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Exosome of mouse lung telocyte line as well as extraction method and application of exosome

An extraction method and exosome technology, applied in the field of biomedicine, can solve the problems of increasing tumorigenicity and immunogenicity, limiting the basic research and clinical application research of special cell exosomes, and insufficient stability, and achieve good The effect of applying the foreground

Pending Publication Date: 2021-12-07
ZHONGSHAN HOSPITAL FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0020] The technical problems to be solved by the present invention are: 1. The cells used in cell therapy, such as stem cells, have insufficient stability. If they are cultured in vitro for a long time, the transformation of cell characteristics will easily occur, and this instability will increase its tumorigenicity and The risk of immunogenicity and other issues; 2. Obtaining exosomes from cells cultured in vitro has high requirements on the number and density of cells, while primary cells cannot reach the number and density of cells for obtaining exosomes due to in vitro culture, thus Limiting the basic research and clinical application research on the exosomes of tetroid cells

Method used

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  • Exosome of mouse lung telocyte line as well as extraction method and application of exosome
  • Exosome of mouse lung telocyte line as well as extraction method and application of exosome
  • Exosome of mouse lung telocyte line as well as extraction method and application of exosome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0199] Extraction and identification of exosomes (TCs-exo) from mouse lung specific cell line:

[0200] 1. Extraction of exosomal TCs-exo:

[0201] (1) When culturing TCs to about 75% of the cell density, discard the complete medium containing FBS, wash 2-3 times with PBS, and add serum-free medium for 24 hours;

[0202] (2) Collect cell culture medium and centrifuge at 300 × g for 10 min;

[0203] (3) Centrifuge at 2000×g for 10min (precipitate=dead cells);

[0204] (4) Centrifuge at 10,000×g for 30min (precipitate=cell debris);

[0205] (5) 100,000×g ultracentrifugation for 70min (precipitation=exosome+interfering protein);

[0206] (6) PBS rinse, 100,000×g ultracentrifugation for 70min (precipitate=exosome);

[0207] (7) Dissolve the exosomes with an appropriate amount of PBS, and store them in a -80°C refrigerator for later use.

[0208] 2. TEM observation of exosomes:

[0209] Observation and morphological analysis of exosomes using TEM, the results are as follows ...

Embodiment 2

[0215] In vitro cell experiments were conducted to investigate the effects of the secretions and exosomes of the special network cells on the lipopolysaccharide (LPS)-induced acute injury model of HBE cells:

[0216] 1. HBE was treated with LPS to construct a cell model of acute lung injury, and the biological behavior changes of HBE were observed in the presence of secretions (including exosomes) of mouse lung special network cells:

[0217] 1.1 Co-culture of mouse lung specific cell line and HBE (eg image 3 shown):

[0218] (1) The mouse lung special network cell line is in the upper layer of the co-culture chamber, and HBE is in the lower layer of the co-culture chamber, and adheres overnight; the diameter of the co-culture chamber is 0.4 μm, and the cells cannot penetrate, but the mouse lung special network cells The secretions (including exosomes) can pass through the pores and act on HBE.

[0219] (2) Move the upper chamber so that it is in the same culture well as th...

Embodiment 3

[0257] miRNA expression and protein expression in TCs-derived exosomes:

[0258] 1. Collection and RNA sequencing of TCs-exo exosomes treated with different reagents:

[0259] 1.1 Cell processing reagents and concentrations:

[0260]

[0261]

[0262] 1.2 Experimental steps, such as Figure 14 shown:

[0263] (1) Cell adhesion and grouping:

[0264] Group V: add DMSO solvent as a control group;

[0265] LPS group: DMSO solvent was added to the adherent cells, and then 1 μg / mL LPS was added to obtain the LPS group;

[0266] LY+LPS group: DMSO solvent was added to adherent cells, 0.05 μmol / L LY was added 2 h before LPS stimulation, and 1 μg / ml LPS was added to obtain LY+LPS group;

[0267] LY+PMA+LPS group: 0.05 μmol / L LY was added 2 h before LPS stimulation, 0.05 μmol / L PMA 0.1 μmol / L was added 1 h before LPS stimulation, and 1 μg / mL LPS was added to obtain LY+PMA+LPS group;

[0268] (2) The above four groups of cells were cultured in serum-free medium for 24h respe...

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Abstract

The invention discloses an exosome of a mouse lung telocyte line as well as an extraction method and application of the exosome. The exosome is extracted from a mouse lung telocyte line, CD9 and CD63 proteins are expressed on the surface of the exosome, and CD81 proteins are not expressed on the surface of the exosome. Signal pathways related to a target gene of differential expression miRNA in the exosome of the mouse lung telocyte line comprise an mTOR signal pathway and an HIF1alpha signal pathway, and experiments show that the exosome generated by the mouse lung telocyte line can transmit related regulation signals to human bronchial epithelial cells; and the corresponding or excitation or inhibition effect of a treatment reagent is exerted, so that the a protection effect is achieved on a lipopolysaccharide-induced human bronchial epithelial cell acute lung injury cell model. The exosome of the mouse lung telocyte line can be used for preparing medicines for treating or preventing acute lung injury, and has a good application prospect.

Description

technical field [0001] The invention relates to an exosome of a mouse lung specific network cell line, an extraction method and application thereof, and belongs to the technical field of biomedicine. Background technique [0002] Telocytes (TCs) are a kind of mesenchymal cells located in many organs of humans and mammals [1,2]. The cells have been found in tissues and organs such as lung, heart, intestine, placenta, urinary tract, pleura, exocrine pancreas, salivary gland, breast, myometrium, skeletal muscle and skin [3]. Lung specific network cells have a variety of functions including promoting the development and regeneration of lung tissue, remodeling of extracellular matrix, formation of new blood vessels and maintaining the integrity of the vascular basement membrane [4]. We isolated mouse lung special network cells in the early stage and constructed a mouse lung special network cell line (patent number: ZL 201710272011.9). Previous studies on the cell model and anim...

Claims

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Application Information

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IPC IPC(8): C12N5/071A61K35/42A61P11/00
CPCC12N5/0688A61K35/42A61P11/00C12N2509/00
Inventor 宋东莉王向东唐莉
Owner ZHONGSHAN HOSPITAL FUDAN UNIV
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