CN106556691B - A method of detection electronics tobacco product influences cellular superoxide hydrogen enzyme enzyme activity - Google Patents
A method of detection electronics tobacco product influences cellular superoxide hydrogen enzyme enzyme activity Download PDFInfo
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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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
Abstract
The present invention discloses a kind of method for influencing on cellular superoxide hydrogen enzyme enzyme activity of detection electronics tobacco product, includes the following steps: that tested material pre-treatment, the preparation of human lung cancer cell A549 single cell suspension, calculatings of human lung cancer cell A549 cell concentration, the grouping of human lung cancer cell A549 cell inoculation, tested material, tested material are incubated for, tested material is incubated for that product are collected, tested material is incubated for cracking centrifugation, the measurement of standard curve, the measurement of tested material incubation product, determination of protein concentration and the calculating of catalase enzyme activity of product.The present invention enables this method accurately and effectively to detect the method that electronics tobacco product influences cellular superoxide hydrogen enzyme enzyme activity the optimal setting for being effectively treated, detecting dosage of sample and the correct selection of target cell.
Description
Technical field
The invention belongs to tobacco product biological effect assessment technique field, in particular to a kind of detection electronics tobacco product pair
The method that cellular superoxide hydrogen enzyme enzyme activity influences.
Background technique
As concern of the people to health is higher and higher, to tobacco product, more stringent requirements are proposed, not only to have preferably
Mouthfeel, and to reduce the bad impression of human body as far as possible.Electronics tobacco product avoids traditional cigarette product burns and is produced
Harm, has become the new direction that foreign tobacco company turns to from traditional cigarette product at this stage brought by raw complex mixture
One of.Make to be made of nicotine, fragrance matter etc. by electric heating or electronic atomized mode in its use process of electronics tobacco product
Liquid be converted into the mixture similar to cigarette smoke, then by mixing " smog " transmission such as glycerine/nicotines after atomization
To consumer, makes one the nicotine that inhalation dose does not wait and obtain the physiology impression for being similar to suction traditional cigarette.Electronic cigarette is ground
Hair personnel combine different components according to different proportion at tobacco tar product formula design initial stage and deploy, and being formed has different-style
Primary election formula carries out constantly screening adjustment to formula in conjunction with Chemical Evaluation and sensory evaluation and forms final product formula.So
And primary election formula is large number of, all carrying out sensory evaluation takes time and effort, and sensory evaluation lays particular emphasis on the style mouthfeel to product
It is screened, how product primary election to be formulated into screening using biology test index, reduce the bad impression of human body to obtain
Product formula still belongs to the exploratory stage, without unified standard method at present.
Body is when by various unfavorable stimulations, internal high activity molecule such as active oxygen radical (reactive oxygen
Species, ROS) it generates excessively, degree of oxidation exceeds the removing of oxide, and oxidative system and antioxidant system are unbalance, to lead
Cause tissue damage.Hydrogen peroxide (H2O2) it is important one kind in active oxygen radical, catalase (CAT) can be catalyzed
Hydrogen oxide resolves into oxygen and water, is measured to it, can be used for the further screening of product primary election formula, to obtain reduction human body
The product formula of bad impression.
Summary of the invention
The object of the present invention is to provide a kind of method for being influenced on cellular superoxide hydrogen enzyme enzyme activity of detection electronics tobacco product,
Safety evaluation for electronics tobacco product provides reference.
The present invention discloses a kind of method that detection electronics tobacco product influences cellular superoxide hydrogen enzyme enzyme activity, including following
Step:
A method of detection electronics tobacco product influences cellular superoxide hydrogen enzyme enzyme activity, comprising 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) cell inoculation of human lung cancer cell A549 single cell suspension；
(5) grouping of tested material；
(6) setting of tested material detection dosage；
(7) preparation of tested material culture product；
(8) incubation of tested material；
(9) tested material is incubated for the collection of product；
(10) tested material is incubated for the cracking centrifugation of product；
(11) measurement of standard curve；
(12) tested material is incubated for the measurement of product；
(13) determination of protein concentration；
(14) catalase enzyme activity calculates.
In the preferred embodiment of the invention, above-mentioned method, comprising the following specific steps
(1) pre-treatment of tested material: drawing a certain amount of electronic cigarette stoste, trains according to electronic cigarette stoste and serum-free cell
After the ratio that the volume ratio for supporting base is 10~90% carries out mixed diluting, places -48h, sterilizing filter filtration sterilization for 24 hours and obtain
To prepare liquid, -80 DEG C are saved for use；
(2) it the preparation of human lung cancer cell A549 HPF single cell suspension: after human lung cancer cell A549 recovery, is inoculated into thin
In born of the same parents' culture bottle, it is placed in 37 DEG C, 5vt%CO2It is cultivated in incubator, inverted microscope observation culture cell converges and form feelings
Condition when converging rate, removes the culture medium in culture bottle to 90% when cell length, is washed twice with phosphate buffer, is added appropriate
0.25% trypsin solution, wherein trypsase/phosphate buffer concentration unit is quality: volume；Single layer is incubated for
About 1-2min is hanged at fiber fibroblast culture medium, forms single cell suspension；
(3) calculating of the cell concentration of human lung cancer cell A549 HPF single cell suspension: blood counting chamber counting method pair is used
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) cell inoculation of human lung cancer cell A549 HPF single cell suspension: people's lung after step (3) are counted is at fibre
Dimension cell single cell suspension is added fibroblast culture medium and is diluted to 2.0 × 105A/mL, being inoculated into diameter is that 60mm is thin
In born of the same parents' culture dish, tissue culture plate is placed in 37 DEG C, 5vt%CO by every ware 4mL2It is cultivated for 24 hours in incubator；
(5) grouping of tested material: tested material is divided into two groups: cell controls group and test sample group；The composition of each group are as follows:
Cell controls group is to plant human lung cancer cell A549 on Fibroblast Growth Medium；Test sample group is in fibroblast
Human lung cancer cell A549 is planted on growth medium and adds tested material；
(6) setting of tested material detection dosage: by tested material, i.e. electronic cigarette stoste is divided into 5 non-zero-doses: 25mg/mL,
50mg/mL,100mg/mL,150mg/mL,200mg/mL；
(7) preparation of tested material culture product: diameter is the fibroblast in 60mm Tissue Culture Dish in removing step (4)
Culture medium, then be grouped by step (5), the composition of each group are as follows: cell controls group is fibroblast culture medium+people's lung into fibre
Tie up cell；Test sample group is fibroblast culture medium+human lung cancer cell A549+tested material, and makes test sample group most
Final concentration is respectively the dosage of step (6) setting；Every boreliquid volume is complemented into 4mL with fibroblast culture medium；
(8) incubation of tested material: being that 60mm Tissue Culture Dish is placed in 37 DEG C, 5vt% by the diameter after step (7) sample-adding
CO2It is incubated for for 24 hours in incubator；
(9) tested material is incubated for the collection of product: the culture medium in removal step (8) is washed one time with phosphate buffer PBS,
Appropriate 0.25% trypsin solution is added, wherein trypsase/phosphate buffer concentration unit is quality: volume；
Single layer is incubated for about 1-2min, is hanged with Fibroblast culture solution, 1000rpm low-temperature centrifugation 10min；
(10) tested material is incubated for the cracking centrifugation of product: 100 μ L cell pyrolysis liquids are added in the cell that collection step (9) obtains,
Ice bath cracking, collects cell, 1600rpm low-temperature centrifugation 10min takes supernatant to wait for subsequent measurements after cracking；
(11) measurement of standard curve: the prepared 5mmol/L hydrogenperoxide steam generator of 0,12.5,25,50,75 μ L is taken extremely
In 1.5mL centrifuge tube, it is separately added into catalase detection buffer and is diluted to last volume as 100 μ L mixing, 4 μ L is respectively taken to add
Enter 96 orifice plates, the 200 prepared developing solutions of μ L are added, measure absorbance A 520 at 520nm after 25 DEG C of incubation at least 15min；
(12) tested material is incubated for the measurement of product: 40 μ L sample prepare liquids being added in different centrifuge tubes, blank control group adds
Enter 40 μ L catalases detection buffer；It is then separately added into the 250mmol/L hydrogenperoxide steam generator of 10 μ L, is mixed rapidly,
25 DEG C of reaction 5min；The oscillation of 450 μ L catalase reaction terminate liquids is added and mixes to terminate and reacts；It is added in plastic centrifuge tube
40 μ L catalases detect buffer, add the above-mentioned reaction system that 10 μ L have been terminated and mixed, and mix, and therefrom take final
96 orifice plates are added in 10 μ L of reaction system liquid, add the prepared colour developing working solution of 200 μ L, enzyme mark after 25 DEG C of incubation 15min
Instrument measures absorbance at 520nm；
(13) determination of protein concentration: measurement sample protein concentration；
(14) catalase enzyme activity calculates:
A. standard curve is drawn according to the standard solution absorbance value of step (11) measurement:
A520=k [hydrogen peroxide micromole number]+b, the value of k and b are calculated by standard curve；
B. hydrogen peroxide molal quantity remaining in sample is calculated:
Residual hydrogen peroxide micromole number=(A520-b)/k
C. catalase enzyme activity calculates:
[sample catalase enzyme activity]=([blank control residual hydrogen peroxide micromole number]-[sample remnants mistake
Hydrogen oxide micromole number]) × [extension rate]/(μ of 5min × 40 L × [protein concentration]).
In the preferred embodiment of the invention, the pre-treating method of tested material can also catch in step (1) for cambridge filter
Collection method:
Using linear type smoking machine, 100 mouthfuls of electronic cigarettes of continuous sucking are trapped, respectively with the cambridge filter that diameter is 44mm
Before suction, the quality of Fume collector is weighed after suction, and the quality of electronic cigarette total granules is calculated according to following formula (1)
The English name of mTPM, electronic cigarette total granules are Total particulate matter, abbreviation TPM:
MTPM=m1-m0 (1)
In formula:
m0: the quality of Fume collector before aspirating, unit are milligram (mg)；
m1: the quality of Fume collector after suction, unit are milligram (mg)；
After trapping, the cambridge filter after trapping is put into triangular flask, dimethyl sulfoxide is added, makes the total grain of electronic cigarette
Concentration of the phase object in dimethyl sulfoxide is 40mg/mL；This triangular flask is placed in ultrasound 30min in Ultrasound Instrument；Again with sterile filter
Dimethyl sulfoxide extract liquor is collected by filtration in paper, dispenses into 1mL cryopreservation tube, and -80 DEG C save for use.
In the preferred embodiment of the invention, the pre-treating method of tested material can also be full fume exposure in step (1)
Bottle method of trapping:
Using linear type smoking machine, 100 mouthfuls of electronic cigarettes of continuous sucking, using the full flue gas that serum-free cell culture medium is housed
Exposure bottle trapping, trapping concentration is 20 mouthfuls/mL, with 0.22 μm of sterilizing filter filtration sterilization after trapping, obtains prepare liquid, -80
It DEG C saves stand-by.
The invention has the advantages that:
The present invention is to the optimal setting for being effectively treated, detecting dosage of sample and the correct selection of target cell, so that originally
Method can accurately and effectively detect the method that electronics tobacco product influences cellular superoxide hydrogen enzyme enzyme activity.
Specific embodiment
Below in conjunction with specific example, the present invention will be described in detail, but is not intended to limit the present invention.
The present embodiment influences human lung cancer cell A549 catalase enzyme activity for 4 kinds of commercially available tobacco juice for electronic smoke of foreign countries
Method.
(1) pre-treatment of tested material: drawing a certain amount of electronic cigarette stoste, trains according to electronic cigarette stoste and serum-free cell
After the ratio that the volume ratio for supporting base is 1:5 carries out mixed diluting, places for 24 hours, with 0.22 μm of sterilizing filter filtration sterilization, obtain
Prepare liquid, -80 DEG C save for use；
(2) it the preparation of human lung cancer cell A549 HPF single cell suspension: after human lung cancer cell A549 recovery, is inoculated into thin
In born of the same parents' culture bottle, it is placed in 37 DEG C, 5vt%CO2It is cultivated in incubator, inverted microscope observation culture cell converges and form feelings
Condition when converging rate, removes the culture medium in culture bottle to 90% when cell length, is washed twice with phosphate buffer, is added appropriate
0.25% trypsin solution, wherein trypsase/phosphate buffer concentration unit is quality: volume；Single layer is incubated for
About 1min is hanged at fiber fibroblast culture medium, forms single cell suspension；
(3) calculating of the cell concentration of human lung cancer cell A549 HPF single cell suspension: blood counting chamber counting method pair is used
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) cell inoculation of human lung cancer cell A549 HPF single cell suspension: people's lung after step (3) are counted is at fibre
Dimension cell single cell suspension is added fibroblast culture medium and is diluted to 2.0 × 105A/mL, being inoculated into diameter is that 60mm is thin
In born of the same parents' culture dish, tissue culture plate is placed in 37 DEG C, 5vt%CO by every ware 4mL2It is cultivated for 24 hours in incubator；
(5) grouping of tested material: tested material is divided into two groups: cell controls group and test sample group；The composition of each group are as follows:
Cell controls group is to plant human lung cancer cell A549 on Fibroblast Growth Medium；Test sample group is in fibroblast
Human lung cancer cell A549 is planted on growth medium and adds tested material；
(6) setting of tested material detection dosage: by tested material, i.e. electronic cigarette stoste is divided into 5 non-zero-doses: 25mg/mL,
50mg/mL,100mg/mL,150mg/mL,200mg/mL；
(7) preparation of tested material culture product: diameter is the fibroblast in 60mm Tissue Culture Dish in removing step (4)
Culture medium, then be grouped by step (5), the composition of each group are as follows: cell controls group is fibroblast culture medium+people's lung into fibre
Tie up cell；Test sample group is fibroblast culture medium+human lung cancer cell A549+tested material, and makes test sample group most
Final concentration is respectively the dosage of step (6) setting；Every boreliquid volume is complemented into 4mL with fibroblast culture medium；
(8) incubation of tested material: being that 60mm Tissue Culture Dish is placed in 37 DEG C, 5vt% by the diameter after step (7) sample-adding
CO2It is incubated for for 24 hours in incubator；
(9) tested material is incubated for the collection of product: the culture medium in removal step (8) is washed one time with phosphate buffer PBS,
Appropriate 0.25% trypsin solution is added, wherein trypsase/phosphate buffer concentration unit is quality: volume；
Single layer is incubated for about 2min, is hanged with Fibroblast culture solution, 1000rpm low-temperature centrifugation 10min；
(10) tested material is incubated for the cracking centrifugation of product: 100 μ L cell pyrolysis liquids are added in the cell that collection step (9) obtains,
Ice bath cracking, collects cell, 1600rpm low-temperature centrifugation 10min takes supernatant to wait for subsequent measurements after cracking；
(11) measurement of standard curve: the prepared 5mmol/L hydrogenperoxide steam generator of 0,12.5,25,50,75 μ L is taken extremely
In 1.5mL centrifuge tube, it is separately added into catalase detection buffer and is diluted to last volume as 100 μ L mixing, 4 μ L is respectively taken to add
Enter 96 orifice plates, the 200 prepared developing solutions of μ L are added, measure absorbance at 520nm after 25 DEG C of incubation at least 15min；
(12) tested material is incubated for the measurement of product: 40 μ L sample prepare liquids being added in different centrifuge tubes, blank control group adds
Enter 40 μ L catalases detection buffer；It is then separately added into the 250mmol/L hydrogenperoxide steam generator of 10 μ L, is mixed rapidly,
25 DEG C of reaction 5min；The oscillation of 450 μ L catalase reaction terminate liquids is added and mixes to terminate and reacts；It is added in plastic centrifuge tube
40 μ L catalases detect buffer, add the above-mentioned reaction system that 10 μ L have been terminated and mixed, and mix, and therefrom take final
96 orifice plates are added in 10 μ L of reaction system liquid, add the prepared colour developing working solution of 200 μ L, enzyme mark after 25 DEG C of incubation 15min
Instrument measures absorbance at 520nm；
(13) determination of protein concentration: measurement sample protein concentration；
(14) catalase enzyme activity calculates:
A. standard curve is drawn according to the standard solution absorbance value of step (11) measurement:
A520=k [hydrogen peroxide micromole number]+b, the value of k and b are calculated by standard curve；
B. hydrogen peroxide molal quantity remaining in sample is calculated:
Residual hydrogen peroxide micromole number=(A520-b)/k
C. catalase enzyme activity calculates:
[sample catalase enzyme activity]=([blank control residual hydrogen peroxide micromole number]-[sample remnants mistake
Hydrogen oxide micromole number]) × [extension rate]/(μ of 5min × 40 L × [protein concentration]).
Influence of the 14 kinds of tobacco juice for electronic smoke of table to HPF cellular superoxide hydrogen enzyme enzymatic activity
It can be seen from test result under the conditions of the sample treatment that the method for the present invention provides, detecting dosage, sample
There is influence to the catalase enzymatic activity of HPF cell in detection dosage range in 1#, 2#, 3#, 4#, and have dosage effect pass
System, there are significant difference (p < 0.05) compared with 0 dosage group；Sample 4# is in detection dosage range compared with sample 1#, 2#, 3#
There are significant difference (p < 0.05)；The above results show that catalase enzymatic activity of the different tobacco juice for electronic smoke to HPF cell
Influence have differences.
Claims (3)
1. a kind of method that detection electronics tobacco product influences cellular superoxide hydrogen enzyme enzyme activity, which is characterized in that including following
Specific steps:
(1) pre-treatment of tested material: a certain amount of electronic cigarette stoste is drawn, according to electronic cigarette stoste and serum-free cell culture medium
Volume ratio be 10~90% ratio carry out mixed diluting after, place -48h, sterilizing filter filtration sterilization for 24 hours, obtain to
Liquid is surveyed, -80 DEG C save for use；
(2) preparation of human lung cancer cell A549 single cell suspension: after human lung cancer cell A549 recovery, it is inoculated into Tissue Culture Flask
In, it is placed in 37 DEG C, 5vt%CO2It is cultivated in incubator, converge and the form situation of inverted microscope observation culture cell, to thin
Born of the same parents are long when converging rate, to remove the culture medium in culture bottle to 90%, is washed twice with phosphate buffer, is added appropriate 0.25%
Trypsin solution, wherein trypsase/phosphate buffer concentration unit is quality: volume；Single layer is incubated for 1-2min, uses
It is hanged at 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 (2)
The cell concentration for obtaining human lung cancer cell A549 single cell suspension is calculated, and every milliliter of human lung cancer cell A549 list is calculated
The viable count of cell suspending liquid；
(4) cell inoculation of human lung cancer cell A549 single cell suspension: the human lung cancer cell A549 list after step (3) are counted
Cell suspending liquid is added fibroblast culture medium and is diluted to 2.0 × 105A/mL, being inoculated into diameter is 60mm Tissue Culture Dish
In, tissue culture plate is placed in 37 DEG C, 5vt%CO by every ware 4mL2It is cultivated for 24 hours in incubator；
(5) grouping of tested material: tested material is divided into two groups: cell controls group and test sample group；The composition of each group are as follows: cell
Control group is to plant human lung cancer cell A549 on Fibroblast Growth Medium；Test sample group is in fibroblastic growth
Human lung cancer cell A549 is planted on culture medium and adds tested material；
(6) setting of tested material detection dosage: by tested material, i.e. electronic cigarette stoste is divided into 5 non-zero-doses: 25mg/mL,
50mg/mL,100mg/mL,150mg/mL,200mg/mL；
(7) preparation of tested material culture product: diameter is the Fibroblast cell-culture in 60mm Tissue Culture Dish in removing step (4)
Base, then be grouped by step (5), the composition of each group are as follows: cell controls group is fibroblast culture medium+people's lung into fiber finer
Born of the same parents；Test sample group is fibroblast culture medium+human lung cancer cell A549+tested material, and makes the final dense of test sample group
Degree is respectively the dosage of step (6) setting；Every boreliquid volume is complemented into 4mL with fibroblast culture medium；
(8) incubation of tested material: being that 60mm Tissue Culture Dish is placed in 37 DEG C, 5vt%CO by the diameter after step (7) sample-adding2Culture
It is incubated for for 24 hours in case；
(9) tested material is incubated for the collection of product: the culture medium in removal step (8) is washed one time with phosphate buffer PBS, is added
Appropriate 0.25% trypsin solution, wherein trypsase/phosphate buffer concentration unit is quality: volume；Single layer
It is incubated for 1-2min, is hanged with Fibroblast culture solution, 1000rpm low-temperature centrifugation 10min；
(10) tested material is incubated for the cracking centrifugation of product: 100 μ L cell pyrolysis liquids, ice bath is added in the cell that collection step (9) obtains
Cracking, collects cell, 1600rpm low-temperature centrifugation 10min takes supernatant to wait for subsequent measurements after cracking；
(11) measurement of standard curve: take the prepared 5mmol/L hydrogenperoxide steam generator of 0,12.5,25,50,75 μ L to 1.5mL
In centrifuge tube, it is separately added into catalase detection buffer and is diluted to last volume as 100 μ L mixing, 4 μ L is respectively taken to be added 96
Orifice plate is added the 200 prepared developing solutions of μ L, measures absorbance at 520nm after 25 DEG C of incubation at least 15min；
(12) tested material is incubated for the measurement of product: 40 μ L sample prepare liquids being added in different centrifuge tubes, 40 μ are added in blank control group
L catalase detects buffer；It is then separately added into the 250mmol/L hydrogenperoxide steam generator of 10 μ L, is mixed rapidly, 25 DEG C anti-
Answer 5min；The oscillation of 450 μ L catalase reaction terminate liquids is added and mixes to terminate and reacts；40 μ L mistakes are added in plastic centrifuge tube
Hydrogen oxide enzyme detects buffer, adds the above-mentioned reaction system that 10 μ L have been terminated and mixed, and mixes, therefrom takes end reaction body
It is that 96 orifice plates are added in 10 μ L of liquid, adds the prepared colour developing working solution of 200 μ L, microplate reader measures after 25 DEG C of incubation 15min
Absorbance at 520nm；
(13) determination of protein concentration: measurement sample protein concentration；
(14) catalase enzyme activity calculates:
A. standard curve is drawn according to the standard solution absorbance value of step (11) measurement:
A520=k [hydrogen peroxide micromole number]+b, the value of k and b are calculated by standard curve；
B. hydrogen peroxide molal quantity remaining in sample is calculated:
Residual hydrogen peroxide micromole number=(A520-b)/k
C. catalase enzyme activity calculates:
[sample catalase enzyme activity]=([blank control residual hydrogen peroxide micromole number]-[sample remnants peroxidating
Hydrogen micromole number]) × [extension rate]/(μ of 5min × 40 L × [protein concentration]).
2. the method according to claim 1, wherein the pre-treating method of tested material can also be in step (1)
Cambridge filter method of trapping:
Using linear type smoking machine, 100 mouthfuls of electronic cigarettes of continuous sucking are trapped with the cambridge filter that diameter is 44mm, respectively at pumping
Before suction, the quality of Fume collector is weighed after suction, and the quality mTPM of electronic cigarette total granules is calculated according to following formula (1),
The English name of electronic cigarette total granules is Total particulate matter, abbreviation TPM:
MTPM=m1-m0 (1)
In formula:
m0: the quality of Fume collector before aspirating, unit are milligram (mg)；
m1: the quality of Fume collector after suction, unit are milligram (mg)；
After trapping, the cambridge filter after trapping is put into triangular flask, dimethyl sulfoxide is added, makes electronic cigarette total granules
Concentration in dimethyl sulfoxide is 40mg/mL；This triangular flask is placed in ultrasound 30min in Ultrasound Instrument；Again with aseptic filter paper mistake
Dimethyl sulfoxide extract liquor is collected in filter, is dispensed into 1mL cryopreservation tube, and -80 DEG C save for use.
3. the method according to claim 1, wherein the pre-treating method of tested material is full flue gas in step (1)
Exposure bottle method of trapping:
Using linear type smoking machine, 100 mouthfuls of electronic cigarettes of continuous sucking, using the full fume exposure that serum-free cell culture medium is housed
Bottle trapping, trapping concentration are 20 mouthfuls/mL, with 0.22 μm of sterilizing filter filtration sterilization after trapping, obtain prepare liquid, -80 DEG C of guarantors
It deposits stand-by.
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| CN107828854A (en) * | 2017-09-21 | 2018-03-23 | 云南中烟工业有限责任公司 | The method that detection electronics smoke sol water extract influences on cellular superoxide hydrogenase activity |
| CN108204952A (en) * | 2017-12-14 | 2018-06-26 | 云南中烟工业有限责任公司 | It is a kind of based on catalase activity for detecting the method that gum base type chewing tobacco damages cellular oxidation |
| 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 |
| CN109609592A (en) * | 2018-11-28 | 2019-04-12 | 云南中烟工业有限责任公司 | A method of detection heating does not burn Cigarette grain phase to the influence of cellular superoxide hydrogenase activity |
| CN113281397B (en) * | 2021-05-19 | 2023-04-25 | 中国科学技术大学 | Method for tracking lysosomal content in monosysins |
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