CN106520903A - Method for detecting influences of buccal cigarette product on lipid peroxidation of cells - Google Patents
Method for detecting influences of buccal cigarette product on lipid peroxidation of cells Download PDFInfo
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- CN106520903A CN106520903A CN201611056537.5A CN201611056537A CN106520903A CN 106520903 A CN106520903 A CN 106520903A CN 201611056537 A CN201611056537 A CN 201611056537A CN 106520903 A CN106520903 A CN 106520903A
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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
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—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
- G01N33/502—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 non-proliferative effects
- G01N33/5038—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 non-proliferative effects involving detection of metabolites per se
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Abstract
The invention discloses a method for detecting the influences of a buccal cigarette product on lipid peroxidation of cells. The method comprises the following steps: (1) preprocessing test samples; (2) preparing a human oral keratinocyte single cell suspension; (3) calculating the cell concentration of the human oral keratinocyte single cell suspension; (4) inoculating the human oral keratinocyte single cell suspension with cells; (5) grouping the test samples; (6) setting the test dosage of the test samples; (7) preparing a test sample culture product; (8) incubating the test samples; (9) collecting test sample incubated products; (10) cracking and centrifuging the test sample incubated products; (11) diluting a standard product; (12) detecting the test samples; and (13) calculating the malondialdehyde content. The effective treatment of the samples, the optimal setting of the detection dosage and the correct selection of the target cells make the method accurately and effectively detect the influences of the buccal cigarette product on the lipid peroxidation of the cells.
Description
Technical field
The invention belongs to tobacco product biological effect assessment technique field, more particularly to a kind of detection mouth is containing tobacco product pair
The method of cytolipin Peroxidation Effects.
Background technology
With concern more and more higher of the people to health, tobacco product is put forward higher requirement, will not only have preferably
Mouthfeel, and the bad impression of human body to be reduced as far as possible.Mouth containing tobacco product avoids traditional cigarette product burns and is produced
The harm brought by raw complex mixture, has become the new direction that foreign tobacco company is turned to from traditional cigarette product at this stage
One of.According to the component and Morphological Features of product, suck tobacco product and be divided into the tradition mouth containing tobacco product of the Snus types containing raw tobacco material
With the Novel buccal cigarette product without raw tobacco material (such as tobacco lozenge).Buccal cigarette research staff will at the product formula design initial stage
Different components are allocated according to different proportion combination, form the primary election formula with different-style, in conjunction with Chemical Evaluation and sense organ
Evaluating carries out constantly screening to adjust to formula forming final product formula.But primary election formula is large number of, is all felt
Official evaluates and takes time and effort, and sensory evaluation lays particular emphasis on the style mouthfeel to product and screens, and how to be referred to using biology test
Mark is filled a prescription into screening to product primary election, to obtain the product formula for reducing the bad impression of human body, still belongs to the exploratory stage at present, without system
One 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, and MDA (MAD) is that a kind of important effect of lipid peroxidation is biological
Mark, is measured to which, can be used for the further screening of product primary election formula, to obtain the product for reducing the bad impression of human body
Product are filled a prescription.
The content of the invention
It is an object of the invention to provide a kind of detection mouth method to cytolipin Peroxidation Effects containing tobacco product, is electronics
The safety evaluation of tobacco product provides reference.
A kind of detection mouth method to cytolipin Peroxidation Effects containing tobacco product, comprises the following steps：
(1) pre-treatment of tested material；
(2) preparation of human mouth horn cell single cell suspension；
(3) calculating of the cell concentration of human mouth horn cell single cell suspension；
(4) the cell inoculation of human mouth horn cell 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) the cracking centrifugation of tested material incubation 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：1.0g mouth containing tobacco products are accurately weighed, 30mL serum free mediums is added, in 37 DEG C
Under, 24h is stood, extraction first carries out initial filter except Slag treatment, then is removed with 0.45 μm of organic membrane filtration with qualitative filter paper after extraction
Bacterium, obtains prepare liquid；
(2) preparation of human mouth horn cell single cell suspension：After the recovery of human mouth horn cell, cell training is inoculated into
In foster bottle, 37 DEG C, 5vt%CO are placed in2Cultivate in incubator, inverted microscope observes converging and form situation for cultured cells,
Whne cell length to 90% when converging rate, the culture medium in blake bottle is removed, washed twice with phosphate buffer, added 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 OKM cell culture mediums, forms single cell suspension；
(3) calculating of the cell concentration of human mouth horn cell single cell suspension：With blood counting chamber counting method to step
Suddenly (2) obtain the cell concentration of human mouth horn cell single cell suspension and are calculated, and calculate every milliliter of human mouth cutin
The viable count of cell single cell suspension；
(4) the cell inoculation of human mouth horn cell single cell suspension：Human mouth cutin after step (3) is counted is thin
Born of the same parents' single cell suspension adds OKM cell culture mediums to 2.0 × 105Individual/mL, then be 4mL/ wares by inoculum concentration, it is inoculated into
In a diameter of 60mm Tissue Culture Dish, Tissue Culture Plate is placed in into 37 DEG C, 5vt%CO2Culture 24h in incubator；
(5) packet of tested material：Tested material is divided into into two groups：Cell controls group and detection sample group；The composition of each group is：
Cell controls group is that human mouth horn cell is planted on the kerationcyte culture medium of OKM oral cavities；Detection sample group is to give birth in OKM cells
Human mouth horn cell is planted on long culture medium and adds tested material；
(6) tested material detects the setting of dosage：Tested material, i.e. buccal cigarette sample extraction liquid are divided into into 5 non-non- zero-doses,
Respectively：12.5 product extracts, 25% sample extraction liquid, 50% sample extraction liquid, 75% sample extraction liquid, 100% sample extraction
Take liquid；
(7) preparation of tested material culture product：The OKM cell culture mediums in 60mm Tissue Culture Plates in step (4) are removed, then
It is grouped by step (5), the composition of each group is：Cell controls group is OKM cell culture mediums+human mouth horn cell；Detection
Sample sets are OKM cell culture mediums+human mouth horn cell+tested material, and the ultimate density of detection sample group is respectively walked
Suddenly the dosage that (6) set；And 2 tissue cultures are supported product and will complement to 4mL per ware liquid volume with OKM cell culture mediums；
(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：OKM cell culture mediums are removed, is washed one time with phosphate buffer, added 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 OKM cell culture fluids, 1000rpm low-temperature centrifugation 10min；
(10) the cracking centrifugation of tested material incubation 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 1600g low-temperature centrifugation 10min take supernatant and treat subsequent measurements；
(11) standard items dilution：Appropriate MDA distilled water diluting is taken to 1,2,5,10,20,50 μm of ol/L, for making
Make calibration curve；
(12) tested material sample determination：The step of 0.1mL sample prepare liquids, variable concentrations gradient is added in different centrifuge tubes
Suddenly the standard dilutions prepared by (11), are subsequently separately added into 0.2mL MDAs detection working solution, 100 DEG C of boiling water after mixing
Bath 15min, 1000rpm room temperatures centrifugation 10min, take in 200 μ L of supernatant, 96 orifice plates of addition, ELIASA 532nm mensuration absorbances；
(13) mda content is calculated：Calibration 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.
In the preferred embodiment of the invention, the OKM cells described in step (5) are
Oral Keratinocyte Medium cells.
Beneficial effect of the present invention：
Correct selection of the present invention to the effective process, the Optimal Setting of detection dosage and target cell of sample so that this
Method accurately and effectively detection mouth can contain tobacco product 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.
Impact of the present embodiment for the commercially available mouth containing tobacco product of 2 kinds of foreign countries to human mouth horn cell lipid peroxidation is surveyed
Examination.
The present embodiment includes following preparation process：
(1) pre-treatment of tested material：1.0g mouth containing tobacco products are accurately weighed, 30mL serum free mediums is added, in 37 DEG C
Under, 24h is stood, extraction first carries out initial filter except Slag treatment, then is removed with 0.45 μm of organic membrane filtration with qualitative filter paper after extraction
Bacterium, obtains prepare liquid；
(2) preparation of human mouth horn cell single cell suspension：After the recovery of human mouth horn cell, cell training is inoculated into
In foster bottle, 37 DEG C, 5vt%CO are placed in2Cultivate in incubator, inverted microscope observes converging and form situation for cultured cells,
Whne cell length to 90% when converging rate, the culture medium in blake bottle is removed, washed twice with phosphate buffer, added 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 OKM cell culture mediums, forms single cell suspension；
(3) calculating of the cell concentration of human mouth horn cell single cell suspension：With blood counting chamber counting method to step
Suddenly (2) obtain the cell concentration of human mouth horn cell single cell suspension and are calculated, and calculate every milliliter of human mouth cutin
The viable count of cell single cell suspension；
(4) the cell inoculation of human mouth horn cell single cell suspension：Human mouth cutin after step (3) is counted is thin
Born of the same parents' single cell suspension adds OKM cell culture mediums to 2.0 × 105Individual/mL, then be 4mL/ wares by inoculum concentration, it is inoculated into
In a diameter of 60mm Tissue Culture Dish, Tissue Culture Plate is placed in into 37 DEG C, 5vt%CO2Culture 24h in incubator；
(5) packet of tested material：Tested material is divided into into two groups：Cell controls group and detection sample group；The composition of each group is：
Cell controls group is that human mouth horn cell is planted on the kerationcyte culture medium of OKM oral cavities；Detection sample group is to give birth in OKM cells
Human mouth horn cell is planted on long culture medium and adds tested material；
(6) tested material detects the setting of dosage：Tested material, i.e. buccal cigarette sample extraction liquid are divided into into 5 non-non- zero-doses,
Respectively：12.5 product extracts, 25% sample extraction liquid, 50% sample extraction liquid, 75% sample extraction liquid, 100% sample extraction
Take liquid；
(7) preparation of tested material culture product：The OKM cell culture mediums in 60mm Tissue Culture Plates in step (4) are removed, then
It is grouped by step (5), the composition of each group is：Cell controls group is OKM cell culture mediums+human mouth horn cell；Detection
Sample sets are OKM cell culture mediums+human mouth horn cell+tested material, and the ultimate density of detection sample group is respectively walked
Suddenly the dosage that (6) set；And 2 tissue cultures are supported product and will complement to 4mL per ware liquid volume with OKM cell culture mediums；
(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：OKM cell culture mediums are removed, is washed one time with phosphate buffer, added 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 OKM cell culture fluids, 1000rpm low-temperature centrifugation 10min；
(10) the cracking centrifugation of tested material incubation 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 1600g low-temperature centrifugation 10min take supernatant and treat subsequent measurements；
(11) standard items dilution：Appropriate MDA distilled water diluting is taken to 1,2,5,10,20,50 μm of ol/L, for making
Make calibration curve；
(12) tested material sample determination：The step of 0.1mL sample prepare liquids, variable concentrations gradient is added in different centrifuge tubes
Suddenly the standard dilutions prepared by (11), are subsequently separately added into 0.2mL MDAs detection working solution, 100 DEG C of boiling water after mixing
Bath 15min, 1000rpm room temperatures centrifugation 10min, take in 200 μ L of supernatant, 96 orifice plates of addition, ELIASA 532nm mensuration absorbances；
(13) MDA MDA cubages：Calibration curve is drawn according to standard liquid absorbance, MDA is calculated and is contained
Amount.
Impact of the 12 kinds of buccal cigarette extracts of table to MDA changes of contents in human lung cancer cell A549 HOK
By result of the test as can be seen that under the conditions of sample treatment that the inventive method is given, detection dosage, sample
1#, 2# are present on the MDA contents of HOK cells in the range of detection dosage to be affected, and has dose-effect relationship, with 0 dosage group phase
Than there is significant difference (p<0.05)；There is significant difference (p compared with 2# sample rooms in 1# samples<0.05)；Result above shows,
Impact of the different buccal cigarettes to the MDA contents of HOK cells has differences.
Claims (2)
1. a kind of detection mouth method to cytolipin Peroxidation Effects containing tobacco product, it is characterised in that comprise the following steps：
(1) pre-treatment of tested material；
(2) preparation of human mouth horn cell single cell suspension；
(3) calculating of the cell concentration of human mouth horn cell single cell suspension；
(4) the cell inoculation of human mouth horn cell 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) the cracking centrifugation of tested material incubation 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：1.0g mouth containing tobacco products are accurately weighed, 30mL serum free mediums are added, it is at 37 DEG C, quiet
24h is put, is extracted, first carried out initial filter after extraction with qualitative filter paper and remove Slag treatment, then it is degerming with 0.45 μm of organic membrane filtration, obtain
Prepare liquid；
(2) preparation of human mouth horn cell single cell suspension：After the recovery of human mouth horn cell, Tissue Culture Flask is inoculated into
In, it is placed in 37 DEG C, 5vt%CO2Cultivate in incubator, converging and form situation for inverted microscope observation cultured cells treats thin
Born of the same parents' length removes the culture medium in blake bottle to 90% when converging rate, 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 OKM cell culture mediums, form single cell suspension；
(3) calculating of the cell concentration of human mouth horn cell single cell suspension：With blood counting chamber counting method to step (2)
The cell concentration for obtaining human mouth horn cell single cell suspension is calculated, and calculates every milliliter of human mouth horn cell list
The viable count of cell suspending liquid；
(4) the cell inoculation of human mouth horn cell single cell suspension：Human mouth horn cell list after step (3) is counted
Cell suspending liquid adds OKM cell culture mediums to 2.0 × 105Individual/mL, then be 4mL/ wares by inoculum concentration, it is inoculated into diameter
In for 60mm Tissue Culture Dish, Tissue Culture Plate is placed in into 37 DEG C, 5vt%CO2Culture 24h in incubator；
(5) packet of tested material：Tested material is divided into into two groups：Cell controls group and detection sample group；The composition of each group is：Cell
Control group is that human mouth horn cell is planted on the kerationcyte culture medium of OKM oral cavities；Detection sample group is to train in OKM cell growths
Human mouth horn cell is planted on foster base and adds tested material；
(6) tested material detects the setting of dosage：Tested material, i.e. buccal cigarette sample extraction liquid are divided into into 5 non-non- zero-doses, respectively
For：12.5 product extracts, 25% sample extraction liquid, 50% sample extraction liquid, 75% sample extraction liquid, 100% sample extraction liquid；
(7) preparation of tested material culture product：The OKM cell culture mediums in 60mm Tissue Culture Plates in step (4) are removed, then by step
Suddenly (5) are grouped, and the composition of each group is：Cell controls group is OKM cell culture mediums+human mouth horn cell；Detection sample
Group is OKM cell culture mediums+human mouth horn cell+tested material, and makes the ultimate density of detection sample group be respectively step (6)
The dosage of setting；And 2 tissue cultures are supported product and will complement to 4mL per ware liquid volume with OKM cell culture mediums；
(8) incubation of tested material：60mm Tissue Culture Dish after step (7) is loaded is placed in 37 DEG C, 5vt%CO2Incubate in incubator
Educate 24h；
(9) tested material is incubated the collection of product：OKM cell culture mediums are removed, is washed one time with phosphate buffer, added 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 OKM cell culture fluids, 1000rpm low-temperature centrifugation 10min；
(10) the cracking centrifugation of tested material incubation 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 is taken to 1,2,5,10,20,50 μm of ol/L, for making mark
Directrix curve；
(12) tested material sample determination：The step of 0.1mL sample prepare liquids, variable concentrations gradient are added in different centrifuge tubes
(11) standard dilutions prepared by, are subsequently separately added into 0.2mL MDAs detection working solution, 100 DEG C of boiling water baths after mixing
15min, 1000rpm room temperature is centrifuged 10min, takes in 200 μ L of supernatant, 96 orifice plates of addition, ELIASA 532nm mensuration absorbances；
(13) mda content is calculated：Calibration curve is drawn according to standard liquid absorbance, mda content is calculated.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201611056537.5A CN106520903A (en) | 2016-11-25 | 2016-11-25 | Method for detecting influences of buccal cigarette product on lipid peroxidation of cells |
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| CN201611056537.5A CN106520903A (en) | 2016-11-25 | 2016-11-25 | Method for detecting influences of buccal cigarette product on lipid peroxidation of cells |
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Cited By (3)
| 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 |
| CN108120801A (en) * | 2017-12-14 | 2018-06-05 | 云南中烟工业有限责任公司 | It is a kind of that the method influenced is aoxidized on cytolipin for detecting gum base type chewing tobacco |
| 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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| CN102043020A (en) * | 2009-10-26 | 2011-05-04 | 贵阳医学院 | Method for sieving active ingredients playing protection role in cranial nerve from erigeron breviscapus |
| CN104789633A (en) * | 2015-04-22 | 2015-07-22 | 云南中烟工业有限责任公司 | Test method for detecting cytotoxicity of buccal tobacco products |
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2016
- 2016-11-25 CN CN201611056537.5A patent/CN106520903A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102043020A (en) * | 2009-10-26 | 2011-05-04 | 贵阳医学院 | Method for sieving active ingredients playing protection role in cranial nerve from erigeron breviscapus |
| CN104789633A (en) * | 2015-04-22 | 2015-07-22 | 云南中烟工业有限责任公司 | Test method for detecting cytotoxicity of buccal tobacco products |
Cited By (3)
| 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 |
| CN108120801A (en) * | 2017-12-14 | 2018-06-05 | 云南中烟工业有限责任公司 | It is a kind of that the method influenced is aoxidized on cytolipin for detecting gum base type chewing tobacco |
| 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: 20170322 |
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| RJ01 | Rejection of invention patent application after publication |