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Retinal pigment epithelium epithelial-mesenchymal transition model and application thereof

A retinal pigment and epithelial cell technology, applied in the field of retinal pigment epithelial cell epithelial-mesenchymal transition model, can solve the problems that the cell model cannot be widely used, and the connection between cells is difficult to be destroyed. The method is simple and easy to implement , good repeatability

Active Publication Date: 2015-12-02
TONGJI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In this model, RPE cells were treated with 2mMEGTA for 48 hours, and after the intercellular connection was interrupted, it was found that the cells at the edge had motility and migration ability, and the morphology also developed towards mesenchymal cells, but the disadvantage was that the intercellular connection in the center was difficult. is destroyed, so no EMT transition occurs
Therefore, this cell model cannot be widely used

Method used

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  • Retinal pigment epithelium epithelial-mesenchymal transition model and application thereof
  • Retinal pigment epithelium epithelial-mesenchymal transition model and application thereof
  • Retinal pigment epithelium epithelial-mesenchymal transition model and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Cell culture:

[0038] The culture of ARPE-19 cells was divided into two parts: in the cell dish and in the EMT model. When cultured in a cell dish, culture in DMEM-F12 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptavidin double antibody. Cells per dish (10cm culture dish, cell density is 1×107cells / dish). When doing EMT model, ARPE-19 cells were digested with trypsin, and then cultured in serum-free DMEM-F12 medium containing N2 and non-essential amino acids (NEAA) in a bacterial dish, at 12h and 24h respectively After 36h, 48h, and 72h, cell samples were collected or dissolved in trizol, and detected by Q-PCR, ELISA, Western blot and other methods. The cells in two dishes were added with S3I (Stat3 inhibitor) and U0126 respectively at 24h, and the samples were collected at 48h. In the experiment of detecting stat3 activation, the P-STAT3-TA-LUC reporter gene plasmid was first transfected into ARPE-19 with lipofectaminer2000, and after 48 ho...

Embodiment 2

[0053] To investigate the inhibitory effect of mir-24 on the EMT of ARPE-19.

[0054] 1. Cell culture

[0055] The main body of cell culture is mir-24-ARPE-19 cells, which are divided into two parts, one of which is to verify the effect of mir-24 on the EMT, proliferation and migration of ARPE-19 cells, as well as the effect of stat3 signaling pathway. One part is to clarify the targeting effect of mir-24 and inflammatory factor CHI3L1.

[0056] The details are as follows: Validation of the effect of mir-24 on the EMT of ARPE-19 cells: cultured in serum-free DMEM / F12 containing N2 and NEAA, petridish. To verify the effect of mir-24 on the proliferation and migration of ARPE-19 cells: culture in DMED / F12 culture medium with 10% fetal bovine serum, celldish, scratch after 24 hours, change to serum-free culture medium and continue to culture. To verify the effect of mir24 on the stat3 signaling pathway in the EMT process of ARPE-19 cells: cultured in DMED / F12 medium with 10% fe...

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Abstract

The invention relates to a retinal pigment epithelium epithelial-mesenchymal transition model and application thereof. After being digested with pancreatin, ARPE-19 (acute retinal pigment epitheliitis 19) cells are transferred to a serum-free DMEM-F12 medium containing N2 and non-essential amino acids and are cultured in a Petri dish, and the retinal pigment epithelium epithelial-mesenchymal transition model is acquired. The model is usable for researching a molecular mechanism of retinal pigment epithelium to which epithelial-mesenchymal transition occurs and finding intervened targeting of epithelial-mesenchymal transition.

Description

technical field [0001] The invention relates to a cell model, in particular to a retinal pigment epithelial cell epithelial-mesenchymal transition model and its application. Background technique [0002] Proliferative vitreoretinopathy (PVR) is the most important reason for the failure of rhegmatogenous retinal detachment surgery. Because of its refractory and recurrence, it has always been a research hotspot by scholars from all over the world. The incidence of PVR is 5%-20% %. PVR is a proliferative disease. The basic pathological process of PVR is that after a retinal hole occurs, the blood-retinal barrier is destroyed, proteins and active mediators (such as transforming growth factor (TGF)-β, and fibroblast growth factor (FGF)) are released in the vitreous, and retinal pigment epithelial cells (retinal pigment epithelial, RPE) are exposed. Loss of contact with photoreceptors, RPE dissociation, migration, cytokines stimulate RPE proliferation, cell phenotype changes, se...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N5/10
Inventor 吕立夏徐国彤
Owner TONGJI UNIV
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