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Lactic dehydrogenase determination-based cigarette smoke cell toxicity evaluation method

A lactate dehydrogenase, cigarette smoke technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problem of no unified method and procedure, and achieve fast and easy operation, stable results and high sensitivity Effect

Inactive Publication Date: 2014-05-28
CHINA NAT TOBACCO QUALITY SUPERVISION & TEST CENT
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Problems solved by technology

How to scientifically and accurately evaluate the harmfulness of cigarette products and tobacco additives, there is no uniform method and procedure in the world

Method used

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  • Lactic dehydrogenase determination-based cigarette smoke cell toxicity evaluation method
  • Lactic dehydrogenase determination-based cigarette smoke cell toxicity evaluation method
  • Lactic dehydrogenase determination-based cigarette smoke cell toxicity evaluation method

Examples

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example 1

[0037] Example 1: In order to investigate the cytotoxicity of two particles of cigarette samples to the A549 cell line, the following experiments were carried out:

[0038] Prepare the solution used in the experiment according to the above requirements. Cigarette samples were smoked using a rotary 20H smoking machine (Germany, Borgwaldt Company). The smoking mode was in accordance with ISO requirements, and the particulate phase components of cigarettes were captured by Cambridge filters. A total of 195 mg of smoke particulate matter was captured by smoking 20 cigarettes, and 19.5 mL of dimethyl sulfoxide (DMSO) was added after taking out the Cambridge filter, and extracted by shaking for half an hour to obtain smoke with a final concentration of 10 mg / mL Aerosol extract. Diluted with cell culture medium, prepared a series of smoke exposure solutions with different concentrations to infect A549 cells.

[0039] CHO cell lines were cultured in 1640 medium containing 10% fetal ...

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Abstract

The invention discloses a lactic dehydrogenase determination-based cigarette smoke cell toxicity evaluation method, comprising the following steps: 1) preparation of an experiment reagent; 2) cell inoculation; 3) cigarette smoke contamination; 4) lactate dehydrogenase (LDH) determination; 5) result and analysis. The cell toxicity of a smoke particulate matter and a smoke gas phase matter can be respectively inspected by preparation of a smoke particulate matter extracting solution and a smoke gas phase matter absorption liquid; the lactic dehydrogenase determination-based cigarette smoke cell toxicity evaluation method is applicable to a plurality of adherent culture cells, and can be used for inspecting the cell toxicity of the cigarette smoke on different cell lines. The detection sensitivity is improved by selection of cell lysis buffer and optimization of reaction time of LDH matrix liquid. The optimal contamination concentration is determined by optimizing the cigarette smoke contamination concentration, so as to obtain an optimal dose effect curve. The damage degree to a cell membrane caused by the smoke particulate matter and the smoke gas phase matter of the cigarette can be reflected by determination of the LDH, and the cell toxicity mechanism of the cigarette smoke is announced. In addition, the evaluation method also has the advantages of being rapid, simple and convenient to operate, high in sensitivity, and stable in result.

Description

technical field [0001] The invention relates to the research field of in vitro toxicological evaluation of cigarette smoke, in particular to a method for measuring the cytotoxicity of cigarette smoke particulate matter or gas phase matter based on lactate dehydrogenase (LDH). Background technique [0002] Carrying out in vitro toxicology experiments is an important method to evaluate the health hazards of smoking. Among them, cytotoxicity plays an important role in many pathological processes including cancer and inflammation, and cytotoxicity test is an important way to understand the mechanism of interaction between compounds and cells and tissues. How to scientifically and accurately evaluate the harmfulness of cigarette products and tobacco additives, currently there is no uniform method and procedure in the world. Detection of cytotoxicity can be performed by various biological endpoints, such as counting the number of cells by measuring the activity of acid phosphatas...

Claims

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Application Information

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IPC IPC(8): C12Q1/32C12Q1/02
Inventor 陈欢杨进刘洋刘彤韩书磊吴帅宾付立伟侯宏卫胡清源
Owner CHINA NAT TOBACCO QUALITY SUPERVISION & TEST CENT
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