CN103088105A - MTT (thiazolyl blue) cell toxicity test method of biological assessment of total particle matter in cigarette smoke - Google Patents
MTT (thiazolyl blue) cell toxicity test method of biological assessment of total particle matter in cigarette smoke Download PDFInfo
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Abstract
The invention relates to an MTT (thiazolyl blue) cell toxicity test method of biological assessment of a total particle matter in cigarette smoke and belongs to the technical field of safety assessment of tobacco and cigarette smoke. The MTT cell toxicity test method is characterized by comprising the following steps of: inoculating and culturing immortalized human bronchial epithelial cells (BEAS-2B cells) and contaminating the total particle matter in the cigarette smoke; detecting the cell survival rate by adopting an MTT method; and analyzing and evaluating the cell toxicity of the total particle matter in the cigarette smoke according to a test result. Compared with the prior art, the MTT cell toxicity test method has the following characteristics that BEAS-2B cells are human cells and are target organ source cells acting on a human body by the smoke; a BEAS-2B cell system is used for carrying out cell toxicity assessment on the cigarette smoke and has the strong pertinence; in a testing process, steps of washing the cells for a plurality of times are reduced; and a formaldehyde fixing step does not need to be carried out so that a testing period is shortened, the operation is rapid and convenient, the sensitivity is high and the result is stable and reliable. Moreover, the MTT test method disclosed by the invention is further applicable to a smoke cell toxicity test of various cell systems and the commonality is strong.
Description
Technical field
The present invention relates to a kind of Cytotoxic test method of MTT of cigarette smoke total particulate matter biological assessment, belong to tobacco and cigarette smoke safety evaluation technical field.
Background technology
The aerosol that cigarette smoke is comprised of several thousand kinds of chemical substances wherein has the part chemical substance to be defined as carcinogens or auxiliary carcinogens, smoking and some important diseases of the mankind exist close dependency.The in vitro toxicity experimental study of cigarette smoke has become estimates smoking to the powerful measure of Health hazard, and wherein, the vitro cytotoxicity test is playing a very important role aspect evaluating cigarette flue gas and low hazard cigarette goods hazardness.The principle that the method for carrying out adopting when cigarette smoke cytotoxicity is estimated is measured based on cell survival rate mostly.International tobacco scientific research Cooperation Centre (CORESTA) recommendation is selected suitable mammalian cell and is adopted the cytotoxicity of neutral red test method evaluation cigarette smoke total particulate matter.But the neutral red test operation steps is comparatively loaded down with trivial details, the dyeing in process of the test, and a plurality of cleaning steps increased the possibility of error.In addition, present correlative study both domestic and external adopts rodent zooblast system (as Chinese hamster ovary celI) to carry out the toluylene red cell toxicity test usually.And the main toxicity of flue gas still acts on human body, exists certain limitation with zooblast evaluating cigarette flue gas total particulate matter cytotoxicity.
The final purpose of toxicology test is to pay close attention to human health.For the lower characteristics of cigarette flue gas toxity, when carrying out the Cytotoxic evaluation of flue gas, need to select clone with strong points and highly sensitive test method; When detecting, the toxicity of carrying out the high-throughput sample also require simultaneously the testing method can be easier, quick, accurate.
The MTT(tetrazolium bromide) method is a kind of method that detects cell survival and growth that is widely used in.Its detection principle is that the succinodehydrogenase in the viable cell plastosome can make exogenous MTT be reduced to water-insoluble bluish voilet crystallization first a ceremonial jade-ladle, used in libation (Formazan) and be deposited in cell, and dead cell is without this function.With the first a ceremonial jade-ladle, used in libation in dimethyl sulfoxide (DMSO) (DMSO) dissolved cell and measure its absorbance value, can indirectly reflect viable cell quantity.In certain cell count scope, the amount that the MTT crystallization forms is directly proportional to cell count.Compare with the toluylene red method, the method can be saved some cleaning steps, and is highly sensitive, easy and simple to handle.The BEAS-2B cell is the Immortalized human bronchial epithelial cell, acts on the target organ derived cell of human body for flue gas, is usually used in the correlative study of respiratory tract disease.Adopt the BEAS-2B cell to carry out the cytotoxicity test of cigarette smoke, have more specific aim, can solve preferably the problem of species difference.
Summary of the invention
Purpose of the present invention is just for existing weak point in prior art, and special exploitation is a kind of for the Cytotoxic testing method of cigarette smoke total particulate matter.Present method end user's bronchial epithelial cell (BEAS-2B cell) carries out the cytotoxicity of MTT cell toxicity test evaluating cigarette flue gas total particulate matter, not only easy and simple to handle, highly sensitive, and result can reflect the cytotoxicity of cigarette smoke total particulate matter comparatively truly.
Purpose of the present invention can realize by following technique measures:
1) test preparation reagent:
(1) cell growth medium: the LHC-8 substratum, add gentamicin sulphate before use, final concentration is 100 units/mL;
(2) digestion stop buffer: DMEM-1640+10% foetal calf serum;
(3) 0.01 M phosphate buffered saline(PBS) (PBS): 0.2 g KCl, 0.2 g KH
2PO
4, 8 g NaCl, 2.9 g Na
2HPO
412H
2O adds ultrapure water to 1 L, and pH 6.9 ~ 7.2, autoclaving;
(4) MTT solution: take 250 mg MTT, put into small beaker, add 50 mL PBS and stir 30 min on the electromagnetic force agitator, with the millipore filter degerming of 0.22 μ m, packing ,-20 ℃ of preservations;
(5) 0.05% pancreatin solution: take the 0.1g pancreatin, add 200 mL PBS ice-water baths to stir 30 min, the millipore filter degerming of 0.22 μ m, packing ,-20 ℃ of preservations;
(6) sodium dodecyl sulfate solution: take a certain amount of sodium laurylsulfonate and be dissolved in sterilized water, making its concentration is 10mg/mL, with the millipore filter degerming of 0.22 μ m, and 4 ℃ of preservations.
2) cell inoculation culture: the human bronchial epithelial cell that is in exponential phase of growth of amplification cultivation (BEAS-2B cell) is through trysinization, and the preparation single cell suspension is inoculated in 96 porocyte culture plates every hole 1 * 10 after counting
4Individual cell, be placed in incubator (37 ℃, 5% CO
2) in hatch 24 h.
3) cigarette smoke total particulate matter (TPM) contamination: remove the solution in 96 well culture plates, if four class experimental group, be respectively: blank group, solvent control group, positive controls and flue gas total particulate matter group, and add in the requsst listed below the dimethylsulfoxide extraction liquid sample of cell growth medium, dimethyl sulfoxide (DMSO), sodium laurylsulfonate and flue gas total particulate matter in respective aperture.The blank group is inoculating cell not, only adds the Growth of Cells substratum; The solvent control group adds dimethyl sulfoxide (DMSO), and add-on is suitable with TPM group highest detection dosage; Flue gas total particulate matter group adds the flue gas total particulate matter dimethylsulfoxide extraction liquid sample of various dose, and the dosage that makes TPM in every mL cell culture medium is 0,10,15,20,30,40,50,60,70 μ g successively; Positive controls adds sodium dodecyl sulfate solution.The cell growth medium that adds in every hole and the cumulative volume of various tested materials are 200 μ L, continue to be placed in incubator and hatch 24 h.
4) the MTT cytotoxicity detects: directly add MTT solution in the Tissue Culture Plate after processing to contamination 24 h, every hole 20 μ L are placed in incubator and hatch 4 h; Discard the solution in culture plate, add DMSO solution 150 μ L/ holes, be placed in concussion 15 min in the microwell plate vibrator; Detect every hole light absorption value at 490 nm wavelength places on microplate reader.Calculate relative light absorption value, light absorption value represents cell survival rate relatively.
5)) results and analysis: according to light absorption value, calculate cell survival rate according to formula (1), and draw the lower Cytotoxic dose-effect relationship curve of cigarette smoke total particulate matter exposure.Formula is as follows:
In formula:
X---cell survival rate;
OD
n---the average light absorption value of TPM group;
OD
0---the average light absorption value of acellular control group;
OD
c---the average light absorption value of solvent control group.
The present invention's feature compared to existing technology is as follows: adopt human bronchial epithelial cell (BEAS-2B cell) to carry out cell toxicity test, the BEAS-2B cell is comparatively responsive to the toxicity of smoke condensate, can effectively tell the cytotoxicity of TPM; Adopt MTT dyeing to carry out, reduced the process of cleaning for several times in whole process of the test.The present invention be advantageous in that: the BEAS-2B cell is human archeocyte, is the target organ derived cell that flue gas acts on human body, adopts BEAS-2B clone to carry out the Cytotoxic evaluation of cigarette smoke, and specific aim is stronger; Not only reduced the repeatedly step of washed cell, also need not to carry out the formaldehyde fixing step, shortened test period, made operation more fast and convenient, susceptibility is higher, and result is more reliable and more stable.The present invention adopts mtt assay to carry out cell survival rate and detects, and according to the cytotoxicity of test result analysis evaluating cigarette flue gas total particulate matter, has started a kind of new method that is used for the detection of cigarette smoke total particulate matter cytotoxicity.
In addition, MTT testing method involved in the present invention also is applicable to the smoke cytotoxicity test of various kinds of cell system, and versatility is stronger.
Description of drawings
Fig. 1 is the cigarette smoke cytotoxicity dose-effect relationship figure of the embodiment of the present invention 1.
Fig. 2 is the cigarette smoke cytotoxicity dose-effect relationship figure of the embodiment of the present invention 2.
Embodiment:
The present invention is described further with the following Examples, but does not limit the present invention.
The a pair of Kentucky of embodiment reference cigarette 3R4F carries out Cytotoxic evaluation
1, experimental section
1.1 key instrument and reagent
CO
2Incubator; Inverted microscope; Microplate reader; 96 porocyte culture plates; Tissue Culture Flask; Flow cytometer.
The LHC-8 substratum; Foetal calf serum; Phosphate buffered saline buffer; Trypsinase; The MTT dyestuff; Dimethyl sulfoxide (DMSO) (cell cultures level).
1.2 the preparation of cigarette smoke total particulate matter (TPM)
With cigarette balance 48 hours in thermostatic constant wet chamber, choose the weight cigarette consistent with resistance to suction.Press the iso standard method, aspirate respectively 20 cigarette on smoking machine, cambridge filter adds DMSO to soak by 10 mg/mL, supersound extraction 20 min, and the filter with 0.22 μ l filters at last, divides to install in cryopreservation tube, is stored in-80 ℃ of Ultralow Temperature Freezers standby.
The preparation of MTT solution
The final concentration that common MTT is made into is 5 mg/mL, must make solvent with PBS or physiological saline.Be sub-packed in the EP pipe, the aluminium-foil paper parcel is stored in-20 ℃ of refrigerators standby.
1.3 subject cell
Human bronchial epithelial cell (Human bronchial epithelial cells, BEAS-2B cell).
1.4 test method
During test, well-grown BEAS-2B cell is inoculated in 96 porocyte culture plates every hole 1 * 10
4Individual cell is placed in incubator (37 ° of C, 5% CO
2) in hatch 24 h; Discard the solution in culture plate, blank group, solvent control group, positive controls and TPM group are set respectively, and add corresponding tested material, after continuing to hatch 24 h; Directly add MTT solution, every hole 20 μ l are placed in incubator and hatch 4 h; Discard the solution in culture plate, add DMSO solution 150 μ L/ holes, be placed in concussion 10 min in the microwell plate vibrator; Measure every hole light absorption value of 490 nm wavelength on microplate reader, and carry out interpretation of result.
2, result
As shown in Figure 1, the flue gas total particulate matter sample of 3R4F cigarette has comparatively significantly restraining effect to the BEAS-2B cell, in the concentration range of 0 to 70 μ g/mL, along with the increase of TPM sample concentration, cell survival rate reduces thereupon, presents significant dose-effect relationship.
Certain model reference cigarette of two pairs of domestic developments of embodiment carries out Cytotoxic evaluation, and embodiment is with reference to embodiment 1.
As shown in Figure 2, the flue gas total particulate matter sample of this model reference cigarette has comparatively significantly restraining effect to the BEAS-2B cell, in the concentration range of 0 to 70 μ g/mL, along with the increase of TPM sample concentration, cell survival rate reduces thereupon, presents significant dose-effect relationship.
Claims (6)
1. the MTT cell toxicity test method of a cigarette smoke total particulate matter biological assessment is characterized in that: said method comprising the steps of:
1) test preparation reagent,
2) cell inoculation culture,
3) cigarette smoke total particulate matter (TPM) contamination,
4) the MTT cytotoxicity detects,
5) results and analysis.
2. the MTT cell toxicity test method of cigarette smoke total particulate matter biological assessment according to claim 1, it is characterized in that: test preparation reagent has following:
(1) cell growth medium: the LHC-8 substratum, add gentamicin sulphate before use, final concentration is 100 units/mL;
(2) digestion stop buffer: DMEM-1640+10% foetal calf serum;
(3) 0.01 M phosphate buffered saline(PBS) (PBS): 0.2 g KCl, 0.2 g KH
2PO
4, 8 g NaCl, 2.9 g Na
2HPO
412H
2O adds ultrapure water to 1 L, pH 6.9-7.2, autoclaving;
(4) MTT solution: take 250 mg MTT, put into small beaker, add 50 mL PBS and stir 30 min on the electromagnetic force agitator, with the millipore filter degerming of 0.22 μ m, packing ,-20 ℃ of preservations;
(5) 0.05% pancreatin solution: take the 0.1g pancreatin, add 200 mL PBS ice-water baths to stir 30 min, the millipore filter degerming of 0.22 μ m, packing ,-20 ℃ of preservations;
(6) sodium dodecyl sulfate solution: take a certain amount of sodium laurylsulfonate and be dissolved in sterilized water, making its concentration is 10mg/mL, with the millipore filter degerming of 0.22 μ m, and 4 ℃ of preservations.
3. the MTT cell toxicity test method of cigarette smoke total particulate matter biological assessment according to claim 1, it is characterized in that: cell inoculation culture process is as follows: the human bronchial epithelial cell that is in exponential phase of growth of amplification cultivation (BEAS-2B cell) is through trysinization, the preparation single cell suspension, be inoculated in 96 porocyte culture plates every hole 1 * 10 after counting
4Individual cell, be placed in incubator (37 ℃, 5% CO
2) in hatch 24 h.
4. the MTT cell toxicity test method of cigarette smoke total particulate matter biological assessment according to claim 1, it is characterized in that: cigarette smoke total particulate matter contamination process is as follows: remove the solution in 96 well culture plates, if four class experimental group, be respectively: blank group, solvent control group, positive controls and flue gas total particulate matter group, and add in the requsst listed below the dimethylsulfoxide extraction liquid sample of cell growth medium, dimethyl sulfoxide (DMSO), sodium laurylsulfonate and flue gas total particulate matter in respective aperture; The blank group is inoculating cell not, only adds the Growth of Cells substratum; The solvent control group adds dimethyl sulfoxide (DMSO), and add-on is suitable with TPM group highest detection dosage; Flue gas total particulate matter group adds the flue gas total particulate matter dimethylsulfoxide extraction liquid sample of various dose, and the dosage that makes TPM in every mL cell culture medium is 0,10,15,20,30,40,50,60,70 μ g successively; Positive controls adds sodium dodecyl sulfate solution; The cell growth medium that adds in every hole and the cumulative volume of various tested materials are 200 μ L, continue to be placed in incubator and hatch 24 h.
5. the MTT cell toxicity test method of cigarette smoke total particulate matter biological assessment according to claim 1, it is characterized in that: MTT cytotoxicity testing process is as follows: directly add MTT solution in the Tissue Culture Plate after processing to contamination 24 h, every hole 20 μ L are placed in incubator and hatch 4 h; Discard the solution in culture plate, add DMSO solution 150 μ L/ holes, be placed in concussion 15 min in the microwell plate vibrator; Detect every hole light absorption value at 490 nm wavelength places on microplate reader; Calculate relative light absorption value, light absorption value represents cell survival rate relatively.
6. the MTT cell toxicity test method of cigarette smoke total particulate matter biological assessment according to claim 1, it is characterized in that: result is with minute lower as follows: according to light absorption value, calculate cell survival rate according to formula (1), and draw the lower Cytotoxic dose-effect relationship curve of cigarette smoke total particulate matter exposure, formula is as follows:
In formula:
X---cell survival rate;
OD
n---the average light absorption value of TPM group;
OD
0---the average light absorption value of acellular control group;
OD
c---the average light absorption value of solvent control group.
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| CN103604917A (en) * | 2013-11-28 | 2014-02-26 | 云南烟草科学研究院 | Screening method of positive substances for pyrolyzate trapping substance cytotoxicity test |
| CN104293876A (en) * | 2014-09-23 | 2015-01-21 | 云南中烟工业有限责任公司 | Cigarette filter tip water extract cytotoxicity test positive control setting method |
| CN106442250A (en) * | 2016-08-01 | 2017-02-22 | 上海伽誉生物科技有限公司 | Anti-pollution testing device and method |
| CN106591413A (en) * | 2016-11-25 | 2017-04-26 | 云南中烟工业有限责任公司 | Method of detecting influence on early apoptosis of cells due to total particulate matters in cigarette smoke |
| CN110736696A (en) * | 2019-11-21 | 2020-01-31 | 上海烟草集团有限责任公司 | method for determining cigarette smoke cytotoxicity under exposure of gas-liquid interface |
| CN111337671A (en) * | 2020-04-15 | 2020-06-26 | 江西省交通工程集团建设有限公司 | Method and device for detecting smoke suppression effect of asphalt based on biological immunotoxicity and application of method and device |
| CN112375802A (en) * | 2020-11-06 | 2021-02-19 | 中国科学院城市环境研究所 | Reagent or kit for evaluating toxicity effect of resveratrol interfering with pulmonary epithelial cell polycyclic aromatic hydrocarbon and application and detection thereof |
| CN113621557A (en) * | 2021-09-02 | 2021-11-09 | 中国烟草总公司郑州烟草研究院 | Human liver microsome and cell co-culture system and construction method and application thereof |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103604917A (en) * | 2013-11-28 | 2014-02-26 | 云南烟草科学研究院 | Screening method of positive substances for pyrolyzate trapping substance cytotoxicity test |
| CN103604917B (en) * | 2013-11-28 | 2015-10-28 | 云南烟草科学研究院 | The positive thing screening method of thermal cracking products trapping thing cell toxicity test |
| CN104293876A (en) * | 2014-09-23 | 2015-01-21 | 云南中烟工业有限责任公司 | Cigarette filter tip water extract cytotoxicity test positive control setting method |
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| CN111337671A (en) * | 2020-04-15 | 2020-06-26 | 江西省交通工程集团建设有限公司 | Method and device for detecting smoke suppression effect of asphalt based on biological immunotoxicity and application of method and device |
| CN112375802A (en) * | 2020-11-06 | 2021-02-19 | 中国科学院城市环境研究所 | Reagent or kit for evaluating toxicity effect of resveratrol interfering with pulmonary epithelial cell polycyclic aromatic hydrocarbon and application and detection thereof |
| CN113621557A (en) * | 2021-09-02 | 2021-11-09 | 中国烟草总公司郑州烟草研究院 | Human liver microsome and cell co-culture system and construction method and application thereof |
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Application publication date: 20130508 |