CN106769920A - A kind of method for detecting cigarette smoke condensates to cytolipin Peroxidation Effects - Google Patents
A kind of method for detecting cigarette smoke condensates to cytolipin Peroxidation Effects Download PDFInfo
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- CN106769920A CN106769920A CN201611056861.7A CN201611056861A CN106769920A CN 106769920 A CN106769920 A CN 106769920A CN 201611056861 A CN201611056861 A CN 201611056861A CN 106769920 A CN106769920 A CN 106769920A
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Abstract
The present invention discloses a kind of detection cigarette smoke condensates to the method for cytolipin Peroxidation Effects, comprises the following steps：(1) pre-treatment of tested material；(2) preparation of human lung cancer cell A549 single cell suspension；(3) calculating of the cell concentration of human lung cancer cell A549 single cell suspension；(4) the cell inoculation of human lung cancer cell A549 single cell suspension；(5) packet of tested material；(6) tested material detects the setting of dosage；(7) preparation of tested material culture product；(8) incubation of tested material；(9) tested material is incubated the collection of product；(10) tested material is incubated the cracking centrifugation of product；(11) standard items dilution；(12) tested material sample determination；(13) mda content is calculated.The present invention is to effective treatment of sample, the correct selection of the Optimal Setting and target cell of detection dosage so that this method can accurately and effectively detect cigarette smoke condensates to cytolipin Peroxidation Effects.
Description
Technical field
The invention belongs to tobacco product biological effect assessment technique field, more particularly to a kind of detection total grain of cigarette smoke
Method of the phase thing to cytolipin Peroxidation Effects.
Background technology
As people are to the concern more and more higher of health, requirement higher is proposed to tobacco product, not only had preferably
Mouthfeel, and to reduce the bad impression of human body as far as possible.Cigarette product research staff is first in the design of tobacco tar product formula
Different components are combined and allocated by the phase according to different proportion, form the primary election formula with different-style, in conjunction with Chemical Evaluation and
Sensory evaluation carries out constantly screening adjustment to formula and forms final product formula.But primary election formula is large number of, all enters
Row sensory evaluation takes time and effort, and sensory evaluation lays particular emphasis on and the style mouthfeel of product is screened, and how to be surveyed using biology
Examination index is formulated into screening to product primary election, and the product formula of the bad impression of human body is reduced to obtain, and the exploratory stage is still belonged at present,
Without unified standard method.
Body when various unfavorable stimulations are subjected to, internal high activity molecule such as active oxygen radical (reactive oxygen
Species, ROS) and active nitrogen free radical (reactive nitrogen species, RNS) generation is excessively, degree of oxidation surpasses
Go out the removing of oxide, oxidative system and antioxidant system are unbalance, so as to cause tissue damage.This phenomenon is referred to as oxidisability
Damage, oxidative damage is a kind of broad spectrum activity damage mechanisms, MDA (MAD) is that a kind of important effect of lipid peroxidation is biological
Mark, is measured to it, can be used for the further screening of product primary election formula, and the product of the bad impression of human body is reduced to obtain
Product are formulated.
The content of the invention
It is an object of the invention to provide a kind of method for detecting cigarette smoke condensates to cytolipin Peroxidation Effects,
For the safety evaluation of electronics tobacco product provides reference.
A kind of detection cigarette smoke condensates are comprised the following steps to the method for cytolipin Peroxidation Effects：
(1) pre-treatment of tested material；
(2) preparation of human lung cancer cell A549 single cell suspension；
(3) calculating of the cell concentration of human lung cancer cell A549 single cell suspension；
(4) the cell inoculation of human lung cancer cell A549 single cell suspension；
(5) packet of tested material；
(6) tested material detects the setting of dosage；
(7) preparation of tested material culture product；
(8) incubation of tested material；
(9) tested material is incubated the collection of product；
(10) tested material is incubated the cracking centrifugation of product；
(11) standard items dilution；
(12) tested material sample determination；
(13) mda content is calculated.
In the preferred embodiment of the invention, above-mentioned method, including step in detail below：
(1) pre-treatment of tested material：With reference to National Standard of the People's Republic of China GB/T 19609-2004《Cigarette is with often
Rule analysis determines TPM and tar with smoking machine》Cigarette smoke condensates sample is prepared, sample concentration is 10mg/mL；
(2) preparation of human lung cancer cell A549 HPF single cell suspensions：After human lung cancer cell A549 recovery, it is inoculated into thin
In born of the same parents' blake bottle, 37 DEG C, 5vt%CO are placed in2Cultivated in incubator, inverted microscope observes converging and form feelings for cultured cells
Condition, it is long to 90% when converging rate whne cell, the culture medium in blake bottle is removed, washed twice with phosphate buffer, add appropriate
0.25% trypsin solution, the wherein concentration unit of trypsase/phosphate buffer are quality：Volume；Individual layer is incubated
About 1-2min, is hanged with into fiber fibroblast culture medium, forms single cell suspension；
(3) calculating of the cell concentration of human lung cancer cell A549 HPF single cell suspensions：With blood counting chamber counting method pair
The cell concentration that step (2) obtains human lung cancer cell A549 single cell suspension is calculated, and calculates every milliliter of people's lung into fibre
Tie up the viable count of cell single cell suspension；
(4) the cell inoculation of human lung cancer cell A549 HPF single cell suspensions：By the people's lung after step (3) counting into fibre
Dimension cell single cell suspension adds fibroblast culture medium to be diluted to 2.0 × 105Individual/mL, is inoculated into 96 hole cell culture
In plate, it is inoculated into 60mm Tissue Culture Dish, per ware 4mL, Tissue Culture Plate is placed in 37 DEG C, 5vt%CO2Culture in incubator
24h；
(5) packet of tested material：Tested material is divided into two groups：Cell controls group and detection sample sets；The composition of each group is：
Cell controls group is to plant human lung cancer cell A549 on Fibroblast Growth Medium；Detection sample sets are in fibroblast
Human lung cancer cell A549 is planted on growth medium and tested material is added；
(6) tested material detects the setting of dosage：Tested material, i.e. electronic cigarette stoste are divided into 5 non-non- zero-doses：20μg/
mL、40μg/mL、60μg/mL、80μg/mL、100μg/mL；
(7) preparation of tested material culture product：Fibroblast in removal step (4) in a diameter of 60mm Tissue Culture Plates
Culture medium, then be grouped by step (5), the composition of each group is：Cell controls group is fibroblast culture medium+people's lung into fibre
Dimension cell；Detection sample sets are fibroblast culture medium+human lung cancer cell A549+tested material, and make detection sample sets most
Final concentration is respectively the dosage of step (6) setting；And 2 tissue cultures are supported product fibroblast culture medium and will be mended per ware liquid volume
Enough to 4mL；
(8) incubation of tested material：A diameter of 60mm Tissue Culture Dish after step (7) is loaded is placed in 37 DEG C, 5vt%
CO224h is incubated in incubator；
(9) tested material is incubated the collection of product：Removal fibroblast culture medium, is washed one time with phosphate buffer PBS, plus
Enter appropriate 0.25% trypsin solution, the wherein concentration unit of trypsase/phosphate buffer is quality：Volume；It is single
Layer is incubated about 1-2min, is hanged with Fibroblast culture solution, 1000rpm low-temperature centrifugations 10min；
(10) tested material is incubated the cracking centrifugation of product：The cell that (9) step is obtained, adds 100 μ L cell pyrolysis liquids, carefully
Cellular lysate need to be carried out in ice bath, and cell is collected after cracking, and 1600rpm low-temperature centrifugation 10min take supernatant and treat subsequent measurements；
(11) standard items dilution：Appropriate MDA distilled water diluting to 1,2,5,10,20,50 μM is taken, is marked for making
Directrix curve；
(12) tested material sample determination：0.1mL samples prepare liquid, step (11) is added to prepare not in different centrifuge tubes
With the standard dilutions of concentration gradient, 0.2mL MDAs detection working solution, 100 DEG C of boiling water baths after mixing are then separately added into
15min, 1000rpm room temperature be centrifuged 10min, take 200 μ L of supernatant add 96 orifice plates in, ELIASA 532nm mensuration absorbances；
(13) mda content is calculated：Standard curve is drawn according to standard liquid absorbance, mda content is calculated.
In the preferred embodiment of the invention, the cell pyrolysis liquid described in step (10) is bought in green skies company, is one
Plant the lysate of the cell lysis under the conditions of non denatured.
Beneficial effect of the present invention：
The present invention is to effective treatment of sample, the correct selection of the Optimal Setting and target cell of detection dosage so that this
Method can accurately and effectively detect cigarette smoke condensates to cytolipin Peroxidation Effects.
Specific embodiment
The present invention is described in detail below in conjunction with instantiation, but is not intended to limit the present invention.
The present embodiment is for a kind of smoke's total particulate matter of domestic commercial cigarettes to human lung cancer cell A549 lipid peroxidation
Influence test.
(1) pre-treatment of tested material：With reference to National Standard of the People's Republic of China GB/T 19609-2004《Cigarette is with often
Rule analysis determines TPM and tar with smoking machine》Cigarette smoke condensates sample is prepared, sample concentration is 10mg/mL；
(2) preparation of human lung cancer cell A549 single cell suspension：After human lung cancer cell A549 recovery, cell training is inoculated into
Support in bottle, be placed in 37 DEG C, 5vt%CO2Cultivated in incubator, converge and the form situation of inverted microscope observation cultured cells,
It is long to 90% when converging rate whne cell, the culture medium in blake bottle is removed, washed twice with phosphate buffer, add appropriate
0.25% trypsin solution, the wherein concentration unit of trypsase/phosphate buffer are quality：Volume；Individual layer is incubated
About 2min, is hanged with into fiber fibroblast culture medium, forms single cell suspension；
(3) calculating of the cell concentration of human lung cancer cell A549 single cell suspension：With blood counting chamber counting method to step
Suddenly (2) obtain the cell concentration of human lung cancer cell A549 single cell suspension and are calculated, and calculate every milliliter of people's lung into fiber
The viable count of cell single cell suspension；
(4) the cell inoculation of human lung cancer cell A549 single cell suspension：By the people's lung after step (3) counting into fiber finer
Born of the same parents' single cell suspension adds fibroblast culture medium to be diluted to 2.0 × 105Individual/mL, is inoculated into 96 porocyte culture plates,
It is inoculated into a diameter of 60mm Tissue Culture Dish, per ware 4mL, Tissue Culture Plate is placed in 37 DEG C, 5vt%CO2Training in incubator
Support 24h；
(5) packet of tested material：Tested material is divided into two groups：Cell controls group and detection sample sets；The composition of each group is：
Cell controls group is to plant human lung cancer cell A549 on Fibroblast Growth Medium；Detection sample sets are in fibroblast
Human lung cancer cell A549 is planted on growth medium and tested material is added；
(6) tested material detects the setting of dosage：Tested material, i.e. electronic cigarette stoste are divided into 5 non-non- zero-doses：20μg/
mL、40μg/mL、60μg/mL、80μg/mL、100μg/mL；
(7) preparation of tested material culture product：Fibroblast cell-culture in removal step (4) in 60mm Tissue Culture Plates
Base, then be grouped by step (5), the composition of each group is：Cell controls group is fibroblast culture medium+people's lung into fiber finer
Born of the same parents；Detection sample sets are fibroblast culture medium+human lung cancer cell A549+tested material, and make the final dense of detection sample sets
Degree is respectively the dosage of step (6) setting；And 2 tissue cultures are supported product fibroblast culture medium and will be complemented to per ware liquid volume
4mL；
(8) incubation of tested material：60mm Tissue Culture Dish after step (7) is loaded is placed in 37 DEG C, 5vt%CO2Culture
24h is incubated in case；
(9) tested material is incubated the collection of product：Removal fibroblast culture medium, is washed one time with phosphate buffer PBS, plus
Enter appropriate 0.25% trypsin solution, the wherein concentration unit of trypsase/phosphate buffer is quality：Volume；It is single
Layer is incubated about 2min, is hanged with Fibroblast culture solution, 1000rpm low-temperature centrifugations 10min；
(10) tested material is incubated the cracking centrifugation of product：The cell that (9) step is obtained, adds 100 μ L cell pyrolysis liquids, carefully
Cellular lysate need to be carried out in ice bath, and cell is collected after cracking, and 1600rpm low-temperature centrifugation 10min take supernatant and treat subsequent measurements；
(11) standard items dilution：Appropriate MDA distilled water diluting to 1,2,5,10,20,50 μM is taken, is marked for making
Directrix curve；
(12) tested material sample determination：0.1mL samples prepare liquid, step (11) is added to prepare not in different centrifuge tubes
With the standard dilutions of concentration gradient, 0.2mL MDAs detection working solution, 100 DEG C of boiling water baths after mixing are then separately added into
15min, 1000rpm room temperature be centrifuged 10min, take 200 μ L of supernatant add 96 orifice plates in, ELIASA 532nm mensuration absorbances；
(13) mda content is calculated：Standard curve is drawn according to standard liquid absorbance, mda content is calculated.
What the smoke's total particulate matter of the domestic commercial cigarettes of 11 kinds of table changed to human lung cancer cell A549 HPF mda contents
Influence
By result of the test as can be seen that under the conditions of the sample treatment, detection dosage that the inventive method is given, compareing
There is influence in sample sets, and have dose-effect relationship in the range of detection dosage on the mda content of HPF cells, with 0 dosage
Group is compared has significant difference (p<0.05), show that test system is normal；And sample 1# is right in the range of detection dosage, with 0 dose
Amount group is compared without significant difference (p>0.05)；Result above shows that the method can effectively detect cigarette smoke granule phase substance pair
The influence of cytolipin peroxidating.
Claims (2)
1. it is a kind of to detect cigarette smoke condensates to the method for cytolipin Peroxidation Effects, it is characterised in that including following
Step：
(1) pre-treatment of tested material；
(2) preparation of human lung cancer cell A549 single cell suspension；
(3) calculating of the cell concentration of human lung cancer cell A549 single cell suspension；
(4) the cell inoculation of human lung cancer cell A549 single cell suspension；
(5) packet of tested material；
(6) tested material detects the setting of dosage；
(7) preparation of tested material culture product；
(8) incubation of tested material；
(9) tested material is incubated the collection of product；
(10) tested material is incubated the cracking centrifugation of product；
(11) standard items dilution；
(12) tested material sample determination；
(13) mda content is calculated.
2. method according to claim 1, it is characterised in that including step in detail below：
(1) pre-treatment of tested material：With reference to National Standard of the People's Republic of China GB/T 19609-2004《Cigarette is with conventional point
Analysis determines TPM and tar with smoking machine》Cigarette smoke condensates sample is prepared, sample concentration is 10mg/mL；
(2) preparation of human lung cancer cell A549 single cell suspension：After human lung cancer cell A549 recovery, Tissue Culture Flask is inoculated into
In, it is placed in 37 DEG C, 5vt%CO2Cultivated in incubator, converge and the form situation of inverted microscope observation cultured cells treat thin
Born of the same parents are long when converging rate, to remove the culture medium in blake bottle to 90%, is washed twice with phosphate buffer, adds appropriate 0.25%
Trypsin solution, the wherein concentration unit of trypsase/phosphate buffer are quality：Volume；Individual layer is incubated about 1-2min,
Hanged with into fiber fibroblast culture medium, form single cell suspension；
(3) calculating of the cell concentration of human lung cancer cell A549 single cell suspension：With blood counting chamber counting method to step (2)
The cell concentration for obtaining human lung cancer cell A549 single cell suspension is calculated, and calculates every milliliter of human lung cancer cell A549 list
The viable count of cell suspending liquid；
(4) the cell inoculation of human lung cancer cell A549 single cell suspension：Human lung cancer cell A549 list after step (3) is counted
Cell suspending liquid adds fibroblast culture medium to be diluted to 2.0 × 105Individual/mL, is inoculated into 96 porocyte culture plates, inoculation
To in 60mm Tissue Culture Dish, per ware 4mL, Tissue Culture Plate is placed in 37 DEG C, 5vt%CO2Culture 24h in incubator；
(5) packet of tested material：Tested material is divided into two groups：Cell controls group and detection sample sets；The composition of each group is：Cell
Control group is to plant human lung cancer cell A549 on Fibroblast Growth Medium；Detection sample sets are in fibroblastic growth
Human lung cancer cell A549 is planted on culture medium and tested material is added；
(6) tested material detects the setting of dosage：Tested material, i.e. electronic cigarette stoste are divided into 5 non-non- zero-doses：20μg/mL、40
μg/mL、60μg/mL、80μg/mL、100μg/mL；
(7) preparation of tested material culture product：Fibroblast cell-culture in removal step (4) in a diameter of 60mm Tissue Culture Plates
Base, then be grouped by step (5), the composition of each group is：Cell controls group is fibroblast culture medium+people's lung into fiber finer
Born of the same parents；Detection sample sets are fibroblast culture medium+human lung cancer cell A549+tested material, and make the final dense of detection sample sets
Degree is respectively the dosage of step (6) setting；And 2 tissue cultures are supported product fibroblast culture medium and will be complemented to per ware liquid volume
4mL；
(8) incubation of tested material：A diameter of 60mm Tissue Culture Dish after step (7) is loaded is placed in 37 DEG C, 5vt%CO2Culture
24h is incubated in case；
(9) tested material is incubated the collection of product：Removal fibroblast culture medium, is washed one time with phosphate buffer PBS, is added suitable
The trypsin solution of amount 0.25%, the wherein concentration unit of trypsase/phosphate buffer are quality：Volume；Individual layer is incubated
About 1-2min is educated, is hanged with Fibroblast culture solution, 1000rpm low-temperature centrifugations 10min；
(10) tested material is incubated the cracking centrifugation of product：The cell that (9) step is obtained, adds 100 μ L cell pyrolysis liquids, cell to split
Solution need to be carried out in ice bath, and cell is collected after cracking, and 1600rpm low-temperature centrifugation 10min take supernatant and treat subsequent measurements；
(11) standard items dilution：Appropriate MDA distilled water diluting to 1,2,5,10,20,50 μM is taken, it is bent for making standard
Line；
(12) tested material sample determination：What in different centrifuge tubes prepared by addition 0.1mL samples prepare liquid, step (11) is different dense
The standard dilutions of gradient are spent, 0.2mL MDAs detection working solution, 100 DEG C of boiling water baths after mixing is then separately added into
15min, 1000rpm room temperature be centrifuged 10min, take 200 μ L of supernatant add 96 orifice plates in, ELIASA 532nm mensuration absorbances；
(13) mda content is calculated：Standard curve is drawn according to standard liquid absorbance, mda content is calculated.
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| CN201611056861.7A CN106769920A (en) | 2016-11-25 | 2016-11-25 | A kind of method for detecting cigarette smoke condensates to cytolipin Peroxidation Effects |
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| CN201611056861.7A CN106769920A (en) | 2016-11-25 | 2016-11-25 | A kind of method for detecting cigarette smoke condensates to cytolipin Peroxidation Effects |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107764716A (en) * | 2017-10-21 | 2018-03-06 | 云南中烟工业有限责任公司 | A kind of detection method of cigarette smoke to cellular water Permeability |
| CN107828848A (en) * | 2017-09-21 | 2018-03-23 | 云南中烟工业有限责任公司 | Detect method of the electronics smoke sol water extract to cytolipin Peroxidation Effects |
| CN109593818A (en) * | 2018-11-28 | 2019-04-09 | 云南中烟工业有限责任公司 | A method of detection heating does not burn Cigarette grain phase to cytolipin Peroxidation Effects |
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| CN1284167A (en) * | 1997-10-09 | 2001-02-14 | 卢米特斯特有限公司 | Method and appts for measuring lipid peroxidation in biological fluids and suspensions of tissues |
| CN101461560A (en) * | 2008-01-29 | 2009-06-24 | 延吉烟悦科技有限公司 | Fire-cured tobacco type low-coke tar cigarette and technique for preparing the same |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107828848A (en) * | 2017-09-21 | 2018-03-23 | 云南中烟工业有限责任公司 | Detect method of the electronics smoke sol water extract to cytolipin Peroxidation Effects |
| CN107764716A (en) * | 2017-10-21 | 2018-03-06 | 云南中烟工业有限责任公司 | A kind of detection method of cigarette smoke to cellular water Permeability |
| CN107764716B (en) * | 2017-10-21 | 2020-08-14 | 云南中烟工业有限责任公司 | Method for detecting influence of cigarette smoke on cell water permeability |
| CN109593818A (en) * | 2018-11-28 | 2019-04-09 | 云南中烟工业有限责任公司 | A method of detection heating does not burn Cigarette grain phase to cytolipin Peroxidation Effects |
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Application publication date: 20170531 |