Method for screening anti-endometritis drugs in dairy cows, cell model and application

An endometritis and screening method technology, which is applied in the fields of screening, cell models and uses of anti-dairy cow endometritis drugs, can solve the problems such as the lack of ideal research strategies for anti-dairy cow endometritis drugs, and achieves high-throughput Screening and detection, rapid screening and detection, the effect of eliminating interference

Active Publication Date: 2019-02-01
LANZHOU INST OF ANIMAL SCI & VETERINARY PHARMA OF CAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, there is still no ideal research strategy for the in vitro activity detection and accurate and effective evaluation methods of anti-cow endometritis drugs

Method used

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  • Method for screening anti-endometritis drugs in dairy cows, cell model and application
  • Method for screening anti-endometritis drugs in dairy cows, cell model and application
  • Method for screening anti-endometritis drugs in dairy cows, cell model and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] The specific steps of the method for in vitro screening and detection of anti-cow endometritis medicine of the present invention are as follows:

[0065] 1. Cultivation of dairy cow endometrial epithelial cells: Inoculate and culture dairy cow endometrial epithelial cells in logarithmic growth phase in 96 microwell plates, in saturated humidity, 5% CO 2 Pre-cultivate in an incubator, the medium is DMEM / F12 culture solution and 10% endotoxin-free fetal bovine serum (Gibco Company). Cell suspension in 5×10 4 Inoculate at a concentration of 1 / ml, 100 μl per well.

[0066] 2. Induction of cell inflammatory response: After 24 hours of cell inoculation and culture, the cell metabolism fluid in the culture plate was discarded. Dissolve the bacterial endotoxin LPS (O111:B4, Sigma Company) in fresh DMEM / F12 culture solution to a concentration of 10 μg / ml, and add it to all wells except the four wells A1, A2, B1 and C1 , 100 μl / well; A1, A2, B1 and C1 four wells continued to a...

Embodiment 2

[0075] In this embodiment, the theory of the present invention (integrin-linked protein kinase (ILK) expression in dairy cow endometrium epithelial cells is extremely significantly increased in the inflammatory response by immunoblotting and laser near-infrared molecular imaging system )authenticating:

[0076] The dairy cow endometrial epithelial cell culture, inflammatory response induction process and detection method are consistent with the method in Example 1. After the total protein was extracted from the treated cells by conventional methods, polyacrylamide gel electrophoresis and semi-dry transfer were performed, and anti-integrin-linked protein kinase antibody (Anti-ILK antibody) and anti-actin antibody (anti-ACTB antibody) for Western blot detection, the results were obtained by film exposure method, the results can be found in figure 2 . In addition, laser near-infrared molecular imaging technology was used to detect the expression changes of integrin-linked prot...

Embodiment 3

[0078]This embodiment uses laser near-infrared molecular imaging technology to the theory of the present invention (after endometritis occurs in dairy cows, the expression of integrin-linked protein kinase in endometrial epithelial cells is extremely significantly increased, and the expression of anti-inflammatory drugs on the protein The amount has a significant down-regulation effect) for verification:

[0079] The process of culturing dairy cow endometrial epithelial cells, inducing inflammatory response and drug intervention is consistent with the method in the present invention. The treated cells were detected by laser near-infrared molecular imaging with IRDye800-labeled integrin-linked protein kinase antibody (Anti-ILKantibody-IRDye800) and ALexa680-labeled internal control protein β-actin antibody (anti-β-actin antibody-ALexa680). The results were obtained by laser scanning, the results can be found in Figure 4 . It can be seen from the figure that the emission opti...

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Abstract

The invention provides a method for in vitro screening and detecting anti-endometritis drugs in dairy cows. The method for in vitro screening and detecting the anti-endometritis drugs in the dairy cows comprises the steps that after in vitro cultured dairy cow endometrial epithelial cells are induced to have an inflammatory reaction, drugs to be detected are added, and the anti-inflammatory activity of the drugs to be detected is accurately reflected by detecting the expression change of inflammatory proteins in cells relative to internal reference proteins; if the relative expression quantityof the inflammatory proteins is reduced, the drugs to be detected has anti-inflammatory activity, and the multiple of the relative expression quantity is the magnitude of the relative anti-inflammatory activity of the drugs to be detected; otherwise, the anti-inflammatory activity does not exist; and the inflammatory proteins are integrin linked-kinase. According to the method for in vitro screening and detecting the anti-endometritis drugs in the dairy cows, the anti-endometritis activity in the dairy cows of the drugs to be detected can be simply, conveniently and rapidly screened and detected in vitro. When a multi-hole board is used, high throughput screening and detection can be realized, and a screening and detection result has important reference value for modern clinic applicationand modernization development of traditional Chinese medicines.

Description

technical field [0001] The invention relates to a screening method and a cell model of an anti-cow endometritis drug. Background technique [0002] Preclinical drug activity testing is an important process to determine whether a drug is suitable for disease treatment. Dairy cow endometritis is a high incidence of postpartum dairy cows, and the creation and screening of anti-inflammatory drugs for this disease is very important. In order to find safe and effective anti-inflammatory drugs, it is often necessary to select a suitable inflammatory model to screen and evaluate the anti-inflammatory effect. With the rapid development of cell culture technology, inflammatory cell models are widely used in the study of inflammatory mechanisms at home and abroad, and it is considered that they can be used in the research of drug anti-inflammatory activity screening. With the advent of the "antibiotic-free" era and the increasing demand for green and healthy milk sources, the applica...

Claims

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Application Information

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IPC IPC(8): G01N33/50G01N33/58G01N33/68G01N21/64
CPCG01N21/6428G01N33/5008G01N33/582G01N33/68G01N2800/7095G01N2201/129
Inventor 张世栋严作廷王东升杨峰武小虎
Owner LANZHOU INST OF ANIMAL SCI & VETERINARY PHARMA OF CAAS
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