CN112538459A - Method for separating exosome in liver cancer tissue - Google Patents

Abstract

A method for isolating exosomes from liver cancer tissue, comprising: immediately soaking a fresh HCC tissue specimen after operation in PBS containing streptomycin and penicillin double antibody after the tissue specimen is separated from the body; under the aseptic operation condition, cutting the tissue blocks into tissue fragments with the size of 1-3 mm by using scissors; rinsing blood with PBS containing double antibodies, placing the blood in a DMEM/F12 culture medium, adding type I and type IV collagenase and hyaluronidase to degrade extracellular matrix, and adding DNase to digest genomic DNA released by dead cells; incubating for 30-60 minutes by using a constant-temperature shaking table; filtering the digestive juice with 70 μm filter to remove undigested tissue debris; obtaining the exosome by a gradient centrifugation method. The method of the invention can be directly separated from the liver cancer tissue specimen after the operation, and can obtain the exosome which accurately reflects the characteristics of the HCC tumor.

Description

Method for separating exosome in liver cancer tissue

[ technical field ] A method for producing a semiconductor device

The invention belongs to the technical field of biological medicines, and particularly relates to a separation method of exosomes in liver cancer tissues.

[ background of the invention ]

The exosome is formed by sprouting from the limiting membrane of intracellular multivesicular bodies (MVBs) to the cavity, is a vesicular body released to the outside of cells after being directly fused with a plasma membrane through the MVBs, has the diameter of 30-120nm and has a lipid bilayer structure. Many cells of the body, such as immune cells like T, B lymphocytes and dendritic cells, blood cells like erythrocytes and mast cells, liver cells, epithelial cells, neurons, stem cells, tumor cells and the like can release exosomes, and are stably present in body fluids of the body, such as serum, urine, ascites and the like, and are particularly enriched in tumor microenvironment. Exosomes can carry a large number of components closely related to their origin and function, such as proteins, mrnas, non-coding RNAs and lipids, and are not easily degraded by the external environment. The exosome is taken up and internalized by the receptor cell and regulates the biological function of the receptor cell through the modes of macropinocytosis, receptor-mediated endocytosis and the like, so the exosome is an important medium for cell-cell substance and information transmission, and the active substance encapsulated by the exosome is an important object for tumor diagnosis, treatment and research of the tumor occurrence and development rules.

The exosomes in liquid specimens such as body fluid of tumor patients or tumor cell line culture solution can be easily separated by the current technical means, but the compositions of the exosomes from the sources are complex, the exosomes are not high-purity exosomes from tumor cells, the characteristics of tumors cannot be comprehensively reflected, and the exosomes directly separated from tumor tissue samples are more representative.

The current technical means can only separate the exosomes in the peripheral blood of the human primary hepatocellular carcinoma (HCC) cell line or HCC patients, but the exosomes obtained by the method are not the exosomes from HCC in the true sense. Because solid tumors tend to be very heterogeneous, established commercial tumor cell lines can only represent the biological characteristics of a certain population or a certain subset of cells in a solid tumor, and cannot reflect the overall characteristics of the tumor; in addition, although exosomes can exist stably in body fluid such as peripheral blood, trace exosomes secreted by tumor cells growing in local parts of the body become very small in content after being diluted by a blood system, and meanwhile, the blood serum is also mixed with exosomes released by other non-tumor cells of the body.

[ summary of the invention ]

The technical problem to be solved by the invention is to provide a method for separating exosomes in liver cancer tissues, which can be directly separated from a liver cancer tissue specimen after operation and can obtain exosomes which accurately reflect the characteristics of HCC tumors.

The invention is realized by the following steps:

a method for separating exosomes in liver cancer tissues comprises the following steps:

the method comprises the following steps: immediately soaking a fresh HCC tissue specimen after operation in PBS containing streptomycin and penicillin double antibody once the HCC tissue specimen is separated from the body, so that the tissue is in a sterile environment and the activity state of the separated cells is kept;

step two: under the aseptic operation condition, cutting the tissue blocks into tissue fragments with the size of 1-3 mm by using scissors so as to facilitate subsequent digestion;

step three: rinsing blood with PBS containing double antibodies, placing the blood in a DMEM/F12 culture medium, adding type I and type IV collagenase and hyaluronidase to degrade extracellular matrix, loosening connection between cells so as to release exosomes, and adding DNase to digest genomic DNA released by dead cells;

step four: incubating for 30-60 minutes by a constant temperature shaking table to enable the tissue to be dissociated into single cell suspension, and fully releasing exosomes in the tumor tissue;

step five: filtering the digestive juice with 70 μm filter to remove undigested tissue debris;

step six: obtaining the exosome by a gradient centrifugation method.

Further, in the third step, the type I collagenase is 50-100U/ml, the type IV collagenase is 20-50U/ml, the hyaluronidase is 20-50U/ml, and the DNase is 0.1 mg/ml.

Further, in the fourth step, the constant temperature shaking table is 37 ℃.

Further, the sixth step specifically includes:

centrifuging at 300g for 10 minutes and 2000g for 20 minutes, and removing cell precipitates to obtain supernatant;

the resulting supernatant was centrifuged at 16,500g for 45 minutes and 100,000g for 2 hours at 4 ℃ to obtain exosomes.

The invention has the advantages that:

1. the exosome obtained by the method is directly separated from an HCC tissue sample and is an exosome derived from tumor cells in the true sense;

2. the exosome obtained by the method can more accurately and comprehensively reflect the characteristics of the tumor cells;

3. the exosome obtained by the method is an exosome derived from high-purity tumor cells, and is not mixed with exosomes produced by non-tumor cell secretion in body fluids such as blood and the like.

[ description of the drawings ]

The invention will be further described with reference to the following examples with reference to the accompanying drawings.

FIG. 1 is a schematic flow diagram of the process of the present invention.

FIG. 2 is a schematic diagram showing the particle size comparison of exosomes extracted from patient plasma (A) and liver cancer tissue specimens (B) measured by nano-flow technique.

FIG. 3 is a schematic diagram showing the morphological comparison of exosomes extracted from plasma (A) and liver cancer tissue specimen (B) of a patient observed by a projection electron microscope.

FIG. 4 is a schematic representation of Western blot detection of the expression of exosome-specific proteins and liver cancer-specific proteins.

[ detailed description ] embodiments

As shown in fig. 1, a method for separating exosomes from liver cancer tissue comprises the following steps:

the method comprises the following steps: immediately soaking a fresh HCC tissue specimen after operation in PBS containing streptomycin and penicillin double antibody once the HCC tissue specimen is separated from the body, so that the tissue is in a sterile environment and the activity state of the separated cells is kept;

step two: under the aseptic operation condition, cutting the tissue blocks into tissue fragments with the size of 1-3 mm by using scissors so as to facilitate subsequent digestion;

step three: after rinsing and washing blood with PBS containing double antibody, placing the blood in DMEM/F12 culture medium, simultaneously adding 100U/ml type I, 50U/ml type IV collagenase and 50U/ml hyaluronidase to degrade extracellular matrix, loosening connection between cells so as to facilitate the release of exosomes, and adding 0.1mg/ml DNase to digest genomic DNA released by dead cells;

step four: incubating for 30-60 minutes at 37 ℃ by using a shaking table to dissociate the tissues into single cell suspension and fully release exosomes in the tumor tissues;

step five: filtering the digestive juice with 70 μm filter to remove undigested tissue debris;

step six: obtaining exosomes by a gradient centrifugation method, which specifically comprises the following steps:

centrifuging at 300g for 10 minutes and 2000g for 20 minutes, and removing cell precipitates to obtain supernatant;

the resulting supernatant was centrifuged at 16,500g for 45 minutes and 100,000g for 2 hours at 4 ℃ to obtain exosomes.

Randomly selecting three patients with liver cancer, extracting exosomes (5 ml of supernatant used for extracting exosomes after removing tissue fragments and cell precipitates) from liver cancer tissues of the patients according to the method, simultaneously extracting exosomes from 5ml of plasma of the same patient, and comparing the extraction effects of the exosomes from two sources. Firstly, the concentration and the particle size of exosomes are measured by adopting a nano-flow technology, the result shows that the particle sizes of the exosomes are all about 80nm (as shown in figure 2), and the concentration of the exosomes extracted from the liver cancer tissue of a patient is obviously higher than that of the exosomes extracted from plasma (as shown in table 1); secondly, observing the form of the exosome by adopting a projection electron microscope, and displaying that the exosome is a disk-shaped vesicle with a double-layer membrane structure (as shown in figure 3); western blot is adopted again to detect the expression of the exosome marker protein and the liver cancer specific protein, and the result shows that exosome extracted from the liver cancer tissue of a patient can detect high-level exosome marker proteins TSG101, CD63 and CD9, and can detect liver cancer specific proteins AFP and GPC3, while exosome extracted from plasma has obviously low content of exosome marker protein and hardly detects AFP and GPC3 protein (as shown in figure 4).

TABLE 1 exosome concentration and particle size measurement results

The exosome obtained by the method is directly separated from an HCC tissue sample, is an exosome derived from tumor cells in a real sense, can more accurately and comprehensively reflect the characteristics of the tumor cells, is an exosome derived from high-purity tumor cells, and is an exosome generated by non-tumor cell secretion in body fluids such as blood and the like without being doped.

The above description is only an example of the preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (4)

1. A method for separating exosomes in liver cancer tissues is characterized in that: the method comprises the following steps:

the method comprises the following steps: immediately soaking a fresh HCC tissue specimen after operation in PBS containing streptomycin and penicillin double antibody once the HCC tissue specimen is separated from the body, so that the tissue is in a sterile environment and the activity state of the separated cells is kept;

step two: under the aseptic operation condition, cutting the tissue blocks into tissue fragments with the size of 1-3 mm by using scissors so as to facilitate subsequent digestion;

step three: rinsing blood with PBS containing double antibodies, placing the blood in a DMEM/F12 culture medium, adding type I and type IV collagenase and hyaluronidase to degrade extracellular matrix, loosening connection between cells so as to release exosomes, and adding DNase to digest genomic DNA released by dead cells;

step four: incubating for 30-60 minutes by a constant temperature shaking table to enable the tissue to be dissociated into single cell suspension, and fully releasing exosomes in the tumor tissue;

step five: filtering the digestive juice with 70 μm filter to remove undigested tissue debris;

step six: obtaining the exosome by a gradient centrifugation method.

2. The method of claim 1, wherein said method comprises the steps of: in the third step, the type I collagenase is 50-100U/ml, the type IV collagenase is 20-50U/ml, the hyaluronidase is 20-50U/ml, and the DNase is 0.1 mg/ml.

3. The method of claim 1, wherein said method comprises the steps of: in the fourth step, the constant temperature shaking table is 37 ℃.

4. The method of claim 1, wherein said method comprises the steps of: the sixth step specifically includes:

centrifuging at 300g for 10 minutes and 2000g for 20 minutes, and removing cell precipitates to obtain supernatant;

the resulting supernatant was centrifuged at 16,500g for 45 minutes and 100,000g for 2 hours at 4 ℃ to obtain exosomes.

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