Biomarker for auxiliary diagnosis of preeclampsia and application thereof

Abstract

The invention discloses a biological marker for auxiliary diagnosis of preeclampsia and application of the biological marker, and belongs to the technical field of biology. It is found for the first time that the EDIL3 protein or / and the O-fucose on the EDIL3 protein can change the migration, invasion and proliferation capacity of trophoblast cells, experiments prove that deletion of the O-fucose on the EDIL3 inhibits secretion of the EDIL3, deletion of the O-fucose on the EDIL3 inhibits migration, invasion, angiogenesis and proliferation capacity of the trophoblast cells, and the EDIL3 protein or / and the O-fucose on the EDIL3 protein can change the migration, invasion and proliferation capacity of the trophoblast cells. The deletion of the O-fucose of the EDIL3 leads to phenotypes of blood pressure rise, proteinuria, fetal rat intrauterine development limitation and placenta development deficiency of pregnant rats, and the EDIL3 protein or / and the O-fucose on the EDIL3 protein can be used as a biological marker for auxiliary diagnosis of preeclampsia, so that auxiliary diagnosis and clinical treatment of preeclampsia are facilitated, and the application prospect is very good.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a biological marker for assisting in the diagnosis of preeclampsia and an application thereof. Background Art

[0002] Preeclampsia is a systemic syndrome characterized by symptoms such as hypertension and proteinuria at ≥20 weeks of pregnancy. The basic pathological change of preeclampsia is systemic arteriolar vasospasm, which involves multiple organs and can cause adverse outcomes such as intrauterine growth restriction, placental abruption, and even miscarriage. At present, the pathogenesis of preeclampsia is generally believed to be the result of multiple factors. The main causes include abnormal activation of vascular endothelial cells, oxidative stress, abnormal trophoblast cell invasion, and impaired spiral artery remodeling. Among them, abnormal trophoblast cell invasion is considered to be an important link in its pathogenesis. Trophoblast cells are located in the outermost layer of mature embryos, mediating the recognition and adhesion between the embryo and the uterine wall, promoting embryo implantation and placental development, and establishing maternal-fetal circulation to ensure adequate nutrition and oxygen supply to the fetus. Insufficient invasion of trophoblast cells into the uterine environment often causes pregnancy-related diseases such as preeclampsia and intrauterine growth restriction.

[0003] Although many studies have been devoted to clarifying the etiology of preeclampsia, its specific pathogenesis has not yet been fully elucidated. Therefore, early prediction and clinical diagnosis of patients at risk of preeclampsia need to be further improved. To date, the diagnostic criteria for preeclampsia include clinical parameters and laboratory tests; the determination of angiogenic factors [pro-angiogenic vascular endothelial growth factor (VEGF) and placental growth factor (PlGF)] and anti-angiogenic factors [such as tyrosine kinase-1 (sFlt-1)] is still the main clinical detection method, as well as abnormal Doppler examination with relatively low predictive value. Therefore, it is very important to study new diagnostic methods for preeclampsia, which can be used for preventive intervention and clinical treatment.

[0004] Protein glycosylation is one of the important ways of post-translational modification of proteins. During pregnancy, sugar chains carry huge biological information and participate in sperm-egg recognition, embryonic development and implantation. Protein O-fucosylation is a highly selective reaction. The enzymes that catalyze protein O-fucosylation are O-fucosyltransferases (poFUTs), including poFUT1 and poFUT2. poFUT1 / 2 are located in the endoplasmic reticulum. PoFUT1 and poFUT2 can transfer the donor substrate GDP-Fucose to the hydroxyl groups of Ser and Thr residues of protein polypeptide chains containing epidermal growth factor-like repeats (EGF-like repeats) and TSRs repeats (thrombospondin type-1 repeats). At present, studies have reported that abnormal expression and site mutations of poFUT1 can cause embryonic development disorders and placental dysfunction. EDIL3 protein is an endogenous anti-inflammatory glycoprotein expressed and secreted by a variety of cells. Studies have shown that its domain contains a specific O-fucosylation modification site ( Figure 1 AC shows that literature 1, Schürpf T, et al. The RGD finger of Del-1 is a unique structural feature critical for integrin binding. FASEB J. 2012; 26 (8): 3412-3420. Literature 2, Schneider M, et al. Biological functions of fucose in mammals. Glycobiology. 2017; 27 (7): 601-618.). Studies have reported that O-fucose at this site is beneficial to maintaining the stability of the EGF2 domain on EDIL3. In addition, studies have shown that in breast cancer and liver cancer tissues, EDIL3 can promote the migration and invasion of cancer cells; at the same time, in the absence of exogenous growth factors, EDIL3 protein accumulates around new blood vessels in embryonic and tumor tissues, indicating that EDIL3 has an angiogenic effect; however, the role of EDIL3 in embryonic development and the role of O-fucose on EDIL3 in the function of trophoblast cells have not yet been clarified. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a biological marker for assisting the diagnosis of preeclampsia and its application.

[0006] The object of the present invention is to achieve the following:

[0007] The present invention provides a biological marker for assisting diagnosis of preeclampsia, wherein the biological marker is EDIL3 protein and / or O-fucose on the EDIL3 protein.

[0008] Furthermore, the O-fucose attachment site is Thr 88 of the EDIL3 protein.

[0009] The present invention also provides the use of the above-mentioned biological markers in the preparation of reagents or kits for auxiliary diagnosis of preeclampsia.

[0010] Furthermore, the detection methods of the reagent or kit include enzyme-linked immunosorbent assay, protein immunoblotting, immunoprecipitation, immunofluorescence, Transwell migration and invasion assay, matrix gel tube formation assay and EdU proliferation assay.

[0011] Furthermore, the pathological samples detected are serum samples and placental tissue samples.

[0012] Furthermore, the placental tissue includes chorionic trophoblast cells, syncytial trophoblast cells and extravillous trophoblast cells.

[0013] Furthermore, when the EDIL3 protein or / and the O-fucose synthesis on the EDIL3 protein is reduced, the proliferation, migration and invasion capabilities of trophoblastic cells are inhibited, leading to preeclampsia.

[0014] Furthermore, when the EDIL3 protein or / and the O-fucose synthesis on the EDIL3 protein is reduced, the angiogenesis ability of trophoblast cells is inhibited, leading to preeclampsia.

[0015] The beneficial effects of the present invention compared with the prior art are as follows:

[0016] The present invention discovers for the first time that EDIL3 protein or / and O-fucose on EDIL3 protein can change the ability of trophoblast cells to migrate, invade and proliferate. The present invention proves through experiments that the loss of O-fucose on EDIL3 inhibits the secretion of EDIL3, the loss of O-fucose on EDIL3 inhibits the migration, invasion, angiogenesis and proliferation ability of trophoblast cells, the loss of O-fucose on EDIL3 leads to the phenotype of increased blood pressure, proteinuria, restricted intrauterine growth of fetal mice and placental development defects in pregnant mice, and EDIL3 protein or / and O-fucose on EDIL3 protein can be used as a biological marker for auxiliary diagnosis of preeclampsia, which will be helpful for the diagnosis of preeclampsia and has very good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention, the drawings related to the embodiments are briefly introduced below.

[0018] Figure 1is the expression of EDIL3 protein in the serum of normal pregnant women and preeclampsia patients; Figure 1 In A, three epidermal growth factor-like repeat structures (EGF1-3) in the EDIL3 protein are marked, EGF2 is represented in cyan, and the O-fucose ligand in EGF2 is shown in orange; Figure 1 B is the sequence conservation analysis of the O-fucose modification site region of EDIL3 protein among species; Figure 1 C is a schematic diagram of the synthesis of O-fucose on EDIL3 protein catalyzed by poFUT1; Figure 1 D and Figure 1 E is the analysis of EDIL3 levels in the serum of normal pregnant women and preeclampsia patients by ELISA and Western blot, respectively; Figure 1 F, the expression of poFUT1 in the serum of normal pregnant women and preeclampsia patients was analyzed by ELISA; Figure 1 G shows the correlation analysis between the expression of poFUT1 and EDIL3 in the serum of normal pregnant women and patients with preeclampsia, ns=no significance, **p<0.01, ***p<0.001.

[0019] Figure 2 The results show the effect of O-fucose deficiency on the secretion of EDIL3 protein in trophoblast cells (HTR8 / SVneo); wherein, Figure 2 A and Figure 2 B is the analysis of EDIL3 mRNA level and protein expression level detected by q-PCR and Western blot, respectively; Figure 2 C is the expression level of secretory EDIL3 in the culture medium of trophoblast cells (HTR8 / SVneo) detected by ELISA; Figure 2 D shows the localization and expression of EDIL3 and O-fucose in cells by cell immunofluorescence staining, the scale bar represents 5 μm; Figure 2 E and Figure 2 F is the change of O-fucose on EDIL3 protein detected by co-immunoprecipitation Co-IP and Western blot; Figure 2 GH and Figure 2 IJ were detected by Western blot and ELISA respectively. WT and poFUT1 R240A The content of secreted EDIL3 in HTR8 / SVneo cells after plasmid expression, *p<0.05, **p<0.01, ***p<0.001.

[0020] Figure 3The results show that EDIL3 protein affects the migration, invasion, angiogenesis and proliferation of trophoblast cells through O-fucosylation modification. Figure 3 A is a Transwell experiment to detect the effect of EDIL3 protein on the migration and invasion ability of trophoblast cells HTR8 / SVneo; Figure 3 B. Western blot analysis of the effect of EDIL3 protein on the expression of proteins related to EMT behavior of trophoblast cells (E-cadherin, N-cadherin, Vimentin and Snail); Figure 3 C. Co-tube formation assay to detect the effect of EDIL3 protein on the angiogenesis ability of trophoblast cells HTR8 / SVneo, BF: white field; IF: fluorescent field, red arrow: HTR8 / SVneo, blue arrow: HUVEC; Figure 3 D is Western blot analysis of the effect of EDIL3 protein on the expression of angiogenesis-related proteins (CD31, CD34 and VE-cadherin); Figure 3 E The proliferation ability of trophoblast cells HTR8 / SVneo was evaluated by EdU proliferation assay; Figure 3 F The effect of EDIL3 protein on the expression of proliferation-related proteins (Cyclin D1, Cyclin E1 and PCNA) was analyzed by Western blot. The scale bar represents 50 μm. *p<0.05, **p<0.01, ***p<0.001.

[0021] Figure 4 In a mouse model, the loss of O-fucose in EDIL3 in the early placenta can induce abnormal placental development and preeclampsia-like phenotypes in pregnant mice. Figure 4 A is a schematic diagram of constructing a mouse model of preeclampsia; Figure 4 B is a pregnant mouse injected with Ad-Vector, Ad-EDIL3 WT 、Ad-EDIL3 T88A and Ad-sh-poFUT1 adenovirus at E7.5, 9.5, 11.5, 14.5, and 17.5; Figure 4 C is Ad-Vector, Ad-EDIL3 WT 、Ad-EDIL3 T88A and systolic blood pressure of pregnant mice in the Ad-sh-poFUT1 group at E7.5, 9.5, 11.5, 14.5, and 17.5; Figure 4 D. Detection of Ad-Vector and Ad-EDIL3 by BCA method WT 、Ad-EDIL3 T88A and 24h proteinuria of pregnant mice in the Ad-sh-poFUT1 group; Figure 4 E. Determination of Ad-Vector, Ad-EDIL3 by ELISA WT 、Ad-EDIL3 T88A Figures FH show the placenta weight, crown-rump length and body weight of the fetuses in each group at E18.5 after cesarean section; Figure I shows the sFLT-1 levels in pregnant mice from Ad-Vector, Ad-EDIL3 WT 、Ad-EDIL3 T88A Representative images of fetuses and uteri in the Ad-sh-poFUT1 and Ad-vector groups; Figure J shows fetal skeletons stained with Alcian blue (cartilage) and Alizarin red (bone) at E18.5; Figure K shows kidney tissue of E18.5 pregnant mice stained with H&E, with the scale representing 50 μm; Figure L shows kidney tissue of E18.5 pregnant mice stained with Ad-vector, Ad-EDIL3 WT 、Ad-EDIL3 T88A H&E staining of placental tissues in the FUT1 and Ad-sh-poFUT1 groups; JZ, junctional zone; LZ, labyrinthal zone; TGC, trophoblast giant cell; SpT, spongy trophoblast; GlyT, glycogenotrophoblast; CT, cytotrophoblast; MS, maternal sinusoid; the scale bar represents 50 μm; Figure M detected the expression of trophoblast-specific protein α (TPBPA) in placental tissue by immunohistochemistry staining, the scale bar represents 50 μm; Figure N detected the expression of CD34 in placental tissue by tissue immunofluorescence, FC: fetal capillaries; MBS: maternal blood space; LT: labyrinthal trophoblast; S-TGC: sinusoidal trophoblast giant cell, the scale bar represents 50 μm; Figure O detected the involvement of trophoblast cell infiltration in the process of placental angiogenesis by tissue immunofluorescence, *p<0.05, **p<0.01, ***p<0.001. DETAILED DESCRIPTION

[0023] The present invention is described in detail below with reference to the embodiments, but the embodiments of the present invention are not limited thereto. Obviously, the embodiments described below are only partial embodiments of the present invention. For those skilled in the art, other similar embodiments obtained without creative labor all fall within the protection scope of the present invention.

[0024] The cells, tissues and main reagents used in the examples are as follows:

[0025] 1. Cells: Human chorionic trophoblast cells (HTR8 / SVneo) were purchased from ATCC cell bank.

[0026] 2. Tissue: Human serum samples and placental tissue were obtained from the Laboratory Department of the Second Affiliated Hospital of Dalian Medical University and have passed the ethical application.

[0027] 3. Reagents:

[0028]

[0029]

[0030] Example 1: ELISA detection of EDIL3 and poFUT1 expression in serum of normal pregnant women and preeclampsia patients

[0031] 1. All reagents should be equilibrated at room temperature for at least 30 minutes before use;

[0032] 2. Dilution of the standard: first use the standard / sample diluent to make the standard EDIL3 protein lyophilized powder and the standard poFUT1 protein lyophilized powder to 150μL, then vortex mix for 30 seconds to obtain the standard stock solution. Take 5 clean EP tubes, add 150μL of the standard diluent to each tube, and mark them with 8ng / mL, 4ng / mL, 2ng / mL, 1ng / mL, and 0.5ng / mL respectively. Take 150μL of the standard stock solution and add it to the tube marked with 8ng / mL. After mixing, take out 150μL and add it to the next tube, and so on until the last tube. Zero well: directly add the standard / sample diluent;

[0033] 3. Dilution of biotin antigen: extract 1mL of biotin antigen diluent into concentrated biotin antigen, vortex mix for 15 seconds until the lyophilized powder is completely dissolved, then transfer all the liquid into the diluent bottle, vortex mix again for 30 seconds to obtain the biotin antigen working solution;

[0034] 4. Dilution of Avidin-HRP: Centrifuge at low speed (so that the concentrated solution sinks to the bottom of the tube), transfer all the concentrated Avidin-HRP into the corresponding diluent and vortex mix for 30 seconds to obtain the Avidin-HRP working solution;

[0035] 5. Serum samples: Let the blood coagulate naturally at room temperature for 10-20 minutes, and centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully, and if precipitation occurs during storage, centrifuge again;

[0036] 6. No sample is added to the blank wells. Only color developing reagents A, B and stop solution are added for zero adjustment. Standard wells: Add 50μL of diluted standard to each well, add 50μL of standard / sample diluent to the zero well, and then add 50μL of biotin antigen working solution (zero wells must be made and used as the last point of the standard curve). Sample wells: Add 50μL of sample (add directly, high concentration can be diluted 2-5 times with sample diluent), and then add 50μL of biotin antigen working solution;

[0037] 7. Shake gently, cover with sealing film, and incubate in a 37°C incubator for 30 minutes. Dilute the 25-fold concentrated washing solution with distilled water 25 times and set aside. First wash: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and discard it, repeat this 5 times, and pat dry. Add 50μL of avidin-HRP to the standard wells and sample wells, shake gently, cover with sealing film, and incubate in a 37°C incubator for 30 minutes. Second wash: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and discard it, repeat this 5 times, and pat dry;

[0038] 8. Color development: First add 50μL of color developer A to each well, then add 50μL of color developer B, gently shake and mix, and color development at 37℃ in the dark for 10 minutes. Termination: Add 50μL of stop solution to each well to terminate the reaction (the blue color immediately turns yellow). Determination: Zero the blank well and measure the absorbance (OD value) of each well in sequence at a wavelength of 450nm. The determination should be performed within 10 minutes after adding the stop solution. Calculation: Calculate the regression equation of the standard curve based on the concentration and OD value, use ELISAcalc for calculation, and use the logistic curve (four parameters) as the fitting model.

[0039] The results are as follows Figure 1 As shown in D, the results showed that the expression of EDIL3 in the serum of patients with preeclampsia was significantly decreased compared with that in the serum of normal pregnant women.

[0040] The results are as follows Figure 1 As shown in F, the results showed that the expression of O-fucosyltransferase (poFUT1) in the serum of preeclampsia patients was significantly decreased compared with that in the serum of normal pregnant women.

[0041] Example 2: Real-time fluorescence quantitative gene amplification (q-PCR) detection of EDIL3 levels in trophoblast cells HTR-8 / SVneo

[0042] 1. Construction of EDIL3 overexpression plasmid and EDIL3 mutant plasmid (T88A)

[0043] (1) Obtaining the target gene fragment: The coding region sequence of the EDIL3 (NM_005711.5) gene was downloaded from NCBI, and the target gene amplification primers were designed using Premier 5.0 software. The amplification system was shown in Table 1, and the reaction system was shown in Table 2. A 1% agarose gel was prepared. After amplification, agarose electrophoresis was performed at a voltage of 60 V for 30 min, and the amplified fragments were cut and recovered.

[0044] Table 1 Amplification system

[0045]

[0046] Table 2 Reaction system

[0047]

[0048]

[0049] (2) The EDIL3 DNA fragment and the empty plasmid pcDNA3.1 were digested with enzymes respectively. The enzyme digestion system is shown in Table 3 below:

[0050] Table 3 Enzyme digestion system

[0051] Bam HI XbarⅠ 10Xbuffer <![CDATA[PCR ddH2O]]> Total pcDNA3.1 plasmid (μL) 1.0 1.0 2.0 14.0 20 EDIL3 DNA fragment 1.0 1.0 3.5 0 35

[0052] (3) Place the prepared enzyme digestion reaction system mixed solution in a 37°C incubator for 24 hours.

[0053] (4) Prepare 1% agarose gel and perform electrophoresis after enzyme digestion at 60 V for 30 min.

[0054] (5) After the electrophoresis, take a picture of the gel and then recover the gel.

[0055] (6) The EDIL3 amplified fragment after restriction digestion was ligated with the empty plasmid pcDNA3.1.

[0056] Table 4 Ligation reaction system

[0057] Reagents System (μL) T4 DNA ligase 1.0 10Xbuffer 1.5 Snippet 9.5 Plasmids 3.0 Total 15

[0058] (7) After the mixture is prepared, place it in a 4°C refrigerator and leave it to connect overnight (24 hours).

[0059] (8) Take a small amount of the ligation product for enzyme digestion identification.

[0060] Table 5 Enzyme digestion identification system

[0061] Reagents System (μL) Bam HI 1.0 XbarⅠ 1.0 10Xbuffer 2.0 Connect products 4.0 <![CDATA[PCR ddH2O]]> 12 Total 20

[0062] (9) The enzyme digestion mixture was placed in a 37°C incubator for 2 h, and 1% agarose gel was prepared at the same time and allowed to stand at room temperature for 45 min.

[0063] (10) After the enzyme digestion, perform gel electrophoresis at 60 V for 30 min and take a photo of the results.

[0064] (11) The recombinant pcDNA3.1-EDIL3 overexpression plasmid identified by the above enzyme digestion was transformed, cloned and amplified, and then the plasmid was extracted using a plasmid extraction kit and transfected into trophoblast cells to obtain recombinant cells.

[0065] 2. Total RNA Extraction

[0066] (1) Take out the recombinant trophoblast cells to be treated from the incubator, discard the culture medium, wash them 1-2 times with autoclaved PBS, and discard the PBS;

[0067] (2) Add 500 μL Trizol to the culture dish and pipette the cells with an autoclaved pipette tip;

[0068] (3) Transfer the liquid after blowing into a new autoclaved EP tube, add 100 μL of chloroform, vortex for 15-20 seconds, and let it stand on ice for 5 minutes;

[0069] (4) centrifugation at 12000 rpm for 10 min in a low-temperature ultracentrifuge;

[0070] (5) Take a new autoclaved EP tube, add 250 μL of isopropanol, carefully transfer the upper clear liquid after centrifugation to the EP tube, invert to mix, and store at -20°C overnight;

[0071] (6) The next day, centrifuge the mixture at 12,000 rpm for 15 min in a low-temperature ultracentrifuge, carefully discard the supernatant, and retain the white precipitate;

[0072] (7) Add 1 mL of 70% ethanol to each EP tube and invert to mix thoroughly to remove the organic solvent;

[0073] (8) Centrifuge at 12,000 rpm for 10 min in a low-temperature ultracentrifuge, discard the supernatant, and mark the location of the precipitate with a marker pen. Then, use autoclaved filter paper to carefully absorb the liquid in the EP tube and leave it at room temperature for several minutes until the RNA precipitate becomes transparent.

[0074] (9) Add 10 μL of DEPC water to the EP tube and gently blow the marked area to fully dissolve the RNA. Measure the concentration and purity of the RNA using a micro-nucleic acid quantifier and store at -80°C.

[0075] 3. Reverse transcription to generate cDNA

[0076] (1) Take out and thaw RNA in advance. Place 1 μg of RNA template in an autoclaved EP tube, fill it up to 7 μL with DEPC water, mix gently, and place on ice.

[0077] (2) The reaction system is added in the order shown in the table below:

[0078] Table 6 Reverse transcription system

[0079] composition volume 2×TS Reaction Mix 10μL Transcript RT / RI Enzyme Mix 1μL gDNA Remover 1μL Random Primer (EDIL3 Primer) 1μL <![CDATA[RNA+DEPC H2O]]> 7μL Overall system 20μL

[0080] The reaction conditions were as follows: 42 °C for 15 min, 85 °C for 5 s, and 4 °C for 10 min.

[0081] (3) Obtain cDNA.

[0082] 4. PCR reaction

[0083] The reaction system for PCR amplification of cDNA is shown in the following table.

[0084] Table 7 PCR reaction system

[0085]

[0086]

[0087] The reaction conditions were as follows: pre-denaturation at 94°C for 30 s, denaturation at 94°C for 5 s, annealing at 60°C for 30 s, and amplification for 40 cycles.

[0088] Table 8 PCR primer sequences

[0089]

[0090] The results are as follows Figure 2 As shown in A, the results showed that the mRNA level of EDIL3 in trophoblast cells was increased after transfection of EDIL3 overexpression plasmid and mutant plasmid (T88A).

[0091] Example 3: Western blot detection of EDIL3 expression in placental tissue serum and cells

[0092] 1. Extraction of Total Serum Protein

[0093] After the collected blood is naturally coagulated, the coagulated blood sample is centrifuged (about 2000-3000g, 10-15 minutes) to separate the serum from the clot. The upper serum is gently aspirated, and the total protein in the serum sample is quantified using a protein concentration determination method (such as Bradford method, BCA method, etc.), and the collected serum is stored at -80°C.

[0094] 2. Extraction of Total Cell Protein

[0095] (1) Each group of trophoblast cells HTR-8 / SVneo in Example 2 was taken out of the incubator, the supernatant was discarded, and the cells were washed twice with PBS;

[0096] (2) Then, according to the amount of cells, add appropriate cell lysis buffer RIPA and protease inhibitors (100:1) and lyse on ice for 10 minutes;

[0097] (3) Use egg white scraper to scrape the cells, place them in a 1.5 mL EP tube, and store them at -80°C for later use.

[0098] 3. SDS-PAGE electrophoresis

[0099] (1) Glue preparation: Align the glass plates and place them on the gel preparation rack, screwing them to position 1.0 and ensuring that there is no leakage. Prepare 10% separation gel and 6% concentrated gel and shake thoroughly. First, slowly add the separation gel along the inner surface of the glass plate, then seal with anhydrous ethanol and solidify at room temperature for 30 minutes. Discard the ethanol, wipe off the residual ethanol, then add the concentrated gel, insert the comb, and solidify at room temperature for 30 minutes. After the colloid solidifies, carefully remove the comb and place it in the electrophoresis solution for later use.

[0100] (2) Sample loading: Calculate the sample loading volume based on the protein quantification results, and the loading volume is 25-40 μg;

[0101] (3) Electrophoresis: Keep the electrophoresis in an ice bath and use a voltage of 100 V. After the protein sample enters the separation gel from the stacking gel, change the voltage to 120 V, observe the position of the protein Maker in real time, and terminate the electrophoresis.

[0102] (4) Transfer: Carefully disassemble the glass plate and cut the gel block according to the target molecular weight. Cut the filter paper and cellulose acetate membrane (NC membrane) according to the size of the cut gel. Place the gel block in the order of sponge pad, filter paper, membrane, gel, filter paper, and sponge pad, taking care not to create bubbles, into the transfer tank filled with transfer solution. Transfer the gel at a voltage of 150 V and a current of 250 mA for an appropriate time. After the transfer is completed, stain with Ponceau red to confirm the transfer status.

[0103] (5) Primary antibody incubation: prepare primary antibodies (EDIL3 1:1000; His 1:1000; GAPDH 1:2000), incubate overnight at 4°C, remove from the membrane the next day, and wash the membrane four times with TBST, each time for 10 min;

[0104] (6) Secondary antibody incubation: Incubate with HRP-labeled secondary antibody (goat anti-rabbit IgG 1:3000) at room temperature for 1 h. Wash the membrane 4 times with TBST, 10 min each time.

[0105] (7) Luminescent development: Mix luminescent solution A and solution B at a ratio of 1:1 and place in a dark place. Wipe the NC membrane dry with filter paper, cover with luminescent solution, place on Bio-Rad imager for color development, and analyze the bands with Image Lab.

[0106] The results are as follows Figure 2 As shown in B, the expression of EDIL3 protein in trophoblast cells increased after transfection of EDIL3 overexpression plasmid and mutant plasmid (T88A).

[0107] Example 4: Immunoprecipitation (IP) to detect O-fucose on EDIL3

[0108] 1. Alkyne-labeled O-fucose

[0109] (1) Prepare the medium containing alkyne-labeled O-fucose (25-50 μM) in advance, add the trophoblast cells with different treatments and incubate them in a 37°C incubator for 24-72 h;

[0110] (2) Remove the cells, discard the culture medium, and wash with PBS three times, 5 min each time;

[0111] (3) Add 200 μL of cell lysis buffer to the culture dish, repeatedly pipette and lyse on ice for 15 min, scrape the cell protein sample and place it in a 1.5 mL EP tube;

[0112] (4) Ultrasonicate the protein sample for 1-2 seconds;

[0113] (5) Centrifuge at 18,000 g for 5 min at 4°C, transfer the supernatant to a new EP tube to remove cell debris, determine the protein concentration, and store at -20°C;

[0114] Magnetic bead pretreatment: 50 μL of magnetic beads were placed in a 1.5 mL EP tube, 200 μL of Lysis Buffer was added to resuspend the magnetic beads, and detection antibodies (His and EDIL3) were added. The mixture was fully mixed and rolled on a shaker for 10 min to allow the magnetic beads and antibodies to fully bind to form a magnetic column antibody complex.

[0115] Discard the supernatant, wash twice with 200 μL Lysis Buffer, and discard the supernatant. Mix the total protein obtained in step (1) with the magnetic bead antibody complex obtained in step (3), gently blow and mix with a pipette, and then place on a shaker and incubate overnight;

[0116] On the next day, place the EP tube on a magnetic rack and collect the supernatant in the EP tube;

[0117] The magnetic beads-antigen-antibody complex was washed three times with 200 μL Lysis Buffer, the supernatant was discarded, and the protein on the magnetic beads was digested with trypsin and collected in an EP tube.

[0118] 2. Interaction between azide compounds and alkynes

[0119] (1) Add each component according to the system in the table below to a total volume of 160 μL and vortex for 5 seconds.

[0120] Table 9 Click iT reaction system

[0121]

[0122] (2) Add 10 μL copper(II) sulfate (CuSO4) (Component B) and shake for 5 seconds.

[0123] (3) Add 10 μL of Component C, shake for 5 seconds, and let stand for 2-3 minutes.

[0124] (4) Add 20 μL of Component D and shake for 5 seconds. The liquid turns bright orange. Continue shaking for 25 minutes.

[0125] (5) Add 600 μL methanol, mix well, and shake for 5 seconds.

[0126] (6) Add 150 μL of chloroform and shake for 5 seconds.

[0127] (7) Add 400 μL ddH2O and shake for 5 seconds.

[0128] (8) Centrifuge at 18,000 g for 5 min at 4°C and transfer the supernatant to another EP tube.

[0129] (9) Add 450 μL methanol, mix well, and shake for 5 seconds. Centrifuge at 18,000 g for 5 minutes at 4°C and discard the supernatant. Repeat the wash once.

[0130] (10) Allow to dry naturally at room temperature, add Loading Buffer and boil for 10 min.

[0131] (11) Load samples for Western blot experiment.

[0132] The results are as follows Figure 2 As shown in EF, poFUT1 can catalyze the O-fucosylation modification at Thr 88 of EDIL3 protein.

[0133] Example 5: Transwell migration and invasion assay

[0134] 1. Laying glue in the chamber: melt matrigel in a 4°C refrigerator, dilute matrigel with serum-free medium DMEM / F-12 at a ratio of 1:9, gently pipette to mix, add 50 μL matrigel to the Transwell chamber, try not to create bubbles, and place the chamber in a 24-well plate in a 37°C incubator.

[0135] 2. Cell counting: Take out the cells to be treated from the incubator, digest them with trypsin, centrifuge at 800 rpm for 4 min, resuspend them in serum-free medium, count them 3 times with a counting plate, and adjust the cell concentration to 2x10 5 Pieces / mL;

[0136] 3. Add 700 μL of DMEM / F-12 medium containing 10% FBS to the 24-well plate and gently place it into the Transwell chamber. Pipette 200 μL of cell suspension from the cell suspension and add it to the upper chamber and place it in a 37°C incubator to continue culturing for several hours;

[0137] 4. Crystal violet staining: remove the chamber and rinse it lightly with PBS; fix it with methanol for 15 minutes and rinse it twice with PBS, each time for 5 minutes; stain it with 0.5% crystal violet solution for 15 minutes and rinse it twice with PBS, each time for 5 minutes; use a cotton swab to gently wipe off the cells on the inside of the chamber; randomly select 3 different fields of view under an inverted microscope to take pictures and count the number of cells; repeat the experiment three times and calculate the results.

[0138] The results are as follows Figure 3 As shown in A, it shows that transfection with EDIL3 overexpression plasmid can promote the migration and invasion ability of trophoblast cells, while transfection with EDIL3 mutant plasmid (T88A) will inhibit the migration and invasion ability of trophoblast cells compared with the overexpression group.

[0139] Example 6: Matrigel tube formation experiment

[0140] 1. Spreading gel: Melt matrigel in a 4℃ refrigerator, spread matrigel into a 96-well plate with a pre-cooled pipette tip, 50μL matrigel per well, try not to create bubbles, and culture in a 37℃ incubator for 1-2h.

[0141] 2. Take out the cells to be treated from the incubator, digest them with trypsin, centrifuge at 800 rpm for 4 min, resuspend them in serum-free medium, count them 3 times with a counting plate, and adjust the cell concentration to 5×10 4 The cells were placed on the prepared matrigel, and the formation of tubules was observed under a microscope and photographed at 6h and 12h.

[0142] The results are as follows Figure 3 As shown in C, it shows that transfection with EDIL3 overexpression plasmid can promote the angiogenesis ability of trophoblast cells, while transfection with EDIL3 mutant plasmid (T88A) will inhibit the angiogenesis ability of trophoblast cells compared with the overexpression group.

[0143] Example 7: EdU proliferation assay

[0144] 1. Cell culture: Take trophoblast cells in logarithmic growth phase, 5×10 3 Cells were seeded in 96-well plates.

[0145] 2. EdU labeling: After the cells adhere to the wall, dilute the EdU solution with fresh DMEM / F-12 complete medium at a ratio of 1000:1 to prepare 50μM EdU medium, add 100μL to each well and incubate in a 37℃ incubator for 2 hours, then discard the medium. Wash the cells twice with PBS, 5 minutes each time.

[0146] 3. Cell fixation: Add 50 μL 4% paraformaldehyde to each well and incubate at room temperature for 30 min, then discard the fixative; add 50 μL 2 mg / mL glycine solution to each well and incubate on a shaker for 5 min; discard the glycine solution, add PBS and wash on a shaker for 5 min; discard PBS, add 100 μL 0.1% Triton X-100 and incubate on a shaker for 10 min; wash once with PBS for 5 min.

[0147] 4. Apollo staining: Add 100 μL of 1× Apollo staining reaction solution to each well and incubate on a shaker at room temperature in the dark for 30 min; discard the staining solution, add 100 μL of 0.1% Triton X-100 to each well, wash on a shaker for 3 times, each time for 10 min, and discard the permeabilization agent.

[0148] 5. Live cell labeling: Prepare 1×Hoechest33342 reaction solution with deionized water at a ratio of 100:1, add 100 μL to each well, incubate on a shaker at room temperature and away from light for 30 minutes, then discard the reaction staining solution, add 100 μL PBS to wash twice, and observe and take pictures under an inverted microscope.

[0149] The results are as follows Figure 3 As shown in E, it shows that transfection with EDIL3 overexpression plasmid can promote the proliferation ability of trophoblast cells, while transfection with EDIL3 mutant plasmid (T88A) will inhibit the proliferation ability of trophoblast cells compared with the overexpression group.

[0150] Example 8: Animal Experiment

[0151] 1. Select clean-grade adult KM mice, the room temperature is 20-25℃, the humidity is 40-70%, the light is moderate, the environment is clean and quiet, and they can freely eat SPF-grade granular feed and drinking water.

[0152] 2. Confirmation of pregnancy: Every evening at 7:00, the females and males were put together in a 2:1 cage ratio. The vaginal plug was observed at 6:00 the next morning. If a vaginal plug was seen, it was recorded as pregnancy day 0.5 (E0.5).

[0153] 3. Experimental groups:

[0154] The green fluorescent (GFP)-labeled mouse EDIL3WT overexpression, mouse EDIL3T88A and mouse poFUT1 shRNA adenovirus used in the present invention were constructed and the virus titer was determined by Zhenjiang abm Biological Co., Ltd.; 40 pregnant mice were randomly divided into 4 groups, Ad-Vector pregnant mouse control group (n=10), Ad-EDIL3WT pregnant mouse experimental group (n=10), Ad-EDIL3T88A pregnant mouse experimental group (n=10), Ad-sh-poFUT1 pregnant mouse experimental group (n=10). Adenovirus (1×109 PFU, 100 μL) was injected into the tail vein at E 8.5. The mice were killed at E18.5 and the fetuses were removed by cesarean section.

[0155] 4. Mouse tail vein injection steps:

[0156] (1) Fix the mouse and expose the tail vein;

[0157] (2) Disinfect the mouse tail skin with alcohol to make the dilation of the tail vein clear;

[0158] (3) Select a clear blood vessel at the distal end of the tail vein for needle insertion. After aspirating blood, slowly inject 100 μL of adenovirus (1×109 PFU) in the syringe into the vein within 1 min. During the administration, pay attention to the condition of the mice.

[0159] (4) Remove the syringe and apply pressure with a cotton swab to stop bleeding for 3 minutes; observe the condition of the mouse for 10 minutes.

[0160] The blood pressure and body weight of pregnant mice were monitored using the tail-cuff method starting from E7.5 and measured every 2 days (E7.5, E9.5, E11.5, E13.5, E15.5, and E17.5).

[0161] Place the mice in a standard metabolic cage, install a filter screen to filter the feces, and collect the mouse urine in a 5mL centrifuge tube. Fasting, free water intake, collect 24h fasting urine and record the urine volume, take the supernatant and directly determine the urine total protein content by BCA method.

[0162] Collection of tissue specimens

[0163] (1) At E18.5, the mice were anesthetized by intraperitoneal injection of 10% chloral hydrate solution, and the fetuses were removed by cesarean section. The fetal weight, number of fetuses, and wet weight of the placenta were recorded. The appearance of the fetuses was observed for gross malformations and intrauterine embryo resorption.

[0164] (2) The kidneys and placentas of pregnant mice were collected, fixed with 4% neutral formaldehyde for 24 h, embedded in paraffin, and subjected to pathological section examination.

[0165] 5. HE staining

[0166] (1) Slice the slices and place them in xylene I for 10 min; xylene II for 10 min; anhydrous ethanol I for 5 min; anhydrous ethanol II for 5 min; 95% alcohol for 5 min; 90% alcohol for 5 min; 80% alcohol for 5 min; 70% alcohol for 5 min; wash with running water;

[0167] (2) Hematoxylin staining of cell nuclei: sections were stained with hematoxylin for 3-8 min and then washed with running water;

[0168] (3) Eosin staining of cytoplasm: Slice into eosin staining solution for 1-3 min and then rinse with running water;

[0169] (4) Dehydration and sealing: Dehydrate the sections in 95% alcohol I for 5 min; 95% alcohol II for 5 min; anhydrous ethanol I for 5 min; anhydrous ethanol II for 5 min; xylene I for 5 min; and xylene II for 5 min until they are transparent. Remove the sections from the xylene and air dry them, then seal the sections with a neutral resin. Observe and photograph them under a microscope for analysis.

[0170] 6. Alecine Blue & Alizarin Red Staining

[0171] (1) E18.5 fetal mice were skinned and defatted and then fixed with 95% ethanol for 5 days;

[0172] (2) Isolation and fixation of skeleton: acetone treatment for 2-7 days;

[0173] (3) Staining: Use forceps to place the fetus into a sample bottle containing 40 ml of Alcian blue-alizarin red dye solution and stain for 4 hours. After 2-3 days, observe whether the paws and tongue are red. While staining, observe whether the whole body is blue. If it starts to turn blue, take it out and wash it with pure water. Then, put it in 1% KOH for 2 days to make it transparent (specifically, the skeleton is mainly transparent).

[0174] (4) Transparency and photography: The fetuses were removed and placed in 20% glycerol for one week until the skeleton and muscles were mainly clear. Finally, they were transferred to 50% glycerol and photographed with a camera.

[0175] (5) Storage: The samples were stored in 50% glycerol.

[0176] 7. Immunohistochemical Staining

[0177] (1) Paraffin sections were baked at 65℃ for 1-2h and then placed in: xylene I for 10min, then xylene II for 10min, 100% ethanol I for 5min, 100% ethanol II for 5min, 95% ethanol for 5min, 85% ethanol for 5min, and 70% ethanol for 5min; then rinsed with slow water flow for 10min, not directly flushing the sections. Rinse with PBS 3 times, 5min each time.

[0178] (2) Antigen repair: Place the sections in antigen repair buffer and repair them in a microwave oven for 20 minutes. Take them out and cool them naturally to room temperature, then rinse them with PBS three times, 5 minutes each time.

[0179] (3) Tissue sections were blocked with immunostaining blocking solution at room temperature for 1 h. The blocking solution was discarded, excess liquid was wiped off with filter paper, and primary antibody solution (CD34 1:100; CK7 1:100; α-SMA 1:100) was evenly added to the tissue sections and incubated overnight at 4°C.

[0180] (4) Rewarm for 1 hour, recover the primary antibody solution, wash with PBS three times, 10 minutes each time, wipe dry, add the prepared secondary antibody evenly, and incubate at room temperature for 1 hour;

[0181] (5) Wash with PBS three times, 5 min each time, and stain the nucleus with DAPI for 20 min; wash with PBS twice, 5 min each time, add anti-fluorescence quenching sealing medium, and take pictures under a fluorescence microscope.

[0182] The results are as follows Figure 4 As shown in the figure, we detected the indicators closely related to preeclampsia, including pregnant weight, pregnant blood pressure, placental weight, fetal size, fetal weight, 24h proteinuria and expression of soluble FMS-like tyrosine kinase 1 (sFLT-1). WT Compared with the EDIL3 group T88A Deficiency of poFUT1 and EDIL3 leads to preeclampsia-like symptoms, including weight loss, increased systolic blood pressure, proteinuria, and increased sFLT-1 levels in pregnant mice. T88A Pregnancy outcomes in sh-poFUT1 and sh-poFUT1 include placental malformation, fetal growth restriction, and early embryo resorption, as evidenced by reduced placental weight, lower crown-rump length (CRL), and fetal weight ( Figure 4 BI). Studies have reported that EDIL3 can inhibit osteoclastogenesis and inhibit inflammatory bone loss. Alcian blue and alizarin red staining of E18.5 fetal mouse bones, compared with Ad-Vector and Ad-EDIL3 WT Compared with the group, although Ad-EDIL3 T88A The fetal growth of the treated mice was significantly restricted due to the defects of placental development, but Ad-EDIL3 T88A However, Ad-sh-poFUT1 treated fetuses had shorter skeletal elements, lacked physiological curvature of the dorsal spine, had a reduced thorax, and had severe delays in endochondral bone formation in the cartilage of the occipital region, limbs, thorax, humerus, and femur, suggesting that the lack of O-fucose can delay skeletal maturation in fetal mice ( Figure 4 J) HE staining showed that Ad-EDIL3 T88AAfter treatment, the open area of ​​glomerular capillaries in the renal tissue of the mother mice decreased, and the glomeruli shrank and became smaller. At E18.5, the glomeruli of the mother mice treated with Ad-sh-poFUT1 were ischemic and dilated, the glomerular space was narrowed, and the endothelial cells were swollen ( Figure 4 K) H&E staining of the placenta showed that, in addition, compared with the Ad-Vector group and Ad-EDIL3 WT Compared with the group, Ad-EDIL3 T88A Treatment with Ad-sh-poFUT1 significantly changed the structure and morphology of the placental labyrinth trophoblast, and the area of ​​the labyrinth responsible for the exchange of placental tissue was severely reduced ( Figure 4 L). Placental histology and pathology suggest dysfunction of the labyrinthal trophoblast cells, manifested by nuclear condensation, nuclear fragmentation, nuclear dissolution, and decreased chromatin basophilia ( Figure 4 L). To determine whether EDIL3 is required for the development of spongiotrophoblast cells and glycotrophoblast cells in the junctional zone of placental tissue, the expression of trophoblast-specific protein α (TPBPA) in E18.5 placentas was detected by immunohistochemistry. TPBPA was expressed in spongiotrophoblast cells and also in glycotrophoblast cells in the junctional zone. WT Compared with the group, Ad-EDIL3 T88A The expression of TPBPA was reduced in the placenta tissue of Ad-sh-poFUT1 and Ad-sh-poFUT1. In addition, although TPBPA expression was detectable in the labyrinth layer of sh-poFUT1, it was almost absent in the junctional zone. These results indicate that EDIL3 is required for the development of the spongiotrophoblast subset marked by TPBPA in the junctional zone. Figure 4 M) Tissue immunofluorescence showed that compared with the control group and Ad-EDIL3 WT Compared with the group, Ad-EDIL3 T88A The number of placental neovascularization and CD34 expression decreased after treatment with Ad-sh-poFUT1 ( Figure 4 N). Figure O shows that trophoblast cell infiltration participates in spiral artery remodeling during placental angiogenesis by tissue immunofluorescence, and α-smooth muscle actin (α-SMA)-positive vascular smooth muscle cells (VSMCs) are displaced, indicating that spiral artery remodeling is normal. EDIL3 WT The vascular morphology of the group was similar to that of the control group, but at E18.5, EDIL3 T88A The vascular smooth muscle lining of the sh-poFUT1 group was thickened and the vascular morphology was narrowed. The scale bar represents 50 μm. *p<0.05, **p<0.01, ***p<0.001.

[0183] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A biological marker for assisting the diagnosis of preeclampsia, characterized in that: The biological marker is EDIL3 protein and / or O-fucose on EDIL3 protein.

2. The biomarker according to claim 1, characterized in that The O-fucose attachment site is Thr 88 of the EDIL3 protein.

3. Use of the biological marker described in claim 1 or 2 in the preparation of a reagent or kit for auxiliary diagnosis of preeclampsia.

4. The use according to claim 3, characterized in that: The detection methods of the reagent or kit include enzyme-linked immunosorbent assay, protein immunoblotting, immunoprecipitation, immunofluorescence, Transwell migration and invasion assay, matrix gel tube formation assay and EdU proliferation assay.

5. The use according to claim 3, characterized in that: The pathological samples tested were serum samples and placental tissue samples.

6. The use according to claim 5, characterized in that: The placental tissue includes chorionic trophoblast cells, syncytial trophoblast cells and extravillous trophoblast cells.

7. The use according to claim 3, characterized in that: When the EDIL3 protein or / and the O-fucose synthesis on the EDIL3 protein is reduced, the proliferation, migration and invasion abilities of trophoblast cells are inhibited, leading to preeclampsia.

8. The use according to claim 3, characterized in that: When EDIL3 protein or / and O-fucose synthesis on EDIL3 protein is reduced, the angiogenesis ability of trophoblast cells is inhibited, leading to preeclampsia.

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