Method of screening action target of cardiac glycoside medicine using protein composition technology

A proteomics and drug action technology, applied in the field of medicine, can solve the problems of affecting cell signal transduction, can not fully explain the inhibition or activation of Na-KATPase, etc., and achieve the effect of huge economic and social benefits

Inactive Publication Date: 2009-11-04
SHANDONG UNIV
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  • Abstract
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  • Claims
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Problems solved by technology

At present, it is believed that this type of drug exerts positive inotropic effect through the inhibitory pathway of Na-KATPase on the myocardial cell membrane, but this cannot fully explain the opposite effects such as inhibition or activation of Na-KATPase at different drug concentrations. Recent studies have found that the drug participates in apoptosis at the level of cardiomyocytes and affects cell signal transduction and intervention to improve cardiac function, and part of the mechanism is not related to the inhibition of Na-KATPase

Method used

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  • Method of screening action target of cardiac glycoside medicine using protein composition technology
  • Method of screening action target of cardiac glycoside medicine using protein composition technology
  • Method of screening action target of cardiac glycoside medicine using protein composition technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] (1) To establish a model of the effect of cardiac glycosides on cardiomyocytes.

[0032] Take 10 newborn 1-2 day old Wistar rats, perform thoracotomy, take out the heart, wash it with D-Hanks solution, add 0.125% trypsin and 0.02% disodium diethylenetetraacetate, bathe in water at 37°C for 5 minutes, pipette for 1 Add 0.125% trypsin, place in a water bath at 37°C for 10 minutes, then pipette for 1 to 2 minutes, naturally precipitate, absorb the supernatant, filter it with a 400-mesh metal filter, and transfer it to a 10ml centrifuge Add an equal volume of DMEM culture medium to the centrifuge tube. Repeat the digestion 5 times until the myocardial tissue block is basically digested into single cells; then add DMEM culture medium containing 15% fetal bovine serum to break up the cell clusters into a single cell suspension; adjust the number of cells to 1×105 / ml, and The single cell suspension was inoculated into the culture flask; and the culture container was placed at...

Embodiment 2

[0045] (1) To establish the effect model of digitalis drugs on cardiomyocytes.

[0046] Cardiomyocyte culture is as described in Example 1, the difference is that in the drug action group, digoxin is replaced by helicanthinogen, and the drug concentration is 1 × 10 -9 mol / L, acting for 24 hours respectively, to create a cardiomyocyte model of cardiac glycosides.

[0047] (2) Preparation of protein samples by two-dimensional electrophoresis of cardiomyocytes, the same as in Example 1.

[0048] (3) two-dimensional gel electrophoresis, with embodiment 1

[0049] (4) image collection and analysis, with embodiment 1

[0050] After the gel was stained with Coomassie Brilliant Blue, it was scanned with a gel scanner to obtain a digital image, which was analyzed with PDQUEST software. The image analysis process included the clipping of the map, the detection of protein spots, the matching between different gels, and the quantitative analysis. Normalize and use the standard molecula...

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Abstract

The invention relates to a method for screening cardiac glycoside drug action targets by proteomics technology, which belongs to the field of medical technology. Including: cardiomyocyte culture and the establishment of cardiac glycoside drugs on cardiomyocyte models, preparation of cardiomyocyte two-dimensional electrophoresis protein samples, two-dimensional gel electrophoresis, image acquisition and analysis, obtaining differentially expressed proteins of cardiac glycoside drugs on cardiomyocytes, After in-gel digestion of the differential proteins, use mass spectrometry to determine the mass fingerprint of the differential protein peptides; enter the sequence database to identify and analyze the differential proteins, and deduce their DNA sequences. Perform mass spectrometry analysis on characteristic proteins, and screen out differential proteins by comparing with mass spectrum libraries; verify the biological functions of differentially expressed proteins. Using proteomics technology to screen the drug targets of cardiac glycosides against heart failure can be directly used to guide the development of new high-efficiency and low-toxicity anti-heart failure drugs.

Description

(1) Technical field [0001] The invention relates to a method for screening the target protein of cardiac glycoside drugs acting on cardiomyocytes by using proteomics technology, in particular to a method for screening target proteins of cardiac glycoside drugs acting on cardiomyocytes, and belongs to the technical field of medicine. (2) Background technology [0002] Coronary heart disease, hypertension, chronic valvular heart disease, congenital heart disease and other important cardiovascular system diseases can develop heart failure to a certain extent, and become the main cause of death of the above diseases. The prognosis of heart failure is poor, the total mortality rate is about 15-20%, and the annual mortality rate of severe patients can reach 50%. Therefore, effective treatment of heart failure is a global public health problem. Cardiac glycosides are currently the first choice for the treatment of heart failure, but the effective range and safety range of this typ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N35/00G01N21/84G01N1/34B01D57/02
Inventor 邱洁高海青梁英贾继辉陈春燕马亚兵
Owner SHANDONG UNIV
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