CN107083365A - A kind of Chinese Lungs gland cell system and its application - Google Patents

A kind of Chinese Lungs gland cell system and its application Download PDF

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CN107083365A
CN107083365A CN201710426801.8A CN201710426801A CN107083365A CN 107083365 A CN107083365 A CN 107083365A CN 201710426801 A CN201710426801 A CN 201710426801A CN 107083365 A CN107083365 A CN 107083365A
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cell
chinese
lungs
cafq1
cell system
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CN107083365B (en
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路丽明
孙攀
郑宁
张�杰
赵丹丹
杨玉琴
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Shanghai Chest Hospital
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    • 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
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    • C12N5/0693—Tumour cells; Cancer cells
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    • 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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    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57423—Specifically defined cancers of lung
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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Abstract

The invention discloses a kind of Chinese Lungs gland cell system CAFQ1 and its application, its deposit number is：CCTCC NO：C201774.Cell line of the present invention has similar protein expression to primary tumor tissue, while also having Chromosomal Abnormal Karyotype, the tumor characteristic with external one-tenth knurl ability.The cell line of the present invention can in vitro continuous passage culture and keep the characteristic of adenocarcinoma of lung tumour cell constant, be suitable as researching and developing the cell material of the treatment of adenocarcinoma of lung related immune, diagnosis and medicine.The cell line of the present invention includes ALK:Kras is mutated, and the two mutation can cause normal pulmonary epithelial cells to be mutated, the formation of tumour, promotes tumor cell proliferation.Therefore, this cell line can be used as studying ALK:The strong vehicles cells that Kras studies for the targeted drug of target.

Description

A kind of Chinese Lungs gland cell system and its application
Technical field
The present invention relates to microorganism medical domain, more specifically to a kind of Chinese Lungs gland cell system CAFQ1 and It is applied.
Background technology
Lung cancer is one of death rate highest tumour in current world wide, is accounted in death caused by tumour 19.4%.In Europe, with the reduction and the increase of female smoker of the Chinese number of smoking, the incidence of disease of male is dropping year by year Low, the incidence of disease of women increases year by year, wherein, 80%-85% is non-small cell lung cancer, and 5 year life cycle was less than 11%.2014 Show in the epidemiology of lung cancer research of Asia：43.4% is women in lung cancer, and average age of onset is 60 years old, 52.6% Non-smoking history, 51.4% has EGFR mutation.Lung cancer is broadly divided into：Non-small cell lung cancer and ED-SCLC.Non-small cell lung cancer Gland cancer, squamous carcinoma, maxicell lung cancer are broadly divided into, and adenocarcinoma of lung is most common type in lung cancer, accounts for 40%.Drive gene Mutation, plays vital effect such as EGFR, ALK, RET, ROS1 in the formation of adenocarcinoma of lung infantile tumour.Therefore, The efficiency of lung cancer therapy is greatly improved using the medicine for driving gene as target spot.But, do not have in most lung cancer The isogenic mutation of EGFR, the medicine of target spot class is acted on it less, and traditional chemotherapy and radiation is still the master of such lung cancer Want treatment means.Therefore, for such common lung cancer, we should increase more research inputs.
In the research of the lung cancer in the whole world, the stabilization set up, up to hundred kinds of the cell line passed on for a long time, but it is hereditary The Chinese ethnic group of background and Asia differs greatly.The heterogeneity and complexity of tumour be not agnate and geographical origins same Its cell characteristics of the tumour of kind organization type are also differed.For the adenocarcinoma of lung of significantly more efficient Study of China China people Pathogenesis and treatment method, the method that we use tumour cell original cuiture, newly-built one plant of Chinese Lungs gland cancer are thin Born of the same parents are that this plant of cell line has ALK：Kras is mutated, while having no EGFR mutation, is provided newly for China human lung cancer research Model, provide more preferable platform for the Mechanism Study of adenocarcinoma of lung.
The content of the invention
First technical problem to be solved by this invention is to provide Chinese Lungs gland cell system CAFQ1.
Second technical problem to be solved by this invention is to provide Chinese Lungs gland cell system CAFQ1 and controlled in preparation Treat the application in adenocarcinoma of lung medicine.
3rd technical problem to be solved by this invention is to provide Chinese Lungs gland cell system CAFQ1 and is preparing lung Application in gland cancer diagnostic reagent.
4th technical problem to be solved by this invention is to provide Chinese Lungs gland cell system CAFQ1 and is preparing lung Application in gland cancer animal model.
5th technical problem to be solved by this invention is to provide Chinese Lungs gland cell system CAFQ1 as medicine Target spot is preparing the application in suppressing adenocarcinoma of lung medicine.
In order to solve above-mentioned first technical problem, the invention provides Chinese Lungs gland cell system CAFQ1, its feature It is, its deposit number is：CCTCC NO：C201774.
In order to solve above-mentioned second technical problem, prepared the invention provides Chinese Lungs gland cell system CAFQ1 Treat the application in adenocarcinoma of lung medicine.
In order to solve above-mentioned 3rd technical problem, prepared the invention provides Chinese Lungs gland cell system CAFQ1 Application in adenocarcinoma of lung diagnostic reagent.
In order to solve above-mentioned 4th technical problem, prepared the invention provides Chinese Lungs gland cell system CAFQ1 Application in adenocarcinoma of lung animal model.
In order to solve above-mentioned 5th technical problem, medicine is used as the invention provides Chinese Lungs gland cell system CAFQ1 Thing target spot is preparing the application in suppressing adenocarcinoma of lung medicine.
The claimed cell line of the present invention is named as Chinese Lungs gland cell system CAFQ1, is deposited in Chinese Typical Representative training Support thing collection (preservation address：The Wuhan University's Chinese Typical Representative culture of Wuhan City, Hubei Province Wuchang District Luo Jia Shan road 16 is protected Tibetan center postcode 430072), preservation date is on May 23rd, 2017, and deposit number is CCTCC NO：C201774.
Beneficial effects of the present invention：(1) cell line of the present invention has similar protein expression to primary tumor tissue, simultaneously Also there is Chromosomal Abnormal Karyotype, the tumor characteristic with external one-tenth knurl ability.(2) cell line of the invention can be continuous in vitro Secondary Culture and keep the characteristic of adenocarcinoma of lung tumour cell constant, be suitable as the treatment of research and development adenocarcinoma of lung related immune, diagnosis and The cell material of medicine.(3) cell line of the invention includes ALK:Kras is mutated, and the two mutation can cause on normal lung Chrotoplast is mutated, the formation of tumour, promotes tumor cell proliferation.Therefore, this cell line can be used as studying ALK:Kras The strong vehicles cells studied for the targeted drug of target.
Brief description of the drawings
Fig. 1 is cellular morphology (100X) under a kind of Chinese Lungs gland cell system CAFQ1 inverted microscopes of the invention.
Fig. 2 is a kind of Chinese Lungs gland cell system CAFQ1 growth and proliferation of cell curve of the invention.
Fig. 3 is a kind of Chinese Lungs gland cell system CAFQ1 chromosome karyotype analysis figure of the invention.
Fig. 4 is a kind of Chinese Lungs gland cell system CAFQ1 nude mice by subcutaneous of the invention into knurl figure.
Embodiment
With reference to specific embodiment, the present invention is expanded on further.Experimental method used in following embodiments for example without Specified otherwise, is conventional method.Material, reagent used etc. in following embodiments, unless otherwise specified, can be from business way Footpath is obtained.It should be understood that these embodiments are only illustrative of the invention and is not intended to limit the scope of the invention.
Embodiment 1.
The separating treatment of primary tumor cell
It is primary thin that the fresh Chinese tumor tissue obtained from hospital's operating table carries out tumour in the case where answering gnotobasis Born of the same parents' is separately cultured.
1. first inserting tumor tissue in 50ml centrifuge tubes, washed 5 times with being rocked containing 10 times of dual anti-PBS, wash nothing Watery blood.Tissue block is put into culture dish, excess tissue, such as fat or slough is cut off.
2. tissue is moved in another plate, about 1mm is frittered into intersection scalpel3The tissue block of size.Take 70um Sieves, be placed on 50ml centrifuge tube.Gently being pressurizeed with the core of syringe makes tissue enter culture medium by mesh, Absorption culture medium blows over screen cloth and sweeps away cell.
3. add 5ml pancreatin/clostridiopetidase A II in the culture dish of remaining tissue block, 37 DEG C of incubators digest 2 hours, during which At interval of piping and druming in 15 minutes once.After the completion of digestion, 70um strainer filterings 1 time, the cell liquid under filter adds PBS to 5ml, 1200rpm, 5 minutes centrifuge washings are once.
Cultivated 4. centrifugation gained cell is transferred in culture dish, while tissue block is put into other culture dishes, stand training Support.12-24 hours definitely are stood, the slow culture dish that takes out adds nutrient solution, continues to cultivate, stands 2-3 days.Carefully taken out after 3 days Blake bottle examines and removes the fragment of floating, changes nutrient solution and continues to cultivate.
The in vitro culture of primary tumor cell
1. interval 24-48 hours.
2. smooth Microscopic observation cell growth status, changes appropriate incubation liquid in good time.Such as seeing after adherent cell growth can be appropriate Increase Culture liquid measure, with needed for meeting cell growth.
3. the condition of culture of culture of the present invention is：In 37 DEG C, 5%CO2Cultivated under environment, culture medium is RPMI1640+ 10%FBS Cell Basal Mediums.
The Colony Culture of the primary tumor cell of embodiment 2.
1. the digestion of cell pancreatin is prepared into single cell suspension, cell is diluted to 1 × 10/mL by cell count.Specifically Do not walk as follows：
A) postdigestive cell is diluted to 1 × 105Individual/mL.
B) 1 × 10 is taken5Individual/mL cell suspension 200uL, add to 20mL, as 1 × 103Individual/mL.
C) by 1 × 103Individual/mL cell suspension 200uL, add to 20mL, as 1 × 10/mL.
2. adding 100uL 1 × 10/mL cell suspensions in the microtiter culture plate of 96 orifice plates, it can be formed per hole One colony.
3. being inoculated with after a few houres, the culture hole of only one of which cell is found out by microscope.This some holes is marked, tracking is seen Examine, PH, which declines, represents cell growth, is confirmed by micro- sem observation.After cell is covered with, pancreatin digestion 6 orifice plates of access are passed It is commissioned to train foster.
The Chinese Lungs gland cell system CAFQ1 of embodiment 3. Testing and appraisal
1. the morphological observation of primary tumor cell strain
Tumour cell form majority is in shuttle-type and oval type under light microscopic, and minority is in polygonal, and cell the two poles of the earth are tapering, nucleus Substantially, a small number of is in multinuclear, and kernel is high-visible, the big characteristic of caryoplasm ratio.It is random that the cell of exponential phase has sprawled CAFQ1 It is generally oval epithelioid cell to stretch, and polygonal, form is relatively regular.As shown in Figure 1.
2. immunofluorescence detects cellular labeled proteins
1) 1mL complete medium suspends after trypsin digestion cell, fully blows and beats into single cell suspension, carries out cytometer Number.24 orifice plate access 5 × 10 as needed4Individual cell.It is added dropwise before cell suspension first in culture medium a small amount of every Kong Lijia, makes Slide is sticked together with culture dish reduces cell entrance, is hiked up to prevent slide.
2) the fresh PBS of slide for climbing cell is washed twice.The paraformaldehyde fixation 40min that PBS adds 4% is blotted, PBS is washed twice after methanol is fixed, and 5 minutes every time, blots unnecessary PBS.0.2% Triton X-100, are dissolved in PBS, room Temperature is placed 15 minutes, carries out cell membrane punching.Punching liquid is blotted, PBS is added, washes twice.Instill lowlenthal serum closing 1h.
3) primary antibody is dissolved in lowlenthal serum, drips to and climbed on Cell sheet glass, 4 ° of refrigerator overnights are incubated.PBS is washed within second day Three times, fluorescence secondary antibody 1h is incubated at room temperature, lucifuge is noted.PBS is washed three times, with 90% glycerine mounting.
Chinese Lungs gland cell system CAFQ1 of the present invention immunofluorescence dyeing result shows, Chinese Lungs adenocarcinoma cell It is that CAFQ1 has similar protein expression to show as TTF1, CEA, vimentin and pan- to the pulmonary adenocarcinoma in source Keratin is positive.
2. primary tumor cell strain growth curve detection
1) single cell suspension is made in cell pancreatin digestion, is counted with tally after concentration of cell suspension, is configured to experiment The cell concentration 1 × 10 needed3Individual/mL.
2) inoculating cell suspension (the 100ml/ holes) in 96 orifice plates.By culture plate be placed on incubator preculture (at 37 DEG C, 5%CO2Under conditions of).
3) the CCK-8 solution for adding 10ml to every hole (is careful not in hole generate bubble, they can influence the reading of OD values Number).
4) culture plate is incubated 1-4 hours in incubator.
5) absorbance at 450nm is determined with ELIASA.
Chinese Lungs gland cell system CAFQ1 of the present invention growth curve is as shown in Fig. 2 its exponential phase is 6.5 days.
Embodiment 4,
Cell karyotyping detection cell caryogram feature, chromosome quantitative etc..
1) cell in exponential phase is added into colchicine, makes colchicine final concentration 0.1ug/ml, continue to train Support 3h；
2) 10ml tip centrifuge tubes will be transferred to after cell dissociation, 1000rpm centrifuges 10min, removes supernatant, adds 0.075mol/L KC110ml, 37 DEG C are incubated after 30min, then the addition Fresh fixative (glacial acetic acid into centrifuge tube：Methanol= 3:1) 1ml, is mixed, 1000rpm, is centrifuged 10min, is abandoned supernatant；
3) Fresh fixative 10ml is slowly added into, gently blows even, room temperature fixes 20min, centrifugation removes supernatant, then fix； 1000rpm, centrifuges 10min, abandons supernatant；
4) Fresh fixative 250ul is added, gently blows even, in the height apart from slide 15cm, drips suspension, air Fully dry, fresh Giemsa dyeing, running water is rinsed, it is transparent to dry rear dimethylbenzene, resinene closing, Microscopic observation.
The chromosome structure and number of Chinese Lungs gland cell system of the present invention show exception, in heteroploid or many times 37 metaphase phases are analyzed under body, mirror, nucleus is more, larger, rounded；37 split coil method chromosome numbers are 43-57 bars.As shown in Figure 3.
Embodiment 5.
To heterogenous animal Inoculation cell suspension, one-tenth knurl ability is observed.
1) the hind leg root injection of skin Chinese Lungs adenocarcinoma cell sterilized to the nude mice for 34 week old that numbering is 1,2,3 It is CAFQ1 cell suspensions, every 1x107Pressed lightly on after individual cell, injection, do not make outflow.
2) the 12nd day, 3 nude mices occurred in that white projection, the 40th day in cell infusion region, it is seen that tumour is in lobate Growth, tumour major diameter is 12mm-15mm, and nude mice is put to death after anesthesia.
Chinese Lungs gland cell system of the present invention can be prepared in nude mice by subcutaneous into knurl and filter out with suppression and kill Hinder the antibody of CAFQ1 cells, the main component of Antilung gland cancer medicine is used as using this antibody.
Embodiment 6.
Tumour cell genome sequencing
Cell count 1x106Individual cell of the present invention, using QINGEN DNA extraction kit, extracts its DNA, carries out deep Degree sequencing.
ALK is detected in Chinese Lungs gland cell system of the present invention:Kras gene mutations, have no EGFR mutation.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art Member, under the premise without departing from the principles of the invention, can also make some improvements and modifications, these improvements and modifications also should be regarded as Protection scope of the present invention.

Claims (5)

1. Chinese Lungs gland cell system CAFQ1, it is characterised in that its deposit number is：CCTCC NO：C201774.
2. applications of the Chinese Lungs gland cell system CAFQ1 described in claim 1 in treatment adenocarcinoma of lung medicine is prepared.
3. applications of the Chinese Lungs gland cell system CAFQ1 described in claim 1 in adenocarcinoma of lung diagnostic reagent is prepared.
4. applications of the Chinese Lungs gland cell system CAFQ1 described in claim 1 in adenocarcinoma of lung animal model is prepared.
5. Chinese Lungs gland cell system CAFQ1 described in claim 1 is as drug target in suppression adenocarcinoma of lung medicine is prepared Application.
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108118031A (en) * 2017-12-27 2018-06-05 上海市胸科医院 A kind of drug resistance of lung cancer cell line and preparation method thereof
CN111733135A (en) * 2020-06-16 2020-10-02 华东医院 Chinese lung squamous carcinoma cell line and application thereof
CN113512531A (en) * 2021-06-04 2021-10-19 广东省实验动物监测所 Lung adenocarcinoma cell line and application thereof
CN113817684A (en) * 2021-09-30 2021-12-21 天津医科大学总医院 Chinese lung adenocarcinoma cell line with high lymph node metastasis potential and application thereof
CN116904521A (en) * 2023-09-13 2023-10-20 四川大学华西医院 Mutant lung adenocarcinoma cell line, construction method and application

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CN105567639A (en) * 2014-10-13 2016-05-11 上海市肿瘤研究所 Human lung adenocarcinoma cell LC-21 and application thereof

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CN105567639A (en) * 2014-10-13 2016-05-11 上海市肿瘤研究所 Human lung adenocarcinoma cell LC-21 and application thereof

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108118031A (en) * 2017-12-27 2018-06-05 上海市胸科医院 A kind of drug resistance of lung cancer cell line and preparation method thereof
CN111733135A (en) * 2020-06-16 2020-10-02 华东医院 Chinese lung squamous carcinoma cell line and application thereof
CN111733135B (en) * 2020-06-16 2023-03-10 华东医院 Chinese lung squamous carcinoma cell line and application thereof
CN113512531A (en) * 2021-06-04 2021-10-19 广东省实验动物监测所 Lung adenocarcinoma cell line and application thereof
CN113512531B (en) * 2021-06-04 2022-05-31 广东省实验动物监测所 Lung adenocarcinoma cell line and application thereof
CN113817684A (en) * 2021-09-30 2021-12-21 天津医科大学总医院 Chinese lung adenocarcinoma cell line with high lymph node metastasis potential and application thereof
CN113817684B (en) * 2021-09-30 2023-09-19 天津医科大学总医院 Chinese lung adenocarcinoma cell line with high lymph node metastasis potential and application thereof
CN116904521A (en) * 2023-09-13 2023-10-20 四川大学华西医院 Mutant lung adenocarcinoma cell line, construction method and application
CN116904521B (en) * 2023-09-13 2024-01-26 四川大学华西医院 Mutant lung adenocarcinoma cell line, construction method and application

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