1. A biological marker for predicting preeclampsia, wherein the biological marker is Gal α 1,3GalNAc or/and α 1, 3N-acetylgalactosaminyltransferase.

2. The biomarker of claim 1, wherein the Gal α 1,3GalNAc is detected by a reagent comprising soybean lectin.

3. The biological marker according to claim 2, wherein said pathological sample to be tested is placental tissue.

4. The biological marker of claim 3, wherein said placental tissue comprises villous cytotrophoblast cells, syncytiotrophoblast cells, and extravillous trophoblast cells.

5. The biomarker of claim 4, wherein the detected pathological sample is extravillous trophoblast cells.

6. The biomarker of any of claims 1 to 5, wherein the detection method comprises immunohistochemistry, lectin blotting, western immunoblotting, Transwell migration invasion assay, matrigel tube assay, and EdU proliferation assay.

7. Use of Gal α 1,3GalNAc or/and α 1, 3N-acetylgalactosaminyltransferase according to claim 1 in the preparation of a diagnostic reagent or diagnostic kit for the prediction of preeclampsia.

Background

Preeclampsia is a systemic syndrome which is characterized in that symptoms such as hypertension, proteinuria and the like appear in more than or equal to 20 weeks of gestation; the basic pathological changes in preeclampsia are systemic arteriolar vasospasm, involving multiple organs, often resulting in the adverse consequences of restricted intrauterine growth, premature placental stripping, and even abortion. The major causes of preeclampsia include: endothelial cell activation, intravascular inflammation, oxidative stress, abnormal trophoblast invasion, damaged spiral artery recasting, and the like; at present, the pathogenesis of preeclampsia is generally considered to be the result of the combined action of multiple factors, wherein abnormal invasion of trophoblast cells is considered to be the central link of the pathogenesis of the preeclampsia. The trophoblast cells are positioned on the outermost layer of a mature embryo, mediate recognition between the embryo and the uterine wall, and promote embryo implantation and placenta development. Trophoblast cells have two biological functions, secretion and migration invasion. Trophoblast cells include Choriotrophoblast (CTB) and Syncytrophoblast (STB), where CTB can differentiate into extravillous trophoblast cells with high invasive potential (EVT), EVT can invade the endometrium and maternal spiral arteries, participate in embryo implantation, placenta establishment, and placental vascular remodeling, establishing the maternal-fetal circulation to ensure adequate nutrition and oxygen supply to the fetus. Insufficient EVT invasion into the uterine environment often causes pregnancy related diseases such as preeclampsia, fetal intrauterine growth restriction, and the like.

Although much research is currently being conducted to elucidate the cause of preeclampsia and predict the disease before symptoms of preeclampsia appear, the pathogenesis of preeclampsia is unclear. Several predictive markers have been studied, but according to the WHO definition none have been selected as predictive or diagnostic markers for PE. Therefore, further research is necessary. Whereas to date, diagnostic criteria for PE include clinical parameters and laboratory examinations, 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) ] remain the most prominent detection methods on the market, as well as abnormal doppler with relatively low predictive value. Therefore, the study of preeclampsia diagnostic biological markers is of particular importance.

Disclosure of Invention

In view of the above, the present invention provides a biological marker for predicting preeclampsia and application thereof. Glycosylation of proteins is one of the important modes of post-translational modification of proteins, and is involved in various processes such as embryo implantation and ontogeny. In the process of pregnancy, the sugar chain carries huge biological information to participate in the processes of sperm and egg recognition, embryo development, implantation and the like. The invention discovers for the first time that the expression of Gal alpha 1,3GalNAc (glycosidic bond oligosaccharide) in preeclampsia placental tissues is obviously improved compared with normal gestational tissues, and Gal alpha 1,3GalNAc can be catalytically synthesized by alpha 1, 3N-acetylgalactosamine aminotransferase (GTA), so that Gal alpha 1,3GalNAc and GTA are used as biological markers for diagnosing preeclampsia, and the influence of the biological markers on the migration invasion, the placental angiogenesis and proliferation capacity of trophoblast cells is proved at the same time, thereby being beneficial to the prediction and diagnosis of preeclampsia, deeply understanding the embryo implantation mechanism and providing a new theoretical basis for the clinical treatment of preeclampsia with sugar as a target.

The purpose of the invention is realized by the following modes:

the invention provides a biological marker for predicting preeclampsia, wherein the biological marker is Gal alpha 1,3GalNAc or/and alpha 1, 3N-acetylgalactosamine aminotransferase.

Further, the Gal α 1,3GalNAc is specifically recognized by Soybean lectin (SBA) and detected by a reagent containing Soybean lectin.

Further, the detected pathological sample is placenta tissue.

Further, the placental tissue includes Choriotrophoblast (CTB), Syncytiotrophoblast (STB) and extravillous trophoblast (EVT).

Further, the pathological sample to be tested is extravillous trophoblast cells (EVT).

Further, the detection method comprises Immunohistochemistry (IHC), Lectin blot (Lectin blot), Western blot (Western blot), Transwell migration invasion assay, matrigel tube-forming assay and EdU proliferation assay.

In another aspect, the invention provides the use of Gal α 1,3GalNAc or/and α 1, 3N-acetylgalactosamine aminotransferase in the preparation of a diagnostic reagent or diagnostic kit for the prediction of preeclampsia.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention discovers for the first time that the expression of Gal alpha 1 and 3GalNAc in preeclamptic placental tissues is obviously improved compared with normal gestational tissues, Gal alpha 1 and 3GalNAc can be catalytically synthesized by alpha 1, 3N-acetylgalactosamine aminotransferase (GTA), and Gal alpha 1,3GalNAc and GTA can be used as biological markers for diagnosing preeclampsia.

2. The invention further detects the expression of the trophoblast cells Gal alpha 1 and 3GalNAc through Lectin blot and cell immunofluorescence, thereby proving that the transfection of GTA siRNA can inhibit the expression of Gal alpha 1 and 3GalNAc in trophoblasts, and the transfection of GTA cDNA on the basis can promote the synthesis of Gal alpha 1 and 3 GalNAc; the early pregnancy villus explant is used as a primary model, a Transwell migration invasion experiment, a matrigel tube forming experiment and an EdU proliferation experiment prove that the GTA inhibits the migration invasion, angiogenesis and proliferation capacity of trophoblast cells by promoting the synthesis of the trophoblast cells Gal alpha 1 and 3 GalNAc.

3. The invention is helpful for the prediction and diagnosis of preeclampsia, deeply understands the embryo implantation mechanism and provides a new theoretical basis for the treatment of preeclampsia with sugar as a target clinically.

Drawings

In order to more clearly illustrate the embodiments of the present invention, the drawings to which the embodiments relate will be briefly described below.

FIG. 1 is a graph of Gal α 1,3GalNAc and GTA expression in placental tissue from patients with normal pregnancy and preeclampsia. Wherein 1st trimester is the normal early pregnancy stage, 3rd trimester is the normal late pregnancy stage, and Pre-eclampsia is preeclampsia.

FIG. 2 is a graph showing the expression of Gal α 1,3GalNAc and GTA in different experimental groups by human choriotrophoblast cells (HTR 8/SVneo).

FIG. 3 is a graph of the migratory invasion, vascularization and proliferative capacity of human choriotrophoblast cells (HTR8/SVneo) in different experimental groups.

Detailed Description

The present invention is described in detail below with reference to examples, but the embodiments of the present invention are not limited thereto, and it is obvious that the examples in the following description are only some examples of the present invention, and it is obvious for those skilled in the art to obtain other similar examples without inventive exercise and falling into the scope of the present invention.

Example 1

Materials and methods

Clinical samples (placental tissue) were obtained from women presenting preeclamptic symptoms during pregnancy and healthy women of normal pregnancy, paraffin embedded.

Detecting the expression of Gal alpha 1,3GalNAc and GTA in the placenta tissue of normal pregnant women and preeclamptic patients.

1. Immunohistochemical method for detecting positioning and expression of Gal alpha 1,3GalNAc and GTA in placenta tissue

(1) Dewaxing: the prepared paraffin sections were placed in the following solutions in order: soaking in xylene I for 10min, then soaking in xylene II for 10min, soaking in 100% ethanol I for 5min, soaking in 100% ethanol II for 5min, soaking in 95% ethanol for 5min, soaking in 85% ethanol for 5min, and soaking in 70% ethanol for 5 min; then rinsing with slow water flow for 10min, and soaking in PBS for 3 times for 5 min.

(2) Antigen retrieval: placing the slices in citrate buffer solution, covering, and thawing in microwave oven for 20 min. Taking out and naturally cooling to room temperature.

(3) Soaking in PBS for 5min for 3 times; the slices were wiped dry and placed in a brown box and 3% hydrogen peroxide was added dropwise, incubated at room temperature in the dark for 20 min.

(4) Sealing goat serum: soaking in PBS for 5min for 3 times; wiping off and dripping goat serum, and sealing in a brown box for 1 h.

(5) Primary antibody incubation: the excess liquid was blotted on filter paper and primary antibody was raised overnight at 4 deg.C (rabbit anti-human GTA 1: 150; lectin SBA1: 300).

(6) Rewarming: the next day, the mixture was taken out and left at room temperature for 1 hour.

(7) PBS was washed 3 times for 10min each, after which PBS was spun off, the liquid on the back and front sides was blotted dry and biotinylated secondary antibody was added and incubated for 1h at room temperature.

(8) Washing with PBS for 5min for 3 times, adding streptavidin labeled with horseradish enzyme, and incubating at room temperature for 1 h.

(9) PBS washing 3 times for 10min, DAB color development, from 5s, microscope observation.

(10) Washing with flowing water for 10min, and staining with hematoxylin.

(11) Dewatering after flushing for 10min with running water: 70% ethanol (2min) -85% ethanol (2min) -95% ethanol (2min) -absolute ethanol I (2min) -absolute ethanol II (2min) -xylene I (2 min).

(12) Sealing: the neutral gum was overlaid on a glass slide and the sample determined to be bubble free. After air-drying, the film was photographed under a microscope (shown in FIG. 1A).

2. Expression of Gal alpha 1,3GalNAc and GTA detected by Western blot and Lectin blot

Extraction of Total cellular protein

Taking out the cells from the incubator, discarding the supernatant, and washing with PBS 2 times;

appropriate cell lysates were then added according to the cell amount and incubated on ice for 10 minutes.

Scraping cells with protein, placing in a 1.5ml lep tube, sealing with a sealing film, and boiling in boiling water for 15 minutes. Storing at-20 deg.C for use.

Extraction of placental tissue proteins

Taking out placenta tissue from a refrigerator at the temperature of-80 ℃, putting the placenta tissue on ice, shearing appropriate tissue, putting the tissue into an EP tube, adding tissue protein lysate, and mixing the tissue protein lysate with the volume ratio of 100:1 adding protease inhibitor, and grinding on ice.

Incubate at 4 ℃ for 4-6h, vortexe for 15 seconds every 30 min. The tissue protein lysate was centrifuged at 10000g for 10min at 4 ℃ and the supernatant was collected in a new EP tube.

The protein was quantified using BCA protein quantification kit, and protein lysate was added and boiled for denaturation for 15 min.

SDS-PAGE electrophoresis is carried out, and the specific steps are as follows:

preparing glue: the glass plate is aligned and placed on the glue making frame and screwed to the 1.0 gear, and no leakage is ensured. Preparing 10% separation gel and 6% concentration gel, and shaking thoroughly. The separation gel is slowly added along the inner surface of the glass plate, and then sealed with absolute ethyl alcohol liquid, and solidified for 30min at room temperature. Discarding ethanol, wiping off residual ethanol, adding concentrated gel, inserting into comb, coagulating at room temperature for 30min, carefully pulling out comb after gel coagulation, and placing in electrophoresis solution for use.

Loading: calculating the sample loading volume of the sample according to the quantitative result of the protein, wherein the sample loading amount is 25-40 mu g;

electrophoresis: and (3) keeping an ice bath in the electrophoresis process, carrying out electrophoresis by using 100V voltage, changing the voltage to 120V after the protein sample enters the separation gel from the concentrated gel, observing the position of the protein marker in real time, and stopping electrophoresis.

Film transfer: the glass plate was carefully disassembled to cut a gel mass according to the molecular weight of interest, and filter paper and cellulose acetate film (NC film) were cut according to the size of the gel cut. According to the sequence of the spongy cushion, the filter paper, the membrane, the glue, the filter paper and the spongy cushion, the spongy cushion is placed into a membrane transferring groove filled with membrane transferring liquid with attention paid not to generate bubbles. After the film is transferred for a suitable time at a voltage of 150V and a current of 250mA, ponceau red can be used for dyeing, and the condition of the film transfer can be confirmed.

Primary antibody incubation: preparing primary antibody (rabbit anti-human GTA 1: 1000; rabbit anti-human CD 311: 1000; rabbit anti-human VEGFR 21: 1000; rabbit anti-human GAPDH 1: 2000; lectin SBA1: 8000), incubating at 4 deg.C overnight, taking out the primary antibody the next day, washing membrane with TBST for 4 times, each time for 10 min.

And (3) secondary antibody incubation: HRP-labeled secondary antibodies (goat anti-rabbit IgG 1: 3000; streptavidin 1:12000) were incubated for 1h at room temperature. TBST membrane washing was performed for 10min 4 times.

And (3) luminescence development: mixing the luminescent liquid A and the luminescent liquid B in a ratio of 1:1, mixing uniformly and placing in dark. The NC membrane was wiped with filter paper, coated with a luminescent solution, placed in a Bio-Rad imager for color development, and analyzed by Image Lab software for bands.

As can be seen from FIG. 1, there were only traces of Gal α 1,3GalNAc and GTA in the placental tissues of the 1st trimester (early pregnancy stage) and 3rd trimester (late pregnancy stage) experimental groups, and there were significant increases of Gal α 1,3GalNAc and GTA in the placental tissues of preeclamptic patients, i.e., there were significant increases of Gal α 1,3GalNAc and GTA in the placental tissues of preeclamptic patients compared to normal gestational tissues.

Example 2: western blot and cell immunofluorescence detection of expression of Gal alpha 1,3GalNAc in trophoblast cells

Climbing sheets: after trypsinizing human choriotrophoblast cells (HTR8/SVneo), centrifugation was performed at 800rpm for 4min, fresh medium was resuspended, and the cell suspension was placed in a petri dish with a slide on which the cells were grown.

Collecting the slices: the culture medium was removed from the dish and washed 3 times for 3min each with PBS.

Fixing: add 4% paraformaldehyde to the dish with a tilt and fix for 20 min.

The paraformaldehyde was discarded and the wash was performed 3 times for 3min with PBS.

And (3) sealing: selecting climbing pieces, placing into a wet box, adding immunostaining sealing liquid, and sealing for 1 h.

Primary antibody incubation: the slide was clamped from the blocking solution, blotted dry with filter paper, and incubated with primary antibody overnight at 4 deg.C (rabbit anti-human GTA 1: 150; lectin SBA1: 300).

The following day the slide was taken out of the refrigerator wet box and placed in a porcelain well plate and washed 6 times with PBS for 5min each time.

And (4) dropwise adding a fluorescence-labeled secondary antibody, incubating at room temperature for 1h, and washing with PBS for 5min for 6 times.

DAPI staining of nuclei: the slide was stained with DAPI dropwise at room temperature for 10min (DAPI 1: 4000).

Sealing: washing with PBS for 3 times, each for 3 min; and sealing the anti-fluorescence quencher, and observing and photographing under an inverted fluorescence microscope.

As shown in FIG. 2, the GTA siRNA transfection inhibited the expression of Gal α 1,3GalNAc in trophoblasts, while GTA cDNA transfection promoted the synthesis of Gal α 1,3 GalNAc.

Example 3: early pregnancy villus explant culture

(1) Spreading glue: melting matrigel in a refrigerator at 4 ℃, diluting matrigel with serum-free medium DMEM/F-12 at a ratio of 1:2, gently blowing and mixing, adding 50 μ L matrigel to each well of a 96-well plate, avoiding bubbles as much as possible, and placing in an incubator at 37 ℃.

(2) The villous tissue in the early gestation period was obtained from healthy women who had suffered an abortion in the gynecological operating room of the affiliated hospital for non-medical reasons, and the blood clots were immediately rinsed clean with pre-chilled sterile PBS.

(3) In a sterile operating station, villus tissue is placed in a large dish containing PBS and cut into 2-5mm fragments.

(4) Carefully transferred to matrigel in 96-well plates, anchored in the center of matrigel.

(5) After anchoring for 4-6h, complete medium was added, and the morphology was observed under an inverted microscope and recorded as 0h by photography.

(6) The medium was discarded, transfection was performed in groups, and serum-free medium containing either Scramble (untreated group) or GTA siRNA was added.

(7) After 6h of transfection, DMEM/F-12 complete medium containing 10% FBS was added to terminate the transfection.

(8) Plates were removed at 24h, 48h post-transfection for kinetic observation and photographed, and the distance of exogenous migration of EVTs from the villous end was measured using Photoshop CS6 software.

As can be seen from fig. 3(a), the results show: the distance of ectotrophic trophoblast cells (EVTs) that migrated exogenously was significantly increased in the GTA siRNA-transfected group compared to the Scramble control group.

Example 4: transwell migration invasion experiment

(1) Paving glue in the small chamber: matrigel was thawed in a 4 ℃ freezer, diluted 1:9 with serum-free medium DMEM/F-12, gently whipped, mixed well, 50 μ Lmatrigel was added to the Transwell chamber, with minimal air bubbling, and the chamber was placed in a 24-well plate in a 37 ℃ incubator.

(2) Cell counting: taking out cells of the Scramble, GTA siRNA, no-load Vector and GTA cDNA group from the incubator, digesting the cells by pancreatin, centrifuging the cells at 800rpm for 4min, resuspending the cells by using a serum-free medium, and counting the cells on a plateCount 3 times, adjust cell concentration to 2 × 10⁵one/mL.

(3) Adding 700. mu.L of DMEM/F-12 medium containing 10% FBS to a 24-well plate, gently placing the plate in a Transwell chamber, sucking 200. mu.L of cell suspension from the cell suspension, adding the cell suspension to the Transwell chamber, placing the cell suspension in an incubator at 37 ℃ and continuing the incubation for several hours

(4) Crystal violet dyeing: taking out the chamber, and lightly rinsing the chamber with PBS; methanol is fixed for 15min, and the chamber is rinsed 2 times with PBS for 5min each time; dyeing with 0.5% crystal violet dye solution for 15min, rinsing the chamber with PBS for 2 times, each time for 5 min; gently rubbing off the cells inside the chamber with a cotton swab; randomly selecting 3 different field photographs under an inverted microscope and calculating the number of cells; the experiment was repeated three times and the results were counted.

As can be seen from fig. 3(B), the Transwell migratory invasion assay confirmed that GTA inhibits trophoblastic invasion, angiogenesis and proliferative capacity by promoting the synthesis of trophoblastic cells Gal α 1,3 GalNAc.

Example 5: matrigel tube forming experiment

(1) Spreading glue: the matrigel was thawed in a 4 ℃ freezer, spread in a 96-well plate with a precooled tip, 50. mu.L of matrigel per well, with as little air bubbles as possible, and incubated in an incubator at 37 ℃ for 1-2 h.

(2) Taking out cells HTR8/SVneo of the Scramble, GTA siRNA and GTA cDNA experimental groups from the incubator, digesting by pancreatin, and centrifuging at 800rpm for 4 min; resuspending in serum-free medium, counting for 3 times, and adjusting cell concentration to 5 × 10⁴Per mL; the cells were placed on the prepared matrigel and the formation of the tubules was observed under a microscope at 2h and 12h and photographed.

As seen in FIG. 3(C), HTR8/SVneo cells were significantly enhanced in tube forming ability after transfection with GTA siRNA as compared with the Scramble group; the tube forming ability of the GTA siRNA + GTA cDNA co-processing group is inhibited compared with that of the GTA siRNA group

Example 6: EdU proliferation assay

(1) Cell culture: taking the HTR8/SVneo cells of the Scramble, GTA siRNA, No-load Vector and GTA cDNA group in the logarithmic growth phase, and taking 5X 10 cells per well³Individual cells were seeded in 96-well plates.

(2) EdU labeling: after the cells adhered to the wall, the EdU solution was diluted with fresh DMEM/F-12 complete medium at a ratio of 1000:1 to prepare 50. mu.M EdU medium, 100. mu.L of the EdU medium was added to each well, and the mixture was incubated at 37 ℃ for 2 hours, and the medium was discarded. The cells were washed 2 times with PBS for 5min each.

(3) Cell fixation: adding 50 μ L of 4% paraformaldehyde into each well, incubating at room temperature for 30min, and discarding the stationary liquid; adding 50 μ L of 2mg/mL glycine solution into each well, and incubating for 5min in a shaking table; removing the glycine solution, and adding PBS to wash for 5min in a shaking table; discarding PBS, adding 100 μ L of 0.1% Triton X-100, and incubating for 10min in a shaker; PBS wash 1 time, 5 min.

(4) Apollo staining: adding 100 μ L of 1 × Apollo staining reaction solution into each well, and incubating for 30min in a shaking table at room temperature in a dark place; discard staining solution and add 100 μ L0.1% Triton X-100 to each well, shake wash 3 times, 10min each time, discard penetrant.

(5) Live cell labeling: preparing 1 XHoechest 33342 reaction liquid by deionized water according to the proportion of 100:1, adding 100 mu L of reaction liquid into each hole, incubating for 30min by a shaking table in the dark at room temperature, discarding the reaction staining liquid, adding 100 mu L of PBS, washing for 2 times, and observing and photographing under an inverted microscope.

As can be seen from fig. 3(D), EdU proliferation experiments demonstrated that the proliferation of HTR8/SVneo cells was significantly promoted by down-regulating GTA expression, and compared to the GTA siRNA group, HTR8/SVneo cells were reduced in proliferation potency after GTA siRNA + GTA cDNA co-treatment, indicating that GTA cDNA was able to inhibit the proliferation potency of HTR8/SVneo cells.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.