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Method for testing induction of tobacco products on vascular endothelial cell apoptosis

A technology of tobacco products and vascular endothelium, which is applied in the field of in vitro toxicology research of tobacco products to achieve the effects of optimized process, simple operation and high sensitivity

Inactive Publication Date: 2018-02-23
ZHENGZHOU TOBACCO RES INST OF CNTC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to solve the problem that there is no universally applicable test method for inducing apoptosis of human vascular endothelial cells for tobacco products at present, and specially establishes a detection method that can induce apoptosis of human vascular endothelial cells for different types of tobacco product solutions. Methods The total particulate matter in cigarette and electronic cigarette smoke and snus solution were prepared, and then the corresponding solution was used to expose human vascular endothelial cells, and the cell apoptosis test established by Annexin V and flow cytometry method

Method used

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  • Method for testing induction of tobacco products on vascular endothelial cell apoptosis
  • Method for testing induction of tobacco products on vascular endothelial cell apoptosis
  • Method for testing induction of tobacco products on vascular endothelial cell apoptosis

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preparation example Construction

[0032] 1) Preparation of different tobacco product solutions

[0033] a) Preparation of total particulate matter solution in cigarette or e-cigarette smoke: use RM20H rotary smoking machine and Cambridge filter to capture total particulate matter in cigarette mainstream smoke, and use linear smoking machine and Cambridge filter to capture e-cigarette mainstream smoke Gas total particulate matter, cut the Cambridge filter that has captured the total particulate matter into pieces, put it into a conical flask, add an appropriate volume of DMSO to make the final concentration of the cigarette total particulate matter solution 10 mg / mL, and e-cigarette The concentration of the total particle phase was 250 mg / mL, shaken at room temperature for 30 min, filtered the DMSO solution with a 0.2 μm sterile filter, and stored at -80 °C.

[0034] b) Preparation of pouch-type snus solution: Dissolve the contents of the snus pouch with deionized water and DMSO to a solution with a final conce...

Embodiment 1

[0039] Cut the Cambridge filters that captured the total particulate matter of cigarette and e-cigarette aerosol respectively into pieces and put them into conical flasks, add an appropriate volume of DMSO to make the final concentration of the total particulate matter solution of cigarettes 10 mg / mL, and electronically The concentration of the total particulate matter in the smoke was 250 mg / mL. After shaking at room temperature for 30 min, the solution was filtered through a 0.2 μm sterile filter and divided into packages, and stored at -80°C until use.

[0040] The expanded and cultured cells were digested with 0.05% trypsin and prepared into 1x10 5 cells / mL of cell suspension. Add 3.5 mL of cell suspension to three 6 cm cell culture dishes and store at 37 °C, 5 % CO 2 Incubate under the condition for 24 h. After the cells were cultured for 24 hours, the medium was aspirated, and 3 mL of negative control medium was added to the control group (CK), and 450 μL of cigarette ...

Embodiment 2

[0042] Dissolve the contents of a certain snus with deionized water and DMSO to a solution with a final concentration of 250 mg / mL; shake the suspension overnight at 37 °C, collect the supernatant after centrifugation, and filter with a 0.2 μm sterile filter After aliquoting, store at -80°C until use.

[0043] The expanded and cultured cells were digested with 0.05% trypsin and prepared into 1x10 5 cells / mL of cell suspension. Add 3.5 mL of cell suspension to three 6 cm cell culture dishes and store at 37 °C, 5 % CO 2 Incubate under the condition for 24 h. After the cells were cultured for 24 hours, the culture medium was aspirated, and 3 mL of negative control medium was added to the control group (CK), and 45 μL of buccal DMSO solution, 15 μL of DMSO and 2940 μL of cell culture medium were added to the buccal DMSO solution treatment group 1200 μL of snus solution, 60 μL of DMSO and 1740 μL of cell culture medium were added to the snus solution treatment group. at 37°C, 5...

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Abstract

The invention discloses a method for testing induction of tobacco products on vascular endothelial cell apoptosis. The method is characterized in that tobacco product solutions are prepared and then are subjected to centrifugation and purification of filtration membranes, poisoning and exposure on human vascular endothelial cells with appropriate density which are inoculated and are cultured overnight are carried out, then Annexin V-FITC and PI dyeing on cell suspensions prepared through digestive cells can be carried out, and finally, cells are detected by using a flow-type cytometer so as toquickly analyze a cell apoptosis condition. The method has the advantages of simpleness and convenience in operation, high sensitivity, reliability in results and the like, and can accurately detectan induction condition of tobacco products of different types on the human vascular endothelial cell apoptosis.

Description

technical field [0001] The present invention relates to in vitro toxicology research of tobacco products, specifically a detection method that can be used for different tobacco products to induce apoptosis of vascular endothelial cells. Background technique [0002] There are currently many types of tobacco products, including cigarettes, snus, and electronic cigarettes. Different tobacco products have their own characteristics. For example, cigarettes generate aerosols through the burning of tobacco leaves, snus is infiltrated through the mouth, and electronic cigarettes generate aerosols by heating the liquid. However, these tobacco products all contain nicotine, alcohols, aldehydes and other compounds, and long-term ingestion in the body will lead to the risk of cardiovascular disease in the human body. Therefore, the development of a detection method applicable to the apoptosis of vascular endothelial cells induced by various tobacco products is helpful to the developme...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/50G01N15/14G01N1/30
CPCG01N33/5064G01N1/30G01N15/14
Inventor 乔梁峻谢复炜华辰凤李翔康彧尚平平赵阁赵俊伟郭军伟刘惠民
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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