CN102618619A - Method for testing in vitro cytotoxicity of cigarette smoke - Google Patents

Abstract

The invention discloses a method for testing in vitro cytotoxicity of cigarette smoke. The method is characterized by comprising the following steps of: 1) preparing a laboratory reagent; 2) performing cell inoculation culture; 3) performing cigarette smoke contamination; 4) dyeing with water-soluble tetrazolium-1 (WST-1); and 5) obtaining a result and analyzing. Compared with the prior art, the method is characterized in that a test step for washing cells for multiple times is eliminated in the whole test process, so that the method is easy and convenient to operate; a step of replacing a nutrient solution is not required during dyeing with the WST-1, and a diluted nutrient solution can be directly added for reacting; formazan produced by the WST-1 is water-soluble, so that a subsequent dissolution step is eliminated; and absorbance detection can be finished in 2 hours after WST-1 dyeing, so that the test period is shortened, and the method is quick in comparison with the conventional testing method. The measuring method is quick and convenient to operate, and also has the advantages of high sensitivity and stable result; and the method can be applied to smoke cytotoxicity test of multiple cell lines.

Description

A kind of cigarette smoke vitro cytotoxicity testing method

 

Technical field

The present invention relates to the external toxicological evaluation research field of cigarette smoke, is a kind of method of testing the cigarette smoke vitro cytotoxicity specifically.

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Background technology

The external toxicologic study of cigarette smoke has become estimates smoking to one of important means of Health hazard, at present, concentrates on the cytotoxicity and the genotoxicity aspect of smoke condensate mostly about the external toxicologic study of cigarette smoke.How science thinks poorly of the hazardness that endangers cigarette product and tobacco additive agent exactly, does not also have unified method and program at present in the world.In biological experiment research, the mensuration of carrying out cell survival rate is the experimental technique of a key.The principle that the method for carrying out adopting when cigarette smoke cytotoxicity is estimated is at present measured based on cell survival rate mostly, but relevant test operation step is comparatively loaded down with trivial details, the cycle is longer.Develop new testing method satisfy have susceptibility, safety, characteristics such as quick, easy be a kind of demand.

WST-1 (a kind of water-soluble tetrazolium salts) dyestuff is a kind of fast high-sensitive degree detection reagent that is widely used in cell proliferation and cell survival rate; Under the situation that electron coupling reagent exists, can be generated orange-yellow Jia Za by more Intramitochondrial desaturase reduction.Cell proliferation is many more, and then color is dark more; Cell survival rate is low more, and then color is shallow more.It is water miscible that WST-1 produces De Jia Za, can save follow-up dissolving step, and the WST-1 dye stability is strong simultaneously, and linearity range is wide during its color reaction, and is highly sensitive, and pair cell does not have overt toxicity.

Cigarette smoke is a kind of mixture system of complicacy, and toxicity is lower, when carrying out the Cytotoxic evaluation of flue gas, needs highly sensitive TP; When carrying out the toxotest of batch cigarette sample, also require testing method quick, easy simultaneously.

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Summary of the invention

The object of the invention is directed against the problem of above-mentioned existence just, and based on the principle of WST-1 dye colour reaction and the advantage of himself, according to the characteristics of cigarette smoke cytotoxicity, the measuring method of a kind of cigarette smoke vitro cytotoxicity of foundation.

The objective of the invention is to realize through following technical scheme:

1) preparation experiment reagent: have following several kinds:

(1) grown cultures liquid: RPMI-1640+10% foetal calf serum+2 mM L-glutaminate+100 IU/ml penicillium mould+100 μ g/ml Streptomycin sulphates.

(2) diluted sample nutrient solution: RPMI-1640+5% foetal calf serum+2 mM L-glutaminate+100 IU/ml penicillium mould+100 μ g/ml Streptomycin sulphates.

(3) WST-1 dye liquor :-20 ℃ of preservations, face with preceding 37 ℃ of incubations and melt, avoid precipitating.

(4) positive control: sodium lauryl sulphate (SDS) (200 μ g/ml, 100% cell lethality concentration).

(5) negative control: 2% (v/v) DMSO 99.8MIN. (DMSO).

 

2) cell inoculation is cultivated: after the Chinese hamster ovary cell Chinese hamster ovary celI of amplification cultivation digests, and preparation cell suspension (1 * 10 ⁵Individual/ml).Every hole adds 100 μ l grown cultures liquid as blank in 36 holes of the most peripheral of 96 orifice plates, and all the other every holes add 100 μ l cell suspensions, in 37 ℃, 5% CO ₂Cultivate 24 h under the condition.

 

3) cigarette smoke contamination: use the diluted sample nutrient solution that flue gas TPM sample is adjusted to different concns, 8 non-zero-doses are set.Behind cell cultures 24 h; Nutrient solution is removed in suction; 96 orifice plates (removing most peripheral 36 holes) every row 6 holes are that the one group of contamination of flue gas TPM and positive control that is set to negative control, 8 various dose is respectively handled; Every hole adds the dilution nutrient solution that contains negative control, flue gas TPM or positive control of 100 μ l, and every hole adds 100 μ l dilution nutrient solution as blank, in 37 ℃, 5% CO in 36 holes of the most peripheral of 96 orifice plates ₂Cultivate 24 h under the condition.

 

4) WST-1 dyeing: behind flue toxicity contaminated 24 h, every hole adds 10 μ l through the WST-1 of incubation dye liquor, and cell is in 37 ℃, 5% CO ₂Hatch 2 h under the condition, quick oscillation 1 min under the room temperature uses ELIASA to detect the light absorption value of every hole in 450 nm wavelength, and reference wavelength is 630 nm.

 

5) result and analysis

Original light absorption value A:A=A _{450 nm}– A _{630 nm}

Through gauged light absorption value A _{(through blank correction)}: A _{(through blank correction)}=A – A _(blank)

Relative light absorption value C:C=

* 100%

(

: the MV of light absorption value)

Light absorption value is represented cell survival rate relatively.

The present invention compares prior art and has following characteristics: in entire test, reduced the repeatedly testing sequence of washed cell, made that operation is more easy; Need not to change the step of nutrient solution during WST-1 dyeing, the WST-1 dye liquor can directly add the dilution nutrient solution and react; It is water miscible that WST-1 produces De Jia Za, has saved follow-up dissolving step, and existing testing method is carrying out needing to use extraction solution that the product of color reaction is dissolved before light absorption value detects; Can accomplish light absorption value in 2 h of WST-1 dyeing back and detect, shorten test period with respect in the past testing method, quicker; Characteristics based on the WST-1 good stability; After adding WST-1 generation color reaction; Can read light absorption value repeatedly at different time, make the test duration more flexible, the test result good reproducibility; And be the condition that the continuous action of pair cell toxic effect provides continuous monitoring after the contamination of test flue gas TPM, help finding best detection time; Linearity range is wide during the WST-1 color reaction, and is highly sensitive.This measuring method also has the advantage that operation is more fast and convenient, susceptibility is higher, the result is more stable, and this method goes for the smoke cytotoxicity test of various kinds of cell system simultaneously.

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Description of drawings

Fig. 1 is a smoke cytotoxicity dose-effect relationship curve.

From Fig. 1, can find out: cell survival rate and flue gas TPM concentration are tangible dose-effect relationship, and cell survival rate increases with poisoning dosage and reduces.

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Embodiment

Reference cigarette 3R4F estimates to the Kentucky

After the Chinese hamster ovary celI of amplification cultivation digests, preparation cell suspension (1 * 10 ⁵Individual/ml).Every hole adds 100 μ l grown cultures liquid as blank in 36 holes of the most peripheral of 96 orifice plates, and all the other every holes add 100 μ l cell suspensions, in 37 ℃, 5% CO ₂Cultivate 24 h under the condition.Behind cell cultures 24 h; Nutrient solution is removed in suction, and 96 orifice plates (removing most peripheral 36 holes) every row 6 holes are that the one group of contamination of flue gas TPM and positive control that is set to negative control, 8 various dose (10,50,75,100,120,140,160,200 μ g/ml) is respectively handled.Every hole adds the dilution nutrient solution that contains negative control, flue gas TPM or positive control of 100 μ l, and every hole adds 100 μ l dilution nutrient solution as blank, in 37 ℃, 5% CO in 36 holes of the most peripheral of 96 orifice plates ₂Cultivate 24 h under the condition.Contaminate behind 24 h, every hole adds 10 μ l through the WST-1 of incubation dye liquor, and cell is in 37 ℃, 5% CO ₂Hatch 2 h under the condition, quick oscillation 1 min under the room temperature uses ELIASA to detect the light absorption value of every hole in 450 nm wavelength, and reference wavelength is 630 nm.

 

The cytotoxicity test result

Original light absorption value

* *: dash area is a blank

Calibrated light absorption value

Relative light absorption value

Claims (6)

1. cigarette smoke vitro cytotoxicity testing method is characterized in that: may further comprise the steps:

1) preparation experiment reagent,

2) cell inoculation is cultivated,

3) cigarette smoke contamination,

4) WST-1 dyeing,

5) result and analysis.

2. cigarette smoke vitro cytotoxicity testing method according to claim 1, it is characterized in that: the experiment reagent of preparation has following:

(1) grown cultures liquid: RPMI-1640+10% foetal calf serum+2 mM L-glutaminate+100 IU/ml penicillium mould+100 μ g/ml Streptomycin sulphates;

(2) diluted sample nutrient solution: RPMI-1640+5% foetal calf serum+2 mM L-glutaminate+100 IU/ml penicillium mould+100 μ g/ml Streptomycin sulphates;

(3) WST-1 dye liquor :-20 ℃ of preservations, face with preceding 37 ℃ of incubations and melt, avoid precipitating;

(4) positive control: sodium lauryl sulphate (SDS) (200 μ g/ml, 100% cell lethality concentration);

(5) negative control: 2% (v/v) DMSO 99.8MIN. (DMSO).

3. cigarette smoke vitro cytotoxicity testing method according to claim 1, it is characterized in that: the cell inoculation culturing process is following: after the Chinese hamster ovary cell Chinese hamster ovary celI of amplification cultivation digests, preparation cell suspension (1 * 10 ⁵Individual/ml); Every hole adds 100 μ l grown cultures liquid as blank in 36 holes of the most peripheral of 96 orifice plates, and all the other every holes add 100 μ l cell suspensions, in 37 ℃, 5% CO ₂Cultivate 24 h under the condition.

4. cigarette smoke vitro cytotoxicity testing method according to claim 1 is characterized in that: cigarette smoke contamination process is following: use the diluted sample nutrient solution that flue gas TPM sample is adjusted to different concns, 8 non-zero-doses are set; Behind cell cultures 24 h; Nutrient solution is removed in suction; 96 orifice plates (removing most peripheral 36 holes) every row 6 holes are that the one group of contamination of flue gas TPM and positive control that is set to negative control, 8 various dose is respectively handled; Every hole adds the dilution nutrient solution that contains negative control, flue gas TPM or positive control of 100 μ l, and every hole adds 100 μ l dilution nutrient solution as blank, in 37 ℃, 5% CO in 36 holes of the most peripheral of 96 orifice plates ₂Cultivate 24 h under the condition.

5. cigarette smoke vitro cytotoxicity testing method according to claim 1, it is characterized in that: the WST-1 dyeing course is following: behind flue toxicity contaminated 24 h, every hole adds 10 μ l through the WST-1 of incubation dye liquor, and cell is in 37 ℃, 5% CO ₂Hatch 2 h under the condition, quick oscillation 1 min under the room temperature uses ELIASA to detect the light absorption value of every hole in 450 nm wavelength, and reference wavelength is 630 nm.

6. cigarette smoke vitro cytotoxicity testing method according to claim 1 is characterized in that:

The result with analyze as follows:

Original light absorption value A:A=A _{450 nm}– A _{630 nm}

Through gauged light absorption value A _{(through blank correction)}: A _{(through blank correction)}=A – A _(blank)

Relative light absorption value C:C= * 100%

(

: the MV of light absorption value)

Light absorption value is represented cell survival rate relatively.