CN106367468A - Method of detecting influence on human oral cell [gamma] H2AX level due to tobacco chewing gum - Google Patents
Method of detecting influence on human oral cell [gamma] H2AX level due to tobacco chewing gum Download PDFInfo
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- CN106367468A CN106367468A CN201610729383.5A CN201610729383A CN106367468A CN 106367468 A CN106367468 A CN 106367468A CN 201610729383 A CN201610729383 A CN 201610729383A CN 106367468 A CN106367468 A CN 106367468A
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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
- G01N33/502—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 non-proliferative effects
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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
- G01N33/5044—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
Abstract
The invention discloses a method of detecting influence on human oral cell [gamma] H2AX level due to tobacco chewing gum. The method includes the steps of: 1) freezing to-be-tested tobacco chewing gum in liquid nitrogen; 2) instantly crushing the tobacco chewing gum by means of a crusher; 3) freezing the crushed tobacco chewing gum in the liquid nitrogen; 4) soaking the crushed tobacco chewing gum in artificial saliva; 5) centrifugally separating an extract liquid; 6) detecting a supernatant liquid by means of an osmotic pressure measuring instrument and regulating the osmotic pressure to be consistent with that of a cell culture medium; 7) measuring the content of nicotine in the extract liquid; 8) culturing human oral mucosa epithelial cells as a detection object; 9) adding the extract liquid of the tobacco chewing gum to the cultured human oral mucosa epithelial cells; and 10) culturing the cells for 4 hours, and detecting the change situation of the [gamma] H2AX of the cells by means of a high-content imaging instrument.
Description
Technical field
The present invention relates to biological technology application, it is applied especially to gum base type tobacco product for human mouth cell
The mensure of rh2ax impact.
Background technology
Gum base type smoke-free tobacco product (tobacco chewing gum) be one kind add in gum base a certain amount of
A kind of mouth smoke-free tobacco product that tobacco extract, spice and some other edible additive are made.Gum base type Nicotiana tabacum L.
The occupation mode of product is to put in mouth to chew, and the leachable of chewing can converge gathering in the oral cavity with saliva, is practised according to consumption
Used difference spues or swallows.Because this product mainly acts on oral cavity, directly contacted with Stomatocyte, therefore having must
Set up a kind of its method on the impact of human mouth cell γ h2ax level of detection, to be estimated to its safety.
Existing tobacco product method for evaluating safety is primarily directed to traditional cigarette, the typically flue gas to traditional cigarette
Carry out trapping and carry out follow-up test biology again.But gum base type tobacco product does not produce flue gas, and directly acts on mouth
The security assessment method in chamber, therefore traditional cigarette is not particularly suited for this product.And packed mouth containing tobacco product is typically using being attached to
Mode between gingiva and lip uses, and its inclusions is leachable using common infusion method, and gum base type tobacco product is due to glue
The presence of base, direct infusion method major part inclusions are difficult to dissolution, thus biological safety test cannot accurately be carried out.
Content of the invention
The present invention is directed to the feature of gum base type product, in conjunction with biological detection method, establishes a kind of detection gum base type cigarette
The method that straw-made articless affect on human mouth cell γ h2ax level, the safety evaluation for gum base type tobacco product provides reference.
The present invention disclose a kind of detection gum base type tobacco product γ thin on human mouth h2ax level impact method, including with
Lower step:
(1) described gum base type tobacco product to be measured is placed in freezing 20-40min in liquid nitrogen；
(2) the described gum base type tobacco product freezing step (1), is pulverized with pulverizer immediately, collects after pulverizing
Unqualified gum base type tobacco product；
(3) again described unqualified gum base type tobacco product is placed in freezing in liquid nitrogen；
(4) 20-40min is soaked in the described unqualified gum base type tobacco product artificial saliva that step (3) obtains, every 2-
5min stirs once；The temperature of described artificial saliva is 36-38 DEG C；
(5) the extracting solution centrifugation 5-10min that step (4) is obtained；
(6) supernatant being obtained with osmotic tester determination step (5), adjusts its osmotic pressure and cell culture medium one
Cause, obtain gum base type tobacco product extracting solution；
(7) measure the nicotine content in extracting solution；
(8) culture human mouth mucosal epithelial cells are as detection object；
(9) the gum base type tobacco product extracting solution obtaining in step (6) is added to cultured human mouth in step (8)
In mucosal epithelial cells；
(10) after the short cultivation of 4 hours, detect cell γ h2ax situation of change with high intension imager.
Preferably, the cultural method in step (8) includes step in detailed below:
(8-1) extraction of oral cavity mucous membrane tissue: take the oral cavity mucous membrane tissue of less than 6 years old Patients with Cleft Lip and Palate；
(8-2) with the concentration containing penicillin be 100ug/ml, streptomycin be 50ug/ml phosphate buffer rinsing step
(8-1) oral cavity mucous membrane tissue 2-3 time；
(8-3) shearing of piece of tissue: remove the sub-mucosal tissues in (8-2) with eye scissorss, and the mucosa after pruning is cut
Become the fritter of about 5mm × 5mm, be subsequently adding 0.25% neutral protease；
(8-4) the oral mucosa fritter of step (8-3) is digested 16-18h at 4 DEG C, then by surface epithelium and epithelium
Lower floor is separately；
(8-5) separation of epithelial cell: the epithelial layer that step (8-4) is obtained, plus people 0.25% trypsin is at 36 DEG C
Lower digestion 15m in, every 5m in piping and druming is vibrated 1 time, is subsequently adding the dmem/f12 culture medium containing 10% hyclone, blows and beats into
Single cell suspension, centrifugation, rinsed 1-2 time with phosphate buffer (pbs), be then added to add in advance somatomedin
In keratinocyte culture medium；
(8-6) original cuiture of epithelial cell: the epithelial cell that step (8-5) is obtained is made after single cell suspension with carefully
Born of the same parents count, with 1-2 × 105The density of individual/ml cell is inoculated in 25cm2In culture bottle, put into 37 DEG C, 5%co2Train in incubator
Support；Culture fluid is changed first, every 2d changes culture fluid once later after 3d；
(8-7) Secondary Culture of epithelial cell: pass on when the epithelial cell of step (8-6) reaches cover bottom of bottle 80%
Culture, with 0.25% trypsinization, treats that epithelial cell becomes round, with the dmem/f12 training containing 10% hyclone
Foster base terminates digestion, centrifugation, adds the keratinocyte culture medium adding somatomedin in advance, makes epithelial cell dense
Degree reaches 5 × 104Individual/ml；Single cell suspension is planted in 96 porocyte culture plates, inoculum concentration is 200 μ l/ holes, will plant
96 good porocyte culture plates are positioned over 37 DEG C, 5%co2CO2 gas incubator in culture 24h；
Preferably, step (10) includes step in detailed below:
(10-1) incubation of tested material: the gum base type tobacco product extracting solution obtaining in step (6) is added step (8-7)
In the epithelial cell obtaining, using sample nicotine amount as detection dosage Classification Index；Blank control group only adds culture medium, solvent
Matched group adds artificial saliva；Tissue Culture Plate is positioned over 37 DEG C, 5%co2CO2 gas incubator in culture 24h；
(10-2) human mouth mucosal epithelial cells are fixed: with the cell in phosphate buffer washing step (10-1), then
Fix cell 10min with 4% paraformaldehyde solution (prepared before use), then wash 2 times with pbs；
(10-3) human mouth mucosal epithelial cells film is changed thoroughly: by the cell in step (10-2) with containing 0.2%tritonx-
100 phosphate buffer changes 10min, phosphate buffer washed cell 2 times thoroughly；
(10-4) human mouth mucosal epithelial cells γ h2ax dyeing: with the γ h2ax antibody containing 10 μ g/ml fitc labellings
Phosphate buffer dyes to the cell in step (10-3), and lucifuge dyes 2h, then with pbs washed cell 2 times；
(10-5) human mouth mucosal epithelial cells hoechst dyeing: with the phosphate buffer pair containing 5 μ g/ml hoechst
Nucleus in step (10-4) carry out lucifuge dyeing 15min；
(10-6) high intension imaging system detection: select fitc the and hoechst fluorescence dual pathways in step (10-5)
Cell is scanned obtaining picture；
(10-7) graphical analyses: using high intension analysis software, the picture in (10-6) is analyzed, measures hoechst
The average diameter of passage cell core, to arrange the diameter parameters of individual cells core, is joined according to the distance between nucleus setting distance
Number；According to the above software that sets, picture is identified, and calculate individual cells core fitc and hoechst passage single thin
Karyon Mean Fluorescence；With the individual cells core Mean Fluorescence of hoechst passage as comparison, compare fitc passage single thin
The change of karyon Mean Fluorescence, evaluates dna degree of injury with it.
Beneficial effect of the present invention
1st, the present invention is difficult to the feature extracted using direct infusion method in view of gum base type tobacco product, using super
Pulverize after (about -196 DEG C) freezings of low temperature, this for the gum base material sticking sticky paste can be effectively ground into powder by this, then will
Powder again with nitrogen ultra low temperature freezing after, be added rapidly in room temperature (36-38 DEG C) artificial saliva, using from deep cooling rapidly to
This shock heating effect of room temperature, promotes powder to crush further because drastically expanding with heat and contract with cold, and improves the contact with extracting solution for the powder
Area, thus improve the efficiency of extraction chemistry material from powder.
2nd, because gum base type tobacco product adds the shadows such as more sugar or salinity generally for strengthening mouth comfort
Ring the material of liquid infiltration pressure, cause cell if directly adding extracting solution and being easily caused due to the problem of osmotic pressure in cell
Death, the therefore present invention have adjusted the osmotic pressure of extracting solution before processing cell, thus improving the accuracy of experiment.
3rd, for simulating actual conditions of trying one's best, the present invention employs artificial saliva when extracting, and simulates close to population
The temperature in chamber and edible time, have selected human mouth cell in detection object, and in order to as far as possible close to the reality of human mouth cell
Border situation, the present invention has cultivated normal person's Buccal mucosa cell as detection object using the method for original cuiture, compares
More conventionally employed zooblast or the detection of cancerous cell, improve the reliability of test.
4th, general dna injury experiment haves the characteristics that complex operation and does not have intuitive, and the present invention adopts γ h2ax
Immunofluorescence high intension method, the method has sensitivity, quick (within 4h) and intuitively feature, can rapidly and efficiently detect greatly
Batch gum base type tobacco product affects on the γ h2ax of human mouth cell.
Specific embodiment
1. a kind of gum base type of random choose tobacco product (n-2, nicotine content 2mg/), random choose 2 puts into sample
In QC, sample cell is placed in freezing 30min in liquid nitrogen.
2. sample cell is taken out from liquid nitrogen, further takes out gum base type tobacco product (n-2), immediately with Sample Grinder by its
Pulverize, collected with sample cell unqualified after pulverizing.
3. will be equipped with the unqualified sample cell of gum base type tobacco product and be again placed in liquid nitrogen freezing 30min；
4. take out sample cell, the artificial saliva adding 10ml to be preheated to 37 DEG C immediately from liquid nitrogen, soak the glue after pulverizing
Fundamental mode tobacco product unqualified (n-2), using the extraction conditions of 37 DEG C, 30min, and stirs extraction every 2min in immersion process
Liquid is so that sample and extracting solution are fully contacted the dissolution thus beneficial to inclusions.
5. extracting solution is placed in 15ml centrifuge tube, 10min is with precipitation solid thing for centrifugation.
6. draw supernatant with pipettor to be placed in new centrifuge tube, and measure the infiltration of extracting solution using osmotic tester
Pressure, adjusts its osmotic pressure consistent with cell culture medium.
7. measure the concentration of the nicotine in extracting solution, to verify extraction efficiency.
8. what people's Normal oral mucosa was organized draws materials: selects less than 6 years old Patients with Cleft Lip and Palate, retains and cut in Unilateral Cleft Lip Repair
The unnecessary oral cavity mucous membrane tissue removing, is immediately placed in the sterile tube holding dmem/f12 culture medium, is sent to laboratory treatment.
9. the flushing of piece of tissue: with containing dual anti-(the final concentration of 100ug/ml of penicillin, streptomycin on superclean bench
For 50ug/ml) pbs (phosphate buffer) repeatedly rinse 3 times, remove piece of tissue on attachment blood stains and antibacterial.
10. the shearing of piece of tissue: remove sub-mucosal tissues with eye scissorss, be cut into the fritter of about 5mm × 5mm, add
0.25%dispase (neutral protease).
The digestion of 11. piece of tissue: fritter is put into 4 DEG C of refrigerator digestion 16-18h, with ophthalmic tweezers by surface epithelium and epithelium
Lower floor is separately.
The separation of 12. epithelial cells: prune epithelial layer and become 1mm3Fritter, plus 36 DEG C of people's 0.25% trypsin digestion
15m in (5m in piping and druming vibration 1 time), removes and does not digest completely cell block, add the dmem/f12 containing 10% hyclone
Culture medium terminates digestion, blows and beats into single cell suspension with suction pipe.1000r/min is centrifuged 5min, abandons supernatant, is rinsed once with pbs,
Add the ksfm culture medium (keratinocyte culture medium) adding somatomedin in advance.
The original cuiture of 13. epithelial cells: human mouth mucosal epithelial cells use cell counting after making single cell suspension, with
1-2×105The density of individual/ml cell is inoculated in 25cm2In culture bottle, put into 37 DEG C, 5%co2Cultivate in incubator.First after 3d
Secondary change liquid, every 2d changes liquid once later.
The Secondary Culture of 14. epithelial cells: Secondary Culture when cell reaches cover bottom of bottle 80%, with 0.25% pancreas
Protease digestion, Microscopic observation cell retraction situation, treat that cell becomes round, with the dmem/f12 culture containing 10% hyclone
Base terminates digestion, and 1000r/min is centrifuged 5min, abandons supernatant, adds and adds the ksfm culture medium of somatomedin in advance (cutin is formed
Cell culture medium), make cell concentration reach 5 × 104Individual/ml.Single cell suspension is planted in 96 porocyte culture plates, connects
The amount of kind is 200 μ l/ holes, and planted 96 porocyte culture plates are positioned over 37 DEG C, 5%co2CO2 gas incubator in culture
24h.
15. test packets: test is divided into three groups, respectively blank control group, solvent control group and test sample group, often
Individual test group do 3 parallel.
The incubation of 16. tested materials: the gum base type tobacco product extracting solution obtaining in (6) is added the human mouth in (14) stick
In film epithelial cell, using sample nicotine amount as detection dosage Classification Index；Blank control group only adds culture medium, solvent control
Group adds artificial saliva.Tissue Culture Plate is positioned over 37 DEG C, 5%co2CO2 gas incubator in culture 24h；
17. human mouth mucosal epithelial cells are fixed: with the cell in phosphate buffer washing step (16), then with 4%
Paraformaldehyde solution (prepared before use) fixes cell 10min, then washs 2 times with pbs；
18. human mouth mucosal epithelial cells films are changed thoroughly: by the cell in step (17) with containing 0.2%tritonx-100's
Phosphate buffer changes 10min, phosphate buffer washed cell 2 times thoroughly；
19. human mouth mucosal epithelial cells γ h2ax dyeing: with the phosphorus of the γ h2ax antibody containing 10 μ g/ml fitc labellings
Acid buffer dyes to the cell in step (18), and lucifuge dyes 2h, then with pbs washed cell 2 times；
20. human mouth mucosal epithelial cells hoechst dyeing: with the phosphate buffer containing 5 μ g/ml hoechst to step
(19) nucleus in carry out lucifuge dyeing 15min；
21. high intension imaging system detections: select fitc the and hoechst fluorescence dual pathways that the cell in step (20) is entered
Row scanning obtains picture；
22. graphical analyses: using high intension analysis software, the picture in (21) is analyzed, measures hoechst passage
Nuclear average diameter, to arrange the diameter parameters of individual cells core, arranges distance parameter according to the distance between nucleus.
According to the above software that sets, picture is identified, and calculates the individual cells of the fitc and hoechst passage of individual cells core
Core Mean Fluorescence.With the individual cells core Mean Fluorescence of hoechst passage as comparison, compare fitc passage individual cells
The change of core Mean Fluorescence, evaluates dna degree of injury with it.
Table 1 be variable concentrations (concentration nicotine concentration identify, respectively 12.5 μ g/ml, 25 μ g/ml, 50 μ g/ml,
100 μ g/ml) extracting solution process human mouth cell, measure the impact to human mouth mucosal epithelial cells γ h2ax for the extracting solution.By
Table 1 can be seen that the n-2 type gum base type tobacco product γ h2ax that inducing cell produces in the range of study dosage and study dosage
Between no dose-effect relationship, positive controls micronuclear rateses γ h2ax compared with negative control group dramatically increases it was demonstrated that this test
System is normal, and detection data is effective.Table 1 n-2 type gum base type tobacco product γ h2ax testing inspection result
Claims (3)
1. a kind of method that detection gum base type tobacco product affects on human mouth cell γ h2ax level is it is characterised in that include
Following steps:
(1) described gum base type tobacco product to be measured is placed in freezing 20-40min in liquid nitrogen；
(2) the described gum base type tobacco product freezing step (1), is pulverized with pulverizer immediately, collects broken after pulverizing
Last gum base type tobacco product；
(3) again described unqualified gum base type tobacco product is placed in freezing in liquid nitrogen；
(4) 20-40min is soaked in the described unqualified gum base type tobacco product artificial saliva that step (3) obtains, every 2-5min
Agitation is once；The temperature of described artificial saliva is 36-38 DEG C；
(5) the extracting solution centrifugation 5-10min that step (4) is obtained；
(6) supernatant being obtained with osmotic tester determination step (5), it is consistent with cell culture medium to adjust its osmotic pressure,
Obtain gum base type tobacco product extracting solution；
(7) measure the nicotine content in extracting solution；
(8) culture human mouth mucosal epithelial cells are as detection object；
(9) the gum base type tobacco product extracting solution obtaining in step (6) is added to cultured human mouth mucosa in step (8)
In epithelial cell；
(10) after the cultivation of 4 hours, detect cell γ h2ax situation of change with high intension imager.
2. method according to claim 1 is it is characterised in that the cultural method in step (8) comprises the following steps:
(8-1) extraction of oral cavity mucous membrane tissue: take the oral cavity mucous membrane tissue of less than 6 years old Patients with Cleft Lip and Palate；
(8-2) with the concentration containing penicillin be 100ug/ml, streptomycin be 50ug/ml phosphate buffer rinsing step (8-1)
Oral cavity mucous membrane tissue 2-3 time；
(8-3) shearing of piece of tissue: remove the sub-mucosal tissues in (8-2) with eye scissorss, and the mucosa after pruning is cut into about
The fritter of 5mm × 5mm, is subsequently adding 0.25% neutral protease；
(8-4) the oral mucosa fritter of step (8-3) is digested 16-18h at 4 DEG C, then by surface epithelium and propria lamina
Separately；
(8-5) separation of epithelial cell: the epithelial layer that step (8-4) is obtained, plus people 0.25% trypsin disappears at 36 DEG C
Change 15min, every 5min piping and druming is vibrated 1 time, is subsequently adding the dmem/f12 culture medium containing 10% hyclone, blows and beats into unicellular
Suspension, centrifugation, rinsed 1-2 time with phosphate buffer, the cutin being then added to add in advance somatomedin is formed carefully
In born of the same parents' culture medium；
(8-6) original cuiture of epithelial cell: the epithelial cell that step (8-5) is obtained is made and used cytometer after single cell suspension
Number, with 1-2 × 105The density of individual/ml cell is inoculated in 25cm2In culture bottle, put into 37 DEG C, 5%co2Cultivate in incubator；3d
Change culture fluid afterwards first, every 2d changes culture fluid once later；
(8-7) Secondary Culture of epithelial cell: pass on training when the epithelial cell of step (8-6) reaches cover bottom of bottle 80%
Support, with 0.25% trypsinization, treat that epithelial cell becomes round, with the dmem/f12 culture medium containing 10% hyclone
Terminate digestion, centrifugation, add the keratinocyte culture medium adding somatomedin in advance, so that epithelial cell concentration is reached
To 5 × 104Individual/ml；Single cell suspension is planted in 96 porocyte culture plates, inoculum concentration is 200 μ l/ holes, by planted
96 porocyte culture plates are positioned over 37 DEG C, 5%co2CO2 gas incubator in culture 24h.
3. method according to claim 1 is it is characterised in that step (10) includes step in detail below:
(10-1) incubation of tested material: the gum base type tobacco product extracting solution obtaining in step (6) is added step (8-7) to obtain
Epithelial cell in, using sample nicotine amount as detection dosage Classification Index；Blank control group only adds culture medium, solvent control
Group adds artificial saliva；Tissue Culture Plate is positioned over 37 DEG C, 5%co2CO2 gas incubator in culture 24h；
(10-2) human mouth mucosal epithelial cells are fixed: with the cell in phosphate buffer washing step (10-1), then with 4%
Paraformaldehyde solution fixes cell 10min, then washs 2 times with phosphate buffer；
(10-3) human mouth mucosal epithelial cells film is changed thoroughly: by the epithelial cell in step (10-2) with containing 0.2%tritonx-
100 phosphate buffer changes 10min thoroughly, then uses phosphate buffer washed cell 2 times；
(10-4) human mouth mucosal epithelial cells γ h2ax dyeing: with the phosphoric acid of the γ h2ax antibody containing 10 μ g/ml fitc labellings
Buffer dyes to the epithelial cell in step (10-3), and lucifuge dyes 2h, then washs epithelial cell 2 with phosphate buffer
Secondary；
(10-5) human mouth mucosal epithelial cells hoechst dyeing: with the phosphate buffer containing 5 μ g/ml hoechst to step
(10-4) epithelial nucleus in carry out lucifuge dyeing 15min；
(10-6) high intension imaging system detection: select fitc the and hoechst fluorescence dual pathways to the epithelium in step (10-5)
Cell is scanned obtaining picture；
(10-7) graphical analyses: using high intension analysis software, the picture in step (10-6) is analyzed, measures hoechst
The average diameter of passage cell core, to arrange the diameter parameters of individual cells core, is joined according to the distance between nucleus setting distance
Number；According to the above software that sets, picture is identified, and calculate individual cells core fitc and hoechst passage single thin
Karyon Mean Fluorescence；With the individual cells core Mean Fluorescence of hoechst passage as comparison, compare fitc passage single thin
The change of karyon Mean Fluorescence, evaluates dna degree of injury with it.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106970051A (en) * | 2017-03-16 | 2017-07-21 | 国家烟草质量监督检验中心 | A kind of method that DNA Damage is caused based on high intension technology quantitative analysis tar |
| CN106979939A (en) * | 2017-03-16 | 2017-07-25 | 国家烟草质量监督检验中心 | A kind of method that DNA Damage is caused based on high intension technology quantitative analysis nitrosamine or nitrosamine metabolin |
| CN107064086A (en) * | 2017-03-16 | 2017-08-18 | 国家烟草质量监督检验中心 | The method that one kind causes DNA Damage based on high intension technology quantitative analysis benzo [a] pyrene |
| CN107064087A (en) * | 2017-03-16 | 2017-08-18 | 国家烟草质量监督检验中心 | A kind of method that metabolin based on high intension technology quantitative analysis benzene or benzene causes DNA Damage |
| CN107153054A (en) * | 2017-03-16 | 2017-09-12 | 国家烟草质量监督检验中心 | A kind of method that DNA Damage is caused based on high intension technology quantitative analysis nicotine |
| CN107513552A (en) * | 2017-10-21 | 2017-12-26 | 云南中烟工业有限责任公司 | The detection method that a kind of cigarette smoke influences on the cell expression quantity of aquaporin 5 |
| CN107586830A (en) * | 2017-10-21 | 2018-01-16 | 云南中烟工业有限责任公司 | The detection method that a kind of cigarette smoke influences on the cell mRNA expression quantity of aquaporin 5 |
| CN107607511A (en) * | 2017-10-21 | 2018-01-19 | 云南中烟工业有限责任公司 | The detection method that a kind of cigarette smoke influences on the Subcellular Localization of aquaporin 5 |
| CN108130356A (en) * | 2017-12-14 | 2018-06-08 | 云南中烟工业有限责任公司 | It is a kind of to be used to detect method of the gum base type chewing tobacco to DNA Damage |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102507789A (en) * | 2011-11-17 | 2012-06-20 | 中国烟草总公司郑州烟草研究院 | Method for detecting nicotine release behavior of gum base type smoke-free tobacco product |
-
2016
- 2016-08-26 CN CN201610729383.5A patent/CN106367468A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102507789A (en) * | 2011-11-17 | 2012-06-20 | 中国烟草总公司郑州烟草研究院 | Method for detecting nicotine release behavior of gum base type smoke-free tobacco product |
Non-Patent Citations (1)
| Title |
|---|
| CAROLINA GARCIA-CANTON等: ""Assessment of the in vitro γH2AX assay by High Content Screening as a novel genotoxicity test"", 《MUTATION RESEARCH》 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106970051A (en) * | 2017-03-16 | 2017-07-21 | 国家烟草质量监督检验中心 | A kind of method that DNA Damage is caused based on high intension technology quantitative analysis tar |
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| CN107513552B (en) * | 2017-10-21 | 2021-02-02 | 云南中烟工业有限责任公司 | Method for detecting influence of cigarette smoke on aquaporin 5 cell expression quantity |
| CN107586830B (en) * | 2017-10-21 | 2021-02-02 | 云南中烟工业有限责任公司 | Method for detecting influence of cigarette smoke on mRNA expression quantity of aquaporin 5 cells |
| CN108130356A (en) * | 2017-12-14 | 2018-06-08 | 云南中烟工业有限责任公司 | It is a kind of to be used to detect method of the gum base type chewing tobacco to DNA Damage |
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