CN104004715A - Human leukemia HL-60 cell drug-resistance cell line HL-60/RS cell and preparation method thereof - Google Patents
Human leukemia HL-60 cell drug-resistance cell line HL-60/RS cell and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a human leukemia HL-60 cell drug-resistance cell line HL-60/RS cell and a preparation method thereof. The preservation number of the cell line HL-60/RS is CCTCC No. C201430. The preparation method is characterized in that a human leukemia HL-60 cell line is used as a parent cell, adriamycin is used for simulating an internal chemotherapy process, and a gradual long-term intermittent repeated drug concentration impact increasing and continuous induction method is used for establishing the leukemia multidrug-resistance cell line HL-60/RS. The preparation method has the beneficial effects that the leukemia multidrug-resistance cell line HL-60/RS is established by adopting the concentration impact increasing and continuous induction method, and experiments prove that the established drug-resistance cell line has the characteristics of wide drug-resistance spectrum, stable drug resistance and the like; the drug-resistance cell line has high drug resistance and can be used for researching the sensitivity, drug resistance and arsenic resistance of tumor cells to anti-cancer drugs and screening resistance-reversal agents with reliability.
Description
Technical field
The present invention relates to human leukemia HL-60 cell's drug-resistant cell strain HL-60/RS cell and preparation method thereof.
Background technology
Leukemia is one of between twenty and fifty main malignant tumour of harm, chemotherapy remains leukemic main methods for the treatment of at present, but the drug tolerance of cancer therapy drug induction in chemotherapy, particularly acquired multidrug resistance (multidrug resistance, MDR) phenomenon is to hinder the major obstacle of leukemia treating effect, is also a unsolved difficult problem still at present.White arsenic is the effective constituent of Chinese medicine arsenic, in the treatment of various leukemia and noumenal tumour, is used widely, and has obtained good clinical efficacy.Studies confirm that white arsenic is not the substrate of drug-resistant protein P-gp and suppresses P-gp and express, not with conventional chemotherapy medicine generation cross resistance, therefore alone or obtain good efficacy with conventional chemotherapy Drug combination in the treatment of resistance, recurrent and Refractory Leukemia.In clinical application arsenic trioxide in treatment leukemia/neoplastic process, find that part leukemia/tumour cell also can produce the tolerance to white arsenic gradually, obtain anti-arsenic, mechanism and not clear with the relation of MDR.Current conventional employing Zorubicin multi-medicine drug resistance of leukaemia clone induction, overexpression P-gp (as K562/ADM etc.) in the same old way to white arsenic sensitivity, not anti-arsenic.Therefore, the mechanism of the MDR of research induced by chemotherapeutic agents and with the relation of anti-arsenic, and prevent the generation of anti-arsenic, inquire into the resistance leukemia cell cellular elements mechanism responsive or tolerate to it, can be to solve containing arsenic chemotherapeutics resistance problem and the clinical As2O3 that whether selects provides experimental basis or seeks new treatment target spot as the leukemic medicine of resistance.
Summary of the invention
Object of the present invention is exactly for above-mentioned defect of the prior art, for analyzing the variation of the biological character after leukemia cell's chemotherapy resistance, screen and judge the susceptibility of chemotherapeutics, analyze leukemia cell's multidrug resistance and anti-arsenic mechanism, the relation of research multidrug resistance and anti-arsenic, the generation of MDR and/or anti-arsenic in prevention leukemia chemotherapy process, in-vitro screening resistance adjusting agent, provide a kind of can with the multi-medicine drug resistance of leukaemia clone HL-60/RS of white arsenic crossing drug resistant.
To achieve these goals, technical scheme provided by the invention is: human leukemia HL-60 cell's drug-resistant cell strain HL-60/RS cell, the deposit number of described cell line HL-60/RS is CCTCC NO:C201430, the preservation time is on April 16th, 2014, preservation place be Chinese Typical Representative culture collection center (address for China. Wuhan. Wuhan University).
Second object of the present invention has been to provide the preparation method of above-mentioned human leukemia HL-60 cell's drug-resistant cell strain HL-60/RS cell, to be parental cell with human leukemia HL-60 cell, adopt Zorubicin analogue body chemotherapy process, progressively improve the method that drug level, long-term, intermittent repeated stock add successive induction, acquired multi-medicine drug resistance of leukaemia clone HL-60/RS is set up in induction.
Its feature is for to have high drug-resistance to white arsenic, and the arsenic transfer related protein such as high expression level MRP1, MRP2 and ASNA1.
Further, the preparation method of above-mentioned human leukemia HL-60 cell's drug-resistant cell strain HL-60/RS cell, comprises the following steps:
1) the human leukemia HL-60 cell of taking the logarithm vegetative period is seeded in to contain with volume percent and counts in the RPMI RPMI-1640 of 15% new-born calf serum, 100U/mL penicillin, 100 μ g/mL Streptomycin sulphates, is placed in 37 DEG C, 5% CO
2, cultivate under saturated humidity environment, obtain cell culture system;
2) in the cell culture system obtaining in step 1), add Zorubicin, initial impact concentration is 0.1mg/L, in the time of cellular-restoring normal growth, in addition drug-induced with concentration again, so repeat time then raising impact drug level until cell can normally be survived and breed under this concentration, carry out second and take turns impact induced, the amplitude that drug level increases is 0.1mg/L, repeated intermittent impact induced in a manner described, and increasing along with impact induced number of times and resistance, every amplitude of taking turns drug level increase improves gradually, initial stage amplitude is little, later stage amplitude strengthens, every amplitude of taking turns the increase of impact induced drug level is 0.1mg/L, 0.2 mg/L, 0.3 mg/L, 0.5 mg/L, 0.6 mg/L, 0.8 mg/L, 1.0 mg/L, 1.5 mg/L, 2.0 mg/L, 2.5 m g/L, 3.0 mg/L, 3.5 mg/L, 4.0 mg/L, 5.0mg/L, in every wheel, increase the number of times that impacts concentration according to cell state and the decision of resistance stability with amplitude, general 1-3 time, the impact induced time is 20 months, can in the cell culture system that contains 40.0mg/L Zorubicin, maintain and survive and propagation to final human leukemia HL-60 cell, withdraw induced drug and be placed in without medicine system and cultivate, it is stable that resistance keeps, and obtains having the HL-60/RS cell of resistance.
institute's with medicament source:
New-born calf serum: Lanzhou Rong Ye biotechnology company product, lot number 20100925;
Penicillin: Huabei Pharmaceutic Co., Ltd's product, lot number 1105403;
Streptomycin sulphate: Huabei Pharmaceutic Co., Ltd's product, lot number 1112106;
RPMI RPMI-1640: Gibco BRL company of U.S. product, lot number 870532;
Zorubicin: Wanle Pharmaceutical Co Ltd, Shenzhen's product, lot number 1103E1.
Principle of the present invention is, be parental cell with human leukemia HL-60 cell, adopt Zorubicin (Adriamycin, ADM) analogue body chemotherapy process, progressively improve the methods induction that drug level, long-term, intermittent repeated stock add successive induction and set up acquired multi-medicine drug resistance of leukaemia clone HL-60/RS, and observe biological characteristics and the susceptibility to white arsenic.
Concrete enforcement technology is as follows: 1. logarithmic phase HL-60 cell is seeded in the RPMI-1640 nutrient solution containing 15% new-born calf serum, 100U/mL penicillin, 100mg/mL Streptomycin sulphate, is placed in 37 DEG C, 5% CO
2, cultivate in saturated humidity incubator.2. in cell culture system, add Zorubicin, initial impact concentration is 0.1mg/L, in the time of cellular-restoring normal growth, in addition drug-induced with concentration again, so repeat time then raising impact drug level until cell can normally be survived and breed under this concentration, carry out second and take turns impact induced, the amplitude that drug level increases is 0.1mg/L.So repeated intermittent impact induced, and along with the increasing of impact induced number of times and resistance, every amplitude of taking turns drug level and increasing also improves gradually, and initial stage amplitude is less, and later stage amplitude strengthens, every amplitude of taking turns the increase of impact induced drug level is 0.1,0.2,0.3,0.5,0.6,0.8,1.0,1.5,2.0,2.5,3.0,3.5,4.0,5.0mg/L, lasts 20 months, and final HL-60 cell can be containing maintaining survival and propagation in the culture system of 40.0mg/L Zorubicin.After removing induced drug, in without medicine system, cultivate HL-60 cell long-period stable maintenance resistance.Mdr cell after induction is recovery after frozen 3 months in liquid nitrogen, and it is stable that cells resistance maintains.The final HL-60/RS cell with resistance obtaining.
To in the above described manner, the HL-60/RS clone with multidrug resistance that adopts " progressively improving concentration impact+successive induction " to set up, carry out biological characteristics detection:
2.1 Antibiotic Resistances:
Collect HL-60 cell and the HL-60/RS cell of logarithmic growth, adopt mtt assay to detect the susceptibility to Zorubicin and cis-platinum, vincristine(VCR), Etoposide, cytosine arabinoside and 5 FU 5 fluorouracil, cell is by 1 × 10
5/ ml inoculates 96 porocyte culture plates, adds the medicine of respective concentration, is placed in CO
2incubator is cultivated 48h and 72h, before cultivating termination, the every hole of 4h adds 10 μ l MTT solution (5mg/ml), continue to cultivate, every hole adds the 10% SDS solution termination reaction containing 0.01mol/L HCl, 37 DEG C of juxtapositions are spent the night, automatically microplate reader is measured absorbancy (A) value at 570nm wavelength, and calculates inhibiting rate and the half amount of suppression (IC of relative medicine
50) value and resistance multiple.The tolerance of HL-60/RS cell to Zorubicin and Antibiotic Resistance as shown in Figure 1, with IC
50relatively, HL-60/RS cell is 85.68 times of its parent HL-60 cell to the tolerance of Zorubicin.HL-60/RS cell has cross resistance in various degree to cis-platinum, vincristine(VCR), Etoposide and cytosine arabinoside, but to the susceptibility of 5 FU 5 fluorouracil without obvious change.
2.2 morphological changes:
Collect good HL-60 cell and the HL-60/RS cell of growth conditions, Wright-Gimsa dyeing, om observation, simultaneously observation of cell modification of surface morphology under observation of cell device and nuclear change, scanning electronic microscope under transmission electron microscope, the light microscopic of HL-60 cell and HL-60/RS cell and electron microscopic morphology feature are as shown in Figure 2.With contrast the comparison of HL-60 cell, HL-60/RS cellular form has no notable difference, karyon is compared with the circular of homogeneous or oval, diameter and increases, core/slurry ratio significantly increases, without class leaflet core or class band form nucleus.Under scanning electron microscope HL-60/RS cell surface more smooth, lack microvillus; In ultrastructure, kytoplasm is finer and close, and chromatin densification in nucleus, heterochromatin increase.Observe and show from morphology, the HL-60/RS cell phenotype that brings out resistance through ADM is more original and inmature, and differentiation degree is lower.
2.3 resistance proteins:
Collect the HL-60 cell that growth conditions is good and the HL-60/RS cell that tolerates different concns ADM, cell concn is adjusted to 10
6individual/ml, adds mouse-anti people P-gp monoclonal antibody MRK-16 as primary antibodie while detecting P-gp, and room temperature lucifuge is hatched 20min, resuspended after PBS washing, then adds sheep anti mouse PE-IgG2a anti-as two, and room temperature lucifuge is hatched 20min, PBS washing.While detecting BCRP, add the mouse-anti people BCRP antibody of FITC mark.Flow cytometer detects respectively the positive expression rate of P-gp and BCRP.The expression of HL-60 cell and HL-60/RS cells resistance albumen changes as shown in Figure 3, result shows, the expression of P-gp and BCRP is increased and is increased with HL-60 cell tolerance ADM concentration, in the time that the dosage of HL-60 cell tolerance ADM reaches 5mg/L, survivaling cell just almost 100% is expressed P-gp, and BCRP positive rate is 6.2%.While reaching the highest tolerance concentration (40mg/L), P-gp
+and BCRP
+cell is respectively 100% and 24.2%, is respectively 78.7 times and 25.0 times of HL-60 cell.
2.4 drug efflux functions (ADM content in cell):
HL-60 and HL-60/RS cell are suspended from PBS, add the ADM of final concentration 30mg/L, 37 DEG C of lucifuges are hatched 30min, and cold PBS washs after 2 times, cell is divided into two portions: a part of cell is resuspended in the cold PBS of 1ml, FCM detects ADM accumulation (fluorescence intensity) in cell.Another part cell is placed in 37 DEG C of continuation lucifuges and hatches 60min, cold PBS washing 2 times, and FCM detects the ADM amount (fluorescence intensity) retaining in cell, calculates outer output.The outer row function of HL-60 cell and HL-60/RS cell drug (ADM content in cell) changes as shown in Figure 4, and in HL-60/RS cell, the content (fluorescence intensity) of ADM is only 59% of HL-60 cell; Continue to hatch 60min without medicine, only in 63% HL-60/RS mdr cell, can measure ADM, and the ADM content that can measure is only for 25.7% of HL-60 cell, approximately has 66.5% ADM to be discharged from extracellular, and contrast HL-60 cell only discharges 22.6%.The above results proves that the P-gp of HL-60/RS cell high expression level and the drug efflux activity of BCRP are one of main mechanisms of its resistance.
2.5 white arsenic susceptibility:
Collect HL-60 cell and HL-60/RS cell, and select to be all classical multidrug resistance leukemia K 562/ADM cell of ADM induction and the responsive K562 cell of parent thereof for contrast, by 1.0 × 10
5individual/mL is inoculated in 96 well culture plates, adds the As of different concns
2o
3, 37 DEG C, 5%CO
2and cultivate 24-72h under saturated humidity condition, and from cultivating terminal 4h, add 10 μ L MTT solution (5mg/mL), continue to cultivate 4h, add 10%SDS, 37 DEG C of hold over night.Full-automatic microplate reader is measured the absorbancy (A value) in every hole in 570nm wavelength place, calculate cell proliferation inhibition rate and half-inhibition concentration (IC
50).As shown in Figure 5, result shows HL-60 cell and the HL-60/RS cell susceptibility (with K562/ADM cell and the comparison of K562 cell) to white arsenic, K562/ADM cell compared with K562 cell to As
2o
3more responsive, 24, the IC of 48 and 72 hours
50value is respectively 0.75,0.82 and 0.80 times of K562 cell; And but in contrast, HL-60/RS cell far beyond HL-60 cell to As
2o
3tolerance, IC
50value is respectively 7.39,10.87 and 12.89 times of HL-60 cell.HL-60/RS mdr cell is to As
2o
3susceptibility far below K562/ADM mdr cell, its IC
50value is respectively 9.51,9.78 and 10.89 times of K562/ADM cell, but their parental cell HL-60 and K562 cell are to As
2o
3susceptibility close.Result discloses, although HL-60/RS cell and K562/ADM cell are Zorubicin induction MDR cell that select, mdr1/P-gp high expression level, to As
2o
3susceptibility but completely contrary.
2.6 arsenic translocators are expressed:
Collect respectively HL-60 cell, HL-60/R cell, K562 cell and K562/ADM cell, extract RNA and protein, adopt respectively real-time fluorescence quantitative RT-PCR method and Western blot method to detect and arsenic transfer related protein ABCCl(MRPl), ABCC2(MRP2) and the expression level of the ATP enzyme (arsenite-stimulated ATPase, ASNA1) of arsenic activation.1. quantitative RT-PCR: TRIZOL method extracting cell total rna, cDNA is synthesized in reverse transcription, the total system 25 μ l of quantitative PCR, comprise 2 × SYBR Green buffer, 12.5 μ l upstream primer 1 μ l, downstream primer 1 μ l, cDNA 2 μ l, PCR primer sequence is as shown in table 1.
Table 1
。
Reaction conditions: 95 DEG C, 10 sec, then 95 DEG C of 5sec, 60 DEG C of 30sec totally 40 circulations, the specificity of melt curve analysis assay products is carried out in amplification after finishing.Quantitative gene expression adopts relatively threshold value method to analyze.2. collecting cell PBS washing, RIPA lysate extracting total protein, after BCA method is carried out quantitatively to protein, add 5 × sample-loading buffer, mix, 100 DEG C are boiled 5 min and make protein denaturation, each sample is got respectively 50 μ g and is carried out loading, 8-10% SDS-PAGE gel electrophoresis separates, be transferred to pvdf membrane, after spending the night, 5 % skim-milk room temperatures sealings 1h or 4 DEG C add respectively anti-MRP1 antibody, anti-MRP2 antibody, anti-ASNA1 antibody and anti-β-actin antibody, 4 DEG C are spent the night or incubated at room 1-2h, with PBS-T washing 3 × 5min, add respectively anti-incubated at room 2 h of the goat-anti rabbit of IRDye700DX mark and the sheep anti mouse two of IRDye800CW mark, after PBS-T washing 5 × 10min, PBS washs 10-30min, Odyssey infrared laser scanner scans and analyzes.The expression level of HL-60 cell and HL-60/RS cell MRPl, MRP2 and ASNA1, and with K562/ADM cell and K562 cell more as shown in Figure 6, result shows, the K562/ADM cell of resistance and HL-60/RS cell MRP(MRP1 and MRP2) expression all higher than its parent's sensitive cells, and HL-60/RS cell is higher than K562/ADM cell; In HL-60/RS cell, the expression of ASNA1 is far above K562/ADM cell, HL-60 cell and K562 cell, is respectively 7.34 times of K562/ADM cell, 2.45 times of HL-60 cell, 6.16 times of K562 cell.And the expression amount of K562/ADM cell is 0.83 times of K562 cell.Expression and its susceptibility to white arsenic of two couples of cell MRP1, MRP2 and ASNA1 are basically identical.Show that HL-60/RS cell is associated because of the expression of MRP1 and the MRP2, particularly ASNA1 of the relevant transporter of arsenic to white arsenic tolerance.
Beneficial effect of the present invention is: the present invention induces the multi-medicine drug resistance of leukaemia HL-60/RS clone of foundation, it is the induction method that adopts " progressively improving concentration impact+successive induction ", be different from current tumor drug resistance research concentration gradient incremental methods and the heavy dose of modes of intermittently impacting in short-term of adopting, the drug-resistant cell strain of foundation has the wide and resistance of Antibiotic Resistance and the feature such as stablizes more; The multidrug resistance of HL-60/RS cell is similar with at present the most classical K562/ADM cell, and high expression level drug transporter P-gp, MRP and BCRP; With drug-resistant leukemia cell comparisons such as K562/ADM, except having the resistance of wide spectrum, also white arsenic is had to high drug-resistance, and the arsenic transfer related protein such as high expression level MRP1, MRP2 and ASNA1.The feature of HL-60/RS clone be multidrug resistance and with white arsenic cross tolerance and high expression level ASNA1 gene, can be used for the mechanism of susceptibility, resistance and the anti-arsenic of study tumor cell to cancer therapy drug, and screening has the reversal agent of drug resistance of reliability; Can the detection measurable susceptibility to arsenical class medicine, particularly recurrent of clinical ASNA1 gene, intractable and resistance leukemia use the pharmacological agent of arsenical class instead.
Brief description of the drawings
Fig. 1 is tolerance and the Antibiotic Resistance of HL-60/RS clone provided by the invention to Zorubicin, wherein, and A: the resistance under different inductive conditions; B: Antibiotic Resistance.
Fig. 2 is light microscopic and the electron microscopic morphology feature of HL-60 cell and HL-60/RS cell.
Fig. 3 is that the expression of HL-60 cell and HL-60/RS cells resistance albumen changes, wherein, and the expression level of A:Western P-gp that blot surveys, MRP and BCRP; B: the dynamic change of P-gp and BCRP in Induction Process.
Fig. 4 is that the outer row function of HL-60 cell and HL-60/RS cell drug (ADM content in cell) changes.
Fig. 5 is HL-60 cell and the susceptibility of HL-60/RS cell to white arsenic, is the result relatively obtaining with K562/ADM cell and K562 cell.
Fig. 6 is the expression level of HL-60 cell and HL-60/RS cell MRPl, MRP2 and ASNA1, and with K562/ADM cell and K562 cell comparison (A: the expression level of mRNA that quantitative RT-PCR is surveyed; B:Western protein expression level that blot surveys).
the translator of Chinese of the English mark in accompanying drawing is as follows:
ADM(Zorubicin); Tolerant ADM conc.(tolerates doxorubicin concentration); 50% inhibitory concentration(IC50, half-inhibition concentration); Cisplatin(cis-platinum); 5-Fluorourid(5-Fluracil); Daunorubicin(daunorubicin); Cytrarabine(cytosine arabinoside); Vincristine(vincristine(VCR)); Etoposide(Etoposide); Relative expression(relative expression quantity); Positive rate(positive rate); MFI(average fluorescent strength); P-gp (P-glycoprotein); BCRP(breast drug-resistance protein); MRP1(multidrug-associated protein 1); MRP2(multidrug-associated protein 2); The ATP enzyme that ASNA1(arsenic activates).
Embodiment
embodiment 1:
Taking Zorubicin as inductor, HL-60 cells cell is set up multiple medicine-resistant cell line.1. HL-60 is seeded in RPMI 1640 nutrient solutions containing 15% new-born calf serum, 100U/mL penicillin, 100 μ g/mL Streptomycin sulphates, is placed in 37 DEG C, 5 % CO
2, cultivate in saturated humidity incubator.2. in cell culture system, add Zorubicin, initial impact concentration is 0.1mg/L, in the time that survivaling cell restore normal growth, in addition drug-induced with concentration again, so repeat time then raising impact drug level until cell can normally be survived and breed under this concentration, carry out second and take turns impact induced, the amplitude that drug level increases is 0.1mg/L.So repeated intermittent impact induced, and along with the increasing of impact induced number of times and resistance, every amplitude of impacting drug level and increasing of taking turns also improves gradually, initial stage amplitude is less, and later stage amplitude strengthens, every amplitude of taking turns the increase of impact induced drug level is followed successively by 0.1,0.2,0.3,0.5,0.6,0.8,1.0,1.5,2.0,2.5,3.0,3.5,4.0,5.0mg/L, last 20 months, final HL-60 cell can, containing maintaining survival and propagation in the culture system of 40.0mg/L Zorubicin, build up multi-medicine drug resistance of leukaemia clone HL-60/RS cell.3. HL-60/RS cell is cultivated in without Zorubicin culture system, HL-60 cell long-period stable maintenance resistance.Recovery after frozen 3 months in liquid nitrogen, it is stable that cells resistance maintains.The Resistance index of the HL-60/RS cell that induction is set up is 85.68, and cis-platinum, vincristine(VCR), Etoposide and cytosine arabinoside are had to cross resistance; High expression level drug-resistant protein P-gp and BCRP.
embodiment 2:
It is identical with embodiment 1 that mode is set up in the induction of HL-60/RS cell.HL-60/RS cytotostatic carries out the mensuration to white arsenic susceptibility after growing latter 1 month.HL-60/RS cell (selecting HL-60 sensitive cells, K562/ADM multidrug resistance cell and parent K562 cell thereof for contrast), by 1.0 × 10
5individual/mL is inoculated in 96 well culture plates, adds the As of 0.1-5.0mg/L
2o
3, 37 DEG C, 5%CO
2and cultivate 24-72h under saturated humidity condition, mtt assay is measured cell inhibitory effect and IC50.HL-60/RS cell is to white arsenic height tolerance, and tolerance is 12.89 times of parent HL-60 cell, be 10.89 times of K562/ADM mdr cell.
embodiment 3:
It is identical with embodiment 1 that mode is set up in the induction of HL-60/RS cell.HL-60/RS cell and contrast HL-60 cell, K562 cell and K562/ADM cell, adopt TRIZOL method extracting cell total rna and RIPA lysate extracting total protein, use respectively the expression of real-time fluorescence quantitative RT-PCR method and the detection of Western blot method and arsenic transfer related protein MRPl, MRP2 and ASNA1.HL-60/RS cell MRP(MRP1 and MRP2) expression apparently higher than HL-60 cell and K562/ADM cell; The expression of HL-60/RS cell ASNA1 is significantly higher than HL-60 cell and K562/ADM cell, is respectively 7.34 times and 2.45 times of HL-60 cell of K562/ADM cell.The expression amount of K562/ADM cell is 0.83 times of K562 cell.The expression level of MRP and ASNA1 is consistent with the susceptibility of white arsenic.
Finally it should be noted that: the foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, although the present invention is had been described in detail with reference to previous embodiment, for a person skilled in the art, its technical scheme that still can record aforementioned each embodiment is modified, or part technical characterictic is wherein equal to replacement.Within the spirit and principles in the present invention all, any amendment of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Sequence table
<110> Lanzhou University
<120> multi-medicine drug resistance of leukaemia clone HL-60/RS and preparation method thereof
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Sequence table
<110> Lanzhou University
<120> multi-medicine drug resistance of leukaemia clone HL-60/RS and preparation method thereof
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Claims (3)
1. human leukemia HL-60 cell's drug-resistant cell strain HL-60/RS cell, is characterized in that, the deposit number of described clone HL-60/RS is CCTCC NO:C201430, and the preservation time is on April 16th, 2014, and preservation place is Chinese Typical Representative culture collection center.
2. the preparation method of human leukemia HL-60 cell's drug-resistant cell strain HL-60/RS cell claimed in claim 1, it is characterized in that, to be parental cell with human leukemia HL-60 cell, adopt Zorubicin analogue body chemotherapy process, progressively improve the method that drug level, long-term, intermittent repeated stock add successive induction, acquired multi-medicine drug resistance of leukaemia clone HL-60/RS is set up in induction.
3. the preparation method of human leukemia HL-60 cell's drug-resistant cell strain HL-60/RS cell according to claim 2, is characterized in that, comprises the following steps:
1) the human leukemia HL-60 cell of taking the logarithm vegetative period is seeded in to contain with volume percent and counts in the RPMI RPMI-1640 of 15% new-born calf serum, 100U/mL penicillin, 100 μ g/mL Streptomycin sulphates, is placed in 37 DEG C, 5%CO
2, cultivate under saturated humidity environment, obtain cell culture system;
2) in the cell culture system obtaining in step 1), add Zorubicin, initial impact concentration is 0.1mg/L, in the time of cellular-restoring normal growth, in addition drug-induced with concentration again, so repeat time then raising impact drug level until cell can normally be survived and breed under this concentration, carry out second and take turns impact induced, the amplitude that drug level increases is 0.1mg/L, repeated intermittent impact induced in a manner described, and increasing along with impact induced number of times and resistance, every amplitude of taking turns drug level increase improves gradually, initial stage amplitude is little, later stage amplitude strengthens, every amplitude of taking turns the increase of impact induced drug level is 0.1mg/L, 0.2 mg/L, 0.3 mg/L, 0.5 mg/L, 0.6 mg/L, 0.8 mg/L, 1.0 mg/L, 1.5 mg/L, 2.0 mg/L, 2.5 m g/L, 3.0 mg/L, 3.5 mg/L, 4.0 mg/L, 5.0mg/L, in every wheel, it is 1-3 time with the number of times of amplitude increase impact concentration, the impact induced time is 20 months, can in the cell culture system that contains 40.0mg/L Zorubicin, maintain and survive and propagation to final human leukemia HL-60 cell, withdraw induced drug and be placed in without medicine system and cultivate, it is stable that resistance keeps, and obtains having the HL-60/RS cell of resistance.
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|---|---|---|---|
| CN201410217030.8A CN104004715B (en) | 2014-05-21 | 2014-05-21 | Human leukemia HL-60 cell's drug-resistant cell strain HL-60/RS cell and preparation method thereof |
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| CN108048402A (en) * | 2018-01-25 | 2018-05-18 | 四川大学华西第二医院 | A kind of people's Pancytopenia glucocorticoid drug-resistant cell strain and its construction method and application |
| CN108315302A (en) * | 2018-01-25 | 2018-07-24 | 四川大学华西第二医院 | A kind of people's B-lineage Acute Lymphocyte Leukemia glucocorticoid drug-resistant cell strain and its construction method and application |
| CN108315303A (en) * | 2018-01-30 | 2018-07-24 | 复旦大学附属中山医院 | A method of preparing Human gallbladder carcinoma gemcitabine medicine-resistant cell line |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108048402A (en) * | 2018-01-25 | 2018-05-18 | 四川大学华西第二医院 | A kind of people's Pancytopenia glucocorticoid drug-resistant cell strain and its construction method and application |
| CN108315302A (en) * | 2018-01-25 | 2018-07-24 | 四川大学华西第二医院 | A kind of people's B-lineage Acute Lymphocyte Leukemia glucocorticoid drug-resistant cell strain and its construction method and application |
| CN108315303A (en) * | 2018-01-30 | 2018-07-24 | 复旦大学附属中山医院 | A method of preparing Human gallbladder carcinoma gemcitabine medicine-resistant cell line |
| CN108315303B (en) * | 2018-01-30 | 2021-03-30 | 复旦大学附属中山医院 | Method for preparing human gallbladder cancer gemcitabine drug-resistant cell line |
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