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Exosome of LDLR mutant, preparation method of exosome, and application of exosome in preparing drug against hyperlipidaemia

A technology of exosomes and receptors, which is applied in the field of exosomes overexpressing human low-density lipoprotein receptor gene mutants and its preparation, and the preparation of drugs for the treatment of dyslipidemia, which can solve the problem of immunogenic therapeutic genes Short expression duration and other issues, to achieve the effect of lowering LDL-C level, low immunogenicity, and treating dyslipidemia

Inactive Publication Date: 2019-07-16
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The present invention solves the problems of immunogenicity therapy and short duration of gene expression in the prior art when using adenovirus as a carrier for treatment, and provides an exosome that overexpresses LDLR mutants and its preparation and application

Method used

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  • Exosome of LDLR mutant, preparation method of exosome, and application of exosome in preparing drug against hyperlipidaemia
  • Exosome of LDLR mutant, preparation method of exosome, and application of exosome in preparing drug against hyperlipidaemia
  • Exosome of LDLR mutant, preparation method of exosome, and application of exosome in preparing drug against hyperlipidaemia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: LDLR muteins promote LDL uptake

[0034] Inoculate the primary hepatic cells of LDLR- / - mice that are growing well in a cell culture dish, and culture them in a medium containing fetal bovine serum for 18-24 hours, until the primary hepatic cells of LDLR- / - mice grow and fuse to When 30-50%, the adenovirus overexpressing mLDLR and the GFP control adenovirus were transfected into LDLR- / - mouse liver primary cells. After 6 hours of transfection, fresh DMEM medium was replaced to continue culturing for 48 hours. Add 30mg / ml red fluorescent probe-labeled Dil-LDL, and incubate in the incubator for 4-5h in the dark. The uptake of intracellular Dil-LDL was observed by confocal microscopy.

[0035] figure 1 Fluorescent photographs (400 times) of adenovirus transfection of LDLR- / -hepatic primary cells and Dil-LDL uptake provided by Example 1 of the present invention; wherein figure 1 (a) is the fluorescence map of adenovirus GFP overexpressing mLDLR; figure 1 (b)...

Embodiment 2

[0036] Example 2: Obtaining of exosomes overexpressing LDLR gene mutants

[0037] (1) HepG2 cells were inoculated into DMEM medium containing 10% fetal bovine serum for culture.

[0038] (2) When the HepG2 cells grew to 30-50% confluent, the mLDLR overexpression adenovirus was transfected into the HepG2 cells. The mLDLR in this step is the termination mutation of c.G2164T relative to SEQ ID NO:1.

[0039] (3) Six hours after the mLDLR-overexpressing adenovirus was transfected into HepG2 cells, the serum-free DMEM medium was replaced to continue culturing. After 48 hours, the supernatant was collected and centrifuged to remove floating living cells, and the supernatant containing exosome cells was collected.

[0040] (4) Separating and purifying the exosome-containing cell supernatant collected in step (3) by ultracentrifugation, and harvesting the HepG2 cell exosomes overexpressing mLDLR.

[0041] The mLDLR overexpressing adenovirus described in step (2) can be prepared by m...

Embodiment 3

[0051] Example 3: LDLR muteins bind LDL

[0052] The nitrocellulose membrane was placed on a clean filter paper, and the cell supernatant liquid containing overexpressed mLDLR and GFP control adenovirus exosomes obtained in Example 2 were used to spot the sample on the nitrocellulose membrane, and the sample was covered with the nitrocellulose membrane. After absorption, incubate the nitrocellulose membrane with 25ug / ml LDL solution for 2h and perform Western blot. LDLR imaging results show that the sample is successfully attached to the nitrocellulose membrane, as shown in Figure 4 As shown in (a); APOB imaging results also show that the LDLR mutant protein has the ability to bind LDL, as Figure 4 (b) shown.

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Abstract

The invention discloses an exosome of an LDLR mutant, a preparation method of the exosome, and an application of the exosome in preparing drug against hyperlipidaemia, and relates to the technical field of drugs for treating dyslipidemia. The preparation method comprises the following steps: inoculating a medium containing fetal bovine serum with hepatocytes, transfecting the hepatocytes with adenoviruses over-expressing a low-density lipoprotein receptor gene mutant that is terminating mutation caused by mutation of a single base in an O-linked sugar chain domain of the low-density lipoprotein receptor gene; then replacing the serum-free medium and continuously conducting culturing after transfection, and collecting supernatant; centrifuging the supernatant to remove floating living cells, thereby obtaining the supernatant containing the exosome; and condutcing separating and purifying to obtain the cell exosome over-pressing the low-density lipoprotein receptor gene mutant. The exosome of the present invention is a natural vector produced by an endogenous cell. The exosome is used as a transport vector, and has the unique advantages of low immunogenicity, high transport efficiency, good stability, high targetability and ability to pass the blood-brain barrier.

Description

technical field [0001] The present invention relates to the technical field of drugs for treating dyslipidemia, in particular to a cell exosome overexpressing LDLR mutants and its preparation and application, in particular to overexpressing human low-density lipoprotein receptor gene mutants (mutant low-density lipoprotein receptor gene mutants) , mLDLR) cell exosomes, a preparation method thereof, and an application for preparing a drug for treating dyslipidemia. Background technique [0002] Dyslipidemia mainly includes elevated total cholesterol (Tch) and low-density lipoprotein cholesterol (LDL-C), triglyceride (TG) and / or decreased high-density lipoprotein cholesterol (HDL-C). Homozygous Familial Hypercholesterolemia (HoFH) is a monogenic disease that causes dyslipidemia. HoFH patients have decreased expression of low-density lipoprotein receptor (LDLR), serum Tch and LDL- C levels are significantly increased and lead to atherosclerosis and cardiovascular and cerebrova...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861C12N5/10A61K35/407A61P3/06
CPCA61K35/407A61P3/06C12N5/067C12N15/86C12N2510/00C12N2710/10043
Inventor 凃欣周颖超谢强
Owner HUAZHONG UNIV OF SCI & TECH
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