CN103792218A - Method for detecting genetic toxicity of total particulate matters in main stream smoke of cigarette by adopting binuclear method - Google Patents
Method for detecting genetic toxicity of total particulate matters in main stream smoke of cigarette by adopting binuclear method Download PDFInfo
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- CN103792218A CN103792218A CN201410059881.4A CN201410059881A CN103792218A CN 103792218 A CN103792218 A CN 103792218A CN 201410059881 A CN201410059881 A CN 201410059881A CN 103792218 A CN103792218 A CN 103792218A
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Abstract
The invention discloses a method for detecting the genetic toxicity of total particulate matters in main stream smoke of a cigarette by adopting a binuclear method. The method is characterized by comprising the following steps: selecting BEAS-2B cells of a human bronchial epithelial cell line BEAS-2B as an evaluation system; inoculating the cells into a culture dish; adding a total particulate matter sample in cigarette smoke for exposing; carrying out culture and methanol solution immobilization on the exposed cells in sequence; dyeing by adopting a DAPI solution and photographing and counting under a fluorescence microscope, wherein the number of cells containing micronucleus in 1,000 binucleated cells is obtained for each sample; comparing with a control sample, and analyzing the increasing quantity of the micronucleus, so as to judge the degree of the genetic toxicity of smoke. According to the invention, the genetic toxicity of the cigarette smoke is evaluated through smoke acting on target organ derived cells, so that the pertinency is high; a metabolism activation system (rat liver S9 mixed solution) is not required to be added in the experiment process, and fluorescence staining is directly performed in the cell culture dish without need of transferring to a glass slide; the method has the characteristics of simplicity and convenience in operation, high sensitivity, reliable result and the like.
Description
Technical field
The present invention relates to tobacco and cigarette smoke safety evaluatio technical field, specifically a kind of double-core method that adopts detects the genotoxic method of cigarette mainstream smoke total particulate matter, that the cell micronucleus rate causing by mensuration cigarette smoke TPM is carried out, for the genetoxic of evaluating cigarette flue gas.
Background technology
In cigarette smoke, have 69 kinds of compounds to be defined as carcinogenic substance, some material is the tumor promotion factor or auxiliary carcinogenic substance in addition, therefore about the toxicology assessment of cigarette smoke becomes one of hot issue of smoking and health field.Relevant genotoxic in vitro toxicology test is commonly used to the aerocolloidal mutagenesis potentiality of evaluation of flue gas.Micronucleus test is a kind of evaluation method of commonly using in the genetoxic test of chemical substance or potpourri, and cigarette smoke and smoke condensate all can cause the appearance of cell micronucleus.Such as, by BALB/c-3T3 cell or V79 cell be exposed to the obvious increase that all can find micronuclear rates in main flume and smoke condensate [list of references 1:Gu Z W, Whong W Z, Wallace W E,
et al. Induction of micronuclei in BALB/c-3T3 cells by selected chemicals and complex mixtures[J] .Mutation Research, 1992, 279:217-222. list of references 2:Channarayappa, Nath J, Ong T. Clastogenic and aneuploidogeniceffects of cigarette smoke condensate, Mitomycin C and vincristine sulfate[J] .Mutagenesis, 1992, 7 (6): 457-260. list of references 3:Veltel D, Hoheneder A. Characterzation of cigarettesmoke-induced micronuclei in vitro[J] .Exp.Toxic Pathol, 1996, 48:548-550.].In the world, CORESTA in vitro toxicology task force also recommend to select micronucleus test method to carry out the genetoxic of evaluating cigarette flue gas.Based on this, domestic tobacco scientific and technical personnel adopt Chinese hamster ovary cell (Chinese hamster ovary celI) to carry out the genetoxic of evaluating cigarette flue gas TPM as the exposure system of in-vitro micronucleus test conventionally, and in experiment, have designed respectively the processing procedure (YQ 4 tobaccos and tobacco smoke security biological assessment the 3rd part: in-vitro micronucleus test) of adding metabolism activation system and do not add metabolism activation system.
Summary of the invention
The object of the invention is for some adopt mammalian cell to test the features such as the genotoxic method of cigarette smoke TPM is easy not at present, detect the principle that should derive from emphatically human tissue cell according to genetic toxicology, and the method that the one of special exploitation adopts human bronchial epithelial cell line (BEAS-2B cell) to carry out cigarette smoke genetic toxicity test, this method has been omitted the processing procedure of adding metabolism activation system, can reflect preferably the external genetoxic of cigarette smoke, simple to operate, highly sensitive, with strong points, reliable results.
The object of the invention is to be achieved through the following technical solutions: a kind of double-core method that adopts detects the genotoxic method of cigarette mainstream smoke total particulate matter, comprise the following steps: first in cultured BEAS-2B cell, add the extract sample of cigarette smoke TPM (TPM) to expose, the cell exposing is fixed through cultivation and cold methanol, then use the fluorescent dye of DAPI solution, selection is evenly distributed, the good region of dyeing, the counting of taking pictures under fluorescent microscope, in 1000 dikaryocytes of each sample counting, contain the cell quantity of micronucleus, with the control sample comparison that does not add the exposure of cigarette smoke TPM, judge that cigarette smoke causes the quantity that cell micronucleus increases, and then judge the genotoxic size of cigarette smoke, concrete steps are as follows:
A. cell is cultivated and inoculation: BEAS-2B cell is inoculated in cell culture fluid and is placed in CO
2incubator is hatched, and adopts 0.05% pancreatin solution vitellophag and prepares cell suspending liquid when Growth of Cells reaches when 70%-80% converges, and after the cell suspending liquid counting that will prepare, is seeded in diameter 35mm double dish, and the cell quantity that makes every ware is 1.5 × 10
5-2.0 × 10
5individual, adding nutrient solution to cumulative volume is 2mL, in CO
2in incubator, continue to cultivate 24h;
B. cigarette smoke TPM exposes: pass through smoking machine smoking cigarette according to state's calibration method, and prepare dimethyl sulfoxide (DMSO) (DMSO) the extract sample of TPM, remove the nutrient solution in double dish, if four class experimental group: cell control group, solvent control group, positive controls and TPM group, in each group, each detection dosage is all established 3 parallel wares; TPM group is according to cell median lethal dose IC
50, get 1/2 IC
50, 1/4 IC
50, 1/8I C
50with 1/16 IC
504 dosage, add respectively the TPM sample of various dose, and it is consistent to make to organize the concentration of DMSO in interior double dish by the each TPM concentration of DMSO polishing group; Cell control group only adds cell culture fluid; Solvent control group adds DMSO, and the DMSO amount adding is organized 1/2 IC with TPM
50the volume number of dosage is consistent; Positive controls adds 2 μ L endoxan solution; Each group adds after tested material, adds 12 μ L cytochalasin B solution in every ware simultaneously, finally with cell culture fluid, liquor capacity in every ware is complemented to 2 mL, continues at CO
2in incubator, cultivate 24 h;
C. micronucleus test: remove the solution in double dish, with PBS solution rinsing 2 times, discard after washing lotion, fix 10 min with approximately 2 mL methanol solutions, inversion is dried, to the DAPI 1.5 mL lucifuges that add 1 μ g/mL in double dish 10 min that dye, with distilled water flushing 3 times, dark place nature dries or is air-dry; Under fluorescent microscope, burst of ultraviolel is observed, and selects to be evenly distributed, and the good region of dyeing, takes pictures and picture count, contains the quantity of micronucleus in 1000 dikaryocytes of each sample counting; Select the dikaryocyte caryoplasm of observing to be separated from each other, micronucleus is not overlapping with main core, is easy to differentiate;
D. data processing and analysis: compared with solvent control group, whether statistical study micronuclear rates has dose-response relationship and have significant difference, the genetoxic of evaluating cigarette flue gas by TPM group.
In the present invention, test cell nutrient solution is LHC-8 nutrient culture media.
DMSO used is that cell is cultivated level.
Cytochalasin B solution adopts DMSO to be mixed with 1 mg/mL and preserves-20 ℃ of conditions.The cytochalasin B solution of 6 μ g/mL be by cytochalasin B and cell culture fluid in proportion mixed preparing form
Endoxan solution adopts PBS to be mixed with 0.2 mg/mL, after filtration sterilization, preserves-20 ℃ of conditions.
The compound method of DAPI dyeing liquor is: be mixed with the storage liquid of 1 mg/mL with distilled water ,-20 ℃ keep in Dark Place, when use with the working fluid of distilled water diluting to 1 μ g/mL.
The test philosophy of foundation of the present invention is: first in cultured cell, directly add the extract sample of TPM to expose, the cell exposing is fixed through cultivation and cold methanol, then use the fluorescent dye of DAPI solution, under fluorescent microscope, in counting cells, contain again the cell quantity of micronucleus, with the control sample comparison that does not add the exposure of cigarette smoke TPM, judge that cigarette smoke causes the quantity that cell micronucleus increases, and then judge the genotoxic size of cigarette smoke.
The present invention adopts BEAS-2B cell as exposure system, in experimentation without adding S9 metabolism activation system, while adopting DAPI fluorescent dye test, can directly in Tissue Culture Dish, carry out, to microslide, reflect more objectively the toxic action of cigarette smoke without transitional cell, and the genetoxic of energy quantitative evaluation cigarette smoke, compared with prior art have simple to operate, highly sensitive, the feature of reliable results, has started a kind of new for cigarette smoke genetic toxicity method for measuring.
Accompanying drawing explanation
Fig. 1 is the histogram that embodiment 1 cigarette smoke causes the micronucleus increment rate of BEAS-2B cell.
Fig. 2 is the histogram that embodiment 2 cigarette smokes cause the micronucleus increment rate of BEAS-2B cell.
Fig. 3 is the histogram that embodiment 3 cigarette smokes cause the micronucleus increment rate of BEAS-2B cell.
?
Embodiment
The present invention is described further with the following Examples, but does not limit the present invention.
Embodiment 1
Domestic certain reference cigarette is evaluated.By reference cigarette balance 48 hours in climatic chamber, choose the cigarette that weight is consistent with resistance to suction.Press national standard method, aspirate respectively 20 cigarette on smoking machine, cambridge filter adds DMSO to soak by 10 mg/mL TPM, and ultrasound wave extracts 20 min, finally uses Filter paper filtering, divides and installs in cryopreservation tube, is stored in-70 ℃ of ultra low temperature freezers for subsequent use.
Test cell is inoculated in cell culture fluid, at CO
2in cell culture incubator, at 37 ℃, cultivate, when cell proliferation is counted during to finite concentration, and cell is transferred in 35 mm double dish, concentration is 1.0 × 10
5cell/ware, adds nutrient solution to cumulative volume 2 mL, in CO
2in cell culture incubator, at 37 ℃, cultivate 24 h, according to the median lethal dose IC calculating in cell toxicity test
50be 32 μ g/mL, 4 detection dosage of TPM group be set and be respectively 2.0 μ g/mL, 4.0 μ g/mL, 8.0 μ g/mL and 16.0 μ g/mL, remove the nutrient solution in double dish, the every ware of cell control group adds 2 mL cell culture fluids, the every ware of solvent control group adds 3.2 μ L DMSO, it is the endoxan solution of 0.2 mg/mL that the every ware of positive controls adds 2 μ L concentration, 16.0 μ g/mL TPM organize every ware and add 3.2 μ L TPM, 8.0 μ g/mL TPM organize every ware and add 1.6 μ L TPM and 1.6 μ L DMSO, 4.0 μ g/mL TPM organize every ware and add 0.8 μ L TPM and 2.4 μ L DMSO, 2.0 μ g/mL TPM organize every ware and add 0.4 μ L TPM and 2.8 μ L DMSO, each group adds after tested material, all with cell culture fluid, liquor capacity in double dish is complemented to 2 mL, in every ware, add 12 μ L cytochalasin B solution to make the final concentration of cytochalasin B is 6 μ g/mL simultaneously, continue at CO
2in incubator, cultivate.
The cell exposing is cultivated after 24 h, and sucking-off nutrient solution is also used respectively PBS solution rinsing 2 times, and then with fixing Tissue Culture Dish 10 min through exposure of 90% methanol solution, naturally dries or air-dry.In air-dry double dish, add the DAPI solution of 100 mg/mL to soak 3 min, then use distilled water flushing double dish 30 s, dark place nature dries or is air-dry, first under 10 times of mirrors, observe, selection is evenly distributed, the good region of dyeing, then count under fluorescent microscope, in 1000 cells of each sample counting containing the cell quantity of micronucleus.The results are shown in Figure 1.As can be seen from Figure 1 along with the increase of fume exposure concentration, cigarette smoke causes that cell micronucleus rate also increases thereupon, is obvious dosage-response relation.
The present embodiment is same as embodiment 1 substantially, is only that cigarette sample is the reference cigarette containing certain adjuvant.The results are shown in Figure 2.
Embodiment 3
The present embodiment is same as embodiment 1 substantially, is only that cigarette sample is domestic certain brand cigarette.The results are shown in Figure 3.
Claims (5)
1. one kind adopts double-core method to detect the genotoxic method of cigarette mainstream smoke total particulate matter, it is characterized in that: this assay method comprises the following steps: first in cultured BEAS-2B cell, add the extract sample of cigarette smoke TPM (TPM) to expose, the cell exposing is fixed through cultivation and cold methanol, then use the fluorescent dye of DAPI solution, selection is evenly distributed, the good region of dyeing, the counting of taking pictures under fluorescent microscope, in 1000 dikaryocytes of each sample counting, contain the cell quantity of micronucleus, with the control sample comparison that does not add the exposure of cigarette smoke TPM, judge that cigarette smoke causes the quantity that cell micronucleus increases, and then judge the genotoxic size of cigarette smoke.
2. employing double-core method according to claim 1 detects the genotoxic method of cigarette mainstream smoke total particulate matter, it is characterized in that: the concrete steps of this detection method are as follows:
A. cell is cultivated and inoculation: BEAS-2B cell is inoculated in cell culture fluid and is placed in CO
2incubator is hatched, and adopts 0.05% pancreatin solution vitellophag and prepares cell suspending liquid when Growth of Cells reaches when 70%-80% converges, and after the cell suspending liquid counting that will prepare, is seeded in diameter 35 mm double dish, and the cell quantity that makes every ware is 1.5 × 10
5-2.0 × 10
5individual, adding nutrient solution to cumulative volume is 2 mL, in CO
2in incubator, continue to cultivate 24 h;
B. cigarette smoke TPM exposes: pass through smoking machine smoking cigarette according to state's calibration method, and prepare dimethyl sulfoxide (DMSO) (DMSO) the extract sample of TPM, remove the nutrient solution in double dish, if four class experimental group: cell control group, solvent control group, positive controls and TPM group, in each group, each detection dosage is all established 3 parallel wares; TPM group is according to cell median lethal dose IC
50, get 1/2 IC
50, 1/4 IC
50, 1/8I C
50with 1/16 IC
504 dosage, add respectively the TPM sample of various dose, and it is consistent to make to organize the concentration of DMSO in interior double dish by the each TPM concentration of DMSO polishing group; Cell control group only adds cell culture fluid; Solvent control group adds DMSO, and the DMSO amount adding is organized 1/2 IC with TPM
50the volume number of dosage is consistent; Positive controls adds 2 μ L endoxan solution; Each group adds after tested material, adds 12 μ L cytochalasin B solution in every ware simultaneously, finally with cell culture fluid, liquor capacity in every ware is complemented to 2 mL, continues at CO
2in incubator, cultivate 24 h;
C. micronucleus test: remove the solution in double dish, with PBS solution rinsing 2 times, discard after washing lotion, fix 10 min with approximately 2 mL methanol solutions, inversion is dried, to the DAPI 1.5 mL lucifuges that add 1 μ g/mL in double dish 10 min that dye, with distilled water flushing 3 times, dark place nature dries or is air-dry; Under fluorescent microscope, burst of ultraviolel is observed, and selects to be evenly distributed, and the good region of dyeing, takes pictures and picture count, contains the quantity of micronucleus in 1000 dikaryocytes of each sample counting; Select the dikaryocyte caryoplasm of observing to be separated from each other, micronucleus is not overlapping with main core, is easy to differentiate;
D. data processing and analysis: compared with solvent control group, whether statistical study micronuclear rates has dose-response relationship and have significant difference, the genetoxic of evaluating cigarette flue gas by TPM group.
3. employing double-core method according to claim 2 detects the genotoxic method of cigarette mainstream smoke total particulate matter, it is characterized in that: cytochalasin B solution adopts DMSO to be mixed with 1 mg/mL and preserves-20 ℃ of conditions.
4. employing double-core method according to claim 2 detects the genotoxic method of cigarette mainstream smoke total particulate matter, it is characterized in that: endoxan solution adopts PBS to be mixed with 0.2 mg/mL, after filtration sterilization, preserves-20 ℃ of conditions.
5. employing double-core method according to claim 2 detects the genotoxic method of cigarette mainstream smoke total particulate matter, it is characterized in that: the compound method of DAPI dyeing liquor is: the storage liquid that is mixed with 1 mg/mL with distilled water,-20 ℃ keep in Dark Place, when use with the working fluid of distilled water diluting to 1 μ g/mL.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104122189A (en) * | 2014-08-07 | 2014-10-29 | 云南中烟工业有限责任公司 | In-vitro cell micronuclei method for detecting total particulate matters of main stream smoke of cigarettes |
| CN104237495A (en) * | 2014-09-23 | 2014-12-24 | 云南中烟工业有限责任公司 | Method for setting positive control of in vitro micronucleus test using aqueous extract of cigarette filter |
| CN105606576A (en) * | 2016-02-01 | 2016-05-25 | 清华大学 | Method for detecting specific mRNA in cell adopting molecular probe |
| CN106546722A (en) * | 2016-11-25 | 2017-03-29 | 云南中烟工业有限责任公司 | It is a kind of for detecting method that electronics tobacco product is affected on cell micronucleus rate |
| CN106591413A (en) * | 2016-11-25 | 2017-04-26 | 云南中烟工业有限责任公司 | Method of detecting influence on early apoptosis of cells due to total particulate matters in cigarette smoke |
| CN108449993A (en) * | 2015-08-19 | 2018-08-24 | 内奥利诊断公司 | The individual prediction technique of the DNA break genotoxicity effect of chemical reagent or biochemical reagents |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104122189A (en) * | 2014-08-07 | 2014-10-29 | 云南中烟工业有限责任公司 | In-vitro cell micronuclei method for detecting total particulate matters of main stream smoke of cigarettes |
| CN104122189B (en) * | 2014-08-07 | 2017-01-25 | 云南中烟工业有限责任公司 | In-vitro cell micronuclei method for detecting total particulate matters of main stream smoke of cigarettes |
| CN104237495A (en) * | 2014-09-23 | 2014-12-24 | 云南中烟工业有限责任公司 | Method for setting positive control of in vitro micronucleus test using aqueous extract of cigarette filter |
| CN104237495B (en) * | 2014-09-23 | 2016-04-27 | 云南中烟工业有限责任公司 | A kind of cigarette filter water extract micronucleus test in vitro positive control method to set up |
| CN108449993A (en) * | 2015-08-19 | 2018-08-24 | 内奥利诊断公司 | The individual prediction technique of the DNA break genotoxicity effect of chemical reagent or biochemical reagents |
| CN105606576A (en) * | 2016-02-01 | 2016-05-25 | 清华大学 | Method for detecting specific mRNA in cell adopting molecular probe |
| CN106546722A (en) * | 2016-11-25 | 2017-03-29 | 云南中烟工业有限责任公司 | It is a kind of for detecting method that electronics tobacco product is affected on cell micronucleus rate |
| CN106591413A (en) * | 2016-11-25 | 2017-04-26 | 云南中烟工业有限责任公司 | Method of detecting influence on early apoptosis of cells due to total particulate matters in cigarette smoke |
| CN106546722B (en) * | 2016-11-25 | 2019-01-25 | 云南中烟工业有限责任公司 | A method of cell micronucleus rate is influenced for detecting electronics tobacco product |
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