Method for screening medicaments injuring endothelial cells by active nitrogen and application

An endothelial cell and screening method technology, which is applied in the field of drug screening of endothelial cell damage caused by reactive nitrogen, can solve the problems of endothelial cell damage, the instability of reactive nitrogen small molecules, and the establishment of a model of reactive nitrogen damage to endothelial cells, so as to avoid blindness. sexual effect

Inactive Publication Date: 2010-02-24
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, since active nitrogen is a small molecule unstable substance, its existence time alone is extremely short. To establish a relevant cell model, it is necessary to select an appropriate active nitrogen as a modeling substance.
However, in the current research, there is no literature report about a certain reactive nitrogen inducer that can be used as a clear source of reactive nitrogen to cause damage to endothelial cells, and there is no in vitro study of the damage model of reactive nitrogen to endothelial cells under pathological conditions, and Related reports on the application of this model in pharmacology

Method used

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  • Method for screening medicaments injuring endothelial cells by active nitrogen and application
  • Method for screening medicaments injuring endothelial cells by active nitrogen and application
  • Method for screening medicaments injuring endothelial cells by active nitrogen and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 GSNO acts on EA.hy926 cells to create a reactive nitrogen injury model and use it for drug screening

[0024] Take the logarithmic growth of EA.hy926 cells, add an appropriate amount of trypsin-EDTA digestion solution to make the adherent cells fall off, collect the cells, count them, and prepare a cell suspension (5×10 4 / mL), add 100 μL to each well of a 96-well plate, at 37°C, 5% CO 2 Cultivate for 24h. Dissolve GSNO in sterile normal saline to prepare a solution with a mother liquor of 100mmol / L. After sterilizing with a 0.22μm filter membrane, add it to the cell culture medium to make the final concentrations 0.1, 0.5, 1, and 2mM, respectively. Use the solvent as a negative control, put it into the incubator and continue to cultivate for 24 or 48 hours under the same conditions. Add 10 μL of MTT (5 mg / mL, dissolved in D-Hanks’) 4 hours before the end of the culture, aspirate the supernatant at the end of the test, add 150 μL DMSO, measure the OD value o...

Embodiment 2

[0028] Example 2 SIN-1 acts on HUVEC cells to create a reactive nitrogen injury model and is used for drug screening

[0029] Using a method similar to that of Example 1, a reactive nitrogen damage model in which SIN-1 acts on HUVEC cells was obtained. The specific method is as follows: SIN-1 was dissolved in sterile physiological saline to prepare a solution with a mother liquor of 1 mol / L, and after sterilized by 0.22 μm filter membrane, it was added to the cell culture medium of HUVEC to make the final concentration 1 mM. The cells were placed in the incubator for 24 hours to obtain the reactive nitrogen damage model.

[0030] Select 6 samples for cell test, named 9#~14# respectively, all the samples to be tested are dissolved in the culture medium, and the 9# sample is a substance that does not have the function of scavenging active oxygen and active nitrogen, which is a negative control. The 10# sample is Hemoglobin, which is currently recognized as an active nitrogen sc...

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Abstract

The invention discloses a method for screening medicaments injuring endothelial cells by active nitrogen. Exogenous active nitrogen inducers are added to endothelial cells cultured in vitro in a certain concentration, and after reacting for a certain time, the active nitrogen inducers injure the endothelial cells so as to establish a model of injuring the endothelial cells by active nitrogen. By utilizing the cell model, different medicaments are added, and medicaments which can lighten the injury of the active nitrogen to the endothelial cells are screened in vitro by observing generating conditions of corresponding ROS and 3-NT and using a cell survival rate as a target spot. The invention avoids the blindness of the integral level screening of the cells, reduces the workload, has specific medicament action mechanism and the advantages of immediacy, rapidness and convenience and is suitable for mass screening of heart cerebrovascular candidate medicaments.

Description

technical field [0001] The invention belongs to the technical field of cell biology and pharmacology, relates to an endothelial cell model and its establishment method and application, in particular to a drug screening method for reactive nitrogen-induced endothelial cell damage, and the application of related drug screening based on the model . Background technique [0002] Drug screening models are an important and necessary condition for finding and discovering good therapeutic drugs. The drug screening model based on specific targets at the molecular and cellular levels has the characteristics of less material consumption, fast and sensitive, clear drug mechanism of action, and large-scale screening. It is currently the main mode of drug screening. [0003] In the past ten years, there have been many views on the role of reactive nitrogen (·NO) on endothelial cells, but it is generally believed that it is a small molecule substance with multiple biological activities an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02C12N5/08C12N5/06
Inventor 韩峰楼宜嘉刘启兵刘璐璐
Owner ZHEJIANG UNIV
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