CN108004294A - A kind of method for detecting gum base type chewing tobacco cytotoxicity - Google Patents

A kind of method for detecting gum base type chewing tobacco cytotoxicity Download PDF

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Publication number
CN108004294A
CN108004294A CN201711341226.8A CN201711341226A CN108004294A CN 108004294 A CN108004294 A CN 108004294A CN 201711341226 A CN201711341226 A CN 201711341226A CN 108004294 A CN108004294 A CN 108004294A
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China
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cell
concentration
gum base
base type
chewing tobacco
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管莹
夭建华
李雪梅
高茜
米其利
朱洲海
徐玉琼
陆舍铭
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China Tobacco Yunnan Industrial Co Ltd
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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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics

Abstract

The present invention relates to a kind of method for detecting gum base type chewing tobacco cytotoxicity, belongs to tobacco and tobacco product security biological assessment technical field.This method includes sample pre-treatments, the preparation of single cell suspension, the cell concentration of single cell suspension calculate, tested material exposure, be incubated tested material, dyestuff is incubated, measure light absorption value, calculates cell inhibitory rate.The dissolution mode of integrated survey gum base type chewing tobacco of the present invention, route of exposure, establish gum base type chewing tobacco sample-pretreating method, target cell is acted on according to gum base type chewing tobacco and have chosen human mouth horn cell HOK as gum base type chewing tobacco cell toxicity test detection cell, and sample detection dosage is determined, the cell toxicity test detection method of suitable gum base type chewing tobacco is formd, is had a good application prospect.

Description

A kind of method for detecting gum base type chewing tobacco cytotoxicity
Technical field
The invention belongs to tobacco and tobacco product security biological assessment technical field, and in particular to one kind is used to detect The method of gum base type chewing tobacco cytotoxicity.
Background technology
Neutral RBC Toxicity method has the characteristics that easy, quick, sensitive, is to apply more detection cytotoxicity at present Method.International tobacco scientific research Cooperation Centre CORESTA, which is organized in, has set up the external toxicity test of cigarette smoke for 2002 Working group, by substantial amounts of literature survey and research work, which recommends using dimethyl diaminophenazine chloride cell toxicity test to cigarette The potential cytotoxicity of smoke condensate is detected, and the detection method is widely used by domestic and international tobacco company at present.
The increasingly stringent and smoking of tobacco supervision legislation and going deep into for health research, result in tobacco company and seek risk Novel tobacco product lower, without environment flue gas.Mouth containing tobacco product avoids complicated mixed caused by traditional cigarette product burns Harm caused by compound, has become one of new direction that foreign tobacco company turns to from traditional cigarette product at this stage.According to The component and Morphological Features of product, mouth containing tobacco product are divided into traditional Snus types buccal cigarette containing raw tobacco material and without raw tobacco materials Novel buccal cigarette（Such as gum base type chewing tobacco）.Gum base type chewing tobacco be one kind using tobacco or tobacco extract as active principle, with can Edible matrix is carrier, the novel tobacco product by the mode of chewing to human body delivery of nicotine.Tobacco or tobacco in mastication processes Extract and other food additives are released slowly into saliva, its delivery mode is different from traditional cigarette and containing raw tobacco material Snus type buccal cigarettes, are adapted to the cytotoxicity of gum base type chewing tobacco to try therefore, it is necessary to be established for gum base type chewing tobacco own characteristic Test detection method, it is ensured that the security of product.
The content of the invention
The purpose of the present invention is to solve the deficiencies in the prior art, the security of evaluation gum base type chewing tobacco, there is provided a kind of Method for detecting gum base type chewing tobacco cytotoxicity, this method are on the basis of traditional cigarette product cell toxicity test method It is upper to improve a kind of obtained test method for being suitable for the detection of gum base type chewing tobacco cytotoxicity.
To achieve the above object, the technical solution adopted by the present invention is as follows：
A kind of method for detecting gum base type chewing tobacco cytotoxicity, includes the following steps：
Step（1）, sample pre-treatments：Gum base type chewing tobacco product is added in 37 DEG C of oral cavity kerationcyte culture medium, matrix The quality of type chewing tobacco product and the volume ratio of oral cavity kerationcyte culture medium are 1g：10mL；After grinding 10min at 37 DEG C, Filter afterwards, serum is added into filtrate so that the final volume percentage concentration of serum is 10%, obtains prepare liquid；
Step（2）, the preparation of single cell suspension：By human mouth horn cell HOK in 37 DEG C, 5%CO2Oral cavity is used in incubator Kerationcyte culture medium culture, culture medium is removed when cell confluency rate is 80~90%, with the phosphate buffer that pH value is 7.2 Wash twice, abandon washing lotion；It is 0.25% to add concentration（w/v）Trypsin solution carry out individual layer and be incubated 1~2min, per 25cm2It is raw It is 0.25% that the cell of long area, which needs to add concentration,（w/v）1~2mL of trypsin solution, individual layer be incubated after add oral cavity Kerationcyte culture medium has hanged cell, obtains single cell suspension；
Step（3）, the cell concentration calculating of single cell suspension：With blood counting chamber counting method to step（2）Gained is unicellular The cell concentration of suspension is calculated, and calculates the viable count of every milliliter of single cell suspension；
Step（4）, cell inoculation：By step（3）Single cell suspension after counting is diluted to oral cavity kerationcyte culture medium 1.0~1.5 × 105A/mL, then be inoculated into by inoculum concentration for 200 μ L/ holes in Tissue Culture Plate, Tissue Culture Plate is put afterwards In 37 DEG C, 5%CO2Culture 24h in incubator；
Step（5）, tested material exposure：Tested material is divided into four groups：Blank control group, cell controls group, positive controls and detection Sample sets；
Removing step（4）Culture medium in middle Tissue Culture Plate, then be grouped as stated above, every group of multiple multiple holes, at the same time Required in the corresponding hole of blank control group acellular；Oral cavity cutin is added in blank control group and the corresponding hole of cell controls group Cell culture medium；Add the sodium dodecyl sulfate solution of 200 μ g/mL in the corresponding hole of positive controls；In detection sample sets Oral cavity kerationcyte culture medium and various dose step are added in corresponding hole（1）Gained prepare liquid, to the volume hundred of prepare liquid Point concentration more than 0%, less than or equal to 60% in the range of；Blank control group, cell controls group, positive controls and detection sample The final volume of group is 200 μ l/ holes；
Step（6）, it is incubated tested material：By step（5）Tissue Culture Plate after sample-adding is placed in 37 DEG C, 5%CO2It is incubated in incubator 24h；
Step（7）, dyestuff incubation：In step（6）Detection sample sets, blank control group, cell controls group and the positive after incubation Control group removes cell culture medium, adds concentration and is the 200 μ L/ holes of neutral red solution of 100 μ g/mL, then is placed in 37 DEG C, 5%CO2 Neutral red solution is removed after 4h is incubated in incubator, it is 1% to add concentration（v/v）200 μ L/ holes of formalin, after fixed 1min Formalin is removed, prepared 200 μ L/ holes of dimethyl diaminophenazine chloride extract is added afterwards, vibrates 10min afterwards；
The dimethyl diaminophenazine chloride extract is 50 according to volume ratio by absolute ethyl alcohol, glacial acetic acid and aseptic deionized water:1:49 ratios It is formulated；
Step（8）, measure light absorption value：By step（7）Detection sample sets, blank control group, cell controls group and sun after processing Property control group, light absorption value is detected with microplate reader under 540nm wavelength；
Step（9）, calculate cell inhibitory rate：According to step（8）Gained light absorption value, calculates cell inhibitory rate according to the following formula：
In formula：
Work as ODnDuring average light absorption value porous for positive controls, X is the cell inhibitory rate of positive controls；Work as ODnFor detection During the porous average light absorption value of a certain concentration of sample sets, X is the cell inhibitory rate under the detection sample concentration；
ODo--- step（8）The porous average light absorption value of gained blank control group；
ODc--- step（8）The porous average light absorption value of gained cell controls group；
First, the cell inhibitory rate of positive controls is calculated；When the cell inhibitory rate of positive controls is not more than 90%, then table Bright experiment is invalid, needs again according to step（1）- step（8）Tested；When the cell inhibitory rate of positive controls is more than 90% When, then show that experiment is effective, then calculate the cell inhibitory rate under detection each concentration of sample sets and draw cell inhibitory rate-concentration Curve, curve obtained characterizes the gum base type chewing tobacco cytotoxicity.
It is further preferred that step（1）The lapping mode is specially：Grinding rod grinds inverse after 1min clockwise Hour hands grind 1min, alternately, grinding rate 30rpm/min.
It is further preferred that step（1）The filter method is first carries out initial filter with qualitative filter paper, then with 0.45 μ The organic membrane filtrations of m.
It is further preferred that step（4）The Tissue Culture Plate is 96 porocyte culture plates.
It is further preferred that step（4）In, add oral cavity horn cell culture in the corresponding hole of detection sample sets Base and various dose step（1）Gained prepare liquid, the concentration expressed in percentage by volume to prepare liquid is respectively 10%, 20%, 30%, 40%, 50%、60%。
It is further preferred that step（5）During packet, each concentration, blank control group, cell pair of sample sets are detected 8 multiple holes are respectively provided with according to group and positive controls.
It is further preferred that step（7）The vibration is carried out in micropore plate oscillator, and rotating speed is 1000rpm。
Compared with prior art, the present invention its advantage is：
The present invention is the improvement carried out on the basis of the detection method of mouth containing tobacco product cytotoxicity, with sucking tobacco product cell toxicant Property detection method compare, the user of Snus type buccal cigarette buccal type of the mouth containing tobacco product pre-treatment simulation containing raw tobacco material Method, is that extraction is stood at 37 DEG C, is suitable for Snus type buccal cigarettes, and gum base type chewing tobacco is using edible matrix as carrier, is led to Cross novel tobacco product of the chewing mode to human body delivery of nicotine, stood at 37 DEG C extraction differed with the occupation mode that it is chewed compared with Greatly, its real use state can not effectively be simulated.Therefore, the exposure that the present invention passes through integrated survey gum base type chewing tobacco product Approach, the mode of action, establish buccal cigarette product sample pre-treating method, and people is have chosen according to mouth containing tobacco product effect target cell Oral cavity horn cell HOK determines sample detection dosage as gum base type chewing tobacco product cell toxicity test detection cell, Form the cell toxicity test detection method of suitable gum base type chewing tobacco product.Effective processing, the optimization of detection dosage of sample Setting and the correct selection of target cell so that this method can accurately and effectively detect the cell toxicant of gum base type chewing tobacco product Property.
Brief description of the drawings
Fig. 1 is cell inhibitory rate curve map.
Embodiment
With reference to embodiment, the present invention is described in further detail.
It will be understood to those of skill in the art that the following example is merely to illustrate the present invention, and it should not be regarded as limiting this hair Bright scope.In the examples where no specific technique or condition is specified, according to the described technology of document in the art or condition Or carried out according to product description.Production firm person is not specified in material therefor or equipment, is that can be obtained by buying Conventional products.
Kerationcyte culture medium OKM in oral cavity of the present invention, phosphate buffer are common commercial prod.
For the present invention unless otherwise stated, percentage sign represents percentage by volume, ratio is volume ratio.
Embodiment 1
A kind of method for detecting gum base type chewing tobacco cytotoxicity, includes the following steps：
Step（1）, sample pre-treatments：Gum base type chewing tobacco product is added in 37 DEG C of oral cavity kerationcyte culture medium, matrix The quality of type chewing tobacco product and the volume ratio of oral cavity kerationcyte culture medium are 1g：10mL；After grinding 10min at 37 DEG C, Filter afterwards, serum is added into filtrate so that the final volume percentage concentration of serum is 10%, obtains prepare liquid；
Step（2）, the preparation of single cell suspension：By human mouth horn cell HOK in 37 DEG C, 5%CO2Oral cavity is used in incubator Kerationcyte culture medium culture, culture medium is removed when cell confluency rate is 80~82%, with the phosphate buffer that pH value is 7.2 Wash twice, abandon washing lotion；It is 0.25% to add concentration（w/v）Trypsin solution carry out individual layer and be incubated 1min, per 25cm2Growth It is 0.25% that the cell of area, which needs to add concentration,（w/v）Trypsin solution 1mL, individual layer be incubated after add oral cavity cutin Cell culture medium has hanged cell, obtains single cell suspension；
Step（3）, the cell concentration calculating of single cell suspension：With blood counting chamber counting method to step（2）Gained is unicellular The cell concentration of suspension is calculated, and calculates the viable count of every milliliter of single cell suspension；
Step（4）, cell inoculation：By step（3）Single cell suspension after counting is diluted to oral cavity kerationcyte culture medium 1.0×105A/mL, then be inoculated into by inoculum concentration for 200 μ L/ holes in Tissue Culture Plate, Tissue Culture Plate is placed in 37 afterwards ℃、5%CO2Culture 24h in incubator；
Step（5）, tested material exposure：Tested material is divided into four groups：Blank control group, cell controls group, positive controls and detection Sample sets；
Removing step（4）Culture medium in middle Tissue Culture Plate, then be grouped as stated above, every group of multiple multiple holes, at the same time Required in the corresponding hole of blank control group acellular；Oral cavity cutin is added in blank control group and the corresponding hole of cell controls group Cell culture medium；Add the sodium dodecyl sulfate solution of 200 μ g/mL in the corresponding hole of positive controls；In detection sample sets Oral cavity kerationcyte culture medium and various dose step are added in corresponding hole（1）Gained prepare liquid, to the volume hundred of prepare liquid Point concentration more than 0%, less than or equal to 60% in the range of；Blank control group, cell controls group, positive controls and detection sample The final volume of group is 200 μ l/ holes；
Step（6）, it is incubated tested material：By step（5）Tissue Culture Plate after sample-adding is placed in 37 DEG C, 5%CO2It is incubated in incubator 24h；
Step（7）, dyestuff incubation：In step（6）Detection sample sets, blank control group, cell controls group and the positive after incubation Control group removes cell culture medium, adds concentration and is the 200 μ L/ holes of neutral red solution of 100 μ g/mL, then is placed in 37 DEG C, 5%CO2 Neutral red solution is removed after 4h is incubated in incubator, it is 1% to add concentration（v/v）200 μ L/ holes of formalin, after fixed 1min Formalin is removed, prepared 200 μ L/ holes of dimethyl diaminophenazine chloride extract is added afterwards, vibrates 10min afterwards；
The dimethyl diaminophenazine chloride extract is 50 according to volume ratio by absolute ethyl alcohol, glacial acetic acid and aseptic deionized water:1:49 ratios It is formulated；
Step（8）, measure light absorption value：By step（7）Detection sample sets, blank control group, cell controls group and sun after processing Property control group, light absorption value is detected with microplate reader under 540nm wavelength；
Step（9）, calculate cell inhibitory rate：According to step（8）Gained light absorption value, calculates cell inhibitory rate according to the following formula：
In formula：
Work as ODnDuring average light absorption value porous for positive controls, X is the cell inhibitory rate of positive controls；Work as ODnFor detection During the porous average light absorption value of a certain concentration of sample sets, X is the cell inhibitory rate under the detection sample concentration；
ODo--- step（8）The porous average light absorption value of gained blank control group；
ODc--- step（8）The porous average light absorption value of gained cell controls group；
First, the cell inhibitory rate of positive controls is calculated；When the cell inhibitory rate of positive controls is not more than 90%, then table Bright experiment is invalid, needs again according to step（1）- step（8）Tested；When the cell inhibitory rate of positive controls is more than 90% When, then show that experiment is effective, then calculate the cell inhibitory rate under detection each concentration of sample sets and draw cell inhibitory rate-concentration Curve, curve obtained characterizes the gum base type chewing tobacco cytotoxicity.
Embodiment 2
A kind of method for detecting gum base type chewing tobacco cytotoxicity, includes the following steps：
Step（1）, sample pre-treatments：Gum base type chewing tobacco product is added in 37 DEG C of oral cavity kerationcyte culture medium, matrix The quality of type chewing tobacco product and the volume ratio of oral cavity kerationcyte culture medium are 1g：10mL；After grinding 10min at 37 DEG C, Filter afterwards, serum is added into filtrate so that the final volume percentage concentration of serum is 10%, obtains prepare liquid；
Step（2）, the preparation of single cell suspension：By human mouth horn cell HOK in 37 DEG C, 5%CO2Oral cavity is used in incubator Kerationcyte culture medium culture, culture medium is removed when cell confluency rate is 88~90%, with the phosphate buffer that pH value is 7.2 Wash twice, abandon washing lotion；It is 0.25% to add concentration（w/v）Trypsin solution carry out individual layer and be incubated 2min, per 25cm2Growth It is 0.25% that the cell of area, which needs to add concentration,（w/v）Trypsin solution 2mL, individual layer be incubated after add oral cavity cutin Cell culture medium has hanged cell, obtains single cell suspension；
Step（3）, the cell concentration calculating of single cell suspension：With blood counting chamber counting method to step（2）Gained is unicellular The cell concentration of suspension is calculated, and calculates the viable count of every milliliter of single cell suspension；
Step（4）, cell inoculation：By step（3）Single cell suspension after counting is diluted to oral cavity kerationcyte culture medium 1.5×105A/mL, then be inoculated into by inoculum concentration for 200 μ L/ holes in 96 porocyte culture plates, Tissue Culture Plate is placed in afterwards 37℃、5%CO2Culture 24h in incubator；
Step（5）, tested material exposure：Tested material is divided into four groups：Blank control group, cell controls group, positive controls and detection Sample sets；
Removing step（4）Culture medium in middle Tissue Culture Plate, then be grouped as stated above, every group of multiple multiple holes, at the same time Required in the corresponding hole of blank control group acellular；Oral cavity cutin is added in blank control group and the corresponding hole of cell controls group Cell culture medium；Add the sodium dodecyl sulfate solution of 200 μ g/mL in the corresponding hole of positive controls；In detection sample sets Oral cavity kerationcyte culture medium and various dose step are added in corresponding hole（1）Gained prepare liquid, to the volume hundred of prepare liquid Point concentration more than 0%, less than or equal to 60% in the range of；Blank control group, cell controls group, positive controls and detection sample The final volume of group is 200 μ l/ holes；
Step（6）, it is incubated tested material：By step（5）Tissue Culture Plate after sample-adding is placed in 37 DEG C, 5%CO2It is incubated in incubator 24h；
Step（7）, dyestuff incubation：In step（6）Detection sample sets, blank control group, cell controls group and the positive after incubation Control group removes cell culture medium, adds concentration and is the 200 μ L/ holes of neutral red solution of 100 μ g/mL, then is placed in 37 DEG C, 5%CO2 Neutral red solution is removed after 4h is incubated in incubator, it is 1% to add concentration（v/v）200 μ L/ holes of formalin, after fixed 1min Formalin is removed, prepared 200 μ L/ holes of dimethyl diaminophenazine chloride extract is added afterwards, vibrates 10min afterwards；
The dimethyl diaminophenazine chloride extract is 50 according to volume ratio by absolute ethyl alcohol, glacial acetic acid and aseptic deionized water:1:49 ratios It is formulated；
Step（8）, measure light absorption value：By step（7）Detection sample sets, blank control group, cell controls group and sun after processing Property control group, light absorption value is detected with microplate reader under 540nm wavelength；
Step（9）, calculate cell inhibitory rate：According to step（8）Gained light absorption value, calculates cell inhibitory rate according to the following formula：
In formula：
Work as ODnDuring average light absorption value porous for positive controls, X is the cell inhibitory rate of positive controls；Work as ODnFor detection During the porous average light absorption value of a certain concentration of sample sets, X is the cell inhibitory rate under the detection sample concentration；
ODo--- step（8）The porous average light absorption value of gained blank control group；
ODc--- step（8）The porous average light absorption value of gained cell controls group；
First, the cell inhibitory rate of positive controls is calculated；When the cell inhibitory rate of positive controls is not more than 90%, then table Bright experiment is invalid, needs again according to step（1）- step（8）Tested；When the cell inhibitory rate of positive controls is more than 90% When, then show that experiment is effective, then calculate the cell inhibitory rate under detection each concentration of sample sets and draw cell inhibitory rate-concentration Curve, curve obtained characterizes the gum base type chewing tobacco cytotoxicity.
Wherein, step（1）The lapping mode is specially：Grinding rod is ground counterclockwise after grinding 1min clockwise 1min, alternately, grinding rate 30rpm/min；The filter method is first to carry out initial filter with qualitative filter paper, then is used 0.45 μm of organic membrane filtration.
Embodiment 3
A kind of method for detecting gum base type chewing tobacco cytotoxicity, includes the following steps：
Step（1）, sample pre-treatments：Gum base type chewing tobacco product is added in 37 DEG C of oral cavity kerationcyte culture medium, matrix The quality of type chewing tobacco product and the volume ratio of oral cavity kerationcyte culture medium are 1g：10mL；After 37 DEG C of thermostat water bath It is middle to grind 10min with mill, filter afterwards, serum is added into filtrate so that the final volume percentage concentration of serum is 10%, obtain prepare liquid；
Step（2）, the preparation of single cell suspension：By human mouth horn cell HOK in 37 DEG C, 5%CO2Oral cavity is used in incubator Kerationcyte culture medium culture, culture medium is removed when cell confluency rate is 83~87%, with the phosphate buffer that pH value is 7.2 Wash twice, abandon washing lotion；It is 0.25% to add concentration（w/v）Trypsin solution carry out individual layer and be incubated 1.5min, per 25cm2It is raw It is 0.25% that the cell of long area, which needs to add concentration,（w/v）Trypsin solution 1.5mL, individual layer be incubated after add oral cavity Kerationcyte culture medium has hanged cell, obtains single cell suspension；
Step（3）, the cell concentration calculating of single cell suspension：With blood counting chamber counting method to step（2）Gained is unicellular The cell concentration of suspension is calculated, and calculates the viable count of every milliliter of single cell suspension；
Step（4）, cell inoculation：By step（3）Single cell suspension after counting is diluted to oral cavity kerationcyte culture medium 1.2×105A/mL, then be inoculated into by inoculum concentration for 200 μ L/ holes in 96 porocyte culture plates, Tissue Culture Plate is placed in afterwards 37℃、5%CO2Culture 24h in incubator；
Step（5）, tested material exposure：Tested material is divided into four groups：Blank control group, cell controls group, positive controls and detection Sample sets；Blank control group and cell controls group add oral cavity kerationcyte culture medium；Positive controls add the 12 of 200 μ g/mL Sodium alkyl sulfate solution；Detection sample sets add various dose step（1）Gained prepare liquid；
Removing step（4）Culture medium in middle Tissue Culture Plate, then be grouped as stated above, every group of multiple multiple holes, at the same time Required in the corresponding hole of blank control group acellular；Oral cavity cutin is added in blank control group and the corresponding hole of cell controls group Cell culture medium；Add the sodium dodecyl sulfate solution of 200 μ g/mL in the corresponding hole of positive controls；In detection sample sets Oral cavity kerationcyte culture medium and step are added in corresponding hole（1）Gained prepare liquid, to the concentration expressed in percentage by volume point of prepare liquid Wei 10%, 20%, 30%, 40%, 50%, 60%；Blank control group, cell controls group, positive controls and the end for detecting sample sets Volume is 200 μ l/ holes；
During packet, each concentration, blank control group, cell controls group and the positive controls that detect sample sets are respectively provided with 8 again Hole.
Step（6）, it is incubated tested material：By step（5）Tissue Culture Plate after sample-adding is placed in 37 DEG C, 5%CO2In incubator It is incubated 24h；
Step（7）, dyestuff incubation：In step（6）Detection sample sets, blank control group, cell controls group and the positive after incubation Control group removes cell culture medium, adds concentration and is the 200 μ L/ holes of neutral red solution of 100 μ g/mL, then is placed in 37 DEG C, 5%CO2 Neutral red solution is removed after 4h is incubated in incubator, it is 1% to add concentration（v/v）200 μ L/ holes of formalin, after fixed 1min Formalin is removed, adds prepared 200 μ L/ holes of dimethyl diaminophenazine chloride extract afterwards, afterwards the 1000rpm in micropore plate oscillator Vibrate 10min；
The dimethyl diaminophenazine chloride extract is 50 according to volume ratio by absolute ethyl alcohol, glacial acetic acid and aseptic deionized water:1:49 ratios It is formulated；
Step（8）, measure light absorption value：By step（7）Detection sample sets, blank control group, cell controls group and sun after processing Property control group, with microplate reader under 540nm wavelength（Dimethyl diaminophenazine chloride method）Detect light absorption value；
Step（9）, calculate cell inhibitory rate：According to step（8）Gained light absorption value, calculates cell inhibitory rate according to the following formula：
In formula：
Work as ODnDuring average light absorption value porous for positive controls, X is the cell inhibitory rate of positive controls；Work as ODnFor detection During the porous average light absorption value of a certain concentration of sample sets, X is the cell inhibitory rate under the detection sample concentration；
ODo--- step（8）The porous average light absorption value of gained blank control group；
ODc--- step（8）The porous average light absorption value of gained cell controls group；
First, the cell inhibitory rate of positive controls is calculated；When the cell inhibitory rate of positive controls is not more than 90%, then table Bright experiment is invalid, needs again according to step（1）- step（8）Tested；When the cell inhibitory rate of positive controls is more than 90% When, then show that experiment is effective, then calculate the cell inhibitory rate under detection each concentration of sample sets and draw cell inhibitory rate-concentration Curve, curve obtained characterizes the gum base type chewing tobacco cytotoxicity.
Wherein, step（1）The lapping mode is specially：Grinding rod is ground counterclockwise after grinding 1min clockwise 1min, alternately, grinding rate 30rpm/min；The filter method is first to carry out initial filter slagging-off with qualitative filter paper, then It is degerming with 0.45 μm of organic membrane filtration.
1 cell toxicity test of table is loaded table
Application example
Using 3 method of the embodiment of the present invention, 201510193142.9 methods respectively to commercially available three kinds of gum base types chewing tobacco product Cytotoxicity is detected, and does parallel laboratory test twice, the results are shown in Table 2.
2 three kinds of gum base type type mouth containing tobacco product cell toxicity test testing results of table
Wherein, it is cell controls group when prepare liquid concentration is 0%.
The cell inhibitory rate of positive controls is more than 90%, it was demonstrated that test system is effectively, normally.
Cell inhibitory rate curve is drawn according to the cell inhibitory rate under sample difference test concentrations.Shown in the result is shown in Figure 1.
Sample treatment, detection cell, the detection dosage provided it can be seen from upper table result in the method for the present invention Under the conditions of, there is dose-effect relationship, positive control cell between three kinds of gum base type chewing tobacco product cell inhibitory rates and detection dosage Inhibiting rate is more than 90%, it was demonstrated that this test system is normal, and detection data are effective.And use in 201510193142.9 inventions and fit Shown together in the methods and results of Snus type buccal cigarettes, in the range of detection dosage three kinds of gum base type chewing tobacco product cell inhibitory rates with Without dose-effect relationship between detection dosage, this is probably due to using in 201510193142.9 three kinds of tested gum base type chewing tobacco Inclusion release is incomplete, it is impossible to truly reflects its cytotoxicity.
The signified dose-effect relationship that has of the invention is generic term in the art, i.e., with the rising of dosage, suppresses Rate is regular to be increased or reduces.
The present invention is the improvement carried out on the basis of the detection method of mouth containing tobacco product cytotoxicity, thin with mouth containing tobacco product The detection method of cellular toxicity compares, and mouth containing tobacco product pre-treatment Snus type buccal cigarette buccal type of the simulation containing raw tobacco material makes It is that extraction is stood at 37 DEG C with method, is suitable for Snus type buccal cigarettes, and gum base type chewing tobacco is using edible matrix as load Body, the novel tobacco product by the mode of chewing to human body delivery of nicotine, the occupation mode that extraction is chewed with it is stood at 37 DEG C Differ larger, can not effectively simulate its real use state.
The route of exposure of integrated survey gum base type chewing tobacco product of the present invention, the mode of action, establish buccal cigarette product sample Pre-treating method, human mouth horn cell HOK is have chosen as gum base type chewing tobacco product according to mouth containing tobacco product effect target cell Cell toxicity test detection cell, and sample detection dosage is determined, form the cell toxicant of suitable gum base type chewing tobacco product Property method for testing and detecting.The correct selection of effective processing of sample, the optimal setting of detection dosage and target cell so that we Method can accurately and effectively detect the cytotoxicity of gum base type chewing tobacco product.
Basic principle, main feature and the advantages of the present invention of the present invention has been shown and described above.The technology of the industry Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this The principle of invention, without departing from the spirit and scope of the present invention, various changes and modifications of the present invention are possible, these changes Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its Equivalent thereof.

Claims (7)

  1. A kind of 1. method for detecting gum base type chewing tobacco cytotoxicity, it is characterised in that include the following steps：
    Step（1）, sample pre-treatments：Gum base type chewing tobacco product is added in 37 DEG C of oral cavity kerationcyte culture medium, matrix The quality of type chewing tobacco product and the volume ratio of oral cavity kerationcyte culture medium are 1g：10mL；After grinding 10min at 37 DEG C, Filter afterwards, serum is added into filtrate so that the final volume percentage concentration of serum is 10%, obtains prepare liquid；
    Step（2）, the preparation of single cell suspension：By human mouth horn cell HOK in 37 DEG C, 5%CO2Oral cavity angle is used in incubator Cell plastid medium culture, culture medium is removed when cell confluency rate is 80~90%, is washed with the phosphate buffer that pH value is 7.2 Twice, washing lotion is abandoned；It is 0.25% to add concentration（w/v）Trypsin solution carry out individual layer and be incubated 1~2min, per 25cm2Growth It is 0.25% that the cell of area, which needs to add concentration,（w/v）1~2mL of trypsin solution, individual layer be incubated after add oral cavity angle Cell plastid culture medium has hanged cell, obtains single cell suspension；
    Step（3）, the cell concentration calculating of single cell suspension：With blood counting chamber counting method to step（2）Gained is unicellular The cell concentration of suspension is calculated, and calculates the viable count of every milliliter of single cell suspension；
    Step（4）, cell inoculation：By step（3）Single cell suspension after counting is diluted to oral cavity kerationcyte culture medium 1.0~1.5 × 105A/mL, then be inoculated into by inoculum concentration for 200 μ L/ holes in Tissue Culture Plate, Tissue Culture Plate is put afterwards In 37 DEG C, 5%CO2Culture 24h in incubator；
    Step（5）, tested material exposure：Tested material is divided into four groups：Blank control group, cell controls group, positive controls and detection Sample sets；
    Removing step（4）Culture medium in middle Tissue Culture Plate, then be grouped as stated above, every group of multiple multiple holes, at the same time Required in the corresponding hole of blank control group acellular；Oral cavity cutin is added in blank control group and the corresponding hole of cell controls group Cell culture medium；Add the sodium dodecyl sulfate solution of 200 μ g/mL in the corresponding hole of positive controls；In detection sample sets Oral cavity kerationcyte culture medium and various dose step are added in corresponding hole（1）Gained prepare liquid, to the volume hundred of prepare liquid Point concentration more than 0%, less than or equal to 60% in the range of；Blank control group, cell controls group, positive controls and detection sample The final volume of group is 200 μ l/ holes；
    Step（6）, it is incubated tested material：By step（5）Tissue Culture Plate after sample-adding is placed in 37 DEG C, 5%CO2It is incubated in incubator 24h；
    Step（7）, dyestuff incubation：In step（6）Detection sample sets, blank control group, cell controls group and the positive after incubation Control group removes cell culture medium, adds concentration and is the 200 μ L/ holes of neutral red solution of 100 μ g/mL, then is placed in 37 DEG C, 5%CO2 Neutral red solution is removed after 4h is incubated in incubator, it is 1% to add concentration（v/v）200 μ L/ holes of formalin, after fixed 1min Formalin is removed, prepared 200 μ L/ holes of dimethyl diaminophenazine chloride extract is added afterwards, vibrates 10min afterwards；
    The dimethyl diaminophenazine chloride extract is 50 according to volume ratio by absolute ethyl alcohol, glacial acetic acid and aseptic deionized water:1:49 ratios It is formulated；
    Step（8）, measure light absorption value：By step（7）Detection sample sets, blank control group, cell controls group and sun after processing Property control group, light absorption value is detected with microplate reader under 540nm wavelength；
    Step（9）, calculate cell inhibitory rate：According to step（8）Gained light absorption value, calculates cell inhibitory rate according to the following formula：
    In formula：
    Work as ODnDuring average light absorption value porous for positive controls, X is the cell inhibitory rate of positive controls；Work as ODnFor detection During the porous average light absorption value of a certain concentration of sample sets, X is the cell inhibitory rate under the detection sample concentration；
    ODo--- step（8）The porous average light absorption value of gained blank control group；
    ODc--- step（8）The porous average light absorption value of gained cell controls group；
    First, the cell inhibitory rate of positive controls is calculated；When the cell inhibitory rate of positive controls is not more than 90%, then table Bright experiment is invalid, needs again according to step（1）- step（8）Tested；When the cell inhibitory rate of positive controls is more than 90% When, then show that experiment is effective, then calculate the cell inhibitory rate under detection each concentration of sample sets and draw cell inhibitory rate-concentration Curve, curve obtained characterizes the gum base type chewing tobacco cytotoxicity.
  2. 2. the method according to claim 1 for detecting gum base type chewing tobacco cytotoxicity, it is characterised in that step（1） The lapping mode is specially：Grinding rod grinds 1min counterclockwise after grinding 1min clockwise, and alternately, grinding rate is 30rpm/min。
  3. 3. the method according to claim 1 for detecting gum base type chewing tobacco cytotoxicity, it is characterised in that step（1） The filter method is first carries out initial filter with qualitative filter paper, then with 0.45 μm of organic membrane filtration.
  4. 4. the method according to claim 1 for detecting gum base type chewing tobacco cytotoxicity, it is characterised in that step（4） The Tissue Culture Plate is 96 porocyte culture plates.
  5. 5. the method according to claim 1 for detecting gum base type chewing tobacco cytotoxicity, it is characterised in that step（4） In, add oral cavity kerationcyte culture medium and various dose step in the corresponding hole of detection sample sets（1）Gained prepare liquid, extremely The concentration expressed in percentage by volume of prepare liquid is respectively 10%, 20%, 30%, 40%, 50%, 60%.
  6. 6. the method according to claim 1 for detecting gum base type chewing tobacco cytotoxicity, it is characterised in that step（5） During packet, each concentration, blank control group, cell controls group and the positive controls that detect sample sets are respectively provided with 8 multiple holes.
  7. 7. the method according to claim 1 for detecting gum base type chewing tobacco cytotoxicity, it is characterised in that step（7） The vibration is carried out in micropore plate oscillator, rotating speed 1000rpm.
CN201711341226.8A 2017-12-14 2017-12-14 A kind of method for detecting gum base type chewing tobacco cytotoxicity Pending CN108004294A (en)

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Publication number Priority date Publication date Assignee Title
US3106210A (en) * 1957-11-18 1963-10-08 Reynolds Metals Co Smoking tobacco
CN104789633A (en) * 2015-04-22 2015-07-22 云南中烟工业有限责任公司 Test method for detecting cytotoxicity of buccal tobacco products
CN105852191A (en) * 2016-05-26 2016-08-17 云南中烟工业有限责任公司 Gum base type chewing tobacco and preparation method thereof

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Publication number Priority date Publication date Assignee Title
US3106210A (en) * 1957-11-18 1963-10-08 Reynolds Metals Co Smoking tobacco
CN104789633A (en) * 2015-04-22 2015-07-22 云南中烟工业有限责任公司 Test method for detecting cytotoxicity of buccal tobacco products
CN105852191A (en) * 2016-05-26 2016-08-17 云南中烟工业有限责任公司 Gum base type chewing tobacco and preparation method thereof

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