CN107312752A - A kind of lung cancer cell line and its application - Google Patents
A kind of lung cancer cell line and its application Download PDFInfo
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Abstract
The invention provides a kind of lung cancer cell line and its application, lung cancer cell line is named as human lung cancer cell line ZLC 001, and preserving number is：CCTCC NO：C201754.The present inventor's lung cancer cell line ZLC 001 comes from the tumour cell of a Chinese Pleural Fluid of Patients With Lung Cancer, STR testing results prove that it is unique, and do not occur cross pollution with other cells during original cuiture, Clone formation is strong with Tumor formation, can be used as the ideal cell line applied in terms of lung cancer research and lung cancer detection kit developing, drug screening.
Description
Technical field
The present invention relates to biology and oncology technical field, more particularly to a kind of lung cancer cell line and its application.
Background technology
At present, lung cancer is morbidity and mortality highest tumour in the world, and non-small cell lung cancer (NSCLC) accounts for all
About the 85% of lung cancer.Predicted according to the cancer data of China, 73.33 Wan Xinfa cases of lung cancer will be had in 2015, are had
61.02 ten thousand people die from lung cancer, therefore, and lung cancer has become the key factor for threatening health of the masses.
The clinical manifestation of lung cancer is more complicated, the presence or absence of sings and symptoms, weight and the morning and evening of appearance, depending on tumour
Happening part, histological type, whether there is transfer and whether there is complication, and reaction degree and tolerance difference.Lung cancer
Early symptom is often relatively slight, or even can be without any discomfort.Central type carcinoma of lung symptom occurs early and again, and peripheral type carcinoma of lung symptom occurs
It is late and relatively light or even asymptomatic, often it is found in physical examination.The main underwent operative of Early stage NSCLC (I, II phase), about
25% operation case receives chemotherapy and/or radiotherapy, and the NSCLC patient of most of III phases and IV phases typically can receiving
Treatment, radiotherapy.At present, 5 years survival rates of the early stage of lung cancer can reach 55%, but 5 years survival rates of advanced lung cancer only have
4%.Because there are not clinical symptoms, existing DISTANT METASTASES IN when most of lung cancer (57%) are made a definite diagnosis in the early stage of lung cancer.
With some developments on lung cancer molecular biology research, the generation of some oncogenes and tumor suppressor gene in tumour
Very important effect is played in development, these discoveries fundamentally change the therapeutic scheme of Patients with Non-small-cell Lung.Cause
This, target therapeutic agent, such as angiogenesis inhibitors, EGF-R ELISA (EGFR) inhibitor and anaplastic lymphoma swash
Enzyme (ALK) inhibitor also becomes the pith of NSCLC treatments.In addition, immunization therapy is by targetting apoptosis
The drug receptor of effect has been approved for treating certain form of NSCLC in T cell.
For the cancer therapy drug research of the pathogenesis and targeted therapy and chemotherapy of understanding non-small cell lung cancer in depth, it is necessary to build
Found suitable research model.However, the NSCLC Patient cells system having built up at present all derives from western countries, but one mostly
A little epidemiology, molecular studies and clinical manifestation find that yellow and white people have phase in the various aspects of non-small cell lung cancer
As the female ratio of big difference, including non-smoking, EGFR catastrophes and tyrosine kinase inhibitor (TKI) clinical drug are anti-
It should wait.Moreover, the clinical practice of going deep into the treatment concept of the accurate medical science of tumour contributes to there is provided more lung cancer cell lines
The research and development and application of targeted drug.The lung cancer cell line of Chinese people source is set up, the pathogenesis and treatment for lung cancer provide new
Model, be worth with important theory and actual application.
The content of the invention
The present invention lacks the lung cancer cell line of Chinese people source for domestic at present, and derives from Chinese there is provided a kind of
Lung cancer cell line.
From a patients with lung cancer, (patient is 44 years old Chinese women to the present inventor's lung cancer cell line ZLC-001, and tumour is located at the right side
In lung, inferior lobe, the thoracoscope wall of the chest endometrial biopsy pathology prompting (right side parietal pleura) gland cancer, phase clinical stages 4.) hydrothorax, centrifugation
After take tumour cell, through original cuiture and after building and being tied to form work(, human lung cancer cell line ZLC-001 is named as, on May 31st, 2017
The China typical culture collection center positioned at Wuhan, China Wuhan University is preserved in, preserving number is：CCTCC NO：C201754.
The present inventor's lung cancer cell line ZLC-001 cells are observed under inverted light phase contrast microscope in flat irregular many
Angular, cell volume is big, and core is big, and endochylema enriches, and Cell tracking is close, adherent growth, the characteristics of meeting galandular epithelium like cell.
Growth is very fast after adherent, with good cultured and amplified in vitro.Chromosome number is distributed between 60~69 mostly, and mode is
61, meet the feature of malignant tumour.
The cells such as the present inventor's lung cancer cell line ZLC-001 STR sequencing results and ATCC, DSMZ preserve the database in storehouse
Carry out inquiry contrast, identical STR testing results do not found, it was demonstrated that it is unique, and do not occur during original cuiture and
The cross pollution of other cells.Immunohistochemical experiment finds that human lung cancer cell line ZLC-001 cells are positive in CK7, and CK5/6 is cloudy
Property, NSE feminine genders, p63 feminine genders, TTF1 is negative and Vimentin is positive.
Colony formation finds that the cloning efficiency of human lung cancer cell line's ZLC-001 cells is relevant with inoculum density, connects
The Cell clonality planted when density is 4500 cells is stronger.
Nude mice (nude mouse) finds that human lung cancer cell line ZLC-001 has stronger Tumor formation into knurl experimental result.
Invention further provides the daughter cell of described lung cancer cell line.The daughter cell is basic or all remains
The characteristic of parental cell.
Answering in the cell model as lung cancer genesis mechanism research is tied up to invention further provides described lung carcinoma cell
With.Because the present inventor's lung cancer cell line ZLC-001 is to originate to set up from Chinese, and it is that the time is shorter to build, and character is stable,
Using the lung cancer cell line as research model, have very great help for the primary lung cancer genesis mechanism for understanding Chinese.
The application set up in mammal lung cancer model is tied up to present invention also offers described lung carcinoma cell.The described food in one's mouth
Newborn animal is nude mouse or nude rat.By the way that the human lung cancer cell line ZLC-001 cells of certain cell quantity are inoculated in
The position such as nude mouse or the subcutaneous of nude rat, liver, abdominal cavity or tail vein, obtains the animal model of lung cancer.
The application extracted in lung cancer specific tumour mark is tied up to present invention also offers described lung carcinoma cell.Pass through
Research is compared with normal pneumonocyte and other kinds of cancer cell, it can be found that the molecular labeling of lung cancer, for this
Molecular labeling can carry out the application in terms of follow-up disease detection and drug development.
Screening is tied up to present invention also offers described lung carcinoma cell or assesses the application in treatment lung-cancer medicament.First,
By adding different pharmaceutical into the human lung cancer cell line ZLC-001 culture mediums, observation cell state change, acquisition tentatively has
The drug candidate of effect.Then, drug candidate is administered to the animal model of above-mentioned lung cancer, observation and the survival of non-dispenser group animal
Phase, tumor size, transfer case etc., screening obtain the medicine of potential treatment lung cancer.
Present invention also offers the application that described lung carcinoma cell is tied up in exploitation lung cancer detection kit.It was found that lung cancer is special
After the tumor markers of the opposite sex, the detection reagent of the generation of detection lung cancer or development can be developed according to the tumor markers
Box.
The present inventor's lung cancer cell line ZLC-001 comes from a Chinese Pleural Fluid of Patients With Lung Cancer, and STR testing results are proved
It is unique, and the cross pollution with other cells does not occur during original cuiture, and Clone formation and Tumor formation by force, can
To be used as the ideal cell line applied in terms of lung cancer research and lung cancer detection kit developing, drug screening.
Brief description of the drawings
Fig. 1 is the Morphology observation figure of the present inventor's lung cancer cell line ZLC-001 cells；
Fig. 2 is the present inventor's lung cancer cell line ZLC-001 cell growth curve figure；
Fig. 3 is the karyotype distribution map of the present inventor's lung cancer cell line ZLC-001 cells；
Fig. 4 is STR analysis result figures, wherein, figure A~F is the result figure of not iso-allele respectively；
Fig. 5 is human lung cancer cell line's ZLC-001 Immunohistochemical analysis result figures, wherein, figure A~F is respectively
CK7、CK5/6、NSE、p63、TTF1、Vimentin；
Fig. 6 is the present inventor's lung cancer cell line ZLC-001 cell clonal formation experimental result pictures, wherein, figure A, B and C connect
Kind of cell number is respectively 500,1500 and 4500；
Fig. 7 is tumor formation in nude mice result figure.
Embodiment
Embodiment 1
From a patients with lung cancer, (patient is 44 years old Chinese women, and tumour is located in right lung, inferior lobe, thoracoscope wall of the chest inner membrance
Biopsy pathology points out (right side parietal pleura) gland cancer, phase clinical stages 4.) hydrothorax.By hydrothorax low-speed centrifugal, 1000rpm, 5 points
Clock.Supernatant discarding, adds appropriate PBS, and soft piping and druming cell, is resuspended it, centrifuges again repeatedly, 1000rpm, 5 points
Clock.Supernatant discarding, adds 10mL (GIBCO companies) containing 10%FBS, 1% is dual anti-, and (GIBCO is public for 1% nonessential amino acid
Department), the primitive cell culture base of DMEM/F12 cell culture mediums (GIBCO companies), soft piping and druming is mixed, and suspension is transferred to
In 10cm culture dishes, 5%CO is put into2, in 37 DEG C of cell culture incubators, fresh culture is changed after 5~7 days.In succeeding generations, profit
Had differences with the digestion power of fibroblast and tumour cell, constantly remove fibroblast, until culture dish in without
Method can continue passage growth to macroscopic fibroblast.Build and be tied to form after work(, be named as human lung cancer cell line ZLC-
001, the China typical culture collection center being preserved on May 31st, 2017 positioned at Wuhan, China Wuhan University, preserving number
For：CCTCC NO：C201754.
Embodiment 2
Human lung cancer cell line's ZLC-001 cells of Secondary Culture are taken, under an optical microscope (Japanese Olympus IMT-2
Inverted microscope) observation living cell growth situation.As shown in figure 1, cell Optical Morphology picture, it is seen that cell growth is vigorous, the back of the body
Scape is clear, and impurity is rare, and cell is in flat irregular polygonal, and cell volume is big, and core is big, and endochylema enriches, and Cell tracking is tight
It is close, adherent growth, the characteristics of meeting galandular epithelium like cell.
Embodiment 3
Cell Counting Kit abbreviation CCK kits, are the alternative of mtt assay, are a kind of (water-soluble based on WST
Tetrazolium salts, chemical name：2- (2- methoxyl group -4- nitre phenyl) -3- (4- nitre phenyl) -5- (2,4- disulfobenzenes) -2H- tetrazolium list sodium
Salt) be widely used in cell propagation and cytotoxicity fast high-sensitive degree detection kit.
Human lung cancer cell line's ZLC-001 cells that growth conditions are good are taken, is digested through pancreatin, cell suspension is made, and count
Number.Inoculating cell suspension (the 100 μ L/ holes) in 96 orifice plates.Culture plate is placed on preculture in incubator (in 37 DEG C, 5%CO2
Under conditions of).Detected respectively 6 hours, 24 hours, 48 hours, 72 hours, 96 hours, 120 hours.To every during detection
Hole adds 5 μ L CCK solution, and culture plate is incubated 4 hours in incubator, and the absorbance at 450nm is determined with ELIASA.
As shown in Fig. 2 growth is very fast after the present inventor's lung cancer cell line ZLC-001 is adherent, with good cultured and amplified in vitro.
Embodiment 4
Human lung cancer cell line's ZLC-001 cells are taken, are planted in six orifice plates, after cultivating 48 hours, with 0.01mg/mL autumn
Narcissus element processing 16h, when microscopy it was observed that when M phases cell proportion is more than 50%, collecting M phase cells.It is low using KCl hypotonic mediums
Processing M 15~20min of phase cell are oozed, cell, glass are fixed at room temperature as fixer with methanol/glacial acetic acid (volume ratio 3: 1)
Film-making on piece.Slide is placed in 30~60s of digestion in 0.02% trypsin solution, rinsed through PBS, Giemsa dyeing is dried, completed
Film-making.In micro- Microscopic observation, chromosome number in each cell is calculated, 20 cells is randomly selected and is calculated.Such as Fig. 3 institutes
Show, chromosome number is distributed between 60~69 mostly, mode is 61, meet the feature of malignant tumour.
Embodiment 5
STR (short tandem repeat, STR) is also known as microsatellite DNA.It is general long by 2 by one
~6bp core sequence is formed through the arrangement of multiple tandem sequence repeats, and number of repetition is mostly between 10~60 times.Core sequence between individual
The number of repetition of row is in variability, thus the number of repetition of one group of STR sequence is almost unique in Different Individual, is
The main method that cell biology is identified cell identity and source.Collect the human lung cancer cell line ZLC- of fresh cultured
001 cell, (Qiagen companies, name of article QIAamp DNA Mini are purchased from Qiagen genomic DNAs Mini Kit
Kit, article No. is 51304) to extract cell genomic dna, and entering performing PCR with the STR primers of 5' ends fluorescence labeling is expanded, and gained is produced
Thing is sequenced.Wherein the primer sequence and copy number of STR bit point are as shown in table 1 and Fig. 4.Above-mentioned sequence and ATCC, DSMZ etc.
Cell preserve storehouse database carry out inquiry contrast, identical STR testing results are not found, it is possible thereby to prove its be it is unique,
And do not occur cross pollution with other cells during original cuiture.
The copy number of the STR bit point of table 1.
Embodiment 6
SABC detects human lung cancer cell line's ZLC-001 cellular labeled proteins.SABC dye is carried out using S-P methods
Color, 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. IR619, DAKO are public by CK7
Department), TTF1 (article No. MAB-0599, Foochow steps new company), NSE (article No. ZM-0203, Beijing company of Zhong Shan Golden Bridge),
Vimentin (IR630, DAKO company).
Tumour cell SABC：Cell (human lung cancer cell line ZLC-001 cells) to be detected shift to an earlier date 24 hours creep plates in
Sterilize on slide, after 24 hours, rinsed 2 times with PBS, ice paraformaldehyde fixes 15 minutes, is rinsed with PBS
3 times, each 2 minutes, it is soaked in standby in PBS.Slide needed for taking plus 0.5%Triton X-100 (DPBS matches somebody with somebody) are incubated 20 minutes,
PBS rinses 3 times, each 2 minutes, then adds the incubation at room temperature 10 minutes of 50 μ L peroxidase blocking solutions (reagent A),
PBS is rinsed 3 times, 3 minutes every time, removes PBS liquid, the normal nonimmune animal blood serums (reagent B) of 50 μ L is added dropwise, at room temperature
It is incubated 10 minutes；Except serum deprivation, 50 μ L primary antibodies are added dropwise, are incubated 60 minutes at room temperature, PBS is flushed three times, 3~5 minutes every time；Drop
Plus secondary antibody, it is incubated 10 minutes at room temperature, PBS is flushed three times, 3 minutes every time.Polyperoxidase-anti-mouse/ is added dropwise
Rabbit IgG, are incubated 30 minutes at room temperature, and PBS is rinsed 3 times, every time 2 minutes.DAB solution, colour developing 5 are prepared in dilution
~10 minutes, Microscopic observation.Clear water is rinsed, and haematoxylin is redyed, conventional dehydration, transparent, mounting.
As a result as shown in figure 5, human lung cancer cell line ZLC-001 cells are positive in CK7, CK5/6 is negative, and NSE is negative, p63
Feminine gender, TTF1 is negative and Vimentin is positive.The immunohistochemistry results prompting human lung cancer cell line ZLC-001 of cell is gland
Cancer source.
Embodiment 7
Human lung cancer cell line's ZLC-001 cells that growth conditions are good are taken, cell suspension are made after being digested through pancreatin, and count
Number.Cell suspension is inoculated in 6 orifice plates with 500,1500,4500 cell per wells.6 orifice plates are placed in incubator, stood
Culture 2 weeks, changes weekly liquid 2 times.After 2 weeks, cell culture fluid is discarded, after being rinsed with PBS, absolute methanol fixes 10min, Ran Houyong
0.1% violet staining 10min, washes away dyeing liquor, drying at room temperature, and photograph to record.As shown in fig. 6, human lung cancer cell line
The cloning efficiency of ZLC-001 cells has relation, cell clonal formation when inoculum density is 4500 cells with inoculum density
Ability is stronger.
Embodiment 8
Human lung cancer cell line's ZLC-001 cells that growth conditions are good are taken, cell suspension are made after being digested through pancreatin, 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 is such as
It is accessible after 2 weeks after inoculation to arrive lump shown in Fig. 7, with the increase of time, lump increase.
Claims (8)
1. lung cancer cell line, it is characterised in that be named as human lung cancer cell line ZLC-001, preserving number is：CCTCC NO：
C201754。
2. the daughter cell of lung cancer cell line as claimed in claim 1.
3. the application that lung carcinoma cell as claimed in claim 1 is tied up in the cell model as lung cancer genesis mechanism research.
4. lung carcinoma cell as claimed in claim 1 ties up to the application set up in mammal lung cancer model.
5. application as claimed in claim 4, it is characterised in that described mammal is nude mouse or nude rat.
6. lung carcinoma cell as claimed in claim 1 ties up to the application extracted in lung cancer specific tumour mark.
7. lung carcinoma cell as claimed in claim 1 ties up to screening or assesses the application in treatment lung-cancer medicament.
8. the application that lung carcinoma cell as claimed in claim 1 is tied up in exploitation lung cancer detection kit.
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| CN102586188A (en) * | 2011-12-31 | 2012-07-18 | 广州呼吸疾病研究所 | Cell line from human lung large cell cancer and preparation method thereof |
| 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 |
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| CN102586188A (en) * | 2011-12-31 | 2012-07-18 | 广州呼吸疾病研究所 | Cell line from human lung large cell cancer and preparation method thereof |
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