Biomarker associated with preeclampsia and use thereof

Abstract

A biomarker associated with preeclampsia, the biomarker comprising MICB+ & MCAM+ subtype exosomes. A use of a reagent in the preparation of a product for predicting the risk of developing preeclampsia, the reagent being used for measuring MICB+ & MCAM+ subtype exosomes. A kit for predicting the risk of developing preeclampsia, the kit comprising a reagent for measuring the content of MICB+ & MCAM+ subtype exosomes.

Description

A biomarker related to preeclampsia and its application Technical Field

[0001] The present invention relates to the field of molecular biology, and in particular to a biomarker associated with preeclampsia and applications thereof. Background Art

[0002] Preeclampsia, a type of hypertensive disorder during pregnancy, is a multi-system progressive disease unique to pregnancy. It is also called preeclampsia. It usually manifests as new-onset hypertension and proteinuria, or new-onset hypertension and end-organ dysfunction with or without proteinuria after 20 weeks of pregnancy. The incidence rate is 6%-10%. The disease can continue to progress. If not treated effectively in time, it may lead to a poor prognosis and even the death of the mother and fetus. Therefore, early detection and symptomatic treatment of this disease are very important. Currently, the diagnosis of preeclampsia mostly relies on a combination of clinical symptoms and laboratory test indicators. At the time of diagnosis, only symptomatic treatment can be given until delivery. There is a lack of relevant specific serological predictive markers to predict the occurrence and development of the disease.

[0003] Exosomes are a type of extracellular vesicle that act as messengers for intercellular communication, transmitting molecular signals. They carry macromolecules such as proteins, nucleic acids, and lipids from their source cells. Through surface proteins, they target other tissues and organs, delivering signaling molecules and thereby participating in the regulation of various signaling molecules. Recent studies have found that these vesicles are closely associated with the development and progression of preeclampsia, and can even serve as serological biomarkers for the prediction of preeclampsia and therapeutic targets. However, exosomes are complex entities with complex physical properties and physiological functions, and the functions of individual exosomes or certain subtypes, as well as their role in disease, remain unclear.

[0004] Summary of the Invention

[0005] The present invention aims to provide a biomarker related to preeclampsia and its application to address the problems of the prior art. The present invention provides a biomarker related to preeclampsia that can be used to effectively predict whether a pregnant woman has preeclampsia.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a biomarker associated with preeclampsia, wherein the biomarker includes MICB + &MCAM + Subtypes of exosomes.

[0008] The present invention provides the use of a reagent for detecting the content of the above-mentioned biomarker in the preparation of a product for predicting the risk of preeclampsia.

[0009] Preferably, the product comprises a reagent, a chip or a kit.

[0010] The present invention provides a kit for predicting the risk of preeclampsia, which comprises a reagent for detecting the content of the above-mentioned biomarker.

[0011] Preferably, the reagent includes MICB antibody and MCAM antibody; the MICB antibody is used to detect MICB + &MCAM + MICB, a marker protein on the surface of exosomes, is detected by the MCAM antibody. + &MCAM + MCAM, a marker protein on the surface of exosomes.

[0012] Preferably, the MICB antibody is a PE-labeled MICB antibody; and the MCAM antibody is a PE-labeled MCAM antibody.

[0013] Preferably, the sample to be detected in the kit is serum.

[0014] Preferably, the volume ratio of the serum, PE-labeled MICB antibody and PE-labeled MCAM antibody is 10:1:1.

[0015] The present invention discloses the following technical effects:

[0016] The present invention discovered MICB for the first time + &MCAM + A novel application of exosome subtypes as predictive markers for preeclampsia has been demonstrated through experiments demonstrating significantly higher levels of this subtype in the serum of patients with preeclampsia compared to those with normal pregnancies. A receiver operating characteristic curve analysis using this differential protein combination of exosome subtypes yielded an AUC value of 0.952. This suggests that this serum exosome subtype can serve as an early screening and predictive marker for preeclampsia, addressing the current lack of specific serological markers for early screening and prediction of preeclampsia. Therefore, the biomarkers provided by this invention have excellent clinical value for early screening and prediction of preeclampsia, addressing the current gaps in early screening and prediction of disease progression. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0018] FIG1 is a graph showing the test results of total exosome content in serum samples of pregnant women with preeclampsia and normal pregnant women in Example 1;

[0019] Figure 2 shows the test results of 5 samples in Example 2, where A is the test result of the total exosome content of the serum sample, B is the flow cytometry test result, P1 is the exosome with fluorescence (positive exosome), and P2 is the exosome without fluorescence;

[0020] FIG3 shows a single exosome-MICB in serum samples of pregnant women with preeclampsia and serum samples of normal pregnant women in Example 3. + &MCAM + The detection results of subtype exosomes, where A is the detection result of the total exosome content of the serum sample, B is the flow cytometry detection result, P1 is the fluorescent exosome (positive exosome), and P2 is the non-fluorescent exosome;

[0021] Figure 4 shows the evaluation of MICB + &MCAM + The results of constructing receiver operating curves and calculating AUC values ​​for exosome subtypes as markers for the prediction of preeclampsia. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0027] Example 1

[0028] The serum of pregnant women with preeclampsia and normal pregnancy was obtained from the Department of Obstetrics of the Third Affiliated Hospital of Guangzhou Medical University. In this example, the serum exosomes of 30 pregnant women before 20 weeks of gestation were selected for the relevant serum exosome research. After follow-up investigation, it was found that 11 of the 30 pregnant women were diagnosed with preeclampsia and 19 were normal.

[0029] The method for extracting total exosomes from the serum of 30 pregnant women is as follows:

[0030] 1. Pipette 1 mL of serum into a 1.5 mL centrifuge tube. Centrifuge at 300 × g for 10 minutes in a high-speed centrifuge. Collect the supernatant and transfer it to a new centrifuge tube. Discard the pellet to remove free blood cells from the sample.

[0031] 2. Place the supernatant treated in step 1 in a high-speed centrifuge at 2000 × g for 10 minutes. Collect the supernatant and transfer it to a new centrifuge tube. Discard the pellet to remove free cell debris.

[0032] 3. Place the supernatant treated in step 2 in a high-speed centrifuge at 10,000 × g for 10 minutes. Collect the supernatant and transfer it to a new centrifuge tube. Discard the pellet to remove apoptotic bodies from the sample.

[0033] 4. Add PBS to the supernatant treated in step 3 to a total volume of 10 mL, centrifuge at 10,0000×g for 90 min at 4°C, remove the supernatant, and the precipitate is rich in the serum exosomes we need.

[0034] 5. Pipette 1 mL of 1× PBS solution and evenly blow the centrifugal precipitate. After it is fully suspended in PBS, transfer the suspension to a new 1.5 mL centrifuge tube. This solution is the exosome-rich solution.

[0035] 6. The total exosome content of the exosome-rich solution was tested. The test results are shown in Table 1 and Figure 1.

[0036] Table 1 Detection results of total exosome related parameters and content

[0037] As shown in Table 1 and Figure 1, the average size of exosomes in the extracted serum was 93.18 nm, and the concentration was 6.35×10 12 particles / mL.

[0038] Example 2 Construction of a single exosome detection method for serum samples of preeclampsia and normal pregnancy

[0039] The exosome-rich solution (EV) sample extracted in Example 1 was mixed with a PE-labeled CD9 antibody (manufacturer: Bioss, catalog number: BC03075793). The CD9 antibody specifically recognizes the CD9 protein in the exosome membrane. CD9 protein is a recognized marker protein of the exosome membrane. Therefore, this protein was used as the target protein to explore the construction of a single exosome detection method. The specific experimental steps are as follows:

[0040] 500 μL of the exosome-rich solution extracted in Example 1 was taken and divided into 5 tubes, 100 μL in each tube, and 0 μL, 1 μL, 2.5 μL, 5 μL and 10 μL of PE-labeled CD9 antibody were added respectively, incubated in the dark for 30 min, added 1000 μL PBS, mixed, centrifuged at 52000 rpm for 24 min, the supernatant was removed, and the solution was resuspended in 1000 μL PBS. Centrifuged again, the supernatant was removed, and the solution was resuspended in 100 μL PBS. The ratio of fluorescent particles in 5 samples was detected by the machine, of which 10 μL of PE-labeled CD9 antibody detected CD9. + The exosome subtype had the best effect, and its detection results are shown in Table 2 and Figure 2.

[0041] Table 2 CD9 + Detection results of exosome-related parameters and content

[0042] As shown in Table 2 and Figure 2, the CD9 detected by 10 μL PE-labeled CD9 antibody + The average number of exosome subtypes was 80.83, and the concentration was 1.28×10 9 pieces / mL.

[0043] Example 3 Single exosome-MICB in serum samples of preeclampsia and normal pregnancy + &MCAM + Detection of exosome subtypes

[0044] 1. The exosome (EV) sample extracted in Example 1 was mixed with PE-labeled MICB antibody (manufacturer: Abcam, Catalog No.: ab300485) and PE-labeled MCAM antibody (manufacturer: Cell Signaling, Catalog No.: 68706). MICB and MCAM antibodies specifically recognize MICB and MCAM proteins in the exosome membrane. The specific experimental steps are as follows:

[0045] Take 100 μL of the exosome-rich solution extracted in Example 1, add 10 μL of PE-labeled MICB antibody and 10 μL of PE-labeled MCAM antibody, incubate in the dark for 30 min, add 1000 μL of PBS, mix, centrifuge at 52000 rpm for 24 min, remove the supernatant, resuspend in 1000 μL of PBS, centrifuge again, remove the supernatant, resuspend in 100 μL of PBS, and detect the ratio of MICB+ & MCAM+ subtype exosome fluorescent particles on the machine. The test results are shown in Table 3 and Figure 3.

[0046] Table 3 MICB + &MCAM + Detection results of exosome-related parameters and content

[0047] As shown in Table 3 and Figure 3, the MICB detected by MICB antibody and MCAM antibody + &MCAM + The average exosome particle size was 79.85 and the concentration was 1.33×10 9 pieces / mL.

[0048] In order to evaluate MICB + &MCAM + Exosome subtypes were used as markers for the prediction of preeclampsia, and receiver operating curves were constructed to calculate the AUC values. + &MCAM + The receiver operating curve analysis of the subtype exosome results showed that its AUC value could reach 0.952, so it can be judged that MICB + &MCAM + The subtype of exosomes has better performance in predicting preeclampsia and is a better early screening and prediction marker for preeclampsia.

[0049] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art shall fall within the scope of protection defined by the claims of the present invention.

Claims

1. A biomarker associated with preeclampsia, characterized in that: The biomarkers include MICB + &MCAM + Subtypes of exosomes.

2. Use of a reagent for detecting the content of the biomarker described in claim 1 in the preparation of a product for predicting the risk of preeclampsia.

3. The use according to claim 2, characterized in that: The product includes a reagent, a chip or a kit.

4. A kit for predicting the risk of preeclampsia, characterized in that: The kit comprises reagents for detecting the content of the biomarker according to claim 1.

5. The kit according to claim 4, characterized in that The reagent includes MICB antibody and MCAM antibody; the MICB antibody is used to detect MICB + &MCAM + MICB is a marker protein on the surface of exosomes. The MCAM antibody is used to detect MICB + &MCAM + MCAM, a marker protein on the surface of exosome subtypes.

6. The kit according to claim 5, characterized in that The MICB antibody is a PE-labeled MICB antibody; the MCAM antibody is a PE-labeled MCAM antibody.

7. The kit according to claim 4, characterized in that The sample to be detected in the kit is serum.

8. The kit according to claim 7, characterized in that The volume ratio of the serum, PE-labeled MICB antibody and PE-labeled MCAM antibody is 10:1:1.

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