Application of biomarker in prediction or auxiliary diagnosis of preeclampsia

Abstract

The invention belongs to the technical field of molecular diagnosis, and particularly relates to application of a biomarker in prediction or auxiliary diagnosis of preeclampsia. The invention provides an application of a reagent for detecting expression levels of Caspase3, BAX and IL-8 proteins in preparation of a product for predicting or assisting in diagnosing preeclampsia. Through micro-plasma Olink proteomics and clinical test verification, it is found that the risk of preeclampsia of a pregnant woman can be noninvasively, accurately and effectively predicted by detecting the protein expression levels of Caspase3, BAX and IL-8 in plasma; by using the kit provided by the invention, the occurrence of preeclampsia can be predicted or diagnosed in an auxiliary manner before clinical symptoms appear, so that the early discovery rate of diseases is greatly improved, an earlier intervention and treatment opportunity is provided for doctors, and the clinical application prospect is good.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular diagnosis, and specifically relates to an application of a biomarker in predicting or assisting in the diagnosis of preeclampsia. Background Art

[0002] Preeclampsia (PE) is a pregnancy-specific disease characterized by hypertension (≥140 / 90 mm Hg) and proteinuria (≥300 mg / L / 24 h) after 20 weeks of gestation, or with kidney, eye and other organ damage. PE is an important factor leading to maternal and perinatal morbidity and mortality, occurring in 2 to 4 cases per 100 pregnancies worldwide. It manifests in two different forms, divided according to gestational age at diagnosis or delivery: early-onset preeclampsia (EOPE, <34 weeks) and late-onset preeclampsia (LOPE, >34 weeks). Compared with late-onset preeclampsia, early-onset preeclampsia is thought to be associated with a higher incidence of hemolysis, elevated liver enzymes and low platelet count syndrome (HELLP syndrome), abnormal uterine artery Doppler waveforms, atherosclerosis, placental lesions, small gestational age and fetal growth restriction.

[0003] As the leading cause of maternal and perinatal morbidity and mortality worldwide, the disease has a higher risk of occurrence in the assisted reproductive population. However, due to the complex mixed nature of preeclampsia and the lack of effective early pregnancy biomarkers, many patients miss the critical opportunity for early intervention. At present, clinical intervention for preeclampsia still remains in the passive treatment stage after the onset of symptoms, mainly focusing on symptom management, and timely delivery as the most effective treatment. Once preeclampsia is diagnosed, blood pressure control is the main means of clinical intervention, and the only effective treatment is termination of pregnancy, which can lead to premature birth in about 1 / 3 of patients. Therefore, biological markers for predicting and diagnosing preeclampsia are of great clinical significance.

[0004] In previous studies, the inventor's research group found that compared with the normal pregnancy control group, the expression level of interleukin 8 (IL8) was significantly increased in the late pregnancy group of patients with preeclampsia. Compared with the late pregnancy group with preeclampsia, predicting the risk of preeclampsia earlier before the onset of clinical symptoms has important clinical significance. It can be used to predict and intervene in patients who may develop preeclampsia early. Therefore, it is urgent to develop a protein marker that can predict the risk of preeclampsia. Summary of the invention

[0005] In view of the technical problems existing in the above-mentioned prior art, the purpose of the present invention is to use a biomarker in the prediction or auxiliary diagnosis of preeclampsia. The biomarkers are Caspase3, BAX and IL-8. The kit provided by the present invention can predict or assist in the diagnosis of the occurrence of preeclampsia before the onset of clinical symptoms, thereby greatly improving the early detection rate of the disease and providing doctors with earlier intervention and treatment opportunities.

[0006] The first aspect of the present invention provides an application of a reagent for detecting the content of a marker in a biological sample in the preparation of a product for predicting or assisting in the diagnosis of preeclampsia, wherein the biomarkers are Caspase3, BAX and IL-8.

[0007] Furthermore, when the Caspase3 content in the biological sample was ≥1000 U / L, the BAX content was ≥20 ng / mL, and the IL-8 content was ≥2000 pg / mL, the patient was predicted to be at risk of preeclampsia.

[0008] Furthermore, the biological sample is blood.

[0009] Furthermore, the biological sample is plasma in blood.

[0010] Furthermore, the product is a reagent, a chip, a test strip or a test kit.

[0011] A second aspect of the present invention provides a kit, which includes the above-mentioned reagent for detecting the content of a marker in a biological sample.

[0012] Furthermore, the reagents include antibodies to Caspase 3, antibodies to BAX, antibodies to IL-8, standards for Caspase 3, standards for BAX, and standards for IL-8.

[0013] Furthermore, the kit further comprises a sample diluent and / or a detection buffer.

[0014] In summary, compared with the prior art, the present invention has the following advantages and effects: (1) The present invention proposes for the first time that Caspase3, BAX and IL-8 can be used as biomarkers for predicting or evaluating preeclampsia. The biomarkers provided by the present invention are reliable markers for predicting and screening out high-risk groups for preeclampsia, and can predict the occurrence of preeclampsia or assist in the diagnosis of preeclampsia before the onset of clinical symptoms. The experimental results of the present invention show that measuring the levels of Caspase3, BAX and IL-8 proteins at 12 to 24 weeks of gestation can predict the possibility of preeclampsia in pregnant patients, greatly improving the early detection rate of preeclampsia, providing doctors with earlier intervention and treatment opportunities, and facilitating closer prediction of the occurrence of complications in asymptomatic patients and early intervention to reduce maternal perinatal mortality.

[0015] (2) Reagents for detecting the content of markers Caspase3, BAX and IL-8 in biological samples can be prepared into products for predicting or assisting in the diagnosis of preeclampsia. The present invention discloses a kit that is easy to operate and easy to popularize in primary medical institutions and families, thereby expanding the screening scope of the preeclampsia population and reducing the missed diagnosis rate. Compared with traditional screening methods, the kit provided by the present invention has higher specificity and accuracy. Clinical trials have found that reagents for detecting the content of biomarkers can accurately predict the possibility of preeclampsia, and the test sample is plasma, which has the excellent characteristics of being non-invasive, simple, and highly accurate, and is easily accepted by patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a heat map of differentially expressed proteins (DEPs) in four protein groups from Olink proteomic sequencing of plasma and serum; Figures A, B, C, and D are cardiac metabolic group, inflammatory group, neurological group, and oncology group, respectively; each column represents a different sample, and the first row from top to bottom indicates that the patient type of the corresponding sample is Control (not suffering from eclampsia) or PE (suffering from preeclampsia), and the second row indicates that the corresponding sample is Plasma or Serum; each row represents a different protein, showing the expression of the protein in different samples, red indicates high expression, blue indicates low expression, and the darker the color, the higher the expression level; the tree diagram on the left indicates the similarity of proteins, and the shorter the branches, the more similar the expression patterns of these proteins in different samples.

[0017] Figure 2 Figure A is a heat map of the top 20 proteins with the greatest differences in plasma and serum between normal pregnancy controls and EOPE patients; Figure 2 Figure B shows the expression of BAX, CASP3 (Caspase3), DECR1, MAGED1 and EPCAM proteins.

[0018] Figure 3The protein levels of Caspase3 (CASP3), BAX and IL-8 (CXCL8) in plasma of pregnant women at 12-24 weeks of gestation; the biomarkers detected in Figures A, B and C are Caspase3, BAX and IL-8, respectively; NC is the group without preeclampsia, and PE is the group with preeclampsia; ** indicates p <0.01, * indicates p <0.05. DETAILED DESCRIPTION

[0019] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

[0020] In the description of the present invention, unless otherwise specified, all reagents used are commercially available and all methods used are conventional techniques in the art.

[0021] In some embodiments of the present invention, human Bcl-2 associated X protein (BAX) ELISA kit, human cysteine ​​protease-3 (caspase-3) ELISA kit and human interleukin 8 (IL-8 / CXCL8) ELISA kit were all purchased from Gilead Biotech Co., Ltd.

[0022] Preeclampsia (PE) is a pregnancy-specific disease. Once diagnosed, the only effective treatment is termination of pregnancy, which can lead to premature birth in about 1 / 3 of patients. Biological markers of preeclampsia are helpful to more closely predict the occurrence of complications in asymptomatic patients and intervene as early as possible to reduce maternal perinatal mortality. In previous studies, the inventor's research team found that compared with the normal pregnancy control group, the expression level of interleukin 8 (IL8) in the late pregnancy group with preeclampsia was significantly increased. Compared with the late pregnancy group with preeclampsia, it is of great clinical significance to predict the risk of preeclampsia earlier before the onset of clinical symptoms. It can predict and intervene in patients who may develop preeclampsia early. Therefore, it is urgent to develop a protein marker that can predict the risk of preeclampsia.

[0023] The present invention provides an application of a biomarker in predicting or assisting in the diagnosis of preeclampsia. Relying on a large number of clinical samples from the Guangzhou Maternal and Child Critical Care Center, the present invention uses micro-plasma Olink proteomics and clinical trials to verify that by detecting the protein content of cysteine ​​protease (Caspase3, CASP3‌), BAX and interleukin-8 (IL-8) in plasma, the risk of developing preeclampsia in pregnant women can be predicted non-invasively, accurately and effectively; the use of the kit provided by the present invention can predict or assist in the diagnosis of the occurrence of preeclampsia before the onset of clinical symptoms, thereby greatly improving the early detection rate of the disease, providing doctors with earlier intervention and treatment opportunities, and has good prospects for clinical application.

[0024] Example 1: Biomarker Discovery 1. Patient registration and data collection Between January 2017 and October 2022, patients were screened by inclusion and exclusion criteria, and eligible patients were enrolled and data were collected.

[0025] The screening criteria for patient registration are: pregnant women with singleton pregnancy at 12 to 24 weeks, diagnosed with normal pregnancy or preeclampsia. The detailed inclusion and exclusion criteria are shown in Tables 1 and 2:

[0026] Table 1 Inclusion and exclusion criteria for patients with normal pregnancy Table 2 Inclusion and exclusion criteria for patients with preeclampsia Collection of patient data: All medical records of eligible patients were reviewed and informed consent was obtained from all participating patients.

[0027] 2. Collection of serum and plasma and Olink proteomics sequencing Thirteen representative samples were selected from the collected samples, including four serum samples (two samples from normal pregnancy patients and two samples from preeclampsia patients) and nine plasma samples (four samples from normal pregnancy patients). Using microplasma Olink proteomics technology, 1536 proteins, including 384 cardiovascular metabolic proteomes, 384 inflammatory response proteomes, 384 neurology proteomes and 384 oncology proteomes, were sequenced.

[0028] 3. Olink proteomic sequencing analysis According to Olink proteomics Figure 1Based on the sequencing results, the present invention identified 158 differentially expressed proteins (DEPs) among 1536 proteins between the preeclampsia group and the control group, including 34 DEPs from the cardiac metabolism group, 40 DEPs from the inflammatory response group, 43 DEPs from the oncology group and 41 DEPs from the neurology group.

[0029] In order to find and verify more early pregnancy biomarkers, this paper combined proteins from all four proteomics research directions and focused on the top 20 proteins with the greatest differences ( Figure 2 Among these proteins, the present invention focuses on the apoptosis proteins BAX, CASP3 (Caspase3), DECR1 and MAGED1, and the epithelial protein EPCAM ( Figure 2 Figure B), which indicates that excessive apoptosis may predict the occurrence of preeclampsia in early pregnancy, and there are literature reports that patients with preeclampsia in late pregnancy have elevated apoptosis-related proteins, so the present invention focuses on the apoptosis proteins BAX and CASP3.

[0030] Example 2: Validation and application of biomarkers Plasma samples were collected from 10 pregnant women without preeclampsia and 11 pregnant women diagnosed with preeclampsia. The levels of Caspase3, BAX and IL-8 in plasma samples of 10 pregnant women without preeclampsia (NC group) and 11 pregnant women diagnosed with preeclampsia (PE group) were compared using human Bcl-2-associated X protein (BAX) ELISA kit containing BAX antibody, human cysteine ​​protease-3 (Caspase-3) ELISA kit containing Caspase-3 antibody and human interleukin 8 (IL-8 / CXCL8) ELISA kit containing IL-8 antibody (Table 3), and correlation analysis was performed ( Figure 3 ).

[0031] Table 3 Levels of Caspase3, BAX and IL-8 As can be seen from Table 3, the Caspase3 content in the peripheral blood plasma of patients with preeclampsia was ≥1000 U / L, the BAX content was ≥20 ng / mL, and the IL-8 content was ≥2000 pg / mL.

[0032] from Figure 3It can be seen that compared with the NC group, the expression of Caspase3, BAX and IL-8 in peripheral blood plasma of patients in the PE group was increased and statistically significant. When the Caspase3 content in peripheral blood plasma was ≥1000U / L, the BAX content was ≥20ng / mL and the IL-8 content was ≥2000pg / mL, the patient was at risk of preeclampsia.

[0033] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of a reagent for detecting the content of a marker in a biological sample in the preparation of a product for predicting or assisting in the diagnosis of preeclampsia, characterized in that: The biomarkers are Caspase3, BAX and IL-8.

2. The use according to claim 1, characterized in that: When the Caspase3 content in the biological sample was ≥1000 U / L, the BAX content was ≥20 ng / mL, and the IL-8 content was ≥2000 pg / mL, the patient was predicted to be at risk of preeclampsia.

3. The use according to claim 1, characterized in that: The biological sample is blood.

4. The use according to claim 3, characterized in that: The biological sample is plasma in blood.

5. The use according to claim 1, characterized in that: The product is a reagent, a chip, a test strip or a test kit.

6. A kit, characterized in that: The kit comprises the reagent for detecting the content of a marker in a biological sample as described in claim 1.

7. The kit according to claim 6, characterized in that The reagents include Caspase 3 antibody, BAX antibody, IL-8 antibody, Caspase 3 standard substance, BAX standard substance and IL-8 standard substance.

8. The kit according to claim 6, characterized in that The kit further comprises a sample diluent and / or a detection buffer.

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