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MTT (thiazolyl blue) cell toxicity test method of biological assessment of total particle matter in cigarette smoke

A technology for biological evaluation and cigarette smoke, applied in the field of tobacco and cigarette smoke safety evaluation, to achieve high sensitivity, reduce the steps of washing cells, and shorten the test cycle

Inactive Publication Date: 2013-05-08
ZHENGZHOU TOBACCO RES INST OF CNTC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the main toxicity of smoke still acts on the human body, and there are certain limitations in using animal cells to evaluate the cytotoxicity of total particulate matter in cigarette smoke.

Method used

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  • MTT (thiazolyl blue) cell toxicity test method of biological assessment of total particle matter in cigarette smoke
  • MTT (thiazolyl blue) cell toxicity test method of biological assessment of total particle matter in cigarette smoke
  • MTT (thiazolyl blue) cell toxicity test method of biological assessment of total particle matter in cigarette smoke

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Cytotoxicity Evaluation of Kentucky Reference Cigarette 3R4F

[0034] 1. Experimental part

[0035] 1.1 Main instruments and reagents

[0036] CO 2 Incubator; Inverted microscope; Microplate reader; 96-well cell culture plate; Cell culture flask; Flow cytometer.

[0037] LHC-8 medium; fetal bovine serum; phosphate buffer; trypsin; MTT dye; dimethyl sulfoxide (cell culture grade).

[0038] 1.2 Preparation of total particulate matter (TPM) in cigarette smoke

[0039] The cigarettes were balanced in a constant temperature and humidity chamber for 48 hours, and cigarettes with consistent weight and draw resistance were selected. According to the ISO standard method, smoke 20 cigarettes on a smoking machine, soak the Cambridge filter in DMSO at 10 mg / mL, extract it ultrasonically for 20 min, and finally filter it with a 0.22 μl filter tip, dispense it into cryopreservation tubes, and store Store in -80°C ultra-low temperature freezer for later use.

[0040] P...

Embodiment 2

[0048] Example 2 The cytotoxicity evaluation of a certain type of reference cigarette developed in China is carried out, and the specific implementation method refers to Example 1.

[0049] Depend on figure 2 It can be seen that the smoke total particulate matter sample of this type of reference cigarette has a relatively obvious inhibitory effect on BEAS-2B cells. In the concentration range of 0 to 70 μg / mL, with the increase of TPM sample concentration, the cell survival rate decreased significantly. Decreased with it, showing a significant dose-effect relationship.

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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

[0001] technical field [0002] The invention relates to a test method of MTT cytotoxicity for the biological evaluation of total particulate matter in cigarette smoke, and belongs to the technical field of safety evaluation of tobacco and cigarette smoke. Background technique [0003] Cigarette smoke is an aerosol composed of thousands of chemical substances, some of which have been identified as carcinogens or auxiliary carcinogens. Smoking is closely related to the occurrence of some important human diseases. The in vitro toxicity test of cigarette smoke has become a powerful means to evaluate the health hazards of smoking. Among them, the in vitro cytotoxicity test plays a very important role in evaluating the hazards of cigarette smoke and low-hazard cigarette products. Most of the methods used to evaluate the cytotoxicity of cigarette smoke are based on the principle of cell viability measurement. The International Cooperation Center for Tobacco Research (CORESTA) rec...

Claims

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Application Information

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IPC IPC(8): C12Q1/02G01N21/31
Inventor 毛健李鹏卢斌斌孙世豪谢剑平张建勋宗永立
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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