CN110426516A - The protein marker of auxiliary identification Rituximab drug-resistant type ABC-DLBCL cell and its application - Google Patents
The protein marker of auxiliary identification Rituximab drug-resistant type ABC-DLBCL cell and its application Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/06—Anti-neoplasic drugs, anti-retroviral drugs, e.g. azacytidine, cyclophosphamide
Abstract
The invention discloses the protein marker of auxiliary identification Rituximab drug-resistant type ABC-DLBCL cell and its applications.Present invention protection for detect the substances of specific markers preparation for detect ABC-DLBCL cell to be measured whether the application in the kit for being Rituximab drug-resistant type ABC-DLBCL cell.Marker first -1 is any one or combination in 8 protein.Marker first -2 is any one or combination in 45 protein.Marker second -1 is any one or combination in 5 protein.Marker second -2 is any one or combination in 77 protein.Marker third is any one or combination in 21 protein.Marker fourth is any one or combination in 29 protein.The present invention successfully constructs Rituximab drug-resistant cell strain, it was found that multiple protein markers.The cell model and result of study for further research Rituximab drug resistance and overcome drug resistance to provide foundation.
Description
Technical field
The present invention relates to the protein marker of auxiliary identification Rituximab drug-resistant type ABC-DLBCL cell and its applications.
Background technique
Non-Hodgkin lymphoma (Non-Hodgkin lymphoma) is common lymphoid tissue malignant tumour, is first 10
One of cancer mortality cause of disease, in recent years, China's non-Hodgkin lymphoma disease incidence are also in obvious ascendant trend.It wherein diffuses big
B cell lymphoma (DLBCL) is the hypotype of the most common non-Hodgkin lymphoma (NHL), accounts about annual new diagnosis NHL case
32.5%, be one group in many-sided malignant tumour with very big heterogeneity such as clinical manifestation, tissue morphology and prognosis.
DLBCL can occur at any position of any organ and body, and clinical manifestation is the aggressive course of disease.According to origin of cell difference
DLBCL point is activating B cell sample DLBCL (ABC-DLBCL), Germinal center B cell sample DLBCL (GCB-DLBCL) and unfiled
Type, ABC-DLBCL poor prognosis and easily infringement marrow.But in the East Asia crowd including Chinese, ABC-DLBCL institute accounting
Example is significantly larger than American-European countries.
DLBCL unified first-line treatment scheme is that (R refers to RTX Metro to rituximab combination R-CHOP chemotherapy at present
China, C refer to that CTX cyclophosphamide, H refer to that ADM adriamycin, O refer to that VCR vincristine, P refer to Pre prednisone).
Rituximab, corresponding English name are Rituximab.Although rituximab combination R-CHOP chemotherapy can significantly improve
The final result (total cure rate about 60%) of DLBCL patient, but bad (the long-term nothing of the first-line treatment prognosis of ABC-DLBCL patient
Progress existence cannot obtain completely slow less than 50%), and in the drug resistant patient of first-line treatment more than 70% conventional two wires scheme
Solution.
ABC-DLBCL and GCB-DLBCL is because signal path difference leads to Different therapeutical effect, and latest edition NCCN guide is also bright
Really point out that first visit patient must take tissue division origin of cell.Therefore, how drug resistance ABC- after screening R-CHOP Regimen Chemotherapy
DLBCL patient is clinical major issue urgently to be resolved.
Summary of the invention
The object of the present invention is to provide auxiliary identification Rituximab drug-resistant type ABC-DLBCL cell protein marker and
It is applied.
The present invention also protects the substance for detecting specific markers for detecting ABC-DLBCL cell to be measured to be in preparation
Application in the no kit for Rituximab drug-resistant type ABC-DLBCL cell；The specific markers are for following (a) or (b)
Or (c) or (d) or (e) or (f) or (g) or (h):
(a) protein markers first -1；
(b) protein markers first -2；
(c) protein markers second -1；
(d) protein markers second -2；
(e) protein marker third；
(f) protein marker fourth；
(g) protein markers first -1, protein markers second -1, the group of protein marker third and protein marker fourth
It closes；
(h) protein markers first -2, protein markers second -2, the group of protein marker third and protein marker fourth
It closes；
Protein markers first -1 described above is any one or any combination (8 albumen in following protein
Matter): Nicotinate-nucleotide pyrophosphorylase；PCN；Zizimin-3；NAPRTase；
Immunoglobulin heavy constant gamma 1；GPI；Valine--tRNA ligase,mitochondrial；
HSPA1A；
Protein markers first -2 described above is any one or any combination (45 albumen in following protein
Matter): Nicotinate-nucleotide pyrophosphorylase；PCN；Zizimin-3；NAPRTase；
Immunoglobulin heavy constant gamma 1；GPI；Valine--tRNA ligase,mitochondrial；
HSPA1A；FLN-A；Vimentin isoform 1；Tubulin-specific chaperone D；CD48protein；
Adenosine deaminase isoform 1；GK；Thioredoxin；PC4protein；Phosphoinositide 3-
kinase adapter protein 1；BCL2-like 1isoform 3；Putative deoxyribonuclease
TATDN1；Epididymis secretory sperm binding protein；Protein CutA；VAV-2；UPD；
Lymphocyte-specific protein 1；Bcl2-L-5；Gasdermin-D；MTHFS；Family with sequence
similarity 49,member B,isoform CRA_a；T-cell leukemia/lymphoma 1A,isoform CRA_
b；Unconventional myosin-Ig；ABC50protein；CSTB protein；Sialophorin(GpL115,
leukosialin,CD43),isoform CRA_a；Pyruvate kinase；EBF2protein；CNP；Tubulin
5beta；TAP1；Testicular tissue protein Li 180；Squalene monooxygenase；PMI；RICH-
1；Proto-oncogene c-Rel；Polymerase(DNA directed),epsilon 3(P17subunit),isoform
CRA_a；Unconventional myosin-Id；
Protein markers second -1 described above is any one or any combination (5 albumen in following protein
Matter): EEF1A1protein；Protein unc-45homolog A；LPCAT-1；ENO2protein；Fanconi anemia,
complementation group I；
Protein markers second -2 described above is any one or any combination (77 albumen in following protein
Matter): ANT 2；Protein kinase C substrate 80K-H isoform 1；ColGalT 1；Heat shock
protein 90Bb；PMVK protein；Atlastin-3；TRG-8；NACHT,LRR and PYD domains-
containing protein 2；Guanidinoacetate N-methyltransferase；Histone
acetyltransferase 1；Cathepsin S；N-alpha acetyl transferase 40；Minor
histocompatibility antigen H13；EH-domain containing 1,isoform CRA_b；ARHGAP45；
Abhydrolase domain-containing protein 11；Oligosaccharyl transferase subunit
DAD1；Pleckstrin homology domain-containing family F member 1；Protein YIF1A；
Ubiquitin-conjugating enzyme E2S；Reticulon；MRE11A protein；Thymidine
phosphorylase；GMF-gamma；B-cell receptor-associated protein 29；PKC-B；Aspartate
aminotransferase；TDP43isoform H；RNA-binding protein Raly；RNA demethylase
ALKBH5；Methyltransferase-like protein 1；Torsin-1A-interacting protein 1；
Probable bifunctional dTTP/UTP pyrophosphatase/methyltransferase protein；
DCN1-like protein；Polycomb protein SUZ12；Thymidine kinase；PTDSS1protein；
Signal peptidase complex subunit 3；A-LAP；N-terminal kinase-like protein；
Stromal interaction molecule 1；DNA ligase；Ubiquitin-protein ligase G2；PHD
finger protein 6,isoform CRA_d；Oligosaccharyl transferase 48kDa subunit；
Porphobilinogen deaminase；Transmembrane protein 126A；PPIase FKBP11；
Oligoribonuclease,mitochondrial；Hypoxia up-regulated protein 1；Leukocyte
antigen CD37；Signal peptidase complex catalytic subunit SEC11；Importin
subunit alpha；Immunoglobulin heavy constant gamma 3；hRAD50；Mitochondrial
import inner membrane translocase subunit TIM50；Very-long-chain 3-oxoacyl-CoA
synthase；Centromere protein V；tRNA 2'-phosphotransferase 1；DNA(cytosine-5)-
methyltransferase；DEAD(Asp-Glu-Ala-Asp)box polypeptide 41,isoform CRA_a；
Checkpoint protein；Mesencephalic astrocyte-derived neurotrophic factor；AP
complex subunit sigma；Transmembrane protein 214；RFC37；LRP chaperone MESD；
PTPRC-associated protein；Pro-interleukin-16；RNA helicase；Glucosamine 6-
phosphate N-acetyltransferase；Protein FAM49A；EEF1A1protein；Protein unc-
45homolog A；LPCAT-1；ENO2protein；Fanconi anemia,complementation group I；
Protein markers third described above are any one or any combination (21 protein) in following protein:
Serine/threonine-protein kinase PRP4homolog；Choline dehydrogenase,isoform
CRA_a；Leucine-rich repeat-containing protein 57；3-hydroxybutyrate
dehydrogenase,type 2,isoform CRA_b；Epiplakin；T-lymphocyte activation antigen
CD86；Transmembrane protein 223；Salt-tolerant protein；SLAMF1protein；
PDDC1protein；Myosin IC,isoform CRA_a；Iron-responsive element binding protein
2,isoform CRA_a；Protein kinase C and casein kinase substrate in neurons
protein 1；Tether-containing UBX domain for GLUT4；Biogenesis of lysosome-
related organelles complex 1subunit 3；Stratifin；TNF alpha-induced protein 2；
IL-27B；Transmembrane protein 65；hIRIP；BAI1-associated protein2-like protein
1；
Protein markers fourth described above is any one or any combination (29 protein) in following protein:
Activating signal cointegrator 1complex subunit 2,isoform CRA_a；UDP-N-
acetylglucosamine--dolichyl-phosphate N-acetylglucosaminephosphotransferase；
Etoposide induced 2.4mRNA,isoform CRA_a；Mitochondrial chaperone BCS1；
POLD3protein；Met proto-oncogene(Hepatocyte growth factor receptor),isoform
CRA_c；Putative bifunctional UDP-N-acetylglucosamine transferase and
deubiquitinase ALG13；Ribosome-binding protein 1；Cytochrome b-245beta
polypeptide isoform 1；Mirror-image polydactyly 1isoform 4；Uncharacterized
protein C3orf38；Histone H2B；Actin,cytoplasmic 1；Ceramide synthase 2；Serine/
threonine-protein kinase26；CD22protein；Bromodomain adjacent to zinc finger
domain,1A,isoform CRA_c；Histone lysine demethylase PHF8；ATP synthase
mitochondrial F1complex assembly factor 1；Reticulophagy regulator 3；
Cytochrome c oxidase subunit 2；Endonuclease domain-containing 1protein；KDEL
receptor 1；FSAP；M1G-methyltransferase；Myopathy with excessive autophagy
protein；GPx-7；GINS complex subunit 4；PH domain-containing family F member 2.
The present invention also protects the substance for detecting specific markers in preparation for detecting whether subject has occurred
Application in the drug resistant kit of Rituximab；The specific markers are protein markers first -1 or protein markers
First -2；The subject is ABC-DLBCL patient.
The subject is the ABC-DLBCL patient not treated.
The subject is the ABC-DLBCL patient for having carried out Rituximab treatment.Before being treated with the subject
ABC-DLBCL cell is compared, if carrying out specific marker in the ABC-DLBCL cell at a certain moment after Rituximab treatment
The abundance of object (protein markers first -1) is 5 times or more, and the moment, Rituximab drug resistance occurred for subject.It is tested with this
ABC-DLBCL cell before person treats is compared, if carrying out the ABC-DLBCL at a certain moment after Rituximab treatment
The abundance of specific markers (protein markers first -2) is 2 times or more in cell, and the moment, Rituximab occurred for subject
Drug resistance.
The subject is the ABC-DLBCL patient for having carried out rituximab combination R-CHOP chemotherapy.With the subject
ABC-DLBCL cell before being treated is compared, if the ABC-DLBCL for carrying out a certain moment after Rituximab treatment is thin
The abundance of specific markers (protein markers first -1) is 5 times or more in born of the same parents, and it is resistance to that Rituximab has occurred for moment subject
Medicine.Compared with the ABC-DLBCL cell before the subject treats, if carrying out a certain moment after Rituximab treatment
ABC-DLBCL cell in specific markers (protein markers first -2) abundance be 2 times or more, the moment, subject sent out
Raw Rituximab drug resistance.
The present invention also protects a kind of method for preparing Rituximab drug-resistant type ABC-DLBCL cell, includes the following steps:
Parental cell is cultivated and passed on, during the cultivation process using Rituximab processing (specially from low concentration to high concentration
Substep cultivate), detect parental cell and every time passage after cell in specific markers abundance；The specific markers are
Protein markers first -1 or protein markers first -2；When the specific markers are protein markers first -1, with parent
Cell is compared, if the abundance of specific markers is 5 times or more in the cell after passage, the cell after the passage is
Rituximab drug-resistant type ABC-DLBCL cell；When the specific markers are protein markers first -2, with parental cell phase
Than if the abundance of specific markers is 2 times or more in cell after passage, cell after the passage is Rituximab drug resistance
Type ABC-DLBCL cell；The parental cell is ABC-DLBCL cell.
The parental cell is non-Rituximab drug-resistant type ABC-DLBCL cell.
The parental cell is SU-DHL-2 cell.
The parental cell is the SU-DHL-2 cell line that ATCC number is CRL-2956.
When specific markers are the combination of multiple proteins, abundance is the abundance that 2 times or more refers to every kind of protein
It is 2 times or more.
When specific markers are the combination of multiple proteins, abundance is 5 times or more the abundance for referring to every kind of protein
It is 5 times or more.
The present invention also protects the substance for detecting specific markers in preparation for detecting whether subject has occurred
Application in the drug resistant kit of Rituximab；The specific markers are protein markers second -1 or protein markers
Second -2；The subject is ABC-DLBCL patient.
The subject is the ABC-DLBCL patient not treated.
The subject is the ABC-DLBCL patient for having carried out Rituximab treatment.Before being treated with the subject
ABC-DLBCL cell is compared, if carrying out specific marker in the ABC-DLBCL cell at a certain moment after Rituximab treatment
The abundance of object (protein markers second -1) is 1/5th hereinafter, Rituximab drug resistance has occurred for moment subject.With this
ABC-DLBCL cell before subject treats is compared, if carrying out the ABC- at a certain moment after Rituximab treatment
The abundance of specific markers (protein markers second -2) is half hereinafter, moment subject has sent out in DLBCL cell
Raw Rituximab drug resistance.
The subject is the ABC-DLBCL patient for having carried out rituximab combination R-CHOP chemotherapy.With the subject
ABC-DLBCL cell before being treated is compared, if the ABC-DLBCL for carrying out a certain moment after Rituximab treatment is thin
The abundance of specific markers (protein markers second -1) is 1/5th hereinafter, moment subject has occurred in born of the same parents
Rituximab drug resistance.Compared with the ABC-DLBCL cell before the subject treats, if after carrying out Rituximab treatment
The a certain moment ABC-DLBCL cell in specific markers (protein markers second -2) abundance be half hereinafter,
The moment, Rituximab drug resistance occurred for subject.
The present invention also protects a kind of method for preparing Rituximab drug-resistant type ABC-DLBCL cell, includes the following steps:
Parental cell is cultivated and passed on, during the cultivation process using Rituximab processing (specially from low concentration to high concentration
Substep cultivate), detect parental cell and every time passage after cell in specific markers abundance；The specific markers are
Protein markers second -1 or protein markers second -2；When the specific markers are protein markers second -1, with parent
Cell is compared, if the abundance of specific markers is 1/5th hereinafter, the cell after the passage is in the cell after passage
Rituximab drug-resistant type ABC-DLBCL cell；When the specific markers are protein markers second -2, with parental cell phase
Than if the abundance of specific markers is half hereinafter, the cell after the passage is Rituximab in cell after passage
Drug-resistant type ABC-DLBCL cell；The parental cell is ABC-DLBCL cell.
The parental cell is non-Rituximab drug-resistant type ABC-DLBCL cell.
The parental cell is SU-DHL-2 cell.
The parental cell is the SU-DHL-2 cell line that ATCC number is CRL-2956.
When specific markers are the combination of multiple proteins, abundance is that half or less refers to every kind of protein
Abundance is half or less.
When specific markers are the combination of multiple proteins, abundance refers to every kind of protein for 1/5th or less
Abundance is 1/5th or less.
Any description above abundance concretely detected abundance of high performance liquid chromatography-mass spectrometry.
The present invention also protects the substance for detecting specific markers in preparation for detecting whether subject has occurred
Application in the drug resistant kit of Rituximab；The specific markers are protein markers third；The subject is ABC-
DLBCL patient.
The subject is the ABC-DLBCL patient not treated.
The subject is the ABC-DLBCL patient for having carried out Rituximab treatment.
The subject is the ABC-DLBCL patient for having carried out rituximab combination R-CHOP chemotherapy.
If detecting specific markers third in the ABC-DLBCL cell of subject, which has occurred Rituximab
Drug resistance.
The present invention also protects a kind of method for preparing Rituximab drug-resistant type ABC-DLBCL cell, includes the following steps:
Parental cell is cultivated and passed on, during the cultivation process using Rituximab processing (specially from low concentration to high concentration
Substep is cultivated), it detects and whether there is specific markers in the cell after passing on every time；The specific markers are protein marker
Object third；If detecting specific markers third in the cell after passage, the cell after the passage is Rituximab drug-resistant type ABC-
DLBCL cell.
The parental cell is non-Rituximab drug-resistant type ABC-DLBCL cell.
The parental cell is SU-DHL-2 cell.
The parental cell is the SU-DHL-2 cell line that ATCC number is CRL-2956.
The present invention also protects the substance for detecting specific markers in preparation for detecting whether subject has occurred
Application in the drug resistant kit of Rituximab；The specific markers are protein markers fourth；The subject is ABC-
DLBCL patient.
The subject is the ABC-DLBCL patient not treated.
The subject is the ABC-DLBCL patient for having carried out Rituximab treatment.
The subject is the ABC-DLBCL patient for having carried out rituximab combination R-CHOP chemotherapy.
If specific markers fourth is not detected in the ABC-DLBCL cell of subject, which has occurred
Rituximab drug resistance.
The present invention also protects a kind of method for preparing Rituximab drug-resistant type ABC-DLBCL cell, includes the following steps:
Parental cell is cultivated and passed on, during the cultivation process using Rituximab processing (specially from low concentration to high concentration
Substep is cultivated), it detects and whether there is specific markers in the cell after passing on every time；The specific markers are protein marker
Object fourth；If specific markers fourth is not detected in the cell after passage, the cell after the passage is Rituximab drug-resistant type
ABC-DLBCL cell.
The parental cell is non-Rituximab drug-resistant type ABC-DLBCL cell.
The parental cell is SU-DHL-2 cell.
The parental cell is the SU-DHL-2 cell line that ATCC number is CRL-2956.
ABC-DLBCL cell, full name are activating B cell type Diffuse Large B-Cell Lymphoma cell.
The substance of any description above detection specific markers are as follows: for using cell as the substance of sample preparation protein solution
And/or the instrument of the substance and/or high performance liquid chromatography-mass spectrometry for carrying out FASP enzymatic hydrolysis.
The present inventor uses external low concentration gradient incremental method, successfully constructs Rituximab drug-resistant cell strain, and
Primary Study is carried out to resistance mechanism by Label-free technology, it was found that multiple protein markers.The cell model and
Result of study is further research Rituximab drug resistance and drug resistance is overcome to provide foundation.
Detailed description of the invention
Fig. 1 is SDS-PAGE electrophoresis.
Fig. 2 is peptide fragment ion score distribution map.
Fig. 3 is identification protein relative molecular mass distribution figure.
Fig. 4 is peptide section sequence staple diagram.
Fig. 5 is protein sequence coverage distribution map.
Fig. 6 is identification peptide fragment distributed number figure.
Fig. 7 is the volcano B/A figure.
Fig. 8 is that GO analyzes result.
Fig. 9 is KEGG path analysis result.
Figure 10 is B/A differential expression protein interactive network result.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.
SU-DHL-2 cell, that is, people's diffusivity large B cell lymphoid tumor cell.SU-DHL-2 cell line: ATCC, CRL-2956.
SU-DHL-2 cell line, also known as SU-DHL-2-WT.SU-DHL-2 cell line (being commonly referred to as SUDHL2 cell in document), belongs to
In activating B cell type Diffuse Large B-Cell Lymphoma cell (ABC-DLBCL cell).
The building of embodiment 1, drug-resistant cell strain
Condition of culture: 37 DEG C, 5%CO2Cell incubator in.
1, using the RPMI-1640 culture medium culture SU-DHL-2 cell line containing 10% fetal calf serum to logarithmic growth phase.
2, after completing step 1,1000rpm is centrifuged 5min, reject supernatant, be added it is new containing 1 μ g/mL Rituximab and
The RPMI-1640 culture medium of 10% fetal calf serum is cultivated 24 hours.
3, after completing step 2,1000rpm is centrifuged 5min, and the new RPMI- containing 10% fetal calf serum is added in reject supernatant
1640 culture mediums, culture cell to logarithmic growth phase.
4, after completing step 3,1000rpm is centrifuged 5min, reject supernatant, be added it is new containing 1 μ g/mL Rituximab and
The RPMI-1640 culture medium of 10% fetal calf serum is cultivated 24 hours.
5, after completing step 3,1000rpm is centrifuged 5min, and the new RPMI- containing 10% fetal calf serum is added in reject supernatant
1640 culture mediums, culture cell to logarithmic growth phase.
6, after completing step 5,1000rpm is centrifuged 5min, reject supernatant, be added it is new containing 5 μ g/mL Rituximab and
The RPMI-1640 culture medium of 10% fetal calf serum is cultivated 24 hours.
7, after completing step 6,1000rpm is centrifuged 5min, and the new RPMI- containing 10% fetal calf serum is added in reject supernatant
1640 culture mediums, culture cell to logarithmic growth phase.
8, after completing step 7,1000rpm is centrifuged 5min, reject supernatant, be added it is new containing 5 μ g/mL Rituximab and
The RPMI-1640 culture medium of 10% fetal calf serum is cultivated 24 hours.
9, after completing step 8,1000rpm is centrifuged 5min, and the new RPMI- containing 10% fetal calf serum is added in reject supernatant
1640 culture mediums, culture cell to logarithmic growth phase.
10, after completing step 9,1000rpm is centrifuged 5min, reject supernatant, be added it is new containing 10 μ g/mL Rituximab and
The RPMI-1640 culture medium of 10% fetal calf serum is cultivated 24 hours.
11, after completing step 10,1000rpm is centrifuged 5min, reject supernatant, is added new containing 10% fetal calf serum
RPMI-1640 culture medium, culture cell to logarithmic growth phase.
12, after completing step 11,1000rpm is centrifuged 5min, and reject supernatant is added new containing 10 μ g/mL Rituximab
With the RPMI-1640 culture medium of 10% fetal calf serum, cultivate 24 hours.
13, after completing step 12,1000rpm is centrifuged 5min, reject supernatant, is added new containing 10% fetal calf serum
RPMI-1640 culture medium, culture cell to logarithmic growth phase.
14, after completing step 13,1000rpm is centrifuged 5min, and reject supernatant is added new containing 20 μ g/mL Rituximab
With the RPMI-1640 culture medium of 10% fetal calf serum, cultivate 24 hours.
15, after completing step 14,1000rpm is centrifuged 5min, reject supernatant, is added new containing 10% fetal calf serum
RPMI-1640 culture medium, culture cell to logarithmic growth phase.
16, after completing step 15,1000rpm is centrifuged 5min, and reject supernatant is added new containing 20 μ g/mL Rituximab
With the RPMI-1640 culture medium of 10% fetal calf serum, cultivate 24 hours.
17, after completing step 16,1000rpm is centrifuged 5min, reject supernatant, is added new containing 10% fetal calf serum
RPMI-1640 culture medium, culture cell to logarithmic growth phase.
18, after completing step 17,1000rpm is centrifuged 5min, and reject supernatant is added new containing 50 μ g/mL Rituximab
With the RPMI-1640 culture medium of 10% fetal calf serum, cultivate 24 hours.
19, after completing step 18,1000rpm is centrifuged 5min, reject supernatant, is added new containing 10% fetal calf serum
RPMI-1640 culture medium, culture cell to logarithmic growth phase.
20, after completing step 19,1000rpm is centrifuged 5min, and reject supernatant is added new containing 50 μ g/mL Rituximab
With the RPMI-1640 culture medium of 10% fetal calf serum, cultivate 24 hours.
21, after completing step 20,1000rpm is centrifuged 5min, reject supernatant, is added new containing 10% fetal calf serum
RPMI-1640 culture medium, culture cell to logarithmic growth phase.
22, after completing 21,1000rpm is centrifuged 5min, reject supernatant, be added it is new containing 100 μ g/mL Rituximab and
The RPMI-1640 culture medium of 10% fetal calf serum is cultivated 24 hours.
23, after completing step 22,1000rpm is centrifuged 5min, reject supernatant, is added new containing 10% fetal calf serum
RPMI-1640 culture medium, culture cell to logarithmic growth phase.
24, after completing step 23,1000rpm is centrifuged 5min, and reject supernatant is added new containing 100 μ g/mL Rituximab
With the RPMI-1640 culture medium of 10% fetal calf serum, cultivate 24 hours.
25, after completing step 24,1000rpm is centrifuged 5min, reject supernatant, is added new containing 10% fetal calf serum
RPMI-1640 culture medium, culture cell to logarithmic growth phase.
216, after completing step 25,1000rpm is centrifuged 5min, and reject supernatant is added new containing 128 μ g/mL
The RPMI-1640 culture medium of Rituximab and 10% fetal calf serum are cultivated 24 hours.
27, after completing step 26,1000rpm is centrifuged 5min, reject supernatant, is added new containing 10% fetal calf serum
RPMI-1640 culture medium, culture cell to logarithmic growth phase.
28, after completing step 27,1000rpm is centrifuged 5min, and reject supernatant is added new containing 128 μ g/mL Rituximab
With the RPMI-1640 culture medium of 10% fetal calf serum, cultivate 24 hours.
By above-mentioned steps, obtain being named as SU-DHL-2- to the 128 drug resistant cell strains of μ g/mL Rituximab
R。
Embodiment 2, label-free proteomics (Label-free) method
Cell sample are as follows: cell sample A1, cell sample A2, cell sample A3, cell sample B1, cell sample B2, thin
Born of the same parents' sample B 3.Cell sample A1, cell sample A2, cell sample A3 are SU-DHL-2-WT, represent 3 biology and repeat.Carefully
Born of the same parents' sample B 1, cell sample B2, cell sample B3 are SU-DHL-2-R, represent 3 biology and repeat.
One, the preparation of protein solution
Cell sample is taken, addition SDT lysate is ultrasonic (80W, work 10s, interval 15s, recycles 10 times), then boiling water
15min is bathed, then 14000g is centrifuged 40min, takes supernatant, as protein solution.
SDT lysate: 4g/100ml SDS, 100mM Tris-HCl, 1mM DTT, pH7.6.
Quantification of protein is carried out using BCA method (BCA quantification kit, P0012, the green skies).Dispense sample, -80 DEG C of guarantors
It deposits.
Each cell sample obtains 500 μ L protein solutions.Protein solution is followed successively by according to principle corresponding with cell sample
Protein solution A1, protein solution A2, protein solution A3, protein solution B1, protein solution B2, protein solution B3.
The protein concentration of protein solution A1 is 20 μ g/ μ L.The protein concentration of protein solution A2 is 23 μ g/ μ L.Protein solution
The protein concentration of A3 is 18 μ g/ μ L.The protein concentration of protein solution B1 is 31 μ g/ μ L.The protein concentration of protein solution B2 is 26 μ
g/μL.The protein concentration of protein solution B3 is 23 μ g/ μ L.
Two, SDS-PAGE electrophoresis
Each protein solution (containing 20 μ g albumen) is taken respectively, 5 × sample-loading buffer is added, then boiling water bath 5min is carried out
12.5%SDS-PAGE electrophoresis (constant current 14mA, 90min), then carries out coomassie brilliant blue staining.
Electrophoretogram is shown in Fig. 1.
Three, FASP is digested
The protein solution for taking 30 μ L step 1 to obtain, it is 100mM that DTT, which is added, to concentration, and boiling water bath 5min is subsequently cooled to
Room temperature.Then 200 μ L UA buffer are added and mix, are then transferred into 10kD ultra-filtration centrifuge tube, 14000g centrifugation
15min, reject supernatant add 200 μ L UA buffer and mix, and 14000g is centrifuged 15min, reject supernatant.Then
100 μ L IAA buffer, first 600rpm are added and vibrate 1min, then room temperature is protected from light 30min, and 14000g is centrifuged 15min,
Reject supernatant.Then 100 μ L UA buffer, 14000g are added and are centrifuged 15min, then 100 μ L are added in reject supernatant
UA buffer, 14000g are centrifuged 15min, reject supernatant.Then 100 μ L 25mM NH are added4HCO3Aqueous solution, 14000g from
Then 100 μ L 25mM NH are added in heart 15min, reject supernatant4HCO3Aqueous solution, 14000g are centrifuged 15min, reject supernatant
Liquid.Then 40 μ L Trypsin buffer, first 600rpm are added and vibrate 1min, then 37 DEG C of standing 16-18h.It is then transferred into
In new collecting pipe, 14000g is centrifuged 15min, collects filtrate.Then 40 μ L 25mM NH are added4HCO3Aqueous solution simultaneously mixes,
14000g is centrifuged 15min, collects filtrate.Then desalination is carried out using C18Cartridge, be then freeze-dried, then with 40 μ L
0.1% (volume ratio) aqueous formic acid is redissolved, as peptide fragment solution.By detecting OD280nmIt is quantitative that value carries out peptide fragment.
UA buffer (pH 8.0): 8M urea, 150mM Tris-HCl, surplus is water.
IAA buffer: being added IAA in UA buffe, and makes its concentration 100mM.
Trypsin buffer: 4 μ g Trypsin are added to 40 μ L 100mM NH4HCO3In aqueous solution.
Every kind of protein solution obtains 40 μ L peptide fragment solution.Peptide fragment solution is followed successively by according to principle corresponding with protein solution
Peptide fragment solution A 1, peptide fragment solution A 2, peptide fragment solution A 3, peptide fragment solution B 1, peptide fragment solution B 2, peptide fragment solution B 3.Peptide fragment solution A 1
Peptide fragment concentration be 0.74 μ g/ μ L.The peptide fragment concentration of peptide fragment solution A 2 is 0.83 μ g/ μ L.The peptide fragment concentration of peptide fragment solution A 3 is
0.51μg/μL.The peptide fragment concentration of peptide fragment solution B 1 is 0.60 μ g/ μ L.The peptide fragment concentration of peptide fragment solution B 2 is 0.66 μ g/ μ L.Peptide
The peptide fragment concentration of section solution B 3 is 1.26 μ g/ μ L.
Four, peptide fragment is identified by high performance liquid chromatography-mass spectrometry and is attributed to protein
The peptide fragment solution that step 3 obtains identifies peptide fragment by high performance liquid chromatography-mass spectrometry and is attributed to protein.
It is separated using the HPLC liquid phase systems Easy nLC of nanoliter flow velocity.A liquid is that 0.1% (volume ratio) formic acid is water-soluble
Liquid, B liquid are the aqueous solution containing 0.1% (volume ratio) formic acid and 84% (volume ratio) acetonitrile.Mobile phase is A liquid or B liquid or both
Mixed liquor.The balance pillar of the mobile phase containing 95% (volume ratio) first is accounted for A liquid, then peptide fragment solution is by autosampler loading
To loading column (Thermo Scientific Acclaim PepMap100,100 μm of * 2cm, nanoViper C18), through excessive
Analyse column (Thermo scientific EASY column, 10cm, ID75 μm, 3 μm, C18-A2) separation.Flow rate of mobile phase is
300nL/min.Elution process: 0min-110min, B liquid account for the volume fraction of mobile phase from 0% linear rise to 55%；
110min-115min, B liquid account for the volume fraction of mobile phase from 55% linear rise to 100%；115min-120min, B liquid account for
The volume parts of mobile phase are 100%.
It is analyzed by mass spectrometry using Q-Exactive mass spectrograph.A length of 120min when analysis, detection mode are cation, female
Ion scan range 300-1800m/z, first mass spectrometric resolution ratio are 70,000at 200m/z, AGC (Automatic gain
Control) target is 1e6, and Maximum IT is 50ms, and it is 60.0s that dynamic, which excludes time (Dynamic exclusion),.
The mass-charge ratio of polypeptide and peptide fragment acquires in following manner: each full scan (full scan) acquire afterwards 20 it is broken
Piece map (MS2scan), MS2Activation Type are HCD, and Isolation window is 2m/z, second order ms resolution ratio
17,500at 200m/z, Normalized Collision Energy are 30eV, Underfill 0.1%.
Five, data are analyzed
Mass spectral analysis initial data is RAW file, carries out checking storehouse mirror using MaxQuant software (version number 1.5.3.17)
Fixed and quantitative analysis.
Peptide fragment ion score distribution map is shown in Fig. 2.Identification protein relative molecular mass distribution figure is shown in Fig. 3.Peptide section sequence is long
Degree distribution map is shown in Fig. 4.Protein sequence coverage distribution map is shown in Fig. 5.Identification peptide fragment distributed number figure is shown in Fig. 6.Peptide is identified altogether
Number of segment 28582, protein amounts are about 3000.
The volcano B/A illustrated example is shown in Fig. 7.In the significant difference analysis of quantitative result, first in Screening Samples group three times
The data repeated in experimental data at least there are two non-null value are for statistical analysis, wherein meeting differential expression multiple greater than 2.0
Times (up-regulation/downward) and P-value are considered as differential expression less than the protein of 0.05 screening criteria.
Compared with SU-DHL-2-WT: the albumen for raising (change > 2 fold) in SU-DHL-2-R has 45 (wherein 5 times
Above 8), it is shown in Table 1；The albumen for lowering (change < 0.5 fold) in SU-DHL-2-R has 77 (wherein 5 times or more 5),
It is shown in Table 2.There are 21 albumen to be not detected in SU-DHL-2-R in SU-DHL-2-WT, is shown in Table 3.In SU-DHL-2-WT, there is 29
A albumen is not detected in SU-DHL-2-R, is shown in Table 4.The present inventor has found in close relations with Rimximab drug resistance
Albumen, these albumen can be used as prediction the drug resistant marker of Rituximab.
Table 1
Table 2
Table 3
Table 4
In Tables 1 and 2, average A is cell sample A1, cell sample A2, cell sample A3 carry out above-mentioned step respectively
Suddenly the average value of the abundance numerical value (LFQ intensity) obtained, average B are cell sample B1, cell sample B2, cell
Sample B 3 carries out the average value for the abundance numerical value (LFQ intensity) that above-mentioned steps obtain respectively, B/A be average B with
The ratio of average A.
The protein name and corresponding gene title of albumen involved in Tables 1 and 2 are shown in Table 5.
The protein name and corresponding gene title of albumen involved in table 3 and table 4 are shown in Table 6.
Table 5
Table 6
B/A differential expression protein interactive network result example is shown in Figure 10.
To after identification differential protein carry out bioinformatic analysis, comprising: Gene Ontology (GO) functional annotation and
Enrichment is analyzed, KEGG access annotates and enrichment analysis, protein clustering analysis (Clustering), protein interaction network
It analyzes (PPI).GO analysis prompt mdr cell biological behaviour shows main concentration B cell differentiation, calcium mucin combines, DNA
Damage, Apoptosis etc. (see Fig. 8).The difference of KEGG path analysis prompt Rituximab mdr cell and parental cell
Gene is mainly obviously enriched in B-cell receptor signal path and HIF-1 signal path (see Fig. 9).By KEGG path analysis and
GO analysis, it has been found that most close access may be B-cell receptor signal path with Rituximab drug resistance relationship, and mechanism can
It can be after Rimximab activates the access, to influence B-cell receptor downstream passages, can inhibit apoptosis of tumor cells, promote tumour thin
Born of the same parents' proliferation and drug resistance.This result of study is to predict Rituximab drug resistance in the future and overcome drug resistance, provides theoretical foundation.
Claims (9)
1. the substance for detecting specific markers is in preparation for detecting whether ABC-DLBCL cell to be measured is Rituximab
Application in the kit of drug-resistant type ABC-DLBCL cell；The specific markers be following (a) or (b) or (c) or (d) or
(e) or (f) or (g) or (h):
(a) protein markers first -1；
(b) protein markers first -2；
(c) protein markers second -1；
(d) protein markers second -2；
(e) protein marker third；
(f) protein marker fourth；
(g) combination of protein markers first -1, protein markers second -1, protein marker third and protein marker fourth；
(h) combination of protein markers first -2, protein markers second -2, protein marker third and protein marker fourth；
Protein markers first -1 described above is any one or any combination in following protein: Nicotinate-
nucleotide pyrophosphorylase；PCN；Zizimin-3；NAPRTase；Immunoglobulin heavy
constant gamma 1；GPI；Valine--tRNA ligase,mitochondrial；HSPA1A；
Protein markers first -2 described above is any one or any combination in following protein: Nicotinate-
nucleotide pyrophosphorylase；PCN；Zizimin-3；NAPRTase；Immunoglobulin heavy
constant gamma 1；GPI；Valine--tRNA ligase,mitochondrial；HSPA1A；FLN-A；Vimentin
isoform 1；Tubulin-specific chaperone D；CD48 protein；Adenosine deaminase
isoform 1；GK；Thioredoxin；PC4 protein；Phosphoinositide 3-kinase adapter
protein 1；BCL2-like 1 isoform 3；Putative deoxyribonuclease TATDN1；Epididymis
secretory sperm binding protein；Protein CutA；VAV-2；UPD；Lymphocyte-specific
protein 1；Bcl2-L-5；Gasdermin-D；MTHFS；Family with sequence similarity 49,
member B,isoform CRA_a；T-cell leukemia/lymphoma 1A,isoform CRA_b；
Unconventional myosin-Ig；ABC50 protein；CSTB protein；Sialophorin(GpL115,
leukosialin,CD43),isoform CRA_a；Pyruvate kinase；EBF2 protein；CNP；Tubulin
5beta；TAP1；Testicular tissue protein Li 180；Squalene monooxygenase；PMI；RICH-
1；Proto-oncogene c-Rel；Polymerase(DNA directed),epsilon 3(P17 subunit),
isoform CRA_a；Unconventional myosin-Id；
Protein markers second -1 described above is any one or any combination in following protein: EEF1A1protein；
Protein unc-45 homolog A；LPCAT-1；ENO2 protein；Fanconi anemia,complementation
group I；
Protein markers second -2 described above is any one or any combination in following protein: ANT 2；Protein
kinase C substrate 80K-H isoform1；ColGalT1；Heat shock protein90Bb；PMVK
protein；Atlastin-3；TRG-8；NACHT,LRR and PYD domains-containing protein 2；
Guanidinoacetate N-methyltransferase；Histone acetyltransferase 1；Cathepsin S；
N-alpha acetyl transferase 40；Minor histocompatibility antigen H13；EH-domain
containing 1,isoform CRA_b；ARHGAP45；Abhydrolase domain-containing protein 11；
Oligosaccharyl transferase subunit DAD1；Pleckstrin homology domain-containing
family F member 1；Protein YIF1A；Ubiquitin-conjugating enzyme E2 S；Reticulon；
MRE11A protein；Thymidine phosphorylase；GMF-gamma；B-cell receptor-associated
protein 29；PKC-B；Aspartate aminotransferase；TDP43 isoform H；RNA-binding
protein Raly；RNA demethylase ALKBH5；Methyltransferase-like protein 1；Torsin-
1A-interacting protein 1；Probable bifunctional dTTP/UTP pyrophosphatase/
methyltransferase protein；DCN1-like protein；Polycomb protein SUZ12；Thymidine
kinase；PTDSS1 protein；Signal peptidase complex subunit 3；A-LAP；N-terminal
kinase-like protein；Stromal interaction molecule 1；DNA ligase；Ubiquitin-
protein ligase G2；PHD finger protein 6,isoform CRA_d；Oligosaccharyl
transferase 48 kDa subunit；Porphobilinogen deaminase；Transmembrane protein
126A；PPIase FKBP11；Oligoribonuclease,mitochondrial；Hypoxia up-regulated
protein 1；Leukocyte antigen CD37；Signal peptidase complex catalytic subunit
SEC11；Importin subunit alpha；Immunoglobulin heavy constant gamma 3；hRAD50；
Mitochondrial import inner membrane translocase subunit TIM50；Very-long-chain
3-oxoacyl-CoA synthase；Centromere protein V；tRNA 2'-phosphotransferase 1；DNA
(cytosine-5)-methyltransferase；DEAD(Asp-Glu-Ala-Asp)box polypeptide 41,
isoform CRA_a；Checkpoint protein；Mesencephalic astrocyte-derived neurotrophic
factor；AP complex subunit sigma；Transmembrane protein 214；RFC37；LRP chaperone
MESD；PTPRC-associated protein；Pro-interleukin-16；RNA helicase；Glucosamine 6-
phosphate N-acetyltransferase；Protein FAM49A；EEF1A1 protein；Protein unc-45
homolog A；LPCAT-1；ENO2 protein；Fanconi anemia,complementation group I；
Protein markers third described above are any one or any combination in following protein: Serine/
threonine-protein kinase PRP4 homolog；Choline dehydrogenase,isoform CRA_a；
Leucine-rich repeat-containing protein 57；3-hydroxybutyrate dehydrogenase,
type 2,isoform CRA_b；Epiplakin；T-lymphocyte activation antigen CD86；
Transmembrane protein 223；Salt-tolerant protein；SLAMF1 protein；PDDC1 protein；
Myosin IC,isoform CRA_a；Iron-responsive element binding protein 2,isoform
CRA_a；Protein kinase C and casein kinase substrate in neurons protein 1；
Tether-containing UBX domain for GLUT4；Biogenesis of lysosome-related
organelles complex 1subunit 3；Stratifin；TNF alpha-induced protein 2；IL-27B；
Transmembrane protein65；hIRIP；BAI1-associated protein 2-like protein 1；
Protein markers fourth described above is any one or any combination in following protein: Activating
signal cointegrator 1 complex subunit 2,isoform CRA_a；UDP-N-
acetylglucosamine--dolichyl-phosphate N-acetylglucosaminephosphotransferase；
Etoposide induced 2.4 mRNA,isoform CRA_a；Mitochondrial chaperone BCS1；POLD3
protein；Met proto-oncogene(Hepatocyte growth factor receptor),isoform CRA_c；
Putative bifunctional UDP-N-acetylglucosamine transferase and deubiquitinase
ALG13；Ribosome-binding protein 1；Cytochrome b-245 beta polypeptide isoform 1；
Mirror-image polydactyly 1 isoform 4；Uncharacterized protein C3orf38；Histone
H2B；Actin,cytoplasmic 1；Ceramide synthase 2；Serine/threonine-protein kinase
26；CD22 protein；Bromodomain adjacent to zinc finger domain,1A,isoform CRA_c；
Histone lysine demethylase PHF8；ATP synthase mitochondrial F1 complex
assembly factor 1；Reticulophagy regulator 3；Cytochrome c oxidase subunit 2；
Endonuclease domain-containing 1 protein；KDEL receptor 1；FSAP；M1G-
methyltransferase；Myopathy with excessive autophagy protein；GPx-7；GINS
complex subunit 4；PH domain-containing family F member 2.
2. the substance for detecting specific markers is in preparation for detecting whether subject has occurred the drug resistant examination of Rituximab
Application in agent box；The specific markers are institute in the protein markers first -1 or claim 1 described in claim 1
The protein markers first -2 stated；The subject is ABC-DLBCL patient.
3. a kind of method for preparing Rituximab drug-resistant type ABC-DLBCL cell, includes the following steps: to carry out parental cell
Culture and passage, are handled using Rituximab during the cultivation process, special in the cell after detecting parental cell and every time passage
The abundance of different marker；The specific markers are in protein markers first -1 described in claim 1 or claim 1
The protein markers first -2；When the specific markers are protein markers first -1, compared with parental cell, if
The abundance of specific markers is 5 times or more in cell after passage, and the cell after the passage is Rituximab drug-resistant type ABC-
DLBCL cell；When the specific markers are protein markers first -2, compared with parental cell, if the cell after passage
The abundance of middle specific markers is 2 times or more, and the cell after the passage is Rituximab drug-resistant type ABC-DLBCL cell；It is described
Parental cell is ABC-DLBCL cell.
4. the substance for detecting specific markers is in preparation for detecting whether subject has occurred the drug resistant examination of Rituximab
Application in agent box；The specific markers are institute in the protein markers second -1 or claim 1 described in claim 1
The protein markers second -2 stated；The subject is ABC-DLBCL patient.
5. a kind of method for preparing Rituximab drug-resistant type ABC-DLBCL cell, includes the following steps: to carry out parental cell
Culture and passage, are handled using Rituximab during the cultivation process, special in the cell after detecting parental cell and every time passage
The abundance of different marker；The specific markers are in protein markers second -1 described in claim 1 or claim 1
The protein markers second -2；When the specific markers are protein markers second -1, compared with parental cell, if
The abundance of specific markers is 1/5th hereinafter, the cell after the passage is Rituximab drug-resistant type in cell after passage
ABC-DLBCL cell；When the specific markers are protein markers second -2, compared with parental cell, if after passage
The abundance of specific markers is half hereinafter, the cell after the passage is Rituximab drug-resistant type ABC-DLBCL in cell
Cell；The parental cell is ABC-DLBCL cell.
6. the substance for detecting specific markers is in preparation for detecting whether subject has occurred the drug resistant examination of Rituximab
Application in agent box；The specific markers are protein markers third described in claim 1；The subject is ABC-
DLBCL patient.
7. a kind of method for preparing Rituximab drug-resistant type ABC-DLBCL cell, includes the following steps: to carry out parental cell
Culture and passage, are handled using Rituximab during the cultivation process, with the presence or absence of special mark in the cell after detecting passage every time
Will object；The specific markers are protein markers third described in claim 1；If detected in the cell after passage
Specific markers third, the cell after the passage are Rituximab drug-resistant type ABC-DLBCL cell.
8. the substance for detecting specific markers is in preparation for detecting whether subject has occurred the drug resistant examination of Rituximab
Application in agent box；The specific markers are protein markers fourth described in claim 1；The subject is ABC-
DLBCL patient.
9. a kind of method for preparing Rituximab drug-resistant type ABC-DLBCL cell, includes the following steps: to carry out parental cell
Culture and passage, are handled using Rituximab during the cultivation process, with the presence or absence of special mark in the cell after detecting passage every time
Will object；The specific markers are protein markers third described in claim 1；If do not detected in the cell after passage
To specific markers fourth, the cell after the passage is Rituximab drug-resistant type ABC-DLBCL cell.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112007159A (en) * | 2020-08-10 | 2020-12-01 | 中国人民解放军陆军军医大学 | Application of PC4 inhibitor in preparation of products for preventing and treating diffuse large B cell lymphoma |
| CN113855799A (en) * | 2021-10-21 | 2021-12-31 | 天津市人民医院 | Application of combination of cideramide and rituximab in treatment of senile relapse refractory B cell lymphoma disease |
| CN117683893A (en) * | 2024-02-04 | 2024-03-12 | 首都医科大学附属北京友谊医院 | Biomarker for predicting drug resistance of BTK inhibitor and application thereof |
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| CN101711286A (en) * | 2007-04-02 | 2010-05-19 | 健泰科生物技术公司 | Biological markers predictive of rheumatoid arthritis response to b-cell antagonists |
| CN103667466A (en) * | 2013-12-02 | 2014-03-26 | 中国人民解放军军事医学科学院基础医学研究所 | Marker for B cell non-hodgkin lymphoma and drug resistance detection thereof |
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| CN101711286A (en) * | 2007-04-02 | 2010-05-19 | 健泰科生物技术公司 | Biological markers predictive of rheumatoid arthritis response to b-cell antagonists |
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| CN112007159A (en) * | 2020-08-10 | 2020-12-01 | 中国人民解放军陆军军医大学 | Application of PC4 inhibitor in preparation of products for preventing and treating diffuse large B cell lymphoma |
| CN113855799A (en) * | 2021-10-21 | 2021-12-31 | 天津市人民医院 | Application of combination of cideramide and rituximab in treatment of senile relapse refractory B cell lymphoma disease |
| CN117683893A (en) * | 2024-02-04 | 2024-03-12 | 首都医科大学附属北京友谊医院 | Biomarker for predicting drug resistance of BTK inhibitor and application thereof |
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