Application of REEP5 in preparation of premature delivery detection product and detection kit

Abstract

The invention discloses application of REEP5 in preparation of a premature delivery detection product and a detection kit, and belongs to the field of biomedicine. In long-term research, the inventor team finds that the exosome protein REEP5 is obviously enriched in the premature fetal membrane exosome and the plasma exosome (protein expression is up-regulated); therefore, in order to timely and accurately judge whether the premature delivery occurs or not, a premature delivery diagnosis marker REEP5 is provided and is used for preparing a premature delivery detection product; meanwhile, the invention provides a premature delivery detection kit. The method provides a reference basis for premature delivery prevention, subsequent interference and the like.

Description

Technical Field

[0001] This invention relates to the application of exosomal protein REEP5, specifically to the application of REEP5 in the preparation of preterm birth detection products and detection kits, wherein exosomal protein REEP5 is used as a marker for preterm birth assessment, which belongs to the biomedical field. Background Technology

[0002] Premature birth refers to delivery between 28 and 37 weeks of gestation; newborns delivered at this time are called premature infants. Premature birth is a leading cause of neonatal and infant mortality and short- and long-term complications in children under five years of age, and has always been a focus of global public health. Despite continuous advancements in clinical medicine, the specific molecular mechanisms of premature birth remain unclear, resulting in limited preventative and treatment options.

[0003] Preterm birth is the leading cause of neonatal death and long-term complications worldwide. Existing predictive methods (such as cervical length measurement, ultrasound, and fFN detection) have insufficient sensitivity / specificity or operational limitations. Currently, there is still a lack of molecular biomarkers that can effectively predict and diagnose preterm birth early. This is a significant cause of perinatal and infant mortality and disability in clinical practice. Therefore, finding early diagnostic biomarkers for preterm birth is of great importance.

[0004] Therefore, a product that can promptly determine whether a birth is premature is needed to provide a reference for prevention and subsequent interference, which is an urgent technical problem to be solved. Summary of the Invention

[0005] Through long-term research, the inventors discovered that the exosomal protein REEP5 is significantly enriched (upregulated protein expression) in both preterm fetal membrane exosomes and plasma exosomes. To provide a timely and accurate diagnostic marker for preterm birth, they proposed REEP5 as a biomarker and its application in the preparation of preterm birth detection products and a diagnostic kit.

[0006] To achieve the above technical objectives, the following technical solution is proposed: The primary objective of this technical solution is to provide the application of REEP5 in exosomes in the preparation of preterm birth detection products, wherein the expression level of REEP5 in exosomes is used as the detection indicator.

[0007] Furthermore, the test is performed on the subject's biological sample, which is plasma.

[0008] Furthermore, the test is performed on the subject's biological sample, which is plasma taken from pregnant women during the 26th to 36th week of pregnancy.

[0009] Furthermore, the detection of REEP5 expression levels employs immunohistochemistry, Western blotting, mass spectrometry, chemiluminescence, and ELISA.

[0010] Furthermore, the testing standards include: The normal value of REEP5 expression level in exosomes is used as the standard value. When the REEP5 expression level in exosomes is higher than the standard value, the biological sample is considered positive, indicating that the pregnant woman corresponding to the biological sample has a higher risk of premature birth. When the REEP5 expression level in exosomes is equal to or lower than the standard value, the biological sample is considered negative, indicating that the pregnant woman corresponding to the biological sample has a lower risk of premature birth. The normal value is the expression level of REEP5 in plasma exosomes of pregnant women at full term.

[0011] The second objective of this technical solution is to provide: a preterm birth detection kit, including reagents for detecting the expression level of REEP5 in exosomes.

[0012] The beneficial technical effects of adopting this technical solution are as follows: I. This invention develops a new diagnostic biomarker for preterm birth - REEP5, and proposes the application of REEP5 in the preparation of preterm birth detection products and detection kits, which can timely and accurately determine whether it is preterm birth, and provide a reference for clinical diagnosis of preterm birth. Second, in this invention, the exosomal protein REEP5 has good stability and a long half-life, and has great potential in the diagnosis of preterm birth. Third, this invention innovatively applies the mature "liquid biopsy" concept in the field of oncology to obstetrics, realizing real-time, dynamic, and non-invasive molecular monitoring of the placenta, a key organ, which is a major shift in the prenatal diagnosis paradigm (a way to diagnose diseases through testing before delivery). Fourth, in this invention, the biological sample is plasma. The blood draw involved in the detection using plasma is a relatively simple, low-risk and minimally invasive method, unlike amniocentesis or other surgical sampling methods, which are more invasive and risky to the human body. Attached Figure Description

[0013] Figure 1 The expression of REEP5 in the fetal membrane tissue of preterm and full-term women in Example 3 (14 preterm women and 11 full-term women). Figure 2 Exosome characteristics in plasma from preterm and full-term pregnant women in Example 4: Exosome particle size analysis diagram; Figure 3 Exosome characteristics in plasma from preterm and full-term pregnant women in Example 4: Exosome identification images under a transmission electron microscope; Figure 4 To identify the characteristics of exosomes in the plasma of preterm and full-term pregnant women in Example 4: Western blotting was used to detect the expression of exosome markers in extracted exosomes and positive controls; Figure 5 Identification and verification of preterm birth-related proteins in fetal membrane-derived exosomes in Example 4: Principal component analysis of all fetal membrane exosome samples; Figure 6 Identification and verification of preterm birth-related proteins in exosomes derived from fetal membranes in Example 4: Volcano plot of significantly differentially expressed proteins; Figure 7 Identification and validation of preterm birth-related proteins in exosomes derived from fetal membranes in Example 4: GO analysis pathways of the top 10 differentially expressed proteins; Figure 8 Identification and verification of preterm birth-related proteins in exosomes derived from fetal membranes in Example 4: heatmap of the first 20 upregulated proteins; Figure 9 Identification and verification of preterm birth-related proteins in exosomes derived from fetal membranes in Example 4: Scatter plot of the expression levels of the first 20 upregulated proteins; Figure 10 Identification and validation of preterm birth-related proteins in plasma-derived exosomes in Example 4: Principal component analysis of total fetal membrane exosome samples; Figure 11 Identification and verification of preterm birth-related proteins in plasma-derived exosomes in Example 4: Volcano plot of significantly differentially expressed proteins; Figure 12 Identification and validation of preterm birth-related proteins in plasma-derived exosomes in Example 4: GO analysis of the top 10 differentially expressed proteins; Figure 13 For the identification and verification of preterm birth-related proteins in plasma-derived exosomes in Example 4: heatmap showing the expression levels of the top 20 upregulated proteins; Figure 14 For the identification and verification of preterm birth-related proteins in plasma-derived exosomes in Example 4: scatter plot showing the expression levels of the top 20 upregulated proteins; Figure 15 In Example 4, REEP5 was enriched in both preterm fetal membranes and tissue exosomes: Venn diagram of preterm upregulated proteins in plasma and fetal membranes; Figure 16 In Example 4, REEP5 was enriched in both preterm fetal membranes and tissue exosomes: Venn diagram of preterm downregulated proteins in plasma and fetal membranes; Figure 17 Example 4 shows that REEP5 was enriched in preterm fetal membranes and tissue exosomes: a heatmap of differentially expressed proteins in plasma and fetal membranes between preterm and full-term deliveries; Figure 18 In Example 4, REEP5 was enriched in both preterm fetal membranes and tissue exosomes: a scatter plot of differentially expressed proteins in plasma between preterm and full-term deliveries; Figure 19 In Example 4, REEP5 was enriched in both preterm fetal membranes and tissue exosomes: a scatter plot of differentially expressed proteins in fetal membranes between preterm and full-term deliveries; Figure 20 The expression of exosomal protein REEP5 in fetal membrane tissue in Example 4 (Western protein immunoblotting). Figure 21 The results of the test in a pregnant woman at 26 weeks of gestation in Example 5, when the exosomal protein REEP5 was used as a marker (wherein, the expression level of exosomal protein REEP5 in normal full-term pregnant women was used as the standard value). Detailed Implementation

[0014] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0015] In the embodiments described below, the research involved has been approved by the Ethics Committee of West China Second Hospital of Sichuan University (Approval No.: 2023012). Consent and approval from patients have been obtained for all samples and patient data used in this study.

[0016] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; unless otherwise specified, the experimental materials used in the following examples are all purchased from conventional biochemical reagent suppliers.

[0017] The detection methods involved include: Western blot: Representative exosome samples and supernatant were lysed with RIPA lysis buffer and then quantified using a BCA kit. Primary antibodies against CD9 (1:1000, Systems Biosciences, Inc., catalog number EXOAB-CD9A-1), CD63 (1:1000, Systems Biosciences, Inc., catalog number EXOAB-CD63A-1), TSG101 (1:1000, Systems Biosciences, Inc., catalog number EXOAB-TSG101-1), and REEP5 (1:1000, Wuhan Sanying Biotechnology Co., Ltd., catalog number 14643-1-AP) were used. A goat anti-rabbit horseradish peroxidase (HRP) conjugated secondary antibody (1:20000, Systems Biosciences, Inc., catalog number 180202-002) validated for exosome analysis was used as the secondary antibody. Transmission electron microscopy (TEM) examination: A 5 μL exosome sample was placed on a copper grid and incubated at room temperature for 5 minutes, then blotted dry with absorbent paper. One drop of 2% uranium acetate was added to the copper grid, and the sample was incubated at room temperature for 1 minute. Excess liquid was then blotted away with absorbent paper, followed by drying at room temperature for 20 minutes. The sample was then observed using an electron microscope (Tecnai G2 SpiritBio Twin type) at 80 kV. Nanoparticle tracking analysis (NTA): Cryopreserved exosome samples were thawed in a 25°C water bath. The samples were then diluted with 1× phosphate-buffered saline (PBS) for direct nanoparticle tracking analysis. Detection was performed using a nanoparticle size tracking analyzer (Zeta Vision S / N 22-756, manufactured by Particle Matrix Company). Four-dimensional label-free proteomics analysis: The main experimental steps for proteomics analysis included protein extraction, protein quantification, quality control, trypsin digestion, liquid chromatography-mass spectrometry (LC-MS / MS), and data analysis. For plasma samples, all samples were digested with a uniform 7.87 μg of protein based on the minimum total sample volume. All fetal membrane samples were digested with a uniform 4.22 μg of protein based on the minimum total sample volume. Missing values ​​were filled with the minimum quantification value of the detected protein, and the results were used for subsequent differential statistical analysis. Immunohistochemical (IHC) staining: Fetal membrane tissue samples were embedded in paraffin and fixed with 4% paraformaldehyde (PFA). Specifically, after acquisition, samples were immediately placed in pre-cooled 4% paraformaldehyde-phosphate buffer (0.1M PBS, pH 7.4) and fixed at 4°C for 24-48 hours. The fixation time was adjusted according to the size of the tissue block to ensure complete penetration fixation. Immunohistochemical analysis was performed using REEP5 (1:1000, Proteintech, catalog number 14643-1-AP) and the ABC Elite Immunoperoxidase Kit, following the manufacturer's instructions. The H scoring system was used to semi-quantitatively assess protein expression levels by combining staining intensity and the percentage of positive cells. Two independent parameters were evaluated for each slide: staining intensity (0 = negative, 1 = weak, 2 = moderate, 3 = strong) and the percentage of positive cells (quantified as a continuous variable ranging from 0% to 100%). The overall H score is calculated using the following formula: H score = percentage of positive cells × intensity score; Multiplex Immunohistochemical Staining: Multiplex immunohistochemical (mIHC) staining was performed using the Opal 7-color kit (PerkinElmer, product number NEL811001KT). Fetal membrane tissue pathology slides were placed in a 63°C oven for 1 hour. Subsequently, dewaxing, antigen retrieval, endogenous peroxidase removal, and blocking were performed. Primary antibody was incubated at room temperature for 1 hour. After rinsing with TBST (Tween-Tris buffered saline), secondary antibody was incubated at room temperature for 10 minutes. After rinsing with TBST, Opal dye (1:100 dilution) was applied for 10 minutes. The antigen-bound primary and secondary antibody complexes were removed by microwave heating, leaving only the fluorescent dye. This process from primary antibody incubation to microwave heating was repeated continuously until all markers were fully labeled. The primary antibodies used were REEP5 (1:1000, Proteintech, catalog number 14643-1-AP) and E-cadherin (Abacata, catalog number PA073). Finally, DAPI (4',6-diamidinyl-2-phenylindole) staining was performed, and the slides were mounted with fluorescent anti-quenching mounting medium. Multiplex immunohistochemical imaging and inForm analysis: Slides were scanned using an AKOYA PhenoImagerHT microscope. Acquired images were analyzed using inForm software. Cell segmentation was based on different marker expression patterns. Statistical analysis: Statistical significance was set at p < 0.05. All data were analyzed using GraphPad Prism 8.0, R studio 4.3.3, SPSS 20.0, and Microsoft Excel.

[0018] In addition, the plasma control samples involved were from pregnant women who did not experience preterm labor; the fetal membrane control samples involved were from pregnant women who did not experience preterm labor.

[0019] The following examples illustrate this.

[0020] Example 1 This embodiment provides the application of REEP5 in exosomes in the preparation of preterm birth detection products, wherein the expression level of REEP5 in exosomes is used as the detection index.

[0021] Specifically, the test is performed on the biological sample of the subject, which is plasma. Preferably, the biological sample is plasma collected during the 26th to 36th week of pregnancy.

[0022] For detection methods: Immunohistochemistry, Western blotting, mass spectrometry, chemiluminescence, and ELISA were used to detect REEP5 expression levels.

[0023] For the detection criteria: the normal value of REEP5 expression level in exosomes is used as the standard value. When the REEP5 expression level in exosomes is higher than the standard value, the biological sample is considered positive, indicating that the pregnant woman corresponding to the biological sample is at high risk of preterm birth. When the REEP5 expression level in exosomes is equal to or lower than the standard value, the biological sample is considered negative, indicating that the pregnant woman corresponding to the biological sample is at low risk of preterm birth. The normal value is the REEP5 expression level in plasma exosomes of pregnant women with normal full-term deliveries.

[0024] Example 2 Based on Example 1, this example provides: a preterm birth detection kit, including reagents for detecting the REEP5 expression level in exosomes.

[0025] Example 3 Human reproduction, pregnancy, and embryonic development require sophisticated intercellular communication. Semen, amniotic fluid, blood, and breast milk all contain exosomes with specific functions. During pregnancy, the number of exosomes increases significantly, becoming an active medium for material exchange and signal transmission between the fetus and mother. In addition to maternal tissues, exosomes are also released from the placenta and fetal tissues for communication. Studies have found that with the aging of the fetal membranes, the number, size, and surface markers of exosomes may change. These changes may affect the function of exosomes in intercellular communication within the fetal membranes, leading to fetal membrane dysfunction. Exosomes derived from the fetal membranes may carry markers reflecting dynamic changes at the maternal-fetal interface. These exosomes enter the bloodstream via blood circulation, and extracting exosomes from plasma from the fetal membranes provides a new approach for non-invasive diagnosis of preterm birth. Furthermore, the exosomes involved are derived from the fetal membranes, making them highly specific. The placenta and fetal membranes are organs and tissues closely related to pregnancy and childbirth, and can reflect dynamic changes at the maternal-fetal interface.

[0026] Based on this, in order to further verify that the expression level of REEP5 in fetal membrane tissue in preterm mothers was higher than the standard value, fetal membrane samples were collected from 25 mothers who gave birth at West China Second Hospital of Sichuan University, including 14 preterm mothers and 11 full-term mothers. Immunohistochemistry (IHC) staining was used to detect the expression of REEP5 in fetal membrane tissue of preterm mothers and full-term mothers, respectively. The results are as follows Figure 1 As shown, the REEP5 expression level in the fetal membrane tissue of preterm mothers is higher than that of full-term mothers.

[0027] Example 4 Building upon Examples 1-2, this example further validates that the expression level of REEP5 in exosomes in preterm mothers was higher than the standard value. From January 2023 to December 2024, fetal membrane samples were collected from 10 mothers who delivered at West China Second University Hospital of Sichuan University. The mothers were divided into two groups: 5 preterm mothers (gestational age >28 weeks, <37 weeks) and 5 full-term mothers (gestational age ≥37 weeks). Gestational age was determined based on early pregnancy ultrasound examination results. All included mothers had no obvious infection in the uterus or amniotic cavity and were all singleton pregnancies. No difference in age was observed between the two groups. Two days prior to delivery, blood samples were collected into 4ml BD vacuum blood collection tubes coated with K2-ethylenediaminetetraacetic acid (EDTA) (BD K2E [EDTA] tubes manufactured by BD Biosciences, UK). The whole blood was then centrifuged at 4200×g for 10 minutes to obtain plasma. The plasma samples were stored at -80℃ until later for exosome extraction. A total of 10 plasma samples were collected for exosome isolation. Fetal membrane tissue samples were collected immediately after delivery. A total of 10 fetal membrane samples were collected for exosome isolation. Specifically, it includes the following: I. Isolation of exosomes from samples 1. Isolation of exosomes from fetal membrane samples Exosomes were isolated from fetal membrane samples using continuous ultracentrifugation. The fetal membrane samples were first rinsed three times with phosphate-buffered saline (PBS), and then the fetal membrane tissue was cut into small pieces. These pieces were placed in PBS containing collagenase and deoxyribonuclease I and digested in a 37°C incubator until no granular residue remained. The mixture was then centrifuged at 500×g for 5 min at 4°C, repeated twice, followed by centrifugation at 2000×g for 15 min. The supernatant was filtered through a 0.22 μm filter membrane, and then the sample was ultracentrifuged at 100,000×g for 75 min. The precipitate was resuspended in 3 ml of 25% sucrose solution and centrifuged at 100,000×g for 1.5 h at 4°C. 2 ml of the sucrose layer was collected, PBS was added, and the mixture was centrifuged again at 100,000×g for 1.5 h at 4°C. Finally, the precipitate (exosomes) was resuspended in phosphate buffer. 2. Isolation of plasma exosomes Plasma samples were stored at -80°C until the exosome extraction process was performed. 500 ml of thawed plasma was centrifuged at 4°C first at 2000 × g for 10 min, then at 10000 × g for 30 min. The supernatant was loaded onto a ready-to-use size exclusion column (qEVoriginal), containing exosomes in 1.5 ml of separation buffer. II. After separating and extracting exosomes from the fetal membrane samples, the exosomes were identified using nanoparticle tracking analysis, transmission electron microscopy, and Western blotting. Figure 2-4 As shown. After exosome extraction, proteins were detected using 4D label-free proteomics analysis. Missing values ​​in the results were filled with half the minimum quantitative value of the detected protein for subsequent differential statistical analysis.

[0028] III. Protein markers in fetal exosomes of preterm and full-term pregnant women To validate exosomal proteins that can differentiate between preterm and term delivery, fetal membrane-derived exosomal proteins from 5 preterm and 5 term women were analyzed. Principal component analysis (PCA) excluded one term fetal membrane sample, leaving 5 preterm and 4 term samples for analysis (e.g., ...). Figure 5 The volcano plot shows proteins with significantly different expression between the two groups (such as...). Figure 6 A total of 435 differentially expressed proteins were identified, of which 350 were upregulated and 85 were downregulated. GO analysis revealed that these differentially expressed proteins are mainly involved in ribosome-related signaling pathways, such as cytoplasmic ribosomes, cytoplasmic macroribosome subunits, ribosome subunits, ribosomes, and macroribosome subunits (e.g., ribosomes, macroribosome subunits). Figure 7 Among these 350 upregulated proteins, GNGT2, RPL36A, PPP2R2D, NMNAT1, and HIC1 showed the most significant differences (e.g., Figure 8-9 ).

[0029] IV. Protein markers in plasma exosomes of preterm and full-term pregnant women Since tissue biopsies are not always reliable for disease detection, a similar analysis was performed using plasma-derived exosomal proteomics from the same patients. After excluding one full-term plasma sample by principal component analysis, plasma exosomal proteomics were analyzed from 5 preterm women and 4 full-term women (e.g., ...). Figure 10 ); There were 330 differentially expressed proteins, of which 206 were upregulated and 124 were downregulated (e.g., ...). Figure 11 GO analysis showed that collagen-containing extracellular matrix, extracellular matrix, external coating structures, and endomembrane system signaling pathways were most enriched (e.g., Figure 12 Of the 206 upregulated proteins, the differences were most significant for the top 20 proteins, including SURF4, SDHB, SEC61B, NENF, and VMA21 (e.g., Figure 13-14 ).

[0030] V. Characteristic proteins of exosomes derived from preterm fetal membranes in plasma After identifying exosomal proteins associated with preterm birth in fetal membranes and plasma, the proteomes of preterm exosomal proteins derived from fetal membranes and plasma were compared to identify biomarkers of preterm fetal membrane-derived exosomals in plasma. Specifically, Venn diagrams were used to analyze the overlap between upregulated and downregulated proteins in fetal membranes and plasma (e.g.,...). Figure 15-16 The exosomal protein REEP5 was highly enriched in preterm fetal membranes and plasma, while PIGR, SERPINA5, COL1A2, and MAMDC2 were all lowly enriched in preterm fetal membranes and plasma (e.g., Figure 17-19 ).

[0031] VI. Expression of exosomal protein REEP5 in fetal membrane tissue The expression of exosomal protein REEP5 in fetal membrane exosomes of preterm and normal term pregnant women was detected by Western blotting. The results are as follows Figure 20As shown, compared with women who have given birth at full term, the exosomal protein REEP5 in the fetal membrane tissue of women with premature birth is significantly upregulated.

[0032] Example 5 This embodiment tested a plasma sample from a 27-year-old pregnant woman at 26 weeks of gestation, and used the expression level of REEP5 in exosomes from a plasma sample (taken at 26 weeks of gestation) from a 27-year-old woman with normal full-term delivery (delivered at 39 weeks of gestation) as a control. The specific process is as follows: 1) Sampling Blood samples were collected into vacuum blood collection tubes coated with K2-ethylenediaminetetraacetic acid (EDTA) (4ml BD vacuum blood collection tubes K2E [EDTA] manufactured by BD, UK), and then the whole blood was centrifuged at 4200×g for 10 minutes to obtain plasma. The plasma samples were stored at -80℃ until they were used for exosome extraction. A total of two plasma samples were collected (for parallel experiments) for exosome isolation. 2) Isolation of plasma exosomes Plasma samples were stored at -80°C until the exosome extraction process was performed. 500 ml of thawed plasma was centrifuged at 4°C first at 2000 × g for 10 min, then at 10000 × g for 30 min. The supernatant was loaded onto a ready-to-use size exclusion column (qEVoriginal), containing exosomes in 1.5 ml of separation buffer. 3) Expression of exosomal protein REEP5 The expression of REEP5 in plasma exosomes was detected using 4D label-free proteomics analysis and compared with the control. The results are as follows Figure 21 As shown, compared with women who have given birth at full term, women with premature births have significantly upregulated REEP5 in their plasma exosomes, indicating that women in the 26th week of gestation are at high risk of premature birth and should prepare in advance to deal with premature birth.

Claims

1. The application of exosome-derived REEP5 in the preparation of preterm birth detection products, among which, The expression level of REEP5 in exosomes was used as the detection index.

2. The application according to claim 1, characterized in that, The test is performed on the subject's biological sample, which is plasma.

3. The application according to claim 1, characterized in that, The test is performed on the subject's biological sample, which is plasma taken from pregnant women during the 26th to 36th week of pregnancy.

4. The application according to claim 1, characterized in that, The REEP5 expression level was detected using immunohistochemistry, Western blotting, mass spectrometry, chemiluminescence, and ELISA.

5. The application according to any one of claims 1-4, characterized in that, The testing standards include: The normal value of REEP5 expression level in exosomes is used as the standard value. When the REEP5 expression level in exosomes is higher than the standard value, the biological sample is considered positive; when the REEP5 expression level in exosomes is equal to or lower than the standard value, the biological sample is considered negative. The normal value is the expression level of REEP5 in plasma exosomes of pregnant women at full term.

6. A preterm birth detection kit, characterized in that, This includes reagents for detecting REEP5 expression levels in exosomes.

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