CN109666716A - A method of Cigarette grain phase cytotoxicity of not burning is heated in detection - Google Patents

A method of Cigarette grain phase cytotoxicity of not burning is heated in detection Download PDF

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CN109666716A
CN109666716A CN201811469837.5A CN201811469837A CN109666716A CN 109666716 A CN109666716 A CN 109666716A CN 201811469837 A CN201811469837 A CN 201811469837A CN 109666716 A CN109666716 A CN 109666716A
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管莹
李雪梅
高茜
徐玉琼
汤建国
朱东来
张霞
韩敬美
何东沅
张伟
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China Tobacco Yunnan Industrial Co Ltd
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/5044—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 involving specific cell types

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Abstract

The present invention discloses a kind of method that Cigarette grain phase cytotoxicity of not burning is heated in detection, comprising the following steps: (1) sample pre-treatments；(2) preparation of people's nasal epithelia single cell suspension；(3) cell concentration of people's nasal epithelia single cell suspension calculates；(4) inoculation of people's nasal epithelia single cell suspension；(5) tested material is grouped；(6) setting of tested material detection dosage；(7) preparation of tested material culture product；(8) it is incubated for tested material；(9) dyestuff is incubated for；(10) light absorption value is measured；(11) cell survival rate is calculated；(12) cell survival rate curve is drawn.The present invention enables this method accurately and effectively to detect influence of the cigarette smoke condensates to early apoptosis of cells the optimal setting of sample detection dosage and the correct selection of target cell.Compensation process as the detection of cigarette smoke condensates cytotoxicity is to distinguish influence of the product to meronecrosis and apoptosis.

Description

A method of Cigarette grain phase cytotoxicity of not burning is heated in detection
Technical field
The invention belongs to the safety biological effect assessment technique field of tobacco product, in particular to a kind of detection heating The method for Cigarette grain phase cytotoxicity of not burning.
Background technique
Neutral RBC Toxicity method and thiazole blue laws (MTT) have the characteristics that it is easy, quick, sensitive, be current application compared with The method of more two kinds of toxicology detection cytotoxicities.International tobacco scientific research Cooperation Centre CORESTA is organized in 2002 The external toxicity test job group of cigarette smoke is set up, by a large amount of literature survey and research work, which recommends to adopt The potential cytotoxicity of cigarette smoke condensate is detected with dimethyl diaminophenazine chloride cell toxicity test, the detection method is at present It is widely used by domestic and international tobacco company.
The increasingly stringent and smoking of tobacco supervision legislation is goed deep into health research, results in tobacco company and seeks risk New tobacco products lower, without environment flue gas.Heat non-combustion-type cigarette, it is characterised in that heating tobacco and non-combustion-cigarette Grass provides the consumer with certain tobacco feature impression.Heating does not burn cigarette products when avoiding traditional cigarette product burns Harm, has become the new side that foreign tobacco company turns to from traditional cigarette product at this stage brought by the complex mixture of generation One of to.
The change of test object leads to the very big differences of appearance such as the pre-treating method, detection cell, detection dosage of sample, These differences can produce bigger effect the accuracy of test result, existing for detecting the neutrality of cigarette smoke cytotoxicity RBC Toxicity method is no longer satisfied the needs for heating cigarette products cytotoxicity detection of not burning.Currently, research both at home and abroad There has been no the accurately and effectively detection methods for the cytotoxicity for heating the cigarette products that do not burn for mechanism and tobacco company.
Summary of the invention
The object of the present invention is to provide a kind of methods that Cigarette grain phase cytotoxicity of not burning is heated in detection, to add Heat does not burn Cigarette grain phase as detection basis, can enhance the accuracy of testing result, and then more effectively evaluate Heat the safety for the cigarette products that do not burn.
RPMI1640 cell culture medium used in the present invention is bought in any goods companies being commercially available in market.
A method of Cigarette grain phase cytotoxicity of not burning is heated in detection, comprising the following steps:
(1) sample pre-treatments: using linear type smoking machine, is 55.0mL, duration 3s, suction frequencies in pumping volume 10 mouthfuls of continuous sucking, to be trapped with the cambridge filter that diameter is 44mm, before suction, after suction under the puffing regimens of 30s The quality for weighing Fume collector calculates the quality mTPM for heating the Cigarette grain phase that do not burn according to formula (1)；
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 the triangular flask of 50mL, dimethyl sulfoxide is added, makes to heat Concentration of the Cigarette grain phase in dimethyl sulfoxide of not burning is 40mg/mL；This triangular flask is placed in Ultrasound Instrument ultrasonic 30min；Dimethyl sulfoxide extract liquor is collected by filtration with aseptic filter paper again, dispenses into the cryopreservation tube of 1mL, -80 DEG C save for use；
(2) preparation of people's nasal epithelia single cell suspension: after people's nasal epithelia RPMI2650 recovery, inoculation To 25cm2In Tissue Culture Flask, 10mLRPMI1640 cell culture medium is added, is placed in 37 DEG C, 5%CO2It is cultivated in incubator, Set micro- sem observation culture cell converge with form situation, when converging rate, remove culture to 70%~90% when cell is long RPMI1640 cell culture medium in bottle is washed twice with phosphate buffer, abandons washing lotion；The pancreas egg of 1mL 0.25% (w/v) is added White enzyme solutions single layer is incubated for 1~2min, is hanged with RPMI1640 cell culture medium, forms single cell suspension；
(3) cell concentration of people's nasal epithelia single cell suspension calculates: with blood counting chamber counting method to step (2) cell concentration of income earner's nasal epithelia single cell suspension calculates, and obtains every milliliter of single cell suspension Viable count；
(4) inoculation of people's nasal epithelia single cell suspension: people's nasal epithelia list after step (3) are counted Cell suspending liquid RPMI1640 cell culture medium to 2.0 × 105A/mL, then be inoculated by inoculum concentration for 200 holes μ L/ In 96 porocyte culture plates, 96 porocyte culture plates are then placed in 37 DEG C, 5%CO2It is cultivated for 24 hours in incubator；
(5) tested material is grouped: tested material being divided into four groups: blank group, cell controls group, positive controls and test sample Group；The composition of each group are as follows: blank group does not plant people's nasal epithelia, only adds RPMI1640 cell culture medium；Cell controls group It plants people's nasal epithelia and adds RPMI1640 cell culture medium；The dodecyl sulphate of 200 μ g/mL is added in positive controls 200 μ L of sodium；Test sample group is added prepare liquid obtained by step (1) and adds RPMI1640 cell culture medium；
(6) tested material detects setting for dosage: the ultimate density of prepare liquid obtained by step (1) is divided as 10 non-zero-doses Not are as follows: 60 μ g/mL, 120 μ g/mL, 180 μ g/mL, 240 μ g/mL, 300 μ g/mL, 360 μ g/mL, 420 μ g/mL, 480 μ g/mL, 540μg/mL,600μg/mL；
(7) preparation of tested material culture product: the culture medium in removing step (4) in 96 porocyte culture plates, then press step (5) it is grouped, the liquid volume in hole every in tissue culture plate is complemented into 200 holes μ l/ with RPMI1640 cell culture medium；It does 8 holes are parallel, in order to avoid edge effect, when data statistics removal 2 hole of edge using intermediate 6 holes data；
(8) it is incubated for tested material: 96 porocyte culture plates after step (7) sample-adding is placed in 37 DEG C, 5%CO2In incubator It is incubated for for 24 hours；
(9) dyestuff is incubated for: blank group, cell controls group, positive controls and test sample group after step (8) are incubated for It is middle that the MTT solution that concentration is 5mg/mL, 20 holes μ L/ are added, then it is placed in 37 DEG C, 5%CO24h is incubated in incubator to move back except 96 holes 200 hole μ L/ of DMSO solution is added in liquid in tissue culture plate, is placed in microplate oscillator and vibrates 10min；
(10) light absorption value is measured: blank group, cell controls group, positive controls and inspection after step (9) dyestuff is incubated for Sample group detects light absorption value under 490nm wavelength with microplate reader；
(11) it calculates cell survival rate: according to gained light absorption value, calculating cell survival rate according to the following formula:
In formula:
X --- cell survival rate；
ODn--- the porous average light absorption value of test sample group；
ODo--- the porous average light absorption value of blank group；
ODc--- the porous average light absorption value of cell controls group；
(12) cell survival rate curve is drawn:
Curve is drawn according to 10 non-zero-doses and the corresponding cell survival rate of 0 dosage, that is, can determine heating detected It does not burn the cytotoxicity of Cigarette grain phase.
The invention has the advantages that:
Integrated survey of the present invention heating is not burnt the handling characteristics and the mode of action of cigarette, and is not burnt volume according to heating Cigarette acts on target cell and chooses people's nasal epithelia RPMI2650 as cell toxicity test detection cell, and sample has been determined Dosage is detected, the cell toxicity test detection method for being suitble to heating not burn cigarette is formd.The present invention is to sample detection dosage Optimal setting and target cell correct selection, enable this method accurately and effectively to detect heating and do not burn the total grain of cigarette The cytotoxicity of phase object.
Detailed description of the invention
Fig. 1 is that the detection heating of embodiment 1 does not burn Cigarette grain phase to the cell survival rate curves of three kinds of cytotoxicities Figure.
Fig. 2 is the cell doubling time figure of three kinds of subject cells used in embodiment 1.
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.
Embodiment 1
The heating trapped using cambridge filter does not burn Cigarette grain phase as sample, respectively with people's nasal epithelia (RPMI 2650), human bronchial epithelial cell (Beas-2b), people's alveolar epithelial cells (HPAEpiC) detect its cytotoxicity.
Specific detection process, comprising the following steps:
(1) sample pre-treatments: using linear type smoking machine, is 55.0mL, duration 3s, suction frequencies in pumping volume 10 mouthfuls of continuous sucking, to be trapped with the cambridge filter that diameter is 44mm, before suction, after suction under the puffing regimens of 30s The quality for weighing Fume collector calculates the quality mTPM for heating the Cigarette grain phase that do not burn according to formula (1)；
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 the triangular flask of 50mL, dimethyl sulfoxide is added, makes to heat Concentration of the Cigarette grain phase in dimethyl sulfoxide of not burning is 40mg/mL；This triangular flask is placed in Ultrasound Instrument ultrasonic 30min；Dimethyl sulfoxide extract liquor is collected by filtration with aseptic filter paper again, dispenses into the cryopreservation tube of 1mL, -80 DEG C save for use；
(2) preparation of single cell suspension: people's nasal epithelia (RPMI2650), human bronchial epithelial cell (Beas- 2b), after people's alveolar epithelial cells (HPAEpiC) recovery, it is inoculated into 25cm2In Tissue Culture Flask, it is thin that 10mL RPMI1640 is added Born of the same parents' culture medium is placed in 37 DEG C, 5%CO2It is cultivated in incubator, converge and the form situation of inverted microscope observation culture cell, When cell length to 70%~90% when converging rate, the RPMI1640 cell culture medium in culture bottle is removed, phosphate buffer is used It washes twice, abandons washing lotion；The trypsin solution single layer that 1mL 0.25% (w/v) is added is incubated for 1~2min, with RPMI1640 cell Culture medium hangs, and forms single cell suspension；
(3) cell concentration of single cell suspension calculates: with blood counting chamber counting method to unicellular outstanding obtained by step (2) The cell concentration of supernatant liquid is calculated, and the viable count of every milliliter of single cell suspension is obtained；
(4) inoculation of single cell suspension: the single cell suspension RPMI1640 cell culture after step (3) are counted Base is diluted to 2.0 × 105A/mL, then be inoculated into 96 porocyte culture plates by inoculum concentration for 200 holes μ L/, it is then that 96 holes are thin Born of the same parents' culture plate is placed in 37 DEG C, 5%CO2It is cultivated for 24 hours in incubator；
(5) tested material is grouped: tested material being divided into four groups: blank group, cell controls group, positive controls and test sample Group；The composition of each group are as follows: blank group does not plant people's nasal epithelia, only adds RPMI1640 cell culture medium；Cell controls group It plants people's nasal epithelia and adds RPMI1640 cell culture medium；The dodecyl sulphate of 200 μ g/mL is added in positive controls 200 μ L of sodium；Test sample group is added prepare liquid obtained by step (1) and adds RPMI1640 cell culture medium；
(6) tested material detects setting for dosage: the ultimate density of prepare liquid obtained by step (1) is divided as 10 non-zero-doses Not are as follows: 60 μ g/mL, 120 μ g/mL, 180 μ g/mL, 240 μ g/mL, 300 μ g/mL, 360 μ g/mL, 420 μ g/mL, 480 μ g/mL, 540μg/mL,600μg/mL；
(7) preparation of tested material culture product: the culture medium in removing step (4) in 96 porocyte culture plates, then press step (5) it is grouped, the liquid volume in hole every in tissue culture plate is complemented into 200 holes μ l/ with RPMI1640 cell culture medium；It does 8 holes are parallel, in order to avoid edge effect, when data statistics removal 2 hole of edge using intermediate 6 holes data；
(8) it is incubated for tested material: 96 porocyte culture plates after step (7) sample-adding is placed in 37 DEG C, 5%CO2In incubator It is incubated for for 24 hours；
(9) dyestuff is incubated for: blank group, cell controls group, positive controls and test sample group after step (8) are incubated for It is middle that the MTT solution that concentration is 5mg/mL, 20 holes μ L/ are added, then it is placed in 37 DEG C, 5%CO24h is incubated in incubator to move back except 96 holes 200 hole μ L/ of DMSO solution is added in liquid in tissue culture plate, is placed in microplate oscillator and vibrates 10min；
(10) light absorption value is measured: blank group, cell controls group, positive controls and inspection after step (9) dyestuff is incubated for Sample group detects light absorption value under 490nm wavelength with microplate reader；
(11) it calculates cell survival rate: according to gained light absorption value, calculating cell survival rate according to the following formula:
In formula:
X --- cell survival rate；
ODn--- the porous average light absorption value of test sample group；
ODo--- the porous average light absorption value of blank group；
ODc--- the porous average light absorption value of cell controls group；
(12) cell survival rate curve is drawn:
Curve is drawn according to 10 non-zero-doses and the corresponding cell survival rate of 0 dosage, that is, can determine heating detected It does not burn the cytotoxicity of Cigarette grain phase.
As seen from Figure 1, the cigarette that do not burn is heated to exist between the survival rate and detection dosage of three kinds of tested cells Dose-effect relationship, heating is not burnt, and there were significant differences (p < 0.05) for cytotoxicity of the Cigarette grain phase to different cells.
Different subject cells is consistent to identical sample tests qualitative results, sample room relative quantification trend one It causes, the above candidate cell could be used for the biological effect test of solvent.There is the tolerance of solvent in different subject cells Significant difference, when study dosage is identical, people's nasal epithelia (RPMI 2650) tolerance is higher, and human bronchial epithelial is thin Born of the same parents (Beas-2b) take second place, and people's alveolar epithelial cells (HPAEpiC) is lower.Fig. 2 is the cell doubling time of detection cell, In test period for 24 hours, the proliferation of 2650 cell of RPMI is very fast.
The route of exposure of cigarette products in conclusion integrated survey of the present invention heating is not burnt, the mode of action establish Cigarette products pre-treating method, and the feature short according to the tolerance height and cell doubling time of test cell of not burning are heated, It has chosen people's nasal epithelia RPMI2650 conduct and heats the cigarette products cell toxicity test detection cell that do not burn, and really Determine sample detection dosage, forms the cell toxicity test detection method for being suitble to heating not burn cigarette products.Sample has The correct selection of effect processing, the optimal setting of detection dosage and target cell enables this method accurately and effectively to detect and adds Hot Cigarette grain phase cytotoxicity of not burning.

Claims (1)

  1. A kind of method of Cigarette grain phase cytotoxicity 1. detection heating is not burnt, which comprises the following steps:
    (1) sample pre-treatments: using linear type smoking machine, is 55.0mL, duration 3s in pumping volume, suction frequencies are Under the puffing regimens of 30s, 10 mouthfuls of continuous sucking, is trapped with the cambridge filter that diameter is 44mm, claimed before suction, after suction The quality for measuring Fume collector calculates the quality mTPM for heating the Cigarette grain phase that do not burn according to formula (1)；
    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 the triangular flask of 50mL, dimethyl sulfoxide is added, makes to heat non-ignitable Burning concentration of the Cigarette grain phase in dimethyl sulfoxide is 40mg/mL；This triangular flask is placed in ultrasound 30min in Ultrasound Instrument； Dimethyl sulfoxide extract liquor is collected by filtration with aseptic filter paper again, dispenses into the cryopreservation tube of 1mL, -80 DEG C save for use；
    (2) it the preparation of people's nasal epithelia single cell suspension: after people's nasal epithelia RPMI2650 recovery, is inoculated into 25cm2In Tissue Culture Flask, 10mLRPMI1640 cell culture medium is added, is placed in 37 DEG C, 5%CO2It cultivates, is inverted in incubator Converge and the form situation of micro- sem observation culture cell when converging rate, remove culture bottle to 70%~90% when cell length In RPMI1640 cell culture medium, with phosphate buffer wash twice, abandon washing lotion；The tryptose of 1mL 0.25% (w/v) is added Enzyme solutions single layer is incubated for 1~2min, is hanged with RPMI1640 cell culture medium, forms single cell suspension；
    (3) cell concentration of people's nasal epithelia single cell suspension calculates: with blood counting chamber counting method to step (2) institute The cell concentration for obtaining people's nasal epithelia single cell suspension calculates, and obtains the living cells of every milliliter of single cell suspension Number；
    (4) inoculation of people's nasal epithelia single cell suspension: people's nasal epithelia after step (3) are counted is unicellular Suspension RPMI1640 cell culture medium to 2.0 × 105A/mL, then 96 holes are inoculated by inoculum concentration for 200 holes μ L/ In tissue culture plate, 96 porocyte culture plates are then placed in 37 DEG C, 5%CO2It is cultivated for 24 hours in incubator；
    (5) tested material is grouped: tested material being divided into four groups: blank group, cell controls group, positive controls and test sample group； The composition of each group are as follows: blank group does not plant people's nasal epithelia, only adds RPMI1640 cell culture medium；The plantation of cell controls group People's nasal epithelia simultaneously adds RPMI1640 cell culture medium；The lauryl sodium sulfate of 200 μ g/mL is added in positive controls 200μL；Test sample group is added prepare liquid obtained by step (1) and adds RPMI1640 cell culture medium；
    (6) tested material detects setting for dosage: the ultimate density of prepare liquid obtained by step (1) is respectively as follows: as 10 non-zero-doses 60μg/mL、120μg/mL、180μg/mL、240μg/mL、300μg/mL、360μg/mL、420μg/mL、480μg/mL、540μg/ mL,600μg/mL；
    (7) preparation of tested material culture product: the culture medium in removing step (4) in 96 porocyte culture plates, then by step (5) into Row grouping, complements to 200 holes μ l/ for the liquid volume in hole every in tissue culture plate with RPMI1640 cell culture medium；
    (8) it is incubated for tested material: 96 porocyte culture plates after step (7) sample-adding is placed in 37 DEG C, 5%CO2It is incubated in incubator 24h；
    (9) dyestuff is incubated for: being added in the blank group, cell controls group, positive controls and test sample group after step (8) are incubated for Enter the MTT solution that concentration is 5mg/mL, 20 holes μ L/, then is placed in 37 DEG C, 5%CO24h is incubated in incubator to move back except 96 hole cells 200 hole μ L/ of DMSO solution is added in liquid in culture plate, is placed in microplate oscillator and vibrates 10min；
    (10) light absorption value is measured: blank group, cell controls group, positive controls and detection sample after step (9) dyestuff is incubated for Product group detects light absorption value under 490nm wavelength with microplate reader；
    (11) it calculates cell survival rate: according to gained light absorption value, calculating cell survival rate according to the following formula:
    In formula:
    X --- cell survival rate；
    ODn--- the porous average light absorption value of test sample group；
    ODo--- the porous average light absorption value of blank group；
    ODc--- the porous average light absorption value of cell controls group；
    (12) cell survival rate curve is drawn:
    Curve is drawn according to 10 non-zero-doses and the corresponding cell survival rate of 0 dosage, that is, can determine that heating detected is non-ignitable Burn the cytotoxicity of Cigarette grain phase.
CN201811469837.5A 2018-11-28 2018-11-28 A method of Cigarette grain phase cytotoxicity of not burning is heated in detection Pending CN109666716A (en)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111796086A (en) * 2020-07-03 2020-10-20 同济大学 Characterization method of acute and chronic dose-effect relationship of personal care product

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Application publication date: 20190423