Method for rapid detection of platelet stress to glutamate

Abstract

The present application discloses a method for rapidly detecting the stress state of platelets to glutamate, comprising the following steps: obtaining specific antibodies to the amino acid transporter of platelets; adding the specific antibodies to a platelet suspension to form a sample to be detected; detecting the specific signal of the sample by flow cytometry; and statistically analyzing the specific signal to confirm the stress state of the sample. The method of the present application utilizes the advantages of flow cytometry and can analyze a single platelet, thereby analyzing the overall stress state of the platelets of the individual corresponding to the blood sample from a statistical perspective, providing reference data for subsequent identification and prediction of the patient's condition.

Description

Technical Field

[0001] The present application relates to the field of medical testing technology, and in particular to a method for rapidly detecting the stress state of platelets to glutamate. Background Art

[0002] Platelets are non-nuclear membrane cells, belonging to non-traditional cells, susceptible to extracellular microenvironmental stimulation and undergo different degrees of skeleton reorganization. Changes in platelet cell phenotype can reflect the stress state of the human body to a certain extent. Platelet EAAT2 (Excitatory Amino Acid Transporter 2) dominates the glutamate uptake of platelets and is mainly distributed on the platelet membrane and the α-granule membrane of the platelet. When the real-time state of platelets changes, the distribution of EAAT2 membrane protein will change significantly with the skeleton reorganization of platelets. The existing technology commonly uses immunoelectron microscopy (Immunoelectron microscopy, IEM) and immunofluorescence (Immunofluorescence, IF) to detect EAAT2 membrane protein, but these two technologies are very cumbersome in the processing of blood samples, not only the detection cycle is long, the experimental scheme is also inflexible, and too cumbersome or too much intervention operations may have a great impact on the detection results of EAAT2 membrane protein, the detection results are not accurate enough, and it is not suitable for promotion in clinical laboratories. Summary of the invention

[0003] The purpose of the present application is to at least partially overcome the deficiencies of the prior art and to provide a method for rapidly detecting the stress state of platelets to glutamate with high timeliness and simple operation.

[0004] In order to achieve the above technical objectives, the technical solutions adopted in this application are as follows:

[0005] A method for rapidly detecting the stress state of platelets to glutamate comprises the following steps:

[0006] Obtain antibodies specific for platelet amino acid transporters;

[0007] adding the specific antibody to a platelet suspension to form a sample to be tested;

[0008] detecting a specific signal of the sample by flow cytometry;

[0009] Statistical analysis of the specific signals confirmed the stress status of the samples.

[0010] Specifically, the amino acid transporter is a membrane protein.

[0011] Optionally, the specific antibody comprises a first antibody capable of immunologically binding to the extracellular fragment of the amino acid transporter, and a colorimetric marker capable of immunologically binding to the first antibody.

[0012] Further optionally, the specific antibody also includes a third antibody that can immunologically bind to the pan-platelet marker CD61.

[0013] Preferably, the amino acid transporter specifically refers to excitatory amino acid transporter 2.

[0014] Furthermore, the platelet suspension is prepared by:

[0015] Freshly collected blood was anticoagulated with sodium citrate;

[0016] Centrifuge at 200 g for 10 min at room temperature;

[0017] Harvesting plasma yields platelet-rich plasma (PRP);

[0018] The platelet-rich plasma was diluted with Tyrode's solution containing HEPES at a ratio of 1:20 to obtain a platelet suspension.

[0019] Optionally, before adding the specific antibody to the platelet suspension, a stress regulator is added according to the needs of statistical analysis.

[0020] Further optionally, the stress regulator includes an activator, inhibitor, interfering agent or anti-interfering agent for the platelets.

[0021] Specifically, the stress regulator includes thrombin as an activator, an excitatory amino acid transporter inhibitor as an inhibitor, and an inhibitor for preventing fibrin polymerization and thus preventing blood clot formation.

[0022] Furthermore, after adding the specific antibody to the platelet suspension, the cells are fixed with 1% paraformaldehyde for 30 minutes to prepare the sample to be tested.

[0023] Furthermore, the specific signal is associated with the relationship between the excitatory amino acid transporter and platelets, and the cell analysis results of the flow cytometer are statistically analyzed to confirm the cell phenotype of the platelets under different stress conditions.

[0024] Compared with the prior art, this application has the following advantages:

[0025] (1) The method of the present application utilizes the advantages of flow cytometry and can analyze individual platelets, thereby analyzing the overall stress of the platelets of the individual corresponding to the blood sample from a statistical perspective, providing reference data for subsequent identification and prediction of the patient's condition;

[0026] (2) The method of the present application utilizes the association between platelets and the EAAT2 membrane protein, providing a blood test method that can detect stroke, neurodegenerative diseases, etc. in real time, thereby increasing the possibility of clinical promotion;

[0027] (3) The sample preparation process of the method of the present application is simple, the interference with platelets is reduced to the lowest possible level, and the state of cell stress is maintained as much as possible, which is conducive to reflecting more realistic test results, and also improves the test efficiency, which is conducive to application in clinical testing;

[0028] (4) The samples prepared by the method of the present application can simulate stress or drug intervention scenarios by adding stress regulators, providing more abundant reference data for subsequent identification and prediction of the patient's condition, which is conducive to improving the accuracy of judgment and prediction. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a schematic diagram of the expression pattern of platelet EAAT2 membrane protein statistically analyzed using the method of the present application for rapidly detecting the stress status of platelets to glutamate, mainly showing the expression changes of platelet EAAT2 membrane protein before and after thrombin activation and under the conditions of reactivation after inhibition.

[0030] Figure 2 The four different platelet subpopulations counted by the method of the present application for rapidly detecting the stress status of platelets to glutamate mainly show the distribution of platelets in the sample in four regions.

[0031] Figure 3 The results of testing specific samples using the method of the present application for rapidly testing the stress status of platelets to glutamate mainly show the cell stress status reflected by different blood samples. DETAILED DESCRIPTION

[0032] The present application is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0033] The present application provides a method for rapidly detecting the stress state of platelets to plasma glutamate, comprising the following steps:

[0034] Obtain antibodies specific for platelet amino acid transporters;

[0035] adding the specific antibody to a platelet suspension to form a sample to be tested;

[0036] detecting a specific signal of the sample by flow cytometry;

[0037] Statistical analysis of the specific signals confirmed the stress status of the samples.

[0038] Specifically, the trans-plasma membrane transport of glutamate guided by the excitatory amino acid transporter plays a core role in physiology, and the imbalance of intracellular and extracellular glutamate concentrations is related to pathological conditions, and the difference between intracellular and extracellular glutamate concentrations is related to the state of the excitatory amino acid transporter on the cell membrane, and the difference between intracellular and extracellular glutamate concentrations also affects the distribution and state of the excitatory amino acid transporter. For platelets, platelets are prone to cytoskeleton reorganization due to irritants in the blood, and the cytoskeleton reorganization is accompanied to a certain extent by changes in the distribution and expression of excitatory amino acid transporters on the cell membrane, which may specifically cause the excitatory amino acid transporters in the cytoplasm to be recruited to the cell membrane surface or cause the transporters already located on the cell membrane to structurally flip to the correct functional position, thereby causing changes in the number of membrane proteins that can normally perform the transport function.

[0039] The excitatory amino acid transporter EAAT2 (Excitatory Amino Acid Transporter 2) on platelets is the most important EAAT subtype found so far for platelet uptake of excitatory amino acids (mainly glutamate). Glutamate is the most critical excitatory amino acid neurotransmitter in the mammalian nervous system, but excessive glutamate can cause excitotoxic damage to nerves. Extracellular glutamate in the brain can only rely on excitatory amino acid transporters for recycling. In the blood circulation, platelet EAAT2 plays a very important regulatory role in the homeostasis of plasma glutamate. Therefore, detecting the expression of platelet EAAT2 membrane protein can reflect to a certain extent the platelet's induction of glutamate concentration fluctuations in the plasma microenvironment, or the state of glutamate uptake from platelets can reflect to a certain extent the current stress state of the human body to plasma glutamate stimulation.

[0040] One possible implementation is to select a specific antibody that can specifically recognize the extracellular segment structure of EAAT2, commonly known as the "primary antibody". Furthermore, a secondary antibody that can recognize the primary antibody, a colorimetric marker that can immunologically bind to the primary antibody, is used to stain the entire platelet. Since flow cytometry is used, the staining referred to in this embodiment should generally be understood as fluorescent staining. When the sample preparation is completed and the sample is tested, the flow cytometer stimulates the secondary antibody on the surface of the platelet to emit fluorescence, and then identifies the individual platelet according to the intensity of the fluorescence or the matching band and records the corresponding identification value, thereby achieving the analysis of a single platelet, and further forming data reflecting the overall state of the platelets in the sample through statistical analysis. In this embodiment, the flow cytometric primary antibody Rabbit Polyclonal EAAT2 / GLT1Antibody and secondary antibodies Anti-rabbit IgG (H+L), F(ab')2Fragment (Alexa Fluor Conjugate) also includes a third antibody for labeling platelets - direct anti-platelet marker CD61 (CD61-PE).

[0041] The preparation process of the samples to be tested is as follows:

[0042] After collecting fresh blood samples, sodium citrate anticoagulation is added, and then centrifugation is performed at 200g for 10 minutes at room temperature to obtain platelet-rich plasma (PRP). The obtained PRP is allowed to stand at room temperature for 30 minutes and then diluted with HT buffer (HEPES / Tyrode buffer, Tyrode's Solution, HEPES-buffered, Tyrode's solution containing HEPES or HEPES-buffered Tyrode's solution). PRP can be diluted 20 times according to the ratio of platelets to antibodies.

[0043] According to the needs of statistical analysis, an unactivated group and an activated group can be set. In the unactivated group, PRP was directly added with CD61-PE and EAAT2 primary antibodies and stained for 15 minutes; in the activated group, PRP was added with GPRP tetrapeptide, and the platelets were first activated with an activator (such as thrombin) for 2 minutes, and then CD61-PE and EAAT2 primary antibodies were added to stain at room temperature in the dark for 15 minutes, and then secondary antibodies Anti-rabbit IgG (H+L), F(ab')2Fragment (Alexa Fluor Conjugate) staining for 20 minutes. After staining, the cells were fixed with 1% paraformaldehyde for 30 minutes. Furthermore, different intervention groups can be set by adding other stress regulators, such as setting an inhibition and then activation group: using the EAATs inhibitor DL-TBOA to intervene in the activation process of platelets by thrombin, or using other inhibitors to inhibit fibrin polymerization and platelet aggregation. The aforementioned stress regulator can also be an interfering agent or anti-interfering agent for platelets or EAAT2 (or even other protein elements) used to simulate the occurrence of specific symptoms, or a drug added during simulation treatment, or a downstream stressor induced by a drug.

[0044] The process of the on-machine detection and the parameters set in this embodiment are as follows:

[0045] CD61 fluorescence is selected for PE channel identification, EAAT2 fluorescence is selected for APC channel identification, APC gain is between 20 and 350, PE gain is about 300, and compensation is adjusted appropriately according to actual conditions. Prepared samples to be tested can be tested on a flow cytometer within 24 hours.

[0046] The present application uses a flow cytometry primary antibody and a corresponding secondary antibody that can specifically recognize the extracellular segment of EAAT2, combined with a blood sample processing process for thrombin-activated platelets under low-calcium conditions, to clearly capture the EAAT2 membrane protein signal of platelets at rest and during activation.

[0047] Test results:

[0048] For blood samples from the same source, the flow cytometry results of the unactivated group, activated group, and inhibition-reactivated group are shown in the figure below. Figure 1 shown.

[0049] Figure 1 The left column of the scatter plot shows the FSC-SSC scatter plot of a sample, where FSC represents the size of the cell and SSC represents the granularity of the cell. Since the aforementioned method for preparing the sample to be tested enriches platelets, it is impossible to see the clustering difference of the cells from the FSC-SSC scatter plot, but it can be seen that the density of the cells in the circled part has changed significantly in different experimental groups. Further, the values ​​in the FSC-SSC scatter plot are logarithmically processed, and the gate is set to obtain the clustering scatter plot in the right column to more significantly show the changes in the target cells (platelets). According to the changes produced after platelet stress, two groups of cells are clustered in the cluster scatter plot. Further classification can classify these cells into four areas, namely Q1 area on the upper left, Q2 area on the upper right, Q3 area on the lower right, and Q4 area on the lower left. The number of cells in Q1 area and Q2 area are counted respectively to form the histogram on the right: the number of cells in Q1 area increases sharply after changing from an unactivated state to an activated state, and there is a certain increase in the state of first inhibition and then activation; the number of cells in Q2 area can also be seen to increase after changing from an unactivated state to an activated state, and the increase is quite significant in the state of first inhibition and then activation.

[0050] Further verification and analysis, the present application uses flow cytometry to quantify four different platelet subsets marked by CD61 (pan-platelet marker) and EAAT2 membrane protein, which can intuitively present four different stages of cell phenotypes related to the basic state and activation state of platelets: resting state (stable), stressed state (hyperactivity), overexcited state (overreaction), and disintegration state (decay). Figure 2 As shown, it is believed that the Q1 region aggregates the over-excited platelets with enhanced EAAT2 expression, the Q2 region aggregates the stressed platelets with enhanced EAAT2 expression, the Q3 region aggregates the resting (unactivated) platelets, and the Q4 region aggregates the disintegrated platelets with attenuated EAAT2 expression. According to the proportion of cells in the four regions, the condition of the individual or patient corresponding to the sample can be judged and predicted.

[0051] One possible method for discrimination and prediction is to divide the collected blood samples into two groups, one group without adding platelet activators, set as the unactivated group, and the other group with adding thrombin as a platelet activator, set as the activated group. After the two samples are subjected to flow cytometric analysis under the same conditions, the obtained cluster scatter plots are compared, and the expression of EAAT2 membrane protein in the blood samples before and after activation can be clearly seen: Figure 3 As shown, the EAAT2 membrane protein of the activated group of blood sample 1 and blood sample 2 is highly expressed in platelet cells, but the subpopulation distribution of cells with high expression of EAAT2 membrane protein is different; in blood sample 3, the expression of unactivated EAAT2 membrane protein in platelet cells has been enhanced, and the number of platelets with high expression of EAAT2 has further increased after activation; in blood sample 4, the activated EAAT2 membrane protein has been highly expressed in platelets, but the fluorescence signal of EAAT2 membrane protein is weakened. Therefore, the stress degree and buffering potential of platelets corresponding to the four blood samples under glutamate toxicity stress can be analyzed.

[0052] This application can accurately obtain various parameters of cells (such as target protein fluorescence signal, cell size and granularity, etc.) from different angles by taking advantage of the flow cytometry advantage. The obtained data is rich and reliable, and can also easily realize the visualization charts of multiple parameters according to actual needs. In addition, the operation of this application is simple and the experimental cycle is short. It can not only clearly display the expression of EAAT2 membrane protein of resting platelets and activated platelets, but also flexibly add intervention groups to obtain the results after different interventions under the same experimental conditions, which greatly broadens the research space and application scope of this indicator. The experimental cycle can be completed within two hours, which can meet the clinical urgent need to grasp the patient's platelet stress status and the degree of glutamate toxicity damage, and is very suitable for promotion and application in clinical medicine. It can be further applied to the diagnosis and treatment links related to stroke, neurodegenerative diseases and cancer.

[0053] In summary, the present application provides a method for rapidly detecting the stress state of platelets to glutamate, comprising the following steps: preparing specific antibodies for the amino acid transporter of platelets; adding the specific antibodies to a platelet suspension to form a sample to be detected; detecting the specific signal of the sample by flow cytometry; and statistically analyzing the specific signal to confirm the stress state of the sample. The method of the present application utilizes the advantages of flow cytometry and can analyze a single platelet, thereby analyzing the overall stress state of the individual platelets corresponding to the blood sample from a statistical perspective, providing reference data for subsequent identification and prediction of the patient's condition.

[0054] The above embodiments are preferred implementation modes of the present application, but are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present application should be equivalent replacement methods and are included in the protection scope of the present application.

Claims

1. A method for rapidly detecting platelet stress to glutamate, characterized in that: The following steps are involved: Obtaining specific antibodies for platelet amino acid transporters; the specific antibodies include a first antibody that can immunologically bind to the extracellular segment structure of the amino acid transporter, and a colorimetric marker that can immunologically bind to the first antibody; the specific antibodies also include a third antibody that can immunologically bind to the pan-platelet marker CD61; the amino acid transporter is an excitatory amino acid transporter 2 membrane protein; adding the specific antibody to a platelet suspension to form a sample to be tested; Detecting specific signals of the sample by flow cytometry; The specific signal is statistically analyzed to confirm the stress condition of the sample, including associating the specific signal with the relationship between the excitatory amino acid transporter 2 membrane protein and the platelet state, and statistically analyzing the cell analysis results of the flow cytometer to confirm the cell phenotype of the platelets under different stress conditions: resting state, stressed state, overexcited state and disintegrated state.

2. The method according to claim 1, characterized in that The preparation method of the platelet suspension is: Freshly collected blood was anticoagulated with sodium citrate; Centrifuge at 200 g for 10 min at room temperature; Harvesting plasma yields platelet-rich plasma; The platelet-rich plasma was diluted with Tyrode's solution containing HEPES at a ratio of 1:20 to obtain a platelet suspension.

3. The method according to claim 2, characterized in that Before adding the specific antibody to the platelet suspension, a stress regulator is added according to the needs of statistical analysis.

4. The method according to claim 3, characterized in that The stress regulator includes an activator, an inhibitor, an interfering agent or an anti-interfering agent for the platelets.

5. The method according to claim 4, characterized in that The stress regulators include thrombin as an activator, excitatory amino acid transporter inhibitors as inhibitors, or inhibitors for preventing fibrin polymerization and thus blood clot formation.

6. The method according to claim 2, characterized in that After adding the specific antibody to the platelet suspension, the cells were fixed with 1% paraformaldehyde for 30 min to prepare the sample to be tested.

Images