CN106244663A - A kind of detect the method that human mouth apoptosis is affected by gum base type tobacco product - Google Patents
A kind of detect the method that human mouth apoptosis is affected by gum base type tobacco product Download PDFInfo
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- CN106244663A CN106244663A CN201610728636.7A CN201610728636A CN106244663A CN 106244663 A CN106244663 A CN 106244663A CN 201610728636 A CN201610728636 A CN 201610728636A CN 106244663 A CN106244663 A CN 106244663A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G—PHYSICS
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- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
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Abstract
The present invention discloses and a kind of detects the method that human mouth apoptosis is affected by gum base type tobacco product, comprises the following steps: described gum base type tobacco product to be measured is placed in liquid nitrogen freezing by (1)；(2) freezing gum base type tobacco product is pulverized with pulverizer immediately；(3) again unqualified gum base type tobacco product is placed in liquid nitrogen freezing；(4) unqualified gum base type tobacco product artificial saliva is soaked；(5) by extracting solution centrifugation；(6) measure the supernatant with osmotic tester, regulate its osmotic pressure consistent with cell culture medium；(7) nicotine content in extracting solution is measured；(8) human mouth mucosal epithelial cells is cultivated as detection object；(9) gum base type tobacco product extracting solution is joined in cultured human mouth mucosal epithelial cells；(10) after less than in the cultivation of 24 hours, by flow cytomery apoptosis situation.
Description
Technical field
The present invention relates to biological technology application, be applied especially to gum base type tobacco product for human mouth apoptosis
The mensuration of impact.
Background technology
Gum base type smoke-free tobacco product (Tobacco chewing gum) be a kind of 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 goods is to put in mouth to chew, and the leachable chewed can converge gathering in the oral cavity with saliva, practises according to consumption
Used difference spues or swallows.Owing to this product mainly acts on oral cavity, directly contacting with Stomatocyte, therefore having must
Set up a kind of detect its on human mouth apoptosis impact method, so that its safety is estimated.
Existing tobacco product method for evaluating safety is primarily directed to traditional cigarette, it is common that the 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
Chamber, therefore the security assessment method of traditional cigarette is not particularly suited for this product.And packed buccal cigarette goods typically use and are attached to
Mode between gingiva and lip uses, and its inclusions uses common infusion method the most leachable, and gum base type tobacco product is due to glue
The existence of base, direct infusion method major part inclusions is difficult to dissolution, thus cannot accurately carry out biological safety test.
Summary of the invention
The present invention is directed to the feature of gum base type product, in conjunction with biological detection method, establish a kind of detection gum base type cigarette
The method that human mouth apoptosis is affected by straw-made articles, the safety evaluation for gum base type tobacco product provides reference.
The present invention discloses and a kind of detects the method that human mouth apoptosis is affected by gum base type tobacco product, including following step
Rapid:
(1) described gum base type tobacco product to be measured is placed in liquid nitrogen freezing 20-40min；
(2) by the described gum base type tobacco product that step (1) is freezing, pulverized with pulverizer immediately, collected after pulverizing
Unqualified gum base type tobacco product；
(3) again described unqualified gum base type tobacco product is placed in liquid nitrogen freezing；
(4) described unqualified gum base type tobacco product artificial saliva step (3) obtained soaks 20-40min, 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 obtained with osmotic tester determination step (5), regulates its osmotic pressure and cell culture medium one
Cause, obtain gum base type tobacco product extracting solution；
(7) nicotine content in extracting solution is measured；
(8) human mouth mucosal epithelial cells is cultivated as detection object；
(9) the gum base type tobacco product extracting solution obtained in step (6) is joined cultured human mouth in step (8)
In mucosal epithelial cells；
(10) after less than in the short cultivation of 24 hours, by flow cytomery apoptosis situation.
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 the phosphate buffer rinsing step of 50ug/mL
(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 scissors, and the mucosa after pruning cuts
Become the fritter of about 5mm × 5mm, be subsequently adding 0.25% neutral protease II；
(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: epithelial layer step (8-4) obtained, adds people 0.25% trypsin at 36 DEG C
Lower digestion 15min, every 5min piping and druming vibration 1 time, is subsequently adding the DMEM/F12 culture medium containing 10% hyclone, blows and beats into single
Cell suspension, centrifugation, rinse 1-2 time with phosphate buffer, be then added to add in advance the cutin shape of somatomedin
Become in cell culture medium；
(8-6) original cuiture of epithelial cell: use thin after the epithelial cell that step (8-5) obtains is made single cell suspension
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%CO2Incubator is trained
Support；Changing culture fluid after 3d first, the most every 2d changes culture fluid once；
(8-7) Secondary Culture of epithelial cell: pass on when the epithelial cell of step (8-6) reaches and covers 80% at the bottom of bottle
Cultivate, with the trypsinization of 0.25%, treat that epithelial cell becomes round, cultivate with the DMEM/F12 containing 10% hyclone
Base terminates digestion, centrifugation, adds the keratinocyte culture medium adding somatomedin in advance, makes epithelial cell concentration
Reach 5 × 105Individual/mL；Being planted to by single-cell suspension liquid in 24 porocyte culture plates, inoculum concentration is 500 μ L/ holes, by kind well
24 porocyte culture plates be positioned over 37 DEG C, 5%CO2CO2 gas incubator in cultivate 24h；
Preferably, step (10) includes step in detailed below:
(10-1) the hatching of tested material: the gum base type tobacco product extracting solution obtained in step (6) is added step (8-7)
In the epithelial cell obtained, using sample nicotine amount as detection dosage Classification Index；Blank group only adds culture medium, solvent
Matched group adds artificial saliva；
(10-2) Buccal mucosa cell digestion: the epithelial cell in step (10-1) and test sample are hatched 24h
After discard culture fluid, with phosphate buffer wash 2 times, use 0.25% trypsin digestion cell, with the DMEM/ containing 10% hyclone
F12 culture medium is centrifugal after terminating digestion collects epithelial cell；
(10-3) Buccal mucosa cell dyeing: the epithelial cell that (10-2) collects is resuspended in Annexin V-
Dyestuff in FITC/PI double dye detection apoptosis kit combines in liquid, and epithelial cell concentration is about 5 × 105Individual/mL；Take
The single cell suspension of 500 μ l, after adding 5 μ l AnnexinV-FITC mixings, adds 5 μ L propidium iodides, mixing；
(10-4) flow cytometer is used after the epithelial cell in (10-3) being reacted 10min with dye mixture room temperature lucifuge
Detect.
Beneficial effect of the present invention
1, the present invention is difficult to use direct infusion method to carry out the feature extracted in view of gum base type tobacco product, uses ultralow
Pulverizing after temperature (about-196 DEG C) freezing, the material of this for gum base glutinous sticky paste can be effectively ground into powder, then by powder by this
End with after nitrogen ultra low temperature freezing, is added rapidly in room temperature (36-38 DEG C) artificial saliva again, utilizes from deep cooling rapidly to often
This shock heating effect of temperature, promotes powder to crush further because drastically expanding with heat and contract with cold, and improves the contact surface of powder and extracting solution
Long-pending, thus improve the efficiency of extraction chemistry material from powder.
2, add more sugar or salt due to gum base type tobacco product and grade shadow generally for strengthening mouth comfort
Ringing the material of liquid infiltration pressure, causing cell if directly adding cell is easily caused by extracting solution due to the problem of osmotic pressure
Death, therefore the present invention have adjusted the osmotic pressure of extracting solution before processing cell, thus improves the accuracy of experiment.
3, for simulating actual conditions as far as possible, the present invention have employed artificial saliva when extracting, and simulates close to population
The temperature in chamber and edible time, have selected human mouth cell on detection object, and in order to as far as possible close to the reality of human mouth cell
Border situation, the present invention use the method for original cuiture cultivated normal person's Buccal mucosa cell as detection object, compare
More conventionally employed zooblast or the detection of cancerous cell, improve the reliability of test.
4, the present invention considers the gum base type tobacco product promptness to impact cell, uses the short incubation period of 24h, simultaneously
Use this more quickly sensitive method of flow cytomery, the most efficient requirement of detection can be met.
Accompanying drawing explanation
Fig. 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 extracting solution impact on human mouth mucosal epithelial cells apoptosis.
Detailed description of the invention
1. random choose one gum base type tobacco product (N-2, nicotine content 2mg/), random choose 2 puts into sample
In QC, sample cell is placed in liquid nitrogen freezing 30min.
2. sample cell is taken out from liquid nitrogen, further take out gum base type tobacco product (N-2), immediately with Sample Grinder by it
Pulverize, with sample cell collect pulverize after unqualified.
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. from liquid nitrogen, take out sample cell, add 10mL immediately and be preheated to the artificial saliva of 37 DEG C, soak the glue after pulverizing
Fundamental mode tobacco product unqualified (N-2), uses 37 DEG C, the extraction conditions of 30min, and extracts every 2min agitation in immersion process
Liquid is so that sample and extracting solution are fully contacted thus the dissolution of beneficially inclusions.
5. being placed in by extracting solution in 15mL centrifuge tube, centrifugal 10min is to precipitate solids.
6. draw supernatant with pipettor to be placed in new centrifuge tube, and use osmotic tester to measure the infiltration of extracting solution
Pressure, regulates its osmotic pressure consistent with cell culture medium.
7. the concentration of the nicotine in mensuration extracting solution, to verify extraction efficiency.
8. the drawing materials of people's Normal oral mucosa tissue: select less than 6 years old Patients with Cleft Lip and Palate, retain in Unilateral Cleft Lip Repair and cut
The unnecessary oral cavity mucous membrane tissue removed, 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 blood stains and the antibacterial of attachment in piece of tissue.
10. the shearing of piece of tissue: remove sub-mucosal tissues with eye scissors, is cut into the fritter of about 5mm × 5mm, adds
0.25%Dispase II (neutral protease II).
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, add people 0.25% trypsin 36 DEG C digestion
15min (5min piping and druming vibration 1 time), removes and does not digests cell block completely, add the DMEM/F12 training containing 10% hyclone
Support base and terminate digestion, blow and beat into single cell suspension with suction pipe.1000r/min is centrifuged 5min, abandons supernatant, rinses once with PBS, adds
Enter to add in advance the KSFM culture medium (keratinocyte culture medium) of somatomedin.
The original cuiture of 13. epithelial cells: human mouth mucosal epithelial cells uses 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%CO2Incubator is cultivated.Head after 3d
Secondary changing liquid, the most every 2d changes liquid once.
The Secondary Culture of 14. epithelial cells: Secondary Culture when cell reaches to cover 80% at the bottom of bottle, with the pancreas of 0.25%
Protease digestion, Microscopic observation cell retraction situation, treat that cell becomes round, cultivate with the DMEM/F12 containing 10% hyclone
Base terminates digestion, and 1000r/min is centrifuged 5min, abandons supernatant, adds KSFM culture medium (the cutin formation adding somatomedin in advance
Cell culture medium), make cell concentration reach 5 × 105Individual/mL.Single-cell suspension liquid is planted in 24 porocyte culture plates, connect
The amount of kind is 500 μ L/ holes, and the 24 porocyte culture plates planted are positioned over 37 DEG C, 5%CO2CO2 gas incubator in cultivate
24h。
15. test packets: test is divided into three groups, and respectively blank group, solvent control group and test sample group, each
Test group do 3 parallel.
Hatching of 16. tested materials: the gum base type tobacco product extracting solution obtained in (6) is added the human mouth in (14) and sticks
In film epithelial cell, using sample nicotine amount as detection dosage Classification Index；Blank group only adds culture medium, solvent control
Group adds artificial saliva.
17. cell dissociations: discard culture fluid after hatching 24h, wash 2 times with PBS, use 0.25% trypsin digestion cell, use
Culture medium containing hyclone is centrifuged collecting cell with centrifuge after terminating digestion.
18. cell dyeings: the cell of collection is resuspended in the dyestuff in Annexin V-FITC/PI double dye detection kit
In conjunction with in liquid, cell concentration is about 5 × 105Individual/mL.Take the cell suspension of 500 μ l, add 5 μ L AnnexinV-FITC mixings
After, add 5 μ LPI (propidium iodide), mixing.Detect with flow cytometer after room temperature lucifuge reaction 10min.
As seen from the figure, there is certain apoptotic effect in N-2 to epithelial cell, draws in maximum concentration group (100 μ g/mL)
Play the apoptosis of about 10%, only cause the apoptosis of about about 2% at 25 μ g/mL and 12.5 μ g/mL concentration, with solvent pair
Compare the most notable according to group.
Claims (3)
1. one kind is detected the method that human mouth apoptosis is affected by gum base type tobacco product, it is characterised in that include following step
Rapid:
(1) described gum base type tobacco product to be measured is placed in liquid nitrogen freezing 20-40min；
(2) by described gum base type tobacco product freezing for step (1), being pulverized with pulverizer immediately, that collects after pulverizing is broken
End gum base type tobacco product；
(3) again described unqualified gum base type tobacco product is placed in liquid nitrogen freezing；
(4) described unqualified gum base type tobacco product artificial saliva step (3) obtained soaks 20-40min, 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 obtained with osmotic tester determination step (5), regulates its osmotic pressure consistent with cell culture medium,
Obtain gum base type tobacco product extracting solution；
(7) nicotine content in extracting solution is measured；
(8) human mouth mucosal epithelial cells is cultivated as detection object；
(9) the gum base type tobacco product extracting solution obtained in step (6) is joined cultured human mouth mucosa in step (8)
In epithelial cell；
(10) after less than in the cultivation of 24 hours, by flow cytomery apoptosis situation.
Method the most according to claim 1, 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 the phosphate buffer rinsing step (8-1) of 50ug/mL
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 scissors, and the mucosa after pruning is cut into about
The fritter of 5mm × 5mm, is subsequently adding 0.25% neutral protease II；
(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: epithelial layer step (8-4) obtained, adds people 0.25% trypsin and disappears at 36 DEG C
Change 15min, every 5min piping and druming vibration 1 time, be subsequently adding the DMEM/F12 culture medium containing 10% hyclone, blow and beat into unicellular
Suspension, centrifugation, rinse 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: use cytometer after the epithelial cell that step (8-5) obtains is made 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%CO2Incubator is cultivated；3d
After change culture fluid first, later every 2d changes culture fluid once；
(8-7) Secondary Culture of epithelial cell: pass on training when the epithelial cell of step (8-6) reaches and covers 80% at the bottom of bottle
Support, with the trypsinization of 0.25%, treat that epithelial cell becomes round, by the DMEM/F12 culture medium containing 10% hyclone
Terminate digestion, centrifugation, add the keratinocyte culture medium adding somatomedin in advance, make epithelial cell concentration reach
To 5 × 105Individual/mL；Being planted to by single-cell suspension liquid in 24 porocyte culture plates, inoculum concentration is 500 μ L/ holes, by planted
24 porocyte culture plates are positioned over 37 DEG C, 5%CO2CO2 gas incubator in cultivate 24h.
Method the most according to claim 1, it is characterised in that step (10) includes step in detail below:
(10-1) the hatching of tested material: the gum base type tobacco product extracting solution obtained in step (6) is added step (8-7) and obtains
Epithelial cell in, using sample nicotine amount as detection dosage Classification Index；Blank group only adds culture medium, solvent control
Group adds artificial saliva；
(10-2) Buccal mucosa cell digestion: abandon after the epithelial cell in step (10-1) is hatched 24h with test sample
Remove culture fluid, wash 2 times with phosphate buffer, use 0.25% trypsin digestion cell, with the DMEM/F12 containing 10% hyclone
Culture medium is centrifugal after terminating digestion collects epithelial cell；
(10-3) Buccal mucosa cell dyeing: the epithelial cell that (10-2) collects is resuspended in AnnexinV-FITC/PI double
Dyestuff in dye detection apoptosis kit combines in liquid, and epithelial cell concentration is about 5 × 105Individual/mL；Take the list of 500 μ l
Cell suspension, after adding 5 μ l AnnexinV-FITC mixings, adds 5 μ L propidium iodides, mixing；
(10-4) carry out with flow cytometer after the epithelial cell in (10-3) being reacted 10min with dye mixture room temperature lucifuge
Detection.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108020659A (en) * | 2017-12-06 | 2018-05-11 | 中国烟草总公司郑州烟草研究院 | A kind of method of in-situ test Smoke Particulate induction of vascular endothelial Apoptosis |
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| 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 |
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2016
- 2016-08-26 CN CN201610728636.7A patent/CN106244663A/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 (2)
| Title |
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| MANASHI BAGCHI等: "Smokeless tobacco, oxidative stress, apoptosis, and antioxidants in human oral keratinocytes.", 《FREE RADICAL BIOLOGY MEDICINE》 * |
| 王丹等: "葛根芩连汤对人口腔黏膜上皮细胞增殖的影响", 《四川解剖学杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108020659A (en) * | 2017-12-06 | 2018-05-11 | 中国烟草总公司郑州烟草研究院 | A kind of method of in-situ test Smoke Particulate induction of vascular endothelial Apoptosis |
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