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High-throughput screening method for screening collagen transcription inhibitor for treating organ fibrosis

A technology for organ fibrosis and screening methods, applied in biochemical equipment and methods, instruments, measuring devices, etc., can solve problems such as organ function decline and organ fibrosis

Active Publication Date: 2021-05-18
XIAMEN BERYL THERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If this repair response is excessive, strong, and out of control, it will cause organ fibrosis and lead to organ function decline

Method used

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  • High-throughput screening method for screening collagen transcription inhibitor for treating organ fibrosis
  • High-throughput screening method for screening collagen transcription inhibitor for treating organ fibrosis
  • High-throughput screening method for screening collagen transcription inhibitor for treating organ fibrosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] A high-throughput screening method for screening collagen transcription inhibitors for the treatment of organ fibrosis, comprising the following steps:

[0025] Step 1: Clone human Col1A1, Col1A2, Col3A1 promoter-2000-100 regions, and insert them into pGL3-basic reporter gene vector (purchased from Promega) individually or jointly to obtain 4 plasmids: pGL3-Col1A1 Prom (1A1 for short), pGL3-Col1A2 Prom (1A2 for short), pGL3-Col3A1 Prom (referred to as 3A1) and pGL3-Col1A1 Prom -Col1A2 Prom (abbreviated as 1A12), the plasmid used for transfection was obtained through transformation of DH5α bacteria and plasmid extraction steps;

[0026] Step 2: 3 μg of the plasmid obtained in step 1 and 1 μg of the internal reference pRL-TK plasmid (purchased from Promega) were transfected into NIH-3T3 cells in a 6-well plate (10 5 cells / well), 12h after transfection, add 5ng / ml TGF-β1 to the medium for activation;

[0027] Step 3: After TGF-β1 was activated for 24 hours, it was pro...

Embodiment 2

[0029] A high-throughput screening method for screening collagen transcription inhibitors for the treatment of organ fibrosis, comprising the following steps:

[0030] Step 1: Clone human Col1A1, Col1A2, Col3A1 promoter-2000-100 regions, and insert them into pGL3-basic reporter gene vector (purchased from Promega) individually or jointly to obtain 4 plasmids: pGL3-Col1A1 Prom (1A1 for short), pGL3-Col1A2 Prom (1A2 for short), pGL3-Col3A1 Prom (referred to as 3A1) and pGL3-Col1A1 Prom -Col1A2 Prom (abbreviated as 1A12), the plasmid used for transfection was obtained through transformation of DH5α bacteria and plasmid extraction steps;

[0031]Step 2: Transfect 3 μg of the plasmid obtained in step 1 and 1 μg of the internal reference pRL-TK plasmid (purchased from Promega) into human skin fibroblast BJ cells in a 6-well plate (10 5 cells / well), 5 ng / ml TGF-β1 was added to the medium for activation 8 hours after transfection, and different concentrations of TGFβR inhibitors G...

Embodiment 3

[0034] Example 3: Drug BRL-2021101 inhibits collagen transcription

[0035] The compound BRL-2021101 was screened out from the self-owned compound library by the above two methods. It inhibits type I collagen transcription IC50=25 μM, the maximum potency is 94% of Galunisertib.

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PUM

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Abstract

The invention discloses a high-throughput screening method for screening a collagen transcription inhibitor for treating organ fibrosis. The method is characterized by comprising the following steps: cloning human Col1A1, Col1A2 and Col3A1 promoter-2000-100 regions, independently or jointly inserting a pGL3-basic reporter gene vector, transforming DH5 alpha bacteria and extracting plasmids to obtain plasmids for transfection; respectively transfecting the obtained plasmids and internal reference pRL-TK plasmids into fibroblasts, and then adding TGF-beta 1 to activate the fibroblasts; or adding a TGF beta R inhibitor at the same time; and after activation, treating according to a luciferase reporter gene detection kit after activation, reading chemiluminescence data by a microplate reader, and obtaining plasmids sensitive to synthesis of collagen induced by TGF-beta1. The practicability of the screening method is verified by the above results. The invention also discloses application of the method in quantitative analysis of the efficacy of drugs for inhibiting transcription of type I / III collagen.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a high-throughput screening method for screening collagen transcription inhibitors for treating organ fibrosis. Background technique [0002] Tissue cell damage caused by any reason can lead to degeneration, necrosis and inflammatory response of tissue cells. If the damage is small, normal parenchymal cells around the damaged cells will undergo hyperplasia and repair, and this repair can completely restore normal structure and function. However, if the damage is large or repeated damage exceeds the regeneration capacity of the parenchymal cells around the damage, the extracellular matrix will proliferate in large quantities to repair the defect tissue, that is, the pathological change of fibrosis occurs. Therefore, fibrosis is essentially a repair response after tissue damage to protect the relative integrity of tissues and organs. Although the proliferating fibrous connective tissue...

Claims

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Application Information

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IPC IPC(8): C12Q1/02G01N21/64
CPCG01N33/5044G01N33/5038G01N21/6486
Inventor 王阳柏旭
Owner XIAMEN BERYL THERAPEUTICS INC
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