CN112147336A - Marker for detecting and evaluating liver regeneration and application thereof - Google Patents
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Abstract
Use of CD63, albumin ALB and endothelial growth factor VEGF or specific antibodies thereof as markers in the preparation of a kit for detection and assessment of liver regeneration. The invention provides a marker CD63+ ALB + VEGF for noninvasive detection and evaluation of liver regeneration by using peripheral blood for the first time, and the marker provided by the invention has the sensitivity of up to 87.5% and the specificity of up to 90% in liver failure and liver regeneration evaluation and is closely related to liver regeneration. In patients with liver failure, the number of exosomes labeled CD63+ ALB + VEGF was significantly higher in the survival group than in the death group. The result indicates that the exosome with VEGF and ALB in the plasma is a more accurate and specific marker for liver regeneration, and the detection technology can become a new noninvasive liver regeneration diagnosis technology.
Description
Technical Field
The invention relates to a biochemical marker, in particular to a marker and a detection kit for detecting and evaluating liver regeneration conditions, belonging to the technical field of medical inspection.
Background
The chronic infection of Hepatitis B Virus (HBV) is about 3.5 million people worldwide, and China accounts for one third (about 1.2 million). The chronic HBV infection gradually develops into severe hepatitis and the liver function failure caused by the severe hepatitis is a clinical type with the most fierce state of illness and the worst prognosis in hepatitis, and the fatality rate is about 40-60%. Since liver failure often affects young and strong years, the average age of onset is about 45 years, and the damage to individuals, families and society is great. One of the major differences between HBV-associated liver failure and end-stage cirrhosis lies in the regenerative potential of the liver, with the end result of death if patients with end-stage cirrhosis are not transplanted, and some patients with liver failure, although extremely ill, may survive liver regeneration. Therefore, for liver failure patients, liver regeneration assessment is particularly critical for clinical treatment scheme selection and prognosis judgment, patient burden reduction and quality of life improvement.
At present, the markers clinically used for liver regeneration evaluation are lack, and cannot well reflect the liver regeneration condition. The main reference index Alpha-fetoprotein (AFP) has certain limitation in clinical application, because the AFP is mainly an important index for reflecting liver cancer clinically, the rising of the level does not necessarily represent liver regeneration, and the dynamic change trend of the AFP needs to be closely observed in combination with clinic. In addition, the AFP content of germ cell tumor patients such as testicular cancer, ovarian tumor, malignant teratoma and the like is also increased, so the AFP can not accurately reflect liver regeneration. Other indicators related to liver regeneration such as Hepatocyte Growth Factor (HGF) and Vascular Endothelial Growth Factor (VEGF) lack tissue specificity because they are also expressed in other tissue organs outside the liver. Minimally invasive liver biopsy is the gold standard for diagnosing liver regeneration, but has a huge risk in performing liver puncture due to low blood coagulation function of patients with liver failure. Therefore, there is an urgent need to find a new noninvasive index for liver regeneration assessment.
Exosomes are natural closed vesicles with a lipid bilayer structure with the diameter of 30-150 nm, can be synthesized and released by various cells, can be used as a medium in intercellular communication, can transport information cargos such as proteins, lipids and nucleic acids, can bring information from tissues into peripheral blood, are called liquid phase biopsy, and are a hotspot of research in recent years.
However, the traditional exosome extraction method has many problems, and the application of exosome in clinical detection is limited. For example, exosomes extracted by the kit extraction method contain other vesicles and macromolecular impurity proteins, which affect the purity of exosomes. 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 following problems: (1) the exosome yield 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 many exosome markers exist, experiments prove that many markers are not suitable for being used as detection markers for liver regeneration, the labeling effect is poor, and the detection result is directly influenced.
Therefore, how to obtain an effective exosome extraction method and sensitively detect liver regeneration markers remains a research hotspot of the technicians in the field.
Disclosure of Invention
The invention aims to provide a novel marker for noninvasive diagnosis of liver regeneration, which can be used for detecting peripheral blood as a detection sample and sensitively and specifically detecting and evaluating the liver regeneration condition.
Another technical problem to be solved by the present invention is to provide a detection kit for detecting the above-mentioned marker.
In order to achieve the purpose, the invention adopts the following technical scheme:
use of CD63, albumin ALB and endothelial growth factor VEGF or specific antibodies thereof as markers in the preparation of a kit for detection and assessment of liver regeneration.
Preferably, the sample to be detected in the kit is peripheral blood, serum or plasma.
Use of a substance for detecting the content of exosomes labeled with albumin ALB and endothelial growth factor VEGF in the preparation of a kit for detecting and evaluating liver regeneration.
A test kit for detecting and assessing liver regeneration, wherein the kit comprises albumin ALB direct-labeled fluorescent antibody, CD63 direct-labeled fluorescent antibody and VEGF direct-labeled fluorescent antibody.
Preferably, the kit further comprises reagents required for exosome extraction acceptable in the art.
Preferably, the Kit further comprises sheath fluid, washing liquor A (Coulter clean starter Kit, merck-millipore) and magnetic microspheres; 70% isopropanol and a disinfectant.
Preferably, the disinfectant is 0.4-0.7% of sodium hypochlorite.
Preferably, the sample to be detected in the kit is peripheral blood, serum or plasma.
A method for extracting CD63, albumin ALB and endothelial growth factor VEGF marked exosome, which comprises the following steps:
(1) carrying out low-speed centrifugal extraction at normal temperature for the first time, wherein the centrifugal force is 2000-3000 g, centrifuging 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, CD63 and VEGF, adding PBS for centrifugation after keeping out of the sun for 30 minutes at room temperature, and centrifuging for 70-120 min at 100000-120000 g by centrifugal force;
(6) and (4) pouring liquid in the tube, and using the tube wall backflow liquid to avoid light and shake for heavy suspension.
The method provided by the invention is a method for obtaining ALB, CD63 and VEGF marked exosomes, is not a disease diagnosis and treatment method, and the ALB, CD63 and VEGF marked exosomes are only intermediate products of disease diagnosis and evaluation.
The invention has the beneficial effects that:
(1) the invention provides a marker CD63+ ALB + VEGF for noninvasive detection and evaluation of liver regeneration by using peripheral blood for the first time, and the marker provided by the invention has the sensitivity of up to 87.5% and the specificity of up to 90% in liver failure and liver regeneration evaluation and is closely related to liver regeneration. In patients with liver failure, the number of exosomes labeled CD63+ ALB + VEGF was significantly higher in the survival group than in the death group. The result indicates that the exosome with VEGF and ALB in the plasma is a more accurate and specific marker for liver regeneration, and the detection technology can become a new noninvasive liver regeneration diagnosis technology.
(2) The exosome extraction and staining method provided by the invention is simple to operate, high in efficiency, high in obtained exosome yield and purity, and most importantly, the complete structure and biological function of the exosome can be maintained, the flow rate of sample particles in the method can reach 1000obj/s after the exosome is stained by an index antibody, the total number of collected particles can reach 100000, and the detection standard of an Amnis quantitative imaging analysis flow cytometer is far exceeded. Provides an advantageous detection basis for noninvasive detection of liver regeneration.
Drawings
FIG. 1A is a flow-through fluorogram of exosome expression of a patient with liver failure;
FIG. 1B is an exosome expression flow scattergram of a liver failure patient;
FIG. 2 is the exosome expression levels of the survival and death groups of patients with liver failure;
FIG. 3 is the level of exosome expression at different stages in patients with liver failure;
FIG. 4 is a ROC curve of the expression profiles of three exosomes, CD9+ AFP, CD63+ ALB, CD63+ ALB + VEGF, in survival and death groups.
Detailed Description
The invention will be further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.
Firstly, extracting exosomes:
1. clinical blood specimens (specimens from whole blood samples of liver failure patients, Beijing Youyan Hospital, university of capital medical science) were centrifuged at 3000 rpm for 10 min. 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 normal temperature with centrifugal force 2000(RCF) for 10min, collecting supernatant, and centrifuging at low speed to remove large cell and macromolecular impurities; centrifugal force can be in the range of 2000-3000 g, 2000 revolutions are used in the embodiment, and the centrifugal force in other ranges is not described in detail.
(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 any more.
(4) And (3) high-speed centrifugation in a third vacuum environment: and (3) pouring the supernatant subjected to 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 is 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 frame, and balancing the number corresponding to the addition of the equivalent liquid to the centrifugal tube;
vacuum was started, when the vacuum value was <50 μm, centrifugation was started, centrifugation parameters: centrifugal force 110000g (RCF), selecting a rotary head: SW40Ti 16U12543, centrifugation time of 70 minutes and centrifugation temperature of 4 ℃; after the centrifugation, the vacuum is released (generally, the centrifugal force in this step is larger than 100000g, 110000g is used in this embodiment). In the step, the centrifugation time is generally longer than 1 hour, and the centrifugation time is set to be 70 min; centrifugal force can be in the range of 100000-120000 g, 110000g is used in the embodiment, and the centrifugal force in other ranges is not described in detail.
(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. In the invention, the flow rate of the sample particles can reach 1000obj/s, and the total number of the collected particles can reach 100000.
Second, exosome staining
(1) Taking 100 microliters of exosomes obtained in the above steps;
(2) adding a direct-labeled antibody according to the instruction, and keeping out of the sun for 30-60 minutes at room temperature;
the direct-labeled antibodies are ALB-AF488, CD63-PE, AFP-PE, CD9-FITC and VEGF-APC, and the detection experiments of the markers are carried out, and the adding amount is 5ul, 20ul and 20ul respectively. Antibody sources: r & D Systems (ALB-AF488), BD Biosciences (AFP-PE, CD9-FITC, CD63-PE, VEGF-APC). Pouring the mixture into a centrifuge tube, adding PBS to the position of 1-2 mm of the edge of the tube, centrifuging 110000g (RCF) for 70 min;
(3) 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.
Three, imaging flow detection
The method has the advantages that the Amnis quantitative imaging analysis flow cytometer sensitivity and image resolution can detect exosomes with diameters ranging from 30 nm to 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, and the flow rate of the sample particles can reach 1000obj/s, and the total number of the collected particles can reach 100000. Far exceeding the minimum detection standard of flow cytometry.
The embodiment of the invention utilizes the following specific parameters: the excitation light wavelength is 488nm (green fluorescence and yellow fluorescence) and 642nm (red fluorescence), the intensity is 50-200 mW, and the scattered light (SSC) intensity is 20 mW. 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, which can detect the exosomes derived from the hepatocyte, and is not described in the embodiment again.
The detection kit comprises:
sheath fluid (calcium magnesium free PBS); washing solution A (Coulter clean Start Kit, merck-millipore); magnetic microspheres; wash B (70% isopropanol); disinfectant (0.4-0.7% sodium hypochlorite); ALB-AF488 direct-labeled fluorescent antibody; CD63-PE direct labeled fluorescent antibody; VEGF-APC direct-labeled fluorescent antibody. Wash A was purchased from Merck-millipore. The reagent is used for imaging flow detection.
Fourthly, the detection result of the invention is as follows:
FIG. 1A, Alb-AF488 means ALB positive particles, exhibiting green fluorescence, CD63-PE means CD63 positive particles, exhibiting yellow fluorescence, VEGF-APC means VEGF positive particles, exhibiting red fluorescence, Merge means particles positive for ALB, CD63 and VEGF, exhibiting an overlap of three colors, i.e., exosomes with CD63, VEGF and ALB; FIG. 1B is a scatter plot showing the number of particles positive for ALB, CD63, and VEGF. The figure clearly shows that the exosome extracted by the inventor has a complete structure, the size of the exosome is 30-100 nm, the exosome has a complete structure and complete functions, and a favorable detection basis is provided for subsequent tests.
FIG. 2 shows the level of exosome expression in the survival and death groups of patients with liver failure. The liver failure patients are divided into a survival group and a death group, the expression levels of three exosomes including CD9+ AFP, CD63+ ALB and CD63+ ALB + VEGF are detected in the two groups of patients respectively, and the exosomes marked with the CD63+ ALB + VEGF are found to have obvious difference in quantity between the survival group and the death group compared with the other groups, so that the exosomes with the VEGF and the ALB can be used as novel markers for liver regeneration and prognosis evaluation of liver failure.
FIG. 3 shows the level of exosome expression at different stages in patients with liver failure. The liver failure patients are divided into four groups of early stage, middle stage and late stage according to PTA levels, expression levels of three exosomes of CD9+ AFP, CD63+ ALB and CD63+ ALB + VEGF are detected in the four groups of patients respectively, the number of exosomes marked with CD63+ ALB + VEGF is found to be obviously reduced in the late stage, and the number of exosomes marked with CD9+ AFP is reduced in the early stage, the middle stage and the late stage of patients in sequence. These results suggest that exosomes bearing VEGF and ALB in plasma are a more accurate and specific marker for liver regeneration and prognostic assessment than exosomes labeled CD9+ AFP.
FIG. 4 shows the ROC curves for the expression profiles of three exosomes, CD9+ AFP, CD63+ ALB, CD63+ ALB + VEGF, in the survival and death groups. The sensitivity of CD9+ AFP was 100% and the specificity was 20% (survival group 9, death group 10); the sensitivity of CD63+ ALB was 75% and the specificity was 50% (survival group 15, death group 12); the sensitivity of CD63+ ALB + VEGF was 87.5% and the specificity was 90% (survival group 13, death group 11).
Claims (9)
- Use of CD63, albumin ALB and endothelial growth factor VEGF or antibodies specific for the same as markers in the preparation of a kit for detection and assessment of liver regeneration.
- 2. The use according to claim 1, wherein: the sample to be detected of the kit is peripheral blood, serum or plasma.
- 3. Use of a substance for detecting the exosome content labeled CD63, albumin ALB and endothelial growth factor VEGF in the preparation of a kit for detecting and assessing liver regeneration.
- 4. A test kit for detecting and assessing liver regeneration, characterized in that: the kit contains albumin ALB direct-labeled fluorescent antibody, CD63 direct-labeled fluorescent antibody and VEGF direct-labeled fluorescent antibody.
- 5. The test kit of claim 4, wherein: the kit also contains reagents required for exosome extraction acceptable in the art.
- 6. The test kit of claim 4, wherein: the kit also contains sheath fluid, washing liquor A and magnetic microspheres; 70% isopropanol and a disinfectant.
- 7. The test kit of claim 6, wherein: the disinfectant is 0.4-0.7% of sodium hypochlorite.
- 8. The test kit of claim 7, wherein: the sample to be detected of the kit is peripheral blood, serum or plasma.
- 9. A method for extracting CD63, albumin ALB and endothelial growth factor VEGF marked exosomes is characterized by comprising the following steps:(1) carrying out low-speed centrifugal extraction at normal temperature for the first time, wherein the centrifugal force is 2000-3000 g, centrifuging 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, CD63 and VEGF, adding PBS for centrifugation after keeping out of the sun for 30 minutes at room temperature, and centrifuging for 70-120 min at 100000-120000 g by centrifugal force;(6) and (4) pouring liquid in the tube, and using the tube wall backflow liquid to avoid light and shake for heavy suspension.
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| CN116539396A (en) * | 2023-02-20 | 2023-08-04 | 深圳裕康医学检验实验室 | Multi-cancer seed staining method based on direct standard fluorescence and multiple immunofluorescence |
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