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Method for inducing apoptosis by thermal stimulation

A technology of inducing cells and heat stimulation, applied in the field of cell biology, can solve the problems of poor repetition and stability, high necrosis rate, low apoptosis rate, etc.

Active Publication Date: 2020-11-17
SHANDONG UNIV OF TRADITIONAL CHINESE MEDICINE
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0005] Aiming at the problem that thermal stimulation has poor repeatability and stability of apoptosis, and the necrosis rate is high and apoptosis rate is low, this study provides a method for thermal stimulation to induce apoptosis. The effect of early apoptosis rate is high, and the effect verification of drugs and the detection of related apoptosis proteins are further realized

Method used

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  • Method for inducing apoptosis by thermal stimulation
  • Method for inducing apoptosis by thermal stimulation
  • Method for inducing apoptosis by thermal stimulation

Examples

Experimental program
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Effect test

Embodiment 1

[0034] A method for thermal stimulation to induce apoptosis of mouse MCM cardiomyocytes, comprising the following steps:

[0035] (1) Cell culture

[0036] Prepare DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin mixture. Take out the MCM cell cryopreservation tube from liquid nitrogen, put it in warm water at 37°C, hold it with tweezers and shake it gently to make it melt quickly, and immediately pour the liquid into a petri dish filled with fresh medium. At 37°C, 5% CO 2 The next experiment was carried out when the cells were cultured in a good state.

[0037] (2) Treatment of mouse MCM cardiomyocytes

[0038] Take mouse MCM cardiomyocytes in the logarithmic growth phase, and use 2×10 per well 5 seeded in 6-well plates at 37°C, 5% CO 2 cultured in an incubator. After the cells grew into a single layer, they were digested with trypsin, and fresh DMEM medium was added to adjust the cell density to 2×10 6 pieces / ml. Pipette 100 μl of the abov...

Embodiment 2

[0050] A method for heat stimulation to induce mouse MCM cell apoptosis, comprising the following steps:

[0051] (3) Induction of apoptosis

[0052] Gradient heating mode of PCR temperature control system was adopted. Place the ep tube in step (2) on the PCR metal heating plate. The heating program is to increase the temperature step by step from 37°C in units of 1°C. The process is: 37°C, 4min; 38.5°C, 4min; 40 °C, 4min; 41.5°C, 4min; 43°C, 60min; 41.5°C, 4min; 40°C, 4min; 38.5°C, 4min; 37°C, 4min. After the heat stimulation, transfer to a new 6-well plate, 500 μl per well of the heat-stimulated cell suspension with adjusted cell density. The 6-well plate was placed at 37°C, 5% CO 2 The culture was continued in the incubator, and after 22 h of culture, the level of apoptosis was analyzed by flow cytometry.

[0053] All the other steps are the same as in Example 1.

Embodiment 3

[0055] A method for heat stimulation to induce apoptosis of U937 cells, comprising the following steps:

[0056] (3) Induction of apoptosis

[0057] Gradient heating mode of PCR temperature control system was adopted. Place the ep tube in step (2) on the PCR metal heating plate. The heating program is to gradually increase the temperature from 37°C with a gradient of 1°C. The process is: 37°C, 1 min; 37.5°C, 1 min ;38°C, 1min; 38.5°C, 1min; 39°C, 1min; 39.5°C, 1min; 40°C, 1min; 40.5°C, 1min; 41°C, 1min; 41.5°C, 1min; °C, 1min; 42.5°C, 1min; 43°C, 1min; 43.5°C, 1min; 44°C, 60min; 43.5°C, 1min; 43°C, 1min; 42.5°C, 1min; 42°C , 1min; 41.5°C, 1min; 41°C, 1min; 40.5°C, 1min; 40°C, 1min; 39.5°C, 1min; 39°C, 1min; 38.5°C, 1min; 38°C, 1min ; 37.5°C, 1 min; 37°C, 1 min. After the heat stimulation, transfer to a new 6-well plate, 500 μl per well of the heat-stimulated cell suspension with adjusted cell density. The 6-well plate was placed at 37°C, 5% CO 2 The culture was continued...

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Abstract

The invention belongs to the technical field of cytobiology, relates to a method for inducing apoptosis by thermal stimulation, and in particular to a method for inducing apoptosis by taking a PCR temperature control system as thermal stimulation. According to the method, a gradient gradual heating mode is adopted, wherein gradient gradual heating means the temperature stays for a period of time after rising; and the temperature is gradually reduced in a gradient manner after rising to a target temperature and being kept for a period of time. A model for inducing apoptosis based on a gradientgradual heating mode of a PCR precise temperature control system is established, so that the early apoptosis rate of the method is more than than the sum of the late apoptosis rate and the necrosis rate, and the method has good reproducibility and stability. The effect verification on drugs and the detection on related apoptotic proteins can be achieved due to the high early apoptosis rate; Apoptosis caused by the thermal stimulation method can be used for simulating fever of an organism caused by various reasons (pathogenic microorganism infection, organism inflammatory response, malignant tumor diseases and the like), and thermal stimulation caused by the fever can cause damage to myocardial cells and the like of the organism.

Description

technical field [0001] The invention belongs to the technical field of cell biology, and relates to a method for inducing cell apoptosis by heat stimulation, specifically, a method for inducing cell apoptosis by using a PCR temperature control system or a similar precise temperature control device as heat stimulation. Background technique [0002] The concept of apoptosis was proposed to describe morphologically different ways of cell death. Apoptosis has been described as a unique cell death pathway in which cells actively participate in self-clearance. For the study of apoptosis, repetitive and stable methods are critical. There are many factors that cause apoptosis, such as chemical factors (hydrogen peroxide, dithiothreitol, lipopolysaccharide, poisons, etc.), physical factors (hypoxia, mechanical damage, high temperature, low temperature, electric current, etc.), biological factors (various pathogens, viruses, parasites, oxidized low-density lipoproteins, fusion prote...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077C12N5/09C12Q1/02
CPCC12N5/0657C12N5/0693G01N33/5005C12N2523/00
Inventor 杨勇杨佳容蓉马青云于钦辉
Owner SHANDONG UNIV OF TRADITIONAL CHINESE MEDICINE
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