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Method for preparing extracellular vesicles and application of extracellular vesicles

A cell and vesicle technology, applied in the field of extracellular vesicle preparation, can solve the problems of lack of application of cell pyroptosis and transformation, and achieve the effect of precise targeted therapy, good activity and high purity

Active Publication Date: 2020-12-01
XUZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Pyroptosis is an important natural immune response of the body and plays an important role in fighting infection. However, the research on the application and transformation of pyroptosis is still lacking

Method used

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  • Method for preparing extracellular vesicles and application of extracellular vesicles
  • Method for preparing extracellular vesicles and application of extracellular vesicles
  • Method for preparing extracellular vesicles and application of extracellular vesicles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Establishment of pyroptosis model:

[0028] Human umbilical vein endothelial cells (Human Umbilical Vein Endothelial Cells, HUVEC) were cultured in a cell incubator at 37°C, 5% CO 2 , 20%O 2 , when the cell density reached more than 80%, lipopolysaccharide (purchased from Sigma) was added to culture (1 μg / ml), after culturing for 5 hours, nigericin (purchased from Invitrogen) was added to continue to cultivate for 45min (5 μmol / L) , to induce pyroptosis in cells and produce pyroptotic bodies, collect the cell supernatant, and record it as cell supernatant A;

[0029] Extraction of pyroptotic bodies:

[0030] (1) Removing cells and cell debris: Centrifuge the cell supernatant A for 10 min under the condition of a centrifugal force of 300 g, discard the precipitate, take the supernatant, and record it as supernatant B;

[0031] (2) Precipitate pyroptotic bodies: centrifuge the supernatant B for 20 min under the condition of a centrifugal force of 2000 g, discard the su...

Embodiment 2

[0034] Identification of pyroptotic bodies:

[0035] (1) Morphological characteristics of pyroptotic bodies:

[0036] The morphology of pyroptotic bodies was observed by transmission electron microscope (TEM). Take the prepared pyroptotic body suspension, place it on the copper mesh coated with amorphous carbon and infiltrate it for 5 minutes, then place it in a petri dish with filter paper, let it dry naturally, then put the sample into the sample chamber of the transmission electron microscope, observation, the result is figure 1 As shown, the pyroptotic body is a uniform vesicle structure with a diameter of 1-5 μm (standard bar = 2 μm).

[0037] (2) Marker detection of pyroptotic bodies:

[0038] The whole protein was extracted from pyroptotic bodies using Shanghai Sangon General Total Protein Extraction Kit (NO.C006225) according to the kit instructions. The specific method is: according to the method in Example 1, the physiological extracellular vesicle precipitate de...

Embodiment 3

[0040] Detect the source and quantity of pyroptotic bodies to judge the damage site and damage degree of the body:

[0041] Experimental animals: healthy adult C57BL / 6 mice (purchased from the Animal Center of Xuzhou Medical University), weighing 22-28 g, were established by tracheal instillation of lipopolysaccharide (LPS) aqueous solution (2 mg / kg) to establish a mouse model of acute lung injury. The blank group was instilled with the same amount of normal saline through the trachea, which was recorded as the 0h group. After 4h, 12h, and 24h after the establishment of the animal model, the mice were sacrificed by cervical dislocation, the skin of the neck was incised, the trachea was exposed, and the lung bronchi were lavaged with sterile normal saline (0.5ml each time, repeated 3 times) to obtain bronchial tubes. alveolar lavage fluid.

[0042]Use the method in Example 1 to obtain pyroptotic bodies, then stain with 1 μg EpCAM-FITC antibody (bronchial epithelial cell marker...

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Abstract

The invention discloses a method for preparing extracellular vesicles and application of the extracellular vesicles. The method comprises the following steps: establishing a cell pyroptosis model to induce pyroptosis of cells; and obtaining pyroptosis corpuscle having a diameter of 1-5 [mu]m and containing cysteine aspartate specific proteinase-1. The preparation method of the pyroptosis corpuscleis convenient to operate and low in production cost, and the obtained pyroptosis corpuscle is high in purity, good in activity, convenient to store, low in toxic and side effects and beneficial to continuous production; meanwhile, the application way is wide, the extracellular vesicles can be used as a specific marker for detecting a damaged part and can also be used for immunotherapy, and the extracellular vesicles can be used as a tumor vaccine, so that accurate targeting treatment of cancer is realized, and development of the tumor vaccine or medicine is promoted.

Description

technical field [0001] The invention belongs to the technical field of preparation of extracellular vesicles, and in particular relates to a method for preparing extracellular vesicles and an application thereof. Background technique [0002] Pyroptosis, also known as cell inflammatory necrosis, is characterized by swelling and rounding of cells, rupture of cell membranes, and release of inflammatory factors. Studies in recent years have confirmed that pyroptosis can occur in many kinds of cells in the body, and pyroptosis will cause the shrinkage of the nucleus and the formation of pyroptotic bodies by exporting vesicles. [0003] Pyroptosis is an important natural immune response of the body and plays an important role in fighting infection. However, the research on the application and transformation of pyroptosis is still lacking. Contents of the invention [0004] The purpose of this section is to outline some aspects of embodiments of the invention and briefly descri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N5/09A61K35/42A61K35/44A61P35/00
CPCC12N5/069C12N5/0625C12N5/0693A61K35/42A61K35/44A61P35/00C12N2509/10C12N2501/90C12N2501/999
Inventor 张昊秦西淳周叶卿贾才力晁志祥秦昊刘修成
Owner XUZHOU MEDICAL UNIV
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