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Improved cigarette smoke in-vitro contamination method

A technology of cigarette smoke and gas, which is applied in the field of in vitro drug exposure of cigarette smoke, and can solve problems such as difficulty in controlling the concentration of cigarette smoke

Inactive Publication Date: 2010-03-17
ZHENGZHOU TOBACCO RES INST OF CNTC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is characterized in that the cells are directly exposed to mainstream cigarette smoke. Although the cigarette smoke is directly exposed, the gas phase part of the cigarette smoke in contact with the cells is still a part of the gas phase substances dissolved in water, and the concentration of the cigarette smoke in contact with the cells is not high. easy to control

Method used

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  • Improved cigarette smoke in-vitro contamination method

Examples

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Effect test

example 1

[0016] The reference cigarette 2R4F was exposed in vitro. According to the method of the present invention, connect the absorption bottle and the smoking machine, after smoking 20 cigarettes, use DMSO to extract TPM to 10 mg / mL, then add calcium-magnesium phosphate buffer solution equal to the volume of DMSO to the absorption bottle to make the concentration For an equal amount of 10 mg / mL, TPM and GVP were mixed in a ratio of 1:1. Chinese hamster ovary cells (CHO) were selected, and TPM, GVP, TPM+GVP were used for cytotoxicity test respectively. After exposure, the results were as follows:

[0017]

[0018] Note: * "EC50" is the half effective concentration of 50% effective eoncentration

[0019] The results showed that GVP did have a biological effect, and the biological effect of TPM+GVP was stronger than that of TPM and GVP alone.

example 2

[0021] In vitro exposure to a domestic flue-cured tobacco brand cigarette. According to the method of the present invention, according to the method, connect the absorption bottle and the smoking machine, after smoking 20 cigarettes, use DMSO to extract TPM to 10mg / mL, then add calcium-free magnesium phosphate equal to the volume of DMSO to the absorption bottle Buffer, make the concentration equal to 10 mg / mL, and mix TPM and GVP in a ratio of 1:1. The concentration of TPM+GVP was formulated as: 25, 50, 100, 125, 250, 500 μg / mL. When the positive control is without activation system: the positive control of TA98 is 40 μg / mL nitrofluorene, and the positive control of TA100 is 10 μg / mL; when there is an activation system: the positive control of TA98 and TA100 is 20 μg / mL anthramine. Negative control is solvent control.

[0022] Then, draw the prepared 0.1mL test substance and 0.1mL bacterial solution and mix them evenly, and pre-incubate for 30min at 37°C; add 0.5mL 5% S9 mi...

example 3

[0026] The reference cigarette 2R4F was selected for in vitro exposure. According to the method of the present invention, connect the absorption bottle and the smoking machine, after smoking 20 cigarettes, use DMSO to extract TPM to 10 mg / mL, then add calcium-magnesium phosphate buffer solution equal to the volume of DMSO to the absorption bottle to make the concentration For an equal amount of 10 mg / mL, TPM and GVP were mixed in a ratio of 1:1.

[0027] Then, the in vitro micronucleus test was carried out, and the CHO was cultured to a concentration of 1 × 10 5 cells / mL, the cell suspension was inoculated in a 50 mL culture flask at 5 mL / bottle. Cells were incubated overnight to allow attachment.

[0028] The extract was diluted with cell culture medium, and TPM and TPM+GVP solutions were prepared in different doses: 50 μg / mL, 75 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL. After the cells grow on the wall, discard the original culture medium, add the test substance (TPM and TP...

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Abstract

The invention relates to an improved cigarette smoke in-vitro contamination method. The biological effect of the cigarette smoke is evaluated by mixing the gaseous phase part (GVP) and the particle phase part (TPM) of the cigarette smoke to carry out the in-vitro contamination method. Specifically, the improved cigarette smoke in-vitro contamination method comprises the following steps: a. collection of the particle phase part and the gaseous phase part of smoke; b. mixing of the particle phase part and the gaseous phase part; and c, in-vitro contamination: diluting TPM+GVP with a proportion of 1:1 into different concentration gradients, and carrying out a cytotoxicity test, a micronucleus test and an Ames test by in-vitro contamination method. The invention has the advantages that the gaseous phase part of cigarette smoke is collected by an absorption bottle; the in-vitro contamination way of combining GVP and TPM can comprehensively and truly reflect the biological effect of the cigarette smoke; based on the conventional TPM contamination way, the contamination of the gaseous phase part of cigarette smoke is added, so that the in-vitro contamination method is more close to the actual cigarette smoke exposure condition; besides, the contamination concentration is easy to control and the operation is simple.

Description

technical field [0001] The present invention relates to an in vitro poisoning method of cigarette smoke, that is, adopting the method of combining cigarette smoke gas phase part (GVP) and particle phase part (TPM) for in vitro poisoning, so that the test object (cell or bacteria) The exposure environment is closer to the actual cigarette smoke exposure. Background technique [0002] In terms of toxicological evaluation of cigarette additives, some foreign research institutions and companies have carried out a lot of research work. Clements et al conducted a study on the in vitro toxicological evaluation of cigarette additives. The research focused on the research design of the in vitro toxicological evaluation of cigarette additives. According to the standard method of genetic toxicology testing, bacterial mutation-Ames test, mammalian cell mutation-mouse lymphocytes Tumor analysis and chromosome structure variation analysis of cultured human cells or Chinese hamster cells,...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/02
Inventor 尚平平李翔聂聪赵乐彭斌刘惠民谢剑平
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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