Method for determining expression quantity of nicotine acetylcholine receptors in rat lymphocytes

A technology of nicotine acetylcholine and lymphocytes, applied in the field of life science research, can solve the problems of limited lymphocytes, different cell densities, and easy doping with red blood cells, etc.

Inactive Publication Date: 2020-05-29
ANHUI UNIVERSITY +1
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Problems solved by technology

[0005] At present, most of the studies related to nicotine addiction based on animal models focus on brain regions, and there is no detection of nAChRs expression in peripheral blood lymphocytes of rats. This is because fresh blood is required for cell separation, and blood obtained from one rat The maximum volume is 10 mL, and the obtained lymphocytes are limited, and the cell density in the peripheral blood of different species is also different, and it is easy to be mixed with red blood cells during gradient centrifugation

Method used

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  • Method for determining expression quantity of nicotine acetylcholine receptors in rat lymphocytes
  • Method for determining expression quantity of nicotine acetylcholine receptors in rat lymphocytes
  • Method for determining expression quantity of nicotine acetylcholine receptors in rat lymphocytes

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Embodiment 1

[0032] Experimental purpose: A method for detecting the expression of nAChRs in peripheral blood lymphocytes of rats by improving the protein sample preparation before WB experiment and the subsequent experimental conditions.

[0033] A, prepare lymphocyte separation liquid, separate lymphocytes, specifically include the following steps:

[0034]Put 5 mL of rat peripheral blood in an anticoagulant tube, centrifuge at 1500 r / min at 22°C for 15 min within 2 h to obtain the cloudy buffy layer, and dilute it with 2 mL of sterile PBS to make a white blood cell diluent;

[0035] Prepare the lymphocyte separation solution: add 2 mL of sterilized 1.5 mol / L sodium chloride solution to 18 mL of Percoll stock solution at a volume ratio of 9:1 to prepare 20 mL of Percoll dilution; take 5.785 mL of Percoll separation solution Add 4.215 mL of 0.9% sterile saline to prepare a cell separation solution of 1.081 g / mL;

[0036] Isolate lymphocytes: Carefully add 2 mL of 1.081 g / mL cell separati...

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Abstract

The invention belongs to the field of life science research, and specifically relates to a method for detecting the expression quantity of nicotine acetylcholine receptors (nAChRs) in peripheral bloodlymphocytes of rats, which is characterized by comprising the following steps: 1) separating peripheral blood lymphocytes of rats; 2) acquiring cell total protein, concentration determination and sample preparation; and 3) detecting the expression quantity of the nAChRs in the lymphocytes by a western blot (WB) experiment. According to the invention, the detection of the protein expression in theanimal peripheral blood in real time can be realized by determining the protein in the peripheral blood lymphocytes, and the kit has important significance for the research of nicotine addiction biomarkers.

Description

technical field [0001] The invention belongs to the field of life science research, specifically a method for detecting the expression of nAChRs in peripheral blood lymphocytes of rats by improving the separation of peripheral blood lymphocytes of rats, the preparation of protein samples before WB experiments and the conditions of WB experiments . Background technique [0002] Nicotine is the main addictive substance in tobacco products. At present, the basis for objectively evaluating the degree of nicotine addiction is not yet clear. The study of biomarkers of nicotine addiction is very important for smoking and health issues. [0003] A large number of previous studies on neurodegenerative diseases have suggested that peripheral blood is an important way to discover biomarkers. Studies on Alzheimer's disease and Parkinson's disease have shown that there are significant differences and consistent differential expression of proteins co-expressed in brain regions and periph...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/6893G01N2800/307
Inventor 程皖燕付亚宁陈欢王安刘勇侯宏卫胡清源
Owner ANHUI UNIVERSITY
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