CN109444396A - It is a kind of not burn the in-vitro micronucleus detection method of Cigarette grain phase for heating - Google Patents
It is a kind of not burn the in-vitro micronucleus detection method of Cigarette grain phase for heating Download PDFInfo
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- 238000010438 heat treatment Methods 0.000 title claims abstract description 27
- 238000001514 detection method Methods 0.000 title claims abstract description 21
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- 239000000463 material Substances 0.000 claims abstract description 19
- 238000000034 method Methods 0.000 claims abstract description 13
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- 239000006143 cell culture medium Substances 0.000 claims description 20
- 239000012980 RPMI-1640 medium Substances 0.000 claims description 19
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- 239000000047 product Substances 0.000 claims description 18
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- 210000001519 tissue Anatomy 0.000 claims description 8
- ZCHPKWUIAASXPV-UHFFFAOYSA-N acetic acid;methanol Chemical compound OC.CC(O)=O ZCHPKWUIAASXPV-UHFFFAOYSA-N 0.000 claims description 6
- 238000004043 dyeing Methods 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 231100000673 dose–response relationship Toxicity 0.000 claims description 4
- 230000000391 smoking effect Effects 0.000 claims description 4
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- WLOKFRZXOVZGIN-UHFFFAOYSA-N 2-(3-methylthiopropyl)malic acid Chemical compound CSCCCC(O)(C(O)=O)CC(O)=O WLOKFRZXOVZGIN-UHFFFAOYSA-N 0.000 claims description 3
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 claims description 3
- 102000004190 Enzymes Human genes 0.000 claims description 3
- 108090000790 Enzymes Proteins 0.000 claims description 3
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 3
- 102000004142 Trypsin Human genes 0.000 claims description 3
- 108090000631 Trypsin Proteins 0.000 claims description 3
- GBOGMAARMMDZGR-UHFFFAOYSA-N UNPD149280 Natural products N1C(=O)C23OC(=O)C=CC(O)CCCC(C)CC=CC3C(O)C(=C)C(C)C2C1CC1=CC=CC=C1 GBOGMAARMMDZGR-UHFFFAOYSA-N 0.000 claims description 3
- 239000008280 blood Substances 0.000 claims description 3
- 210000004369 blood Anatomy 0.000 claims description 3
- 210000000170 cell membrane Anatomy 0.000 claims description 3
- 238000005138 cryopreservation Methods 0.000 claims description 3
- 229960004397 cyclophosphamide Drugs 0.000 claims description 3
- GBOGMAARMMDZGR-JREHFAHYSA-N cytochalasin B Natural products C[C@H]1CCC[C@@H](O)C=CC(=O)O[C@@]23[C@H](C=CC1)[C@H](O)C(=C)[C@@H](C)[C@@H]2[C@H](Cc4ccccc4)NC3=O GBOGMAARMMDZGR-JREHFAHYSA-N 0.000 claims description 3
- GBOGMAARMMDZGR-TYHYBEHESA-N cytochalasin B Chemical compound C([C@H]1[C@@H]2[C@@H](C([C@@H](O)[C@@H]3/C=C/C[C@H](C)CCC[C@@H](O)/C=C/C(=O)O[C@@]23C(=O)N1)=C)C)C1=CC=CC=C1 GBOGMAARMMDZGR-TYHYBEHESA-N 0.000 claims description 3
- 210000000805 cytoplasm Anatomy 0.000 claims description 3
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- 239000001963 growth medium Substances 0.000 claims description 3
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- 239000000975 dye Substances 0.000 abstract 1
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- 241000196324 Embryophyta Species 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
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- 231100000025 genetic toxicology Toxicity 0.000 description 2
- 235000019353 potassium silicate Nutrition 0.000 description 2
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- NTHWMYGWWRZVTN-UHFFFAOYSA-N sodium silicate Chemical compound [Na+].[Na+].[O-][Si]([O-])=O NTHWMYGWWRZVTN-UHFFFAOYSA-N 0.000 description 2
- 235000019505 tobacco product Nutrition 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 244000025254 Cannabis sativa Species 0.000 description 1
- UGFAIRIUMAVXCW-UHFFFAOYSA-N Carbon monoxide Chemical compound [O+]#[C-] UGFAIRIUMAVXCW-UHFFFAOYSA-N 0.000 description 1
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 1
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- 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
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- G01N33/5014—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 toxicity
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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Abstract
The present invention discloses a kind of for the in-vitro micronucleus detection method for heating the Cigarette grain phase that do not burn, comprising the following steps: (1) sample pre-treatments；(2) preparation of people's nasal epithelia single cell suspension；(3) cell concentration of people's nasal epithelia single cell suspension calculates；(4) inoculation of people's nasal epithelia single cell suspension；(5) tested material is grouped；(6) setting of tested material detection dosage；(7) preparation of tested material culture product；(8) it is incubated for tested material；(9) cell is harvested；(10) hypotonic；(11) piece is dripped；(12) it dyes；(13) observation of micronucleus；(14) result judgement.The method of the present invention can accurately and effectively detect heating and not burn Cigarette grain phase to the influence degree of cell micronucleus rate, and then determine that heating is not burnt the genetoxics of cigarette products.
Description
Technical field
The invention belongs to the safety biological effect assessment technique fields of tobacco product, in particular to a kind of for heating
Do not burn the in-vitro micronucleus detection method of Cigarette grain phase.
Background technique
It is clear that micronucleus test detects terminal, and method is easy, is easy to carry out, and is one of genetoxic standard combination test,
In the safety evaluations of the Healthy relevant products such as food, medical product, industrial and agricultural products, the monitoring of genetic damage, specific crowd
Mutational lesions detection and early prediction etc. are widely used.The Organization of Economy and Cooperation Development (OECD) public affairs in 2009
Cloth mammal external micronucleus test policy paper, tentatively establishes the unified standard of the external micronucleus test of mammal.State
Border tobacco scientific research Cooperation Centre CORESTA, which is organized in, has set up the external toxicity test job group of cigarette smoke for 2002, warp
The a large amount of literature survey of working group and research work are crossed, which recommends using micronucleus test in vitro to cigarette smoke condensate
Potential genetoxic is detected.The detection method has been widely used by domestic and international tobacco company at present.
The increasingly stringent and smoking of tobacco supervision legislation is goed deep into health research, results in tobacco company and seeks risk
New tobacco products lower, without environment flue gas.Heat non-combustion-type cigarette, it is characterised in that heating tobacco and non-combustion-cigarette
Grass provides the consumer with certain tobacco feature impression.Heating does not burn cigarette products when avoiding traditional cigarette product burns
Harm, has become the new side that foreign tobacco company turns to from traditional cigarette product at this stage brought by the complex mixture of generation
One of to.
The change of test object causes sample-pretreating method, detection cell, detection dosage also different, for examining
The micronucleus test in vitro for surveying traditional cigarette genetoxic has been unable to meet the need for heating cigarette products genetoxic detection of not burning
It wants, and research institution and tobacco company not yet formulate the standard for heating the cigarette products micronucleus test in vitro that do not burn both at home and abroad at present
Method.
Summary of the invention
The object of the present invention is to provide a kind of for the in-vitro micronucleus detection method for heating the Cigarette grain phase that do not burn.It is logical
It crosses detection heating and does not burn Cigarette grain phase to the influence degree of cell micronucleus rate, heat the cigarette products that do not burn to determine
Genetoxic.The accuracy of testing result can be enhanced, and then more effectively evaluate the safety for heating the cigarette products that do not burn
Property.
Unless otherwise indicated, percentage of the present invention is percentage by volume.
RPMI1640 cell culture medium used in the present invention is bought in any goods companies being commercially available in market.
It is a kind of not burn the in-vitro micronucleus detection method of Cigarette grain phase for heating, comprising the following steps:
(1) sample pre-treatments: using linear type smoking machine, is 55.0mL, duration 3s, suction frequencies in pumping volume
10 mouthfuls of continuous sucking, to be trapped with the cambridge filter that diameter is 44mm, before suction, after suction under the puffing regimens of 30s
The quality for weighing Fume collector calculates the quality mTPM for heating the Cigarette grain phase that do not burn according to formula (1)；
MTPM=m1-m0 (1)
In formula:
m0--- the quality of Fume collector before aspirating, unit are milligram (mg)；
m1--- the quality of Fume collector after suction, unit are milligram (mg)；
After trapping, the cambridge filter after trapping is put into the triangular flask of 50mL, dimethyl sulfoxide is added, makes to heat
Concentration of the Cigarette grain phase in dimethyl sulfoxide of not burning is 40mg/mL；This triangular flask is placed in Ultrasound Instrument ultrasonic
30min；Dimethyl sulfoxide extract liquor is collected by filtration with aseptic filter paper again, dispenses into the cryopreservation tube of 1mL, -80 DEG C save for use；
(2) preparation of people's nasal epithelia single cell suspension: after people's nasal epithelia RPMI2650 recovery, inoculation
To 25cm2In Tissue Culture Flask, 10mLRPMI1640 cell culture medium is added, is placed in 37 DEG C, 5%CO2It is cultivated in incubator,
Set micro- sem observation culture cell converge with form situation, when converging rate, remove culture to 70%~90% when cell is long
RPMI1640 cell culture medium in bottle is washed twice with phosphate buffer, abandons washing lotion；The pancreas egg of 1mL 0.25% (w/v) is added
White enzyme solutions single layer is incubated for 1~2min, is hanged with RPMI1640 cell culture medium, forms single cell suspension；
(3) cell concentration of people's nasal epithelia single cell suspension calculates: with blood counting chamber counting method to step
(2) cell concentration of income earner's nasal epithelia single cell suspension calculates, and obtains every milliliter of single cell suspension
Viable count；
(4) inoculation of people's nasal epithelia single cell suspension: people's nasal epithelia list after step (3) are counted
Cell suspending liquid RPMI1640 cell culture medium to 2.0 × 105A/mL, then 6 holes are inoculated by inoculum concentration for the hole 2mL/
In tissue culture plate, 6 porocyte culture plates are then placed in 37 DEG C, 5%CO2It is cultivated for 24 hours in incubator；
(5) tested material is grouped: tested material being divided into three groups: cell controls group, positive controls and test sample group；Each group
Composition are as follows: cell controls group plant people's nasal epithelia simultaneously plus RPMI1640 cell culture medium；Positive controls are added
The 0.5 μ L of cyclophosphamide solution of 1mg/mL；Test sample group is added prepare liquid obtained by step (1) and adds RPMI1640 cell culture
Base；
(6) tested material detects setting for dosage: the ultimate density of prepare liquid obtained by step (1) is 4 non-zero-dose, respectively
Are as follows: 40 μ g/mL, 80 μ g/mL, 160 μ g/mL, 320 μ g/mL；
(7) preparation of tested material culture product: the culture medium in removing step (4) in 6 porocyte culture plates, then press step
(5) it is grouped, 6 μ of cytochalasin B solution of 1mg/mL is added in cell controls group, positive controls and test sample group
The liquid volume in hole every in tissue culture plate is complemented to the hole 2mL/ with RPMI1640 cell culture medium by L；
(8) it is incubated for tested material: 6 porocyte culture plates after step (7) sample-adding is placed in 37 DEG C, 5%CO2It is incubated in incubator
It educates for 24 hours；
(9) it harvests cell: being added in the cell controls group, positive controls and test sample group after step (8) are incubated for
The trypsin solution that concentration is 0.25% (W/V) gets off the hole of cell from 6 porocyte culture plates is digested；
(10) hypotonic: step (9) postdigestive cell being placed in centrifuge tube, 5min, revolving speed 1000rpm are centrifuged, is taken out
Centrifuge tube simultaneously removes supernatant, and Klorvess Liquid 3mL is added and gently blows and beats cell, it is molten to be added immediately methanol acetic acid after mixing
Liquid 2mL, is placed on centrifuge and is centrifuged 5min, revolving speed 1000rpm；
(11) it drips piece: the supernatant in step (9) centrifuge tube is removed, 300 μ L methanol acetic acid solution are added, gently blow
It beats and is allowed to form cell suspending liquid, by cell suspending liquid drop on glass slide, and be placed in naturally dry in glass frame, Mei Gejian
Dose drips 3 slides；
(12) dye: the slide after step (9) is dried is placed in Gimsa dye liquor after dyeing 15min, is rinsed with water glass
Piece simultaneously is placed on saving backup after natural drying in glass frame；
(13) gained micronucleus slide after dyeing, the visual field that selection is good and cell density is suitable, observation the observation of micronucleus: are taken
1000 dikaryocytes count micronuclear rates, select the dikaryocyte of observation to should be cytoplasm intact, cell membrane sharpness of border,
Nucleus is separated from each other, and micronucleus is easy to differentiate；
(14) result judgement: sample sets be will test compared with cell controls group, micronuclear rates have conspicuousness to increase and have dosage
Reaction relation can be confirmed as positive findings；It, then must be into if statistically difference has conspicuousness, but when without dose-response relationship
Row repeat test, can repetition person can be identified as the positive.
The invention has the advantages that:
The present invention enables the method for the present invention to the optimal setting of sample detection dosage and the correct selection of target cell
Accurately and effectively detection heating does not burn Cigarette grain phase to the influence degree of cell micronucleus rate, and then determines that heating is not burnt
The genetoxic of cigarette products.
Detailed description of the invention
Fig. 1 refers to influence of the given the test agent to RPMI2650 cell micronucleus rate.
Specific embodiment
Below in conjunction with specific example, the present invention will be described in detail, but is not intended to limit the present invention.
Embodiment 1
The heating trapped using cambridge filter does not burn Cigarette grain phase as sample, prepares 2 kinds and heats the cigarette that do not burn
It is thin to detect it with people's nasal epithelia (RPMI2650) using Kentucky reference cigarette 3R4F as control sample by sample 1# and 2#
Cellular toxicity.
Specific detection process, comprising the following steps:
(1) sample pre-treatments: using linear type smoking machine, is 55.0mL, duration 3s, suction frequencies in pumping volume
10 mouthfuls of continuous sucking, to be trapped with the cambridge filter that diameter is 44mm, before suction, after suction under the puffing regimens of 30s
The quality for weighing Fume collector calculates the quality mTPM for heating the Cigarette grain phase that do not burn according to formula (1)；
MTPM=m1-m0 (1)
In formula:
m0--- the quality of Fume collector before aspirating, unit are milligram (mg)；
m1--- the quality of Fume collector after suction, unit are milligram (mg)；
After trapping, the cambridge filter after trapping is put into the triangular flask of 50mL, dimethyl sulfoxide is added, makes to heat
Concentration of the Cigarette grain phase in dimethyl sulfoxide of not burning is 40mg/mL；This triangular flask is placed in Ultrasound Instrument ultrasonic
30min；Dimethyl sulfoxide extract liquor is collected by filtration with aseptic filter paper again, dispenses into the cryopreservation tube of 1mL, -80 DEG C save for use；
(2) preparation of people's nasal epithelia single cell suspension: after people's nasal epithelia RPMI2650 recovery, inoculation
To 25cm2In Tissue Culture Flask, 10mLRPMI1640 cell culture medium is added, is placed in 37 DEG C, 5%CO2It is cultivated in incubator,
Set micro- sem observation culture cell converge with form situation, when converging rate, remove culture to 70%~90% when cell is long
RPMI1640 cell culture medium in bottle is washed twice with phosphate buffer, abandons washing lotion；The tryptose of 1mL0.25% (w/v) is added
Enzyme solutions single layer is incubated for 1~2min, is hanged with RPMI1640 cell culture medium, forms single cell suspension；
(3) cell concentration of people's nasal epithelia single cell suspension calculates: with blood counting chamber counting method to step
(2) cell concentration of income earner's nasal epithelia single cell suspension calculates, and obtains every milliliter of single cell suspension
Viable count；
(4) inoculation of people's nasal epithelia single cell suspension: people's nasal epithelia list after step (3) are counted
Cell suspending liquid RPMI1640 cell culture medium to 2.0 × 105A/mL, then 6 holes are inoculated by inoculum concentration for the hole 2mL/
In tissue culture plate, 6 porocyte culture plates are then placed in 37 DEG C, 5%CO2It is cultivated for 24 hours in incubator；
(5) tested material is grouped: tested material being divided into three groups: cell controls group, positive controls and test sample group；Each group
Composition are as follows: cell controls group plant people's nasal epithelia simultaneously plus RPMI1640 cell culture medium；Positive controls are added
The 0.5 μ L of cyclophosphamide solution of 1mg/mL；Test sample group is added prepare liquid obtained by step (1) and adds RPMI1640 cell culture
Base；
(6) tested material detects setting for dosage: the ultimate density of prepare liquid obtained by step (1) is 4 non-zero-dose, respectively
Are as follows: 40 μ g/mL, 80 μ g/mL, 160 μ g/mL, 320 μ g/mL；
(7) preparation of tested material culture product: the culture medium in removing step (4) in 6 porocyte culture plates, then press step
(5) it is grouped, 6 μ of cytochalasin B solution of 1mg/mL is added in cell controls group, positive controls and test sample group
The liquid volume in hole every in tissue culture plate is complemented to the hole 2mL/ with RPMI1640 cell culture medium by L；
(8) it is incubated for tested material: 6 porocyte culture plates after step (7) sample-adding is placed in 37 DEG C, 5%CO2It is incubated in incubator
It educates for 24 hours；
(9) it harvests cell: being added in the cell controls group, positive controls and test sample group after step (8) are incubated for
The trypsin solution that concentration is 0.25% (W/V) gets off the hole of cell from 6 porocyte culture plates is digested；
(10) hypotonic: step (9) postdigestive cell being placed in centrifuge tube, 5min, revolving speed 1000rpm are centrifuged, is taken out
Centrifuge tube simultaneously removes supernatant, and Klorvess Liquid 3mL is added and gently blows and beats cell, it is molten to be added immediately methanol acetic acid after mixing
Liquid 2mL, is placed on centrifuge and is centrifuged 5min, revolving speed 1000rpm；
(11) it drips piece: the supernatant in step (9) centrifuge tube is removed, 300 μ L methanol acetic acid solution are added, gently blow
It beats and is allowed to form cell suspending liquid, by cell suspending liquid drop on glass slide, and be placed in naturally dry in glass frame, Mei Gejian
Dose drips 3 slides；
(12) dye: the slide after step (9) is dried is placed in Gimsa dye liquor after dyeing 15min, is rinsed with water glass
Piece simultaneously is placed on saving backup after natural drying in glass frame；
(13) gained micronucleus slide after dyeing, the visual field that selection is good and cell density is suitable, observation the observation of micronucleus: are taken
1000 dikaryocytes count micronuclear rates, select the dikaryocyte of observation to should be cytoplasm intact, cell membrane sharpness of border,
Nucleus is separated from each other, and micronucleus is easy to differentiate；
(14) result judgement: sample sets be will test compared with cell controls group, micronuclear rates have conspicuousness to increase and have dosage
Reaction relation can be confirmed as positive findings；It, then must be into if statistically difference has conspicuousness, but when without dose-response relationship
Row repeat test, can repetition person can be identified as the positive.
As seen from Figure 1, tested two heating do not burn cigarette sample compared with reference cigarette 3R4F, caused thin
There were significant differences for born of the same parents' micronuclear rates (p < 0.05), and under same detection dosage, micronuclear rates caused by reference cigarette 3R4F are higher than heating
Do not burn cigarette.
Integrated survey of the present invention heating is not burnt the route of exposure of cigarette products, and it is non-ignitable to establish heating for the mode of action
Cigarette sample pre-treating method is burnt, and has chosen people's nasal epithelia according to cigarette products effect target cell of not burning is heated
(RPMI 2650), which is used as, heats the cigarette products genetic toxicity test detection cell that do not burn, and sample detection dosage has been determined,
Form the genetic toxicity test detection method for being suitble to heating not burn cigarette products.Sample is effectively treated, detects dosage
The correct selection of optimal setting and target cell enables this method accurately and effectively to detect and heats the total grain phase of cigarette of not burning
Object determines that heating is not burnt the genetoxics of cigarette products to the influence degree of cell micronucleus rate.
Claims (1)
1. a kind of for the in-vitro micronucleus detection method for heating the Cigarette grain phase that do not burn, which is characterized in that including following step
It is rapid:
(1) sample pre-treatments: using linear type smoking machine, is 55.0mL, duration 3s in pumping volume, suction frequencies are
Under the puffing regimens of 30s, 10 mouthfuls of continuous sucking, is trapped with the cambridge filter that diameter is 44mm, claimed before suction, after suction
The quality for measuring Fume collector calculates the quality mTPM for heating the Cigarette grain phase that do not burn according to formula (1)；
MTPM=m1-m0 (1)
In formula:
m0--- the quality of Fume collector before aspirating, unit are milligram (mg)；
m1--- the quality of Fume collector after suction, unit are milligram (mg)；
After trapping, the cambridge filter after trapping is put into the triangular flask of 50mL, dimethyl sulfoxide is added, makes to heat non-ignitable
Burning concentration of the Cigarette grain phase in dimethyl sulfoxide is 40mg/mL；This triangular flask is placed in ultrasound 30min in Ultrasound Instrument；
Dimethyl sulfoxide extract liquor is collected by filtration with aseptic filter paper again, dispenses into the cryopreservation tube of 1mL, -80 DEG C save for use；
(2) it the preparation of people's nasal epithelia single cell suspension: after people's nasal epithelia RPMI2650 recovery, is inoculated into
25cm2In Tissue Culture Flask, 10mLRPMI1640 cell culture medium is added, is placed in 37 DEG C, 5%CO2It cultivates, is inverted in incubator
Converge and the form situation of micro- sem observation culture cell when converging rate, remove culture bottle to 70%~90% when cell length
In RPMI1640 cell culture medium, with phosphate buffer wash twice, abandon washing lotion；The tryptose of 1mL 0.25% (w/v) is added
Enzyme solutions single layer is incubated for 1~2min, is hanged with RPMI1640 cell culture medium, forms single cell suspension；
(3) cell concentration of people's nasal epithelia single cell suspension calculates: with blood counting chamber counting method to step (2) institute
The cell concentration for obtaining people's nasal epithelia single cell suspension calculates, and obtains the living cells of every milliliter of single cell suspension
Number；
(4) inoculation of people's nasal epithelia single cell suspension: people's nasal epithelia after step (3) are counted is unicellular
Suspension RPMI1640 cell culture medium to 2.0 × 105A/mL, then 6 hole cells are inoculated by inoculum concentration for the hole 2mL/
In culture plate, 6 porocyte culture plates are then placed in 37 DEG C, 5%CO2It is cultivated for 24 hours in incubator；
(5) tested material is grouped: tested material being divided into three groups: cell controls group, positive controls and test sample group；The structure of each group
Become: cell controls group plants people's nasal epithelia and adds RPMI1640 cell culture medium；1mg/mL is added in positive controls
0.5 μ L of cyclophosphamide solution；Test sample group is added prepare liquid obtained by step (1) and adds RPMI1640 cell culture medium；
(6) tested material detects setting for dosage: the ultimate density of prepare liquid obtained by step (1) is respectively as follows: as 4 non-zero-doses
40μg/mL,80μg/mL,160μg/mL,320μg/mL；
(7) preparation of tested material culture product: the culture medium in removing step (4) in 6 porocyte culture plates, then by step (5) into
The 6 μ L of cytochalasin B solution of 1mg/mL is added in row grouping, cell controls group, positive controls and test sample group, uses
The liquid volume in hole every in tissue culture plate is complemented to the hole 2mL/ by RPMI1640 cell culture medium；
(8) it is incubated for tested material: 6 porocyte culture plates after step (7) sample-adding is placed in 37 DEG C, 5%CO2It is incubated in incubator
24h；
(9) it harvests cell: concentration is added in the cell controls group, positive controls and test sample group after step (8) are incubated for
Get off for the trypsin solution of 0.25% (W/V) by the hole of cell from 6 porocyte culture plates is digested；
(10) hypotonic: step (9) postdigestive cell being placed in centrifuge tube, 5min, revolving speed 1000rpm are centrifuged, takes out centrifugation
Supernatant is managed and removed, Klorvess Liquid 3mL is added and gently blows and beats cell, methanol acetic acid solution is added immediately after mixing
2mL is placed on centrifuge and is centrifuged 5min, revolving speed 1000rpm；
(11) it drips piece: the supernatant in step (9) centrifuge tube is removed, 300 μ L methanol acetic acid solution are added, gently piping and druming makes
Formation cell suspending liquid and be placed in naturally dry in glass frame, each detection agent by cell suspending liquid drop on glass slide
Amount 3 slides of drop；
(12) dye: the slide after step (9) is dried is placed in Gimsa dye liquor after dyeing 15min, is rinsed with water slide simultaneously
It is placed on saving backup after natural drying in glass frame；
(13) gained micronucleus slide after dyeing, the visual field that selection is good and cell density is suitable, observation 1000 observation of micronucleus: are taken
A dikaryocyte counts micronuclear rates, selects the dikaryocyte of observation to should be cytoplasm intact, cell membrane sharpness of border, cell
Core is separated from each other, and micronucleus is easy to differentiate；
(14) result judgement: sample sets be will test compared with cell controls group, micronuclear rates have conspicuousness to increase and have dose response
Relationship can be confirmed as positive findings；If statistically difference has conspicuousness, but when without dose-response relationship, then weight must be carried out
Retrial is tested, can repetition person can be identified as the positive.
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Citations (5)
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|---|---|---|---|---|
| WO2007084485A2 (en) * | 2006-01-13 | 2007-07-26 | Battelle Memorial Institute | Markers for assessing copd-related diseases |
| CN101691598A (en) * | 2009-09-17 | 2010-04-07 | 中国烟草总公司郑州烟草研究院 | Method for measuring cytotoxicity of condensate of main stream smoke of cigarettes |
| CN106520910A (en) * | 2016-11-25 | 2017-03-22 | 云南中烟工业有限责任公司 | Method for detecting influences of electronic cigarette products on cell superoxide dismutase activity |
| 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 |
| CN107828850A (en) * | 2017-09-21 | 2018-03-23 | 云南中烟工业有限责任公司 | A kind of in-vitro micronucleus detection method for electronics smoke sol water extract |
-
2018
- 2018-11-28 CN CN201811433538.6A patent/CN109444396A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007084485A2 (en) * | 2006-01-13 | 2007-07-26 | Battelle Memorial Institute | Markers for assessing copd-related diseases |
| CN101691598A (en) * | 2009-09-17 | 2010-04-07 | 中国烟草总公司郑州烟草研究院 | Method for measuring cytotoxicity of condensate of main stream smoke of cigarettes |
| CN106520910A (en) * | 2016-11-25 | 2017-03-22 | 云南中烟工业有限责任公司 | Method for detecting influences of electronic cigarette products on cell superoxide dismutase activity |
| 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 |
| CN107828850A (en) * | 2017-09-21 | 2018-03-23 | 云南中烟工业有限责任公司 | A kind of in-vitro micronucleus detection method for electronics smoke sol water extract |
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