CN114167057A - Biological marker for diagnosing abortion and application thereof - Google Patents
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Abstract
The invention discloses a biological marker for diagnosing abortion and application thereof, belonging to the technical field of biology. The invention discovers that alpha 1,3mannosylation (alpha 1,3mannosylation, Man alpha 1,3Man) and alpha 1,3 mannosyltransferase (ALG3) are highly expressed in villus tissues of patients suffering from inevitable abortion for the first time, uses Man alpha 1,3Man and alpha 1,3 mannosyltransferase (ALG3) as biological markers for diagnosing abortion, and simultaneously proves that inhibiting the expression of Man alpha 1,3Man and alpha 1,3 mannosyltransferase (ALG3) can promote the differentiation capability of embryo trophoblast cells, promote placental development and reduce abortion incidence. Therefore, the present invention facilitates the prediction and diagnosis of miscarriage and provides an insight into the mechanisms of placental development. Provides a new theoretical basis for clinically diagnosing and treating abortion by taking sugar as a target.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a biological marker for diagnosing abortion and application thereof.
Background
Maintaining pregnancy requires normal development of the embryo and placenta. When the embryo is implanted, trophoblast cells undergo self-proliferation while differentiating into villous or extravillous trophoblast cells. In the human placenta, there are three major trophoblast subgroups: cytotrophoblasts (CTBs), extravillous cytotrophoblasts (EVTs), and Syncytium Trophoblasts (STBs). During trophoblast differentiation, CTB cells have stem cell function and can undergo two distinct differentiations, one is fusion to multinucleated STB cells and the other is undergoing epithelial mesenchymal conversion to EVT cells. STB, the outer layer of placental villi, is the primary site for gas and nutrient exchange between the mother and fetus. It secretes a variety of hormones and proteins, protecting the fetus from pathogens. EVT cells are trophoblast cells that are migratory and invasive and are critical for placental intercalation and fetal development. The proliferation and differentiation of these cells in the placenta may change throughout pregnancy to better accommodate the growing changes in fetal development. Abnormal fetal development can lead to a variety of pregnancy disorders, including abortion, preeclampsia, and intrauterine growth restriction. The normal development of the placenta requires complex regulatory mechanisms, but the underlying mechanisms of human placental development have not been reported in detail.
Early threatened abortion accounts for 80% of spontaneous abortion. The reason for the threatened abortion is complex, is related to factors such as embryo, maternal and environment, is difficult to prevent the threatened abortion from etiology, and has important significance for effectively controlling the development of the threatened abortion to reduce the abortion occurrence rate. Therefore, the observation of the pregnancy outcome by researching the abortion indexes can provide reference for fetus protection in time. For example, human chorionic gonadotropin (β -HCG, CGB), a glycoprotein hormone secreted by syncytiotrophoblast cells, is a specific marker of pregnancy, and low, low or slow HCG levels, considered to be abnormal in pregnancy, are known. Progesterone (P) is a hormone for regulating female reproduction, the maternal serum progesterone value is less than 12.5ng/mL for threatened abortion, and the B ultrasonic examination result indicates the abnormality of a gestational sac and does not recommend fetus protection. Since progesterone secretion is pulse-like, the accuracy of the determination is limited. The above indexes are related to abortion, but have limited value in clinical diagnosis and prognosis evaluation of threatened abortion, so that it is necessary to discuss new indexes combined with the above indexes to apply to abortion, and has certain research significance.
Proteins have a variety of post-translational modifications, including glycosylation, phosphorylation, and ubiquitination. Glycosylation is the attachment of sugar chains to the amino acid side chains of membrane proteins and cell secreted proteins, and is classified into N-glycosylation modification and O-glycosylation modification. The N-glycosylation modification is the formation of N-linked glycans of various structures on the core five-carbon structure (Man α 1-6 (Man α 1-3) Man β 1-4 GlcNAc β 1-4 GlcNAc β 1-Asn-X-Ser/Thr). On this basis, there are classified into high mannose type, complex type and heterozygote type. The high mannose type is one in which only mannose is linked to mannose at the five carbons of the core. The α 1,3 mannosyltransferase (ALG3) is responsible for the synthesis of the first mannosylation into the lumen of the endoplasmic reticulum, i.e., encodes an α -1,3 mannosyltransferase that converts Man5GlcNAc2-PP-Dol to Man6GlcNAc2-PP-Dol at the b-branch of LLO. The glycosylation degree of the protein can change the physicochemical property and the function of the protein under the regulation and control of specific glycosyl transferase. During pregnancy, protein glycosylation is involved in a variety of physiological processes, such as sperm-egg recognition, embryonic development, and embryo implantation.
Disclosure of Invention
In view of the above, the present invention provides a biological marker for diagnosing abortion and its application.
The purpose of the invention is realized by the following modes:
the invention provides a biological marker for diagnosing abortion, wherein the biological marker is Man alpha 1,3Man or/and alpha 1,3 mannosyltransferase (ALG 3).
In another aspect, the invention provides the use of Man α 1,3Man or/and α 1,3 mannosyltransferase (ALG3) in the manufacture of a reagent or kit for the diagnosis of abortion.
Further, the detection method of the reagent or the kit comprises an immunohistochemistry experiment (IHC), a Lectin blot experiment (Lectin blot), a protein immunoblot experiment (Western blot) and an ELISA experiment.
Further, Man α 1,3Man is specifically recognized by Lectin GNA, and detected by Lectin blot (Lectin blot).
Further, the α 1,3 mannosyltransferase (ALG3) was detected by immunohistochemistry assay (IHC), Western blot assay (Western blot) and ELISA.
Further, the detected pathological samples are villus tissue and serum.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention discovers for the first time that alpha 1,3mannosylation (alpha 1,3mannosylation, Man alpha 1,3Man) and alpha 1,3 mannosyltransferase (ALG3) are abnormally highly expressed in villus tissues of patients suffering from inevitable abortion, and Man alpha 1,3Man can be catalytically synthesized by ALG3, so that alpha 1,3mannosylation and ALG3 of proteins are used as biological markers for diagnosing abortion, and simultaneously proves that alpha 1,3mannosylation and ALG3 of proteins influence the differentiation of CTB cells into EVT or STB cells. The method can help to diagnose and prognose threatened abortion, deeply understand an embryo implantation mechanism, and provide a new theoretical basis for clinically diagnosing and treating abortion with sugar as a target.
2. The invention discovers that ALG3 changes alpha 1,3mannosylation, and influences the self-renewal capacity and differentiation capacity of trophoblast cells; ALG3 siRNA reduces Man α 1,3Man synthesis, thereby inhibiting the self-renewal capacity of trophoblast cells and promoting the ability of trophoblast cells to differentiate into EVT and STB cells.
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 showing the comparison of Man α 1,3Man and ALG3 expression in villus tissues of normal women and patients with refractory abortion as detected by Lectin Histochemistry (A), Lectin blot (B), immunohistochemistry (C) and Western immunoblotting (D), wherein NP represents villus tissue of normal early pregnant women and MIS represents villus tissue of patients with abortion.
FIG. 2 is a graph comparing the expression of Man α 1,3Man and ALG3 in different experimental groups (Scramble control group, ALG3 siRNA transfected group, empty vector group and ALG3 cDNA transfected group) by Lectin blot and Western blot (A) and cellular immunofluorescence (B and C) to detect human choriotrophoblast cells (HTR 8/SVneo).
FIG. 3 is a graph showing the expression of proteins involved in the inhibition of the self-renewal ability of trophoblast cells by transfection of ALG3 siRNA, which is promoted by transfection of ALG3 cDNA, by Western blot (A and B) and spheronization experiments (C and D).
FIG. 4 shows the differentiation potency of trophoblast cells in different experimental groups (Scramble control group, ALG 3-transfected siRNA group, empty vector group, and ALG 3-transfected cDNA group), namely, STB cell integration experiments (A and B) and EVT cell migration invasion experiments (C).
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
Clinical samples (villous tissue) were obtained from patients with inevitable abortion and healthy women of normal pregnancy, paraffin embedded.
Expression of Man alpha 1,3Man and ALG3 in villus tissue was detected.
1. Immunohistochemical method for detecting positioning and expression of Man alpha 1,3Man and ALG3 in villus tissue
(1) Dewaxing: the prepared paraffin sections were placed in the following solutions in order: soaking in xylene I for 10min, 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 5 min; then rinsing with slow water flow for 10 min. PBS was used for 5min each 3 times.
(2) Antigen retrieval: placing the slices in citrate buffer solution, and thawing for 20min in microwave oven. 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; dropping goat serum and sealing for 1 h.
(5) Primary antibody incubation: the excess liquid was blotted on filter paper and primary antibody was incubated overnight at 4 deg.C (rabbit anti-human ALG 31: 150; lectin GNA1: 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, the liquid 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 horseradish-labeled streptavidin, and incubating at room temperature for 1 h.
(9) Washing with PBS for 3 times for 10min, DAB developing, and observing under microscope.
(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), anhydrous ethanol I (2min), anhydrous 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 being dried, the film is arranged on a microscope for observation and photographing.
As can be seen from fig. 1A and 1C, α 1,3mannosylation (α 1,3mannosylation, Man α 1,3Man) and α 1,3 mannosyltransferase (ALG3) are abnormally highly expressed in tissues of patients with inevitable abortion.
2. Western blot and Lectin blot for detecting expression of Man alpha 1,3Man and ALG3
(1) Extraction of Total cellular protein
The cells were removed from the incubator, the supernatant discarded, washed 2 times with PBS, added with cell lysate, and incubated on ice for 10 minutes. Cells were scraped with a spatula, placed in a 1.5mL EP tube, sealed with a sealing membrane, and boiled in boiling water for 15 minutes. Storing at-20 deg.C for use.
(2) Extraction of villus tissue protein
Taking out the tissue from a refrigerator at the temperature of minus 80 ℃, putting the tissue on ice, shearing the 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, vortex for 15 seconds every 30 min. The tissue protein lysate was centrifuged at 4 ℃ for 10000g for 10min, the supernatant was collected in a new EP tube, the protein was quantitated using BCA protein quantitation kit, and loading buffer was added and denatured for 15 min.
(3) SDS-PAGE electrophoresis
Preparing acrylamide gel, applying protein sample, observing the position of protein marker in real time, and stopping electrophoresis. The membrane was transferred using a cellulose acetate membrane (NC membrane) at a voltage of 150V and a current of 250mA for an appropriate time. Primary antibody incubation: preparing primary antibody (rabbit anti-human ALG 31: 1000; rabbit anti-human GAPDH 1: 2000; lectin GNA1:2000), incubating at 4 deg.C overnight, taking out 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 1:2000) were incubated at room temperature for 1 h. TBST membrane washing was performed for 10min 4 times. The resulting mixture was put into a luminescent solution, and the mixture was placed in a Bio-Rad imager for color development, and then the band was analyzed by Image Lab.
As can be seen from fig. 1B and fig. 1D, there were only a small amount of Man α 1,3Man and ALG3 in the villus tissue of the control group, and the expression levels of Man α 1,3Man and ALG3 in the villus tissue of the patients with refractory abortion significantly increased, that is, the expression levels of Man α 1,3Man and ALG3 in the villus tissue of the patients with refractory abortion significantly increased compared to the normal control.
Example 2: lectin staining and cellular immunofluorescence detection of Man alpha 1,3Man expression in trophoblast cells
Climbing sheets: after trypsinization, the trophoblast cells were centrifuged at 800rpm for 4min, the fresh medium was resuspended, and the cell suspension was placed in a petri dish with a slide.
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 petri dish and fix for 20 min.
The paraformaldehyde was discarded and the wash was performed 3 times for 3min with PBS.
And (3) sealing: blocking for 1h by immunostaining blocking solution.
Primary antibody incubation: overnight at 4 deg.C (rabbit anti-human ALG 31: 150; lectin GNA1: 300). PBS wash 6 times for 5min each.
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 transfection of ALG3 siRNA can inhibit the expression of Man α 1,3Man in trophoblasts, while the transfection of ALG3 cDNA can promote the synthesis of Man α 1,3 Man.
Example 3: balling test for detecting self-renewal capacity of trophoblast cells
(1) The trophoblast cells in good growth state were digested and centrifuged, the serum-containing medium was removed, and washed twice with PBS.
(2) Preparation of Stem cell Medium resuspended cells (10)4Individual cells): DMEM/F12+1 x B27+20ng/mL bFGF +20ng/mL EGF.
(3) Cell plating: low adhesion 6 well plates were selected with 4 ml of medium per well. The culture was completed in about 10 days, and the state of spherulization was observed.
As can be seen from FIG. 3, transfection of ALG3 siRNA inhibited the self-renewal ability and desiccation of trophoblast cells; however, ALG3 cDNA promotes the self-renewal ability of trophoblast cells and the dryness of trophoblast cells.
Example 4: transwell experiment for detecting migration invasion capacity of trophoblast cells
(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. mu.L matrigel was added to a Transwell chamber, with minimal air bubbles, and the chamber was placed in a 24-well plate in a 37 ℃ incubator.
(2) Cell counting: taking out trophoblast cells of cDNA groups of Scaramble, ALG3 siRNA, Vector and ALG3 from the incubator, digesting with pancreatin, centrifuging at 800rpm for 4min, resuspending with serum-free medium, counting for 3 times by counting plate, and adjusting cell concentration to 8x104one/mL.
(3) After 700. mu.L of DMEM/F-12 medium containing 10% FBS was added to the 24-well plate, the plate was gently placed in a Transwell chamber, and 200. mu.L of cell suspension was aspirated from the cell suspension and added to the upper chamber, which was placed in an incubator at 37 ℃ for several hours.
(4) Crystal violet dyeing: taking out the chamber, and lightly rinsing the chamber with PBS; methanol fixation for 15 min; dyeing with 0.5% crystal violet dye solution for 15min, rinsing the chamber with PBS for 2 times, each time for 1 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 in FIG. 4C, the Transwell migratory invasion assay demonstrates the ability of transfected ALG3 siRNA to promote the differentiation of trophoblast cells into EVT cells and promote migratory invasion; while ALG3 cDNA inhibits trophoblast cell differentiation into EVT cells and decreases migratory invasion capacity.
Example 5: cell fusion experiments to examine the ability of trophoblast cells to differentiate into STB cells
(1) Trophoblast cells are generally treated with adenylate cyclase activator (Forskolin) at a concentration of 50-100nM at about 20% confluence, and spontaneously fuse after 48 hours of treatment.
(2) Identification of syncytia: and detecting the expression condition of the CGB by a Western blot experiment.
As can be seen from FIG. 4A, the transfection of ALG3 siRNA promotes the ability of trophoblast cells to become integrated, i.e., transformed into STB cells, whereas the transfection of ALG3 cDNA inhibits the ability of trophoblast cells to differentiate into STB 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.
Claims (7)
1. A biological marker for use in the diagnosis of abortion, wherein said biological marker is α 1,3 mannose (Man α 1,3Man) or/and α 1,3 mannosyltransferase (ALG 3).
2. Use of α 1,3 mannose (Man α 1,3Man) or/and α 1,3 mannosyltransferase (ALG3) according to claim 1 in the manufacture of a reagent or kit for the diagnosis of abortion.
3. Use according to claim 2, characterized in that the method of detection of the reagent or kit comprises immunohistochemistry, lectin imprinting, western immunoblotting and ELISA assays.
4. Use according to claim 3, characterized in that α 1,3 mannose (Man α 1,3Man) is detected by a lectin blot assay.
5. Use according to claim 3, characterized in that the α 1,3 mannosyltransferase (ALG3) is detected by immunohistochemical experiments, Western immunoblot experiments and ELISA experiments.
6. The use according to claim 4, wherein the lectin is GNA.
7. The use according to claim 3, wherein the pathological sample to be tested is a villus tissue or a serum test.
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