CN105368783A - Human lung cancer paraquat-resistant cell strain - Google Patents
Human lung cancer paraquat-resistant cell strain Download PDFInfo
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- CN105368783A CN105368783A CN201510753784.XA CN201510753784A CN105368783A CN 105368783 A CN105368783 A CN 105368783A CN 201510753784 A CN201510753784 A CN 201510753784A CN 105368783 A CN105368783 A CN 105368783A
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Abstract
The invention provides a human lung cancer paraquat-resistant cell strain. A human lung cancer cell strain A549 is used as an induction object; an in-vitro induction method of periodic exposure and progressive increase of paraquat concentration is adopted; and a paraquat-resistant cell strain A549/PQ is built. The cell strain is collected in CGMCC (China General Microbiological Culture Collection Center) on June 16, 2015; and the collection number is CGMCC NO.10892. The cell strain has the typical paraquat-resistant characteristic; by using the biological characteristic, a study model can be provided for further studying a paraquat toxicity reversion path, extracting a paraquat poisoning evaluation marker, exploring a novel anti-oxidization path and the like; and high scientific research and production and application values are realized.
Description
Technical field
The invention belongs to cell engineering field, relate to a kind of human cell's strain, be specifically related to a kind of people's lung cancer cell strain of resistance to Paraquat and establishment method thereof and application.
Background technology
Paraquat (paraquat, PQ) be the widely used high-effect weedicide in a kind of whole world, it has very strong toxicity to human body, all can cause poisoning through absorptions such as digestive tube, skin, respiratory tract and veins, lack effective remedy measures clinically, mortality is up to 40 ~ 80%.It is the first that the production of China PQ and consumption all occupy the whole world.In recent years the poisoning morbidity of domestic PQ is that multiple increases, and prevention and control situation is very severe.Although in June, 2015, China started to release the production and sales forbidding PQ water liquid gradually, other formulations will continue manufacture and usage.Therefore, paraquat poisoning is still the vital task of China's poisoning control from now on.
PQ is poisoning causes the multiple visceral organ injury of whole body, and wherein lung is the most serious target organ of getting involved, and can occur " paraquat lung ", and namely Early manifestation is acute lung injury, and stage progress is interstitial pulmonary fibrosis, is PQ poisoning patient main causes of death.PQ absorbs in the active of lung tissue and accumulates and is considered to the key that successive induction injury of lung causes patient respiratory exhaustion.But, so far to Paraquat, in transmembrane transport and the never breakthrough of accumulation Mechanism Study of pneumonocyte, do not find corresponding intervening measure yet, become the key issue in Paraquat removing toxic substances research.
In recent years, there is research by the analysis of the relevant resistance mechanism of the mutant plant (Herba Eleusines Indicae, Arabidopis thaliana etc.) to resistance to PQ, set forth PQ in plant, transported relevant important information.This provides new approaches for researching human body PQ toxicity and transmembrane transport mechanism thereof.By building the human body cell strain of resistance to Paraquat, and studying its antagonism Paraquat toxic mechanism mechanism further, being expected to the new way finding to reverse Paraquat toxicity.
So far, there are no the method report setting up Mammals drug-resistant cell strain with Paraquat induction.The foundation of current tumor drug resistance cell strain has two kinds of methods usually, and one is progressively increase drug level, successive induction method, and two is heavy dose of impact intermittent administration methods.It is generally acknowledged, the method for increasing concen-trations continuous induction is by sustained drug effect, makes tumour cell physiology and hereditary property change to adapt to gradually the toxicity of medicine, belongs to acquired resistance.There is no the mammalian cell strain to Paraquat resistance at present both at home and abroad, by the foundation of the cell strain of resistance to Paraquat, may be used for paraquat poisoning study on prevention and provide important fundamental research model.Found by retrieval, at present both at home and abroad also not about with human lung carcinoma cell line A549 for induction object, set up the bibliographical information of people's drug resistance of lung cancer Paraquat cell strain.
Summary of the invention
In view of the deficiencies in the prior art, the object of the present invention is to provide a kind of people's drug resistance of lung cancer Paraquat cell strain and establishment method thereof, may be used for Paraquat toxicological mechanism, transmembrane transport research and excavate the Paraquat toxicity approach that reverses, research and development paraquat poisoning medicine etc. providing cell model.
The people's lung cancer cell line A549 of resistance to Paraquat/PQ of the present invention, in on 06 16th, 2015 at China Committee for Culture Collection of Microorganisms's common micro-organisms center, it is referred to as CGMCC (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, postcode 100101) preservation, Classification And Nomenclature is human lung carcinoma cell, and deposit number is CGMCCNo.10892.
The technical solution used in the present invention is:
The foundation according to the following steps of A549/PQ drug-resistant cell strain of the present invention, adopt human lung carcinoma cell line A549 for induction object, improvement continuous induction method, namely adopts increasing concen-trations, method that the cycle exposes carries out PQ and expose and establish the people's lung cancer cell line A549 of resistance to Paraquat/PQ.This cell strain on 06 16th, 2015 in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, preserving number is CGMCCNo.10892.
Particularly, the people's lung cancer cell line A549 of the resistance to Paraquat/PQ that the present invention relates to sets up acquisition as follows:
(1) human lung carcinoma cell line A549 cell is placed in nutrient solution (the RPMI-1640 substratum containing 10% foetal calf serum), 37 DEG C, 5%CO
2cellar culture under condition.
(2) cell getting step (1) logarithmic phase is used for experiment, adopt increasing concen-trations, the PQ revulsion that cycle exposes sets up cells resistance strain, PQ concentration is in the medium first from 100 μm of ol/L, change liquid after dosing 24h and remove medicine, regrow until cell and to logarithmic phase, again to add same concentrations PQ stimulate, repetition like this 3 times, then open-assembly time is extended to 48h (namely the PQ of 100 μm of ol/L exposes 48h), change liquid, removing medicine, regrow to logarithmic phase until cell, again give the exposure of same medicine same time, as follows repeat 3 times.Increase PQ concentration more subsequently, the scheme of inducing under each concentration is consistent, increases progressively PQ concentration gradient and is followed successively by 100,200,400,600,800 μm of ol/L, cultured continuously 11 months, obtains the A549 cell strain of resistance to PQ, the people's lung cancer cell line A549 of resistance to Paraquat/PQ.
The present invention has the following advantages and effect:
The change of observation by light microscope morphocytology, Flow cytometry cell cycle distribution, CCK-8 method and LDH release experiment detect cell growth curve and to detect in cell PQ concentration etc. to Paraquat resistance, efficient liquid phase chromatographic analysis (HPLC) method, we find, with parental cell ratio, people's lung cancer cell line A549 of resistance to Paraquat/PQ cell volume increases, form is irregular, intercellular substance is little; The doubling time of cell obviously extends; Cell cycle analysis shows resistance group G0/G1 phase cell showed increased, and S phase cell obviously reduces; CCK-8 detects and shows that PQ exposes the descendant's lung cancer cell line A549 of resistance to Paraquat/PQ survival rate apparently higher than parental cell, and it is intermediate-resistant to the Resistance index 7.98 of PQ; LDH release experiment also confirms that people's lung cancer cell line A549 of resistance to Paraquat/PQ cell injury that PQ induces obviously is lighter than parental cell; FCM analysis shows that people's lung cancer cell line A549 of resistance to Paraquat/PQ early apoptosis of cells rate that PQ induces is lower than parental cell; HPLC method records PQ concentration in exposure descendant lung cancer cell types/PQ cell and is starkly lower than parental cell.Show people's lung cancer cell line A549 of resistance to Paraquat/there is obvious biological differences between PQ cell and parental cell, and IC reduces indication mdr cell may there is the mechanism that antagonism PQ accumulates.
The present invention is on increasing concen-trations successive induction method basis, improve further, adopt increasing concen-trations, cycle exposure PQ induction human lung cancer cell A549, namely while taking into full account that concentration increases progressively gradually, under same concentration, adopt again the method extending open-assembly time gradually, for A549 cell-stimulating compensatory approach of being correlated with provides surge time, be conducive to the toxicity that cell little by little adapts to PQ, thus the resistance obtained PQ, improve induction success ratio.Pass through the method, the present invention obtains the typeⅡ pneumocyte (A549/PQ) to PQ resistance first, this cell strain has obvious anti-PQ toxicity characteristic, and in order to study further, application reverses PQ toxicity approach, extraction paraquat poisoning evaluates mark, excavate anti-oxidant new way etc. provides good research application model.
Accompanying drawing explanation
Fig. 1 is the microgram of light microscopic servant lung cancer cell types and the people's lung cancer cell line A549 of resistance to Paraquat/PQ
A:A549 cell; B:A549/PQ cell
Fig. 2 behaves the lung cancer cell line A549 of resistance to Paraquat/PQ and human lung carcinoma cell line A549 cell growth curve figure.
Fig. 3 behaves the lung cancer cell line A549 of resistance to Paraquat/PQ and human lung carcinoma cell line A549 cell doubling time figure.
Fig. 4 is the comparison diagram that PQ induces the people lung cancer cell line A549 of resistance to Paraquat/PQ and human lung carcinoma cell line A549 cell mortality.
A: induce A549 group mortality ratio than P < 0.05 with 400 μm of ol/LPQ; B: induce A549 group mortality ratio than P < 0.05 with 800 μm of ol/LPQ
Fig. 5 is that PQ exposes the descendant lung cancer cell line A549 of resistance to Paraquat/PQ and human lung carcinoma cell line A549 cells survival rate curve figure.Fig. 6 is the comparison diagram that PQ induces the people lung cancer cell line A549 of resistance to Paraquat/PQ and human lung carcinoma cell line A549 cell IC50.
A: with A549 group than P < 0.05
Fig. 7 is the comparison diagram that PQ induces PQ concentration in the people lung cancer cell line A549 of resistance to Paraquat/PQ and human lung carcinoma cell line A549 cell.
A: the HPLC color atlas detected for mark product PQ; B: detect the HPLC color atlas that sample PQ detects; C:LDH release experiment detects the comparison of PQ inducing cell death rate; D:HPLC detects the comparison of PQ concentration in cell; Black arrow: PQ appearance time (6.70min).*: represent that same concentrations PQ exposes between lower two groups and compare P < 0.05; #: represent that same concentrations PQ exposes between lower two groups and compare P < 0.05
Embodiment
The present invention is further illustrated below by embodiment.Embodiments of the invention are only used for the present invention is described, instead of limitation of the present invention, under concept thereof of the present invention, all belong to the scope of protection of present invention to the simple modifications of the inventive method.
The induction of the embodiment 1 people lung cancer cell strain of resistance to Paraquat is set up
The people's lung cancer cell line A549 of resistance to Paraquat/PQ sets up according to the following steps: (1) A549 cell is placed in the 1640 substratum cellar cultures containing 10% foetal calf serum, and condition is 37 DEG C, 5%CO
2.The cell of at every turn taking the logarithm vegetative period is for experiment.(2) the PQ revulsion of employing increasing concen-trations, cycle exposure sets up cells resistance strain: PQ concentration is in the medium first from 100 μm of ol/L, change liquid after dosing 24h and remove medicine, regrow until cell and to logarithmic phase, again to add same concentrations PQ stimulate, so repeat 3 times.Then extend open-assembly time to 48h (namely 100 μm of ol/L expose 48h), change liquid removing medicine, regrow to logarithmic phase until cell, again give the exposure of same medicine same time, as follows repeat 3 times.Increase PQ concentration more subsequently, the scheme of inducing under each concentration is consistent.Increase progressively PQ concentration gradient and be followed successively by 100,200,400,600,800 μm of ol/L, cultured continuously 11 months, obtain the A549 cell strain of resistance to PQ, name as the people's lung cancer cell line A549 of resistance to Paraquat/PQ.
After cell stablizes resistance to 100 μm of ol/L, 200 μm of ol/L, 400 μm of ol/L, 600 μm of ol/L, 800 μm of ol/LPQ successively, this concentration mdr cell frozen is in liquid nitrogen in time.Frozen storing liquid is made up of 20% calf serum, 5%DMSO, 75%DMEM nutrient solution, and frozen process should progressively be lowered the temperature, take DEG C 1h →-70 DEG C, 4 DEG C of 30min →-20 spend the night → order of liquid nitrogen carries out.The recovery of freeze-stored cell is undertaken by following program: at a 25cm
2tissue Culture Flask in add at least 10ml nutrient solution containing 10% calf serum.From liquid nitrogen container, take out cell cryopreservation tube, put into rapidly 37 DEG C of water-baths, submergence cryopreservation tube also constantly shakes, and the cell suspension in pipe is melted rapidly.Cell suspension is transferred in 15ml centrifuge tube, is added the substratum of 5 ~ 10 times, 1000rpm, centrifugal 5min after melting.Abandon substratum, leave cell precipitation, add new substratum, blow and beat into cell suspension, according to 1 × 10
5every bottle is seeded to culturing bottle, puts into incubator, 37 DEG C, 5%CO
2cultivate.
The embodiment 2 people lung cancer cell line A549 of resistance to Paraquat/PQ and typeⅡ pneumocyte morphological observation
The human lung carcinoma cell line A549 taken the logarithm vegetative period and A549/PQ cell, after conventional digestion goes down to posterity, use Leica opticmicroscope in the complete adherent rear observation of cell form of 24h cell, and take pictures with Nikon photographic camera, result as shown in Figure 1.
Embodiment 3 cell counting is drawn the people lung cancer cell line A549 of resistance to Paraquat/PQ and human lung carcinoma cell line A549 cell growth curve and is measured the doubling time
Get 50,000 is in mid log phase, parent A549 cell, people's lung cancer cell line A549 of resistance to Paraquat/PQ cell that upgrowth situation is good, and make cell suspension, be inoculated in culturing bottle respectively, often kind of cell inoculates 21 bottles.Start cell counting after 24h, continuous counter seven days, count three bottles of cells every day, according to count results, draw cell growth curve.Again according to formulae discovery doubling time (DT), DT=t × lg2/ (lgNt-lgN0), t are the cell cultures time, and N0 is the cell quantity mean value recorded after inoculation 24h, Nt is the cell quantity mean value after cultivating th, and result as shown in Figures 2 and 3.
Embodiment 4PI staining for flow cell art detects the people lung cancer cell line A549 of resistance to Paraquat/PQ and human lung carcinoma cell line A549 cell cycle
(1) to take the logarithm respectively vegetative period, parent A549 cell, people's lung cancer cell line A549 of resistance to Paraquat/PQ cell that upgrowth situation is good;
(2) trysinization, 1000rpm × 5min is centrifugal removes supernatant, and 1mlPBS is resuspended, and 1,000rpm × 5min is centrifugal again removes supernatant, adds 1ml precooling dehydrated alcohol, blows and beats mixing gently, 4 DEG C of fixing 12h.
(3) remove supernatant by centrifugal for the cell suspension 1000rpm × 5min fixed, add 0.5ml propidium iodide stain liquid in every tube cell sample, slow and abundant re-suspended cell precipitates, and 37 DEG C of lucifuge temperature bath 30min, flow cytometer detects the cell cycle.
The comparing of the table 1 people lung cancer cell line A549 of resistance to Paraquat/PQ and human lung carcinoma cell line A549 cell cycle distribution (%, n=3,
)
Note: compare with human lung carcinoma cell line A549 group:
ap < 0.05
Embodiment 5CCK-8 method detects PQ and induces the people lung cancer cell line A549 of resistance to Paraquat/PQ and human lung carcinoma cell line A549 cell growth curve and Resistance index
(1) to take the logarithm respectively vegetative period, parent A549 cell, people's lung cancer cell line A549 of resistance to Paraquat/PQ cell that upgrowth situation is good, be inoculated in 96 orifice plates, 5 × 10
3/ hole.
(2) divide into groups: often kind of cell is divided into blank control wells (acellular have substratum), without drug treating hole, the cell hole of PQ400 μm of ol/L process, the cell hole of 800 μm of ol/L process, the cell hole of 1,200 μm of ol/L process, often organizes three multiple holes.
(3) upper its OD value of each survey in microplate reader 450nm place after processing 48h.
(4) formulae discovery cell inhibitory rate, cell inhibitory rate (%)=100-[A (dosing)-A (blank)]/[A (non-dosing)-A (blank)] × 100.GraphPadPrism5 software is utilized to ask IC50.Resistance index equals the IC50 of the IC50/ parent A549 cell of A549/PQ cell.Result as Fig. 4, shown in 5 and 6.
Embodiment 6 serum lactic dehydrogenase Cytotoxicity assays (LDH) detects PQ and induces the people lung cancer cell line A549 of resistance to Paraquat/PQ and human lung carcinoma cell line A549 cell mortality
(1) by logarithmic phase, human lung carcinoma cell line A549 cell, people's lung cancer cell line A549 of resistance to Paraquat/PQ cell that upgrowth situation is good, be inoculated in 96 orifice plates, when making to be detected, cell density is no more than 80 ~ 90% full.
(2) experiment grouping and process: comprise acellular cultivation fluid apertures (background blank control hole), without the compared with control cells hole (sample controls hole) of drug treating, without the cell hole for follow-up cracking (sample maximum enzyme activity control wells) of drug treating, and A549 cell PQ400 μm of ol/L, 800 μm of ol/L disposal hole, A549/PQ cell PQ400 μm of ol/L, 800 μm of ol/L disposal hole.Often organize three multiple holes.
(3) each survey in microplate reader 490nm place OD value on 48h is processed.
(4) each group of absorbancy recorded all should subtracting background blank control wells absorbancy, then according to formulae discovery cell mortality, cell mortality (%)=(processing sample absorbancy-sample controls hole absorbancy)/(absorbancy-sample controls hole absorbancy of cell maximum enzyme activity) × 100%.Result as Fig. 4, shown in 5 and 6.
Embodiment 7 efficient liquid phase chromatographic analysis (HPLC) detects PQ concentration in A549/PQ and A549 cell
(1) cell grouping and process
Be divided into human lung carcinoma cell line A549 group, human lung carcinoma cell line A549+PQ group (400,800 μm of ol/LPQ process 24h), people's lung cancer cell line A549 of resistance to Paraquat/PQ group, people's lung cancer cell line A549 of resistance to Paraquat/PQ+PQ group (400,800 μm of ol/LPQ process 24h) four groups, after conventional digestion, collect 1 × 10
6individual cell, and wash twice with PBS.Add 100 μ l distilled water suspension cells, ultrasonic 20min smudge cells, the first then adding 20 μ l10% is bright, 8000rpm, centrifugal 10min, gets supernatant liquor and transfers 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);
Applied sample amount 20 μ l, moving phase 20mmol/L SODIUM PHOSPHATE, MONOBASIC: acetonitrile=96: 4, wavelength 258nm, column temperature 35 DEG C, flow velocity 1.00ml/min.
2) configuration of standard substance
Take appropriate PQ standard substance in the EP pipe of 1.5ml, end user's lung cancer cell types intracellular fluid dissolves, and is made into the storing solution that concentration is 1000.0 μ g/ml.Used time, with intracellular fluid stepwise dilution storing solution, is made into the PQ series of tasks solution being respectively 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 containing PQ mass concentration.Get the standard substance of 20 μ l, upper machine testing.
3) preparation of typical curve
Record chromatographic peak and peak area, with the peak area of PQ for ordinate zou (y), the concentration of PQ is that X-coordinate (x) carries out linear regression.Calculate standard equation.
4) PQ Concentration Testing in each treatment group cell
After sample process is good, sample introduction 20 μ l is HPLC and analyzes, and obtain each group of concentration of specimens, this concentration is multiplied by 120 μ l and obtains each group of sample P Q quality, then divided by 1 × 10
6obtain the content of PQ in individual cells.All 1 × 10 is expressed as convenience of statistics
6the amount of individual cell in 120 μ l solution.Result as shown in Figure 7.
Claims (1)
1. people's lung cancer cell line A549 of resistance to Paraquat/PQ, is characterized in that, its deposit number is CGMCCNo.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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2015
- 2015-10-30 CN CN201510753784.XA patent/CN105368783B/en active Active
Patent Citations (6)
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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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