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A wnt4/ywhaz co-modified mesenchymal stem cell-derived exosome and its preparation method and application

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

Active Publication Date: 2021-06-25
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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  • A wnt4/ywhaz co-modified mesenchymal stem cell-derived exosome and its preparation method and application
  • A wnt4/ywhaz co-modified mesenchymal stem cell-derived exosome and its preparation method and application
  • A wnt4/ywhaz co-modified mesenchymal stem cell-derived exosome and its preparation method and application

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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-derived exosome and its preparation method and application, belonging to the technical field of skin repair. The present invention modifies MSC-derived exosomes (MSC-exosome) by overexpressing the adenoviral vector of Wnt4 gene and YWHAZ gene in mesenchymal stem cells, so that exosome overexpresses Wnt4 gene and YWHAZ gene, and realizes that MSC-exosome promotes rat Medicinal purposes for regenerative repair of scalded skin tissue.

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 Patents(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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