CN117660349A - Human basal-type breast cancer multi-drug resistant cell strain established by docetaxel induction and construction method and application thereof - Google Patents
Human basal-type breast cancer multi-drug resistant cell strain established by docetaxel induction and construction method and application thereof Download PDFInfo
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Abstract
The invention discloses a docetaxel induced and established human basal type breast cancer multidrug resistance cell strain, a construction method and application thereof, which utilizes a human breast cancer cell strain MDA-MB-231 and combines two means of docetaxel in-vitro and in-vivo induction to obtain a human breast cancer cell strain DXT-231 with strong docetaxel resistance in-vitro and in-vivo, the cell strain shows strong in-vitro and in-vivo drug resistance to docetaxel, the drug resistance index is 29.64 times, and obvious cross drug resistance is generated to paclitaxel, epirubicin, etoposide, cisplatin and carboplatin. Thus, drug resistant tumor cell models were provided for the following related studies: research on biological characteristics of drug-resistant tumor cells, research on tumor multi-drug resistance mechanism, analysis on sensitivity of chemotherapeutic drugs, screening of chemotherapeutic drugs, development of tumor drug resistance reversal drugs, development of more effective tumor treatment methods, and the like.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a human basal-type breast cancer multi-drug resistant cell strain established by docetaxel induction, and a construction method and application thereof.
Background
Basal-like breast cancers (Basal-like breast cancers, BLBCs) typically do not express estrogen receptors or progestin receptors, and also lack HER2 expression, but express Basal cytokeratins (KRT 5 and KRT 6). Unfortunately, the general lack of hormones and HER2 receptors makes this breast cancer subtype unsuitable for endocrine drugs and HER 2-targeted therapies, such as tamoxifen, aromatase inhibitors, and trastuzumab. BLBCs account for 15-20% of all breast cancer cases, have high BRCA1 mutation rate, poor pathological grading, high incidence of brain and/or bone metastasis, short survival time, poor sensitivity to standard chemotherapeutic drugs, easy recurrence, and lack of individualized, targeted therapeutic options.
NCCN guidelines recommend Docetaxel (DTX) for use in adjuvant chemotherapy of BLBCs, neoadjuvant chemotherapy, and treatment of metastatic breast cancer. However, the clinical application of DTX is severely restricted due to the presence of natural resistance and the development of acquired resistance. The known drug resistance mechanism of docetaxel for breast cancer mainly comprises the following steps: tumor cells can enhance drug efflux by upregulating ABC transporter (e.g., P-glycinate) and reduce accumulation of docetaxel. 2. Microtubule dynamic change: docetaxel prevents cell division by stabilizing microtubules. Drug resistant cells may alter the expression or function of tubulin such that the drug does not function effectively. 3. Apoptosis pathway modulation: drug resistant cells may evade drug-induced cell death by modulating apoptotic pathways (e.g., changes in Bcl-2 family proteins). 4. Enhancement of DNA repair ability: enhanced repair of chemotherapy-induced DNA damage is also a common drug resistance mechanism. 5. Epithelial-mesenchymal transition (EMT): in connection with the development of drug resistance, cells acquire more basic, stem cell-like properties. Countermeasures and research directions for drug resistance: 1. predictive markers: biomarkers that can predict docetaxel resistance are studied and identified in order to more accurately select patient treatment regimens. 2. New drugs and treatment strategies: development of new chemotherapeutic or targeted drugs, as well as immunotherapy, especially against molecular markers specific for BLBCs. 3. Pharmaceutical combination: docetaxel is used in combination with other chemotherapeutic drugs or emerging targeted drugs to overcome drug resistance. 4. Nano-drug delivery system: novel drug delivery systems, such as nanoparticles, have been developed to increase drug concentration at tumor sites, reduce side effects, and overcome drug resistance. 5. Personalized treatment: personalized therapeutic strategies based on genomic, transcriptomic, and proteomic data take into account the genetic background of the patient and the molecular characteristics of the tumor. Current and future research is directed to reveal detailed molecular mechanisms of BLBCs resistance to docetaxel and to explore new therapeutic strategies to improve therapeutic efficacy and survival of patients. Establishment of drug resistant cell lines is a cornerstone for studying drug resistance, which is typically accomplished by long-term exposure of BLBCs cells to increasing concentrations of docetaxel. These drug resistant cell models are capable of: providing a continuous, reproducible experimental system to study and identify the molecular mechanisms of drug resistance; for verifying the effects of drug resistance-related genes and proteins; the effect of the new drug or drug combination on overcoming or reversing resistance is tested. Therefore, the establishment of the basal-type breast cancer drug-resistant cell line of docetaxel can provide necessary research models for the related research of tumor drug resistance, and has practical value.
MDA-MB-231 cells are breast cancer cell lines which are isolated and established from hydrothorax of a 51-year-old female breast cancer patient, and lack ER, PR, HER-2 expression, so that the MDA-MB-231 cells are cell lines which are very commonly used for researching basal-type breast cancer. The drug-resistant cell strain of MDA-MB-231 cells for docetaxel is established by a combined in vitro and in vivo induction method for the first time, the drug-resistant cell period established by the method is obviously shortened, and the cell growth speed is high and the drug resistance is stable through participation of an in vivo microenvironment.
The Chinese patent ZL201410183516.4 proposes: the establishment of tumor drug-resistant cell lines generally comprises two methods, namely a gradual increase of drug concentration and continuous induction method and a large-dose impact intermittent administration method. The two methods are compared by research at home and abroad, and it is considered that different induction modes can obtain drug-resistant cell strains with different drug resistance mechanisms, and meanwhile, the drug resistance degree of tumor cells can be influenced, and the continuous induction is easier to generate drug resistance than the impact induction. However, the drug-resistant cells induced by the in vitro continuous induction method need to be maintained in a specific concentration of drug at regular intervals, otherwise, the drug-resistant property is easy to lose. The process of inducing drug resistant cell lines in vivo typically involves implantation of tumor cells into an animal model (typically a mouse) and periodic administration of a chemotherapeutic agent. The method can better simulate the natural environment of human tumor in organism and the development process of drug resistance to a certain extent, and the drug-resistant cell strain is characterized in that: 1. the drug resistance is stronger: in vivo models may result in greater resistance than in vitro models due to the inclusion of more complex biological processes such as tumor microenvironment, blood supply, immune system interactions, etc. Cells not only develop resistance to initial drug administration, but may also exhibit cross-resistance to multiple drugs. 2. Drug resistance is more stable: after continuous drug selection pressure in animals, surviving tumor cells are often genetically altered to stabilize resistance. This means that these cells retain their resistance even after a period of incubation without drug stress. 3. The mechanism of drug resistance may be closer to human tumors: due to the influence of systemic metabolism and tumor microenvironment, the in vivo method-induced drug resistant cells may be more similar to the drug resistance of human tumors after chemotherapy in drug resistance mechanism. 4. Better models are used to predict clinical effects: because of their high biological relevance, in vivo induced drug-resistant cell lines are often considered as better models for predicting the effects of chemotherapeutic drugs in humans. However, it should be noted that the drug-resistant cell lines established by in vivo methods have limitations: 1. high cost and resource consumption: requiring animal facilities, more time and high costs. 2. Ethical issues: animal experiments need to follow strict ethical guidelines to ensure animal welfare. 3. The technical requirements are high: in vivo experiments generally require a higher level of skill and expertise than in vitro experiments. 4. Individual differences: individual differences exist in animal models, which may affect the reproducibility and generalizability of experimental results. Because of the characteristics of in vivo induced drug resistant cell lines in terms of drug resistance, researchers often use this model to study the biological basis of drug resistance and find possible drug resistance reversal strategies. Meanwhile, in order to ensure the accuracy and reliability of the results, researchers have also intensively explored drug resistance mechanisms and verify in-vivo experimental results in combination with in-vitro experiments. Therefore, the invention mainly induces the generation of drug-resistant cells by a combined method in vitro and in vivo, and verifies the multi-drug resistance property in vitro.
Disclosure of Invention
The invention aims to provide a human basal-type breast cancer multi-drug resistant cell strain established by docetaxel induction, and a preparation method and application thereof.
The technical scheme adopted by the invention is as follows: the invention provides a human basal-model breast cancer multidrug resistance cell strain DXT-231 established by docetaxel induction, which has the preservation number of: cctccc NO: C2022302.
culture name (classification designation): docetaxel-resistant human basal-like breast cancer cell line DXT-231;
preservation date: 2022, 9, 28;
preservation unit: china center for type culture collection (China Center for Type Culture Collection);
the preservation unit is abbreviated as: cctccc;
deposit unit address: university of martial arts in chinese;
preservation number: CCTCC NO: C2022302.
The invention provides a construction method of a breast cancer multi-drug resistant cell strain, which comprises the following steps:
(1) Culturing human breast cancer MDA-MB-231 cell strain to logarithmic phase, adding docetaxel into the culture medium to make its action concentration be 0.05 μg/ml, continuously culturing with DMEM culture solution containing 10% special-grade fetal calf serum at 37deg.C, 5% C02 and 95% humidity for 24 hr, and then changing into fresh culture solution to logarithmic phase;
(2) The concentration of docetaxel was suitably increased in 6 times, and the effective concentration of docetaxel was maintained for a period of time after each increase until cells were stably grown and passaged in the concentration until docetaxel-resistant MDA-MB-231 cells (DXT-1) were obtained which were stably grown, passaged, frozen and resuscitated in a culture system containing 1. Mu.g/ml docetaxel.
(3) The resulting docetaxel-resistant MDA-MB-231 (DXT-1) cells were injected into breast fat pads of female BALB/c mice. Docetaxel treatment is carried out for 2-4 times after the growth of the tumor is stabilized, 5mg/kg of docetaxel is added each time, after 1 month, primary tumor cells are separated and placed in a culture flask, and a culture medium with the concentration of 8ug/ml of docetaxel is added for continuous culture and passage for 1 month;
(4) And (3) repeatedly carrying out the treatment of the step (3) on the cells obtained in the step (3) to obtain the docetaxel-resistant human basal type breast cancer cell strain DXT-231 which can stably grow, passaging, freezing and reviving.
Preferably, the suitable increasing concentration of docetaxel is by using 6 concentration gradients of 0.05 μg/ml, 0.1 μg/ml, 0.2 μg/ml, 0.4 μg/ml, 0.8 μg/ml, 1 μg/ml. (initial docetaxel action concentration was 1/100 of docetaxel versus MDA-MB-231 cell IC50, subsequent fold increases in concentration)
The invention also provides application of the multi-drug resistant cell strain in resisting docetaxel, paclitaxel, epirubicin, etoposide, cisplatin and carboplatin.
Determination of the degree of tumor cell resistance is often expressed as a Resistance Index (RI), which is the ratio of the half-maximal inhibitory concentration (50%inhibitory concentration,IC50) of the resistant cells to the half-maximal inhibitory concentration of the parent cells. Snow et al classify the resistance into low (< 5), moderate (5-15) and high (> 15) according to the level of the resistance index.
Preferably, the multidrug resistant cell line has an index of docetaxel resistance of 29.64 times, paclitaxel resistance of 7.95 times, epirubicin resistance of 5.38 times, etoposide resistance of 4.83 times, cisplatin resistance of 4.9 times, and carboplatin resistance of 6.3 times.
The invention establishes a human basal-type breast cancer drug-resistant strain DXT-231 and provides a drug-resistant tumor cell model for the following related researches: research on biological characteristics of drug-resistant tumor cells, research on tumor multi-drug resistance mechanism, analysis on sensitivity of chemotherapeutic drugs, screening of chemotherapeutic drugs, development of tumor drug resistance reversal drugs, development of more effective tumor treatment methods, and the like.
According to the invention, biological characteristics of the breast cancer multi-drug resistant strain DXT-231 are evaluated through cell morphology, growth curve, drug sensitivity test and protein levels of ABC family transporter members MRP1, P-gp and BCRP, and as a result, the DXT-231 drug resistant strain is successfully established, the drug resistance index is 29.64 times, and obvious cross drug resistance is generated to paclitaxel, epirubicin, etoposide, cisplatin and carboplatin.
Drawings
The invention is further illustrated in the following description in conjunction with the accompanying drawings and detailed description.
FIG. 1 is a view of drug resistant cell DXT-231 and parent cell MDA-MB-231 under an inverted phase contrast microscope;
FIG. 2 is a graph showing the growth of drug resistant cells DXT-231, DXT-1 and parent cell MDA-MB-231;
FIG. 3 is survival of drug resistant cells DXT-231, DXT-1 and parent cells MDA-MB-231 in docetaxel, paclitaxel, epirubicin, etoposide, cisplatin and carboplatin media;
FIG. 4 shows Western immunoblotting to detect levels of MRP1, P-gp, BCRP, GAPDH protein, a member of the ABC family of transporter in MDA-MB-231 and DXT-231.
Detailed Description
The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent websites.
Example 1
Induction of docetaxel-resistant human basal-like breast cancer cell line DXT-231.
The invention establishes a human breast cancer multidrug resistance cell strain DXT-231 by adopting an in-vitro induction method of gradually increasing the concentration and intermittent action of docetaxel and an in-vivo induction method of periodically administering docetaxel by combining tumor cells implanted into mice. The method comprises the following specific steps:
1. the human breast cancer MDA-MB-231 cell strain is cultured by DMEM culture solution (containing 10% calf serum, penicillin 100U/ml and streptomycin 100 μg/ml) under the conditions of 37 ℃,5% C02 and 95% humidity;
2. when culturing to 70% -90% adherence rate, discarding supernatant, adding docetaxel to make its action concentration be 0.05mg/l, continuously culturing at 37deg.C under 5% C02 and 95% humidity for 24 hr, changing fresh culture solution, continuously culturing, and making cell stably grow for 2 times after about 4-6 weeks;
3. the concentration of docetaxel is properly increased, the docetaxel is continuously cultured at 37 ℃ under the conditions of 5% C02 and 95% humidity, fresh culture solution is changed after 24 hours, the docetaxel is continuously cultured, and cells stably grow and are passaged for 2 times after about 4-6 weeks;
4. repeating the step 3 until obtaining DXT-1 cell strain which can stably grow, passaged and revived in 1mg/l docetaxel;
5. the resulting docetaxel-resistant MDA-MB-231 (DXT-1) cells were injected into breast fat pads of female BALB/c mice. Docetaxel treatment is carried out for 2-4 times after the growth of the tumor is stabilized, 5mg/kg of docetaxel is added each time, after 1 month, primary tumor cells are separated and placed in a culture flask, and a culture medium with the concentration of 8ug/ml of docetaxel is added for continuous culture and passage for 1 month;
6. and (3) repeating the step (5) on the cells obtained in the step (5), so as to obtain the docetaxel-resistant human basal type breast cancer cell strain DXT-231 with stable growth, passage, freezing and resuscitation.
In the induction process, it is very important to grasp the proper concentration of docetaxel, and it is ideal that a few drug-resistant cells are screened out by increasing the drug concentration and all cells are not dead, so that MDA-MB-231 cells with higher drug resistance are continuously obtained. In the invention, the concentration gradients of 6 (initial docetaxel action concentration is 1/100 of the IC50 of docetaxel to MDA, the subsequent multiple increase concentration) of 0.05 mug/ml, 0.1 mug/ml, 0.2 mug/ml, 0.4 mug/ml, 0.8 mug/ml and 1 mug/ml are adopted, so that a better effect is obtained, and DXT-1 cells are established in 6 months. The obtained DXT-1 cells were injected into mammary fat pads of female BALB/c mice. Docetaxel treatment is carried out for 2-4 times after the tumor growth is stabilized, 5mg/kg is added each time, after 1 month, primary tumor cells are separated and placed in a culture flask, and culture medium with the concentration of 8ug/ml of docetaxel is added for continuous culture and passage for 1 month. And repeating the above steps. Obtaining a docetaxel-resistant human basal-type breast cancer cell strain DXT-231, and preserving the drug-resistant strain with the preservation number: CCTCC NO: C2022302.
Isolation and extraction of primary breast tumor cells:
1. sterile dishes were prepared, poured into 5ml of HBF (HBSS+2% FBS) and placed on ice. The skin is cut off by using sterile scissors, the tumor tissue is separated in a blunt-end sterile scissors, and after the skin is fixed, the photo is taken.
2. The tumors were carefully isolated, removed, photographed, and the tumor size measured with vernier calipers, and tumor tissue was removed from necrotic tissue and placed on a sterile petri dish containing 5ml HBF.
3. A50 ml centrifuge tube was placed in PBS for irrigating the scalpel, the dish was covered with tape, and a 15ml centrifuge tube was used to hold the sample.
4. The left and right hand knives cut up the tissue rapidly, 3ml of digestive fluid washes the blade, resuspended the tissue therein, and the tissue suspension is transferred into a 15ml centrifuge tube, removing adipose tissue as much as possible. The centrifuge tube was wrapped with plastic film and shaken in an oven at 37℃for 2 hours.
5. The sample was removed from the oven, centrifuged at 450g for 5 minutes, the supernatant discarded and 8ml of HBF: NH was added 4 Cl broth, 450g was centrifuged for 5 min and the supernatant discarded.
6. 8ml of 0.25% pancreatin was added, the mixture was blown up and down 30 times, the suspension was transferred to a 50ml centrifuge tube, 16ml of ice HBF was added, and the mixture was centrifuged at 450g for 5 minutes, and the supernatant was discarded.
7. To each sample, 2ml of dispese and 200. Mu.l of DNase were added, and 10ml of ice HBF was added and mixed up and down with stirring.
8. Soaking the filter membrane with HBF, filtering the above tissue suspension into 50ml centrifuge tube, counting cells, and performing flow cytometryThe number of cells per sample was 1X 10 6 And each. Freezing residual cells, wherein the number of cells in each freezing tube is 1×10 7 And each.
After cells were stably resistant to 0.05. Mu.g/ml, 0.1. Mu.g/ml, 0.2. Mu.g/ml, 0.4. Mu.g/ml, 0.8. Mu.g/ml, 1. Mu.g/ml docetaxel in this order, the concentration of resistant cells was frozen in liquid nitrogen in time. The frozen stock solution consists of 20% calf serum, 10% DMES 0 and 70% DMEM culture solution, the temperature should be gradually reduced during the frozen stock process, and the sequence of overnight-liquid nitrogen at 4 ℃ for 30 minutes-20 ℃ for 1 hour-80 ℃ is adopted. Resuscitatory of cryopreserved cells was performed as follows: at a distance of 25cm 2 At least 10ml of a culture medium containing 10% calf serum is added to the cell culture flask; taking out the frozen storage tube filled with cells from liquid nitrogen, immediately placing the frozen storage tube into a water bath at 37 ℃ to gently shake to enable frozen storage objects to defrost within 1 minute, wiping the outer wall of the frozen storage tube by using an alcohol cotton ball, and then taking the frozen storage tube into an ultra-clean bench; transferring the thawed cell suspension into culture flask with culture solution, slightly shaking, mixing, unscrewing bottle cap, placing into carbon dioxide incubator, and placing at 37deg.C and 5% C0 2 Culturing under 95% humidity for about 24 hr, and changing fresh culture solution into 3-5ml for continuous culture.
Experimental example 1
Morphological observation and biological characterization morphological observation were performed on cell lines of DXT-231 established using the method of the invention:
observing the morphology of the living cells by an inverted phase contrast microscope: taking MDA-MB-231 and DXT-231 cells in logarithmic growth phase in one bottle each, changing liquid, observing the form of living cells under an inverted phase contrast microscope, and photographing. The MDA-MB-231 cells are polygonal, have more consistent forms and have uniform intracellular structures; the size and the morphology of the DXT-231 cells are changed, the cells are more slender, and more dark substances are gathered in the cells, especially near the cell membranes; after treatment with 8mg/L docetaxel, MDA-MB-231 cells were wrinkled into clusters, the adherence and the expansion were severely affected, but DXT-231 cells were not significantly changed, and the results are shown in FIG. 1, and the cell morphology was observed by an inverted phase contrast microscope in FIG. 1: the parent cell MDA-MB-231 and drug resistant cell DXT-231 were incubated with a culture medium with or without docetaxel, and the live cell morphology was observed and photographed under an inverted phase contrast microscope (bar=40 μm).
Experimental example 2
Cell growth curve (MTT method):
cell digestion, counting and concentration preparation of 1X 10 3 Per ml of cell suspension, 100 μl of cell suspension (500 cells per well) was added per well in a 96-well cell culture plate; 96-well cell culture plates were placed at 37℃and 5% C0 2 Culturing 1day, 2day, 3day, 4day, 5day, 6day, and 7day in incubator respectively; MTT staining was performed on 96-well plates, 20 μLMTT (5 mg/mL) was added to each well, and culture was continued in an incubator for 4 hours; the culture medium is discarded, 150 mu of LDMSO is added into each hole for dissolution, and the mixture is gently mixed by a shaking table for 10 minutes; lambda=490 nm, the OD value of each well is read by an enzyme-labeled instrument, and the cell growth curve is drawn by taking time as the abscissa and the OD value as the ordinate. Cell growth curves for MDA-MB-231, DXT-1 and DXT-231 (n=3, ANOVA, # p)<0.05 drug resistant cells DXT-231vs parent cells MDA-MB-231, #p<0.05 drug-resistant cells DXT-231vs. drug-resistant cells DXT-1) see FIG. 2, the growth rate of cells is continuously increased, the growth rate of cells of the drug-resistant strain is obviously lower than that of the parent cells from day 3, the growth curves of the two drug-resistant cells DXT-1 and DXT-231 are separated from each other from day 4, and DXT-1 is obviously slower than DXT-231 cells until day 7. FIG. 2 shows the MTT assay for detecting the growth rate of cells: parent cell MDA-MB-231 and drug-resistant cells DXT-1 and DXT-231 are cultured in a culture solution without docetaxel for 7 days, OD value is detected by MTT method, and cell growth curve is drawn (n=3, ANOVA, p)<0.05 drug resistant cells DXT-231vs parent cells MDA-MB-231, #p<0.05 drug resistant cells DXT-231vs. drug resistant cells DXT-1).
Experimental example 3
Drug sensitivity test:
cell digestion, counting and concentration preparation of 1X 10 5 Per ml of cell suspension, 100. Mu.l of cell suspension per well (1X 10 per well) 4 Individual cells); 96-well cell culture plates were placed at 37℃and 5% C0 2 Culturing in an incubator for 24 hours; diluting the drug to the desired concentration with complete medium, changing 200 μl of the corresponding drug-containing medium into each well, placing 96-well cell culture plate at 37deg.C, 5% C0 2 Culturing in an incubator for 24 hours; 96-well plates were subjected toMTT staining, λ=490 nm, OD value was determined.
As shown in FIG. 3, the survival rates of the parent cell MDA-MB-231 and the drug-resistant cells DXT-1, DXT-231 in the docetaxel, paclitaxel, epirubicin, etoposide, cisplatin and carboplatin containing medium were examined (n=3, ANOVA, <0.05 drug-resistant cells DXT-231vs. parent cell MDA-MB-231, #p <0.05 drug-resistant cells DXT-231vs. drug-resistant cells DXT-1), respectively, the survival rates of drug-resistant cells DXT-1, DXT-231 in the six drug containing medium were higher than the parent cell, wherein the survival rate of drug-resistant cells DXT-231 was the highest as shown in Table 1, the IC50 of the drug resistant cells DXT-1 and DXT-231 are obviously higher than that of the parent cell MDA-MB-231, wherein the IC50 of the drug resistant cell DXT-231 is highest, which indicates that the drug resistant cell DXT-231 has stronger drug resistance than DXT-1, and has different degrees of cross tolerance to taxol, epirubicin, etoposide, cisplatin and carboplatin, the index of docetaxel resistance is 29.64 times, the index of taxol resistance is 7.95 times, the index of epirubicin resistance is 5.38 times, the index of etoposide resistance is 4.83 times, the index of cisplatin resistance is 4.9 times and the index of carboplatin resistance is 6.3 times.
Table 1: MTT method for detecting half inhibition concentration (IC 50) of docetaxel, paclitaxel, epirubicin, etoposide, cisplatin and carboplatin on drug-resistant cells DXT-231, and half inhibition concentration (IC 50) of DXT-1 and parent cells MDA-MB-231 in 72 hours
Experimental example 4
Protein immunoblotting is carried out according to the subject group protocol, firstly, protein quantification is carried out on parent cells MDA-MB-231 and drug-resistant cells DXT-1 and DXT-231 by using coomassie brilliant blue, a standard curve is drawn, the loading quantity and loading volume of each group are calculated, and SDS-PAGE electrophoresis, membrane transfer (wet transfer), BSA blocking, primary antibody incubation (MRP 1, P-gp and BCRP primary antibody action concentration is 1:500), secondary antibody incubation, ECL imaging, darkroom exposure and strip scanning are sequentially carried out.
As shown in FIG. 4, the protein levels of ABC family transporter members MRP1, P-gp and BCRP in parent cells MDA-MB-231 and drug-resistant cells DXT-1, DXT-231 respectively were detected by Western immunoblotting, and the protein levels of ABC family transporter members MRP1, P-gp and BCRP in drug-resistant cells DXT-1, DXT-231 were significantly higher than those in parent cells MDA-MB-231, with the protein levels of MRP1, P-gp and BCRP in drug-resistant cells DXT-231 being the highest and significantly higher than those in three of drug-resistant cells DXT-1.
The invention provides a breast cancer multidrug resistance cell strain DXT-231 established by docetaxel induction, which has the preservation number of: CCTCC NO: C2022302. The invention also provides a construction method thereof, namely, a human breast cancer cell strain DXT-231 with strong in-vitro and in-vivo docetaxel drug resistance is obtained by combining two means of in-vitro and in-vivo induction of the human breast cancer cell strain MDA-MB-231, the cell strain not only maintains the basal cell-like characteristics of the parent cell strain, but also shows strong in-vitro and in-vivo drug resistance to the docetaxel, the drug resistance index is 29.64 times, and obvious cross drug resistance is generated to paclitaxel, epirubicin, etoposide, cisplatin and carboplatin.
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. Docetaxel induces an established human basal-type breast cancer multidrug resistance cell line, which is named as a docetaxel-resistant human basal-type breast cancer cell line DXT-231 with the preservation number of: cctccc NO: C2022302.
2. the method for constructing a multidrug resistant cell line according to claim 1, comprising the steps of:
(1) Culturing human breast cancer MDA-MB-231 cell strain to logarithmic phase, adding docetaxel into the culture medium to make its action concentration be 0.05 μg/ml, continuously culturing with DMEM culture solution containing 10% special-grade fetal calf serum at 37deg.C, 5% C02 and 95% humidity for 24 hr, and then changing into fresh culture solution to logarithmic phase;
(2) The concentration of docetaxel was appropriately increased 6 times, and the effective concentration of docetaxel was maintained for a period of time after each increase until cells were able to stably grow and passaged in the concentration until docetaxel-resistant MDA-MB-231 cells (DXT-1) were obtained that were able to stably grow, passaged, cryopreserved and resuscitated in a culture system containing 1. Mu.g/ml docetaxel;
(3) Injecting the obtained docetaxel resistant MDA-MB-231 (DXT-1) cells into a breast fat pad of a female BALB/c mouse; docetaxel treatment is carried out for 2-4 times after the growth of the tumor is stabilized, 5mg/kg of docetaxel is added each time, after 1 month, primary tumor cells are separated and placed in a culture flask, and a culture medium with the concentration of 8ug/ml of docetaxel is added for continuous culture and passage for 1 month;
(4) And (3) repeatedly carrying out the treatment of the step (3) on the cells obtained in the step (3) to obtain the docetaxel-resistant human basal type breast cancer cell strain DXT-231 which can stably grow, passaging, freezing and reviving.
3. The construction method according to claim 2, wherein: the appropriate increase in the concentration of docetaxel was achieved by using 6 concentration gradients of 0.05. Mu.g/ml, 0.1. Mu.g/ml, 0.2. Mu.g/ml, 0.4. Mu.g/ml, 0.8. Mu.g/ml, 1. Mu.g/ml.
4. The use of the multi-drug resistant cell line of claim 1 in docetaxel, paclitaxel, epirubicin, etoposide, cisplatin, carboplatin.
5. The use according to claim 4, characterized in that: the index of the multi-drug resistant cell strain for resisting docetaxel is 29.64 times, the index of resisting paclitaxel is 7.95 times, the index of resisting epirubicin is 5.28 times, the index of resisting etoposide is 4.83 times, the index of resisting cisplatin is 4.9 times, and the index of resisting carboplatin is 6.3 times.
6. The use of the multi-drug resistant cell line of claim 1 in screening and preparing tumor drug resistance reversal drugs.
7. The use of the multi-drug resistant cell line of claim 1 in the selection and preparation of a chemotherapeutic agent.
8. The use of the multi-drug resistant cell line of claim 1 in constructing a drug resistant tumor cell model.
9. Use of the multi-drug resistant cell line of claim 1 in the study of tumor multi-drug resistance mechanisms.
10. The use according to any one of claims 6 to 9, wherein the tumour is breast cancer.
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