CN105969734A - Esophageal cancer cell line and application thereof - Google Patents
Esophageal cancer cell line and application thereof Download PDFInfo
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- CN105969734A CN105969734A CN201610593841.7A CN201610593841A CN105969734A CN 105969734 A CN105969734 A CN 105969734A CN 201610593841 A CN201610593841 A CN 201610593841A CN 105969734 A CN105969734 A CN 105969734A
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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
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- 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
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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
- C12N2503/00—Use of cells in diagnostics
- C12N2503/02—Drug screening
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- 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
Abstract
The invention discloses an esophageal cancer cell line and application thereof. The esophageal cancer cell line is named a human esophageal squamous carcinoma cell line ZEC-134, and the preservation number is CCTCC NO:C2016108. The human esophageal squamous carcinoma cell line ZEC-134 is from pleural effusion of an esophagus cancer patient from China, an STR detection result proves that the human esophageal squamous carcinoma cell line ZEC-134 is unique, and cross contamination with other cells is not generated in the primary culture process; the human esophageal squamous carcinoma cell line ZEC-134 is high in clone formation and tumorigenicity and can serve as an ideal cell line for esophageal cancer research and application in esophageal cancer detection kit development, drug screening and the like.
Description
Technical field
The present invention relates to biology and oncology's technical field, particularly relate to a kind of esophageal carcinoma cell line and application thereof.
Background technology
The esophageal carcinoma is common malignant tumor of digestive tract.The whole world there are about 300,000 people every year and dies from the esophageal carcinoma.Its sickness rate and death
Rate various countries are widely different.China is one of Esophageal Cancer area in the world, and every annual is died of illness about 150,000 people.Man is more than female,
Age of onset is many more than 40 years old.The typical symptom of the esophageal carcinoma is Progressive symmetric erythrokeratodermia acataposis, before this food of difficult dry pharynx, is then
Semiliquid diet, last water and saliva can not be swallowed.In China, Incidence of esophageal cancer occupies general tumour the 5th, and it is dead
Rate occupies the 4th.Two kinds of Main Subtypes of the esophageal carcinoma: scale cancer and adenocarcinoma, wherein scale cancer accounts for 90%.
Patient with esophageal carcinoma clinical manifestation is as follows:
In early days: symptom is the most inconspicuous, discomfort in various degree but may be had to feel, including swallowing food when swallowing thick and stiff food
Choking feeling, burns sample, acupuncture sample or tractive friction sample pain after breastbone.Food is by slow, and has stagnation sense or foreign body sensation.
Stalk is choked to stagnate to feel and is disappeared usually through alleviating after swallowing water.During symptom light time weight, make slow progress.
Middle and advanced stage: the typical symptom of the esophageal carcinoma is Progressive symmetric erythrokeratodermia acataposis, the food of difficult dry pharynx, was semiliquid diet then before this,
Last water and saliva can not be swallowed.Often tell mucoid expectorant, for the saliva swallowed and the secretions of esophagus.Patient gradually becomes thin,
Dehydration, unable.Continuing chest pain or backache is expressed as symptom in late period, cancer has been invaded and has been organized outside esophagus.When cancerous protuberance is blocked caused
Inflammation edema transient remission, or after part cancerous protuberance comes off, obstruction can temporarily alleviate, and constant error thinks that sb.'s illness took a favorable turn.If cancerous protuberance
Invade recurrent laryngeal nerve, may occur in which hoarseness；If compressing cervical sympathetic ganglia, Horner syndrome can be produced；If intrusion trachea,
Bronchus, can form esophagus, trachea or bronchial fistula, acutely chokes and coughs, and respiratory system sense occurs when there is swallowing water or food
Dye.Cachectic states finally occurs.If there being the Organ relative weight such as liver, brain, may occur in which the states such as jaundice, seroperitoneum, stupor.
Treatment main point of surgical intervention, radiotherapy, chemotherapy etc., in recent years, reached based on the Comprehensive Treatment of operation
Arrive bottleneck, and Chemotherapy in Esophageal Cancer slower development, there is no clear and definite conclusion and standard scheme.
For understanding the pathogeny of esophageal squamous cell carcinoma and medicine and the research of other Therapeutic Method in depth, it is necessary to set up suitable diagnostic cast
Type.The most conventional esophageal cancer cell strain is mainly derived from Japan, including KYSE series and TE series.These are thin
Born of the same parents are to pass in vitro for a long time, and some have lost the characteristic of primary tumor tissues.Because race difference, living habit is different,
The reasons such as environmental factors, these cells containing sequences represent the type of China's esophageal carcinoma and the query that feature existence is the biggest.Domestic minority is real
Testing the cell strain set up room less, the widely used cell strain of part is polluted by other cells so that the research of the esophageal carcinoma
Can not carry out in a deep going way.And, along with the accurate medical science of tumor treatment concept go deep into clinical practice, it is provided that the more esophageal carcinoma is thin
Born of the same parents' strain contributes to research and development and the application of targeted drug.Set up the esophageal cancer cell strain in Chinese source, for the morbidity machine of esophageal squamous cell carcinoma
System and treatment provide new model, have important theory and actual application and are worth.
Summary of the invention
The present invention is directed to the current domestic esophageal cancer cell strain lacking Chinese source, and provide one and derive from Chinese food
Pipe cancerous cell line.
From an example patient with esophageal carcinoma, (patient is 54 years old male to the present inventor's esophageal squamous cell carcinoma cell line ZEC-134, and tumor is positioned at food
Pipe stage casing cancer, CT shows mediastinum many groups lymphatic metastasis, both sides tracheo-esophageal ditch lymphatic metastasis, and gastroscopy pathology biopsy shows
PDSCC, visual tumors cell under hydrothorax mirror, phase clinical stages 4.) hydrothorax specimen sampling, through original cuiture
And after building and being tied to form merit, named Human esophageal squamous cell cancer cell line ZEC-134, it is preserved on July 5th, 2016 and is positioned at Wuhan, China
The China typical culture collection center of Wuhan University, preserving number is: CCTCC NO:C2016108.
The present inventor's esophageal squamous cell carcinoma cell line ZEC-134 cell growth vigorous, clear background, impurity is rare, cell be flat not
Regular polygon, cell is the most closely coupled, meets the feature of epithelioid cell.After adherent, growth is very fast, has good body
Outer cultivation amplification property.Chromosome number is distributed between 45～57 mostly, and mode is 52, meets the feature of malignant tumor.
The cells such as the STR sequencing result of the present inventor's esophageal squamous cell carcinoma cell line ZEC-134 and ATCC, DSMZ preserve the number in storehouse
Carry out inquiry contrast according to storehouse, do not find identical STR testing result, it was demonstrated that it is unique, and does not sends out during original cuiture
The raw cross-contamination with other cells.Immunohistochemical experiment finds, Human esophageal squamous cell cancer cell line ZEC-134 cell is CK5/6 sun
Property, CK14 is positive, and p63 is positive, and CgA is negative, and Sy is negative and CD56 is negative.
Colony formation finds, the cloning efficiency of Human esophageal squamous cell cancer cell line ZEC-134 cell and inoculum density have relation,
Inoculum density is that Cell clonality during 4500 cells is stronger.
Nude mice (nude mouse) becomes tumor experimental result to find, Human esophageal squamous cell cancer cell line ZEC-134 Tumor formation is stronger.
Invention further provides the daughter cell of described esophageal carcinoma cell line.Described daughter cell remains parental generation substantially or all
The characteristic of cell.
Invention further provides described esophageal cancer cell and tie up to the application in the cell model as esophageal carcinoma genesis mechanism research.
Owing to the present inventor's esophageal squamous cell carcinoma cell line ZEC-134 is originated foundation from Chinese, and to build be that the time is shorter, and character is stable,
Using this esophageal carcinoma cell line as study model, have very great help for understanding Chinese primary esophageal carcinogenesis mechanism.
Present invention also offers described esophageal cancer cell to tie up to set up the application in mammal esophageal carcinoma model.Described suckling
Animal is nude mouse or nude rat.By the described Human esophageal squamous cell cancer cell line ZEC-134 cell of certain cell quantity is inoculated in
The positions such as subcutaneous, the liver of nude mouse or nude rat, abdominal cavity or tail vein, it is thus achieved that the animal model of the esophageal carcinoma.
Present invention also offers described esophageal cancer cell to tie up to extract the application in esophageal carcinoma specific tumour mark.By with
Normal esophageal cells and other kinds of cancer cell compare research, it appeared that the molecular marker of the esophageal carcinoma, for this
Molecular marker can carry out the application of the aspects such as follow-up disease detection and drug development.
Present invention also offers described esophageal cancer cell and tie up to the application in screening or assessment treatment esophageal carcinoma medicine.First, logical
Crossing interpolation different pharmaceutical in described Human esophageal squamous cell cancer cell line ZEC-134 culture medium, observation of cell state changes, it is thus achieved that preliminary
Effective drug candidate.Then, drug candidate is administered to the animal model of the above-mentioned esophageal carcinoma, observes and non-dispenser treated animal
Survival period, tumor size, transfer case etc., screening obtains the medicine of the potential treatment esophageal carcinoma.
Present invention also offers described esophageal cancer cell to tie up to develop the application in esophageal carcinoma detection kit.The discovery esophageal carcinoma is special
After the tumor markers of the opposite sex, the detection esophageal carcinoma can be developed according to this tumor markers and occur or the detection kit of development.
The present inventor's esophageal squamous cell carcinoma cell line ZEC-134 comes from the hydrothorax of the patient with esophageal carcinoma of an example China, STR testing result
Proving that it is unique, and the cross-contamination with other cells does not occurs during original cuiture, Clone formation is strong with Tumor formation,
Can be as the ideal cell line of the aspect application such as esophageal carcinoma research and the exploitation of esophageal carcinoma detection kit, drug screening.
Accompanying drawing explanation
Fig. 1 is the Morphology observation figure of Human esophageal squamous cell cancer cell line ZEC-134 cell；
Fig. 2 is the cell growth curve figure of Human esophageal squamous cell cancer cell line ZEC-134；
Fig. 3 is the karyotype scattergram of Human esophageal squamous cell cancer cell line ZEC-134 cell；
Fig. 4 is STR analysis result figure, and wherein, figure A～F is the most homoallelic result figure respectively；
Fig. 5 is Human esophageal squamous cell cancer cell line ZEC-134 cellular immunization CYTOCHEMICAL ANALYSIS result figure；
Fig. 6 is Human esophageal squamous cell cancer cell line ZEC-134 cell clonal formation experimental result picture, wherein, and figure A, B and C inoculation
Cell number is respectively 500,1500 and 4500；
Fig. 7 is that mice becomes tumor experimental result picture.
Detailed description of the invention
Embodiment 1
From an example patient with esophageal carcinoma, (patient is 54 years old male, and tumor is positioned at esophago mesomere cancer, and CT shows mediastinum many groups lymph node
Transfer, both sides tracheo-esophageal ditch lymphatic metastasis, gastroscopy pathology biopsy display PDSCC, visible swollen under hydrothorax mirror
Oncocyte, phase clinical stages 4.) the sampling of hydrothorax specimen, centrifuging and taking be deposited in aseptic super-clean bench with PBS clean centrifugal after,
The 10%FBS (GIBCO company), 1% dual anti-DMEM/F12 cell culture medium (GIBCO containing 10mL is added in culture dish
Company), put into 37 DEG C, 5%CO2Incubator in cultivate.Change liquid after 5～7 days, discard what necrosis in piece of tissue came off
Floating fritter, carries out Secondary Culture.During Secondary Culture, utilize fibroblast different from tumor cell digestion power,
Constantly remove fibroblast.After repeatedly passing on, in culture dish, naked eyes cannot observe fibroblast, and can continued propagation
Pass on.Build after being tied to form merit, named Human esophageal squamous cell cancer cell line ZEC-134, it is preserved on July 5th, 2016 and is positioned at China
The China typical culture collection center of Wuhan Wuhan University, preserving number is: CCTCC NO:C2016108.
Embodiment 2
Take the Human esophageal squamous cell cancer cell line ZEC-134 cell of Secondary Culture, under an optical microscope (Japan Olympus IMT-2
Inverted microscope) observe living cell growth situation.As it is shown in figure 1, cell Optical Morphology picture, it is seen that cell growth is vigorous,
Clear background, impurity is rare, and cell is flat irregular polygon, and cell is the most closely coupled, meets the spy of epithelioid cell
Point.
Embodiment 3
Take the Human esophageal squamous cell cancer cell line ZEC-134 cell that growth conditions is good, through trypsinization, make cell suspension, and count
Number.Inoculating cell suspension (100 μ L/ holes, 2000 cells) in 96 orifice plates, adherent latter 4 hours, add 10 μ L CCK8,
Continue to cultivate 2 hours, measure the light absorption value at 490nm by microplate reader, afterwards, detected every 1 day, continue 6 days.
Utilize Graph Pad Software on Drawing growth curve, and be calculated the population doubling time about 60 hours of cell.As in figure 2 it is shown,
After the present inventor's esophageal squamous cell carcinoma cell line ZEC-134 is adherent, growth is very fast, has good cultured and amplified in vitro.
Embodiment 4
Take Human esophageal squamous cell cancer cell line ZEC-134 cell, be planted in six orifice plates, after cultivating 48 hours, use 0.01mg/mL
Colchicine process 16h, when microscopy observes that M phase cell proportion is more than 50%, collect M phase cell.Use KCl
Hypotonic medium Hypotonic treatment M phase cell 15～20min, at room temperature solid as fixative with methanol/glacial acetic acid (volume ratio 3: 1)
Determine cell, film-making on slide.Slide being placed in 0.02% trypsin solution digestion 30～60s, rinses through PBS, Giemsa contaminates
Color, dries, and completes film-making.In basis of microscopic observation, calculate chromosome number in each cell, randomly select 20 cells and enter
Row calculates.As it is shown on figure 3, chromosome number is distributed between 45～57 mostly, mode is 52, meets the feature of malignant tumor.
Embodiment 5
STR (short tandem repeat, STR) is also called microsatellite DNA.General by one long 2～6bp
Core sequence through repeatedly tandem sequence repeats arrangement form, number of repetition is mostly between 10～60 times.The weight of core sequence between individuality
Again count in variability, thus the number of repetition of one group of STR sequence is the most unique in Different Individual, is that cell is raw
The main method that cell identity and source are identified by thing.Human esophageal squamous cell cancer cell line ZEC-134 collecting fresh cultured is thin
Born of the same parents, with Qiagen genomic DNA Mini Kit (purchased from Qiagen company, name of article QIAamp DNA Mini Kit,
Article No. is 51304) extract cell genomic dna, carry out PCR amplification, to gained with 5' end fluorescently-labeled STR primer
Product checks order.Wherein the primer sequence of STR bit point and copy number are as shown in table 1 and Fig. 4.Above-mentioned sequence and ATCC,
The cells such as DSMZ preserve the data base in storehouse and carry out inquiry contrast, do not find identical STR testing result, thus may certify that it is
Uniquely, and the cross-contamination with other cells does not occurs during original cuiture.
The copy number in table 1STR site.
Embodiment 6
SABC detection Human esophageal squamous cell cancer cell line ZEC-134 cellular labeled proteins.S-P method is used to carry out immunohistochemical staining,
Related kit is purchased from Fuzhou Maixin biotechnology Development Co., Ltd.Antibody used be CK5/6 (article No. MAB-0276,
Foochow steps new company), P63 (article No. ZM-04-06, Beijing company of Zhong Shan Golden Bridge), (article No. ZA-0540, in Beijing for CK14
Company of shirt Golden Bridge), CgA (article No. ZA-0507, Beijing company of Zhong Shan Golden Bridge), Sy (article No. IR660, DAKO company),
CD56 (I article No. R628, DAKO company).
Tumor cell SABC: cell to be detected (Human esophageal squamous cell cancer cell line ZEC-134 cell) shifts to an earlier date 24 hours creep plates
On sterilization microscope slide, after 24 hours, rinsing 2 times with PBS, cold acetone fixes 15 minutes, is soaked in PBS
Standby.Taking the incubated at room temperature 10 minutes that required slide adds 50 μ L peroxidase blocking solution (reagent A), PBS buffers
Liquid rinses 3 times, each 3 minutes, removes PBS liquid, drips the normal nonimmune animal serum of 50 μ L (reagent B), under room temperature
Hatch 10 minutes；Removing serum, drip 50 μ L mono-and resist, incubated at room temperature 60 minutes, PBS flushes three times, each 3～5
Minute；Dropping two resists, and incubated at room temperature 10 minutes, PBS flushes three times, each 3 minutes.Dropping
Polyperoxidase-anti-mouse/rabbit IgG, incubated at room temperature 30 minutes, PBS flushing 3 times, each 2 points
Clock.Dilution preparation DAB solution, develops the color 5～10 minutes, Microscopic observation.Clear water rinses, and haematoxylin is redyed, conventional dehydration,
Transparent, mounting.
Result is as it is shown in figure 5, Human esophageal squamous cell cancer cell line ZEC-134 cell is the CK5/6 positive, and CK14 is positive, p63 sun
Property, CgA is negative, and Sy is negative and CD56 is negative.Immunohistochemical result all illustrates Human esophageal squamous cell cancer cell line
ZEC-134 is scale cancer source.
Embodiment 7
Take the Human esophageal squamous cell cancer cell line ZEC-134 cell that growth conditions is good, after trypsinization, make cell suspension, and count
Number.By cell suspension with 500,1500,4500 cell per well are inoculated in 6 orifice plates.6 orifice plates are placed in incubator,
Quiescent culture 2 weeks, changes weekly liquid 2 times.After 2 weeks, discarding cell culture fluid, after rinsing with PBS, absolute methanol fixes 10
Min, then by 0.1% violet staining 10min, washes away dyeing liquor, drying at room temperature, and Taking Pictures recording.As shown in Figure 6,
The cloning efficiency of Human esophageal squamous cell cancer cell line ZEC-134 cell and inoculum density have relation, and inoculum density is 4500 cells
Time Cell clonality stronger.
Embodiment 8
Take the Human esophageal squamous cell cancer cell line ZEC-134 cell that growth conditions is good, after trypsinization, make cell suspension, and count
Number.Cell suspension is inoculated into 5 nude mice (nude mouse) oxters, Continuous Observation respectively with 10,000,000 cells.Result of the test
As it is shown in fig. 7, accessible to lump, increase in time after 2 weeks after Jie Zhong, lump increases.
Claims (8)
1. esophageal carcinoma cell line, it is characterised in that named Human esophageal squamous cell cancer cell line ZEC-134, preserving number is: CCTCC
NO:C2016108.
2. the daughter cell of esophageal carcinoma cell line as claimed in claim 1.
3. esophageal cancer cell as claimed in claim 1 ties up to the application in the cell model as esophageal carcinoma genesis mechanism research.
4. esophageal cancer cell as claimed in claim 1 ties up to set up the application in mammal esophageal carcinoma model.
Apply the most as claimed in claim 4, it is characterised in that described mammal is nude mouse or nude rat.
6. esophageal cancer cell as claimed in claim 1 ties up to extract the application in esophageal carcinoma specific tumour mark.
7. the application during esophageal cancer cell as claimed in claim 1 ties up to screening or assessment treatment esophageal carcinoma medicine.
8. esophageal cancer cell as claimed in claim 1 ties up to develop the application in esophageal carcinoma detection kit.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110419505A (en) * | 2019-07-18 | 2019-11-08 | 深圳大学 | Mouse cancer of the esophagus model and its method for building up |
| CN111662874A (en) * | 2020-06-18 | 2020-09-15 | 上海市胸科医院 | Chinese esophageal squamous carcinoma cell line and application thereof |
| CN115024280A (en) * | 2022-06-08 | 2022-09-09 | 中山大学孙逸仙纪念医院 | Method and model for establishing esophageal cancer in-situ planting tumor formation model |
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| CN102766599A (en) * | 2012-07-06 | 2012-11-07 | 中国人民解放军第二军医大学 | Application of esophageal squamous cell carcinoma primary tumor strain CH-H-1 |
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2016
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102766599A (en) * | 2012-07-06 | 2012-11-07 | 中国人民解放军第二军医大学 | Application of esophageal squamous cell carcinoma primary tumor strain CH-H-1 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110419505A (en) * | 2019-07-18 | 2019-11-08 | 深圳大学 | Mouse cancer of the esophagus model and its method for building up |
| CN110419505B (en) * | 2019-07-18 | 2021-11-26 | 深圳大学 | Mouse esophageal cancer model and establishment method thereof |
| CN111662874A (en) * | 2020-06-18 | 2020-09-15 | 上海市胸科医院 | Chinese esophageal squamous carcinoma cell line and application thereof |
| CN115024280A (en) * | 2022-06-08 | 2022-09-09 | 中山大学孙逸仙纪念医院 | Method and model for establishing esophageal cancer in-situ planting tumor formation model |
| CN115024280B (en) * | 2022-06-08 | 2023-05-12 | 中山大学孙逸仙纪念医院 | Esophageal cancer in-situ planting and tumor forming model building method and model |
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