CN114686435B - Preparation method of estrogen receptor positive mouse breast cancer cell strain - Google Patents
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Abstract
The invention discloses a preparation method of an estrogen receptor positive mouse breast cancer cell strain, which comprises the following steps: after an animal model is constructed, the cells are subjected to continuous passage to obtain the required cell strain. The preparation method is used for inducing and culturing the mouse breast cancer cells, and the positive cell strain of the mouse-derived estrogen receptor can be stably obtained. The mouse breast cancer cell strain with positive mouse estrogen receptor prepared by the preparation method can be applied to the research of a molecular mechanism of estrogen positive breast cancer generation and a drug resistance mechanism of the molecular mechanism to estrogen receptor antagonists, solves the problem that an immunodeficiency mouse model or a humanized mouse model is frequently used at present in the research of tumor immunity, can fill the blank of an estrogen receptor-alpha positive preclinical cell model and an animal model, and is beneficial to the research of tumor immunity related to the estrogen receptor.
Description
Technical Field
The invention relates to the technical field of animal cell lines, in particular to a preparation method of a mouse breast cancer cell line with positive estrogen receptors.
Background
The breast cancer is the most common tumor of women, and the breast cancer surpasses the lung cancer in 2020, so that the breast cancer becomes the cancer with the most new cases worldwide.
Estrogen receptor-alpha (ER-alpha) positive patients account for 70% to 80% of breast cancer patients. Estrogen receptor- α positive breast cancer cells are estrogen dependent for their growth, and thus endocrine therapy plays an important role in the treatment of breast cancer. Endocrine therapy, however, often renders tumor cells resistant and ultimately causes recurrence of the disease. The current research on the molecular mechanism of estrogen receptor-alpha positive breast cancer development and its resistance mechanism to estrogen receptor antagonists is still very limited. One of the major causes of this is the lack of experimental animal models of estrogen receptor- α positive breast cancer.
The in vitro culture of tumor cells and the construction of preclinical cell models have led to important research approaches in the biomedical field. At present, most of animal breast cancer cell tumor models are composed of estrogen receptor-alpha negative breast cancer cells, such as EO771 cells, 4T1 cells and the like. They are not estrogen-dependent and are difficult to use to study estrogen function. However, the conventional mouse model for studying estrogen receptor-alpha positive breast cancer generally adopts human-derived estrogen receptor-alpha positive breast cancer cells. However, due to the immunological rejection between species, an immunodeficient mouse model or a humanized mouse model is generally used. However, the immunodeficient mouse model lacks functional immune cells and is not favorable for studying tumor immunity. In addition, the humanized mouse model is an immunodeficient mouse transplanted with human tissue cells or genetically modified to express a human gene, and is a unique animal model currently used for studying human cells or tissues. However, the humanized mouse model is complex in establishing method, low in reconstruction rate and low in use frequency, so that the research on the tumor immunization method is limited.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a preparation method of a mouse breast cancer cell strain with positive estrogen receptors.
The first purpose of the invention is to provide a preparation method of an estrogen receptor positive mouse breast cancer cell strain.
The second purpose of the invention is to provide a mouse breast cancer cell strain with positive estrogen receptor.
The third purpose of the invention is to provide the application of the estrogen receptor positive mouse breast cancer cell strain in screening drugs for treating estrogen receptor-alpha positive breast cancer.
In order to achieve the purpose, the invention is realized by the following scheme:
a preparation method of a mouse breast cancer cell strain with positive estrogen receptor comprises the following steps:
s1, inducing C57BL/6 mice to generate breast tumors by medroxyprogesterone acetate (MPA) and dimethylbenzene anthracene (DMBA);
s2, when the diameter of the breast tumor of the C57BL/6 mouse in the step S1 reaches 1-1.5 cm, taking the breast tumor with the volume of 2-3 mm 3 The breast tumor tissue block is planted in a breast fat pad of the NSG mouse;
s3, when tumors with the diameter of 1-1.5 cm are generated in the mammary gland of the NSG mouse in the step S2, the volume ratio is 2-3 mm 3 The breast tumor tissue mass of (a) was implanted in the mammary fat pad of another NSG mouse;
s4, when tumors with the diameter of 1-1.5 cm are generated in the mammary gland of the mouse in the step S3, separating tumor tissues to obtain a single cell suspension, and performing adherent culture;
and S5, removing the fibroblasts generated by adherent culture in the step S4, and continuously culturing to obtain a stable cell strain.
Preferably, in step S1, the mice are C57BL/6 mice 6 to 8 weeks old.
Preferably, in step S1, the medroxyprogesterone acetate is a medroxyprogesterone acetate sustained release tablet.
More preferably, the medroxyprogesterone acetate sustained-release tablet is a medroxyprogesterone acetate 90-day sustained-release tablet.
Preferably, in step S1, the dimethylbenzene anthracene is dimethylbenzene anthracene with a concentration of 5 to 10 mg/mL.
More preferably, in step S1, the method for inducing breast tumor formation in C57BL/6 mice by medroxyprogesterone acetate (MPA) and Dimethylbenzanthracene (DMBA) is as follows: after the medroxyprogesterone acetate sustained-release tablet is implanted for one week, the mice are administrated with dimethylbenzanthracene for intragastric administration with the duration of 8 weeks, and 100-200 mu L of DMBA is administrated each time.
Further preferably, the mice are perfused with dimethylbenzanthracene for 1 and 2 weeks weekly, pausing for 3 weeks, for 4 and 5 weeks weekly, for 6 weeks, and for 7 and 8 weeks weekly.
Preferably, in step S2, the mouse mammary gland fat pad is the fourth pair of mammary gland fat pads on the left side of the mouse.
Preferably, in step S4, the specific method for obtaining the single cell suspension is: the tumor tissue was washed with PBS, digested with the digestive juice for 1 to 2 hours, filtered through a sieve of not more than 100 μm and the filtrate was collected.
More preferably, the digestion solution is a complete medium containing 10 to 15mg/L of collagenase type I and 10 to 15mg/L of collagenase type VI.
Further preferably, the complete medium is a DMEM medium containing 10 to 20% by volume Fetal Bovine Serum (FBS).
Further preferably, the filtrate is centrifuged at 1000r/min for 5 minutes and the supernatant is discarded.
Preferably, in step S4, the adherent culture is an adherent culture in a complete medium.
More preferably, the complete medium is a DMEM medium containing 10 to 20% Fetal Bovine Serum (FBS) by volume.
Preferably, in step S5, fibroblasts produced by adherent culture in step S4 are removed by pancreatin.
Preferably, in step S5, the continuous subculture is subcultured every 2 to 3 days.
More preferably, in step S5, the continuous subculture is 40 or more passages.
More preferably, the stable cell line obtained in step S5 (estrogen receptor positive mouse breast cancer cell line) is ER + The mouse breast cancer cell strain DMMC-02 is preserved in China Center for Type Culture Collection (CCTCC) at 3 months and 10 days in 2022, and the preservation number is CCTCC NO: c202268, deposit address: wuhan university in Wuhan city, hubei province.
The invention also claims the mouse breast cancer cell strain with positive estrogen receptor prepared by the preparation method.
The invention also claims the ER obtained by preparation + The application of the mouse breast cancer cell strain DMMC-02 in screening medicines for treating estrogen receptor-alpha positive breast cancer.
Compared with the prior art, the invention has the following beneficial effects:
the invention establishes a preparation method of an estrogen receptor positive mouse breast cancer cell line, and can stably obtain a mouse-derived estrogen receptor positive cell line. The mouse breast cancer cell strain with positive mouse estrogen receptor prepared by the preparation method can be applied to the research of a molecular mechanism of estrogen positive breast cancer generation and a drug resistance mechanism of the molecular mechanism to estrogen receptor antagonists, solves the problem that an immunodeficiency mouse model or a humanized mouse model is frequently used at present in the research of tumor immunity, can fill the blank of an estrogen receptor-alpha positive preclinical cell model and an animal model, and is beneficial to the research of tumor immunity related to the estrogen receptor.
Drawings
FIG. 1 shows the morphology of tumor tissue induced in C57BL/6 mice.
FIG. 2 shows estrogen receptor positivity of induced tumor tissue in C57BL/6 mice, with a scale of 200 μm.
FIG. 3 shows the morphology of the estrogen receptor positive mouse breast cancer cell lines obtained by serial subculture, with a scale of 500pixels.
FIG. 4 is an immunofluorescence chart of ER, CK and Ki-67 expression conditions in an estrogen receptor positive mouse breast cancer cell line obtained by continuous subculture; in the figure, the green color shows estrogen receptor alpha expression, the red color shows CK expression, the silver color shows Ki-67 expression, and the blue color shows DAPI; the scale bar is 50 μm.
FIG. 5 shows the tumor tissue morphology after tumor formation of estrogen receptor positive mouse breast cancer cell lines obtained by continuous subculture.
FIG. 6 is a HE staining pattern after tumorigenesis of an estrogen receptor positive mouse breast cancer cell line obtained by continuous subculture.
FIG. 7 shows the estrogen receptor positive mouse breast cancer cell line CD8 obtained by continuous subculture + Immunofluorescence mapping of T cell infiltration; the green color shown in the figure is CD3 expression, red is CD8 expression, yellow is CD3 and CD8 expression simultaneously, and blue is DAPI; the scale is 50 μm and the positions shown are exaggerated positions.
FIG. 8 shows the isolation and culture of primary cells from MPA/DMBA-induced C57BL/6 mouse tumors.
FIG. 9 shows the isolation of primary cells from MPA/DMBA-induced C57BL/6 mouse tumors, cultured, and the cells after multiple passages.
Detailed Description
The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Example 1 establishment of Estrogen receptor Positive mouse Breast cancer model
1. Experimental methods
MPA (medroxyprogesterone acetate) sustained-release tablets are embedded under the skin of C57BL/6 mice with 6 weeks of age for 90 days. One week later, C57BL/6 mice were subjected to 8-week-duration gavage with DMBA (dimethylphenylanthracene) at a concentration of 5mg/ml, and 200. Mu.L of DMBA was administered each time for 6 gavages. Wherein the gavage is performed once a week in the 1 st and 2 nd weeks, the gavage is suspended in the third week, the gavage is performed once a week in the 4 th and 5 th weeks, the gavage is suspended in the 6 th week, and the gavage is performed once a week in the 7 th and 8 th weeks.
When the diameter of the breast tumor of the C57BL/6 mouse induced by the DMBA reaches 1-1.5 cm, the tumor tissue is taken out from a super clean bench. And taking a part of tumor tissues for embedding and sectioning, observing the tissue morphology by HE staining and identifying the estrogen receptor positive condition by an immunohistochemical test.
2. Results of the experiment
The morphology of the breast tumor tissue of the mice induced by DMBA is shown in figure 1, and the tumor tissue is fixed by paraformaldehyde with the mass (g)/volume (ml) of 4%. The estrogen receptor positivity of the mouse breast tumor tissue induced by DMBA is shown in figure 2.
As shown, the brown color indicates a positive product estrogen receptor, expressed in the cytoplasm and nucleus of most tumor cells.
Example 2 acquisition of Estrogen receptor-Positive mouse Breast cancer cell lines
1. Experimental method
Taking part of tumor tissue in the estrogen receptor positive mouse breast cancer model established in example 1, and cutting the tissue into 2-3 mm 3 And (5) small blocks. DMBA-induced C57BL/6 mouse mammary tumor tissue blocks were implanted in the fourth pair of mammary fat pads on the left side of the first NSG mouse.
When the diameter of the breast tumor of the first NSG mouse reaches 1-1.5 cm, the tumor tissue is cut into 2-3 mm 3 Pieces, and tumor tissue pieces were implanted in the fourth left-hand pair of a second NSG mouseInside the mammary fat pad. When the breast tumor diameter of the second NSG mouse reached 1-1.5 cm, the tumor tissue was removed from the clean bench and minced, and after washing with PBS, 5mL of digestion solution (complete medium containing 10-15 mg/L collagenase type I and 10-15 mg/L collagenase type VI) was added and digested at 37 ℃ for 1 hour.
After digestion, the suspension was filtered through a sieve of not more than 100 μm, and the filtrate was collected in a 50mL centrifuge tube, centrifuged at 1000r/min for 5 minutes in a centrifuge, and the supernatant was discarded.
Washing the centrifuged sediment for 2 times by PBS, centrifuging again at the centrifugation speed of 1000r/min, discarding the supernatant, resuspending the obtained cell sediment, and performing adherent culture in a complete culture medium (DMEM + FBS with the volume ratio of 10%).
After removing fibroblasts from the cells produced by adherent culture using trypsin, the remaining cells were continuously subcultured.
2. Results of the experiment
The morphology of estrogen receptor positive mouse breast cancer cells obtained by serial subculture is shown in fig. 3.
The primary cells obtained by adherent culture are mixed by tumor cells and fibroblasts. Subculturing the tumor cell to obtain ER + The mouse breast cancer cell line still proliferates and is active after passage of more than 40 generations to obtain ER + The mouse breast cancer cell strain DMMC-02 is preserved in the China center for type culture Collection in 2022, 3 months and 10 days, and the preservation number is CCTCC NO: c202268, deposit address: wuhan university in Wuhan city, hubei province.
Example 3 identification of Estrogen receptor-Positive mouse Breast cancer cell lines
1. Experimental methods
The cells obtained in example 2 by continuous subculture (DMMC-02) were seeded on a cell slide, and when the cell density was about 50%, the cell culture medium was aspirated, fixed with 4% by mass (g)/volume (ml) of paraformaldehyde for 10 minutes, and washed 3 times with PBS buffer for 5 minutes each. Adding PBS and 0.5% triton X100 to the cells, treating on ice for 10 minutes, washing with PBS buffer for 5 minutes for 3 times; after blocking the cells with 1% by volume Bovine Serum Albumin (BSA) for 1 hour, the blocking solution was aspirated.
Primary Antibody (Monoclonal Anti-cytokine, pan Antibody produced in mouse (sigma, C2931); ki 67 Monoclonal Antibody (eBioscience, 14-5698-80); ER polyclonal Antibody (proteintech, 21244-1-AP)) was added to the cell slide and incubated overnight at 4 ℃ and then washed 3 times with PBS buffer for 5 minutes each. Then, a fluorescent secondary antibody (Donkey anti-rb 488, donkey anti-mouse 555, donkey anti-rat 647) was added thereto, and after 1 hour of incubation at room temperature, the cells were washed 3 times with PBS buffer for 5 minutes each. Then, DAPI fluorescent dye was added thereto, and after incubation at room temperature for 15 minutes, mounting was performed under a mirror.
Continuously subculturing to obtain estrogen receptor positive mouse breast cancer cell (3 × 10) 6 ) And (3) planting the tumor in a fourth pair of mammary fat pads of a C57BL/6 mouse, observing the shape of tumor tissues after the mouse becomes tumor, taking a part of tumor tissue embedded sections, and carrying out HE (high intensity eosin) staining observation and immunofluorescence staining detection on the tumor tissues.
2. Results of the experiment
The immunofluorescence detection results of the estrogen receptor positive mouse breast cancer cells obtained by continuous subculture are shown in fig. 4. The tumor morphology after tumorigenesis of estrogen receptor positive mouse breast cancer cells obtained by continuous subculture is shown in fig. 5. The HE staining pattern of tumor tissue after tumorigenesis of estrogen receptor positive mouse breast cancer cells obtained by continuous subculture is shown in FIG. 6. The results of immunofluorescence staining examination of tumor tissue after tumorigenesis of estrogen receptor positive mouse breast cancer cells obtained by serial subculture are shown in fig. 7.
After the estrogen receptor positive mouse breast cancer cells obtained by continuous subculture are planted in a mouse mammary gland fat pad, the tumor is formed in about 2 months, and the growth speed of the mouse transplanted tumor is medium.
In summary, by combining the embodiment 1 and the embodiment 2 of the present invention with the embodiment 3 of the present invention, it can be seen that the present invention successfully establishes a preparation method of an estrogen receptor positive mouse breast cancer cell line, and at the same time establishes an estrogen receptor positive mouse breast cancer model, and the experimental method of the present invention can stably obtain an estrogen receptor positive mouse breast cancer cell line, thereby solving the problem of limited tumor immunity research caused by the lack of functional immune cells in the immunodeficiency mouse model and the complicated establishment method, low reconstruction rate and relatively low use frequency of the humanized mouse model. The estrogen receptor positive mouse breast cancer cell strain obtained by the invention can fill the blank of an estrogen receptor-alpha positive preclinical cell model and an animal model, and is beneficial to the research on estrogen receptor-related tumor immunity.
Comparative example 1 isolation of Primary cells from MPA/DMBA-induced C57BL/6 mouse tumors for culture
1. Experimental methods
The establishment of the estrogen receptor positive mouse breast cancer model is consistent with example 1.
When the diameter of the breast tumor of the mouse induced from the C57BL/6 mouse reaches 1-1.5 cm, the tumor tissue is taken out of the super clean bench and cut into pieces, and after being washed with PBS, 5mL of digestive juice (complete medium containing 10-15 mg/L of collagenase type I and 10-15 mg/L of collagenase type VI) is added and digested for 1 hour at 37 ℃.
After digestion, the suspension was filtered through a sieve of not more than 100 μm, and the filtrate was collected in a 50mL centrifuge tube, centrifuged at 1000r/min for 5 minutes in a centrifuge, and the supernatant was discarded.
After washing the centrifuged pellet 2 times with PBS, the pellet was centrifuged again at a centrifugation speed of 1000r/min and the supernatant was discarded, and the obtained cell pellet was resuspended and then subjected to adherent culture in complete medium (DMEM medium + FBS at a volume ratio of 10%).
And removing fibroblasts in the cells obtained by adherent culture by using pancreatin, and carrying out continuous subculture on the rest cells.
2. Results of the experiment
The primary cells obtained by adherent culture are mixed by tumor cells and fibroblasts. The proliferation ability of the tumor cells is weak, and a monoclonal cell population cannot be established. When the tumor cells are used for continuous subculture, the cultured tumor cells tend to age after 2-3 passages (fig. 8), and the cells are basically fibroblasts after multiple passages (fig. 9).
It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Claims (3)
1. A mouse breast cancer cell strain with positive estrogen receptor is characterized in that the mouse breast cancer cell strain is ER + The mouse breast cancer cell strain DMMC-02 is preserved in the China center for type culture Collection in 2022, 3 months and 10 days, and the preservation number is CCTCC NO: c202268, deposit address: wuhan university in Wuhan city, hubei province.
2. The use of the mouse breast cancer cell line of claim 1 as an estrogen receptor positive mouse breast cancer cell line.
3. The use of the mouse breast cancer cell line of claim 1 in screening drugs for treating estrogen receptor- α positive breast cancer.
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