CN106645747A - Method for detecting influence of smoke total particulate matter on cell inflammatory effect factor - Google Patents

Method for detecting influence of smoke total particulate matter on cell inflammatory effect factor Download PDF

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CN106645747A
CN106645747A CN201611056530.3A CN201611056530A CN106645747A CN 106645747 A CN106645747 A CN 106645747A CN 201611056530 A CN201611056530 A CN 201611056530A CN 106645747 A CN106645747 A CN 106645747A
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cell
liquid
human lung
tested material
lung cancer
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CN106645747B (en
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管莹
李雪梅
夭建华
熊国行
米其利
高茜
朱洲海
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China Tobacco Yunnan Industrial Co Ltd
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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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/715—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons

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Abstract

The invention discloses a method for detecting influence of smoke total particulate matter on a cell inflammatory effect factor. The method comprises the following steps of: (1) preprocessing of a tested substance; (2) preparation of human lung fibroblast single-cell suspension; (3) calculation of cell concentration of the human lung fibroblast single-cell suspension; (4) cell inoculation of the human lung fibroblast single-cell suspension; (5) grouping of the tested substance; (6) setting of a detection dosage of the tested substance; (7) preparation of a tested substance culture product; (8) incubation of the tested substance; (9) collection of tested substance incubation products; (10) preparation of standard solution; (11) adding of a sample to be detected; (12) adding of an antibody; (13) adding of binding solution; (14) color development; (15) measurement of a light absorption value; (16) measurement of a cell factor secretion volume. According to the method disclosed by the invention, influence of the cigarette smoke total particulate matter on secretion of the cell inflammatory effect factor can be accurately and effectively detected.

Description

One kind detection smoke's total particulate matter affects method to cellular inflammation effector
Technical field
The invention belongs to tobacco product biological effect assessment technique field, more particularly to a kind of detection smoke's total particulate matter Method is affected on cellular inflammation effector.
Background technology
With concern more and more higher of the people to health, tobacco product is put forward higher requirement, not only to be had preferably Mouthfeel, and to reduce the bad impression of human body as far as possible.Cigarette product research staff is first in the design of tobacco tar product formula Phase allocates different components according to different proportion combination, forms the primary election formula with different-style, in conjunction with Chemical Evaluation and Sensory evaluation carries out constantly screening adjustment to formula and forms final product formula.But primary election formula is large number of, all enters Row sensory evaluation takes time and effort, and sensory evaluation lays particular emphasis on the style mouthfeel to product and screens, and how to be surveyed using biology Examination index is filled a prescription into screening to product primary election, to obtain the product formula for reducing the bad impression of human body, the exploratory stage is still belonged at present, Without unified standard method.
The content of the invention
It is an object of the invention to provide a kind of detection smoke's total particulate matter affects method to cellular inflammation effector, it is electricity The safety evaluation of sub- tobacco product provides reference.
One kind detection smoke's total particulate matter affects method to cellular inflammation effector, 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) standard liquid is prepared；
(11) test sample is treated in addition；
(12) antibody is added；
(13) addition combines liquid；
(14) develop the color；
(15) light absorption value is measured；
(16) cytokine secretion measures fixed.
In the preferred embodiment of the invention, above-mentioned method, including step in detail below：
(1) pre-treatment of tested material：With reference to National Standard of the People's Republic of China GB/T 19609-2004《Cigarette is with often Rule analysis determines TPM and tar with smoking machine》Cigarette smoke condensates sample is prepared, sample concentration is 10mg/mL；
(2) preparation of human lung cancer cell A549 HPF single cell suspensions：After human lung cancer cell A549 recovery, it is inoculated into thin In born of the same parents' blake bottle, 37 DEG C, 5vt%CO are placed 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 blake bottle, 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, 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 12 porocyte culture plates, 12 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 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 stoste are divided into 5 non-non- zero-doses：20μg/ mL、40μg/mL、60μg/mL、80μg/mL、100μg/mL；
(7) preparation of tested material culture product：Remove the Fibroblast cell-culture in 12 porocyte culture plates in step (4) Base, then be grouped by step (5), the composition of each group is：Cell controls group is fibroblast culture medium+people's lung into fiber finer Born of the same parents；Detection sample group is fibroblast culture medium+human lung cancer cell A549+tested material, and makes the final dense of detection sample group Degree is respectively the dosage of step (6) setting；And 2 tissue cultures are supported product fibroblast culture medium and mend the liquid volume in every hole Foot is to 1mL/ holes；
(8) incubation of tested material：12 porocyte culture plates after step (7) is loaded are placed in 37 DEG C, 5vt%CO2Culture 24h is incubated in case；
(9) tested material is incubated the collection of product：Supernatant in aspiration step (8) cell plates, is centrifuged 20min, rotating speed 1000rpm, takes supernatant；
(10) standard liquid is prepared：Gradient dilution method prepares the calibration curve solution of the inflammatory effector factor,
(11) test sample is treated in addition：Blank control wells add the μ L of distilled water 100, remaining hole to add the standard that step (10) is prepared respectively The μ L of testing sample 100 that product solution or step (9) are obtained, lid ELISA Plate overlay film, 37 DEG C are incubated 90 minutes；
(12) antibody is added：Solid-liquid in step (11) is separated, detached liquid is taken, is dried, added per hole anti- The μ L of body running liquid 100, lid ELISA Plate overlay film, 37 DEG C are incubated 60 minutes；
(13) addition combines liquid：Solid-liquid in step (12) is separated, detached liquid is taken, is dried, used phosphate Buffer solution PBS board-washings three times, every time immersion about 2 minutes, the every holes of 350 μ L/, dry and simultaneously pat liquid in hole on blotting paper Pat dry, per the enzyme-added μ L of combination working solution 100 in hole, lid ELISA Plate overlay film, 37 DEG C are incubated 30 minutes；
(14) develop the color：Solid-liquid in step (13) is separated, detached liquid is taken, is dried, with cleaning fluid board-washing five Secondary, each immersion about 2 minutes, 350 μ L/ are dried and patted on blotting paper and pat dry liquid in hole per hole, and per hole substrate is added The μ L of working solution 90, lid ELISA Plate overlay film, 37 DEG C of incubations, observation adds the μ L/ holes of terminate liquid 50 per hole color change, in good time；
(15) light absorption value is measured：Absorbance is detected under ELIASA 450nm；
(16) cytokine secretion measures fixed：Calibration curve is drawn according to standard liquid absorbance, cell factor is calculated Secretory volume.
In the preferred embodiment of the invention, the process for preparation of the calibration curve solution in step (10) is：Antibody is used Antibody working solution is configured to respective concentration, and wherein IL-6 standard curve ranges are 0-500pg/mL, and IL-8 standard curve ranges are 0-2000pg/mL, IL-10 standard curve range is 0-500pg/mL, and the standard curve range of TGF-β 1 is 0-2000pg/mL, TNF-α standard curve range is 0-500pg/mL, and GM-CSF standard curve ranges are 0-500pg/mL, and antibody is bought in Yi Lairui Special company.
In the preferred embodiment of the invention, the antibody working solution in step (12) belongs to commercialization commodity, can be in city Buy in Yi Lairuite companies on.
In the preferred embodiment of the invention, the enzyme in step (13) belongs to commercialization commodity, Neng Gou with reference to working solution Buy on market in Yi Lairuite companies.
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 can accurately and effectively detect that cigarette smoke condensates affect on the secretion of cellular inflammation effector.
Description of the drawings
Fig. 1 is impact of 4 kinds of cigarette smoke condensates to HPF cell IL-6 cytokine secretions；
Fig. 2 is impact of 4 kinds of cigarette smoke condensates to HPF cell IL-10 cytokine secretions.
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 invention for 4 kinds of domestic commercial cigaretteses smoke's total particulate matter to two kinds of human lung cancer cell A549 IL-6, IL-10 The impact test of inflammatory effector cytokine secretion amount.
(1) pre-treatment of tested material：With reference to National Standard of the People's Republic of China GB/T 19609-2004《Cigarette is with often Rule analysis determines TPM and tar with smoking machine》Cigarette smoke condensates sample is prepared, sample concentration is 10mg/mL；
(2) preparation of human lung cancer cell A549 HPF single cell suspensions：After human lung cancer cell A549 recovery, it is inoculated into thin In born of the same parents' blake bottle, 37 DEG C, 5vt%CO are placed 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 blake bottle, 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 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 12 porocyte culture plates, 12 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 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 stoste are divided into 5 non-non- zero-doses：20μg/ mL、40μg/mL、60μg/mL、80μg/mL、100μg/mL；
(7) preparation of tested material culture product：Remove the Fibroblast cell-culture in 12 porocyte culture plates in step (4) Base, then be grouped by step (5), the composition of each group is：Cell controls group is fibroblast culture medium+people's lung into fiber finer Born of the same parents；Detection sample group is fibroblast culture medium+human lung cancer cell A549+tested material, and makes the final dense of detection sample group Degree is respectively the dosage of step (6) setting；And 2 tissue cultures are supported product fibroblast culture medium and mend the liquid volume in every hole Foot is to 1mL/ holes；
(8) incubation of tested material：12 porocyte culture plates after step (7) is loaded are placed in 37 DEG C, 5vt%CO2Culture 24h is incubated in case；
(9) tested material is incubated the collection of product：Supernatant in aspiration step (8) cell plates, is centrifuged 20min, rotating speed 1000rpm, takes supernatant；
(10) standard liquid is prepared：Gradient dilution method prepares the calibration curve solution of the inflammatory effector factor,
(11) test sample is treated in addition：Blank control wells add the μ L of distilled water 100, remaining hole to add the standard that step (10) is prepared respectively The μ L of testing sample 100 that product solution or step (9) are obtained, lid ELISA Plate overlay film, 37 DEG C are incubated 90 minutes；
(12) antibody is added：Solid-liquid in step (11) is separated, detached liquid is taken, is dried, added per hole anti- The μ L of body running liquid 100, lid ELISA Plate overlay film, 37 DEG C are incubated 60 minutes；
(13) addition combines liquid：Solid-liquid in step (11) is separated, detached liquid is taken, is dried, used phosphate Buffer solution PBS board-washings three times, every time immersion about 2 minutes, the every holes of 350 μ L/, dry and simultaneously pat liquid in hole on blotting paper Pat dry, per the enzyme-added μ L of combination working solution 100 in hole, lid ELISA Plate overlay film, 37 DEG C are incubated 30 minutes；
(14) develop the color：Solid-liquid in step (11) is separated, detached liquid is taken, is dried, with cleaning fluid board-washing five Secondary, each immersion about 2 minutes, 350 μ L/ are dried and patted on blotting paper and pat dry liquid in hole per hole, and per hole substrate is added The μ L of working solution 90, lid ELISA Plate overlay film, 37 DEG C of incubations, observation adds the μ L/ holes of terminate liquid 50 per hole color change, in good time；
(15) light absorption value is measured：Absorbance is detected under ELIASA 450nm；
(16) cytokine secretion measures fixed：Calibration curve is drawn according to standard liquid absorbance, cell factor is calculated Secretory volume.
As a result Fig. 1 and Fig. 2 is seen.By result of the test as can be seen that the sample treatment, the detection that are given in the inventive method Under dosage conditions, sample 1#, 2#, 3#, 4# are deposited in the range of detection dosage to the secretory volume of the HPF cell IL-6 inflammatory effector factors In impact, and there is dose-effect relationship, there is significant difference (p compared with 0 dosage group<0.05)；Sample 4# and sample 1#, 2#, There is significant difference between 3#.And on the secretory volume of the HPF cell IL-10 inflammatory effector factors without impact, and the nothing compared with 0 dosage group Significant difference (p>0.05)；The different cigarette smoke condensates of the result above inflammatory effector factor different to HPF cells The impact of secretory volume has differences.

Claims (2)

1. a kind of detection smoke's total particulate matter affects method to cellular inflammation effector, it is characterised in that comprise 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) standard liquid is prepared；
(11) test sample is treated in addition；
(12) antibody is added；
(13) addition combines liquid；
(14) develop the color；
(15) light absorption value is measured；
(16) cytokine secretion measures fixed.
2. method according to claim 1, it is characterised in that including step in detail below：
(1) pre-treatment of tested material：With reference to National Standard of the People's Republic of China GB/T 19609-2004《Cigarette is with conventional point Analysis determines TPM and tar with smoking machine》Cigarette smoke condensates sample is prepared, sample concentration is 10mg/mL；
(2) preparation of human lung cancer cell A549 single cell suspension：After human lung cancer cell A549 recovery, Tissue Culture Flask is inoculated into In, it is placed in 37 DEG C, 5vt%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, 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 12 In porocyte culture plates, 12 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 two groups：Cell controls group and detection sample group；The composition of each group is：Cell Control 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 on culture medium and adds tested material；
(6) tested material detects the setting of dosage：Tested material, i.e. electronic cigarette stoste are divided into 5 non-non- zero-doses：20μg/mL、40 μg/mL、60μg/mL、80μg/mL、100μg/mL；
(7) preparation of tested material culture product：The fibroblast culture medium in 12 porocyte culture plates in step (4) is removed, then It is grouped by step (5), the composition of each group is：Cell controls group is fibroblast culture medium+human lung cancer cell A549；Inspection Survey sample sets are fibroblast culture medium+human lung cancer cell A549+tested material, and distinguish the ultimate density of detection sample group For the dosage of step (6) setting；And 2 tissue cultures are supported product fibroblast culture medium and supply the liquid volume in every hole 1mL/ holes；
(8) incubation of tested material：12 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：Supernatant in aspiration step (8) cell plates, is centrifuged 20min, rotating speed 1000rpm, Take supernatant；
(10) standard liquid is prepared：Gradient dilution method prepares the calibration curve solution of the inflammatory effector factor；
(11) test sample is treated in addition：Blank control wells add the μ L of distilled water 100, remaining hole to add the standard items that step (10) is prepared molten respectively The μ L of testing sample 100 that liquid or step (9) are obtained, lid ELISA Plate overlay film, 37 DEG C are incubated 90 minutes；
(12) antibody is added：Solid-liquid in step (11) is separated, detached liquid is taken, is dried, antibody work is added per hole Make the μ L of liquid 100, lid ELISA Plate overlay film, 37 DEG C are incubated 60 minutes；
(13) addition combines liquid：Solid-liquid in step (12) is separated, detached liquid is taken, is dried, used phosphate-buffered Liquid PBS board-washings three times, every time immersion about 2 minutes, the every holes of 350 μ L/, dry and pat on blotting paper and clap liquid in hole It is dry, per the enzyme-added μ L of combination working solution 100 in hole, lid ELISA Plate overlay film, 37 DEG C are incubated 30 minutes；
(14) develop the color：Solid-liquid in step (13) is separated, detached liquid is taken, is dried, with cleaning fluid board-washing five times, often Secondary immersion about 2 minutes, 350 μ L/ are dried and patted on blotting paper and pat dry liquid in hole per hole, add substrate to work per hole The μ L of liquid 90, lid ELISA Plate overlay film, 37 DEG C of incubations, observation adds the μ L/ holes of terminate liquid 50 per hole color change, in good time；
(15) light absorption value is measured：Absorbance is detected under ELIASA 450nm；
(16) cytokine secretion measures fixed：Calibration curve is drawn according to standard liquid absorbance, cytokine secretion is calculated Amount.
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