High-flux screening cell model of alpha1-AR subtype selective antagonist, and construction method and application thereof

A cell model, 1-AR technology, applied to cells modified by introducing foreign genetic material, biochemical equipment and methods, recombinant DNA technology, etc., can solve the problem of poor screening effect of anti-benign prostatic hyperplasia drugs, etc. To achieve the effect of efficient, fast and rapid screening of the method

Inactive Publication Date: 2014-01-01
GUANGZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a high-throughput screening method for screening anti-benign prostatic hyperplasia drugs

Method used

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  • High-flux screening cell model of alpha1-AR subtype selective antagonist, and construction method and application thereof
  • High-flux screening cell model of alpha1-AR subtype selective antagonist, and construction method and application thereof
  • High-flux screening cell model of alpha1-AR subtype selective antagonist, and construction method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] alpha 1 -Building process of high-throughput screening model for AR subtype-selective antagonists

[0038] S1. Preservation and extraction of plasmids (pGL4.29[luc2P / CRE / Hygro], pGL4.74[hRluc / TK], EX-A0967-M29, EX-Y3321-M29, EX-Y2008-M29).

[0039] S11. Plasmid transformation and storage: Transfer 1 μL of the above plasmid to the bottom of a 1.5 mL centrifuge tube for ice bath, add to 200 μL of competent cells (DH5α), mix well, and place in ice bath for 30 minutes. Heat shock at 42°C for 40s and ice bath for 3min. Add 400 μL of S.O.C medium, shake culture at 37°C for 1 hour, take 50 μL and spread it on the surface of the solid medium plate containing 100ug / mL ampicillin. Cultivate overnight at 37°C, pick a single clone and inoculate it into 500 μL LB liquid medium, culture on a shaker at 37°C for 6 hours, add resistant glycerol containing 100 μg / mL ampicillin, and the final concentration of glycerol is 15-25%.

[0040] S12. Strain cultivation and plas...

Embodiment 2

[0059] In order to determine the suitable reporter gene in the screening method of the present invention, the present invention studies the response of five response factors directly or indirectly related to α1-ARs, the specific method is as follows:

[0060] Will α 1 -ARs (ADRA1A, ADRA1B and ADRA1D) were co-transfected with the reporter gene and the internal reference plasmid (hRluc-TK) respectively. Negative control and blank control were used for drug addition to investigate the fluorescence induced by the reporter gene to phenylephrine in the co-transfection system. The influence of signal ratio, in which the reporter gene selects 5 main response elements that are directly or indirectly related to α1-ARs: ​​cyclic adenosinase response element (CRE), serum response element (SRE) and activin-1 response element ( AP-1), nuclear factor-кB (NF-кB-RE) and nuclear factor of activated T cells (NFAT-RE), respectively inserted into the firefly luciferase vector (luc2P) to co...

Embodiment 3

[0061] Example 3 Scale Selection of Reporting Components

[0062] The difference in the ratio of the reporter element plasmid has an important influence on the difference in luciferase expression value caused by receptor signal transduction after adding phenylephrine stimulation. In order to ensure the stable and optimal expression of luciferase on the screening platform, the ratio of ADRA1D, reporter gene Luc2P-CRE, and internal reference plasmid (hRluc-TK) was optimized, other conditions and methods were fixed, and the ratio of reporter gene and internal reference plasmid was changed. , to detect differences in luciferase expression intensity at different doses. Such as figure 2 As shown, the eukaryotic expression plasmid, reporter gene, and internal reference plasmid are respectively 1:1:1; 1:10:1; 1:50:1; 1:100:1; 10:100:1; 50:100:1 ; 100:100:1 for co-transfection, the results showed ( figure 2 ), according to the plasmid ratio of 1:1:1, a better signal-to-noise r...

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Abstract

The invention particularly discloses a high-flux screening cell model of an alpha1-AR subtype selective antagonist, and a construction method and application thereof. The construction method comprises the following steps: co-transfecting eukaryotic expression plasmids, reporter genes and internal reference plasmids of three alpha1-AR subtypes (alpha1A, alpha1B and alpha1D) to a host cell, thus establishing a transient transfection cell model; successively adding an antagonist to be screened and an agonist into the cell model, and co-culturing for a period of time until cells crack; and performing luciferase activity determination on the cracked products, and determining relative expression quantities of the reporter genes and the internal references, thereby evaluating the inhibition activity of the medicament on the three alpha1-AR subtypes (alpha1A, alpha1B and alpha1D). The method passes model stability / volatility inspection and model verification. The cell model can be used for high-flux screening of an alpha1-AR subtype selective antagonist, and can be further used for screening of potential medicaments for treating benign prostatic hyperplasia.

Description

technical field [0001] The invention relates to the technical field of constructing cell models for high-throughput screening of drugs, in particular to an α 1 -High-throughput screening cell model of AR subtype-selective antagonists and its construction method and application. Background technique [0002] alpha 1 - Adrenoceptor (AR) can be divided into three subtypes according to clonotype (α 1A , α 1B and alpha 1D ), are widely distributed in vascular and nonvascular tissues, and regulate most of the physiological function responses of humans separately or jointly. With different adjustment functions. alpha 1A Regulate smooth muscle contraction, control urination, and control blood pressure; α 1D -AR is associated with aortic and coronary vasoconstriction; in recent years, α 1A Found to be the major dynamic factor causing bladder outlet block, antagonizing alpha 1A Can relieve urinary tract obstruction, and antagonize α 1D -AR can improve lower urinary tract sym...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/10C12Q1/02
Inventor 袁牧许芳黎枭徐静仪朱着何雪兰朱柳陈洪黄碧云
Owner GUANGZHOU MEDICAL UNIV
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