CN104749353B - A kind of method detecting buccal cigarette goods and cell micronucleus rate being affected - Google Patents
A kind of method detecting buccal cigarette goods and cell micronucleus rate being affected Download PDFInfo
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Abstract
The present invention provides a kind of method detecting buccal cigarette goods and cell micronucleus rate being affected, calculate through the preparation of sample-pretreating method, single cell suspension, cell concn, cell inoculation, add tested material, hatch tested material, harvested cell, hypotonic, drip the steps such as sheet, dyeing, Micronuclei, result judgement. The route of exposure of integrated survey buccal cigarette goods of the present invention, the mode of action, establish buccal cigarette goods sample-pretreating method, and have chosen human oral mucosa inoblast hOMF as buccal cigarette goods micronucleus test in vitro detection cell according to buccal cigarette goods effect target cell, and determine sample detection dosage, define the micronucleus test in vitro detection method of applicable buccal cigarette goods.
Description
Technical field
The present invention relates to a kind of method detecting buccal cigarette goods and cell micronucleus rate being affected, belong to tobacco and tobacco product security biological assessment technical field.
Background technology
Genetics terminal according to detection is different, and the genetoxic short term detection method set up at present is more than 200 kinds [1]. Cell micronucleus rate is one of crucial combined index of genetoxic evaluation, micronucleus test due to its endpoint detection clear and definite, method is easy, it is easy to features such as carrying out,, food, pharmaceutical prod, industrial or agricultural product, etc. be widely used [2-5] in the safety evaluation of Healthy relevant products, the monitoring of genetic damage, the mutational lesions detection of specific crowd and early prediction etc. 2009 the Organization for Economic Cooperation and Development (OECD) disclose the external micronucleus test policy paper of Mammals, tentatively establish the unified standard [6] of the external micronucleus test of Mammals. International tobacco scientific research cooperation center CORESTA is organized in 2002 and has set up the external toxicity test job group of cigarette smoke, through a large amount of literature survey of working group and research work, this working group recommends to adopt micronucleus test in vitro to be detected by the genetoxic that cigarette smoke condensate is potential. This detection method is extensively adopted by domestic and international tobacco company at present.
Increasingly the strict and smoking of tobacco supervision legislation and going deep into of health research, result in tobacco company seek risk lower, without the novel tobacco goods of environment flue gas. buccal cigarette goods avoid the harm that the complex mixture that traditional cigarette product burns produces brings, and present stage has become one of new direction that foreign tobacco company turns to from traditional cigarette goods. component according to product and form feature, buccal cigarette goods are divided into tradition containing the Snus type buccal cigarette goods of raw tobacco material and the Novel buccal cigarette goods (such as tobacco lozenge) not containing raw tobacco material. the use-pattern of buccal cigarette goods is different from traditional cigarette inhaling type, buccal cigarette goods directly use through mouth, and its leachable can enter Digestive tract with saliva, use procedure does not produce flue gas, its route of exposure is significantly different from traditional cigarette, the change of detected object causes sample-pretreating method, detection cell, detection dosage is also different, the needs of buccal cigarette goods genetoxic detection can not be met for detecting the micronucleus test in vitro of traditional cigarette genetoxic, and research institution and tobacco company not yet formulate the standard method of buccal cigarette goods micronucleus test in vitro both at home and abroad at present, therefore, it is necessary to set up, for sucking goods own characteristic, the micronucleus test in vitro detection method being applicable to buccal cigarette goods, guarantee the security of product.
The reference related to above:
[1] LynchAM, SasakiJC, ElespuruR, etal.Newandemergingtechnologiesforgenetictoxicitytesting [J] .EnvironMolMutagen, 2011,52 (3): 205-223.
[2] Ministry of Health of the People's Republic of China .GB15193-2003 toxicological evaluation of food safety procedure [S].
[3] The Ministry of Agriculture of the People's Republic of China, MOA .GB15670-1995 agriculture chemical registration toxicology test method [S].
[4] Chinese food Drug Administration .YY/T0127.12-2008 odontology oral cavity medical apparatus biological assessment the 2nd unit: test method micronucleus test [S].
[5] State Administration for Quality Supervision and Inspection and Quarantine .SN/T2178-2008 hazardous substance mammalian erythropoietin micronucleus test method [S].
[6] OrganizationforEconomicCooperationandDevelopment.Guideli neforthetestingofchemicalsdraftproposalforanewguideline4 87:invitromammaliancellmicronucleustest (MNvit) [S], 2009.
Summary of the invention
It is an object of the invention to the security for weighing buccal cigarette goods, a kind of micronucleus test in vitro method that buccal cigarette goods genetoxic detects is provided, the basis of traditional cigarette goods micronucleus test in vitro method is improved a kind of micronucleus test in vitro method being suitable for the detection of buccal cigarette goods genetoxic obtained.
The present invention is realized by following technical proposal: a kind of method detecting buccal cigarette goods and cell micronucleus rate being affected, through following each step:
(1) sample-pretreating method: accurately take 1.0g buccal cigarette goods, add 30mL serum free medium, thermostat water bath leaves standstill 24h at 37 DEG C and extracts, after extraction, first carry out initial filter except Slag treatment with qualitative filter paper, degerming with 0.45 ��m of organic membrane filtration again, obtain liquid to be measured;
(2) preparation of single cell suspension: human oral mucosa's inoblast hOMF cell is in 37 DEG C, 5%CO2, 95%RH(relative air humidity) incubator is cultivated, until cell confluency rate about 80��90% time, remove substratum, wash twice with phosphoric acid buffer, abandon washing lotion; Adding 1mL-2mL concentration is 0.25%(w/v) trypsin solution carry out individual layer and hatch 1��2min, add DMEM/F12 cell culture medium and hang, form single-cell suspension liquid;
(3) cell concn of single-cell suspension liquid calculates: is calculated by the cell concn of step (2) gained single-cell suspension liquid by blood counting chamber counting process, calculates the viable count of every milliliter of single-cell suspension liquid;
(4) cell inoculation: single-cell suspension liquid DMEM/F12 cell culture medium to 1.0��1.5 �� 10 after step (3) is counted5Individual/mL, then be 2mL/ hole by inoculum size, it is inoculated in 6 porocyte culture plates, 6 porocyte culture plates are placed in 37 DEG C, 5%CO2, 95%RH(relative air humidity) cultivate 24h in incubator;
(5) tested material is divided into three groups: negative control group, positive controls and detection sample sets; Negative control group adds DMEM/F12 cell culture medium; Positive controls adds the endoxan solution that concentration is 0.2 �� g/mL; Detection sample sets adds step (1) the gained liquid to be measured of various dose;
(6) dosage setting: divide index using sample nicotine amount as detection dosage, 4 non-zero dosage are set: 1400 �� g/mL, 700 �� g/mL, 350 �� g/mL, 175 �� g/mL;
(7) remove the substratum in 6 porocyte culture plates in step (4), then divide into groups by step (5), in the corresponding hole of negative control group, add DMEM/F12 cell culture medium; The endoxan solution that concentration is 0.2 �� g/mL is added in the corresponding hole of positive controls; In the detection corresponding hole of sample sets, add step (1) gained liquid to be measured, make the dosage that final concentration is respectively step (6) and sets; The final volume of negative control group, positive controls and detection sample sets is 2mL/ hole; Cytochalasin is all added again so that it is the final concentration in each cell culture well is 3 �� g/mL in negative control group, positive controls and detection sample sets.
(8) tested material is hatched: 6 porocyte culture plates after step (7) is added sample are placed in 37 DEG C, 5%CO2, 95%RH(relative air humidity) incubator hatches 24h;
(9) harvested cell: be 0.25%(w/v by concentration) trypsin solution cell is hatched from step (8) after 6 porocyte culture plates hole in digest respectively, obtain cell suspending liquid;
(10) hypotonic: the cell suspending liquid of step (9) is carried out centrifugation, add Klorvess Liquid 3mL that concentration is 0.075mol/L after removing supernatant liquor and blow and beat cell, add methyl alcohol/glacial acetic acid solution 2mL at once after mixed even, then carry out centrifugation, remove supernatant liquor;
(11) drip sheet: by gained supernatant liquor in step (10) for blowing and beating cell, form cell suspending liquid, then cell suspending liquid is dropped in slide glass dries naturally;
(12) dye: after the slide glass after step (11) being dried is positioned in the Gimsa dye liquor of new preparation to dye 15min, then with tap water slide glass and save backup after naturally drying;
(13) Micronuclei: the slide glass of selecting step (12), at least observes 1000 dikaryocytes, and counts micronuclear rates;
(14) result judges: by the micronuclear rates of testing sample group compared with negative control group, and micronuclear rates has significance to increase and have dose-response relationship, can confirm as positive findings.
The serum free medium of described step (1) is commercial products.
Converging rate in described step (2) is observe converging and form situation of culturing cell by inverted microscope.
Methyl alcohol/the glacial acetic acid solution of described step (10) mixes even formulated by the ratio of methyl alcohol and Glacial acetic acid 4:1 by volume.
The centrifugation of described step (10) is centrifugation 5min under the rotating speed of 1000rmp.
The slide glass of described step (11) is parallel 3, every hole.
Selecting of described step (13) is good and the suitable visual field of cell density, and it is intact that the dikaryocyte of observation should be tenuigenin, and cytolemma border is clear, and nucleus is separated from each other, and micronucleus is easy to differentiate.
Step (14) if in statistically difference have significance, but during without dose-response relationship, then must carry out revision test, repetition person can be defined as the positive.
The inventive method is compared with art methods, advantage is: the route of exposure of integrated survey buccal cigarette goods, the mode of action, establish buccal cigarette goods sample-pretreating method, and have chosen human oral mucosa inoblast hOMF as buccal cigarette goods micronucleus test in vitro detection cell according to buccal cigarette goods effect target cell, and determine sample detection dosage, define the micronucleus test in vitro detection method of applicable buccal cigarette goods. Effective process of sample, the optimization setting of detection dosage and the selecting properly of target cell so that present method can detect buccal cigarette goods accurately and effectively on the impact of cell micronucleus rate.
Embodiment
Below in conjunction with specific examples, the present invention is described in detail, but does not limit the present invention.
Embodiment 1
For the detection of camel (Camel) brand peppermint taste Snus type buccal cigarette goods genetoxic.
(1) sample-pretreating method: accurately take 1.0g camel (Camel) brand peppermint taste Snus type buccal cigarette goods, add the commercial serum free medium of 30mL, thermostat water bath leaves standstill 24h at 37 DEG C extract, first initial filter is carried out except Slag treatment with qualitative filter paper after extraction, degerming with 0.45 ��m of organic membrane filtration again, obtain liquid to be measured;
(2) preparation of single cell suspension: human oral mucosa's inoblast hOMF cell is in 37 DEG C, 5%CO2, 95%RH(relative air humidity) incubator is cultivated, observe converging and form situation of culturing cell by inverted microscope, until cell confluency rate about 80��90% time, remove substratum, wash twice with phosphoric acid buffer, abandon washing lotion; Adding 1mL-2mL concentration is 0.25%(w/v) trypsin solution carry out individual layer and hatch 1��2min, add DMEM/F12 cell culture medium and hang, form single-cell suspension liquid;
(3) cell concn of single-cell suspension liquid calculates: is calculated by the cell concn of step (2) gained single-cell suspension liquid by blood counting chamber counting process, calculates the viable count of every milliliter of single-cell suspension liquid;
(4) cell inoculation: single-cell suspension liquid DMEM/F12 cell culture medium to 1.0��1.5 �� 10 after step (3) is counted5Individual/mL, then be 2mL/ hole by inoculum size, it is inoculated in 6 porocyte culture plates, 6 porocyte culture plates are placed in 37 DEG C, 5%CO2, 95%RH(relative air humidity) cultivate 24h in incubator;
(5) tested material is divided into three groups: negative control group, positive controls and detection sample sets; Negative control group adds DMEM/F12 cell culture medium; Positive controls adds the endoxan solution that concentration is 0.2 �� g/mL; Detection sample sets adds step (1) the gained liquid to be measured of various dose;
(6) dosage setting: divide index using sample nicotine amount as detection dosage, 4 non-zero dosage are set: 1400 �� g/mL, 700 �� g/mL, 350 �� g/mL, 175 �� g/mL;
(7) remove the substratum in 96 porocyte culture plates in step (4), then divide into groups by step (5), in the corresponding hole of negative control group, add DMEM/F12 cell culture medium; The endoxan solution that concentration is 0.2 �� g/mL is added in the corresponding hole of positive controls; In the detection corresponding hole of sample sets, add step (1) gained liquid to be measured, make the dosage that final concentration is respectively step (6) and sets; The final volume of negative control group, positive controls and detection sample sets is 2mL/ hole; Cytochalasin is all added again so that it is the final concentration in each cell culture well is 3 �� g/mL in negative control group, positive controls and detection sample sets.
(8) tested material is hatched: 6 porocyte culture plates after step (7) is added sample are placed in 37 DEG C, 5%CO2, 95%RH(relative air humidity) incubator hatches 24h;
(9) harvested cell: be 0.25%(w/v by concentration) trypsin solution cell is hatched from step (8) after 6 porocyte culture plates hole in digest respectively, obtain cell suspending liquid;
(10) hypotonic: the cell suspending liquid of step (9) is carried out centrifugation, add Klorvess Liquid 3mL that concentration is 0.075mol/L after removing supernatant liquor and blow and beat cell, mixed even after add methyl alcohol/glacial acetic acid solution 2mL(methyl alcohol and Glacial acetic acid 4:1 by volume at once ratio mix even), centrifugation 5min under the rotating speed of 1000rmp, removes supernatant liquor again;
(11) dripping sheet: by gained supernatant liquor in step (10) for blowing and beating cell, form cell suspending liquid, then dropped in by cell suspending liquid and naturally dry on slide glass, slide glass is parallel 3, every hole;
(12) dye: after the slide glass after step (11) being dried is positioned in the Gimsa dye liquor of new preparation to dye 15min, then with tap water slide glass and save backup after naturally drying;
(13) Micronuclei: the slide glass of selecting step (12), good and that cell density the is suitable visual field is selected from micronucleus slide, it is intact that the dikaryocyte observed should be tenuigenin, cytolemma border is clear, nucleus is separated from each other, micronucleus is easy to differentiate, and at least observes 1000 dikaryocytes, and counts micronuclear rates;
(14) result judges: by the micronuclear rates of testing sample group compared with negative control group, and micronuclear rates has significance to increase and have dose-response relationship, can confirm as positive findings. If statistically difference has significance, but during without dose-response relationship, then revision test must be carried out, repetition person the positive can be can be defined as.
After improving pre-treating process, detection cell, detection dosage, the genetoxic of camel (Camel) brand peppermint taste Snus type buccal cigarette goods is detected, the results are shown in Table 1:
Table 1 camel (Camel) brand peppermint taste Snus type buccal cigarette goods micronucleus test in vitro detected result
As can be seen from the above results, when the sample treatment that the inventive method provides, detection cell, detection dosage, without dose-effect relationship between camel (Camel) the brand peppermint taste Snus type buccal cigarette goods micronuclear rates that inducing cell produces within the scope of study dosage and study dosage, and micronuclear rates does not have significance to increase compared with negative control group, goods mutagenicity is negative. Positive controls micronuclear rates micronuclear rates significance compared with negative control group increases, and proves that this test system is normal, and detection data are effective.
Claims (7)
1. the method detecting buccal cigarette goods and cell micronucleus rate being affected, it is characterised in that through following each step:
(1) sample-pretreating method: accurately take 1.0g buccal cigarette goods, add 30mL serum free medium, at 37 DEG C, leave standstill 24h extract, after extraction, first carry out initial filter except Slag treatment with qualitative filter paper, degerming with 0.45 ��m of organic membrane filtration again, obtain liquid to be measured;
(2) preparation of single cell suspension: human oral mucosa's inoblast hOMF cell is in 37 DEG C, 5%CO2, 95%RH incubator is cultivated, until cell confluency rate about 80��90% time, remove substratum, wash twice with phosphoric acid buffer, abandon washing lotion; Add 1mL-2mL mass volume ratio be 0.25% trypsin solution carry out individual layer and hatch 1��2min, add DMEM/F12 cell culture medium and hang, form single-cell suspension liquid;
(3) cell concn of single-cell suspension liquid calculates: is calculated by the cell concn of step (2) gained single-cell suspension liquid by blood counting chamber counting process, calculates the viable count of every milliliter of single-cell suspension liquid;
(4) cell inoculation: single-cell suspension liquid DMEM/F12 cell culture medium to 1.0��1.5 �� 10 after step (3) is counted5Individual/mL, then be 2mL/ hole by inoculum size, it is inoculated in 6 porocyte culture plates, 6 porocyte culture plates are placed in 37 DEG C, 5%CO2, cultivate 24h in 95%RH incubator;
(5) tested material is divided into three groups: negative control group, positive controls and detection sample sets; Negative control group adds DMEM/F12 cell culture medium; Positive controls adds the endoxan solution that concentration is 0.2 �� g/mL; Detection sample sets adds step (1) the gained liquid to be measured of various dose;
(6) dosage setting: divide index using sample nicotine amount as detection dosage, 4 non-zero dosage are set: 1400 �� g/mL, 700 �� g/mL, 350 �� g/mL, 175 �� g/mL;
(7) remove the substratum in 6 porocyte culture plates in step (4), then divide into groups by step (5), in the corresponding hole of negative control group, add DMEM/F12 cell culture medium; The endoxan solution that concentration is 0.2 �� g/mL is added in the corresponding hole of positive controls; In the detection corresponding hole of sample sets, add step (1) gained liquid to be measured, make the dosage that final concentration is respectively step (6) and sets; The final volume of negative control group, positive controls and detection sample sets is 2mL/ hole; Cytochalasin is all added again so that it is the final concentration in each cell culture well is 3 �� g/mL in negative control group, positive controls and detection sample sets;
(8) tested material is hatched: 6 porocyte culture plates after step (7) is added sample are placed in 37 DEG C, 5%CO2, 95%RH incubator hatches 24h;
(9) harvested cell: with mass volume ratio be 0.25% trypsin solution cell is hatched from step (8) after 6 porocyte culture plates hole in digest respectively, obtain cell suspending liquid;
(10) hypotonic: the cell suspending liquid of step (9) is carried out centrifugation, add Klorvess Liquid 3mL that concentration is 0.075mol/L after removing supernatant liquor and blow and beat cell, add methyl alcohol/glacial acetic acid solution 2mL at once after mixed even, then carry out centrifugation, remove supernatant liquor;
(11) drip sheet: by gained supernatant liquor in step (10) for blowing and beating cell, form cell suspending liquid, then cell suspending liquid is dropped in slide glass dries naturally;
(12) dye: after the slide glass after step (11) being dried is positioned in the Gimsa dye liquor of new preparation to dye 15min, then with tap water slide glass and save backup after naturally drying;
(13) Micronuclei: the slide glass of selecting step (12), at least observes 1000 dikaryocytes, and counts micronuclear rates;
(14) result judges: by the micronuclear rates of testing sample group compared with negative control group, and micronuclear rates has significance to increase and have dose-response relationship, namely confirms as positive findings.
2. method according to claim 1, it is characterised in that: the serum free medium of described step (1) is commercial products.
3. method according to claim 1, it is characterised in that: described step (2) is converged rate and is obtained with form situation converging of culturing cell by inverted microscope observation.
4. method according to claim 1, it is characterised in that: the methyl alcohol/glacial acetic acid solution of described step (10) mixes even formulated by the ratio of methyl alcohol and Glacial acetic acid 4:1 by volume.
5. method according to claim 1, it is characterised in that: the second time centrifugation of described step (10) is centrifugation 5min under the rotating speed of 1000rmp.
6. method according to claim 1, it is characterised in that: the slide glass of described step (11) is every porocyte suspension parallel 3 samples on slide glass in step (11).
7. method according to claim 1, it is characterized in that: good and that cell density the is suitable visual field is selected in selecting of described step (13) from micronucleus slide, and it is intact that the dikaryocyte of observation should be tenuigenin, and cytolemma border is clear, nucleus is separated from each other, and micronucleus is easy to differentiate.
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| CN106706911B (en) * | 2016-11-25 | 2019-01-25 | 云南中烟工业有限责任公司 | A method of detection mouth containing tobacco product influences cellular superoxide hydrogen enzyme enzyme activity |
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| 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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| CN106554988B (en) * | 2016-11-25 | 2020-06-19 | 云南中烟工业有限责任公司 | Method for detecting influence of buccal tobacco product on cell superoxide dismutase |
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| CN107843725A (en) * | 2017-12-14 | 2018-03-27 | 云南中烟工业有限责任公司 | It is a kind of to be used to detect the method that gum base type chewing tobacco influences cell micronucleus rate |
| CN109444126A (en) * | 2018-11-28 | 2019-03-08 | 云南中烟工业有限责任公司 | A method of detection heating does not burn Cigarette grain phase to cellular superoxide dismutase activity influence |
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| CN101671734A (en) * | 2009-09-24 | 2010-03-17 | 中国烟草总公司郑州烟草研究院 | Cigarette smoke in-vitro micronucleus detection method |
| CN101709329B (en) * | 2009-11-25 | 2012-02-01 | 中国烟草总公司郑州烟草研究院 | Detection method for vitro cell micronucleus of cigarette smoke genetic toxicity |
| 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 |
| 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 |
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