to claim 3 is characterized in that foreshorten to 26-42 days described transfer time.
5. human breast carcinoma high-transfer cell subbreed according to claim 1 is characterized in that the described nude mice rate of transform is 100%.
6. the establishment method of a human breast carcinoma high-transfer cell subbreed according to claim 1 is characterized in that,
(1) foundation of human breast carcinoma orthotopic transplantation knurl model: get 5 of nude mouses, the outer vigorous continuous passage human breast cancer cell strain MDA-MB-231 of incubation growth of collection body, adjusting cell concn is every milliliter 1 * 10
7Individual, every mouse is got 0.2ml, and to be inoculated in the nearly armpit of the right dorsal part of nude mouse subcutaneous;
When transplanted tumor grows to the diameter 15mm left and right sides, the cervical vertebra dislocation method is put to death mouse, strip out transplanted tumor, remove periphery clot and reticular tissue, with the normal saline flushing that contains antibiotic, cut the tumor tissue of well-grown, incarnadine, flesh of fish shape, it is cut into the fritter of the about 1.5mm of diameter, migrate to other nude mouse forward right side mammary fat pad position with No. 20 trochars, set up mammary cancer orthotopic transplantation knurl model;
(2) screening of orthotopic transplantation lung target metastasis model: after setting up orthotopic transplantation knurl model, ocal resection and corresponding mammary gland when treating that knurl is directly grown to 1.5～2.0cm, treat to sacrifice when animal is in moribund condition mouse and carry out system's dissection, get the obvious visible lung of naked eyes metastasis and migrate to other nude mouse mammary fat pad and carry out orthotopic transplantation, carry out high transfer cycle screening by screening mode in the body of mammary gland orthotopic transplantation, ocal resection and corresponding mammary gland, lung metastasis, mammary gland orthotopic transplantation;
Former generation 10 nude mouses, other per generations 4～6 nude mouses, Cycle Screening to lung shifts and to reach 100%, get tumor tissues and set up corresponding high-transfer cell subbreed this moment;
(3) foundation of human breast carcinoma lung target high-transfer cell system and going down to posterity: lotus knurl nude mouse cervical vertebra dislocation method is put to death, and aseptic condition takes out the subcutaneous transplantation knurl down, rejects necrotic tissue, fibrous capsule and blood vessel;
Wash 2 times with containing two anti-PBS, drip 1-2 to tissue and drip the DMEM nutrient solution that contains 10% calf serum, it is cut into small pieces as far as possible with micro-scissors and microforceps;
To shear good tissue block with microforceps and on the 6cm culture dish, evenly place, about each fritter spacing 0.5cm;
In culture dish, inject an amount of nutrient solution then, be placed on 37 ℃, contain 5%CO
2Incubator in.The primary cell of being set up later carries out the routine cultivation with the DMEM nutrient solution that contains 10% calf serum and goes down to posterity.
7. the establishment method of human breast carcinoma high-transfer cell subbreed according to claim 6 is characterized in that, Cycle Screening to the described in the described step (2) during four generations lung shift and to reach 100%, accompany part animal axillary lymph to carry down simultaneously and move.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105505876A (en) * | 2014-09-26 | 2016-04-20 | 上海市肿瘤研究所 | XL-2 human lung cancer highly metastatic cell line and applications |
| CN114403095A (en) * | 2021-12-02 | 2022-04-29 | 山西医科大学 | Modeling method of spontaneous lymph node metastasis model of mouse breast cancer |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105505876A (en) * | 2014-09-26 | 2016-04-20 | 上海市肿瘤研究所 | XL-2 human lung cancer highly metastatic cell line and applications |
| CN114403095A (en) * | 2021-12-02 | 2022-04-29 | 山西医科大学 | Modeling method of spontaneous lymph node metastasis model of mouse breast cancer |
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Application publication date: 20110525 |