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Establishment method of apoptosis model for inducing pig small intestinal epithelial cells by hydrogen peroxide

An epithelial cell and method for establishing a technology, which is applied in the field of cell apoptosis model construction, can solve the problems such as the lack of an apoptotic model suitable for porcine intestinal epithelial cells, the low cell apoptosis rate, and the complicated preparation process, and achieves stable and reliable effect and induction. Short time and low cost effect

Inactive Publication Date: 2018-05-15
WUHAN POLYTECHNIC UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] To construct a cell apoptosis model, it is necessary to select model cells and inducers. Among them, the cells commonly used to construct cell apoptosis models are mostly liver cells, cancer cells, breast cells, neutrophils, and blood cells. The inducers include fusion proteins , monoclonal antibody, lipopolysaccharide, etc. In the actual application process of these inducers, there are disadvantages such as complicated preparation process, complicated induction operation, and low apoptosis rate.
Therefore, there is no method suitable for constructing the apoptosis model of pig small intestinal epithelial cells in the prior art

Method used

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  • Establishment method of apoptosis model for inducing pig small intestinal epithelial cells by hydrogen peroxide
  • Establishment method of apoptosis model for inducing pig small intestinal epithelial cells by hydrogen peroxide
  • Establishment method of apoptosis model for inducing pig small intestinal epithelial cells by hydrogen peroxide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 Effect of concentration of hydrogen peroxide on the viability of IPEC-1 cells

[0062] (1) will 1×10 5 Cells to be induced per well were inoculated into a 96-well plate, and 100 μL of cell culture medium was added to each well, and the cells were evenly dispersed in the well plate by slight shaking. After 3 days of culture, when the cells adhered to the wall and grew to 70%-80% , aspirate the culture medium, and wash the well wall twice with phosphate buffered saline.

[0063] (2) Add the prepared cell culture solution into the orifice plate successively, 100 μL per hole, wherein, the cell culture solution is divided into the control group and test A and B groups, and the concentrations of hydrogen peroxide in the cell culture solution in the three groups are respectively 0 mM, 0.2mM and 0.5mM, each group repeated 8 times.

[0064] (3) After being stimulated by hydrogen peroxide for 3 hours, the culture medium was aspirated, and the cell viability of IPEC-1 ...

Embodiment 2

[0068] Example 2 Effect of concentration of hydrogen peroxide on the activity of lactate dehydrogenase in IPEC-1 cell supernatant

[0069] (1) will 1×10 5 Inoculate each well of cells to be induced into a 12-well plate, add 1mL of cell culture medium to each well, and shake slightly to disperse the cells evenly in the well plate. After 3 days of culture, when the cells adhere to the wall and grow to 70%-80% , aspirate the culture medium, and wash the well wall twice with phosphate buffered saline.

[0070] (2) Add the prepared cell culture solution into the orifice plate successively, 1mL per hole, wherein, the cell culture solution is divided into the control group and test A and B groups, and the concentrations of hydrogen peroxide in the cell culture solution in the three groups are respectively 0mM, 0.2mM and 0.5mM, each group repeated 4 times.

[0071] (3) After being stimulated by hydrogen peroxide for 3 hours, absorb the cell culture supernatant, transfer it to a 1.5 ...

Embodiment 3

[0075] Example 3 Effect of concentration of hydrogen peroxide on expression of apoptosis-related molecules caspase3, p38 and JNK in IPEC-1 cells

[0076] (1) will 1×10 6 Inoculate cells / ml into 60mm culture dishes, add 4mL cell culture medium to each culture dish and shake gently to disperse the cells evenly in the culture dish. After 3 days of culture, wait for the cells to adhere to the wall and grow to 70%-80% Aspirate the culture medium and wash twice with phosphate buffered saline.

[0077] (2) Add the prepared cell culture solution into the well plate in turn, 4mL per hole, wherein the cell culture solution is divided into the control group and the test group B, and the concentrations of hydrogen peroxide in the cell culture solution in the two groups are 0mM and 0.5mM respectively , each group repeated 4 times.

[0078](3) After being stimulated by hydrogen peroxide for 3 hours, remove the culture medium, wash the well wall twice with phosphate buffer solution, add 10...

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Abstract

The invention discloses an establishment method of an apoptosis model for inducing pig small intestinal epithelial cells by hydrogen peroxide. The establishment method comprises the following steps: preparing a cell culture medium; inoculating the pig small intestinal epithelial cells into the cell culture medium for culturing, and carrying out trypsin digestion treatment to obtain to-be-induced cells; and adding the cell culture medium containing the hydrogen peroxide into the to-be-induced cells and carrying out hydrogen peroxide induction. In the technical scheme provided by the invention,a cell apoptosis model is established by taking a pig small intestinal epithelial cell as a model cell for the first time and the hydrogen peroxide is used as an inducer; the establishment method hasthe advantages of short induction time, simple operation, low cost and stable and reliable effects; and the establishment of the cell apoptosis model provides an important way to study a pig intestinal injury mechanism and regulation measures thereof.

Description

technical field [0001] The invention relates to the technical field of cell apoptosis model construction, in particular to a method for establishing a hydrogen peroxide-induced apoptosis model of porcine small intestinal epithelial cells. Background technique [0002] my country is the largest pig raising country in the world, and the pig raising industry has become an important industry related to the national economy and the people's livelihood. One of the major problems in the pig industry is the intestinal health of piglets, which has become a "bottleneck" that restricts the rapid and healthy development of the pig industry. Every year in my country, piglets suffer from intestinal health problems (such as indigestion, diarrhea, growth retardation and even death) The resulting economic losses amounted to 30 billion yuan. Studies have shown that intestinal epithelial cell apoptosis disorder can lead to structural damage and dysfunction of pig intestinal mucosa, and abnorma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0625
Inventor 刘玉兰康萍涂治骁陈少魁
Owner WUHAN POLYTECHNIC UNIVERSITY
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