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High-throughput screening method for screening compounds affecting collagen stability

A screening method and a stable technology, applied in the field of biomedicine, can solve problems such as organ function decline and organ fibrosis

Pending Publication Date: 2021-05-28
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 compounds affecting collagen stability
  • High-throughput screening method for screening compounds affecting collagen stability
  • High-throughput screening method for screening compounds affecting collagen stability

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034]A high-throughput screening method for screening affecting collagen stabilizers, including the following steps:

[0035]Step 1: Type I collagen α1 and mVENUS fusion expression plasmid Mvenus-col1a1 in the removal of the signal peptide, the human type I collagen α2 and MAPPLE fused expression plasmid mapple-col1a2, and the human III collagen of the removal of the signal peptide MVENUS Fusion Expression Plasmid Mvenus-Col3a1 and Removal Signal Peptide Type III Collagen α1 and MAPPLE Follow Expression Plasmid MAPPLE-COL3A1, and the transformed DH5α bacteria and plasmid a large amount of extracts can be used for transfection plasmids;

[0036]Step 2: The NiH-3T3 cells in 6-well plates co-transfected with Mvenus-Col1a1 and MAPPLE-COL1A2 each were 0.1-4 μg, or MVENUS-Col3a1 and MAPPLE-COL3A1 were 0.1-4 μg, and the plasmid was transfected with cells 12 h, and the concentration was added. 0.01 μm P4HA1 inhibitor S4682;

[0037]Step 3: During the drug intervention 24h, the enzyme detector detec...

Embodiment 2

[0039]A high-throughput screening method for screening affecting collagen stabilizers, including the following steps:

[0040]Step 1: Constructing the fusion of the human I-type collagen α1 and Mvenus-N-terminal (2-173AA) fragment of the removal signal peptide, the expression plasmid VN-col1a1, the human I collagen α2 and Mvenus-C end (156-239aa) Fusion expression plasmid Vc-col1a2; Type III collagen α1 and Mvenus-N-terminal fragment fused expression plasmid Mvn-col3a1 and removed signal peptide in human III type collagen α1 and MVENUS-C end fragment fusion expression plasmid MVC-COL3A1, the transformed DH5α bacterium and the plasmid extraction step are obtained by the plasmid which can be used for transfection;

[0041]Step 2: The NiH-3T3 cells in 6-well plates were transferred to VN-COL1A1 and VC-COL1A2 each each were 0.1-4 μg, or 0.1-4 μg of MVN-Col3a1 and MVC-COL3A1, and the plasmid was transferred to 12 h, and the concentration was added. 10 μm P4HA1 inhibitor S4682;

[0042]Step 3: The...

Embodiment 3

[0044]A high-throughput screening method for screening affecting collagen stabilizers, including the following steps:

[0045]Step 1: Fusion expression plasmid Glucn-col1a1 of the human I collagen α1 and Gluc-N-terminal (2 to 416AA) fragment removing the signal peptide, the human I collagen α2 and Gluc-C end (398 ~ 550AA) Fusion expression plasmid glucc-col1a2; human III collagen α1 and Gluc-N-terminal fragment fusion expression plasmid Glucn-col3a1 and Gluc-C-C-C-terminal fragment fusion expression plasmid Glucc-col3a1, the transformed DH5α bacteria and the plasmid of a large amount of granules can be used for transfection;

[0046]Step 2: The NiH-3T3 cells in 6-well plates were transfected with Glucn-Col1a1 and Glucc-col1a2 each 0.1-4 μg, or Glucn-Col3a1 and Glucc-Col3a1 were 0.1-4 μg, and the plasmid was transferred to the cell 12 h, and the concentration was added. 500 μm P4HA1 inhibitor S4682;

[0047]Step 3: After 48 h was administered for 48 hours, the luciferase substrate was added, ...

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Abstract

The invention discloses a high-throughput screening method for screening compounds influencing collagen stability. Based on fluorescence resonance energy transfer or reporter gene fragment complementation, cells are co-transfected through plasmids, P4HA1 inhibitors s4682 with different concentrations are added into a culture medium, after the drugs are added for 24 hours, FRET, or fluorescence data or chemiluminiscence is detected by a microplate reader, FRET, Venus fragment complementation and Gluc fragment complementation tests prove that the IC50 of the s4682 for inhibiting the interaction between collagen chain molecules, and the results prove the practicability of the screening method. The invention also discloses application of the method in quantitative analysis of the efficacy of a drug for reducing the stability of the collagen triple helix and application of the method in quantitative analysis of a compound for increasing the stability of the collagen triple helix.

Description

Technical field[0001]The present invention relates to the field of biomedicine, and more particularly to a high-throughput screening method for screening affecting collagen stabilizers.Background technique[0002]Any cause caused by tissue cell damage, can lead to changes in tissue cells, necrosis and inflammatory response. If the damage is small, the normal substance cells around the cells will have a hyperplasia repair, which can completely restore normal structures and functions. However, if the damage is large or repeatedly damaged, the extracellular matrix will repair a large number of hyperplasia to the defect tissue, i.e., the pathological changes of fibrosis. Therefore, in nature is a repair reaction after tissue suffering from injury to protect the relative integrity of the organizers. Although the proliferation of fiber connective tissue has repaired defects, it does not have the structure and function of the organ substantine cells. If this repair is over, too strong and ou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/50G01N21/64
CPCG01N33/5008G01N21/6402
Inventor 王阳柏旭
Owner XIAMEN BERYL THERAPEUTICS INC
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