CN106591413A - Method of detecting influence on early apoptosis of cells due to total particulate matters in cigarette smoke - Google Patents
Method of detecting influence on early apoptosis of cells due to total particulate matters in cigarette smoke Download PDFInfo
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Abstract
The invention discloses a method of detecting influence on early apoptosis of cells due to total particulate matters in cigarette smoke. The method includes the steps of: 1) pre-treating to-be-detected substances; 2) preparing a human lung fibroblast single cell suspension liquid; 3) calculating cell concentration of the human lung fibroblast single cell suspension liquid; 4) performing cell inoculation to the human lung fibroblast single cell suspension liquid; 5) grouping the to-be-detected samples; 6) setting detection dose of the to-be-detected samples; 7) preparing a to-be-detected sample culture substance; 8) incubating the to-be-detected samples; 9) collecting incubated products of the to-be-detected samples; and 10) calculating influence ratio of early apoptosis of cells. Through optimization of sample detection dose and correct selection on target cells, the method can accurately and effectively detect the influence on early apoptosis of cells due to the total particulate matters in cigarette smoke. As a complementary method for cytotoxicity detection for the total particulate matters in cigarette smoke, the method distinguishes the influence between necrocytosis and cell apoptosis of the product.
Description
Technical field
The invention belongs to tobacco product biological effect assessment technique field, more particularly to a kind of to detect the total grain of cigarette smoke
The method that phase thing affects on early apoptosis of cells.
Background technology
Apoptotic concept is to be proposed first by three scientists such as Kerr for 1972, although apoptosis
(apoptosis) it is very much like with the final result of necrocytosiss (necrosis), but their process has very big with performance
Difference.Apoptosis are an active processes, and it is related to the effect of activation, expression and the regulation and control of series of genes etc., and it is simultaneously
It is not that under pathological conditions, from a kind of phenomenon of bulk damage, but the one kind initiatively striven for better adapt to living environment is dead
Die process.For the detection of cigarette smoke condensates cytotoxicity, international Nicotiana tabacum L. scientific research Cooperation Centre CORESTA tissues
Recommend using neutral RBC Toxicity method, but there is certain defect in neutral RBC Toxicity method.Dimethyl diaminophenazine chloride enter cell and
The range request of crossing of intracellular accumulation has complete cell membrane, and apoptosis early stage surface of cell membrane changes, negatively charged
Phosphatidylserine (PS) be transferred to from cell membrane outside cell membrane, make PS be exposed to membrane surface.Therefore, it is necessary to
Exploitation is a kind of for detecting the method that cigarette smoke condensates affect on early apoptosis of cells, used as cigarette smoke condensates
The compensation process of cytotoxicity detection is distinguishing impact of the cigarette smoke condensates to necrocytosiss and apoptosis.
The content of the invention
It is an object of the invention to provide a kind of method that detection cigarette smoke condensates affect on early apoptosis of cells, is
The safety evaluation of electronics tobacco product provides reference.
A kind of method that detection cigarette smoke condensates affect on early apoptosis of cells, 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) calculating of early apoptosis of cells contributive rate.
In the preferred embodiment of the invention, said 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《Medicated 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' culture bottle, 37 DEG C, 5vt%CO are placed in2Cultivate in incubator, inverted microscope observes converging and form feelings for cultured cells
Condition, whne cell length to 90% when converging rate, removes the culture medium in culture bottle, is washed twice with phosphate buffer, adds appropriate
0.25% trypsin solution, the wherein concentration unit of trypsin/phosphate buffer are quality：Volume；Monolayer is incubated
About 1-2min, with hanging 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
Step (2) obtains the cell concentration of human lung cancer cell A549 single cell suspension and is calculated, and calculates every milliliter of people's lung into fibre
The viable count of dimension cell single cell suspension；
(4) the cell inoculation of human lung cancer cell A549 HPF single cell suspensions：People's lung after step (3) is counted is into fibre
Dimension cell single cell suspension adds fibroblast culture medium to be diluted to 2.0 × 105Individual/mL, then be 1mL/ holes by inoculum concentration,
In being inoculated into 6 porocyte culture plates, 6 porocyte culture plates are 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 to plant human lung cancer cell A549 on Fibroblast Growth Medium；Detection sample group is in fibroblast
Human lung cancer cell A549 is planted on growth medium and adds tested material；
(6) tested material detects the setting of dosage：Tested material, i.e. electronic cigarette stock solution are divided into into 5 non-non- zero-doses：25μg/
mL、50μg/mL、100μg/mL、150μg/mL、200μg/mL；
(7) preparation of tested material culture product：The fibroblast culture medium in 6 porocyte culture plates in step (4) is removed,
It is grouped by step (5) again, the composition of each group is：Cell controls group is fibroblast culture medium+human lung cancer cell A549；
Detection sample group is fibroblast culture medium+human lung cancer cell A549+tested material, and makes the ultimate density point of detection sample group
Not Wei step (6) setting dosage；And 2 tissue cultures are supported product fibroblast culture medium and supply the liquid volume in every hole
2mL/ holes；
(8) incubation of tested material：6 porocyte culture plates after step (7) is loaded are placed in 37 DEG C, 5vt%CO2Incubator
Middle incubation 24h；
(9) tested material is incubated the collection of product：Culture fluid in collection step (8), pancreatin of the attached cell without EDTA
Together with the culture fluid collected after digestion, under 1000rpm rotating speeds, 4 DEG C of centrifugation 5min collect cell；
(10) calculating of early apoptosis of cells contributive rate：With phosphate buffer washing step (9) the gained cell 2 of pre-cooling
Secondary, every time under 1000rpm rotating speeds, 4 DEG C of centrifugation 5min collect cell, add the combination liquid of 100 μ L：Re-suspended cell, then
The dyestuff of 10 μ L is added, is gently mixed, then react 10min under lucifuge, room temperature, the re-suspended cell for adding 400 μ L is mixed
It is even to obtain detection sample, flow cytomery was used in 1 hour, that is, obtain early apoptosis of cells contributive rate.
In the preferred embodiment of the invention, the re-suspended cell Binding Buffer re-suspended cells described in step (10),
The cell is commercialization cell, commercially available where market arbitrarily can buy.
In the preferred embodiment of the invention, described dyestuff is AnnexinV-FITC and PI dyestuffs, the volume ratio of the two
For 1:1, both at commercialization dyestuff, it is commercially available in market is any can buy where, wherein PI dyestuffs be propidium iodide
Dyestuff.
Beneficial effect of the present invention：
Optimal Setting and the correct selection of target cell of the present invention to sample detection dosage so that this method can be accurate
Effectively impact of the detection cigarette smoke condensates to early apoptosis of cells.As the inspection of cigarette smoke condensates cytotoxicity
The compensation process of survey is distinguishing impact of the product to necrocytosiss and apoptosis.
Description of the drawings
Fig. 1 is that given the test agent has dose-effect relationship in the range of detection dosage between early apoptosis of cells rate and detection dosage
Figure.
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 withers for Kentucky reference Medicated cigarette 3R4F cigarette smoke condensates to human lung cancer cell A549 early stage
Die the test of impact.
(1) pre-treatment of tested material：With reference to National Standard of the People's Republic of China GB/T 19609-2004《Medicated 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
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 culture bottle is removed, washed twice with phosphate buffer, added appropriate
0.25% trypsin solution, the wherein concentration unit of trypsin/phosphate buffer are quality：Volume；Monolayer is incubated
About 1min, with hanging 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
Step (2) obtains the cell concentration of human lung cancer cell A549 single cell suspension and is calculated, and calculates every milliliter of people's lung into fibre
The viable count of dimension cell single cell suspension；
(4) the cell inoculation of human lung cancer cell A549 HPF single cell suspensions：People's lung after step (3) is counted is into fibre
Dimension cell single cell suspension adds fibroblast culture medium to be diluted to 2.0 × 105Individual/mL, then be 1mL/ holes by inoculum concentration,
In being inoculated into 6 porocyte culture plates, 6 porocyte culture plates are 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 to plant human lung cancer cell A549 on Fibroblast Growth Medium；Detection sample group is in fibroblast
Human lung cancer cell A549 is planted on growth medium and adds tested material；
(6) tested material detects the setting of dosage：Tested material, i.e. electronic cigarette stock solution are divided into into 5 non-non- zero-doses：25μg/
mL、50μg/mL、100μg/mL、150μg/mL、200μg/mL；
(7) preparation of tested material culture product：The fibroblast culture medium in 6 porocyte culture plates in step (4) is removed,
It is grouped by step (5) again, the composition of each group is：Cell controls group is fibroblast culture medium+human lung cancer cell A549；
Detection sample group is fibroblast culture medium+human lung cancer cell A549+tested material, and makes the ultimate density point of detection sample group
Not Wei step (6) setting dosage；And 2 tissue cultures are supported product fibroblast culture medium and supply the liquid volume in every hole
2mL/ holes；
(8) incubation of tested material：6 porocyte culture plates after step (7) is loaded are placed in 37 DEG C, 5vt%CO2Incubator
Middle incubation 24h；
(9) tested material is incubated the collection of product：Culture fluid in collection step (8), pancreatin of the attached cell without EDTA
Together with the culture fluid collected after digestion, under 1000rpm rotating speeds, 4 DEG C of centrifugation 5min collect cell；
(10) calculating of early apoptosis of cells contributive rate：With phosphate buffer washing step (9) the gained cell 2 of pre-cooling
Secondary, every time under 1000rpm rotating speeds, 4 DEG C of centrifugation 5min collect cell, add the combination liquid of 100 μ L：Re-suspended cell, then
The dyestuff of 10 μ L is added, is gently mixed, then react 10min under lucifuge, room temperature, the re-suspended cell for adding 400 μ L is mixed
It is even to obtain detection sample, flow cytomery was used in 1 hour, that is, obtain early apoptosis of cells contributive rate.
As a result Fig. 1 is seen.By result of the test as can be seen that the subject cell, the detection dosage conditions that are given in the inventive method
Under, there is dose-effect relationship in given the test agent, between early apoptosis of cells rate and detection dosage in the range of detection dosage with cell
Control is compared has significant difference (p<0.05).The method can effectively detect cigarette smoke granule phase substance to early apoptosis of cells
Impact.
Claims (2)
1. a kind of method that detection cigarette smoke condensates affect on early apoptosis of cells, it is characterised in that including following step
Suddenly：
(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) calculating of early apoptosis of cells contributive rate.
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《Medicated 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%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 culture bottle to 90% when converging rate, is washed twice with phosphate buffer, adds appropriate 0.25%
Trypsin solution, the wherein concentration unit of trypsin/phosphate buffer are quality：Volume；Monolayer is incubated about 1-2min,
With hanging into fiber fibroblast culture medium, single cell suspension is formed；
(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, then be 1mL/ holes by inoculum concentration, it is inoculated into 6
In porocyte culture plates, 6 porocyte culture plates are 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
Matched group is to plant human lung cancer cell A549 on Fibroblast Growth Medium；Detection sample group is in fibroblastic growth
Human lung cancer cell A549 is planted in culture medium and adds tested material；
(6) tested material detects the setting of dosage：Tested material, i.e. electronic cigarette stock solution are divided into into 5 non-non- zero-doses：25μg/mL、50
μg/mL、100μg/mL、150μg/mL、200μg/mL；
(7) preparation of tested material culture product：The fibroblast culture medium in 6 porocyte culture plates in step (4) is removed, then is pressed
Step (5) is grouped, and the composition of each group is：Cell controls group is fibroblast culture medium+human lung cancer cell A549；Detection
Sample sets are fibroblast culture medium+human lung cancer cell A549+tested material, and are respectively the ultimate density of detection sample group
The dosage of step (6) setting；And 2 tissue cultures are supported product fibroblast culture medium and the liquid volume in every hole are supplied into 2mL/
Hole；
(8) incubation of tested material：6 porocyte culture plates after step (7) is loaded are placed in 37 DEG C, 5vt%CO2Incubate in incubator
Educate 24h；
(9) tested material is incubated the collection of product：Culture fluid in collection step (8), attached cell is digested with the pancreatin without EDTA
Afterwards together with the culture fluid collected, under 1000rpm rotating speeds, 4 DEG C of centrifugation 5min collect cell；
(10) calculating of early apoptosis of cells contributive rate：With the phosphate buffer washing step (9) of pre-cooling gained cell 2 times,
Every time under 1000rpm rotating speeds, 4 DEG C of centrifugation 5min collect cell, add the combination liquid of 100 μ L：Re-suspended cell, then add
Enter the dyestuff of 10 μ L, gently mix, then react 10min under lucifuge, room temperature, the re-suspended cell for adding 400 μ L is mixed
Detection sample is obtained, flow cytomery was used in 1 hour, that is, obtain early apoptosis of cells contributive rate.
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Cited By (3)
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| CN108004293A (en) * | 2017-12-14 | 2018-05-08 | 云南中烟工业有限责任公司 | A kind of method for detecting gum base type chewing tobacco cell cycle and influencing |
| CN108020659A (en) * | 2017-12-06 | 2018-05-11 | 中国烟草总公司郑州烟草研究院 | A kind of method of in-situ test Smoke Particulate induction of vascular endothelial Apoptosis |
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Application publication date: 20170426 |