CN111733135B - Chinese lung squamous carcinoma cell line and application thereof - Google Patents
Chinese lung squamous carcinoma cell line and application thereof Download PDFInfo
- Publication number
- CN111733135B CN111733135B CN202010547005.1A CN202010547005A CN111733135B CN 111733135 B CN111733135 B CN 111733135B CN 202010547005 A CN202010547005 A CN 202010547005A CN 111733135 B CN111733135 B CN 111733135B
- Authority
- CN
- China
- Prior art keywords
- cell line
- cell
- chinese
- lung
- lung squamous
- 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
- 206010041823 squamous cell carcinoma Diseases 0.000 title claims abstract description 43
- 210000004072 lung Anatomy 0.000 title claims abstract description 42
- 201000005243 lung squamous cell carcinoma Diseases 0.000 claims abstract description 14
- 230000035772 mutation Effects 0.000 claims abstract description 14
- 239000003814 drug Substances 0.000 claims abstract description 13
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims abstract description 11
- 229940079593 drug Drugs 0.000 claims abstract description 8
- 238000004321 preservation Methods 0.000 claims abstract description 7
- 201000005296 lung carcinoma Diseases 0.000 claims abstract description 4
- 230000002401 inhibitory effect Effects 0.000 claims description 5
- 238000010171 animal model Methods 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 238000012216 screening Methods 0.000 claims description 2
- 230000008685 targeting Effects 0.000 claims description 2
- 210000004027 cell Anatomy 0.000 abstract description 86
- 206010028980 Neoplasm Diseases 0.000 abstract description 18
- 210000001519 tissue Anatomy 0.000 abstract description 15
- 210000004881 tumor cell Anatomy 0.000 abstract description 12
- 108090000623 proteins and genes Proteins 0.000 abstract description 8
- 238000000338 in vitro Methods 0.000 abstract description 6
- 102000004169 proteins and genes Human genes 0.000 abstract description 6
- 210000000349 chromosome Anatomy 0.000 abstract description 5
- 208000037068 Abnormal Karyotype Diseases 0.000 abstract description 3
- 238000003745 diagnosis Methods 0.000 abstract description 2
- 238000009169 immunotherapy Methods 0.000 abstract description 2
- 239000000463 material Substances 0.000 abstract description 2
- 239000006285 cell suspension Substances 0.000 description 15
- 208000020816 lung neoplasm Diseases 0.000 description 8
- 201000005202 lung cancer Diseases 0.000 description 7
- 238000011580 nude mouse model Methods 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 241000699660 Mus musculus Species 0.000 description 5
- IAKHMKGGTNLKSZ-INIZCTEOSA-N (S)-colchicine Chemical compound C1([C@@H](NC(C)=O)CC2)=CC(=O)C(OC)=CC=C1C1=C2C=C(OC)C(OC)=C1OC IAKHMKGGTNLKSZ-INIZCTEOSA-N 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 230000005740 tumor formation Effects 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000004663 cell proliferation Effects 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 238000007920 subcutaneous administration Methods 0.000 description 3
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 206010064571 Gene mutation Diseases 0.000 description 2
- 101150105104 Kras gene Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 108010019160 Pancreatin Proteins 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- 230000006978 adaptation Effects 0.000 description 2
- 230000003698 anagen phase Effects 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 229960001338 colchicine Drugs 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 229940055695 pancreatin Drugs 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 230000000391 smoking effect Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 208000031404 Chromosome Aberrations Diseases 0.000 description 1
- 102000029816 Collagenase Human genes 0.000 description 1
- 108060005980 Collagenase Proteins 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 206010071975 EGFR gene mutation Diseases 0.000 description 1
- 101710113436 GTPase KRas Proteins 0.000 description 1
- 102000018317 Keratin-19 Human genes 0.000 description 1
- 108010066302 Keratin-19 Proteins 0.000 description 1
- 206010023774 Large cell lung cancer Diseases 0.000 description 1
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 206010041067 Small cell lung cancer Diseases 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- 208000009956 adenocarcinoma Diseases 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000000424 bronchial epithelial cell Anatomy 0.000 description 1
- 239000002771 cell marker Substances 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 231100000005 chromosome aberration Toxicity 0.000 description 1
- 210000001726 chromosome structure Anatomy 0.000 description 1
- 229960002424 collagenase Drugs 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 230000003583 cytomorphological effect Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 231100000517 death Toxicity 0.000 description 1
- 238000012350 deep sequencing Methods 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 description 1
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 210000003237 epithelioid cell Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000834 fixative Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 238000003125 immunofluorescent labeling Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 210000003141 lower extremity Anatomy 0.000 description 1
- 201000009546 lung large cell carcinoma Diseases 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 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 1
- 230000001338 necrotic effect Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 208000000587 small cell lung carcinoma Diseases 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 230000005945 translocation Effects 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000012070 whole genome sequencing analysis Methods 0.000 description 1
- 239000008096 xylene Substances 0.000 description 1
Images
Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
-
- 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
- C12N5/0688—Cells from the lungs or the respiratory tract
-
- 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
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
-
- 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
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/15—Humanized animals
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0331—Animal model for proliferative diseases
-
- 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
- C12N2503/00—Use of cells in diagnostics
- C12N2503/02—Drug screening
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- Hematology (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Food Science & Technology (AREA)
- Pathology (AREA)
- Environmental Sciences (AREA)
- Toxicology (AREA)
- General Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Animal Behavior & Ethology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Pulmonology (AREA)
- Oncology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a Chinese lung squamous carcinoma cell line, which is named as SAHP1 and has the preservation number as follows: CCTCC NO: C2019266. The cell line of the invention has similar protein expression with the primary tumor tissue, and simultaneously has chromosome abnormal karyotype and tumor characteristics of in vitro tumor forming capability. The cell line of the invention can be continuously subcultured in vitro while keeping the characteristics of the squamous cell lung carcinoma tumor cells unchanged, and is suitable for being used as a cell material for researching and developing squamous lung carcinoma related immunotherapy, diagnosis and medicaments. The cell line of the invention contains Kras-G12V mutation and can be used as a tool cell for researching targeted drugs taking Kras-G12V mutation as a target.
Description
Technical Field
The invention relates to the technical field of tumor biology, in particular to a Chinese lung squamous carcinoma cell line and application thereof.
Background
Lung cancer is one of the most mortality tumors worldwide today, accounting for 19.4% of deaths caused by tumors. In Europe, with the decrease of the number of people smoking in China and the increase of smokers in women, the incidence of men is reduced year by year, the incidence of women is increased year by year, wherein 80-85% of the cases are non-small cell lung cancer, and the 5-year life cycle is less than 11%. Lung cancer epidemiological studies in asian regions 2014 showed: 43.4% of lung cancers are women with an average age of 60 years, and 52.6% have no history of smoking. Lung cancer is mainly classified into: non-small cell lung cancer and small cell lung cancer. Non-small cell lung cancer is mainly classified into adenocarcinoma, squamous cell carcinoma, and large cell lung cancer, wherein squamous cell lung cancer is a common type of lung cancer. K-ras mutations occur in 20-40% of NSCLC patients, and drugs targeting the driver gene greatly improve the efficiency of treatment of squamous cell lung carcinoma. However, most lung squamous carcinomas have no mutation of genes such as k-ras and the like, and target drugs have little effect on the lung squamous carcinomas, so that traditional chemotherapy and radiotherapy are still the main treatment means of the lung carcinomas. Therefore, we should add more research investment for such common lung cancer.
In the global study of lung cancer, stable, long-passaged cell lines have been established in as many as one hundred species, but the genetic background differs greatly from the Chinese race in Asian regions. Due to the heterogeneity and complexity of tumors, tumors of the same tissue type of different ethnic and regional origin also differ in their cellular characteristics.
In order to more effectively research pathogenesis and treatment method of squamous cell lung carcinoma of Chinese, a tumor cell primary culture method is adopted to establish a Chinese squamous cell lung carcinoma cell line, and the cell line has Kras-G12V mutation and does not have EGFR mutation, so that a new model is provided for the research of lung cancer of Chinese, and a better platform is provided for the research of mechanism of squamous cell lung carcinoma.
Disclosure of Invention
The first objective of the present invention is to provide a Chinese lung squamous carcinoma cell line which has similar protein expression with the primary tumor tissue, has chromosome abnormal karyotype, has in vitro tumorigenicity ability and contains Kras-G12V mutation.
The second purpose of the invention is to provide the application of the Chinese lung squamous carcinoma cell line SAHP1 in the preparation of lung squamous carcinoma diagnostic reagents.
The third purpose of the invention is to provide the application of the Chinese lung squamous carcinoma cell line SAHP1 in preparing medicines for treating the lung squamous carcinoma.
The fourth purpose of the invention is to provide the application of the Chinese human lung squamous carcinoma cell line SAHP1 in preparing lung squamous carcinoma animal models.
The fifth purpose of the invention is to provide the application of the Chinese human squamous cell lung carcinoma cell line SAHP1 in the preparation of targeted drugs for inhibiting squamous cell lung carcinoma, which take Kras-G12V mutation as a target.
The sixth purpose of the invention is to provide the progeny cells of the Chinese lung squamous carcinoma cell line SAHP 1.
In order to achieve the first object, the invention provides a Chinese lung squamous carcinoma cell line, which is named as SAHP1 and has the preservation number as follows: CCTCC NO: C2019266.
In order to achieve the second object, the invention provides an application of a Chinese lung squamous carcinoma cell line SAHP1 in preparing a lung squamous carcinoma diagnostic reagent.
In order to achieve the third purpose, the invention provides an application of the Chinese lung squamous carcinoma cell line SAHP1 in preparing a medicament for treating the lung squamous carcinoma.
In order to achieve the fourth object, the invention provides an application of the Chinese lung squamous carcinoma cell line SAHP1 in preparing an animal model of the lung squamous carcinoma.
In order to realize the fifth purpose, the invention provides application of the Chinese lung squamous carcinoma cell line SAHP1 in preparing a lung squamous carcinoma inhibiting targeted drug taking Kras-G12V mutation as a target. The Kras gene mutation is detected by the Chinese human lung squamous carcinoma cell line SAHP1, subcutaneous tumor formation can be carried out on a nude mouse, and an antibody capable of inhibiting and killing SAHP1 cells is prepared and screened, and is used as a main component of an anti-lung squamous carcinoma medicament.
In order to achieve the sixth object, the present invention provides progeny cells of the Chinese lung squamous carcinoma cell line SAHP 1. The progeny cells retain substantially or all of the characteristics of the parent cell.
The cell line to be protected by the invention is named as a Chinese human lung squamous cell line SAHP1, is preserved in a Chinese typical culture collection (preservation address: lo Jia mountain road 16 Wuhan university Chinese typical culture collection center postcode 430072 in Wuchan, wuhan, hubei), has the preservation date of 2019, 12 and 17 days, and has the preservation number: CCTCC NO: C2019266.
The cell line of the invention contains Kras-G12V mutation, which can cause normal lung epithelial cell mutation and tumor formation, and promote tumor cell proliferation.
The invention has the advantages that the cell line has similar protein expression with primary tumor tissues, has chromosome abnormal karyotype and has the tumor characteristics of in vitro tumorigenicity capacity. The cell line of the invention can be continuously subcultured in vitro while keeping the characteristics of the squamous cell lung carcinoma tumor cells unchanged, and is suitable for being used as a cell material for researching and developing squamous lung carcinoma related immunotherapy, diagnosis and medicaments. The cell line of the invention contains Kras-G12V mutation and can be used as a tool cell for researching targeted drugs taking Kras-G12V mutation as a target.
Drawings
FIG. 1 shows the cell morphology (100X) of the human lung squamous carcinoma cell line SAHP1 under an inverted microscope in the present invention.
FIG. 2 is a cell growth and proliferation curve of a human lung squamous carcinoma cell line SAHP1 of the present invention.
FIG. 3 is a chromosome karyotype analysis chart of the human lung squamous carcinoma cell line SAHP1 of the present invention.
FIG. 4 is a graph showing subcutaneous tumor formation in nude mice of a Chinese human lung squamous carcinoma cell line SAHP1 according to the present invention, wherein a is a tumor growth curve, and b is a photograph of subcutaneous tumor formation in nude mice.
Detailed Description
Hereinafter, the technique of the present invention will be described in detail with reference to specific embodiments. It should be understood that the following detailed description is only for the purpose of assisting those skilled in the art in understanding the present invention, and is not intended to limit the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
Example 1.
Isolation treatment of primary tumor cells
Isolated culture of tumor primary cells was performed in a sterile environment on fresh tumor tissue pieces obtained from animal houses.
1. Tumor tissue blocks were first placed in 50ml centrifuge tubes and washed 5 times with 10-fold double antibody in PBS by shaking until there was no blood. The tissue mass is placed in a petri dish and excess tissue, such as fat or necrotic tissue, is excised.
2. The tissue was transferred to another dish and fine cut to approximately 1mm with a cross scalpel 3 Tissue mass of size. A70 um filter sieve was taken and placed in a 50ml centrifuge tube. The tissue is forced through the mesh into the medium by gentle pressure with the core of the syringe, and the medium is aspirated and blown through the screen to flush the cells.
3. 5ml of trypsin/collagenase II was added to the dish of the remaining tissue mass and digested in an incubator at 37 ℃ for 2 hours, while being blown up once every 15 minutes. After digestion was complete, the cell suspension was filtered 1 time through a 70um filter, and the filtered cell suspension was washed once by centrifugation at 5 min and 5ml in PBS at 1200rpm.
4. And transferring the cells obtained by centrifugation to a culture dish for culture, and simultaneously putting the tissue block into other culture dishes for standing culture. And (4) absolutely standing for 12-24 hours, slowly taking out the culture dish, supplementing the culture solution, continuously culturing, and standing for 2-3 days. After 3 days the flask was carefully removed and the floating fragment was removed and the culture was continued by changing the culture broth.
In vitro culture of primary tumor cells
1. The interval is 24-48 hours.
2. Observing the growth condition of the cells under a light mirror, and replacing the proper culture solution at proper time. If the adherent cells grow, the culture fluid amount can be properly increased to meet the requirement of the growth of the cells.
3. The culture conditions of the culture of the invention are as follows: at 37 ℃ C, 5% CO 2 Culturing in environment with LONZA BEBM basic Medium bronchial epithelial cell basic culture Medium.
Monoclonal culture of primary tumor cells
1. Cells were trypsinized to prepare single cell suspensions, cells were counted, and cells were diluted to 1X10 cells/mL. The method comprises the following specific steps:
a) The digested cells were diluted to 1X10 5 one/mL.
b) Get 1 to10 5 200uL of cell suspension/mL, added to 20mL to give 1X10 3 one/mL.
c) Will be 1 × 10 3 200uL of cell suspension/mL, added to 20mL, was 1X 10/mL.
2. 100uL of 1X 10/mL cell suspension was added to a 96-well microtiter plate, and one colony formed per well.
3. After a few hours of inoculation, culture wells with only one cell were found by microscopy. The wells are marked and followed, and a decrease in PH, indicative of cell growth, is confirmed by microscopic observation. After the cells overgrow, pancreatin digestion is connected into a 6-well plate for subculture.
Obtaining a cell line, wherein the cell line is named as a Chinese human lung squamous carcinoma cell line SAHP1, is preserved in the China center for type culture Collection, and has the preservation number: CCTCC NO: C2019266.
Example 2 detection and identification of Chinese Lung squamous carcinoma cell line SAHP1
1. Cytomorphological observation of primary tumor cell strain
Most of tumor cells under a light mirror are fusiform and oval, a few of the tumor cells are polygonal, the two poles of the cells are sharp, the cell nucleus is obvious, a few of the cells are multinuclear, the nucleolus is clearly visible, and the ratio of the nucleoplasm is large. The SAHP1 which is well spread in the logarithmic growth phase is mostly elliptical epithelioid cells in random extension, and the cells are polygonal and have more regular shapes. As shown in fig. 1.
2. Immunofluorescence detection of cell marker proteins
1) After the cells were digested with trypsin, 1mL of complete medium was suspended, and thoroughly blown into a single cell suspension for cell counting. 24-hole plate access 5 x10 as required 4 And (4) cells. Before dropping cell suspension, a small amount of culture medium is added into each hole to make the slide and the culture dish adhere together to reduce cell entry so as to prevent the slide from floating.
2) Slides that were seeded with cells were washed twice with fresh PBS. The PBS was blotted dry and fixed with 4% paraformaldehyde for 40min, and after fixation with methanol, the PBS was washed twice, 5 min each time, and excess PBS was blotted dry. 0.2% Triton X-100, dissolved in PBS, left at room temperature for 15 minutes, and perforated. The punch was blotted dry, PBS was added and washed twice. Dropping goat serum and sealing for 1h.
3) Primary antibody was dissolved in goat serum, dropped onto a slide glass with cells, and incubated overnight in a refrigerator at 4 ℃. The next day, PBS was washed three times and the fluorescent secondary antibody was incubated at room temperature for 1h, taking care to keep out of the light. The PBS was washed three times and mounted with 90% glycerol.
The immunofluorescence staining result of the Chinese lung squamous cell carcinoma cell line SAHP1 shows that the Chinese lung squamous cell carcinoma cell line SAHP1 has similar protein expression with the derived lung squamous cell carcinoma tissue, and the expression shows that the protein expression is keratin-19 positive.
2. Proliferation curve detection of primary tumor cell strain
1) Digesting the cells with pancreatin to prepare single cell suspension, counting the concentration of the cell suspension by using a counting plate, and preparing the cell suspension into the cell concentration 1 multiplied by 10 required by the experiment 4 one/mL.
2) Cell suspensions (200 ul/well) were seeded in 16-well assay plates. The test plate was placed in a cell incubator for one week (the test plate was connected to a real-time cell analyzer) and cell proliferation was dynamically detected.
The growth curve of the Chinese human lung squamous carcinoma cell line SAHP1 is shown in figure 2.
Example 3 karyotyping
Karyotype analysis for detecting karyotype characteristics and chromosome number
1) Adding colchicine into the cells in logarithmic growth phase to make the final concentration of colchicine 0.1ug/ml, and continuously culturing for 3h;
2) After digestion, the cells were transferred to a 10ml tip centrifuge tube, centrifuged at 1000rpm for 10min, the supernatant was removed, 0.075mol/L KC110ml was added, incubated at 37 ℃ for 30min, and then fresh fixative (glacial acetic acid: methanol = 3:1) 1ml, mixed well, 1000rpm, centrifuged for 10min, and the supernatant discarded;
3) Slowly adding 10ml of fresh fixing solution, gently blowing uniformly, fixing at room temperature for 20min, centrifuging, removing supernatant, and fixing again; centrifuging at 1000rpm for 10min, and discarding the supernatant;
4) Adding 250ul of fresh fixing solution, blowing gently and uniformly, dripping a drop of suspension at a height of 15cm from the glass slide, fully drying air, dyeing by fresh Giemsa, washing by tap water, airing, enabling xylene to be transparent, sealing by neutral resin, and observing under a mirror.
The karyotype analysis of the human lung squamous carcinoma cell line shows that the chromosome structure and the number are abnormal: 2n/3n =54-62[96% ], which is between 2-fold and 3-fold karyotypes, and chromosomal aberrations such as complicated deletion and translocation exist in the karyotype. As shown in fig. 3.
Example 4.
The cell suspension is inoculated into the heterogeneous animal body, and the tumor forming capability is observed.
1) Injecting Chinese human lung squamous carcinoma cell line SAHP1 cell suspension into the skin of the root of the sterilized hind limb of 3 nude mice with the numbers of 1, 2 and 3 and the ages of 4 weeks, wherein each cell suspension is 1x10 7 Individual cells, gently pressed after injection, without outflow.
2) On day 12, 3 nude mice all showed white protrusions in the cell injection region, and on day 40, tumors were seen to grow in leaf form, with a tumor length of 12mm-15mm, and the nude mice were sacrificed after anesthesia. As shown in fig. 4a and 4 b.
The Chinese human lung squamous carcinoma cell line can form tumor under the skin of a nude mouse, and can be used for preparing and screening an antibody capable of inhibiting and killing SAHP1 cells, wherein the antibody is used as a main component of an anti-lung squamous carcinoma medicament.
Example 5 Whole genome sequencing of tumor cells
Cell count 1X10 6 The DNA of the cells is extracted by using a DNA extraction kit of QINGEN, and deep sequencing is carried out.
The Kras gene mutation is detected in the Chinese lung squamous cell carcinoma cell line, and the EGFR gene mutation is not found.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, many modifications and adaptations can be made without departing from the principle of the present invention, and such modifications and adaptations should also be considered as the scope of the present invention.
Claims (4)
1. A Chinese lung squamous carcinoma cell line is characterized in that the Chinese lung squamous carcinoma cell line is named as SAHP1, and the preservation number is as follows: CCTCC NO: C2019266.
2. The use of the Chinese human squamous cell lung carcinoma cell line of claim 1 in the preparation of a squamous lung carcinoma animal model.
3. The use of the human lung squamous carcinoma cell line of China as claimed in claim 1 in screening of targeted drugs for inhibiting lung squamous carcinoma targeting Kras-G12V mutation.
4. A progeny cell of a Chinese lung squamous carcinoma cell line according to claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010547005.1A CN111733135B (en) | 2020-06-16 | 2020-06-16 | Chinese lung squamous carcinoma cell line and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010547005.1A CN111733135B (en) | 2020-06-16 | 2020-06-16 | Chinese lung squamous carcinoma cell line and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111733135A CN111733135A (en) | 2020-10-02 |
| CN111733135B true CN111733135B (en) | 2023-03-10 |
Family
ID=72649383
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010547005.1A Active CN111733135B (en) | 2020-06-16 | 2020-06-16 | Chinese lung squamous carcinoma cell line and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111733135B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20130043520A (en) * | 2011-10-20 | 2013-04-30 | (주)차바이오앤디오스텍 | Method for the isolation and proliferation of primary lung cancer cells |
| CN104694476A (en) * | 2013-12-05 | 2015-06-10 | 上海睿智化学研究有限公司 | Human non-small cell lung cancer cell line, and establishment method and application thereof |
| CN107083365A (en) * | 2017-06-08 | 2017-08-22 | 上海市胸科医院 | A kind of Chinese Lungs gland cell system and its application |
-
2020
- 2020-06-16 CN CN202010547005.1A patent/CN111733135B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20130043520A (en) * | 2011-10-20 | 2013-04-30 | (주)차바이오앤디오스텍 | Method for the isolation and proliferation of primary lung cancer cells |
| CN104694476A (en) * | 2013-12-05 | 2015-06-10 | 上海睿智化学研究有限公司 | Human non-small cell lung cancer cell line, and establishment method and application thereof |
| CN107083365A (en) * | 2017-06-08 | 2017-08-22 | 上海市胸科医院 | A kind of Chinese Lungs gland cell system and its application |
Non-Patent Citations (2)
| Title |
|---|
| "Identification of T-cell Receptors Targeting KRAS-Mutated Human Tumors";Qiong J.Wang et al.;《Cancer Immunol Res》;20160331;第4卷(第3期);第204-214页 * |
| "人肺鳞癌细胞系CHLH-1建立";刘鸿程 等;《第二军医大学学报》;20071231;第28卷(第12期);第1375-1377页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111733135A (en) | 2020-10-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107083365B (en) | Chinese lung adenocarcinoma cell line and application thereof | |
| WO2021208130A1 (en) | Culture medium for esophageal squamous cell carcinoma epithelial cells, culture method, and application thereof | |
| CN103243074B (en) | Human colorectal adenocarcinoma tumor cell line as well as preparation method and application thereof | |
| CN108504625B (en) | Mouse fibroblast and application thereof | |
| CN108866000B (en) | Human epidermal growth factor tyrosine kinase inhibitor acquired drug-resistant lung cancer cell line and establishment method and application thereof | |
| CN106367393B (en) | Prostate Carcinoma of Mice circulating tumor cell system and the separation of prostate cancer circulating tumor cell and cultural method | |
| CN110241071A (en) | A kind of normal renal tubule primary cell of people and its Isolation and culture and application | |
| CN102329775B (en) | A kind of human pancreatic cancer cell to gemcitabine resistance and application thereof | |
| CN111733135B (en) | Chinese lung squamous carcinoma cell line and application thereof | |
| CN111321120B (en) | Separation and culture method of circulating tumor cell line of mouse renal clear cell carcinoma and circulating tumor cell of human-derived renal clear cell carcinoma | |
| CN112251410A (en) | Mouse-derived gastric cancer cell line NCCG1, and establishment method and application thereof | |
| CN111662874A (en) | Chinese esophageal squamous carcinoma cell line and application thereof | |
| CN111635912A (en) | Gene combination for inducing liver cells into liver cancer cells and application thereof | |
| CN107217041A (en) | DC cells and T cells with antigenic specificity with high antigen presentation and its preparation method and application | |
| CN115786262B (en) | Human hilar bile duct cancer cell line CBC3T-1 and application thereof | |
| CN112210538A (en) | Human esophageal squamous carcinoma cell line NCCE1, and establishment method and application thereof | |
| CN109536497A (en) | The application of schistosoma japonicum infection and its component in human tumor prevention and treatment | |
| CN108130311B (en) | Human primary colon cancer liver metastasis cell line HCS1220 | |
| CN113234679A (en) | Crizotinib-resistant human lung adenocarcinoma cell strain and preparation and application thereof | |
| CN109929804B (en) | Human ovarian cancer cell line and preparation method and application thereof | |
| CN109486767B (en) | Human epithelial ovarian cancer cell SHOV4 and application thereof | |
| CN113201494B (en) | Mucous membrane melanoma cell and application thereof | |
| CN113046319B (en) | Human acute myeloid leukemia cell strain and application thereof | |
| CN110177868A (en) | Method for cancer stem cell (CSC) amplification | |
| CN117660351A (en) | Human EGFR gene double-site mutant non-small cell lung cancer cell strain HC5720 and application thereof |
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 |