CN114874987A - Myelodysplastic syndrome drug-resistant cell model and construction method and application thereof - Google Patents

Myelodysplastic syndrome drug-resistant cell model and construction method and application thereof Download PDF

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CN114874987A
CN114874987A CN202210197205.8A CN202210197205A CN114874987A CN 114874987 A CN114874987 A CN 114874987A CN 202210197205 A CN202210197205 A CN 202210197205A CN 114874987 A CN114874987 A CN 114874987A
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cells
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aza
myelodysplastic syndrome
resistant cell
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王慧睿
毛慧云
郭淑利
刘思哲
安万花
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Luoyang Central Hospital
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Abstract

The invention belongs to the technical field of cell drug resistance, and relates to a myelodysplastic syndrome drug-resistant cell model, a construction method and application thereof. The invention successfully induces MDS AZA-resistant cell strains SKM-1-AZA and MOIM-13-AZA, has stable growth and drug resistance, and provides experimental basis for further discussing a drug resistance mechanism.

Description

Myelodysplastic syndrome drug-resistant cell model and construction method and application thereof
Technical Field
The invention belongs to the technical field of cell drug resistance, and relates to a myelodysplastic syndrome drug-resistant cell model and a construction method and application thereof.
Background
Myelodysplastic Syndrome (MDS) is a group of clonal hematopoietic stem cell diseases characterized by one or more lines of abnormal and ineffective hematopoiesis with a probability of conversion to acute myeloid leukemia as high as 30%. Studies have found that the onset of MDS is closely associated with epigenetic abnormalities, with DNA methylation abnormalities being the most common epigenetic change in MDS. Epigenetic treatments represented in recent years by the demethylated drug azacitidine (AZA) have shown good efficacy in MDS. However, the phenomenon of demethylation drug resistance is inevitable, most patients develop drug resistance within 2 years of developing a therapeutic response, and the median Overall Survival (OS) of high-risk and low-risk MDS patients is 4.3 and 14 months, respectively, when few other treatment options exist for this group of patients, and the patients eventually die as the disease progresses. However, the mechanism of resistance to demethylating drugs is currently unknown.
Disclosure of Invention
The invention aims to solve the technical problem of providing a myelodysplastic syndrome drug-resistant cell model, a construction method and application thereof aiming at the defects of the prior art, wherein the cell model is established by adopting a method of gradually increasing the drug concentration and intermittently inducing and is induced by azacitidine. The invention successfully induces MDS AZA-resistant cell strains SKM-1-AZA and MOIM-13-AZA, has stable growth and drug resistance, and provides experimental basis for further discussing a drug resistance mechanism.
The invention provides a method for constructing a drug-resistant cell model of myelodysplastic syndrome, which is induced by azacitidine.
Further, the cell model comprises an SKM-1-AZA cell strain and a MOIM-13-AZA cell strain.
Further, the method for constructing the myelodysplastic syndrome drug-resistant cell model specifically comprises the following steps:
s1, culturing SKM-1 cells, carrying out passage according to the concentration of 1:2-3 every 2-3d, and taking cells in logarithmic growth phase for later use;
s2, adding azacitidine with concentration of 0.1 mu mol/L for the first time into the cells in logarithmic growth phase obtained in S1, replacing the new culture medium with azacitidine with the same concentration after 48h, and performing 1:2, passage;
s3, treating the cells growing to the logarithmic phase in S2 by increasing the concentration of the medicament, wherein each increase is 0.1 mu mol/L, until the cells can finally tolerate azacitidine by 2 mu mol/L, maintaining the cells for half a month at the concentration of 2 mu mol/L, and successfully establishing MDS drug-resistant cell strains SKM-1-AZA and MOIM-13-AZA;
referring to the method of S1-S3, MOIM-13-cells were used to establish MDS resistant cell line MOIM-13-AZA.
Furthermore, in S1, the culture processes of SKM-1 and MOIM-13 cells are both as follows: culturing in RPMI-1640+ 15% fetal calf serum culture medium, adding 1% double antibody and 1% glutamine, and incubating, and subculturing at a concentration of 1:2-3 every 2-3 d.
Further, in S1, the incubation conditions are: 5% CO 2 ，37℃。
The invention also provides a myelodysplastic syndrome drug-resistant cell model constructed by the construction method in claim 1.
Further, the cell model comprises an SKM-1-AZA cell strain and a MOIM-13-AZA cell strain.
The invention also provides an application of azacitidine as defined in claim 1 in constructing a myelodysplastic syndrome drug-resistant cell model.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention adopts a medicament gradual increasing method to establish MDS drug-resistant cell strains under the action of demethylation medicament azacitidine (AZA) in vitro, and provides a model and a theoretical basis for further researching the MDS demethylation medicament drug-resistant mechanism.
2. The invention successfully induces the MDS AZA-resistant cell strains SKM-1-AZA and MOIM-13-AZA by gradually increasing the concentration of azacitidine drugs and intermittently inducing, has stable growth and drug resistance, and provides experimental basis for further discussing a drug resistance mechanism.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 shows the effect of azacitidine AZA and the drug concentration-inhibition ratio on day 1 of the cells in the present invention;
FIG. 2 shows the effect of azacitidine AZA and the drug concentration-inhibition ratio on day 3 of the cells in the present invention;
FIG. 3 shows the effect of azacitidine AZA and the drug concentration-inhibition ratio on day 5 of the cells in the present invention;
FIG. 4 is a graph of azacitidine AZA action versus cell growth in accordance with the present invention;
FIG. 5 shows the effect of azacitidine AZA on the apoptosis rate of cells at different stages in the present invention.
Detailed Description
The invention is described in detail below with reference to the figures and the specific embodiments, but the invention should not be construed as being limited thereto. The technical means used in the following examples are conventional means well known to those skilled in the art, and materials, reagents and the like used in the following examples can be commercially available unless otherwise specified.
Example 1
The embodiment provides a myelodysplastic syndrome drug-resistant cell model and a construction method and application thereof.
Materials and methods
1. Cell lines and Primary reagents
Human MDS cell strains (SKM-1 and MOIM-13) purchased from a national experimental cell resource sharing service platform, passage and preservation in a hematological laboratory of a central hospital in Luoyang;
fetal bovine serum (Hycolne, USA), RPMI-1640 medium (Hycolne, USA), penicillin-streptomycin (Invitrogen, USA), glutamine (Invitrogen, USA), Cell Counting Kit-8 (Beijing Sorba science and technology, Inc.), Annexin V-FITC apoptosis detection Kit (Beijing Sorba science and technology, Inc.), and azacitidine (TaKaRa, Inc.).
2. Experimental methods
(1) Cell culture
SKM-1 and MOIM-13 cells used in this experiment were cultured in RPMI-1640+ 15% fetal bovine serum medium, while 1% diabody (penicillin-streptomycin) and 1% glutamine were added. Incubating in cell culture box containing 5% CO2 and saturated humidity at 37 deg.C, observing growth state, subculturing every 2-3 days at 1:2 or 1:3 concentration, and taking cells in logarithmic growth phase for experimental study.
(2) Induction of drug resistant cells
The MDS drug-resistant strain is established by adopting a method of gradually increasing the drug concentration and intermittently inducing. The cell is easy to die after the drug is added in the initial stage, so the first-time dosing concentration is extremely low and is 0.1 mu mol/L, the old culture medium is removed after 48 hours, the new culture medium with the same concentration of azacitidine is replaced, and after the cell is tolerant and stably grows, the cell is subjected to the conditions of 1:2, passage. And observing the growth state of the cells after passage, freezing and storing a part of the cells in the logarithmic growth phase, continuously increasing the concentration of the drugs for one part, increasing the concentration to 0.1 mu mol/L each time, finally tolerating 2 mu mol/L of demethylation drugs AZA by the cells after 8-10 cycles, maintaining and culturing for half a month at the concentration of 2 mu mol/L, and successfully establishing MDS drug-resistant cell strains SKM-1-AZA and MOIM-13-AZA.
(3) Detecting the half inhibition concentration (IC50) of azacitidine to MDS drug-resistant Cell strains and sensitive Cell strains by using a CCK-8(Cell Counting Kit-8 Cell Counting reagent) method, and calculating a drug Resistance Index (RI); the sensitive strain cells refer to normal non-dosed SKM-1 and MOIM-13 cells.
Well-grown log-phase cells were sampled at 1X 10 per well 4 100 μ L of cells were inoculated in a 96-well plate and cultured at 37 ℃ with 5% CO 2 After 12 hours of culture in an incubator, azacitidine with the concentration of 0, 0.0625. mu. mol/L, 0.25. mu. mol/L, 0.50. mu. mol/L, 1.00. mu. mol/L, 4.00. mu. mol/L, 16.00. mu. mol/L, 32.00. mu. mol/L, 64.00. mu. mol/L and 128.00. mu. mol/L is added, 3 more wells are set for each drug concentration, and a control group without azacitidine and a blank group without cells are additionally set. Adding 10 μ L of CCK8 reagent after adding 1d, 3d and 5d respectively, standing at 37 deg.C and 5% CO 2 After the culture in the incubator is continued for 2h, the optical density value at 490nm is detected by an enzyme-linked immunoassay analyzer to reflect the cell activity, a proliferation curve is drawn, and IC50 and RI are calculated. RI ═ drug resistant cell IC 50/parental cell IC 50. The experiment was repeated 3 times, and the samples were takenAnd (4) average value.
(4) Detection of cell growth curves
Cells growing in logarithmic phase are inoculated in 96-well culture plate and 1X 10 4 Cells/well. Adding CCK8 reagent at 1d, 3d and 5d respectively, standing at 37 deg.C and 5% CO 2 After the culture in the incubator is continued for 2h, the optical density value at 490nm is detected by an enzyme-linked immunoassay analyzer to reflect the cell activity and draw a cell growth curve. The experiment was repeated 3 times and the mean was taken.
(5) Flow detection of apoptosis of sensitive and drug-resistant strains
And (3) detecting by using an Annexin V/PI double-staining method. Well-grown log-phase cells were sampled at 5X 10 per well 5 1mL of cells were plated in 12-well plates. At 37 deg.C, 5% CO 2 Culturing in an incubator, and adding azacitidine after 12h, wherein the concentration is 5 mu mol/L. Adjusting cell concentration to 5 × 10 after adding 1d, 3d, and 5d respectively 8 L -1 Pipetting 1mL of cell suspension into 1.5mL of EP tube at 3000 r.min -1 Centrifuging for 5min, and removing supernatant; washing with precooled PBS, centrifuging again, and removing supernatant; l × Binding Buffer resuspends the cells, adds 5 μ L Annexin V-FITC, gently mixes, incubate for 10min at room temperature in the dark; and then adding 5 mu L of PI reagent, uniformly mixing by using a pipette gun, incubating for 5min in a dark place at room temperature, adding PBS to 500 mu L, detecting by using a flow cytometer within 2h, storing a flow cytometry experimental graph, and selecting a two-dimensional graph for result analysis. The experiment was repeated 3 times and the mean was taken.
3. Statistical analysis
Data analysis and mapping were performed using Graphpad Prism 8.0 statistical software, data expressed as mean. + -. standard deviation (x. + -.s), and comparisons between two sample means were performed using the t-test.
Second, experimental results
1. Drug resistance of drug-resistant cells to the demethylating drug azacitidine (AZA)
The detection results of demethylation drug azacitidine (AZA) drug-resistant cell strains SKM-1 and MOIM-13 are induced in vitro, the proliferation inhibition rate shown after the drug is added for 1 day is shown in figure 1, and meanwhile, the IC50 of the cell strains SKM-1 control group and drug-resistant group cells are respectively 2.678 mu mol/L and 86.39 mu mol/L, the RI is 32.26, the IC50 of the MOIM-13 control group and drug-resistant group cells is respectively 2.176 mu mol/L and 16.39 mu mol/L, and the RI is 7.53.
The proliferation inhibition rate shown on day 3 after the drug addition is shown in FIG. 2, and the IC50 of the cell line SKM-1 control group and the drug-resistant group cell are respectively 0.5317 μmol/L and 8.889 μmol/L, RI is 16.72, the IC50 of the MOIM-13 control group and the drug-resistant group cell are respectively 0.475 μmol/L and 13.61 μmol/L, and RI is 28.65.
The proliferation inhibition rate shown on day 5 after the drug addition is shown in FIG. 3, and the IC50 of the cell line SKM-1 control group and drug-resistant group cells are respectively 0.8505 μmol/L and 5.229 μmol/L, RI is 6.12, the IC50 of the MOIM-13 control group and drug-resistant group cells are respectively 0.3859 μmol/L and 4.524 μmol/L, and RI is 11.72.
2. The sensitive cell and the drug-resistant cell line are cultured for 5 days under the same condition, and certain difference is generated in cell proliferation. The growth of drug-resistant cells was slower than that of sensitive cells, the slope of the growth curve was reduced, and there was a significant difference in cell proliferation rate (as shown in FIG. 4).
3. Apoptosis of cells
Detecting the apoptosis of SKM-1, MOIM-13 sensitive strains and drug-resistant strains by a flow cytometer after the dosing on days 1, 3 and 5.
We found that the percentage of apoptosis was less between the sensitive and resistant cells on days 1 and 3 of the drug administration, but the rate of apoptosis was much less in the resistant cells than in the control group after day 5 of the drug administration (as shown in fig. 5).
In conclusion, the invention adopts a method of gradually increasing the concentration of the drug (azacitidine) and intermittently inducing to establish the human MDS drug-resistant cell strain, and has the characteristics of stable and reliable drug resistance; the invention successfully induces human MDS drug-resistant cell lines SKM-1-AZA and MOIM-13-AZA, and discovers that the drug resistance of the cells at different time periods is different, and the successful establishment of the cell model provides an important carrier for the deep research of the azacitidine drug-resistant mechanism.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims (8)

1. A method for constructing a drug-resistant cell model of myelodysplastic syndrome is characterized in that the cell model is induced by azacitidine.
2. The method of claim 1, wherein the cellular model comprises SKM-1-AZA cell line and MOIM-13-AZA cell line.
3. The method for constructing a myelodysplastic syndrome drug-resistant cell model according to claim 1, which comprises the following steps:
s1, culturing SKM-1 cells, carrying out passage according to the concentration of 1:2-3 every 2-3d, and taking cells in logarithmic growth phase for later use;
s2, adding azacitidine with concentration of 0.1 mu mol/L for the first time into the cells in logarithmic growth phase obtained in S1, replacing the new culture medium with azacitidine with the same concentration after 48h, and performing 1:2, passage;
s3, treating the cells growing to the logarithmic phase in S2 by increasing the concentration of the medicament, wherein each increase is 0.1 mu mol/L, until the cells can finally tolerate azacitidine by 2 mu mol/L, maintaining the cells for half a month at the concentration of 2 mu mol/L, and successfully establishing an MDS medicament-resistant cell strain SKM-1-AZA;
referring to the method of S1-S3, MOIM-13-cells were used to establish MDS resistant cell line MOIM-13-AZA.
4. The method for constructing a myelodysplastic syndrome drug-resistant cell model according to claim 3, wherein in S1, the culture process of SKM-1 and MOIM-13 cells is as follows: culturing in RPMI-1640+ 15% fetal calf serum culture medium, adding 1% double antibody and 1% glutamine, and incubating, and subculturing at a concentration of 1:2-3 every 2-3 d.
5. The method for constructing the myelodysplastic syndrome-resistant cell model according to claim 4, wherein in S1, the incubation conditions are as follows: 5% CO 2 ，37℃。
6. A myelodysplastic syndrome drug-resistant cell model constructed according to the construction method of claim 1.
7. The myelodysplastic syndrome-resistant cell model constructed according to the construction method of claim 6, wherein the cell model comprises SKM-1-AZA cell line and MOIM-13-AZA cell line.
8. Use of azacitidine according to claim 1 for constructing a myelodysplastic syndrome drug-resistant cell model.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104321649A (en) * 2012-02-28 2015-01-28 尼斯-索菲亚·安蒂波利斯大学 Test for diagnosing resistance to azacitidine
KR20160092548A (en) * 2015-01-27 2016-08-05 재단법인 아산사회복지재단 Cell Line With Resistance To Hypomethylation Agents, And Uses Thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104321649A (en) * 2012-02-28 2015-01-28 尼斯-索菲亚·安蒂波利斯大学 Test for diagnosing resistance to azacitidine
US20150094217A1 (en) * 2012-02-28 2015-04-02 Université Nice Sophia Antipolis Test for diagnosing resistance to azacitidine
KR20160092548A (en) * 2015-01-27 2016-08-05 재단법인 아산사회복지재단 Cell Line With Resistance To Hypomethylation Agents, And Uses Thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MURASE 等: "Lack of cross-resistance to FF-10501, an inhibitor of inosine-5\'-monophosphate dehydrogenase, in azacitidine-resistant cell lines selected from SKM-1 and MOLM-13 leukemia cell lines", 《PHARMACOL RES PERSPECT》, vol. 4, no. 1, pages 2 - 3 *

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