CN115927187B - Ameitinib resistant cell strain NCI-H1975-AR and application thereof - Google Patents
Ameitinib resistant cell strain NCI-H1975-AR and application thereof Download PDFInfo
- Publication number
- CN115927187B CN115927187B CN202211740359.3A CN202211740359A CN115927187B CN 115927187 B CN115927187 B CN 115927187B CN 202211740359 A CN202211740359 A CN 202211740359A CN 115927187 B CN115927187 B CN 115927187B
- Authority
- CN
- China
- Prior art keywords
- drug
- resistant
- cell
- nci
- ametinib
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 239000003814 drug Substances 0.000 claims abstract description 47
- 208000010507 Adenocarcinoma of Lung Diseases 0.000 claims abstract description 46
- 201000005249 lung adenocarcinoma Diseases 0.000 claims abstract description 46
- 229940079593 drug Drugs 0.000 claims abstract description 44
- 238000004321 preservation Methods 0.000 claims abstract description 7
- 238000012216 screening Methods 0.000 claims abstract description 6
- 230000035772 mutation Effects 0.000 abstract description 18
- 206010059866 Drug resistance Diseases 0.000 abstract description 13
- 206010028980 Neoplasm Diseases 0.000 abstract description 11
- 208000002154 non-small cell lung carcinoma Diseases 0.000 abstract description 11
- 238000011160 research Methods 0.000 abstract description 11
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 abstract description 10
- 230000007705 epithelial mesenchymal transition Effects 0.000 abstract description 8
- 230000009471 action Effects 0.000 abstract description 7
- 238000000034 method Methods 0.000 abstract description 7
- 238000011282 treatment Methods 0.000 abstract description 7
- 239000002246 antineoplastic agent Substances 0.000 abstract description 6
- 229940041181 antineoplastic drug Drugs 0.000 abstract description 6
- 230000000877 morphologic effect Effects 0.000 abstract description 5
- 238000004458 analytical method Methods 0.000 abstract description 4
- 238000002360 preparation method Methods 0.000 abstract description 4
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 238000010206 sensitivity analysis Methods 0.000 abstract description 3
- 230000007246 mechanism Effects 0.000 abstract description 2
- 239000003560 cancer drug Substances 0.000 abstract 2
- 210000004027 cell Anatomy 0.000 description 148
- 238000002474 experimental method Methods 0.000 description 21
- 239000001963 growth medium Substances 0.000 description 20
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 15
- 108090000623 proteins and genes Proteins 0.000 description 15
- 102000004169 proteins and genes Human genes 0.000 description 15
- 239000002609 medium Substances 0.000 description 12
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 description 11
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 description 11
- 239000000243 solution Substances 0.000 description 11
- YOHYSYJDKVYCJI-UHFFFAOYSA-N n-[3-[[6-[3-(trifluoromethyl)anilino]pyrimidin-4-yl]amino]phenyl]cyclopropanecarboxamide Chemical compound FC(F)(F)C1=CC=CC(NC=2N=CN=C(NC=3C=C(NC(=O)C4CC4)C=CC=3)C=2)=C1 YOHYSYJDKVYCJI-UHFFFAOYSA-N 0.000 description 10
- 230000035755 proliferation Effects 0.000 description 9
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 8
- 201000005202 lung cancer Diseases 0.000 description 8
- 208000020816 lung neoplasm Diseases 0.000 description 8
- 230000005012 migration Effects 0.000 description 8
- 238000013508 migration Methods 0.000 description 8
- 238000001514 detection method Methods 0.000 description 7
- 230000009545 invasion Effects 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 238000012258 culturing Methods 0.000 description 6
- 230000003698 anagen phase Effects 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 229920006395 saturated elastomer Polymers 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 239000007983 Tris buffer Substances 0.000 description 4
- 239000011259 mixed solution Substances 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 4
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- 239000006285 cell suspension Substances 0.000 description 3
- 230000003833 cell viability Effects 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 229940121647 egfr inhibitor Drugs 0.000 description 3
- 230000008014 freezing Effects 0.000 description 3
- 238000007710 freezing Methods 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 239000006166 lysate Substances 0.000 description 3
- 108010082117 matrigel Proteins 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 239000011148 porous material Substances 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 238000002331 protein detection Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 102000004022 Protein-Tyrosine Kinases Human genes 0.000 description 2
- 108090000412 Protein-Tyrosine Kinases Proteins 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 229960001686 afatinib Drugs 0.000 description 2
- ULXXDDBFHOBEHA-CWDCEQMOSA-N afatinib Chemical compound N1=CN=C2C=C(O[C@@H]3COCC3)C(NC(=O)/C=C/CN(C)C)=CC2=C1NC1=CC=C(F)C(Cl)=C1 ULXXDDBFHOBEHA-CWDCEQMOSA-N 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000013592 cell lysate Substances 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 238000007747 plating Methods 0.000 description 2
- 239000003531 protein hydrolysate Substances 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 230000008261 resistance mechanism Effects 0.000 description 2
- 229940121644 second-generation egfr tyrosine kinase inhibitor Drugs 0.000 description 2
- 238000011519 second-line treatment Methods 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 229940121646 third-generation egfr tyrosine kinase inhibitor Drugs 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 101150039808 Egfr gene Proteins 0.000 description 1
- 241000237858 Gastropoda Species 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- 239000005411 L01XE02 - Gefitinib Substances 0.000 description 1
- 239000005551 L01XE03 - Erlotinib Substances 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 229940122907 Phosphatase inhibitor Drugs 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 102000013127 Vimentin Human genes 0.000 description 1
- 108010065472 Vimentin Proteins 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 239000012670 alkaline solution Substances 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 238000001516 cell proliferation assay Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 238000012864 cross contamination Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000003235 crystal violet staining Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 108700021358 erbB-1 Genes Proteins 0.000 description 1
- 229960001433 erlotinib Drugs 0.000 description 1
- AAKJLRGGTJKAMG-UHFFFAOYSA-N erlotinib Chemical compound C=12C=C(OCCOC)C(OCCOC)=CC2=NC=NC=1NC1=CC=CC(C#C)=C1 AAKJLRGGTJKAMG-UHFFFAOYSA-N 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 229940121645 first-generation egfr tyrosine kinase inhibitor Drugs 0.000 description 1
- 239000000834 fixative Substances 0.000 description 1
- 229960002584 gefitinib Drugs 0.000 description 1
- XGALLCVXEZPNRQ-UHFFFAOYSA-N gefitinib Chemical compound C=12C=C(OCCCN3CCOCC3)C(OC)=CC2=NC=NC=1NC1=CC=C(F)C(Cl)=C1 XGALLCVXEZPNRQ-UHFFFAOYSA-N 0.000 description 1
- 229950007440 icotinib Drugs 0.000 description 1
- QQLKULDARVNMAL-UHFFFAOYSA-N icotinib Chemical compound C#CC1=CC=CC(NC=2C3=CC=4OCCOCCOCCOC=4C=C3N=CN=2)=C1 QQLKULDARVNMAL-UHFFFAOYSA-N 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- -1 p-EGFR Proteins 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000000751 protein extraction Methods 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 208000000649 small cell carcinoma Diseases 0.000 description 1
- 238000011272 standard treatment Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 210000005048 vimentin Anatomy 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of medical biology, and particularly discloses an ametinib drug-resistant cell strain, which is characterized in that the ametinib drug-resistant cell strain is an ametinib human lung adenocarcinoma cell strain NCI-H1975-AR, and the preservation number is CCTCC NO: C2022291. The drug-resistant cell strain H1975-AR can stably grow and passaged in a culture system with the action concentration of 1umol/L of the amotinib, the drug Resistance Index (RI) of the amotinib is 522.8, and the H1975-AR cell strain has T790M mutation, has no C797S mutation and shows an epithelial-mesenchymal transition phenotype; therefore, the African-resistant human lung adenocarcinoma cell strain H1975-AR constructed by the invention provides a drug-resistant cell model for researching the morphological and biological characteristics of an African-resistant human non-small cell lung cancer cell, the research and analysis of a non-small cell lung cancer drug-resistant mechanism on the African, the research and analysis of a non-small cell lung cancer drug-resistant related signal path, the anti-tumor drug sensitivity analysis, the preparation of an anti-tumor drug, the screening of a tumor drug-resistant reversal drug, the research of a more effective tumor treatment method and the like, and has higher scientific research and production application values.
Description
Technical Field
The invention belongs to the technical field of biological medicine. In particular to an ametinib resistant cell strain H1975-AR and application thereof.
Background
Lung cancer is one of the most common and highest mortality malignancies worldwide, with non-small cell lung cancer in 80% of cases. Because of the disease characteristics of lung cancer, more than half of lung cancer patients are transferred when the disease is diagnosed, the opportunity of radical operation is lost, and the survival rate is greatly reduced. In recent decades, with development of molecular diagnosis technology and development of targeted drugs, the treatment mode of lung cancer is not limited to traditional chemotherapy, and five-year survival rate is obviously improved. Wherein, EGFR-TKI treatment has remarkable curative effect in lung cancer people with EGFR gene activation mutation.
EGFR activating mutation causes EGFR tyrosine kinase domain to be in unstable conformation and dimerized, so that downstream signal channels of EGFR tyrosine kinase domain are activated, and growth and proliferation, invasion and migration and angiogenesis of tumor cells are promoted, so that tumor generation and development are caused. EGFR-TKIs can inhibit EGFR kinase domain activity by binding to the ATP binding site. Current EGFR-TKIs treatment is the first line standard treatment for lung adenocarcinoma patients with EGFR mutations, including the first generation EGFR-TKIs (gefitinib, erlotinib, icotinib), the second generation EGFR-TKIs (afatinib, dactinib) and the third generation EGFR-TKI (octtinib, vomittinib, amotinib). But all patients are still inevitably resistant to drug.
Ametinib is a novel, irreversible, highly selective, third generation EGFR-TKI with inhibitory effect on EGFR-sensitive mutations and T790M mutations. The results of the APOLLO clinical study show that Ameitinib is safe and effective for patients with T790M positive NSCLC who progress after advanced generation or second generation EGFR-TKIs treatment. At present, the ametinib is applied to the second-line treatment of patients with EGFR mutation in the clinical late stage, but the drug resistance mechanism of the ametinib for the second-line treatment is not completely clear, and no drug-resistant lung adenocarcinoma cell lines of the ametinib are reported at home and abroad at present.
Disclosure of Invention
Aiming at the problems and the defects existing in the prior art, the invention aims to provide an Ameitinib resistant cell strain H1975-AR and application thereof.
In order to achieve the aim of the invention, the technical scheme adopted by the invention is as follows:
the first aspect of the invention provides an Ameitinib resistant cell line H1975-AR, which is named as an Ameitinib resistant human lung adenocarcinoma cell line NCI-H1975-AR, and is preserved in China Center for Type Culture Collection (CCTCC), the preservation date is 9 months and 7 days in 2022, the preservation number is CCTCC NO: C2022291, and the preservation address is eight-path 299 university of Wuhan in Wuhan district of Hubei province.
The anti-ametinib human lung adenocarcinoma cell line NCI-H1975-AR is prepared by adopting an in vitro concentration gradient increasing method, namely by gradually increasing the concentration of an ametinib drug and continuously acting on the NCI-H1975 cell line with EGFR21 exon L858R mutation and T790M mutation to induce and establish the human lung adenocarcinoma drug-resistant cell line. The concrete construction method comprises the following steps:
(1) Human lung adenocarcinoma cell line NCI-H1975 (stock of this laboratory) with EGFR21 exon L858R mutation and T790M mutation was placed in RPMI1640 medium containing 10% fetal bovine serum at 37℃and 5% CO 2 Culturing in a saturated humidity incubator, and digesting and passaging by using trypsin digestion liquid (0.25% Trypsin-EDTA);
(2) At 1X 10 6 NCI-H1975 was inoculated into a 60mm dish and after 24 hours of adherence, a complete medium containing 10nmol/M of Ametinib was added and placed at 37℃with 5% CO 2 Culturing in a saturated humidity incubator; changing the culture medium containing the amotinib every 2-3 days (the concentration of the amotinib in the culture medium is unchanged), multiplying the concentration of the amotinib in the culture medium on the basis after the cells can tolerate the concentration of the amotinib, and continuously repeating the steps until NCI-H1975-AR can stably grow and passaging in the culture medium containing 1umol/L of the amotinib, thereby obtaining the amotinib-resistant lung adenocarcinoma of the inventionCell line NCI-H1975-AR.
The anti-ametinib human lung adenocarcinoma cell strain NCI-H1975-AR constructed by the invention can stably grow and passaged in a culture system with the action concentration of 1umol/L of ametinib, has the drug Resistance Index (RI) of 522.8 for the ametinib, and provides a drug resistant cell model for researching the morphological and biological characteristics of the ametinib resistant human non-small cell lung cancer cells, the drug resistance mechanism of the non-small cell lung cancer to the ametinib, the research analysis of the drug resistance related signal path of the non-small cell lung cancer, the anti-tumor drug sensitivity analysis, the preparation of the anti-tumor drug, the screening of the tumor drug resistance reversal drug, the research of more effective tumor treatment methods and the like, and has higher scientific research and production application values.
The second aspect of the invention provides an application of an afatinib-resistant human lung adenocarcinoma cell line NCI-H1975-AR in screening medicaments for reversing tumor resistance.
The third aspect of the invention provides an application of an African-resistant human lung adenocarcinoma cell line NCI-H1975-AR in preparing an anti-tumor medicament.
The fourth aspect of the invention provides the application of the Ameitinib-resistant human lung adenocarcinoma cell line NCI-H1975-AR in constructing an in vitro tumor drug-resistant cell model
According to the above application, preferably, the tumor is lung cancer. More preferably, the lung cancer is non-small cell cancer. Most preferably, the lung cancer is lung adenocarcinoma.
Compared with the prior art, the invention has the following beneficial technical effects:
the anti-ametinib human lung adenocarcinoma cell strain NCI-H1975-AR constructed by the invention can stably grow and passaged in a culture system with the action concentration of 1umol/L of ametinib, the drug Resistance Index (RI) of the ametinib is 522.8, and the H1975-AR cell strain has T790M mutation, has no C797S mutation and shows an epithelial-mesenchymal transition phenotype; therefore, the anti-ametinib human lung adenocarcinoma cell strain NCI-H1975-AR constructed by the invention provides a drug-resistant cell model for researching the morphological and biological characteristics of an ametinib drug-resistant human non-small cell lung cancer cell, the drug-resistant mechanism of the non-small cell lung cancer to the ametinib, the research and analysis of a drug-resistant related signal path of the non-small cell lung cancer, the anti-tumor drug sensitivity analysis, the preparation of an anti-tumor drug, the screening of tumor drug resistance reversal drugs, the research of a more effective tumor treatment method and the like, has higher scientific research and production application values, and is expected to generate good scientific research, economic and social benefits.
Drawings
FIG. 1 is a diagram showing the results of cell morphology observation of an Ameitinib-resistant cell line NCI-H1975-AR and a parent cell line NCI-H1975 thereof according to the present invention; wherein H1975 represents a parent cell line NCI-H1975, and H1975-AR represents an Ameitinib-resistant human lung adenocarcinoma cell line NCI-H1975-AR;
FIG. 2 shows the results of the viability test of the drug-resistant cell line NCI-H1975-AR and the parent cell NCI-H1975 of the invention under the action of different concentrations of Ameitinib; wherein, H1975 represents a parent cell strain NCI-H1975, H1975-AR represents an Ameitinib-resistant human lung adenocarcinoma cell strain NCI-H1975-AR;
FIG. 3 shows the results of the proliferation potency test of the drug-resistant cell line NCI-H1975-AR and the parent cell NCI-H1975 of the invention; wherein A represents the difference of proliferation capacity of a parent cell NCI-H1975 and an Ameitinib drug-resistant cell strain NCI-H1975-AR detected by an SRB cell proliferation experiment; b represents a flat cloning experiment to detect the difference of proliferation capacity of a parent cell NCI-H1975 and an Ameitinib drug-resistant cell strain NCI-H1975-AR; h1975 represents the parent cell line NCI-H1975, H1975-AR represents the Ameitinib-resistant human lung adenocarcinoma cell line NCI-H1975-AR;
FIG. 4 shows the results of the Transwell invasion and migration capacity test of the Ameitinib resistant cell line NCI-H1975-AR and NCI-H1975 parent cells of the present invention; wherein H1975 represents a parent cell line NCI-H1975, and H1975-AR represents an Ameitinib-resistant human lung adenocarcinoma cell line NCI-H1975-AR;
FIG. 5 shows the results of Western blot experiments of EGFR and its downstream proteins expressed in the parent cells of the African drug resistant cell line NCI-H1975-AR and NCI-H1975 of the invention; wherein H1975 represents a parent cell line NCI-H1975, and H1975-AR represents an Ameitinib-resistant human lung adenocarcinoma cell line NCI-H1975-AR;
FIG. 6 shows the results of Western blotting experiments of the EMT-related protein markers of the African drug-resistant cell line NCI-H1975-AR and NCI-H1975 parent cells of the present invention; wherein H1975 represents a parent cell line NCI-H1975, and H1975-AR represents an Ameitinib-resistant human lung adenocarcinoma cell line NCI-H1975-AR; .
Detailed Description
The following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, components, and/or combinations thereof.
The experimental methods in the following examples, in which specific conditions are not specified, are all conventional in the art or according to the conditions suggested by the manufacturer; the reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail with reference to specific embodiments.
Example 1: construction of an Ameitinib-resistant human lung adenocarcinoma cell line NCI-H1975-AR
The anti-ametinib human lung adenocarcinoma cell line NCI-H1975-AR is prepared by adopting an in vitro concentration gradient increasing method, namely by gradually increasing the concentration of an ametinib drug and continuously acting on the NCI-H1975 cell line with EGFR21 exon L858R mutation and T790M mutation to induce and establish the human lung adenocarcinoma drug-resistant cell line. The concrete construction method comprises the following steps:
(1) Human lung adenocarcinoma cell line NCI-H1975 (stock of this laboratory) with EGFR21 exon L858R mutation and T790M mutation was placed in a bovine serum containing 10% of fetalIn RPMI1640 medium at 37℃in 5% CO 2 Culturing in a saturated humidity incubator, and digesting and passaging by using trypsin digestion liquid (0.25% Trypsin-EDTA);
(2) At 1X 10 6 NCI-H1975 was inoculated into a 60mm dish and after 24 hours of adherence, a complete medium containing 10nmol/M of Ametinib was added and placed at 37℃with 5% CO 2 Culturing in a saturated humidity incubator; the culture medium containing the amotinib is replaced every 2-3 days (the concentration of the amotinib in the culture medium is unchanged), when the cell density grows to 80-90%, cells are digested, passaged according to the proportion of 1:3, and after the cells are attached to the wall, the cells are placed in a newly prepared complete culture medium containing the amotinib with the original concentration for continuous culture, and the culture medium is replaced once every 2-3 days. Thirdly, when the cell density grows to 80-90%, digesting the cells, carrying out passage according to the ratio of 1:3, and placing the cells in a complete culture medium with the concentration of 20nmol/L for continuous culture after the cells are attached to the wall; and replacing the culture medium once in 2-3 days, digesting the cells when the cell density grows to 80-90%, carrying out passage according to the ratio of 1:3, and placing the cells in a complete culture medium with the concentration of 20nmol/L of the amotinib for continuous culture. The concentration of the amotinib in the culture medium increases from 10nmol/L to 20nmol/L to 50nmol/L to 100nmol/L to 200nmol/L to 300 nmol/L in the smooth order
Culturing is carried out under the conditions of nmol/L-400 nmol/L-500 nmol/L-600 nmol/L-800 nmol/L-1000 nmol/L. Each drug concentration is passaged for at least 2 times until cells can stably grow in a culture medium containing 1000nmol/L of the amotinib, thus obtaining the amotinib-resistant human lung adenocarcinoma cell strain NCI-H1975-AR, and the cell strain is preserved in China center for type culture collection of university of Wuhan at 9 months of 2022, with the preservation number of CCTCC NO: C2022291 and the preservation address of eight-path No. 299 Wuchang district of Wuhan, hubei province.
Example 2: freezing and resuscitating of Acetinib-resistant human lung adenocarcinoma cell line NCI-H1975-AR
Freezing: the drug-resistant cells obtained in example 1 were placed in serum-free frozen stock solution of the frozen stock tube, and the frozen stock tube was stored in liquid nitrogen. Serum-free frozen stock solutions were purchased from new siramese biotechnology limited, new siramese, su. Freezing at-80 deg.C overnight, and storing in liquid nitrogen.
Resuscitating: the frozen tube containing the cells was removed from the liquid nitrogen and immediately placed in a 37℃water bath with gentle shaking to thaw the frozen material within 1 minute. Placing the thawed cell suspension in a 15mL sterile centrifuge tube, adding 5mL of H1975 culture medium, centrifuging at 1000 rpm/min for 5min, discarding supernatant, adding 8mL of complete culture medium, slightly blowing and mixing to obtain a mixed solution, sucking the mixed solution, placing into a 100mm cell culture dish, placing into a carbon dioxide incubator, and adding 5% CO at 37deg.C 2 Culturing under saturated humidity, changing culture medium for 2-3 days, and digesting when cell density reaches 80-90%, and subculturing at 1:3.
Example 3: morphological observation of Acetinib-resistant human lung adenocarcinoma cell line NCI-H1975-AR
1. Experimental cell line material: the Aftetinib-resistant human lung adenocarcinoma cell line NCI-H1975-AR and the parent human lung adenocarcinoma cell line NCI-H1975 constructed in example 1.
2. The experimental method comprises the following steps: aftetinib resistant cell strain NCI-H1975-AR and parent human lung adenocarcinoma cell strain NCI-H1975 are respectively inoculated into a 60mm culture dish, and after the cells grow to the logarithmic growth phase, the living cell morphology is observed under an inverted phase contrast microscope and photographed.
3. Experimental results:
the results of the morphological observation are shown in FIG. 1. As can be seen from FIG. 1, the drug-resistant cell line NCI-H1975-AR of Ameitinib and the parent cell line NCI-H1975 thereof all have fusiform shapes, and the morphology is not obviously different.
Example 4: determination of drug resistance index of Acetinib-resistant human lung adenocarcinoma cell line NCI-H1975-AR
1. The experimental method comprises the following steps:
aftetinib-resistant human lung adenocarcinoma cell line NCI-H1975-AR and parental cell NCI-H1975 in the logarithmic growth phase were inoculated into 96-well plates at 3000 cells/100. Mu.L/well, respectively. 200. Mu.L of PBS solution was added to the 96-well plate at the edge of each well to reduce medium evaporation during the experiment. After the plating is finished, lightly beating the edge of the 96-well plate, and dividing cells in the holes three times up, down, left and rightEvenly distributed, and placed in a cell incubator for continuous incubation. About 24 hours after cell plating, 100 mu L of prepared complete culture medium containing the amotinib with different concentrations (the concentration gradient of the amotinib in the complete culture medium is set to be 1nM, 10nM, 100nM and 1000 nM) is added into each hole, and 3-6 compound holes are arranged in each concentration to reduce errors; a blank (with complete medium only, without drug in the medium) was also set. After the dosing is finished, the mixture is placed in a cell incubator for further incubation for 72 hours. Cell culture medium was discarded before detection, and each well was washed 2 times with 100 μlpbs solution. A pre-chilled 10% trichloroacetic acid solution (10 g trichloroacetic acid powder in 100mL ddH) was then added at 100. Mu.L/well 2 O), and after standing for 5 minutes, the 4-degree refrigerator was fixed for 1 hour. The trichloroacetic acid solution was discarded and the 96-well plate was washed four times with running water. The action should be gentle when cleaning the orifice plate, avoiding the flow of water to be fast, leading to the shedding of cells fixed on the bottom surface of the orifice plate. And (5) cleaning and airing at room temperature. 0.4% SRB solution (0.4 g SRB powder was dissolved in 1% acetic acid solution and fixed to 100 mL) was added at 100. Mu.L/well, and stained with shaking in the dark for 20min. After staining, 1% acetic acid solution (1 mL 100% acetic acid plus ddH) 2 O and volume to 100 mL) was washed four times to completely remove unbound dye and air dried at room temperature. Finally, 10mM Tris-base alkaline solution (0.1211 g Tris powder in ddH) was added at 100. Mu.L/well 2 O, constant volume to 100 mL), shaking and mixing for 5min to completely dissolve the SRB dye combined with the protein in Tris-base alkali solution. Or dissolving for 30min at room temperature. Absorbance values at 540nm were measured using a microplate reader. After correction for OD values, cell viability per well was calculated according to the formula and drug dose-response plots were drawn using Graphpad Prism 9.0 software and the half maximal inhibitory concentration (IC 50) and drug Resistance Index (RI) of H1975-AR and parental cells NCI-H1975 on amotinib were calculated. Wherein, the calculation formula of the cell viability is: cell viability (%) = experimental OD value/control OD value x 100%; the calculation formula of the drug Resistance Index (RI) is as follows: drug Resistance Index (RI) =drug resistant cell IC 50/parental cell IC50.
2. Experimental results:
the result of the survival rate detection of the Acetinib resistant cell line NCI-H1975-AR under the action of different concentrations of Acetinib is shown in figure 2. As can be seen from FIG. 2, the survival rate of the drug-resistant cell strain NCI-H1975-AR is far greater than that of the parent cell NCI-H1975 under the action of the same concentration of armetidine. The drug resistance index of the African-resistant human lung adenocarcinoma cell line NCI-H1975-AR is 522.8.
Example 5: detection of proliferation capacity of Ameitinib-resistant human lung adenocarcinoma cell line NCI-H1975-AR
1. SRB cell proliferation assay to detect the proliferation potency of the Ameitinib-resistant human lung adenocarcinoma cell line NCI-H1975-AR:
(1) The experimental method comprises the following steps: the parent cell strain NCI-H1975 and the drug-resistant cell strain NCI-H1975-AR in the logarithmic growth phase were inoculated into 96-well plates at 2000 cells/100. Mu.l/well, respectively. After overnight attachment of the cells, the medium was discarded at 0h, 24h, 48h, 72h, 96h, respectively, and the cells were fixed using 10% trichloroacetic acid solution. After the 96-well plate is dried at room temperature, adding 100 mu L of SRB dye liquor (prepared by 1% acetic acid) of 0.4% (w/v) into each well, pouring out the dye liquor after dyeing for 30min, washing for 4 times by 1% (v/v) acetic acid, removing unbound dye, and drying at room temperature; the dye bound to the cellular protein was dissolved with 100. Mu.L of unbuffered Tris-base lye (10 mM, pH=10.5) and shaken on a horizontal shaker for 20min, and the absorbance was measured using an microplate reader at 540 nm. Growth was plotted using Graphpad Prism 9.0 software and the control cell proliferation was observed.
(2) Experimental results:
the detection results are shown in FIG. 3A. As can be seen from FIG. 3A, the drug-resistant cell line NCI-H1975-AR has a weaker proliferation capacity than the parent cell line NCI-H1975.
2. The proliferation capacity of the Ameitinib-resistant cell line NCI-H1975-AR is detected by a plate cloning experiment:
(1) The experimental method comprises the following steps:
african-resistant human lung adenocarcinoma cell line NCI-H1975-AR in the logarithmic growth phase and parent cell NCI-H1975 thereof are selected, inoculated into a 6-pore plate according to the density of 500-1000 cells/pore, and are put into a cell incubator for incubation for 7-14 days after being gently shaken and uniformly mixed. Cell growth was observed and the culture was stopped by discarding the medium when macroscopic clones appeared in the well plate. After rinsing 2 times with PBS, fixation was performed for 30 minutes using paraformaldehyde. The fixative was discarded, the crystal violet stained, then dried at room temperature and counted.
2. Experimental results:
the detection results are shown in FIG. 3B. As can be seen from FIG. 3B, the drug-resistant cell line NCI-H1975-AR has a weaker proliferation capacity than the parent cell line NCI-H1975.
Example 6: detection of invasion and migration ability of Ameitinib-resistant human lung adenocarcinoma cell line NCI-H1975-AR
1. The experimental method comprises the following steps:
(1) Migration experiment:
a. transwell cells and 24-well plates with a pore size of 8 μm were prepared for the experiments. Drug resistant cell line NCI-H1975-AR in logarithmic growth phase and its parent cell line NCI-H1975 were selected and starved with serum-free medium for 12-24H before the experiment. Cells were resuspended after pancreatin digestion and harvested by centrifugation at 1000rpm for 5 min. The supernatant was discarded and resuspended using serum-free 1640 medium.
b. After the cell is resuspended and thoroughly mixed, the cell concentration is adjusted to (1-10). Times.10 5 Cell suspensions were obtained per cell/mL. 200. Mu.L of the cell suspension was added to the upper chamber of the Tranwell chamber. And 800. Mu.L of medium containing 20% FBS was added to the corresponding wells. The culture medium should cover the lower surface of the Transwell chamber and have no bubbles in front of the lower surface. Placing the cells into a cell culture box for further culture for 24 hours.
c. The medium in the upper chamber and the medium in the corresponding wells was discarded and the culture was terminated. The cells that did not penetrate the upper chamber were gently removed by washing twice with PBS and using a wet cotton swab. After fixation of paraformaldehyde for 30min, crystal violet staining for 30min. Finally, the sample is observed under an inverted microscope, photographed and counted.
(2) Invasion experiments:
placing the Matrigel which is packaged in advance in a 4-degree refrigerator for thawing, using a serum-free 1640 culture medium, and diluting the Matrigel according to a ratio of 1:5. The gel is evenly spread on the upper chamber of a Transwell cell according to 60 mu L/hole, and then is put into a 37-DEG cell incubator for incubation for 2-3 hours so as to make the matrigel gel. The steps of preparation, culture, fixation and staining of the residual cells are the same as those of the Transwell cell migration experiment (i.e., steps a, b and c of the migration experiment).
2. Experimental results:
the detection results are shown in FIG. 4. As can be seen from FIG. 4, the drug resistant cell line NCI-H1975-AR of Azatinib has significantly higher invasion and migration capacity than the parent cell NCI-H1975.
Example 7: EGFR and downstream pathway protein and Epithelial Mesenchymal Transition (EMT) related protein detection in ametinib-resistant human lung adenocarcinoma cell line NCI-H1975-AR
Extracting protein lysate of the African-resistant human lung adenocarcinoma cell line NCI-H1975-AR; and simultaneously, carrying out EGFR, p-EGFR, akt, p-Akt, erk and p-Erk protein immunoblotting experiments and EMT related protein detection on the extracted protein lysate.
1. The experimental method comprises the following steps:
the cells are treated according to specific experimental requirements, and cell lysate is prepared before protein samples are extracted: and adding the protease inhibitor mixed solution and the phosphatase inhibitor mixed solution into the RIPA lysate with the required dosage according to the proportion of 100:1 before using, and uniformly mixing. The whole protein extraction process was performed on ice.
After the cells were treated as required, the medium was discarded, the cells were washed twice with PBS, and the cell lysate was directly added dropwise to the cell surface (six well plates 150-250. Mu.L per well) and the lysate was added. The lysate was evenly distributed on the cell surface by the number of shots (note that cross-contamination between samples was not required). The protein samples were then collected in EP tubes using a spatula after 10min lysis on ice. Centrifuge 12000g for 5min at 4 ℃, take supernatant in new EP tube, add deionized water and 5 x protein loading buffer after subsequent protein concentration determination to configure protein samples to uniform concentration. And then carrying out conventional electrophoresis, membrane transfer and antibody incubation according to a western blotting method, and exposing by a machine.
2. Experimental results:
the results of western blotting of EGFR and its downstream pathway proteins in the African-resistant human lung adenocarcinoma cell line H1975-AR are shown in FIG. 5.
As shown in FIG. 5, 50nmol/L of Ametinib can significantly inhibit the expression of proteins such as p-EGFR, p-Akt, p-Erk and the like in a parent cell H1975; however, no expression of p-EGFR protein was detected in the Ametinib resistant cell line H1975-AR, and 50 to 500nmol/L of Ametinib could not significantly reduce the expression levels of the resistant cell line H1975-AR p-Akt and p-Erk proteins, suggesting that bypass activation exists in the resistant cell line.
The results of the EMT-associated protein detection are shown in FIG. 6. As shown in FIG. 6, compared with the parent cell strain NCI-H1975, the E-cadherein expression of the drug-resistant cell strain H1975-AR is obviously reduced, the Snail expression is obviously increased, and the Vimentin expression is not obviously different; further combining with the results of Transwell migration and invasion experiments, the invasion and migration capacity of the Ameitinib resistant cell strain H1975-AR are enhanced, which suggests that the resistant cell strain NCI-H1975-AR has an epithelial-mesenchymal transition phenotype.
The above-described embodiments are provided to illustrate the gist of the present invention, but are not intended to limit the scope of the present invention. It will be understood by those skilled in the art that various modifications and equivalent substitutions may be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention.
Claims (4)
1. The ametinib drug-resistant cell strain is an ametinib-resistant human lung adenocarcinoma cell strain NCI-H1975-AR, and the preservation number is CCTCC NO: C2022291.
2. Use of an ametinib-resistant cell line according to claim 1 for screening a drug that reverses resistance to lung adenocarcinoma.
3. Use of an ametinib-resistant cell line according to claim 1 for screening and preparing an anti-lung adenocarcinoma drug.
4. Use of an ametinib-resistant cell line according to claim 1 in constructing an extracorporal lung adenocarcinoma drug-resistant cell model.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211740359.3A CN115927187B (en) | 2022-12-30 | 2022-12-30 | Ameitinib resistant cell strain NCI-H1975-AR and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211740359.3A CN115927187B (en) | 2022-12-30 | 2022-12-30 | Ameitinib resistant cell strain NCI-H1975-AR and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115927187A CN115927187A (en) | 2023-04-07 |
| CN115927187B true CN115927187B (en) | 2024-01-23 |
Family
ID=86656012
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211740359.3A Active CN115927187B (en) | 2022-12-30 | 2022-12-30 | Ameitinib resistant cell strain NCI-H1975-AR and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115927187B (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103627674A (en) * | 2013-11-29 | 2014-03-12 | 国家纳米科学中心 | Multidrug-resistant cell strain of human lung adenocarcinoma as well as preparation method and use of cell strain |
| CN111218424A (en) * | 2019-12-12 | 2020-06-02 | 广州医科大学附属第一医院 | Oxitinib drug-resistant cell strain NCI-H1975/AR and application thereof |
| CN111235218A (en) * | 2019-12-12 | 2020-06-05 | 广州医科大学附属第一医院 | Third-generation EGFR-TKI drug-resistant cell strain and application thereof |
| CN111793604A (en) * | 2020-06-05 | 2020-10-20 | 潘慧 | Human non-small cell lung cancer cell strain H1975/OR resistant to oxitinib and application thereof |
| CN112195153A (en) * | 2020-10-09 | 2021-01-08 | 中南大学 | EGFR inhibitor drug-resistant human non-small cell lung cancer cell strain and application thereof |
| AU2020103364A4 (en) * | 2020-11-10 | 2021-01-21 | The First Affiliated Hospital Of Guangzhou Medical University | Ositinib-resistant cell line NCI-H1975/AR and application thereof |
-
2022
- 2022-12-30 CN CN202211740359.3A patent/CN115927187B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103627674A (en) * | 2013-11-29 | 2014-03-12 | 国家纳米科学中心 | Multidrug-resistant cell strain of human lung adenocarcinoma as well as preparation method and use of cell strain |
| CN111218424A (en) * | 2019-12-12 | 2020-06-02 | 广州医科大学附属第一医院 | Oxitinib drug-resistant cell strain NCI-H1975/AR and application thereof |
| CN111235218A (en) * | 2019-12-12 | 2020-06-05 | 广州医科大学附属第一医院 | Third-generation EGFR-TKI drug-resistant cell strain and application thereof |
| CN111793604A (en) * | 2020-06-05 | 2020-10-20 | 潘慧 | Human non-small cell lung cancer cell strain H1975/OR resistant to oxitinib and application thereof |
| CN112195153A (en) * | 2020-10-09 | 2021-01-08 | 中南大学 | EGFR inhibitor drug-resistant human non-small cell lung cancer cell strain and application thereof |
| AU2020103364A4 (en) * | 2020-11-10 | 2021-01-21 | The First Affiliated Hospital Of Guangzhou Medical University | Ositinib-resistant cell line NCI-H1975/AR and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115927187A (en) | 2023-04-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107663539A (en) | Circular rna circ PTGR1 purposes | |
| CN105524924A (en) | Cyclic RNA circ-ZKSCAN1 use | |
| CN108486060A (en) | It is a kind of to be used to treat excretion body of tumour and its preparation method and application | |
| CN104480062A (en) | Separation and culture method for different cellular components of human mammary tissue | |
| CN111793604B (en) | Ornitinib-resistant human non-small cell lung cancer cell strain H1975/OR and application thereof | |
| CN115927187B (en) | Ameitinib resistant cell strain NCI-H1975-AR and application thereof | |
| CN110229901A (en) | Gene hsa_circ_0027089 relevant to triple negative breast cancer diagnosis and treatment and its application | |
| CN104174032A (en) | siRNA molecular composition and application thereof in treatment on pathological scars | |
| CN114032237A (en) | Circular non-coding RNA circSTK39 and application thereof in preventing and treating atherosclerosis | |
| CN113789350A (en) | Construction method of mammal esophageal squamous epithelial immortalized cell line, constructed cell line and organoid thereof | |
| CN110051841B (en) | Application of NAT10 inhibitor in preparation of medicine for inhibiting HIF expression | |
| CN110229900A (en) | Gene hsa_circ_0103520 relevant to breast cancer diagnosis and treatment and its application | |
| CN108465108A (en) | A kind of specific gene target spot prevented or treat glioma | |
| CN116200340B (en) | Aftetinib-resistant human lung adenocarcinoma cell line PC9-AR and application thereof | |
| CN108992457A (en) | The application of miR-144-3p and its target gene in the drug that preparation adjusts human heart Fibroblast Function | |
| CN106636368A (en) | Application of miR-130a to diagnosis, treatment and prognosis of ovarian cancer | |
| CN105396136A (en) | Application of CCN1(Cyr61) to treatment of diseases related to skin injuries and atrophoderma | |
| CN113817684B (en) | Chinese lung adenocarcinoma cell line with high lymph node metastasis potential and application thereof | |
| CN114410589B (en) | Application of ETV4 gene in reprogramming Cheng Zeng-derived scar fibroblast | |
| CN114908172B (en) | Application of APOBEC3B in diagnosis, prognosis prediction and treatment of prostate cancer | |
| CN110257522A (en) | Gene hsa_circ_0045881 relevant to breast cancer diagnosis and treatment and its application | |
| CN109295101A (en) | It is overexpressed miR-125a-5p lentiviral vector construction method and its application | |
| CN108949974A (en) | E3 ubiquitin ligase ASB3 is preparing the application in cancer treatment drug | |
| CN114164270B (en) | Application of CRIP2 in detecting resistance of prostate cancer to docetaxel and reversing resistance of prostate cancer to docetaxel | |
| CN115058517B (en) | Application of PRKD1 gene as non-small cell lung cancer diagnosis or prognosis marker |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |