CN112147058B - Method for detecting hepatocyte-derived exosomes in plasma - Google Patents
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Abstract
The invention improves the existing exosome extraction method, and discloses a method for extracting exosomes from blood plasma. After exosome is extracted, the invention selects hepatocyte marker protein ALB with the best recognition effect and exosome marker protein CD63 to match for extract labeling, then uses the Amnis quantitative imaging analysis flow cytometer of exosome, and uses the best detection parameter to image each cell passing through the flow chamber, thereby realizing multi-parameter quantitative analysis of cell images, obtaining brand new cell morphological statistical data, and accurately and sensitively recognizing exosome of hepatocyte source in plasma. The exosome expression profile derived from the hepatocyte obtained by the invention can be used as a tool for liver regeneration evaluation, and has huge development potential.
Description
Technical Field
The invention relates to a method for detecting exosomes, in particular to a method for detecting exosomes derived from liver cells in plasma.
Background
Exosomes are natural closed vesicles with the diameter of 30-100nm and a lipid bilayer structure, can be synthesized and released by various cell types, can be used as a medium in intercellular communication, can transport information cargos such as proteins, lipids and nucleic acids, play an important role in the occurrence and development of diseases, and are hot spots of research in recent years.
Almost all types of cells in the human body can produce and release exosomes, and therefore plasma is rich in exosomes. Exosomes from different cell sources carry protein molecules derived from corresponding cells, and information in tissue cells is brought into peripheral blood, so that how to separate and identify exosomes from liver cells in plasma is of great significance for diagnosis and treatment of liver diseases. In patients with liver diseases, liver cells are damaged to different degrees, and exosomes derived from liver cells, such as exosomes derived from liver cells, exosomes derived from bile duct epithelial cells, exosomes derived from endothelial cells, exosomes derived from Kuffer cells, exosomes derived from lymphocytes, exosomes derived from hepatic stellate cells, and the like, are present in plasma of the patients. The liver cells are the main constituent cells of liver tissues and account for 60-80% of the total number of the liver cells, and liver diseases are mainly caused by the damage of the structure and the function of the liver cells, so that the detection of exosomes derived from the liver cells can reflect the functional state of the liver in real time, and has guiding significance for the diagnosis and treatment of the liver diseases.
At present, clinical liver regeneration markers are single, and a main index Alpha-fetoprotein (AFP) has certain limitation in liver regeneration evaluation, cannot accurately reflect liver regeneration, and cannot meet clinical requirements. Although minimally invasive liver biopsy is the gold standard for diagnosing liver regeneration, the development of novel, reliable, non-invasive diagnostic methods has been a major research direction. Exosomes carry different proteins and RNA molecules due to different cell sources, and information from tissue cells is brought into peripheral blood, so detection of exosomes in blood is called liquid-phase biopsy, and can reflect the functional state of cells in real time.
In order to study the mechanism of the exosome biological action more deeply, high-quality exosome extraction is the primary step, and methods for extracting and separating exosome from body fluid are various, such as ultracentrifugation, gel exclusion chromatography, kit extraction, immunomagnetic bead method and the like, but various methods have respective advantages and disadvantages, for example, exosome extracted by the kit extraction method also contains other vesicles and macromolecular impurity proteins, which affects the exosome purity. Gel exclusion chromatography requires specialized equipment, is time consuming and is not widely used. The immunomagnetic bead method has low efficiency, and the antibody is expensive and cannot be recovered, so that the immunomagnetic bead method is difficult to widely popularize. Although the traditional centrifugation method is complicated in steps, the method is still the most commonly used method for exosome extraction. The existing differential centrifugation method has relatively simple requirements on experimental equipment and can basically meet the extraction requirements, but has the problem that (1) the yield of exosome is low. The flow cytometer can not detect a target result, because particles with the diameter below 1um of the traditional flow cytometer are difficult to detect, the flow cytometer can only be analyzed by Amnis quantitative imaging, and the exosomes with the diameter ranging from 30 nm to 100nm can be detected by the sensitivity and the image resolution of the Amnis, but the minimum detection parameters of the flow cytometer by the Amnis quantitative imaging analysis are that the flow rate of sample particles reaches at least 100obj/s, the total number of collected particles is at least 20000, and the exosome obtained by the existing extraction method has low yield and is difficult to meet the detection requirement. (2) The extraction steps and methods have certain influence on the physicochemical properties of exosomes, for example, the integrity of exosomes cannot be ensured, and the exosomes are easy to crack and the functionality is influenced in the prior art.
In addition, although a plurality of exosome markers exist, experiments prove that the markers are not suitable for being used as detection markers of exosomes derived from liver cells, the labeling effect is poor, and the detection result is directly influenced.
Therefore, how to obtain an effective exosome extraction method and a method for sensitively distinguishing hepatocyte-derived exosomes in plasma is a research hotspot of a person skilled in the art.
Disclosure of Invention
The technical problem to be solved by the invention is to provide an improved method for accurately and sensitively detecting the hepatocyte-derived exosome in the plasma.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for detecting hepatocyte-derived exosomes in plasma, wherein the method comprises the following steps:
(1) Firstly, carrying out exosome extraction on a plasma sample, carrying out low-speed centrifugal extraction at normal temperature for the first time, centrifuging for 10-20 minutes at a centrifugal force of 2000-3000 g, and taking supernatant;
(2) Centrifuging at a centrifugal temperature of 4 ℃ for 30-60 minutes at a centrifugal force of 10000-12000 g for the second low-speed centrifugation extraction, and taking supernatant;
(3) Performing high-speed centrifugation in a third vacuum environment, pouring the supernatant obtained in the step (2) into a centrifuge tube, then supplementing the solution to a position 1-2 mm away from the tube opening with PBS, starting vacuum, starting centrifugation when the vacuum value is less than 50 mu m, performing centrifugation at 100000-120000 g for more than 1 hour, performing centrifugation at 4 ℃, and stopping centrifugation to release vacuum;
(4) After centrifugation, the supernatant is discarded, and PBS is used for resuspension;
(5) Exosome staining: adding direct standard antibodies ALB-AF488 and CD63-PE, keeping out of the sun for 30 minutes at room temperature, adding PBS for centrifugation, wherein the centrifugal force is 100000-120000 g, and the centrifugation time is 70-120 min;
(6) Pouring out the liquid in the tube, and using the tube wall reflux liquid to avoid light and shake for heavy suspension;
(7) And (4) detecting the extracted exosomes by using an Amnis quantitative imaging analysis flow cytometer to distinguish the hepatocyte-derived exosomes.
Preferably, the centrifugal force in the step (1) is 2000g.
Wherein preferably, the centrifugal force in the step (2) is 10000g.
Wherein the centrifugal force in the step (3) is preferably 110000g.
Wherein the centrifugal force in the step (5) is preferably 110000g.
The preferred centrifugal force is the best centrifugal force recommended by the inventor, and the beneficial effects of the invention can be realized by the centrifugal force within the range of the centrifugal force.
Preferably, the Amnis quantitative imaging analysis flow cytometer detection parameters are as follows: the excitation light wavelength is 400-500 nm and 600-700 nm, the intensity is 50-200 mW, and the scattered light SSC intensity is 15-20 mW.
The invention has the beneficial effects that:
the invention improves the existing exosome extraction method and provides a method for extracting exosomes from blood plasma. The exosome extraction method is simple to operate, high in efficiency, high in yield of the obtained exosome, high in purity, and most importantly, the complete structure and biological function of the exosome can be maintained. After exosome is extracted, the invention selects hepatocyte marker protein ALB with the best recognition effect and exosome marker protein CD63 to match for extract labeling, then uses the Amnis quantitative imaging analysis flow cytometer of exosome, and uses the best detection parameter to image each cell passing through the flow chamber, thereby realizing multi-parameter quantitative analysis of cell images, obtaining brand new cell morphology statistical data, and successfully, accurately and sensitively recognizing exosome of hepatocyte source in plasma. The exosome expression profile derived from the hepatocyte obtained by the invention can be used as a tool for liver regeneration evaluation, and has huge development potential.
Drawings
FIG. 1 shows the enrichment of cellular components of ultracentrifuge extracted particles;
FIG. 2 shows protein fraction analysis of ultracentrifugation extracted particles;
FIG. 3A is a double-staining flow-through fluorescence map of exosomes CD9 and AFP;
FIG. 3B is a double-stain flow-through scattergram of exosomes CD9 and AFP;
FIG. 4A is a double-staining flow-through fluorescence map of exosomes CD63 and ALB;
FIG. 4B is a double-stain scattergram of exosomes CD63 and ALB;
FIG. 5A is a double flow fluorescence plot of exosomes CD9 and NTCP;
FIG. 5B is a double-dye flow scattergram of exosomes CD9 and NTCP;
FIG. 6A is a double-staining flow-through fluorescence map of exosomes CD9 and CK 19;
FIG. 6B is a double-dye flow scattergram of exosomes CD9 and CK 19;
FIG. 7 is a comparison of the expression levels of four hepatocyte-derived exosome markers.
Detailed Description
Example 1 extraction of exosomes in plasma
1. Extraction of exosomes:
1. clinical blood specimens (whole blood samples from patients with chronic acute liver failure, beijing Youtoan Hospital, university of capital medical science) were centrifuged at 3000 rpm for 10min. Taking the plasma into EP tubes, and subpackaging, wherein each tube contains about 1 ml;
2. differential centrifugation purification of plasma exosomes:
(1) Sucking the plasma by a pipette, adding the plasma into a centrifuge tube with each tube being about 1ml, and supplementing the plasma to 12ml by PBS buffer solution;
(2) Centrifuging at low speed at normal temperature with centrifugal force 2000 (RCF) for 10min, collecting supernatant, and centrifuging at low speed to remove large cell and macromolecular impurities; the centrifugal force can be in the range of 2000-3000 g, 2000 revolutions is used in this embodiment, and the details of the centrifugal force in other ranges are not described.
(3) Centrifuging at a centrifugal force of 10000g and a centrifugal temperature of 4 ℃ for 30 minutes at a second low speed for extraction, taking supernatant, and centrifuging at a second low speed for removing cell debris; centrifugal force can be within the range of 10000-12000 g, 10000 revolutions are used in the embodiment, and the centrifugal force in other ranges is not described in detail.
(4) And (3) high-speed centrifugation in a third vacuum environment: pouring the supernatant after low-speed centrifugation into a centrifuge tube, and supplementing the solution to a position 1-2 mm away from the opening of the centrifuge tube with PBS (vacuum pumping is required during high-speed centrifugation, and if the liquid in the centrifuge tube is not full, the tube wall can be flattened due to negative pressure after centrifugation). Opening a centrifugal tube groove, inserting a centrifugal tube, screwing a tube cap, hanging the tube cap on a centrifugal tube rack, and balancing by adding an equal amount of liquid corresponding to the serial number;
vacuum was started, when the vacuum value was <50 μm, centrifugation was started, centrifugation parameters: centrifugal force 110000g (RCF), rotor: SW40Ti 16U12543, centrifuging for 70 minutes at the temperature of 4 ℃; after the centrifugation is finished, the vacuum is released (generally, the centrifugation force in this step is only larger than 100000g, 110000g is used in this embodiment). In the step, the centrifugation time is generally more than 1 hour, and the centrifugation time is set to be 70min; the centrifugal force may be in the range of 100000-120000 g, 110000g is used in this embodiment, and the details of the centrifugal force in other ranges are not described.
(5) After centrifugation, the supernatant was discarded, the pellet was knocked off by knocking the walls of the tube, resuspended in 200. Mu.l PBS, and stored as-80 in EP tubes.
In the prior art, at least 100000g of vacuum high-speed centrifugation is further included after the step (4) in order to elute and obtain exosome with high purity, but the inventor finds that the exosome yield is too low and the structure is damaged after the elution of the second high-speed centrifugation, so the inventor omits the step, and the exosome obtained by the method provided by the invention has high yield and high purity by adjusting the parameters and steps in the method. The flow cytometer requires a minimum flow rate of at least 100obj/s of sample particles and a total number of collected particles of at least 20000. The flow rate of the sample particles in the present invention can reach 1000obj/s, and the total number of the collected particles can reach 100000.
2. Exosome staining
(1) Taking 100 microliters of exosome obtained in the above step;
(2) Adding the direct-labeled antibody according to the instruction and keeping the antibody away from light at room temperature for 30-60 minutes;
the direct standard antibodies were ALB-AF488 and CD63-PE, added at 5ul and 20ul, respectively.
(3) Pouring into a centrifuge tube, adding PBS to the position of 1-2 mm of the edge of the tube, centrifuging 110000g (RCF) for 70min;
(4) Pouring out the liquid in the tube, dipping the tube mouth with toilet paper, shaking and resuspending with the tube wall reflux liquid, and taking care of avoiding light.
3. Imaging streaming detection
The Amnis quantitative imaging analysis flow cytometer sensitivity and image resolution can detect exosomes with the diameter range of 30-100nm, and the inventor selects the following detection parameters under which the exosomes derived from the liver cells can be detected sensitively.
The invention selects Amnis quantitative imaging analysis flow cytometry detection parameters: the flow rate of the sample particles reaches at least 100obj/s, the total number of the collected particles is at least 20000, the flow rate of the sample particles in the invention can reach 1000obj/s, and the total number of the collected particles can reach 100000, which far exceeds the minimum detection standard of the flow cytometer.
The embodiment of the invention utilizes the following specific parameters: the excitation light wavelength is 488nm and 642nm, the intensity is 50-200 mW, and the scattered light (SSC) intensity is 20mW. The excitation light wavelength disclosed by the invention is 400-500 nm and 600-700 nm, the intensity is 50-200 mW, and the scattered light SSC intensity is 15-20 mW, which can detect the exosomes derived from the hepatocyte, and is not described in detail in this embodiment.
The detection method provided by the invention has the following results:
1. as shown in fig. 1 and 2, the particles extracted by the present invention were subjected to proteomic analysis, and cellular component analysis is shown in fig. 1 and 2. Fig. 1 is a diagram of cell component enrichment analysis performed on particles extracted by the method provided by the invention, wherein dots represent that the number of related genes is higher and higher from small to large, the enrichment is lower and lower from red to green, and the uppermost red dot is the largest, which indicates that main components in the extracted particles are exosomes, and the other green dots indicate other cell components or particles. Fig. 2 shows protein component analysis of the extracted particles of the present invention, and fig. 2 shows specific ratios, in which exosome protein accounts for 80% of the extracted protein. FIGS. 1 and 2 show that the exosomes extracted by us have high purity and high extraction yield, and most of the protein in the extracted particles comes from the exosomes.
2. Selection of markers
Many exosome markers exist, but many markers are difficult to detect after an Amnis quantitative imaging analysis flow cytometer, and in order to obtain a marker combination with a good marking effect, the inventor carries out marker detection experiments by utilizing ALB-AF488, AFP-PE, NTCP-APC, CK19-AF647, CD9-FITC and CD63-PE, and the added amounts are 5ul, 20ul and 20ul respectively. Antibody source: r & DSsystems (ALB-AF 488), BD Biosciences (AFP-PE, CK19-AF647, CD9-FITC, CD 63-PE), bioss (NTCP)). The combination of ALB-AF488 and CD63-PE is the best exosome marker of the invention, and the detection result is as follows.
As shown in fig. 3A to 6B, the invention successfully establishes a method for detecting the hepatocyte-derived exosomes in plasma by using the hepatocyte-derived marker proteins ALB, AFP, CK19, NTCP and the exosome-derived marker proteins CD9 and CD63 in combination. From fig. 3A to fig. 6B, it can be seen that the images of the exosomes derived from the hepatocytes in the flow detection, the images of the exosomes in the light field under the optical lens, and the structural integrity of the exosomes extracted by us can be clearly seen, and the size of the exosomes is between 30 nm and 100 nm.
In FIGS. 3A and 3B CD9-FITC refers to CD9 positive particles, exhibiting green fluorescence, AFP-PE refers to AFP positive particles, exhibiting yellow fluorescence, merge refers to CD9 and AFP double positive particles, exhibiting green and yellow, i.e., hepatocyte-derived exosomes; the scatter plot represents the number of dicationic particles.
FIG. 4A and FIG. 4B ALB-AF488 refers to ALB-positive particles, exhibiting green fluorescence, CD63-PE to CD 63-positive particles, exhibiting yellow fluorescence, merge to ALB and CD63 double positive particles, exhibiting green and yellow, i.e., hepatocyte-derived exosomes; the scatter plot represents the number of dicationic particles.
In FIGS. 5A and 5B, CD9-FITC refers to CD 9-positive particles, exhibits green fluorescence, NTCP-APC refers to NTCP-positive particles, exhibits red fluorescence, merge refers to CD9 and NTCP double positive particles, exhibits green and red colors, that is, exosomes derived from hepatocytes; the scatter plot represents the number of dicationic particles.
In FIG. 6A, CD9-FITC indicates CD 9-positive particles exhibiting green fluorescence, CK19-AF647 indicates CK 19-positive particles exhibiting red fluorescence, merge indicates double positive particles of CD9 and AFP, exhibiting green and red colors, that is, exosomes derived from hepatocytes; the scatter plot represents the number of dicationic particles.
The flow fluorescence diagrams of fig. 3A to 6B illustrate that the exosomes extracted by us have complete structure and biological function, the size is between 30 and 100nm, the size and the structure of a single exosome can be clearly seen in images in a bright field, and the exosomes derived from the hepatocytes in plasma are successfully detected by us by applying the hepatocyte and the exosome marker protein. However, because of the sensitivity of the markers, not all markers can be expressed in a good amount, and how to ensure the expression of the exosomes derived from the hepatocytes is another important point of the research of the present invention.
FIG. 7 is a graph comparing the expression levels of four hepatocyte-derived exosome markers. In fig. 7, the number of exosomes derived from hepatocytes detected by the labeling method using the ALB + CD6 combination is the largest and the number of exosomes derived from hepatocytes is the next to CD9+ AFP in the four methods for labeling exosomes derived from hepatocytes, but the AFP expression level is limited in the fluorescence map. The results show that the ALB + CD63 combination expressed the best amount of hepatocyte-derived exosomes in the unlabeled plasma.
The method provided by the invention not only can quickly extract the exosome from the plasma sample, but also can keep the structural and biological functions of the exosome complete, and is convenient for the subsequent identification of the exosome from the hepatocyte. The combination and optimization of the markers enable the method provided by the invention to quickly and sensitively identify the hepatocyte-derived exosomes in the plasma sample, and provide a stable and effective basis for the subsequent expression profile of the hepatocyte-derived exosomes and liver regeneration evaluation.
Claims (6)
1. A method for detecting hepatocyte-derived exosomes in plasma, characterized in that the method comprises the following steps:
(1) Firstly, carrying out exosome extraction on a plasma sample, sucking plasma by using a suction tube, adding the plasma into a centrifuge tube, adding 1ml of the plasma into each tube, supplementing the plasma to 12ml by using PBS buffer solution, carrying out first normal-temperature low-speed centrifugal extraction, carrying out centrifugal force of 2000-3000 g for 10-20 minutes, and taking supernatant;
(2) Centrifuging at a centrifugal temperature of 4 ℃ for 30-60 minutes at a centrifugal force of 10000-12000 g for the second low-speed centrifugation extraction, and taking supernatant;
(3) Performing high-speed centrifugation in a third vacuum environment, pouring the supernatant obtained in the step (2) into a centrifuge tube, then supplementing the solution to a position 1-2 mm away from the tube opening with PBS, starting vacuum, starting centrifugation when the vacuum value is less than 50 mu m, performing centrifugation at 100000-120000 g for more than 1 hour, performing centrifugation at 4 ℃, and stopping centrifugation to release vacuum;
(4) After centrifugation, the supernatant is discarded, and PBS is used for resuspension;
(5) Exosome staining: adding direct standard antibodies ALB-AF488 and CD63-PE, keeping out of the sun for 30 minutes at room temperature, adding PBS for centrifugation, wherein the centrifugal force is 100000-120000 g, and the centrifugation time is 70-120 min;
(6) Pouring out the liquid in the tube, and using the tube wall reflux liquid to avoid light and shake for heavy suspension;
(7) And (3) detecting the extracted exosomes by using an Amnis quantitative imaging analysis flow cytometer to distinguish the exosomes from the hepatocyte source.
2. The method for detecting a hepatocyte-derived exosome in plasma according to claim 1, characterized in that:
in the step (1), the centrifugal force was 2000g.
3. The method for detecting a hepatocyte-derived exosome in plasma according to claim 1, characterized in that:
in the step (2), the centrifugal force is 10000g.
4. The method for detecting a hepatocyte-derived exosome in plasma according to claim 1, characterized in that:
in the step (3), the centrifugal force is 110000g.
5. The method for detecting a hepatocyte-derived exosome in plasma according to claim 1, characterized in that:
in the step (5), the centrifugal force is 110000g.
6. The method for detecting a hepatocyte-derived exosome in plasma according to claim 1, characterized in that:
the detection parameters of the Amnis quantitative imaging analysis flow cytometer are as follows: the excitation light wavelength is 400-500 nm and 600-700 nm, the intensity is 50-200 mW, and the scattered light SSC intensity is 15-20 mW.
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