Applications of ZEB1 (zinc finger E-box binding homeobox 1) in human cardiac fibroblasts

A dirty fibroblast, 1.ZEB1 technology, applied in the application field of ZEB1 in human cardiac fibroblasts, can solve problems such as strengthening cardiac contractility, and achieve the effect of promoting proliferation and good application value

Active Publication Date: 2018-11-30
SOUTH CHINA AGRI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the early stage of myocardial infarction, the synthesis and deposition of extracellular matrix can strengthen the contractility of the heart, form paralyzed scars to prevent heart rupture, and improve heart functi

Method used

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  • Applications of ZEB1 (zinc finger E-box binding homeobox 1) in human cardiac fibroblasts
  • Applications of ZEB1 (zinc finger E-box binding homeobox 1) in human cardiac fibroblasts
  • Applications of ZEB1 (zinc finger E-box binding homeobox 1) in human cardiac fibroblasts

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Experimental program
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Effect test

Embodiment 1

[0038] The cultivation of embodiment 1 human cardiac fibroblasts

[0039] The cell model used in this experiment was human cardiac fibroblasts from ScienCell Research Laboratories, USA. The medium used was cardiac fibroblast medium (Fibroblast Medium-2) provided by ScienCell Research Laboratories, USA.

[0040] (1) Preparation before the experiment: a. Turn on the water bath and adjust the temperature to 35.8-37°C; b. Turn on the ultra-clean bench for ultraviolet sterilization for 30 minutes.

[0041] (2) Take out the cryopreservation tube from the liquid nitrogen, quickly immerse it in a 37°C water bath, and shake it from time to time to melt it as soon as possible.

[0042] (3) Take out the cryopreservation tube from the 37°C water bath, spray 75% alcohol on the outside, transfer it to the ultra-clean bench, carefully open the cover, suck out the cell suspension with a pipette gun, add it to the centrifuge tube and drop it more than 10 times for culture liquid, mix well. ...

Embodiment 2

[0046] Inoculation and transfection of embodiment 2 human cardiac fibroblasts

[0047] (1) When the confluence of cardiac fibroblasts reaches 90% under a microscope, the culture medium is discarded, and the cells are washed twice with preheated PBS containing 1% double-antibody (double-antibody is penicillin and streptomycin);

[0048] (2) Add preheated 0.25% trypsin into the culture flask, put it in the incubator and let it stand for 2 minutes. When most of the cells are observed to float under the microscope, add stop solution (10% FBS in DMEM) to stop the digestion.

[0049] (3) Gently blow the cells with a pipette tip to make a cell suspension, transfer to a 15mL centrifuge tube, and centrifuge at 1000rpm for 5min.

[0050] (4) The cells were washed twice with appropriate amount of PBS.

[0051] (5) An appropriate amount of complete medium was used to resuspend the cell pellet. After the cells were counted, they were evenly distributed into each well, and the complete med...

Embodiment 3

[0055] Embodiment 3qRT-PCR

[0056] (1) Wash the adherent cells 2-3 times with cold PBS, tilt the cell culture dish, suck up the PBS, add 1mL of Trizol, let it stand for 5min, until the cells are completely lysed under the microscope, blow evenly with a pipette tip and transfer to 1.5mL RNase-free EP tube.

[0057] (2) Add 200 μL of pre-cooled chloroform (Trizol interstitial fluid: chloroform is 5:1) to each tube, shake vigorously for 15-30 seconds, place on ice for 5 minutes, then centrifuge at 12,000 rpm, 4°C for 15 minutes; the liquid is divided into three layers: the upper layer is colorless The middle layer is a white aqueous phase containing phenol-chloroform, and the lower layer is light red. Carefully pipette off the upper colorless liquid phase (avoid touching the middle phase). Transfer the upper liquid phase (about 300 μL) into a new 1.5mL EP tube, add an equal amount of isopropanol solution (300 μL), shake it up and down gently 10 times, and let it stand on ice f...

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Abstract

The invention discloses applications of ZEB1 (zinc finger E-box binding homeobox 1) in human cardiac fibroblasts. According to the applications, ZEB1 is adopted as the research object, ZEB1 interference small fragments are synthesized for transfecting the cardiac fibroblasts, the result shows that ZEB1 promotes the proliferation, migration, differentiation and collagen synthesis of the cardiac fibroblasts, and research is further carried out by controlling the functions of miR-590-3p expressed by ZEB1 in the cardiac fibroblasts. After interference of ZEB1, the verification shows that the cellfunction phenotype caused by miR-590-3p can restore, which proves that ZEB1 is an important function target of miR-590-3p in the cardiac fibroblasts, and miR-590-3p can regulate the functions of the fibroblasts through ZEB1. Through the applications of ZEB1 and the ZEB1-targetting miRNA in the cardiac fibroblasts, a very good application value is achieved for researching the fibrosis mechanism after myocardial infarction.

Description

technical field [0001] The invention belongs to the technical fields of cell engineering and genetic engineering, and particularly relates to an application of ZEB1 in human cardiac fibroblasts. Background technique [0002] Myocardial infarction-like blood flow is not smooth, and local myocardial necrosis is caused by long-term myocardial hypoxia and ischemia. In the early stage of myocardial infarction, the synthesis and deposition of extracellular matrix can strengthen the contractility of the heart, form paralyzed scars to prevent heart rupture, and improve heart function to a certain extent; but in the later stages of the disease, due to excessive synthesis of extracellular matrix, It accumulates in large quantities in cardiac tissue, leading to the occurrence of myocardial fibrosis. Cardiac fibroblasts proliferate in large numbers during the process of myocardial fibrosis and differentiate into myofibroblasts at the same time. Myofibroblasts synthesize a large amount ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C07K14/47C12N5/10
CPCC07K14/47C12N5/0656C12N15/113C12N2310/14C12N2310/141C12N2510/00
Inventor 张豪王希龙温丽娟潘金春黄韧杨丰华龚宝勇谭伟江
Owner SOUTH CHINA AGRI UNIV
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