Wnt4/YWHAZ co-modified mesenchymal stem cell (MSC) derived exosome, preparation method therefor and application of Wnt4/YWHAZ co-modified MSC derived exosome

A technology of stem cells and exosomes, applied in the field of skin repair, can solve the problems of poor treatment effect of hucMSC-exosome

Active Publication Date: 2020-01-31
AFFILIATED HOSPITAL OF JINING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But untreated hucMSC-exosomes did not work as well

Method used

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  • Wnt4/YWHAZ co-modified mesenchymal stem cell (MSC) derived exosome, preparation method therefor and application of Wnt4/YWHAZ co-modified MSC derived exosome
  • Wnt4/YWHAZ co-modified mesenchymal stem cell (MSC) derived exosome, preparation method therefor and application of Wnt4/YWHAZ co-modified MSC derived exosome
  • Wnt4/YWHAZ co-modified mesenchymal stem cell (MSC) derived exosome, preparation method therefor and application of Wnt4/YWHAZ co-modified MSC derived exosome

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preparation example Construction

[0043] The present invention provides a method for preparing exosomes derived from mesenchymal stem cells co-modified by Wnt4 / YWHAZ, comprising the following steps:

[0044] 1) Construct Wnt4 overexpression adenovirus and YWHAZ overexpression adenovirus respectively;

[0045] 2) using the Wnt4 overexpression adenovirus and YWHAZ overexpression adenovirus constructed in step 1) to transfect mesenchymal stem cells and culture;

[0046] 3) Collect the cultured supernatant of Wnt4 and YWHAZ overexpressed mesenchymal stem cells, remove floating living cells, separate and purify exosomes in the supernatant, and obtain Wnt4 / YWHAZ co-modified mesenchymal stem cell-derived exosomes body.

[0047] The present invention constructs Wnt4 overexpression adenovirus and YWHAZ overexpression adenovirus respectively.

[0048] In the present invention, the nucleotide sequence of the mRNA variant of Wnt4 in the Wnt4 overexpressing adenovirus is preferably as shown in the sequence table SEQ ID N...

Embodiment 1

[0068] The main materials and sources are as follows:

[0069] MSC culture reagents low-sugar DMEM, fetal bovine serum (Gibco company product), trypsin (Sigma company product), carbon dioxide incubator (Forma company), serum-free medium (Shanghai Yikesai company;

[0070] Transfection reagent: lipo2000 (American lifetechnology company)

[0071] Inverted microscopes, fluorescence microscopes, biological microscopes, electron microscopes, ultra-clean benches, desktop centrifuges;

[0072] Wnt4 overexpression adenovirus vector (Suzhou Forbaio Biotechnology Co., Ltd.), YWHAZ overexpression adenovirus vector (Suzhou Forbaio Biotechnology Co., Ltd.);

[0073] Preparation reagents for Wnt4 and YWHAZ modified exosome: heavy water (D 2 O, Shanghai Chuangsai Company), analytically pure sucrose (Guangzhou Chemical Reagent Factory), fetal bovine serum (Gibico), rabbit anti-human CD81 antibody (U.S. Bioworld Technology Company), rabbit anti-human CD63 antibody (U.S. Epitomics Company), BCA...

Embodiment 2

[0082] Sources of experimental animals and instructions for reagents:

[0083] SD rats (animal experiment center of Jining Medical College, this experiment was approved by the Ethics Committee of the Affiliated Hospital of Jining Medical College);

[0084] Small animal scalding device invented by our research group (authorized patent number: 201420060187.X);

[0085] Immunohistochemical staining reagent (Wuhan Boster Company, operate according to the kit instructions);

[0086] 24-well culture plate, 6-well culture plate (Corning, USA); Roche portable blood glucose meter (vital type)

[0087] HACAT cells (purchased by Shanghai Chinese Academy of Sciences);

[0088] PCNA antibody (Bioworld, USA)

[0089] Biological microscope (Nikon Company, Japan), experimental platform for HE staining and PCNA histochemical staining of pathological sections (Leica Company, Germany).

[0090] The specific implementation steps are described as follows:

[0091] (1) Construction of a deep s...

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Abstract

The invention provides a Wnt4 / YWHAZ co-modified mesenchymal stem cell (MSC) derived exosome, a preparation method therefor and an application of the Wnt4 / YWHAZ co-modified MSC derived exosome and belongs to the technical field of skin repair. According to the Wnt4 / YWHAZ co-modified MSC derived exosome, the MSC derived exosome (MSC-exosome) is modified through overexpression of expression adenovirus vectors of a gene Wnt4 and a gene YWHAZ in mesenchymal stem cells, so that exosome over-expresses the gene Wnt4 and the gene YWHAZ, and the medicinal aim of promoting regenerating repair of rat scalded skin tissue by the MSC-exosome is achieved.

Description

technical field [0001] The invention belongs to the technical field of skin restoration, and in particular relates to a Wnt4 / YWHAZ co-modified mesenchymal stem cell-derived exosome and its preparation method and application. Background technique [0002] Diabetes mellitus (DM) is a lifelong metabolic disease caused by the absolute or relative insufficiency of insulin secretion in the human body. It is an important disease affecting human health. Diabetic foot ulcers are one of the most common and major complications of diabetes. Epidemiological surveys show that about 1 / 4 diabetic patients will develop foot ulcers. Diabetic foot ulcers are the first to develop neuropathy. Later development will lead to foot circulation disorder, sensory disturbance, further toe or foot gangrene, and eventually lead to amputation or severe systemic infection and death. Therefore, the pathogenesis of diabetic skin ulcers is more complicated than other traumas such as burns and is more difficul...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/861A61K35/28A61P17/02
CPCC12N5/0662C12N15/86C07K14/4702A61K35/28A61P17/02C12N2710/10043C12N2710/10343
Inventor 张斌班博张梅别庆丽王森董海新文洁韩书山陈志
Owner AFFILIATED HOSPITAL OF JINING MEDICAL UNIV
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