Method for screening micropore plate of tissue factor approach restrainer and uses thereof

A screening method and technology of tissue factor, applied in in vitro microwell plate screening of tissue factor pathway inhibitors, in the field of tissue factor pathway inhibitors, can solve problems such as unsuitable simultaneous determination of a large number of samples, large amount of reagents used, high price, etc., to achieve Low price, easy operation, and the effect of reducing the amount of reagents

Inactive Publication Date: 2008-06-18
CHINA PHARM UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the chromogenic substrate method reported in the past is expensive, complicated to operate, and consume

Method used

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  • Method for screening micropore plate of tissue factor approach restrainer and uses thereof
  • Method for screening micropore plate of tissue factor approach restrainer and uses thereof
  • Method for screening micropore plate of tissue factor approach restrainer and uses thereof

Examples

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Embodiment 1

[0055] Microplate Screening for Tissue Factor Pathway Inhibitors

[0056] Taking tissue factor as the target, using the microplate screening method described in the present invention, effective TF inhibitors are screened from natural active ingredients, in order to obtain possible compounds for treating thrombotic diseases and discover new anticoagulant drugs. Human vascular endothelial cell line (ECV304) and human peripheral blood mononuclear cells are used to stimulate the expression of TF by inflammatory factors (TNF) or bacterial lipopolysaccharide (LPS), and a certain dose of natural compound is added to co-culture with the cells for a certain period of time. The microwell plate screening method is used to investigate its influence on the procoagulant activity of TF. We screened hydroxygenkwain (IC 50 =0.04μM), kaempferol (IC 50 =0.04μM) and other compounds have strong inhibitory effect on TF expression; ruscogenin (IC 50 =0.02μM), diosgenin (IC 50 =0.043μM), ginsenos...

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Abstract

The invention relates to the field of medicament, in particular to an in-vitro microplate-based screening method of tissue factor (TF) pathway inhibitors, and is characterized in that the invention comprises the following steps: incubating cells and the medicament to be measured together; getting cell lysis solution after freeze thaw, blowing and beating; putting the cell lysis solution on the microplate for incubation; adding human prothrombin complex solution for incubation; adding chromogenic substrate solution in the reactant liquor for incubation; measuring the value of absorbance with a micro-plate reader of 405nm. The invention also discloses some tissue factor pathway inhibitors screened out by using the method, such as hydroxygenkwanin, ruscogenin, quercetinic acid, ginsenoside Rb1, ginsenoside or diosgenin and so on.

Description

technical field [0001] The invention relates to the field of medicines, in particular to an in vitro microwell plate screening method for tissue factor (TF) pathway inhibitors, and the invention also discloses some tissue factor pathway inhibitors screened by the method. Background technique [0002] Tissue factor (TF), coagulation factor III, also known as thromboplastin or thromboplastin (CD142), is a single-chain transmembrane glycoprotein composed of 263 amino acids and belongs to the cytokine superfamily. TF is not only the receptor of coagulation factor VII on the cell surface, but also the cofactor of FVII or activated factor VII (F VIIa). It is the only coagulation factor that exists in cells and tissues other than normal human plasma. Modern studies have shown that the complex of TF and FVIIa is the most important initiator of blood coagulation, and this complex starts the coagulation cascade reaction: when blood vessels are damaged, blood is exposed to the surface ...

Claims

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Application Information

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IPC IPC(8): G01N21/17C12Q1/02
Inventor 余伯阳蒋文雯寇俊萍
Owner CHINA PHARM UNIV
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