CN1236055C - High metastasis human breast cancer cell system and its establishing method - Google Patents
High metastasis human breast cancer cell system and its establishing method Download PDFInfo
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Abstract
The present invention relates to a carcinoma cell line, particularly to an MCF-7 with high metastasis potential and an establishing method thereof, which belongs to the field of animal cell lines. The mechanism of tumor invasion and metastasis are not very clear at present, and therefore, an animal model of tumor metastasis needs to be established for in-depth research. In the past, a nude mouse is adopted as an animal model of tumor metastasis; in the present invention, an SCID mouse is used as an experimental subject. A breast cancer parent MCF-7 cell suspension (5*10<7>/0.2m1) is hypodermically inoculated on the shoulder of the SCID mouse; after 68 days since the MCF-7 cell was inoculated, the SCID mouse is killed for taking out the lung for primary culture; when the cell growing from tissue blocks covers the bottom of a culture bottle, passage is carried out, and the cell is named as LM-MCF-7. When the cell LM-MCF-7 is cultured in vitro for 20 generations, oncocyte is subcutaneously reinjected to the SCID mouse, the method is the same as previous; the tumor forming rate reaches 100%(5/5), and widespread metastasis appears in the lung, kidney, spleen, marrow and lymph node of the SCID mouse.
Description
Technical field
The present invention relates to cancerous cell line, particularly the method for a kind of MCF-7 of high-metastasis tendency and foundation thereof belongs to field of animal cell lines.
Background technology
The invasion and attack of tumour are one of the most basic biological characteristicses of malignant tumour with transfer, also are the major reasons of tumour patient death.Yet cutter system is still not fully aware of at present really about shifting.Therefore, metastases is a focus of oncology studies always.Yet, also mainly be confined at present the observation of the transfer index of correlation of the tumor specimen of excision and some limited transfer characteristics tumor cell line about the research of metastases, and they all can not accurately reflect the whole clinical course that tumor growth shifts, and directly affect the further investigation of metastasis and therapeutic intervention thereof.For this reason, be necessary to set up ideal metastases animal model metastases is carried out deep research.
Used nude mice in the metastases animal model, the characteristics of this strain are congenital athymias in the past, and the T cell function is obviously lost, and the B cell function is normal, and the NK cell viability strengthens, and do not have hair fully.Be widely used in tumor research fields such as human tumor shift factor, different the planting property of tumour and diagnosing tumor treatment.
Summary of the invention
In the present invention, we adopt the SCID mouse as experimental subjects.The SCID mouse is that (Severecombined immunodeficiency, SCID) mouse are almost completely lost T and bone-marrow-derived lymphocyte immunologic function to Reconstruction in Sever Combined Immunodeciency, lack body fluid and cellular immune function.With respect to nude mice, SCID has the following advantages: 1. pathology is to human similar; 2. the tumour cell for external source does not have immunological rejection, has reduced the factor that shifts of disturbing, and the tumour transplatation survival rate is up to 36.9%, and general nude mice is 16.8%; 3. for malignant tumour, rate of transform height not only, and can extensive diffusive, very similar to the human tumor biological behaviour; 4. soak into similar with transfer and the spontaneous transfer of human tumor.
Have the research tumor mass inoculation animal of should choosing to simulate the natural process of metastases, but success ratio is lower.The tumor cell inoculation SCID mouse of cultivating is had good using value, have advantages such as good reproducibility, transfer efficiency height and time weak point.Therefore, we use SCID mouse metastases animal model in this research, successful foundation high-metastasis tendency MCF-7 LM-MCF-7.
Technical scheme of the present invention is that the metastatic lesion lung tissue of utilizing SCID mouse human breast carcinoma is the knurl source as building, method as a kind of new screening high-metastasis tendency tumour cell, adopt the external former foster mode of being commissioned to train, build up a strain and had the MCF-7 of high-metastasis tendency, called after LM-MCF-7.This project is the phasic results of national natural science fund subsidy project (30270322).The invention has the beneficial effects as follows:
1, the SCID mouse model can be used for screening the high-metastasis tendency tumor cell line;
2, the high-metastasis tendency cell strain of Huo Deing can be used for screening the medicine of anti metastasis;
3, by with the comparison of parental cell, can use the high-metastasis tendency cell strain and be used to screen tumor metastasis related gene;
4, can be used for studying the pathogeny of metastases;
5 so the preparation metastases dna vaccination;
6, detect the effect of the vaccine of anti metastasis by the inoculation animal.
Description of drawings
Become knurl knurl piece (A, arrow shows), its pathological tissue (B) behind Fig. 1 .LM-MCF-7 tieback SCID mouse.
Fig. 2. the 70th generation adherent growth LM-MCF-7 cell (A, * 100), cell all is the growth of bunch sample, cell is typical epithelioid cell, its out-of-shape, cell is arranged closely, boundary clear, contact suppresses forfeiture.Contrast is parental cell MCF-7 (B, * 100).
Fig. 3 .LM-MCF-7 cell SCID mouse marrow metastasis (HE * 400, arrow shows).After the cell inoculation 30 days, the spontaneous rate of transform 100% (20/20) of marrow points out this cell to shift based on the blood road.Meet clinical Metastasis in Breast Cancer feature.
Fig. 4 .LM-MCF-7 clone karyomit(e).The result shows that this cell chromosome caryogram variation is big, is polyploid cell, the distortion of karyomit(e) generation number and structure, and the karyomit(e) distribution range is at the 59-65 bar.Numerical chromosome change mainly shows as karyomit(e) monomer, trisome and many bodies.The genetics feature that meets malignant tumour.
Fig. 5. behind the parental cell MCF-7 inoculation SCID mouse 68 days, lung tissue pathology structure observation had the tumor tissues (arrow shows) of early stage transforming growth in lung tissue.(the foster LM-MCF-7 of the acquisition cell of being commissioned to train from this tissue Central Plains).
Embodiment
The concrete grammar of the MCF-7 that the present invention sets up is as follows:
1) inoculation SCID mouse: get mammary cancer parent MCF-7 cell suspension (5 * 10
7/ 0.2ml) subcutaneous vaccination is set up SCID mouse Metastasis in Breast Cancer model in SCID mouse shoulder.The SCID mouse is provided by Tianjin hematology institute of the Chinese Academy of Medical Sciences.
2) confirm metastasis site: the SCID mouse is put to death, the pathological section of each tissue is carried out microscopically observe, the position of establishing its metastasis is lungs, spleen, marrow and lymphoglandula etc.
3) acquisition of the MCF-7 of high-metastasis tendency: inoculation MCF-7 cell after 68 days is put to death the SCID mouse, takes out lungs, carry out former be commissioned to train foster.Adopt the following step: two anti-Hank ' s liquid rinsing tissue block 5 minutes; Use scissors to shred tissue, remove blood, fat, nervous tissue, reticular tissue and necrotic tissue; Lung tissue is cut into 1mm
3Left and right sides fritter; Send into culturing bottle with shearing good fritter with the ophthalmology tweezer, with inoculating needle it is evenly put, at interval 0.5cm; After tissue block is put well,,, build bottle cap up at the bottom of allowing bottle, be placed on 37 ℃ of CO gently with culturing bottle upset
2In the incubator; Placed 3 hours, culturing bottle is slowly overturn to keep flat, and injects 10ml RPMI1640 nutrient solution (containing 20% serum) in bottle, leaves standstill cultivation; Former being commissioned to train changed liquid once in foster per 3 days, removed buoyant tissue block and residual hemocyte.
After the cell that grows in by tissue block can cover with the culturing bottle bottom, carry out passage, concrete steps are as follows: add 1-2ml Digestive system (pancreatin and EDTA) in bottle, after the room temperature 3 minutes, observe tenuigenin retraction, intercellular substance increase, absorb Digestive system, add the PBS Digestive system that culturing bottle is residual and wash off; Remove a bottle interior nutrient solution with the suction pipe suction, a piping and druming bottle parietal cell makes it to break away from bottle wall formation cell suspension repeatedly, and counting is inoculated new culturing bottle respectively.When passage to the third generation, former 20% foetal calf serum of using when supporting of being commissioned to train is reduced to 10%, cell is well-grown still, the form homogeneous more that becomes.
In this experiment, former being commissioned to train of finding to derive from lung tissue supported the cell that goes down to posterity and presented the epithelium sample, the cellular form homogeneous, and contactless inhibition, and growth is very rapid at the bottom of can covering with in about two days bottle, need be gone down to posterity.This clone has been carried out the cultivation more than 100 generations in this laboratory.With this cell called after LM-MCF-7.
4) become the knurl experiment in the LM-MCF-7 cell paste: after the LM-MCF-7 cells in vitro cultivated for 20 generations, it is subcutaneous that oncocyte is recycled into the SCID mouse again, and method is the same.The result shows that tumor formation rate reaches 100%, and occurs extensively shifting at the internal organs such as lungs, kidney, spleen, marrow, lymphoglandula and heart of SCID mouse.
The detection qualification result of the present invention---LM-MCF-7 cell
The present invention adopts the RPMI1640 nutrient solution that contains calf serum to cultivate the LM-MCF-7 cell, and it can external long term growth be gone down to posterity with stable.Through experimental observation and checking, the LM-MCF-7 of growth in vitro has typical epithelium sample form, the forfeiture of contact growth-inhibiting.Genetics research confirms that this cell is a heteroploid, and chromosome number and structural aberration are serious, meet the genetics feature of malignant tumour.This cell SCID mouse inoculation tumor formation rate is 100%, and takes place extensively to shift at internal organs such as lung, kidney, spleen and hearts.After testing, LM-MCF-7 clone still keeps the original biological characteristics of tumor tissue, and fundamental research and clinical intervention study that its foundation can be Metastasis in Breast Cancer mechanism provide rational model.
The evaluation of a.LM-MCF-7 cell phenotype: present the epithelium sample, cellular form homogeneous, contactless inhibition.
B. morphological observation: behind the parental cell MCF-7 inoculation SCID mouse, compare with the one-tenth tumor tissue behind the high-metastasis tendency cell LM-MCF-7 inoculation SCID mouse that derives from parental cell MCF-7, structurally the two is similar, meets the pathological characteristics of mammary cancer.
C. chromosomal evaluation: preparation karyomit(e), observations shows: karyomit(e) is human chromosome, is polyploid.
D. the evaluation in cell source: the antibody of using breast cancer correlation antigen (CA15-3 Breast Antigen) carries out immunohistochemical staining, is positive.Be indicated as human breast cancer cell.
E. the evaluation of cell cycle: use flow cytometer and obtain doubling time cell cycle.
Claims (2)
1. high-metastasis tendency MCF-7 LM-MCF-7 is characterized in that setting up with following method:
1) inoculation SCID mouse: get the subcutaneous vaccination of mammary cancer parent MCF-7 cell suspension in SCID mouse shoulder, the concentration of described cell suspension is 5 * 10
7/ 0.2ml;
2) confirm metastasis site: the SCID mouse is put to death, the pathological section of each tissue is carried out microscopically observe, the position of establishing its metastasis is lungs, spleen, marrow and lymphoglandula;
3) acquisition of the MCF-7 of high-metastasis tendency: inoculation MCF-7 cell after 68 days is put to death the SCID mouse, takes out lungs, carries out formerly being commissioned to train fosterly, adopts the following step: with being added with two anti-Hank ' s liquid rinsing tissue block 5 minutes; Use scissors to shred tissue, remove blood, fat, nervous tissue, reticular tissue and necrotic tissue; Lung tissue is cut into 1mm
3Left and right sides fritter; Send into culturing bottle with shearing good fritter with the ophthalmology tweezer, with inoculating needle it is evenly put, at interval 0.5cm; After tissue block is put well,,, build bottle cap up at the bottom of allowing bottle, be placed on 37 ℃ of CO gently with culturing bottle upset
2In the incubator; Placed 3 hours, culturing bottle is slowly overturn to keep flat, and injects 10ml RPMI1640 nutrient solution in bottle, and described nutrient solution contains 20% serum, leaves standstill cultivation; Former being commissioned to train changed liquid once in foster per 3 days, removed buoyant tissue block and residual hemocyte;
The cell that grows in by tissue block carries out passage after can covering with the culturing bottle bottom, and concrete steps are as follows: add the 1-2ml Digestive system in bottle, after the room temperature 3 minutes, observe tenuigenin retraction, intercellular substance increase, absorb Digestive system, add the PBS Digestive system that culturing bottle is residual and wash off; Remove a bottle interior nutrient solution with the suction pipe suction, a piping and druming bottle parietal cell makes it to break away from a bottle wall formation cell suspension repeatedly, and counting is inoculated new culturing bottle respectively, when passage arrives the third generation, former 20% foetal calf serum of using when supporting of being commissioned to train is reduced to 10%;
The former foster cell that goes down to posterity of being commissioned to train that derives from this lung tissue presents the epithelium sample, cellular form homogeneous, contactless inhibition, and it is very rapid to grow, and at the bottom of just covering with in two days bottle, need go down to posterity, this clone has been carried out the above cultivation of 100 generations, and this clone is LM-MCF-7;
4) become knurl in the LM-MCF-7 cell paste: after the LM-MCF-7 cells in vitro cultivated for 20 generations, it is subcutaneous that oncocyte is recycled into the SCID mouse again, and method is the same; Tumor formation rate reaches 100%, and occurs extensively shifting at lungs, kidney, spleen, marrow, lymphoglandula and the heart of SCID mouse.
2. the establishment method of a high-metastasis tendency MCF-7 LM-MCF-7 is characterized in that being made up of following step:
1) inoculation SCID mouse: get the subcutaneous vaccination of mammary cancer parent MCF-7 cell suspension in SCID mouse shoulder, the concentration of described cell suspension is 5 * 10
7/ 0.2ml;
2) confirm metastasis site: the SCID mouse is put to death, the pathological section of each tissue is carried out microscopically observe, the position of establishing its metastasis is lungs, spleen, marrow and lymphoglandula;
3) acquisition of the MCF-7 of high-metastasis tendency: inoculation MCF-7 cell after 68 days is put to death the SCID mouse, takes out lungs, carries out formerly being commissioned to train fosterly, adopts the following step: with being added with two anti-Hank ' s liquid rinsing tissue block 5 minutes; Use scissors to shred tissue, remove blood, fat, nervous tissue, reticular tissue and necrotic tissue; Lung tissue is cut into 1mm
3Left and right sides fritter; Send into culturing bottle with shearing good fritter with the ophthalmology tweezer, with inoculating needle it is evenly put, at interval 0.5cm; After tissue block is put well,,, build bottle cap up at the bottom of allowing bottle, be placed on 37 ℃ of CO gently with culturing bottle upset
2In the incubator; Placed 3 hours, culturing bottle is slowly overturn to keep flat, and injects 10ml RPMI1640 nutrient solution in bottle, and described nutrient solution contains 20% serum, leaves standstill cultivation; Former being commissioned to train changed liquid once in foster per 3 days, removed buoyant tissue block and residual hemocyte;
The cell that grows in by tissue block carries out passage after can covering with the culturing bottle bottom, and concrete steps are as follows: add the 1-2ml Digestive system in bottle, after the room temperature 3 minutes, observe tenuigenin retraction, intercellular substance increase, absorb Digestive system, add the PBS Digestive system that culturing bottle is residual and wash off; Remove a bottle interior nutrient solution with the suction pipe suction, a piping and druming bottle parietal cell makes it to break away from a bottle wall formation cell suspension repeatedly, and counting is inoculated new culturing bottle respectively, when passage arrives the third generation, former 20% foetal calf serum of using when supporting of being commissioned to train is reduced to 10%;
The former foster cell that goes down to posterity of being commissioned to train that derives from this lung tissue presents the epithelium sample, cellular form homogeneous, contactless inhibition, and it is very rapid to grow, and at the bottom of just covering with in two days bottle, need go down to posterity, this clone has been carried out the above cultivation of 100 generations, and this clone is LM-MCF-7;
4) become knurl in the LM-MCF-7 cell paste: after the LM-MCF-7 cells in vitro cultivated for 20 generations, it is subcutaneous that oncocyte is recycled into the SCID mouse again, and method is the same; Tumor formation rate reaches 100%, and occurs extensively shifting at lungs, kidney, spleen, marrow, lymphoglandula and the heart of SCID mouse.
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| DE102005015953A1 (en) * | 2005-04-07 | 2006-10-12 | Medizinische Hochschule Hannover | Method for enrichment and ex vivo cultivation of breast primary cells |
| 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 |
| CN114015655B (en) * | 2021-12-09 | 2023-09-05 | 北京和合医学诊断技术股份有限公司 | HBRCA-959 cell line and culture method and application thereof |
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