CN104745531A - Method for establishing tumor multi-drug resistant cell mode and human breast cancer multi-drug resistant cell strain established by virtue of method - Google Patents
Method for establishing tumor multi-drug resistant cell mode and human breast cancer multi-drug resistant cell strain established by virtue of method Download PDFInfo
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Abstract
The invention belongs to the field of oncobiology and relates to a method of establishing a tumor multi-drug resistant cell mode in vitro. The method comprises the step of continuously culturing a sensitive tumor cell by taking ROS as an incubation drug to enable the sensitive tumor cell to generate multi-drug resistance. Moreover, the invention further relates to a human breast cancer multi-drug resistant cell strain established by virtue of the method. The cell strain is named MCF-7/ROS and preserved in CCTCC (China Center for Type Culture Collection), wherein the address is 430072, Wuhan University, Wuhan, China; the preservation date is 28th, January, 2015; the preservation number is CCTCC-C201520. The method provided by the invention is simple in process and stable in modeling condition, the modeling cost can be remarkably lowered, and the method has wide applicability. The MCF-7/ROS cell strain established by the method can serve as an advantageous experimental mode for researching related mechanism, target spot exploration and screening of new drugs.
Description
Technical field
The invention belongs to oncobiology field, be specifically related to a kind of method of reacting the outer tumor multi-medicine drug-resistant cell model of chalcogen single-unit modeling construct, also relate to the human breast carcinoma multidrug resistance cell strain set up by this method simultaneously.
Background technology
The outer tumor models of construct is the important experiment basis of research tumorigenesis and intervention, the selection of modeling condition and modeling method is the determinative of model construction, is the Key Platform disclosing similar swollen neoplastic molecular mechanism and the intervention of research tumour medicine.
Develop immunity to drugs after tumor multi-medicine drug-resistant (multidrug resistance, MDR) refers to tumour cell Who Contact Antitumor Drugs:, the multiple antitumor drug that also, mechanism different to structure is different produces the phenomenon of crossing drug resistant.MDR is the modal problem of clinical tumor chemotherapy, fails so far to solve, its reason or not clear with its core mechanism, cannot to carry out target Prevention and Curation relevant.Therefore, the cell model developing multidrug resistance of tumor for further its mechanism of research, explore its therapy target and screening, evaluation related drugs is significant.But the core mechanism that MDR produces not yet is illustrated, research shows: the expression of efflux protein and function raise, cell detoxification system vigor strengthens, antitumor target spot changes and apoptotic responses is lost the factor such as quick and connected each other, interacts, be the phenotypic characteristic of MDR cell, mediated again the formation of MDR.Therefore, find the central factor connecting and dominate above each mechanism, and utilize this factor design, build MDR cell model, not only contribute to the pathologic process illustrating MDR, more will explore for corresponding target spot, drug screening provides more reasonable, efficient evaluation object.
Because core mechanism is failed to understand, the main stream approach building MDR tumor models is both at home and abroad: certain antitumor drug directly selecting Clinical practice, to a kind of tumour cell long-term cultivation of chemosensitivity, obtains the drug-resistant cell strain of this cell to certain antitumour drug.MDR cell model constructed by the method is the main tool studying MDR association area now.The structure of this type of cell strain mainly comprises following several types: drug level incremental method, heavy dose of ballistic method, heavy dose of impact are in conjunction with increasing concen-trations method and transgenosis bound drug sieve method etc.This building mode can simulate the basic condition that clinical MDR produces to a certain extent, but still have many-sided not enough, comprise: (1) pattern layout is difficult to simulate the core mechanism of clinical MDR formation: clinical antineoplastic treatment often uses drug combination, therefore, only select the MDR model that a kind of medicine is set up, although its feature may possess the general character of some clinical MDR, but more can embody a series of individual characteies that this kind of medicine pharmacological action is relevant, the drug-resistant cell strain malignant phenotype obtained has obvious limitation, therefore with the mechanism that this model carries out for experimental subjects, target spot and drug research cannot represent the common situation of other situation MDR, even can ignore the central factor that multidrug resistance is formed, scientific research demand cannot be met.(2) shortcoming that experimental implementation is relevant: conventional construction method complex operation, complexity, toxigenic capacity is high, and the time that builds is general longer, and corresponding mortality is higher, and culture condition is not sought unity of standard, and gained cells resistance degree difference is large.(3) restriction of practicality aspect: MDR cell model is conventional, the necessary platform of research tumor multi-medicine drug-resistant mechanism and medicine, but the kind that conventional model is set up is very limited, commercially available conventional MDR cell strain kind is few, price is high, cannot meet individuation research needs.Secondly, because each modeling can only obtain a kind of drug-induced a kind of cell strain, and gained model only has higher resistance to this kind of medicine, and limited to other drug drug-resistant intensity, therefore, relatively large deviation will be there is with clinical MDR situation in the result being experimental subjects gained with it.In addition, the drug-resistant cell strain speed of growth that antitumor drug induction is set up is general comparatively slow, also greatly limit research and uses.In sum, find general character, core mechanism that resistance is formed, explore MDR model training method advantageously, will the research platform improving this area be contributed to, promote the development of related basic research and drug research.
Summary of the invention
Object of the present invention aims to provide a kind of to react chalcogen (reactive oxygen species, ROS) be the method for the outer tumor multi-medicine drug-resistant cell model of single induction factor construct, also relate to the human breast carcinoma multidrug resistance cell strain MCF-7/ROS set up according to this method simultaneously.
Based on above-mentioned purpose, this invention takes following technical scheme: a kind of method building tumor multi-medicine drug-resistant cell model, comprising with ROS is hatch medicine to carry out continuing to cultivate, make Sensitive Tumor Cells produce the step of multidrug resistance to Sensitive Tumor Cells.
Described ROS is H
2o
2.
Described Sensitive Tumor Cells is human breast cancer cell line Bcap-37.
The concrete steps of described method are:
(1) cell cultures: select certain to the tumor cell line of chemotherapy medicament sensitive, adjustment cell concn, grouping is cultivated for subsequent use;
(2) screening and culturing condition: each group cell uses different ROS concentration to carry out continuing to cultivate respectively, selects the highest ROS concentration as the optimum modeling concentration of this kind of resistant models after 4 weeks in survivaling cell group;
(3) build resistant models: this kind of tumour cell selecting logarithmic phase, adjustment cell concn, use the optimum modeling concentration of step (2) gained to continue culture of tumor cell, go down to posterity by growing state in culturing process;
(4) Cyclic culture of resistance tracking instruction: the evaluation of resistance multiple is carried out to the ROS culturing cell in step (3) every 2-3 week; The relating operation of repeated execution of steps (3), until the resistance that evaluation result prompting cell produces meets expection; Freeze-stored cell.
Step (3) ROS used is H
2o
2, concentration 0.1 μm of ol/L, incubation time is 20 weeks.
In described method, each step used medium is RPMI 1640 substratum containing 10% calf serum.
ROS is changed with replaced medium every other day in step (2) and step (3) culturing cell process.
According to the human breast carcinoma multidrug resistance cell strain of described method establishment, cell strain called after MCF-7/ROS, ROS used are H
2o
2, be deposited in China typical culture collection center, address: China, Wuhan, Wuhan University, 430072; Preservation date: on January 28th, 2015; Deposit number is CCTCC-C201520.
The present invention uses ROS to carry out single factor test modeling as modeling medicine first, directional induction cell long-term with constant dosage, the forming process of simulation clinical tumor resistance.Compared with prior art, this method has following technical superiority:
(1) experimentation is simple, and modeling conditional stability, can significantly reduce modeling cost.
(2) drug-resistant cell strain set up all has crossing drug resistant to multiple antitumor drug, has the advantage that cell proliferation is fast simultaneously, can contribute to the efficiency improving correlative study.
(3) there is suitability widely: experiment shows, be that the model that modeling medicine is set up has the multiple important phenotype similar to clinical multidrug resistance of tumor with ROS, comprise: multidrug-associated protein 1(multidrug resistance-associated protein 1, MRP1) and P-glycoprotein (P-glycoprotein, P-gp) express increase; With excessive activations such as closely-related nuclear factor Nrf2, HIF-1 α of resistance mechanism; Detoxification system represents enzyme system GST π and highly raises; Multidrug resistance specific function molecule PKC α, c-Myc express to be increased.
Meanwhile, contriver also finds in early-stage Study: in multiple commercially tumor drug resistance cell, ROS content is all higher than its sensitive strain, and wherein the ROS content of certain commercially human breast carcinoma mdr cell MCF-7/ADM is 2.77 ± 0.21 times of its sensitive strain.This shows, ROS may be the central factor that multidrug resistance is formed, and promotes that resistance produces by regulation and control P-gp or other functional moleculars.The present invention selects ROS modeling success, and for " ROS can induced tumor multidrug resistance formed " provides important evidence, meanwhile, its resistance mechanism also determines ROS inducing tumor cell and forms multidrug resistance and have broad applicability.In addition, test also confirms, except MCF-7, cultivates K562 cell with conditions of similarity, and it also can be induced to produce resistance.
According to the human breast carcinoma multidrug resistance cell strain MCF-7/ROS that modeling method provided by the invention is set up, 13.99,4.61,5.32,1.73,8.80 are reached respectively to the Resistance index of Zorubicin, taxol, cis-platinum, Fluracil and vincristine(VCR), there is multidrug resistance characteristic, can be research related mechanism, target spot explores and new medicament screen provides more rational experimental model.
Accompanying drawing explanation
Fig. 1 is that Zorubicin (ADM) and taxol (Taxol) are to the IC of each group of cell
50comparison diagram, in figure: * * represents
p< 0.01
vsblank; ## represents
p< 0.01
vs0.1 μm of ol/L Zorubicin group;
Fig. 2 is the microgram of the present inventor's mammary cancer multidrug resistance cell strain MCF-7/ROS;
Fig. 3 is the cell growth curve of MCF-7 and MCF-7/ROS;
Fig. 4 is the expression of MRP1 in MCF-7 cell and location, and wherein Fig. 4-a is DAPI core dye image (blueness) of MCF-7 cell, and Fig. 4-b is the MRP1 colored graph picture (green) of MCF-7 cell, and Fig. 4-c is DAPI and the MRP1 dyeing superimposed image of MCF-7 cell
Fig. 5 is the expression of MRP1 in MCF-7/ROS cell and location, wherein Fig. 5-a is DAPI core dye image (blueness) of MCF-7/ROS cell, Fig. 5-b is the MRP1 colored graph picture (green) of MCF-7/ROS cell, and Fig. 5-c is DAPI and the MRP1 dyeing superimposed image of MCF-7/ROS cell;
Fig. 6 is the expression of P-gp in MCF-7 cell and location, wherein Fig. 6-a is DAPI core dye image (blueness) of MCF-7/ cell, Fig. 6-b is the P-gp colored graph picture (green) of MCF-7 cell, and Fig. 6-c is DAPI and the P-gp dyeing superimposed image of MCF-7 cell;
Fig. 7 is the expression of P-gp in MCF-7/ROS cell and location, wherein Fig. 7-a is DAPI core dye image (blueness) of MCF-7/ROS cell, Fig. 7-b is the P-gp colored graph picture (green) of MCF-7/ROS cell, and Fig. 7-c is DAPI and the P-gp dyeing superimposed image of MCF-7/ROS cell;
Fig. 8 is the expression of HIF-1 α in MCF-7 cell and location; Wherein Fig. 8-a is DAPI core dye image (blueness) of MCF-7 cell, and Fig. 8-b is HIF-1 α colored graph picture (green) of MCF-7 cell, and Fig. 8-c is DAPI and the HIF-1 α dyeing superimposed image of MCF-7 cell;
Fig. 9 is the expression of HIF-1 α in MCF-7/ROS cell and location; Wherein Fig. 9-a is DAPI core dye image (blueness) of MCF-7/ROS cell, and Fig. 9-b is HIF-1 α colored graph picture (green) of MCF-7/ROS cell, and Fig. 9-c is DAPI and the HIF-1 α dyeing superimposed image of MCF-7/ROS cell;
Figure 10 is the expression of Nrf2 in MCF-7 cell and location, wherein Figure 10-a is DAPI core dye image (blueness) of MCF-7 cell, Figure 10-b is the Nrf2 colored graph picture (green) of MCF-7 cell, and Figure 10-c is DAPI and the Nrf2 dyeing superimposed image of MCF-7 cell;
Figure 11 is the expression of Nrf2 in MCF-7/ROS cell and location, wherein Figure 11-a is DAPI core dye image (blueness) of MCF-7/ROS cell, Figure 11-b is the Nrf2 colored graph picture (green) of MCF-7/ROS cell, and Figure 11-c is DAPI and the Nrf2 dyeing superimposed image of MCF-7/ROS cell;
Figure 12 is the Western blot detected result of GST π, PKC α, c-Myc.
Embodiment
The present invention will be further described in conjunction with specific embodiments in the present invention.
Embodiment
one, material
1, cell strain: MCF-7 cell strainHJ2mm, is purchased gained.
2, instrument, software and reagent:
XFS-280A type pressure steam sterilization boiler: Zhejiang Xinfeng Medical Apparatus Co., Ltd.;
PH counts, BT25S, BSA224S-CW electronic balance: Sartorious AG;
Magnetic stirring apparatus: Jiangsu Zhong great instrument plant;
Thermo Barnstead NANO pure DlamondUV/UF ultrapure water system, high-speed low temperature desk centrifuge, ThermoEC250,140,120 vertical electrophoresis apparatus and electric rotary device, the long microplate reader of Multiskan Spectrum all-wave, high-speed low temperature desk centrifuge: Thermo Scientific;
ZHJH-C1112 type Bechtop, HH-6 type thermostat water bath, CO2gas incubator: Shanghai Zhi Cheng analytical instrument Manufacturing Co., Ltd;
KQ-500Z type ultrasonic cleaner: Kunshan Ultrasonic Instruments Co., Ltd.;
TL-2000C type medical detection automatic rotation shaker: Tian Li Medical Devices Co., Ltd. of Jiangyan City;
Micro sample adding appliance: Eppendorf company;
Inverted biologic microscope: Ningbo ShunYu Instruments Co., Ltd;
Laser confocal microscope: Lycra company;
Flow cytometer: BD company;
Gel imaging system, Quantity One image processing software: Bio-Rad company;
SPSS13.0 statistical package: SPSS company;
MTT(bromination-(4,5)-dimethyl-2-thiazolyl-2,5-diphenyltetrazolium bromide), Zorubicin, taxol, cis-platinum, 5 FU 5 fluorouracil, vincristine sulphate, DMSO, DAPI, DCFH-DA:Sigma company;
Calf serum: Hangzhou Sijiqing Biological Engineering Material Co., Ltd.;
RPMI 1640 substratum: Gibco company;
Western blot related reagent and antibody: Wuhan doctor's moral Bioisystech Co., Ltd.
two, construction process
1, cell cultures: the MCF-7 cell strainHJ2mm in vegetative period of taking the logarithm, use RPMI 1640 substratum containing 10% calf serum, adjustment cell concn is 1-2.5 × 10
4individual/mL, obtained cell suspension 5mL are placed in 50mL glass culturing bottle, in 37 DEG C, 5% C0
2overnight incubation in incubator, makes its adherent growth, divides into groups for subsequent use.
2, screening and culturing condition: get 5 groups of cells from step (1) gained cell for subsequent use, uses RPMI 1640 substratum the H giving different concns that contain 10% calf serum
2o
2, continue cultivation 4 weeks; The H that each group of cell is corresponding
2o
2concentration is respectively 0.001 μm of ol/L, 0.01 μm of ol/L, 0.1 μm of ol/L, 1 μm of ol/L, 10 μm of ol/L.Fresh H is changed with replaced medium every other day in culturing process
2o
2.Inverted microscope Continuous Observation cell growth state, the H that the groups of cells of still surviving after 4 weeks is corresponding
2o
2concentration is 0.001 μm of ol/L, 0.01 μm of ol/L, 0.1 μm of ol/L, selects maximum concentration 0.1 μm of ol/L as the optimum modeling concentration of MCF-7 cells resistance model.
3, build resistant models: get 1 group of cell for subsequent use, use RPMI 1640 substratum+0.1 μm of ol/L H containing 10% calf serum
2o
2cultured continuously, takes advantage of the 0.1 μm of ol/L H giving fresh configuration when changing fresh culture every other day in culturing process
2o
2.When Growth of Cells goes down to posterity when about 80%-90% converges: tipping nutrient solution after jog shaking culture bottle, appropriate 2.5g/L trypsinase is added after PBS working fluid rinsing 2 times, under room temperature, digestive process observed by inverted microscope, treat that the cell of individual layer shrinks in flakes, cell becomes round gradually, the RPMI RPMI-1640 added containing 10% calf serum stops digestion, makes single cell suspension.Count under sampling mirror, adjustment cell concn is still 1-2.5 × 10
4individual/mL, often organizes obtained cell suspension 5mL and is placed in new 50mL glass culturing bottle, in 37 DEG C, 5% C0
2in incubator, overnight incubation is adherent, continues afterwards to carry out CMC model according to above-mentioned administering mode.
4, the Cyclic culture of resistance tracking instruction: carry out the evaluation of resistance multiple every 2-3 week to cultured cells, namely mtt assay detects cell inhibitory rate: take out some cells during detection from bottle, with 5 × 10
4individual/mL density is inoculated in 96 well culture plates, and every hole 180 μ L, adds the antitumour drug of various concentration, cultivates 48 hours, and every porocyte adds MTT solution 10 μ L (5 mg/mL), and centrifugal after continuing cultivation 4 h, abandon supernatant.Every hole adds 150 μ L dimethyl sulfoxide (DMSO), and 37 DEG C of concussions surveyed its OD value by microplate reader at 570nm wavelength place after 10 minutes, calculated cell inhibitory rate.When same medicine concentration drag group inhibiting rate is significantly lower than blank group inhibiting rate, tentatively judge that cell produces resistance.Before resistance reaches expection, modeling group needs the relating operation of repeated execution of steps 3 to continue CMC model.Cultivate freeze-stored cell after 20 weeks, be MCF-7/ROS cell.This cell strain is deposited in China typical culture collection center, and deposit number is CCTCC-C201520.
While execution above-mentioned steps 3 and step 4, separately get 5 groups of cells for subsequent use with reference to above-mentioned experiment condition and do parallel test, wherein one group is blank (groups of cells 1), uses the PBS of the RPMI 1640 substratum+same administration volume containing 10% calf serum; All the other are respectively organized and use the RPMI 1640 substratum+relative medicine containing 10% calf serum to carry out cultured continuously.What each group of cell was corresponding hatches medicine in table 1.
The each groups of cells of table 1 and correspondence thereof hatch medicine
three, detection method and result
Adopt the drug-resistance characteristics of following method evaluation model cell.
the resistance of method detection model cell:
Mtt assay detects each concentration antitumour drug to the inhibiting rate of each group of cell, calculates IC
50value, result is as shown in Fig. 1 and table 2.
As can be seen from Figure 1, the multidrug resistance of positive control (groups of cells 2) Zorubicin prolonged incubation induction, adds ROS remover GSH(groups of cells 3 in the process of hatching) significantly can reduce its corresponding IC
50concentration, confirms that ROS is really the important factor of multidrug resistance formation.
Fig. 1 also shows, uses 0.001,0.01 and 0.1 μm of ol/L H
2o
2(respectively corresponding groups of cells 4,5,6) cultured continuously 20 weeks gained cells to the resistance of Zorubicin and taxol all with added H
2o
2concentration is proportionate, and confirms H
2o
2optimum modeling concentration be really 0.1 μm of ol/L.
Known according to table 2, the Resistance index of MCF-7/ROS to Zorubicin, taxol, cis-platinum, 5 FU 5 fluorouracil, vincristine(VCR) is respectively 13.99,4.61,5.32,1.73,8.80, and instruction gained cell strain has the feature of tumor multi-medicine drug-resistant.
Table 2 cell strain is to the IC of each chemotherapeutics
50and Resistance index
3.2 persister morphological observations
By human breast carcinoma multidrug resistance cell strain MCF-7/ROS cell observation of cell form under inverted microscope of logarithmic phase, result as shown in Figure 2: cellular form becomes large, core shrinkage, organoid increases, cell is assembled in flakes, adherent growth.
cell growth curve measures
Through digestion, counting, get MCF-7 respectively, MCF-7/ROS cell makes single cell suspension, and adjusts cell density.Often kind of cell all with 5000/hole kind in 96 orifice plates, 6 multiple holes are set.Simultaneous vaccination 7 piece of 96 orifice plate, is placed in 37 DEG C, 5%CO
21d, 2d, 3d, 4d, 5 d, 6d, 7d are cultivated respectively in incubator; Mtt assay detects the OD value in every hole, take time as X-coordinate, OD value is ordinate zou, draws cell growth curve.Fig. 3 shows, the MCF-7/ROS speed of growth is better than MCF-7 cell.
laser confocal microscope detects expression and the location of MRP1, P-gp, HIF-1 α, Nrf2:
MCF-7 and the MCF-7/ROS cell that phase of taking the logarithm grows, with 1 × 10
4individual cell/ware is inoculated in glass bottom Tissue Culture Dish, in 37 DEG C, 5% C0
2overnight incubation in incubator, abandoning supernatant after cell attachment, PBS washes 3 times, often all over 5min.The paraformaldehyde room temperature adding appropriate 4% afterwards fixes 30 minutes.Discard 4% paraformaldehyde liquid, PBS washes 3 times, adds appropriate 0.l%TritonX-100 (pH 7.4) room temperature and punches 5 minutes.Discard 0.1%Tritonx-100 liquid, PBS washes 3 times, adds appropriate 5% bovine serum albumin 37 DEG C of closed 1-2 hour.Discard confining liquid, PBS washes 3 times, and add the primary antibodie that appropriate 1:400 dilutes, 4 DEG C are spent the night.PBS washes 3 times, and add the FITC mark that appropriate 1:100 dilutes two resist, and hatch 1-2 hour for 37 DEG C.PBS washes 3 times, adds appropriate 10ug/mL DAPI, hatches 10-20 minute for 37 DEG C, carries out scanning imagery after PBS washes 3 times under laser tool focusing microscope.Experimental result is as shown in Fig. 4-11.
As can be seen from the less expression drug efflux proteins of Fig. 4-7, MCF-7 cell, and MCF-7/ROS cell has the drug-resistance characteristics of MRP1, P-gp high expression level, and is mainly positioned on cytolemma, meets multidrug resistance cell clinical characteristics.And as can be seen from Fig. 8-11, upstream nuclear factor Nrf2, HIF-1 α that multidrug resistance is relevant is lower at MCF-7 cells, but in high expression level in MCF-7/ROS cell, and enrichment in nucleus, in state of activation.
detect the expression of GST π, PKC α, c-Myc:
Utilize Western blotting (Western blot) to detect GST π, PKC α, the c-Myc expression of MCF-7 and MCF-7/ROS cell respectively, experimental technique is as follows: often group collects about 1 × 10
7cell, after cracking, BCA protein determination protein content.Sample and equal-volume 2 × loading buffer mix, and 100 DEG C of sex change 5 min ,-20 DEG C frozen for subsequent use.Albumen to be detected selects separation gel by size, get equal protein loading, electrophoresis, transferring film, close, primary antibodie overnight incubation, TBST wash anti-to hatch for 3 times, two, TBST wash film 3 times, chemoluminescence, carry out imaging analysis with gel imaging system, result is as shown in figure 12.As can be seen from the figure, MCF-7/ROS cell has the multidrug resistance cell characteristic of GST π, PKC α, c-Myc high expression level.
Claims (8)
1. build a method for tumor multi-medicine drug-resistant cell model, it is characterized in that, comprising with ROS is hatch medicine to carry out continuing to cultivate, make Sensitive Tumor Cells produce the step of multidrug resistance to Sensitive Tumor Cells.
2. build the method for tumor multi-medicine drug-resistant cell model as claimed in claim 1, it is characterized in that, described ROS is H
2o
2.
3. build the method for tumor multi-medicine drug-resistant cell model as claimed in claim 1, it is characterized in that, described Sensitive Tumor Cells is human breast cancer cell line Bcap-37.
4. build the method for tumor multi-medicine drug-resistant cell model as described in as arbitrary in claim 1-3, it is characterized in that, the concrete steps of described method are:
(1) cell cultures: select certain to the tumor cell line of chemotherapy medicament sensitive, adjustment cell concn, grouping is cultivated for subsequent use;
(2) screening and culturing condition: each group cell uses different ROS concentration to carry out continuing to cultivate respectively, selects the highest ROS concentration as the optimum modeling concentration of this kind of resistant models after 4 weeks in survivaling cell group;
(3) build resistant models: this kind of tumour cell selecting logarithmic phase, adjustment cell concn, use the optimum modeling concentration of step (2) gained to continue culture of tumor cell, go down to posterity by growing state in culturing process;
(4) Cyclic culture of resistance tracking instruction: the evaluation of resistance multiple is carried out to the ROS culturing cell in step (3) every 2-3 week; The relating operation of repeated execution of steps (3), until the resistance that evaluation result prompting cell produces meets expection; Freeze-stored cell.
5. build the method for tumor multi-medicine drug-resistant cell model as claimed in claim 4, it is characterized in that, step (3) ROS used is H
2o
2, concentration 0.1 μm of ol/L, incubation time is 20 weeks.
6. build the method for tumor multi-medicine drug-resistant cell model as claimed in claim 5, it is characterized in that, each step used medium is RPMI 1640 substratum containing 10% calf serum.
7. as described in claim 5 or 6, build the method for tumor multi-medicine drug-resistant cell model, it is characterized in that, in step (2) and step (3) culturing cell process, change ROS with replaced medium every other day.
8. the human breast carcinoma multidrug resistance cell strain of method establishment according to claim 2, it is characterized in that, described cell strain called after MCF-7/ROS, is deposited in China typical culture collection center, address: China, Wuhan, Wuhan University, 430072; Preservation date: on January 28th, 2015; Deposit number is CCTCC-C201520.
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