Application of exosome derived from mesenchymal stem cells in preparation of medicine for treating acute lung injury caused by sulfur mustard

A bone marrow mesenchymal and acute lung injury technology, applied in the field of biomedicine, can solve the problems that have not yet been reported on acute lung injury, and achieve the effect of improving the activity of lung epithelial cells, alleviating lung injury, and repairing lung barrier function

Pending Publication Date: 2021-08-06
THE NAVAL MEDICAL UNIV OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] At present, there are no relevant research reports on the as

Method used

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  • Application of exosome derived from mesenchymal stem cells in preparation of medicine for treating acute lung injury caused by sulfur mustard
  • Application of exosome derived from mesenchymal stem cells in preparation of medicine for treating acute lung injury caused by sulfur mustard
  • Application of exosome derived from mesenchymal stem cells in preparation of medicine for treating acute lung injury caused by sulfur mustard

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1. Obtaining exosomes from bone marrow mesenchymal stem cell supernatant by ultracentrifugation

[0032] A. Bone marrow mesenchymal stem cell culture

[0033] Adding the mesenchymal stem cell culture medium to the bone marrow mesenchymal stem cells, and collecting the culture supernatant after 48 to 72 hours, the formula of the mesenchymal stem cell culture medium is: DMEM / F12 medium, 10% fetal bovine serum, 1% penicillin / streptomycin double antibody.

[0034] B. Exosome isolation

[0035] (1) Collect the culture supernatant of bone marrow mesenchymal stem cells, 2000g, 4°C, 10min, and centrifuge to collect the supernatant;

[0036] (2) 10000g, 4°C, 30min, centrifuge to collect the supernatant;

[0037] (3) 100000g, 4°C, 70min, centrifuge, discard the supernatant, collect the precipitate, resuspend in PBS, repeat the operation;

[0038] (4) The collected precipitate was dissolved in 100 μl PBS to obtain exosomes with high purity.

[0039] according to figur...

Embodiment 2

[0040] Example 2, Western blot identification of exosome marker molecules

[0041] (1) The obtained exosomes were lysed with RIPA lysate, centrifuged at 12000rpm, 4°C for 20min, and the supernatant was collected to collect the total protein. The BCA kit was used to measure and adjust the protein concentration;

[0042] (2) Add protein loading buffer to the protein lysate according to the ratio of 5×protein loading buffer=4:1, shake and mix well, cook at 100°C for 10min;

[0043] (3) Add 10 μl of protein sample to 10% polyacrylamide gel for electrophoresis;

[0044] (4) intercept the corresponding strips and transfer them to the PVDF membrane with an electrotransfer instrument;

[0045] (5) 5% skimmed milk to block non-specific binding sites for 1 h, then incubate overnight at 4°C with CD9, CD63, CD81 and TSG101 antibodies, and wash 3 times with TBST buffer;

[0046] (6) HRP-labeled secondary antibody (Millipore) was incubated at room temperature for 1 h, and then washed 3 ti...

Embodiment 3

[0054] Example 3 Obtaining lung pathological sections and analyzing survival curves after SM-infected mice

[0055] (1) Select 6-8 weeks old ICR mice and randomly assign them to CTRL, SM, SM+NAC, SM+BMSC-Ex groups. Except for CTRL group, other groups are subcutaneously injected with 1,2-propanediol as a solvent to prepare 30mg / kg mustard gas solution;

[0056] (2) After SM exposure, the NAC group was administered NAC (200 mg / kg, once / d, continuously for 7 days) as a positive control group; the BMSC-Ex group was injected with BMSC- Ex, as the experimental group;

[0057] (3) Observe the mouse body weight, diet and death every day, and draw the survival curve;

[0058] (4) On the 5th day, after death with pentobarbital anesthesia, some lung tissues were taken, fixed with 4% paraformaldehyde, and then stained with HE.

[0059] according to figure 2 , the survival rate of mice exposed to SM decreased significantly; after injection of exosomes, the survival rate of mustard gas...

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Abstract

The invention provides application of an exosome derived from mesenchymal stem cells in preparation of a medicine for treating acute lung injury caused by sulfur mustard. The invention particularly relates to separation of bone marrow mesenchymal stem cell supernate to obtain exosomes and application of the exosomes to acute lung injury caused by sulfur mustard. The exosomes can significantly improve survival rate of experimental animals and improve activity of lung cells under the exposure of sulfur mustard, improves changes in lung tight junction proteins caused by sulfur mustard, and resists lung injury effect of sulfur mustard. Therefore, the invention provides a new treatment approach for acute lung injury caused by sulfur mustard.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to the use of exosomes derived from bone marrow mesenchymal stem cells in the preparation of drugs for the treatment of acute lung injury caused by mustard gas and a pharmaceutical composition containing the exosomes. Background technique [0002] Mustard gas (sulfur mustard, SM) is a kind of erosive poison. After poisoning, it can cause blistering of the skin and involvement of multiple organs such as eyes, lungs, liver, and spleen. Lung injury is a leading cause of death, manifesting as short-term acute lung injury dominated by inflammation and long-term pulmonary fibrosis (Wolfe GA, Petteys SM, Phelps JF, Wasmund JB, Plackett TP. Sulfur Mustard Exposure: Review of Acute, Subacute, and Long-Term Effects and Their Management. Journal of special operations medicine: a peer reviewed journal for SOF medical professionals 2019; 19: 81-6.). [0003] Bone marrow mesenchym...

Claims

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Application Information

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IPC IPC(8): A61K35/28A61P11/00C12N5/0775
CPCA61K35/28A61P11/00C12N5/0663C12N2509/10
Inventor 肖凯徐庆强毛冠超孙铭学龚楚楚王振裴志鹏孟文琪岑金凤
Owner THE NAVAL MEDICAL UNIV OF PLA
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