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Application of death domain-associated protein in preparation of medicament for preventing and treating diseases related to vascular smooth muscle cell proliferation and/or vascular smooth muscle cell migration

A technology of vascular smooth muscle and related proteins, which is applied in the field of disease treatment and can solve problems such as the unclear role of DAXX

Active Publication Date: 2016-07-13
HUNAN UNIV OF CHINESE MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although many studies have found that DAXX plays an important role in apoptosis, the role of DAXX in VSMC is still unclear

Method used

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  • Application of death domain-associated protein in preparation of medicament for preventing and treating diseases related to vascular smooth muscle cell proliferation and/or vascular smooth muscle cell migration
  • Application of death domain-associated protein in preparation of medicament for preventing and treating diseases related to vascular smooth muscle cell proliferation and/or vascular smooth muscle cell migration
  • Application of death domain-associated protein in preparation of medicament for preventing and treating diseases related to vascular smooth muscle cell proliferation and/or vascular smooth muscle cell migration

Examples

Experimental program
Comparison scheme
Effect test

experiment example

[0036] In order to study the role of death structure-associated protein DAXX in VSMC, the present invention provides the following experimental examples.

[0037] The plasmid pCDNA3.1-DAXX used in the following experiments was a gift from Dr. Wan Yanping of Nanhua University, and the shRNA-DAXX plasmid and shRNA-PTEN plasmid were purchased from Shanghai Jikai Gene Company. Vascular smooth muscle cells (VSMC) were purchased from the Cell Bank of Central South University.

[0038] Experimental Materials

[0039]The experimental materials used in the following experimental examples are as follows: rabbit monoclonal antibody DAXX (purchased from CellSignaling, product number 4553S), diluted 1:1000 with PBS when used. Rabbit monoclonal antibody p-Akt (purchased from CellSignaling, product number 4060S) was diluted 1:1000 with PBS before use. Rabbit monoclonal antibody Akt (purchased from CellSignaling, product number 4691S) was diluted 1:1000 with PBS before use. Rabbit monoclon...

experiment example 1

[0045] Experimental Example 1: The liposome transfection method was used to overexpress and knock down the expression level of DAXX in VSMC cells to establish a stable cell line.

[0046] experimental method:

[0047] Cell preparation: Cultivate according to the method of routinely culturing VSMC cells. When the cells grow well, digest the cells and dilute the cells with 1.2×10 5 The cells / well were seeded in a 6-well plate, and the next day, when the cells reached 70%-80% confluence, they were ready for transfection.

[0048] Lipofectamine 2000 transfection: Solution I: Take about 4ug of the plasmid, add 240uL optimized medium, mix gently with a pipette, and incubate at room temperature for 5min. Solution II: Take 5uL Liposome 2000, add 245uL optimized medium, mix gently with a pipette gun, and incubate at room temperature for 5min. Add solution I to solution II and incubate at room temperature for 20 minutes. During this period, wash the cells twice with PBS and once with ...

experiment example 2

[0050] Experimental example 2: RT-PCR method to detect the transcription level of related genes.

[0051] Total RNA extraction: take the treated cells, wash them with PBS 3 times, tilt the culture dish, and absorb excess liquid with filter paper. Add 1mL Trizol reagent, gently shake the bottom of the dish until it is completely covered, pipette repeatedly to lyse for 5min, collect the liquid into a 1.5mLEP tube, and lyse at room temperature for 5min. Add 0.2 mL of chloroform, vortex to mix, let stand at room temperature for 3 min, and centrifuge at 12000 rpm for 10 min at 4°C. Carefully draw the supernatant, then transfer it to a 1.5mL enzyme-free EP tube, add 500uL isopropanol, mix well, put it at -20 overnight, and after the RNA is completely precipitated, centrifuge at 4°C, 12000rpm for 10min. Discard the supernatant, add 1 ml of 75% ethanol to wash the precipitate 1-2 times, centrifuge at 7500 g for 5 min, discard the supernatant, and dry at room temperature for 3-5 min. ...

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Abstract

The invention discloses a death domain-associated protein, an activator and / or an expression regulating agent of the death domain-associated protein, and an application of a pharmaceutical composition containing the death domain-associated protein for preventing and treating diseases related to vascular smooth muscle cell proliferation and / or vascular smooth muscle cell migration. According to the invention, the death domain-associated protein can inhibit the vascular smooth muscle cell proliferation and / or the vascular smooth muscle cell migration, and thereby can be used to prevent and treat the diseases related to the vascular smooth muscle cell proliferation and / or the vascular smooth muscle cell migration.

Description

technical field [0001] The present invention relates to the field of treatment of diseases related to proliferation and / or migration of vascular smooth muscle cells. Applications in diseases related to cell proliferation and / or migration. Background technique [0002] When the vascular intima is damaged due to various reasons, an inflammatory response can occur. Various inflammatory factors released by inflammatory cells, including cytokines, can lead to the proliferation of vascular smooth muscle cells, matrix secretion, and the migration of vascular smooth muscle cells (VSMC) to the intima. , causing excessive hyperplasia and hypertrophy of the intima, leading to the occurrence of vascular restenosis. [0003] Percutaneous transluminal coronary angioplasty (PTCA) is currently one of the main means of treating coronary heart disease, but its main disadvantage is that the incidence of restenosis is as high as 30% to 50%. Although intracoronary stents can effectively reduce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/17A61K45/00A61P9/00
CPCA61K38/1761A61K45/06
Inventor 庹勤慧邱飞熊国祚廖端芳
Owner HUNAN UNIV OF CHINESE MEDICINE
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