CN114774362A - Convenient tumor micro-environment exosome extraction kit and extraction method thereof - Google Patents
Convenient tumor micro-environment exosome extraction kit and extraction method thereof Download PDFInfo
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- CN114774362A CN114774362A CN202210512125.7A CN202210512125A CN114774362A CN 114774362 A CN114774362 A CN 114774362A CN 202210512125 A CN202210512125 A CN 202210512125A CN 114774362 A CN114774362 A CN 114774362A
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Abstract
The invention discloses a convenient tumor micro-environment exosome extraction kit, which relates to the technical field of exosome extraction and consists of a reagent A, a reagent B, a reagent C, a 70-micrometer cell filter screen, a 0.22-micrometer filter head and a 1.5-ml centrifuge tube. The invention develops a convenient exosome extraction method for a tumor microenvironment. The extraction of the exosome can be completed within 2 hours, and the exosome with higher purity can be obtained.
Description
Technical Field
The invention belongs to the technical field of exosome extraction, and particularly relates to a convenient tumor micro-environment exosome extraction kit and an extraction method thereof.
Background
The exosome is a nano vesicle with a diameter of 40-180nm, released by cells, a lipid bilayer and rich contents, and widely exists in various cells and body fluids. The exosome secreted by the tumor cell is particularly vigorous, can carry the characteristics of the tumor cell, participates in the genetic information exchange between the tumor cell and the stromal cell, and is an important mode for transmitting biological signals among cells. Researches find that exosome of the tumor microenvironment plays an important role in various pathological processes of tumor occurrence and development, immune response, tumor drug resistance and the like.
However, due to its complex biological origin and the minute size of the nanometer scale, methods for the isolation and purification of exosomes are limited, thus hindering the further development of related studies and related clinical transformation applications. The conventional ultracentrifugation method is long in time consumption and complicated in steps, about 4 hours is needed for each extraction, the requirement on equipment is high, and an ultracentrifuge of 100000g is needed. At present, a kit specially used for extracting tumor micro-environment exosomes is not available in the market.
Disclosure of Invention
The invention aims to provide a convenient tumor micro-environment exosome extraction kit and an extraction method thereof aiming at the existing problems.
The invention is realized by the following technical scheme:
a convenient tumor micro-environment exosome extraction kit comprises a reagent A, a reagent B, a reagent C, a 70-micron cell filter screen, a 0.22-micron filter head and a 1.5-ml centrifuge tube.
A convenient extraction method of a tumor micro-environment exosome extraction kit comprises the following steps:
(1) the obtained tumor tissue was cut into pieces, added with reagent a, and incubated at 37 degrees for 20 minutes;
(2) digesting the tumor tissue into a tumor cell suspension, adding a reagent B, and stopping the digestion reaction;
(3) removing residual connective tissue fragments through a 70-micron cell filter screen, and centrifuging for three minutes at 500g to obtain a supernatant suspension;
(4) filtering the obtained supernatant suspension through a 0.22-micron filter head to remove impurities such as cell fragments, large vesicles and the like;
(5) and finally adding a reagent C, incubating for 30 minutes at 4 ℃, centrifuging at 1500g, removing supernatant, and obtaining precipitate which is the exosome from the tumor microenvironment.
Compared with the prior art, the invention has the following advantages:
1. the invention develops a convenient exosome extraction method for a tumor microenvironment. The extraction of the exosome can be completed within 2 hours, and the exosome with higher purity can be obtained.
2. The characterization and identification of the extracted tumor micro-environment exosomes prove that the high-purity exosomes can be obtained by the kit.
Drawings
FIG. 1 is a nanoparticle tracking analysis;
FIG. 2 is a transmission electron microscope image;
FIG. 3 is a Western blot electrophoretogram.
Detailed Description
For further explanation of the present invention, reference will now be made to the following specific examples.
Example 1
A convenient tumor micro-environment exosome extraction kit comprises a reagent A, a reagent B, a reagent C, a 70-micron cell filter screen, a 0.22-micron filter head and a 1.5ml centrifuge tube.
A convenient extraction method of a tumor micro-environment exosome extraction kit comprises the following steps:
(1) the obtained tumor tissue was cut into pieces, added with reagent a, and incubated at 37 degrees for 20 minutes;
(2) digesting the tumor tissue into a tumor cell suspension, adding a reagent B, and stopping the digestion reaction;
(3) removing residual connective tissue fragments through a 70-micron cell filter screen, and centrifuging for three minutes at 500g to obtain a supernatant suspension;
(4) filtering the obtained supernatant suspension through a 0.22-micron filter head to remove impurities such as cell fragments, large vesicles and the like;
(5) and finally adding a reagent C, incubating for 30 minutes at 4 ℃, centrifuging at 1500g, removing supernatant, and obtaining precipitate which is the exosome from the tumor microenvironment.
Nanoparticle tracking analysis technology shows that the diameters of purified exosomes are mainly distributed in the diameters of 40-180nm, and the purified exosomes have typical exosome size characteristics.
Transmission electron microscope shows that the purified exosome has an obvious membrane, a clear three-dimensional structure, a saucer-like structure or a concave hemisphere-like structure and conforms to the classic electron microscope characteristics of the exosome.
Western blotting showed that the purified exosomes expressed classical markers of exosomes, such as Alix, Flotillin-1, TSG101, CD9 proteins.
The experimental results show that the kit developed by the inventor can effectively obtain high-purity exosomes from the tumor microenvironment.
Claims (2)
1. A convenient tumor micro-environment exosome extraction kit is characterized by comprising a reagent A, a reagent B, a reagent C, a 70-micron cell filter screen, a 0.22-micron filter head and a 1.5ml centrifuge tube.
2. The extraction method of the convenient tumor micro-environment exosome extraction kit according to claim 1, characterized by comprising the following steps:
(1) the obtained tumor tissue was cut into pieces, added with reagent a, and incubated at 37 degrees for 20 minutes;
(2) digesting the tumor tissue into a tumor cell suspension, adding a reagent B, and stopping the digestion reaction;
(3) removing residual connective tissue fragments through a 70-micron cell filter screen, and centrifuging for three minutes at 500g to obtain supernatant suspension;
(4) filtering the obtained supernatant suspension through a 0.22um filter head to remove impurities such as cell fragments, large vesicles and the like;
(5) and finally adding a reagent C, incubating for 30 minutes at 4 ℃, centrifuging at 1500g, removing supernatant, and obtaining precipitate which is the exosome from the tumor microenvironment.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111849903A (en) * | 2020-05-26 | 2020-10-30 | 武汉生之源生物科技股份有限公司 | Kit for separating exosome from cell supernatant and using method thereof |
| CN113215075A (en) * | 2020-01-21 | 2021-08-06 | 武汉生之源生物科技股份有限公司 | Kit for separating exosome from cell supernatant and using method thereof |
| CN113234677A (en) * | 2021-04-27 | 2021-08-10 | 西南医科大学附属医院 | Method for extracting exosome from in-vitro tumor tissue |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113215075A (en) * | 2020-01-21 | 2021-08-06 | 武汉生之源生物科技股份有限公司 | Kit for separating exosome from cell supernatant and using method thereof |
| CN111849903A (en) * | 2020-05-26 | 2020-10-30 | 武汉生之源生物科技股份有限公司 | Kit for separating exosome from cell supernatant and using method thereof |
| CN113234677A (en) * | 2021-04-27 | 2021-08-10 | 西南医科大学附属医院 | Method for extracting exosome from in-vitro tumor tissue |
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