Method and preparation for regulating lipid secretion of retinal pigment epithelial cells and application thereof

A technology of retinal pigment and epithelial cells, applied in the field of regulating lipid secretion of retinal pigment epithelial cells, can solve problems such as lack of intervention in early AMD

Pending Publication Date: 2021-10-29
ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, due to the lack of knowledge about the pathogenesis of early AMD, there is a lack of means to intervene in early AMD

Method used

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  • Method and preparation for regulating lipid secretion of retinal pigment epithelial cells and application thereof
  • Method and preparation for regulating lipid secretion of retinal pigment epithelial cells and application thereof
  • Method and preparation for regulating lipid secretion of retinal pigment epithelial cells and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] 1. Perform transcriptomic analysis to determine differentially expressed gene profiles

[0047] a. RNA sequencing: Human retinal pigment epithelial cells (HRPE, Sciencell,) were cultured to 80%-90% confluence, starved for 12 hours with serum-free medium, and then treated with 50ng / ml human recombinant PDGF-D protein (R&D, 1159- SB-025 / CF) were treated for 18 hours, and RNA was extracted for transcriptome sequencing.

[0048] b. Transcriptomic analysis: 2 μg of total RNA from each sample was used to make an Illumina cDNA library, sequenced on the Illumina HiSeqX-Ten, and the total number of reads (reads) compared to the genome for each sample was between 39 million and 50 million between. Reads were aligned to transcriptome files using TopHat2 and aligned reads were counted for each transcript using Cuffquant. . Differentially expressed genes were determined using DESeq2 (v1.22.2) software. Fold changes were determined from the FPKM of each sample. Differentially ex...

Embodiment 2

[0058] 1. Experimental construction method:

[0059] (1) Construct PDGF-D tissue-specific overexpression mouse model and observe pathological sections

[0060] a. Construct the adeno-associated virus (AAV) of RPE-specific overexpression: Utilize human VMD2 gene transcription initiation site upstream -598bp to +378bp sequence as retinal pigment epithelial cell (RPE) specific promoter, and utilize PDGF- The coding sequence of the D gene (NM_025208) was used as the target fragment to construct the vector and packaged into serotype 8 AAV. Store at -80°C.

[0061] b. Subretinal injection of AAV in mice: topical application of tropicamide for mydriasis, intraperitoneal injection of 4% chloral hydrate (10ml / kg body weight) for anesthesia, and then dripping procainamide for surface anesthesia of the cornea, and using Sodium carboxymethylcellulose prevents corneal desiccation. For subretinal injection, a sterile 5 μl syringe (Hamilton, CAT: 7633-01), 33-gauge needle (Hamilton, CAT: ...

Embodiment 3

[0072] 1. Construct a high-fat diet model (HLD) to simulate early AMD and evaluate the therapeutic potential of targeting PDGF-D, SREBP1, and SCD1

[0073] a. Constructing a high-fat diet model: 12-month-old mice were selected and given a normal diet and a high-fat diet with 4 times the fat content for 1 month.

[0074] b. Detect the expression levels of PDGF-D and lipid synthesis genes in HLD mice: collect normal and HLD mouse eyeballs, separate the retina and choroid, extract RNA according to the method in 2) a, and perform qPCR to detect the expression levels of related genes.

[0075] c. Evaluate the therapeutic potential of targeting PDGF-D on subretinal deposition induced by high-fat diet: while constructing the HLD model, inject PDGF-D antibody CR002 or ShRNA into the mouse vitreous cavity, once a week, a total of 4 times. The mouse eyeballs were taken for paraffin sections and H&E staining, and the number of subretinal deposits in the injected antibody or shRNA and the c...

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Abstract

The invention provides a method and a preparation for regulating lipid secretion of retinal pigment epithelial cells, and application thereof, and the method is characterized in that activation of an SREBP-1 site is inhibited by inhibiting a PDGF-D signal channel, so that the lipid secretion amount of the retinal pigment epithelial cells is reduced. The PDGF-D has an adjusting effect on an SREBP-1 signal channel, and activation of SREBP1 can be inhibited by inhibiting expression of the PDGF-D, so that expression of a lipid synthase gene is reduced, lipid synthesis in RPE is reduced, and formation of lipid deposition under the retina is reduced.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a method, preparation and application for regulating lipid secretion of retinal pigment epithelial cells. Background technique [0002] Age-related macular degeneration (AMD) is the leading cause of blindness in older adults. The current treatment for AMD is mainly to intervene with anti-angiogenesis drugs in the angiogenesis stage of advanced AMD, and the effect is not satisfactory. If it is only effective for some patients, and the duration is short, many patients need long-term, multiple injections of drugs into the vitreous, which often leads to drug resistance and other ocular complications. These facts suggest that we need to conduct more in-depth research on the mechanism of AMD in order to find more effective therapeutic targets. [0003] At present, due to the lack of knowledge about the pathogenesis of early AMD, there is a lack of means to intervene in early AM...

Claims

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Application Information

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IPC IPC(8): A61K45/00A61K39/395A61K48/00A61K31/713A61P9/10A61P27/02
CPCA61K45/00C07K16/22A61K31/713A61P9/10A61P27/02A61K2039/505C07K2317/76
Inventor 李旭日熊振
Owner ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV
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