CN104237495B - A kind of cigarette filter water extract micronucleus test in vitro positive control method to set up - Google Patents
A kind of cigarette filter water extract micronucleus test in vitro positive control method to set up Download PDFInfo
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- CN104237495B CN104237495B CN201410489543.4A CN201410489543A CN104237495B CN 104237495 B CN104237495 B CN 104237495B CN 201410489543 A CN201410489543 A CN 201410489543A CN 104237495 B CN104237495 B CN 104237495B
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Abstract
A kind of cigarette filter water extract of the present invention micronucleus test in vitro positive control method to set up, the method is: that 1) chooses equal number meets the cigarette filter and testing sample group cigarette filter of preparing positive control conditions; Does is 2) compound concentration 200-400? the endoxan aqueous solution of μ g/ml; 3) evenly spraying endoxan aqueous solution for the preparation of on the cigarette filter of positive controls, for subsequent use after its natural drying; 4) by identical method, lixiviate is carried out to positive controls and testing sample group cigarette filter; 5) leaching liquor is as solvent, prepares with powder cell culture medium, by the solution filtration sterilization after preparation; 6) micronucleus test in vitro is carried out to the extract through step 5) process.By the positive control that the method is arranged, can verify the leaching process of cigarette filter and the follow-up micronucleus test in vitro that cigarette filter water extract is carried out whether reliable.
Description
Technical field
The invention belongs to technical field of tobacco, be specifically related to a kind of positive control method to set up when micronucleus test in vitro carries out to cigarette filter water extract.
Background technology
Micronucleus test is a kind of genotoxicity testing method detecting chromosome or mitotic apparatus damage.Akinetic chromosome segment or the whole chromosome lost because spindle is impaired, become micronucleus in the kytoplasm that anaphase of cell division still stays daughter cell.Micronucleus test in vitro is generally utilize the zooblast of in vitro culture to carry out the detection of micronucleus.It is usually as one of the security Biological Detection means of food, medicine, dental material etc.
Because cigarette filter directly contacts with human mouth, there is potential health risk, therefore, be necessary to adopt micronucleus test in vitro means, the potential hazard that it may exist is investigated.When micronucleus test in vitro is carried out to cigarette filter, first need to extract cigarette filter, and then extract is detected.Therefore, need in process of the test to arrange positive control, with verify leaching process and follow-up micronucleus test in vitro whether reliable.
Existing micronucleus test in vitro positive control method to set up has two classes: first method, if tested material can be mixed with solution, then uses and certain densityly knownly has genotoxic reagent and be directly added in cell culture fluid and make positive control; Second method, test if tested material needs to prepare leaching liquor, then positive control is preferably identical and close with tested material character and has genotoxic material, and after the method that this materials'use is identical with tested material is extracted, its leaching liquor should be in test obvious positive reaction.
But existing method be not suitable for the micronucleus test in vitro of cigarette filter leaching liquor, need to prepare cigarette filter leaching liquor this is because carry out micronucleus test in vitro to cigarette filter, therefore, use above-mentioned first method that positive control is set and then cannot verify that whether the process of extraction is reliable.And for use second method, because current existing cigarette filter kind does not find to have genetoxic, and not close with cigarette filter character genotoxic material, therefore for the leaching process of checking cigarette filter and the follow-up micronucleus test in vitro that cigarette filter water extract is carried out whether reliable, be necessary to develop a kind of positive control method to set up for cigarette filter water extract micronucleus test in vitro.
Through literature search, do not find the open report with content same document of the present invention.
Summary of the invention
The object of the present invention is to provide a kind of positive control method to set up when micronucleus test in vitro carries out to cigarette filter water extract.By the positive control that the method is arranged, can verify the leaching process of cigarette filter and the follow-up micronucleus test in vitro that cigarette filter water extract is carried out whether reliable.
The oral contact of filter tip and people during cigarette smoking, carry out extracting the solvent (water or artificial saliva) needing to select and people's saliva character is close to cigarette filter, therefore people is the positive thing added on filter tip is also water miscible.The present invention uses endoxan as the artificial Genotoxic added.No. CAS of endoxan is 50-18-0, is a kind of water-soluble chromosome clastogen, is a kind of positive control agent conventional in micronucleus test in vitro.Therefore, by endoxan is added on filter tip as positive control sample, then prepare water extract and carry out micronucleus test in vitro together with testing sample, the result if positive control is positive, then illustrate that the leaching process of cigarette filter and the follow-up micronucleus test in vitro carried out cigarette filter water extract are reliable.
The present invention realizes by following technical proposals:
That chooses equal number meets the cigarette filter (as the follow-up cigarette filter for the preparation of positive controls) and testing sample group cigarette filter of preparing positive control conditions;
Step (2) compound concentration is the endoxan aqueous solution of 200-400 μ g/ml;
Step (3) is for the preparation of the endoxan aqueous solution of the cigarette filter of positive controls evenly spraying step (2) and preparing, fountain height should make the complete stripping final concentration of the theory of endoxan in filter tip Leaching tests test system be more than or equal to 20 μ g/ml, and make cigarette filter fully absorb cyclophosphamide solution, for subsequent use as positive controls cigarette filter after its natural drying;
The positive controls cigarette filter that step (4) is prepared by the step (3) of identical method to equal number and testing sample group cigarette filter carry out lixiviate;
The leaching liquor that step (5) uses step (4) to obtain, as solvent, is prepared by the method that powder cell culture medium illustrates according to nutrient culture media, is then used for follow-up micronucleus test in vitro by after the solution filtration sterilization after preparation;
Step (6) carries out micronucleus test in vitro to the extract processed through step (5), the concentration of Test extraction liquid in test system is 100%, 50%, 25% and 12.5%4 dosage (test fluid of low concentration uses cell culture fluid dilution to obtain), and the cell open-assembly time of extract is 24 hours.
The cigarette filter preparing positive control conditions that meets in described step (1) refers to: (1) can not react and affect its positive findings because there is comparatively extensive chemical with endoxan for the preparation of the cigarette filter of positive control; (2) cigarette filter for the preparation of positive control can not affect its positive findings because producing stronger suction-operated with endoxan; (3) cell survival rate measured in cell toxicity test for the preparation of the cigarette filter water extract stoste of positive control should be more than or equal to 50%.
Theory complete stripping concentration described in step (3) refers to: the quantity of solvent according to the actual fountain height of prepared cyclophosphamide solution actual concentrations, endoxan and when extracting added by reality, the endoxan supposing to be sprayed on filter tip in lixiviate time the concentration that can calculate under the condition of 100% stripping.
compared with prior art, its beneficial effect is in the present invention:operation is simple for cigarette filter water extract micronucleus test in vitro positive control method to set up of the present invention, economical and practical.The positive thing endoxan of the artificial interpolation that the present invention uses, is a kind of water-soluble chromosome clastogen, is easy to be dissolved in the Extraction solvent distilled water of filter tip.The concentration of the endoxan aqueous solution set by the present invention and proof load can make micronucleus test in vitro to be positive result, and present good dose-effect relationship.By the positive control that the method is arranged, can verify the leaching process of cigarette filter and the follow-up micronucleus test in vitro that cigarette filter water extract is carried out whether reliable.
Accompanying drawing explanation
Fig. 1 prepares artificial positive filter tip with the inventive method as positive control, the water extract of the commercial cigarettes filter tip of 2 kinds of random chooses is carried out to the micronuclear rates figure of the leaching liquor of the variable concentrations that micronucleus test in vitro obtains.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, but content of the present invention is not limited thereto.
Embodiment 1
Prepare artificial positive filter tip with the inventive method as positive control, micronucleus test in vitro is carried out to the commercial cigarettes filter tip of 2 kinds of random chooses.
1, random choose 2 kinds of commercial cigaretteses, randomly draw 20 for the preparation of testing sample group cigarette filter for often kind.Randomly draw 20 meet prepare positive cigarette filter condition Kentucky 3R4F cigarette for the preparation of the cigarette filter of positive controls.Above-mentioned cigarette filter is cut down for subsequent use, the overlapping part switching to tipping paper and cigarette paper during cutting, should be avoided, to prevent cigarette paper and pipe tobacco to bring in filter tip sample.
2, compound concentration is the endoxan aqueous solution of 200 μ g/ml.
3, spraying for the preparation of even on the 3R4F cigarette filter of positive control the endoxan aqueous solution 10mL prepared in step 2, when making the complete stripping final concentration of the theory of endoxan in cigarette filter Leaching tests test system be 20 μ g/ml(extraction, amount of water is 100mL), cigarette filter is made fully to absorb the cyclophosphamide solution of interpolation, for subsequent use as positive cigarette filter control group after cigarette filter natural drying.
4, the positive controls cigarette filter prepared step 3 with 100mL distilled water and 2 kinds of testing sample group cigarette filters (sample 1 and sample 2) leave standstill at 40 DEG C extracts 30min.
5, the water extract obtained by step 4 is as solvent, prepare by the method that DMEM powder cell culture medium illustrates according to nutrient culture media, and add hyclone, volume ratio shared by hyclone body is made to be 10% of extract cumulative volume, then by preparation after solution filtration sterilization, after for follow-up micronucleus test in vitro.
6, use Chinese hamster ovary celI to carry out micronucleus test in vitro to the extract processed through step 5, the concentration of Test extraction liquid in test system is 100%, 50%, 25% and 12.5%4 dosage (test fluid of low concentration uses cell culture fluid dilution to obtain).In cell culture fluid, add cytochalasin B, make its concentration be 3 μ g/mL.Harvesting after exposing 24 hours, makes micronucleus slide with dripping sheet legal system after hypotonic and fixing process.Micronucleus slide carries out Micronuclei to 1000 dikaryocytes under the microscope after Giemsa dyeing.
7, the micronuclear rates under each sample variable concentrations is shown in Fig. 1.From test findings, the extract of the 2 kinds of filter tip samples tested all does not show obvious dose-response relationship under 4 tested concentration, and micronuclear rates and blank group without the significance difference opposite sex (
p>0.05), be therefore negative findings.The micronuclear rates of artificial positive filter tip sample extracting solution increases with test concentrations and reduces, and has comparatively significantly dose-response relationship, and the micronuclear rates of 100%, 50% and 25% 3 dosage and blank group more all have significant difference (
p<0.05), be therefore positive findings.Leaching process and the follow-up micronucleus test in vitro carried out cigarette filter water extract that cigarette filter in the present embodiment is described are reliable.
The compound concentration of endoxan aqueous solution can also be 250 μ g/ml, 300 μ g/ml, 350 μ g/ml, 400 μ g/ml etc.
More than show and describe ultimate principle of the present invention and principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and instructions just illustrates principle of the present invention; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications, and these changes and improvements all fall in the claimed scope of the invention.Application claims protection domain is defined by appending claims and equivalent thereof.
Claims (2)
1. a cigarette filter water extract micronucleus test in vitro positive control method to set up, it is characterized in that, the method comprises the steps:
What step (1) chose equal number meets the cigarette filter and testing sample group cigarette filter of preparing positive control conditions;
Step (2) compound concentration is the endoxan aqueous solution of 200-400 μ g/ml;
Step (3) is for the preparation of the endoxan aqueous solution of the cigarette filter of positive controls evenly spraying step (2) and preparing, fountain height should make the complete stripping concentration of the theory of endoxan in filter tip Leaching tests test system be more than or equal to 20 μ g/ml, and make cigarette filter fully absorb cyclophosphamide solution, for subsequent use as positive controls cigarette filter after its natural drying;
The positive controls cigarette filter that step (4) is prepared step (3) by identical method and testing sample group cigarette filter carry out lixiviate;
The water extract that step (5) uses step (4) to obtain, as solvent, is prepared by the method that powder cell culture medium illustrates according to nutrient culture media, is then used for follow-up micronucleus test in vitro by after the solution filtration sterilization after preparation;
Step (6) extract that step (5) processed carries out micronucleus test in vitro, and the concentration of Test extraction liquid in test system is 100%, 50%, 25% and 12.5%4 dosage, and the cell open-assembly time of extract is 24 hours.
2. cigarette filter water extract micronucleus test in vitro positive control method to set up according to claim 1, is characterized in that: the lixiviate of the cigarette filter described in step (4) adopts distilled water as extract.
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| CN104749353B (en) * | 2015-04-22 | 2016-06-01 | 云南中烟工业有限责任公司 | A kind of method detecting buccal cigarette goods and cell micronucleus rate being affected |
| 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 |
Citations (4)
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|---|---|---|---|---|
| DE3135138A1 (en) * | 1980-11-11 | 1982-06-09 | Baumgartner Papiers S.A., 1020 Renens | CONTROL DEVICE FOR CIGARETTE FILTER |
| CN102095842A (en) * | 2011-02-10 | 2011-06-15 | 云南烟草科学研究院 | Ames test method for biologically evaluating cigarette tipping paper |
| CN103512994A (en) * | 2013-10-14 | 2014-01-15 | 福建中烟工业有限责任公司 | Method for measuring residual volatile components in cigarette filter stick |
| CN103792218A (en) * | 2014-02-21 | 2014-05-14 | 中国烟草总公司郑州烟草研究院 | Method for detecting genetic toxicity of total particulate matters in main stream smoke of cigarette by adopting binuclear method |
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2014
- 2014-09-23 CN CN201410489543.4A patent/CN104237495B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3135138A1 (en) * | 1980-11-11 | 1982-06-09 | Baumgartner Papiers S.A., 1020 Renens | CONTROL DEVICE FOR CIGARETTE FILTER |
| CN102095842A (en) * | 2011-02-10 | 2011-06-15 | 云南烟草科学研究院 | Ames test method for biologically evaluating cigarette tipping paper |
| CN103512994A (en) * | 2013-10-14 | 2014-01-15 | 福建中烟工业有限责任公司 | Method for measuring residual volatile components in cigarette filter stick |
| CN103792218A (en) * | 2014-02-21 | 2014-05-14 | 中国烟草总公司郑州烟草研究院 | Method for detecting genetic toxicity of total particulate matters in main stream smoke of cigarette by adopting binuclear method |
Non-Patent Citations (2)
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| CHO细胞微核的激光扫描细胞仪自动化检测方法;龚婧婧，等;《云南大学学报（自然科学版）》;20120731;第34卷(第4期);第461-467页 * |
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