CN102618619B

CN102618619B - Method for testing in vitro cytotoxicity of cigarette smoke

Info

Publication number: CN102618619B
Application number: CN 201210089227
Authority: CN
Inventors: 李翔; 聂聪; 尚平平; 孙学辉; 杨松; 王宜鹏; 刘惠民
Original assignee: Zhengzhou Tobacco Research Institute of CNTC
Current assignee: Zhengzhou Tobacco Research Institute of CNTC
Priority date: 2012-03-30
Filing date: 2012-03-30
Publication date: 2013-09-18
Anticipated expiration: 2032-03-30
Also published as: CN102618619A

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 cigarette smoke in vitro toxicology evaluation study field, is a kind of method of testing the cigarette smoke vitro cytotoxicity specifically.

Background technology

The research of the in vitro toxicology of cigarette smoke has become estimates smoking to one of important means of Health hazard, and at present, the research of the in vitro toxicology of relevant cigarette smoke concentrates on cytotoxicity and the genotoxicity aspect of smoke condensate mostly.How science thinks poorly of the hazardness of hazard cigarette product and tobacco additive agent exactly, does not also have in the world unified method and program at present.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 at present adopting when cigarette smoke cytotoxicity is estimated is 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, reliability, the characteristics such as quick, easy be a kind of demand.

WST-1(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, in the situation that electron coupling reagent exists, can be generated orange-yellow formazan by more Intramitochondrial desaturase reduction.Cell proliferation is more, and then color is darker; Cell survival rate is lower, and then color is more shallow.It is water miscible that WST-1 produces the De formazan, 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 to cell without overt toxicity.

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

Summary of the invention

Purpose of the present invention is just for the problem of above-mentioned existence, 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 be achieved through the following technical solutions:

1) preparation experiment reagent: have following several:

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

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

(3) WST-1 dye liquor :-20 ℃ of preservations, 37 ℃ of incubations melt before use, avoid precipitating.

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

(5) negative control: 2%(v/v) dimethyl sulfoxide (DMSO) (DMSO).

2) cell inoculation culture: 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.

3) cigarette smoke contamination: use the diluted sample nutrient solution that flue gas total particulate matter sample is adjusted to different concns, 8 non-zero-doses are set.Behind cell cultures 24 h, suck nutrient solution, 96 orifice plates (except most peripheral 36 holes) every row 6 holes are that the one group of contamination of flue gas total particulate matter and positive control that is set to respectively negative control, 8 various dose is processed, every hole adds the dilution nutrient solution that contains negative control, flue gas total particulate matter or positive control of 100 μ l, 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 microplate reader to detect every hole at the light absorption value at 450 nm wavelength places, and reference wavelength is 630 nm.

5) results and analysis

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

Light absorption value A through overcorrection _{(through blank correction)}: A _{(through blank correction)}=A – A _(blank)

Relative light absorption value C:C=

Figure 2012100892279100002DEST_PATH_IMAGE002

* 100%

(

Figure 2012100892279100002DEST_PATH_IMAGE004

: the mean value of light absorption value)

Light absorption value represents cell survival rate relatively.

The present invention has following characteristics compared to existing technology: reduced the repeatedly testing sequence of washed cell in whole process of the test, so 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 the De formazan, 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 finish light absorption value after the WST-1 dyeing in 2 h 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 repeatedly read light absorption value at different time, make the test duration more flexible, the test result good reproducibility, and for after the contamination of test flue gas total particulate matter the continuous action of cytotoxicity being provided the condition of continuous monitoring, be conducive to find best detection time; Linearity range is wide during the WST-1 color reaction, and is highly sensitive.This measuring method has advantages of that also 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.

Description of drawings

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

As can be seen from Figure 1: cell survival rate and flue gas total particulate matter concentration are obvious dose-effect relationship, and cell survival rate increases with poisoning dosage and reduces.

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, suck nutrient solution, 96 orifice plates (except most peripheral 36 holes) every row 6 holes are that the one group of contamination of flue gas total particulate matter and positive control that is set to respectively negative control, 8 various dose (10,50,75,100,120,140,160,200 μ g/ml) is processed.Every hole adds the dilution nutrient solution that contains negative control, flue gas total particulate matter 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 microplate reader to detect every hole at the light absorption value at 450 nm wavelength places, and reference wavelength is 630 nm.

The cytotoxicity test result

Original light absorption value

Figure 2012100892279100002DEST_PATH_IMAGE006

* *: dash area is blank

Calibrated light absorption value

Figure 2012100892279100002DEST_PATH_IMAGE008

Relative light absorption value

Figure 2012100892279100002DEST_PATH_IMAGE010

Claims

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

1) preparation experiment reagent, the experiment reagent of preparation has following:

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

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

(3) WST-1 dye liquor :-20 ℃ of preservations, 37 ℃ of incubations melt before use, avoid precipitating;

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

(5) negative control: 2%(v/v) dimethyl sulfoxide (DMSO) (DMSO);

2) cell inoculation culture, cell inoculation culture process is as follows: 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;

3) cigarette smoke contamination;

4) WST-1 dyeing, the WST-1 dyeing course is as follows: 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 microplate reader to detect every hole at the light absorption value at 450 nm wavelength places, and reference wavelength is 630 nm;

5) results and analysis is as follows:

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

Relative light absorption value C:C= * 100%

Light absorption value represents cell survival rate relatively.

2. cigarette smoke vitro cytotoxicity testing method according to claim 1 is characterized in that: cigarette smoke contamination process is as follows: use the diluted sample nutrient solution that flue gas total particulate matter sample is adjusted to different concns, 8 non-zero-doses are set; Behind cell cultures 24 h, suck nutrient solution, 96 orifice plates are except most peripheral 36 holes, every row 6 holes are that the one group of contamination of flue gas total particulate matter and positive control that is set to respectively negative control, 8 various dose is processed, every hole adds the dilution nutrient solution that contains negative control, flue gas total particulate matter or positive control of 100 μ l, 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.