Preparation method of high-throughput airway inflammatory drug screening cell model based on NF-kappaB signaling pathway, and application of cell model

Abstract

The invention discloses a preparation method of a high-throughput airway inflammatory drug screening cell model based on an NF-kappaB signaling pathway, and an application of the cell model. The preparation method uses a bronchial smooth muscle cell or a bronchial epithelial cell as a source cell, NF-kappaB in the cell is activated by a stimulating factor, and the total NF-kappaB ratio and the phosphorylated NF-kappaB ratio in the cell are detected by a homogeneous time-resolved fluorescence technique to obtain the phosphorylation level of NF-kappaB; and parameters are adjusted to make a ratioof the phosphorylation level of NF-kappaB in a model group cell to the phosphorylation level of NF-kappaB in a normal group cell be equal to or more than 1.2 in order to obtain the cell model. The inflammatory cell model is constructed by screening the influences of the type and the density of the cell and the type, the concentration and the stimulation time of the stimulating factor on the phosphorylation of NF-kappaB and detecting the phosphorylation level of NF-kappaB in the cell through an HTRF technology, so a basis is provided for screening and studying of airway inflammation drugs.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a preparation method and application of a high-throughput airway inflammation drug screening cell model based on NF-κB signaling pathway. Background technique [0002] The activation of the immune system is a very complicated process. Transcription factor nuclear factor-kappa B (NF-κB) can be activated by a variety of stimuli and control different genes and biological immune responses, and has become a widely studied immune molecules. [0003] NF-κB can induce pro-inflammatory cytokine gene transcription and affect autoimmune and acquired immune responses, and is a drug therapy target for various inflammatory diseases. NF-κB is a dimeric protein whose subunits belong to the Rel proto-oncoprotein family. In most cells, including human bronchial epithelial cells, the major inducible NF-κB consists of 50 kD (p50) and 65 kD (RelA, pre-p65) subunits. When cells are stimulated b...

AI Technical Summary

Problems solved by technology

[0006] According to the reported literature on NF-κB detection, current NF-κB detection methods generally include immunohistochemistry, reverse transcriptase-polymerase chain reaction (Reverse transcriptase-polymerase chain reaction, RT-PCR), protein immunoassay Western blot, but the common problem of the three is that the detection steps are cumbersome and time-consuming, and there are certain limitations in the rapid and high-throughput screening of airway inflammation drugs

Immunohistochemical staining is simple to operate, highly sensitive, and specific, and can combine morphology and antigen localization, but there are many factors that affect immunohistochemistry. For paraffin sections, the problem of stripping is common, which can affect staining and results For cell slides and smears, the operation process is cumbersome, the efficiency is low, and the film processing process is easy to introduce a lot of errors, making the results comparable

RT-PCR technology has the characteristics of high sensitivity, high specificity, and high degree of automation, and can also quantify templates, but fluorescent dyes can combine with non-specific double-stranded DNA, which is prone to false positive signals

The main advantage of Western blot is that it can reflect the molecular weight and subtype of proteins, but it is cumbersome and time-consuming in terms of operation and sample pretreatment. The experimental results only have image scanning, which is poor in quantification, and the results cannot be statistically analyzed, which affects the accuracy of the analysis.

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