CN105368783B - A kind of human lung cancer cell strain of resistance to paraquat - Google Patents
A kind of human lung cancer cell strain of resistance to paraquat Download PDFInfo
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Abstract
The present invention use human lung carcinoma cell line A549 for induce object, using the period it is exposed, gradually be incremented by paraquat concentration external evoked method, establish the cell line A549 of resistance to paraquat/PQ.The cell strain was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on June 16th, 2015, and deposit number is CGMCC NO.10892.Cell strain of the invention has typical resistance to paraquat characteristic, this biological characteristics can reverse paraquat toxicity approach, extraction paraquat poisoning to evaluate marker, the anti-oxidant new way of excavation etc. for further research and provide research model, research and production application value with higher.
Description
Technical field
The invention belongs to cell engineering fields, are related to a kind of human cell's strain, and in particular to a kind of human lung cancer resistance to hundred
The withered cell strain of grass and its method for building up and application.
Background technique
Paraquat (paraquat, PQ) is a kind of widely used high-effect herbicide in the whole world, is had to human body very strong
Toxicity, can cause to be poisoned through absorptions such as alimentary canal, skin, respiratory tract and veins, clinically lack effective remedy measures,
Mortality is up to 40~80%.The production of China PQ and dosage rank the first in the world.In recent years country PQ poisoning morbidity is in again
Number increases, and prevention and control situation is very severe.Although in June, 2015 China starts to release the production and sales for gradually forbidding PQ aqueous,
But other dosage forms will continue production and use.Therefore, paraquat poisoning is still that China will be poisoned the vital task of prevention and treatment from now on.
PQ poisoning can cause the multiple visceral organ injuries of whole body, and wherein lung is the target organ of involvement most serious, may occur in which " paraquat
Lung " shows as acute lung injury in early days, and stage progresses to pulmonary interstitial fibrosis, is the main of PQ poisoning patient death
Reason.PQ is in the key that the active intake and accumulation of lung tissue are considered as that successive induction injury of lungs causes patient respiratory failure.
However, paraquat is never broken through in the transmembrane transport and accumulation Mechanism Study of pneumonocyte, is not also found corresponding so far
Intervening measure, it has also become paraquat removing toxic substances research in critical issue.
In recent years, there is research by the analysis of the related resistance mechanism of mutant plant (herba eleusines indicae, arabidopsis etc.) to resistance to PQ, explain
It has stated PQ and has transported relevant important information in plant.This provides new think of for research human body PQ toxicity and its transmembrane transport mechanism
Road.By constructing the human body cell strain of resistance to paraquat, and its antagonism paraquat toxic mechanism mechanism is further studied, is expected to find
Reverse the new way of paraquat toxicity.
So far, it there are no the method report that mammal drug-resistant cell strain is established with paraquat induction.Tumor drug resistance at present
Usually there are two types of methods for the foundation of cell strain, first is that drug concentration, successive induction method are stepped up, second is that large dosage impact interruption
Dose regimen.It is generally believed that the method for increasing concen-trations continuous induction is acted on by sustained drug, make tumour cell physiology and heredity
Characteristic changes gradually to adapt to the toxicity of drug, belongs to acquired resistance.It there is no both at home and abroad at present drug resistant to paraquat
Mammalian cell strain, by the foundation for the cell strain of resistance to paraquat, can be used for paraquat poisoning study on prevention provide it is important
Basic research model.By retrieval find, at present both at home and abroad not yet about with human lung carcinoma cell line A549 be induction object,
Establish the document report of human lung cancer drug resistance paraquat cell strain.
Summary of the invention
In view of the deficiencies in the prior art, the purpose of the present invention is to provide a kind of human lung cancer drug resistance paraquat cell strain and its
Method for building up can be used for paraquat toxicological mechanism, transmembrane transport research and excavate to reverse paraquat toxicity approach, hundred grass of research and development
Withered poisoning treatment's drug etc. provides cell model.
The human lung cancer cell line A549 of resistance to paraquat/PQ of the invention, on 06 16th, 2015 in China Microbiological bacterium
Kind preservation administration committee common micro-organisms center, is referred to as (the address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 CGMCC
Number Institute of Microorganism, Academia Sinica, postcode 100101) preservation, classification naming is human lung carcinoma cell, deposit number CGMCC
No.10892。
The technical solution adopted by the present invention is that:
The foundation according to the following steps of A549/PQ drug-resistant cell strain of the present invention uses human lung carcinoma cell line A549 for induction pair
As improveing continuous induction method, i.e., carrying out PQ exposure using the method for increasing concen-trations, period exposure and establish the resistance to paraquat of human lung cancer
Cell line A549/PQ.The cell strain is common in China Committee for Culture Collection of Microorganisms on 06 16th, 2015
The preservation of microorganism center, deposit number are CGMCC No.10892.
In particular it relates to the human lung cancer cell line A549 of resistance to paraquat/PQ be as follows establish obtain:
(1) human lung carcinoma cell line A549 cell is placed in culture solution (the RPMI-1640 culture medium containing 10% fetal calf serum), and 37
DEG C, 5%CO2Under the conditions of routine culture.
(2) take the cell of step (1) logarithmic growth phase for testing, using increasing concen-trations, the PQ revulsion of period exposure
Cells resistance strain is established, for the concentration of PQ in the medium first since 100 μm of ol/L, liquid removal drug is changed in dosing afterwards for 24 hours, to
Same concentrations PQ stimulation is added in cell again after regrowing to logarithmic phase, be so repeated 3 times, and then extends exposure duration extremely
48h (the PQ exposure 48h of i.e. 100 μm ol/L), changes liquid, removes drug, regrow after cell to logarithmic phase, give phase again
With the exposure of drug same time, as follows it is repeated 3 times.It then is further added by PQ concentration, the scheme induced under each concentration is consistent, passs
Increase PQ concentration gradient and be followed successively by 100,200,400,600,800 μm of ol/L, continuous culture 11 months obtains the A549 cell of resistance to PQ
Strain, the human lung cancer cell line A549 of resistance to paraquat/PQ.
The present invention has the following advantages that and effect:
The variation of optical microphotograph sem observation cytomorphology, Flow cytometry cell cycle distribution, CCK-8 method and LDH
Release experiment detects cell growth curve and detects intracellular PQ to paraquat drug resistance, efficient liquid phase chromatographic analysis (HPLC) method
Concentration etc., it has been found that with parental cell ratio, the human lung cancer cell line A549 of resistance to paraquat/PQ cell volume increases, form is not advised
Then, space between cells is small；The doubling time of cell is obviously prolonged；Cell cycle analysis shows that drug resistance group G0/G1 phase cell obviously increases
It is more, and S phase cell significantly reduces；The human lung cancer cell line A549 of resistance to paraquat/PQ survival rate is bright after CCK-8 detection shows PQ exposure
It is aobvious to be higher than parental cell, it is intermediate-resistant to the Resistance index 7.98 of PQ；LDH release experiment also confirms that people's lung of PQ induction
The cancer cell line A549 of resistance to paraquat/PQ cellular damage is obviously lighter than parental cell；FCM analysis shows people's lung of PQ induction
The cancer cell line A549 of resistance to paraquat/PQ early apoptosis of cells rate is lower than parental cell；HPLC method measures human lung carcinoma cell after exposure
The strain intracellular PQ concentration of A549/PQ is significantly lower than parental cell.Show the human lung cancer cell line A549 of resistance to paraquat/PQ cell and parent
There are apparent biological differences for this iuntercellular, and intracellular concentration reduces what indication mdr cell was accumulated there may be antagonism PQ
Mechanism.
The present invention is on the basis of increasing concen-trations successive induction method, further progress improvement, using increasing concen-trations, period exposure
PQ induces human lung cancer cell A549, that is, while fully considering that concentration is gradually incremented by, and uses gradually extend at the same concentration
The method of exposure duration provides buffer time for the related compensatory approach of A549 cell-stimulating, is conducive to cell and gradually adapts to PQ
Toxicity improve induction success rate to obtain to the drug resistance of PQ.In this way, the present invention is obtained for the first time to PQ drug resistance
TypeⅡ pneumocyte (A549/PQ), which has apparent anti-PQ toxicity characteristic, reverses for further research application
PQ toxicity approach, extraction paraquat poisoning evaluation marker, the anti-oxidant new way of excavation etc. provide good research using mould
Type.
Detailed description of the invention
Fig. 1 is light microscopic servant lung cancer cell types and the human lung cancer cell line A549 of resistance to paraquat/PQ micrograph
A:A549 cell；B:A549/PQ cell
Fig. 2 is the human lung cancer cell line A549 of resistance to paraquat/PQ and human lung carcinoma cell line A549 cell growth curve figure.
Fig. 3 is the human lung cancer cell line A549 of resistance to paraquat/PQ and human lung carcinoma cell line A549 cell doubling time figure.
Fig. 4 is that PQ induces the human lung cancer cell line A549 of resistance to paraquat/PQ and human lung carcinoma cell line A549 cell mortality
Compare figure.
A: A549 group death rate ratio P < 0.05 is induced with 400 μm of ol/L PQ；B: A549 group is induced with 800 μm of ol/L PQ
Death rate ratio P < 0.05
Fig. 5 is the human lung cancer cell line A549 of resistance to paraquat/PQ and human lung carcinoma cell line A549 cells survival rate after PQ exposure
Curve graph.Fig. 6 is the comparison that PQ induces the human lung cancer cell line A549 of resistance to paraquat/PQ and human lung carcinoma cell line A549 cell IC50
Figure.
A: with A549 group ratio P < 0.05
Fig. 7 is that PQ induces the intracellular PQ concentration of the human lung cancer cell line A549 of resistance to paraquat/PQ and human lung carcinoma cell line A549
Comparison figure.
A: for the HPLC chromatogram of mark product PQ detection；B: the HPLC chromatogram of detection sample PQ detection；C:LDH release is real
Test the comparison of detection PQ inducing cell death rate；D:HPLC detects the comparison of intracellular PQ concentration；Black arrow: PQ appearance time
(6.70min).*: indicating the lower two comparison among groups P < 0.05 of same concentrations PQ exposure；#: indicate that same concentrations PQ exposure is two groups lower
Between compare P < 0.05
Specific embodiment
The present invention is further illustrated below by embodiment.The embodiment of the present invention is only used for illustrating the present invention, and
Limitation of the present invention, under concept thereof of the invention to the simple modifications of the method for the present invention belong to the present invention claims
The range of protection.
The induction of the 1 human lung cancer cell strain of resistance to paraquat of embodiment is established
The human lung cancer cell line A549 of resistance to paraquat/PQ is established according to the following steps: (1) A549 cell is placed in containing 10% tire ox blood
Routine culture in 1640 clear culture mediums, condition are 37 DEG C, 5%CO2.The cell of each logarithmic growth phase is for testing.(2)
Establish cells resistance strain using the PQ revulsion of increasing concen-trations, period exposure: the concentration of PQ in the medium is first from 100 μ
Mol/L starts, and liquid removal drug is changed in dosing afterwards for 24 hours, and same concentrations PQ thorn is added again after cell regrows to logarithmic phase
Swash, is so repeated 3 times.Then extend exposure duration to 48h (i.e. 100 μm of ol/L exposure 48h), change liquid and remove drug, to cell
It regrows to logarithmic phase, gives the exposure of identical drug same time again, be as follows repeated 3 times.It is dense to be then further added by PQ
It spends, the scheme induced under each concentration is consistent.It is incremented by PQ concentration gradient and is followed successively by 100,200,400,600,800 μm of ol/L, even
Continuous culture 11 months, obtains the A549 cell strain of resistance to PQ, is named as the human lung cancer cell line A549 of resistance to paraquat/PQ.
When cell successively stablizes 100 μm of ol/L, 200 μm of ol/L, 400 μm of ol/L, 600 μm of ol/L, 800 μm of ol/L PQ
After drug resistance, the concentration mdr cell is frozen in time in liquid nitrogen.Frozen stock solution is trained by 20% calf serum, 5%DMSO, 75%DMEM
Nutrient solution composition, frozen storage process should gradually cool down, and 4 DEG C 30min → -20 DEG C 1h → -70 DEG C overnight → liquid nitrogen sequence is taken to carry out.
The recovery of freeze-stored cell is carried out by following procedure: in a 25cm2Tissue Culture Flask at least 10ml is added containing 10% calf
The culture solution of serum.Cell cryopreservation tube is taken out from liquid nitrogen container, is put into rapidly in 37 DEG C of water-baths, and cryopreservation tube is submerged and is constantly shaken
It is dynamic, melt the cell suspension in pipe rapidly.It after cell suspension melts, is transferred in 15ml centrifuge tube, 5~10 times of training is added
Base is supported, 1000rpm is centrifuged 5min.Culture medium is abandoned, cell precipitation is left, new culture medium is added, is blown and beaten at cell suspension, according to 1
×105Every bottle is seeded to culture bottle, is put into incubator, and 37 DEG C, 5%CO2Culture.
The 2 human lung cancer cell line A549 of resistance to paraquat of embodiment/PQ and typeⅡ pneumocyte morphological observation
Human lung carcinoma cell line A549 and the A549/PQ cell of logarithmic growth phase uses Leica after conventional digestion passage
Optical microscopy observes cellular morphology after cell for 24 hours is completely adherent, and is taken pictures with Nikon camera, as a result as shown in Figure 1.
It is thin that 3 cell counting of embodiment draws the human lung cancer cell line A549 of resistance to paraquat/PQ and human lung carcinoma cell line A549
Intracellular growth curve and measurement doubling time
50,000 are taken to be in mid log phase, the good parent A549 cell of upgrowth situation, the resistance to paraquat of human lung cancer
Cell line A549/PQ cell, is made cell suspension, is inoculated into culture bottle respectively, and every kind 21 bottles of cell inoculation.Start afterwards for 24 hours
Cell count continuous counter seven days, counts three bottles of cells daily, according to count results, draws cell growth curve.Further according to public affairs
Formula calculates doubling time (DT), and DT=t × lg2/ (lgNt-lgN0), t are the cell culture time, and N0 inoculation measures afterwards for 24 hours
Cell quantity average value, Nt are the cell quantity average value cultivated after th, as a result as shown in Figures 2 and 3.
Embodiment 4PI staining for flow cell art detects the human lung cancer cell line A549 of resistance to paraquat/PQ and human lung carcinoma cell line
The A549 cell cycle
(1) difference logarithmic growth phase, the good parent A549 cell of upgrowth situation, the human lung cancer cell strain of resistance to paraquat
A549/PQ cell；
(2) pancreatin digests, and supernatant is removed in 1000rpm × 5min centrifugation, and 1ml PBS is resuspended, again 1,000rpm × 5min from
The heart removes supernatant, 1ml is added, dehydrated alcohol is pre-chilled, gently piping and druming mixes, 4 DEG C of fixed 12h.
(3) supernatant is removed into the cell suspension 1000rpm × 5min fixed centrifugation, 0.5ml is added in every solencyte sample
Cell precipitation slowly and is sufficiently resuspended in propidium iodide stain liquid, and 37 DEG C are protected from light warm bath 30min, and flow cytometer detects cell
Period.
The 1 human lung cancer cell line A549 of resistance to paraquat of table/PQ is compared with human lung carcinoma cell line A549 cell cycle distribution
(%, n=3,)
Note: with human lung carcinoma cell line A549 group ratio:aP < 0.05
Embodiment 5CCK-8 method detects PQ and induces the human lung cancer cell line A549 of resistance to paraquat/PQ and human lung carcinoma cell line A549
Cell growth curve and Resistance index
(1) difference logarithmic growth phase, the good parent A549 cell of upgrowth situation, the human lung cancer cell strain of resistance to paraquat
A549/PQ cell is inoculated into 96 orifice plates, 5 × 103/ hole.
(2) be grouped: every kind of cell is divided into blank control wells (cell-free to have culture medium), without drug-treated hole, 400 μ of PQ
The cell hole of mol/L processing, the cell hole of 800 μm of ol/L processing, the cell hole of 1,200 μm of ol/L processing, every group of three multiple holes.
(3) each its OD value of survey at microplate reader 450nm is gone up after handling 48h.
(4) formula calculate cell inhibitory rate, cell inhibitory rate (%)=100- [A (dosing)-A (blank)]/[A (not plus
Medicine)-A (blank)] × 100.IC50 is sought using 5 software of GraphPad Prism.Resistance index is equal to A549/PQ cell
The IC50 of IC50/ parent's A549 cell.As a result as shown in Fig. 4,5 and 6.
6 lactic dehydrogenase Cytotoxicity assays (LDH) of embodiment detect PQ and induce the human lung cancer cell strain of resistance to paraquat
A549/PQ and human lung carcinoma cell line A549 cell mortality
(1) by logarithmic growth phase, the good human lung carcinoma cell line A549 cell of upgrowth situation, the human lung cancer cell of resistance to paraquat
Strain A549/PQ cell, is inoculated into 96 orifice plates, and it is full to be no more than 80~90% for cell density when making to be detected.
(2) experimental group and processing: including cell-free culture fluid apertures (background blank control hole), without drug-treated
Control cell hole (sample controls hole), cell hole (the sample maximum enzyme activity control for subsequent cracking without drug-treated
Hole) and 400 μm of ol/L of A549 cell PQ, 800 μm of ol/L processing holes, PQ400 μm of ol/L of A549/PQ cell, 800 μm of ol/L
Handle hole.Every group of three multiple holes.
(3) OD value of each survey at microplate reader 490nm is handled on 48h.
(4) each group absorbance measured should all subtract background blank control hole absorbance, then calculate cell according to formula
The death rate, cell mortality (%)=(processing sample absorbance-sample controls hole absorbance)/(suction of cell maximum enzyme activity
Luminosity-sample controls hole absorbance) × 100%.As a result as shown in Fig. 4,5 and 6.
7 efficient liquid phase chromatographic analysis of embodiment (HPLC) detects the intracellular PQ concentration of A549/PQ and A549
(1) cell grouping and processing
It is divided into human lung carcinoma cell line A549 group, (400, the 800 μm of ol/L PQ processing of human lung carcinoma cell line A549+PQ group
For 24 hours), the human lung cancer cell line A549 of resistance to paraquat/PQ group, the human lung cancer cell line A549 of resistance to paraquat/PQ+PQ group (400,800 μ
Mol/L PQ processing is for 24 hours) four groups, 1 × 10 is collected after conventional digestion6A cell, and washed twice with PBS.100 μ l distilled waters are added
Suspension cell, ultrasonic 20min smudge cells, the first that 20 μ l 10% are then added is bright, 8000rpm, is centrifuged 10min, takes supernatant
It is transferred in internal lining pipe, upper machine testing.
Pattern detection
1) chromatographic condition
Chromatographic column: C18 (4.6mm × 150mm, 5 μm)；Guard column: TC-C18 (4.6 × 12.5mm, 5 μm)；
20 μ l of applied sample amount, mobile phase 20mmol/L sodium dihydrogen phosphate: acetonitrile=96: 4, wavelength 258nm, 35 DEG C of column temperature, stream
Fast 1.00ml/min.
2) configuration of standard items
Appropriate PQ standard items are weighed in the EP pipe of 1.5ml, the dissolution of user's lung cancer cell types intracellular fluid is made into
Concentration is the stock solution of 1000.0 μ g/ml.Used time dilutes stock solution with intracellular fluid step by step, is made into mass concentration containing PQ and is respectively
The PQ series of tasks solution of 1.0 μ g/ml, 2.5 μ g/ml, 5.0 μ h/ml, 10.0 μ g/ml, 25.0 μ/ml, 50.0 μ g/ml.Take 20 μ
The standard items of l, upper machine testing.
3) preparation of standard curve
Chromatographic peak and peak area are recorded, using the peak area of PQ as ordinate (y), the concentration of PQ is that abscissa (x) carries out line
Property return.Calculate normal equation.
4) the intracellular PQ Concentration Testing of each processing group
After sample process is good, 20 μ l of sample introduction does HPLC analysis, obtains each group concentration of specimens, which obtains each group multiplied by 120 μ l
Sample P Q mass, then divided by 1 × 106Obtain the content of PQ in individual cells.1 × 10 is represented as convenience of statistical result6A cell
Amount in 120 μ l solution.As a result as shown in Figure 7.
Claims (1)
1. a kind of human lung cancer cell line A549 of resistance to paraquat/PQ, which is characterized in that its deposit number is CGMCC No.10892.
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| CN103740647A (en) * | 2013-11-29 | 2014-04-23 | 南京凯基生物科技发展有限公司 | Human stomach cancer multidrug-resistant cell strain |
| CN103816186A (en) * | 2014-02-28 | 2014-05-28 | 中国人民解放军军事医学科学院附属医院 | Treatment effect of umbilical cord mesenchymal stem cells on paraquat poisoning lung injury |
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| CN101347421A (en) * | 2008-09-17 | 2009-01-21 | 南开大学 | Use of sulphonated calyx [5] arene in aspect of preparing medicament for treating toxicity of Paraguat |
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