CN106047815A - Esophageal cancer cell line and application thereof - Google Patents
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Abstract
The invention discloses an esophageal cancer cell line and application thereof. The esophageal cancer cell line is named as human esophageal squamous cancer cell line ZEC-157, and the preservation number of the esophageal cancer cell line is CCTCC No. C201693. The human esophageal squamous cancer cell line ZEC-157 is from excised tumor tissue of a Chinese esophageal cancer patient, and the STR detecting result proves that the cell line is unique. In addition, the esophageal cancer cell line does not have cross contamination with other cells during primary culture, is high in clone formation ability and tumorigenicity, and can be used as an ideal cell line for esophageal cancer researches, esophageal cancer detection kit development, esophageal cancer medicine 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
Widely different with mortality rate various countries.China is one of Esophageal Cancer area in the world, and every annual is died of illness about 150,000 people.Man is many
Yu Nv, 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,
Then being semiliquid diet, last water and saliva can not be swallowed.In China, Incidence of esophageal cancer occupies general tumour the 5th, its
Mortality 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 when swallowing thick and stiff food
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
Sense.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 semi-fluid then before this
Food, last water and saliva can not be swallowed.Often tell mucoid expectorant, for the saliva swallowed and the secretions of esophagus.Patient is gradually
Become thin, be dehydrated, unable.Continuing chest pain or backache is expressed as symptom in late period, cancer has been invaded and has been organized outside esophagus.Block when cancerous protuberance and drawn
The inflammation edema transient remission risen, 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 invades recurrent laryngeal nerve, may occur in which hoarseness；If compressing cervical sympathetic ganglia, Horner syndrome can be produced；If invading
Trachea-bronchial epithelial cell, can form esophagus, trachea or bronchial fistula, acutely chokes and cough when there is swallowing water or food, and occurs to breathe system
Togetherness contaminates.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, based on the Comprehensive Treatment of operation
Through having reached 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 suitably
Study model.The most conventional esophageal cancer cell strain is mainly derived from Japan, including KYSE series and TE series.These
Cell line passes on the most in vitro, and some have lost the characteristic of primary tumor tissues.Because race difference, living habit is not
With reasons such as, environmental factorss, these cells containing sequences represent the type of China's esophageal carcinoma and feature exists the biggest query.Domestic few
The cell strain that number laboratory has been set up is less, and the widely used cell strain of part is polluted by other cells so that the esophageal carcinoma
Research 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 more eat
Pipe JEG-3 contributes to research and development and the application of targeted drug.Set up the esophageal cancer cell strain in Chinese source, for esophageal squamous cell carcinoma
Pathogenesis and treatment new model is provided, there is important theory and actual application and be 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 China
The esophageal carcinoma cell line of people.
From an example patient with esophageal carcinoma, (patient is 60 years old women to the present inventor's esophageal squamous cell carcinoma cell line ZEC-157, and tumor is positioned at
Esophageal Middle Segment, postoperative pathological display PDSCC, TNM T3N0M0 by stages, the patient clinical 3A phase by stages.) operation
Tumor tissues sampling after excision, through original cuiture and after building and being tied to form merit, named Human esophageal squamous cell cancer cell line ZEC-157, in
On May 11st, 2016 is preserved in the China typical culture collection center being positioned at Wuhan, China Wuhan University, and preserving number is:
CCTCC NO:C201693.
The growth of the present inventor's esophageal squamous cell carcinoma cell line ZEC-157 cell is vigorous, and clear background, impurity is rare, and cell is flat
Flat irregular polygon, cell arrangement is tight, meets the feature of epithelioid cell.After adherent, growth is very fast, has good body
Outer cultivation amplification property.Chromosome number is distributed between 51～65 mostly, and mode is 57, 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-157 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-157 cell is CK5/6 sun
Property, CK14 is positive, and p63 is positive, and CgA is negative, and the weak positive of Sy and CD56 are negative.
Colony formation finds, the cloning efficiency of Human esophageal squamous cell cancer cell line ZEC-157 cell has with inoculum density
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-157 Tumor formation is stronger.
Invention further provides the daughter cell of described esophageal carcinoma cell line.Described daughter cell retains substantially or all
The characteristic of parental cell.
Invention further provides described esophageal cancer cell to tie up in the cell model as esophageal carcinoma genesis mechanism research
Application.Owing to the present inventor's esophageal squamous cell carcinoma cell line ZEC-157 is originated foundation from Chinese, and to build be that the time is shorter,
Character is stable, using this esophageal carcinoma cell line as study model, has for understanding Chinese primary esophageal carcinogenesis mechanism
The biggest help.
Present invention also offers described esophageal cancer cell to tie up to set up the application in mammal esophageal carcinoma model.Described
Mammal be nude mouse or nude rat.By by thin for described Human esophageal squamous cell cancer cell line ZEC-157 of certain cell quantity
Born of the same parents are 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 comparing research with normal esophageal cells and other kinds of cancer cell, it appeared that the molecule mark of the esophageal carcinoma
Note, can carry out the application of the aspects such as follow-up disease detection and drug development for this molecular marker.
Present invention also offers described esophageal cancer cell and tie up to the application in screening or assessment treatment esophageal carcinoma medicine.First
First, by adding different pharmaceutical in described Human esophageal squamous cell cancer cell line ZEC-157 culture medium, observation of cell state changes, obtains
Obtain tentatively effective drug candidate.Then, drug candidate is administered to the animal model of the above-mentioned esophageal carcinoma, observes and non-dispenser group
The survival period of animal, 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.Find food
After the specific tumor markers of pipe cancer, the detection esophageal carcinoma can be developed according to this tumor markers and occur or the inspection of development
Test agent box.
After the excision of the patient with esophageal carcinoma that the present inventor's esophageal squamous cell carcinoma cell line ZEC-157 comes from an example China
Tumor tissues, STR testing result proves that it is unique, and does not occurs the intersection with other cells dirty during original cuiture
Dye, Clone formation is strong with Tumor formation, can be as aspects such as esophageal carcinoma research and the exploitation of esophageal carcinoma detection kit, drug screenings
The ideal cell line of application.
Accompanying drawing explanation
Fig. 1 is the Morphology observation figure of Human esophageal squamous cell cancer cell line ZEC-157 cell；
Fig. 2 is the cell growth curve figure of Human esophageal squamous cell cancer cell line ZEC-157；
Fig. 3 is the karyotype scattergram of Human esophageal squamous cell cancer cell line ZEC-157 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-157 cellular immunization CYTOCHEMICAL ANALYSIS result figure；
Fig. 6 is the immunohistochemical analysis knot of the tumor tissues in source identical with Human esophageal squamous cell cancer cell line ZEC-157
Fruit figure；
Fig. 7 is Human esophageal squamous cell cancer cell line ZEC-157 cell clonal formation experimental result picture, wherein, and figure A, B and C inoculation
Cell number is respectively 500,1500 and 4500；
Fig. 8 is that mice becomes tumor experimental result picture.
Detailed description of the invention
Embodiment 1
From an example patient with esophageal carcinoma, (patient is 60 years old women, and tumor is positioned at Esophageal Middle Segment, and postoperative pathological shows low differentiation squama
Shape cell carcinoma, TNM T3N0M0 by stages, the patient clinical 3A phase by stages.) excision after tumor tissues sampling.Separator well
Fresh human esophageal carcinoma, after cleaning with PBS in aseptic super-clean bench, then with apparatuses such as ophthalmology tweezers, shears in culture dish
Shred and be separated into 0.5～1mm3Fritter be laid at the bottom of ware, and (GIBCO is public to add the 10%FBS containing 10mL in culture dish
Department), 1% dual anti-DMEM/F12 cell culture medium (GIBCO company), put into 37 DEG C, 5%CO2Incubator in cultivate.5
～change liquid after 7 days, discard the downright bad floating fritter come off in piece of tissue, carry out Secondary Culture.During Secondary Culture, utilize
Fibroblast is different from tumor cell digestion power, constantly removes fibroblast.After repeatedly passing on, naked eyes in culture dish
Cannot observe fibroblast, and can pass on by continued propagation.Build after being tied to form merit, named Human esophageal squamous cell cancer cell line ZEC-
157, the China typical culture collection center being positioned at Wuhan, China Wuhan University, preserving number it is preserved on May 11st, 2016
For: CCTCC NO:C201693.
Embodiment 2
Take the Human esophageal squamous cell cancer cell line ZEC-157 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 prosperous
Containing, clear background, impurity is rare, and cell is flat irregular polygon, and cell arrangement is tight, meets the spy of epithelioid cell
Point.
Embodiment 3
Cell index (Cell Index) refers to that living cells interacts with microelectrode in detection plate hole, produces electrical impedance
Changing, xCELLigence cell function analyser, these signals are converted into specific parameter becomes cell index.Cell refers to
Number has been weighed the state growth of cell, diffusion, alteration of form, death well, stress have been waited, and cell index is by many families
Magazine is commented on and adopts.
Take the Human esophageal squamous cell cancer cell line ZEC-157 cell that growth conditions is good, through trypsinization, make cell suspension,
And count.The E-Plate detection plate of xCELLigence cell function analyser adds culture medium and measures background impedance value,
Then in E-Plate detection plate, add 100 μ L cell suspension (5000), in room temperature super-clean bench, place 30min.To add thin
The E-Plate detection plate of born of the same parents puts into (monitor station is placed in advance in incubator) on monitor station, carries out the cell proliferation of Real-time and Dynamic
Detection can obtain cell proliferation curve, keeping count 4～6 days.Utilize Graph Pad Software on Drawing growth curve, and calculate
Obtain the population doubling time about 22 hours of cell.As in figure 2 it is shown, after the present inventor's esophageal squamous cell carcinoma cell line ZEC-157 is adherent
Growth is very fast, has good cultured and amplified in vitro.
Embodiment 4
Take Human esophageal squamous cell cancer cell line ZEC-157 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
Liquid Hypotonic treatment M phase cell 15～20min is at room temperature fixing thin as fixative with methanol/glacial acetic acid (volume ratio 3: 1)
Born of the same parents, film-making on slide.Slide being placed in 0.02% trypsin solution digestion 30～60s, rinses through PBS, Giemsa dyes, and dries in the air
Dry, complete film-making.In basis of microscopic observation, calculate chromosome number in each cell, randomly select 20 cells and calculate.
As it is shown on figure 3, chromosome number is distributed between 51～65 mostly, mode is 57, meets the feature of malignant tumor.
Embodiment 5
STR (short tandem repeat, STR) is also called microsatellite DNA.General long by 2 by one
～the core sequence of 6bp forms through the arrangement of repeatedly tandem sequence repeats, number of repetition is mostly between 10～60 times.Core sequence between individuality
The number of repetition of row is variability, thus the number of repetition of one group of STR sequence is the most unique in Different Individual, is
The main method that cell identity and source are identified by cytobiology.Collect the Human esophageal squamous cell cancer cell line of fresh cultured
ZEC-157 cell, 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 with 5' end fluorescently-labeled STR primer, right
Products therefrom 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 only
One, and during original cuiture, there is not the cross-contamination with other cells.
The copy number of table 1 STR bit point.
| Labelling (Marker) | Allele 1 (Allele 1) | Allele 2 (Allele 2) |
| TH01 | 9 | 9 |
| D12S391 | 19 | 22 |
| D7S820 | 9 | 12 |
| CSF1PO | 10 | 11 |
| FGA | 24 | 24 |
| AMEL | X | X |
| D5S818 | 11 | 11 |
| D2S1338 | 17 | 24 |
| D21S11 | 31 | 31 |
| D18S51 | 14 | 14 |
| TPOX | 8 | 8 |
| VWA | 14 | 20 |
| D8S1179 | 10 | 12 |
| D3S1358 | 15 | 15 |
| D13S317 | 10 | 11 |
| D6S1043 | 12 | 20 |
| D16S539 | 11 | 12 |
| PENTAE | 15 | 18 |
| D19S433 | 13 | 13 |
| PENTAD | 12 | 12 |
Embodiment 6
SABC detection Human esophageal squamous cell cancer cell line ZEC-157 cellular labeled proteins.S-P method is used to carry out SABC
Dyeing, related kit is purchased from Fuzhou Maixin biotechnology Development Co., Ltd.Antibody used is 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 goods
Number R628, DAKO company).
Tumor tissues SABC: tissue (for obtaining the same tumor sample of cell in embodiment 1) wax stone takes off through routine
Wax aquation, use 3%H2O2Deionized water soaks 10 minutes, to block endogenous peroxydase, is then put into 0.01M
In the inflexible rubber acid buffer of pH6.0, pressure cooker spray vapour half a minute carries out antigen retrieval.Dropping one resists, incubated at room temperature 60 minutes,
PBS flushing 3 times, each 2 minutes.Dropping Polymer Helper (polymeric adjuvants), incubated at room temperature 20 minutes,
PBS flushing 3 times, each 2 minutes.Dropping Polyperoxidase-anti-mouse/rabbit IgG, incubates under room temperature
Educate 30 minutes, PBS flushing 3 times, each 2 minutes.Dilution preparation DAB solution, develops the color 5～10 minutes, Microscopic observation.Clearly
Water rinses, and haematoxylin is redyed, conventional dehydration, transparent, mounting.
Tumor cell SABC: cell to be detected (Human esophageal squamous cell cancer cell line ZEC-157 cell) is climbed in advance for 24 hours
Sheet is on sterilization microscope slide, after 24 hours, rinses 2 times with PBS, and cold acetone fixes 15 minutes, is soaked in PBS standby
With.Taking the incubated at room temperature 10 minutes that required slide adds 50 μ L peroxidase blocking solution (reagent A), PBS rinses 3
Secondary, each 3 minutes, remove PBS liquid, drip the normal nonimmune animal serum of 50 μ L (reagent B), incubated at room temperature 10 minutes；Remove
Serum deprivation, drips 50 μ L mono-and resists, and incubated at room temperature 60 minutes, PBS flushes three times, each 3～5 minutes；Dropping two resists, under room temperature
Hatching 10 minutes, PBS flushes three times, each 3 minutes.Dropping Polyperoxidase-anti-mouse/rabbit IgG, room
Hatch under temperature 30 minutes, PBS flushing 3 times, each 2 minutes.Dilution preparation DAB solution, develops the color 5～10 minutes, under mirror
Observe.Clear water rinses, and haematoxylin is redyed, conventional dehydration, transparent, mounting.
As shown in Figure 5 and Figure 6, Human esophageal squamous cell cancer cell line ZEC-157 cell is that CK5/6 is positive to result, and CK14 is positive,
P63 is positive, and CgA is negative, and the weak positive of Sy and CD56 are negative.The immunohistochemistry results with tissue of cell all illustrates people's esophagus
Squamous cell carcinoma system ZEC-157 is scale cancer source.
Embodiment 7
Take the Human esophageal squamous cell cancer cell line ZEC-157 cell that growth conditions is good, after trypsinization, make cell suspension,
And count.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 10min, so
Afterwards by 0.1% violet staining 10min, wash away dyeing liquor, drying at room temperature, and Taking Pictures recording.As it is shown in fig. 7, Human esophageal squamous cell cancer
The cloning efficiency of cell line ZEC-157 cell and inoculum density have relation, and inoculum density is the cell gram during 4500 cells
Grand Forming ability is stronger.
Embodiment 8
Take the Human esophageal squamous cell cancer cell line ZEC-157 cell that growth conditions is good, after trypsinization, make cell suspension,
And count.Cell suspension is inoculated into 5 nude mice (nude mouse) oxters, Continuous Observation respectively with 10,000,000 cells.Test knot
As shown in Figure 8, accessible to lump, increase in time after 2 weeks after inoculation, lump increases fruit.
Claims (8)
1. esophageal carcinoma cell line, it is characterised in that named Human esophageal squamous cell cancer cell line ZEC-157, preserving number is: CCTCCNO:
C201693。
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 answering in the cell model as esophageal carcinoma genesis mechanism research
With.
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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| CN111662874A (en) * | 2020-06-18 | 2020-09-15 | 上海市胸科医院 | Chinese esophageal squamous carcinoma cell line and application thereof |
| CN112790159A (en) * | 2020-10-15 | 2021-05-14 | 河南科技大学 | Method for establishing esophageal cancer PDX mouse model |
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| CN111662874A (en) * | 2020-06-18 | 2020-09-15 | 上海市胸科医院 | Chinese esophageal squamous carcinoma cell line and application thereof |
| CN112790159A (en) * | 2020-10-15 | 2021-05-14 | 河南科技大学 | Method for establishing esophageal cancer PDX mouse model |
| CN112790159B (en) * | 2020-10-15 | 2022-12-20 | 河南科技大学 | Method for establishing esophageal cancer PDX mouse model |
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