CN114167057B - 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, manalpha 1,3 Man) and alpha 1,3 mannosyltransferase (ALG 3) are highly expressed in villus tissues of patients with unavoidable abortion, takes Manalpha 1,3Man and alpha 1,3 mannosyltransferase (ALG 3) as biological markers for diagnosing abortion, and simultaneously proves that inhibiting the expression of Manalpha 1,3Man and alpha 1,3 mannosyltransferase (ALG 3) promotes the differentiation capacity of embryonic trophoblast cells, promotes placenta development and reduces the occurrence rate of abortion. Thus, the present invention is useful for the prediction and diagnosis of abortion and for the deep understanding of the placenta development mechanism. Provides a new theoretical basis for abortion diagnosis and treatment with sugar as a target point clinically.
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
Maintenance of pregnancy requires normal development of the embryo and placenta. When the embryo is implanted, the trophoblast cells undergo self-proliferation while differentiating into villus or extravillus trophoblast cells. In the human placenta, there are three major trophoblast subgroups: cytotrophoblast (CTB), extravillous cytotrophoblast (EVT), and Syncytial Trophoblast (STB). In the trophoblast differentiation process, CTB cells have stem cell function and can undergo two different differentiation processes, one is a STB cell that fuses into multiple nuclei and the other is an EVT cell that undergoes epithelial mesenchymal transition. STB is the primary site of gas and nutrient exchange between the mother and fetus as the outer layer of placenta villi. It secretes a variety of hormones and proteins that protect the fetus from pathogens. EVT cells are migratory and invasive trophoblast cells that are critical for placenta intercalation and fetal development. Proliferation and differentiation of these cells in the placenta may change throughout pregnancy to better accommodate the growth changes in fetal development. Abnormal fetal development can lead to a variety of gestational disorders including abortion, preeclampsia, and intrauterine growth restriction. The normal development of the placenta requires complex regulatory mechanisms, but the underlying mechanisms of human placenta development have not been reported in detail.
Early threatened abortion accounts for 80% of spontaneous abortion. The causes of threatened abortion are complex, and the threatened abortion is difficult to prevent from etiology depending on factors such as embryo, mother and environment, and the effective control of the progress of the threatened abortion is important for reducing the occurrence rate of the threatened abortion. Therefore, the indexes of abortion are studied to observe the pregnancy ending, and a reference can be provided for miscarriage prevention in time. For example, human chorionic gonadotrophin (β -HCG, CGB), a glycoprotein hormone secreted by syngeneic trophoblast cells, is a specific marker of pregnancy, and low, declining or slow growth of HCG levels may be considered for pregnancy abnormalities. Progesterone (P) is a hormone regulating female reproduction, threatened abortion with maternal serum progesterone value less than 12.5ng/mL, B ultrasonic examination result frequently indicates abnormal gestational sac, and miscarriage prevention is not suggested. Because progesterone secretion is pulse-like, the accuracy of judgment is limited. The above indexes such as estrogen and inhibin A are related to abortion, but have limited values in aspects such as clinical diagnosis and prognosis evaluation of threatened abortion, so that it is necessary to investigate the application of the new indexes in combination with the above indexes to abortion, and the method has a certain research significance.
Proteins have a variety of post-translational modifications, including glycosylation modifications, phosphorylation modifications, ubiquitination modifications, and the like. Glycosylation is the attachment of sugar chains to amino acid side chains of membrane proteins and cell secretion proteins, and glycosylation modifications are classified into N-glycosylation modifications and O-glycosylation modifications. N-glycosylation modification is the formation of N-linked glycans of various structures on the core five-carbon structure (Manα1-6 (Manα1-3) Manβ1-4GlcNAcβ1-Asn-X-Ser/Thr). Based on this, high mannose type, complex type and heterozygous type are classified. The high mannose type is to link only mannose to the core five-carbon mannose. The α1,3 mannosyltransferase (ALG 3) is responsible for the synthesis of the first mannosylation into the lumen of the endoplasmic reticulum, i.e. encoding that the α -1,3 mannosyltransferase converts Man5GlcNAc2-PP-Dol to Man6GlcNAc2-PP-Dol at the b-branch of LLO. The degree of glycosylation of a protein can change the physicochemical properties and functions of the protein under the control of a specific glycosyltransferase. During pregnancy, protein glycosylation is involved in a variety of physiological processes, such as sperm-egg recognition, embryo development, and embryo implantation.
Disclosure of Invention
In view of the above, the present invention aims to provide a biological marker for diagnosing abortion and application thereof.
The invention aims at realizing the following steps:
The invention provides a biological marker for diagnosing abortion, which is Manalpha 1,3Man or/and alpha 1,3 mannosyltransferase (ALG 3).
In another aspect the invention provides the use of manα1,3Man or/and α1,3 mannosyltransferase (ALG 3) for the preparation of a reagent or kit for diagnosing abortion.
Further, the detection method of the reagent or the kit comprises an immunohistochemical experiment (IHC), a Lectin blotting experiment (Lectin blot), a Western immunoblotting experiment (Western blot) and an ELISA experiment.
Further, manα1,3 Mans can be specifically recognized by Lectin GNA, and detection can be performed by Lectin blotting (Lectin blot).
Further, the α1,3 mannosyltransferase (ALG 3) was detected by immunohistochemical assay (IHC), western blot assay (Western blot) and ELISA.
Further, the pathological samples detected are villus tissue and serum.
Compared with the prior art, the invention has the following beneficial effects:
1. The invention discovers that alpha 1,3mannosylation (alpha 1,3mannosylation, manalpha 1,3 Man) and alpha 1,3 mannosyltransferase (ALG 3) are abnormally and highly expressed in villus tissues of patients suffering from unavoidable abortion, and Manalpha 1,3Man can be catalyzed and synthesized by ALG3, so that alpha 1,3mannosylation and ALG3 of proteins are used as biological markers for diagnosing abortion, and simultaneously, the alpha 1,3mannosylation and ALG3 of proteins are proved to influence CTB cells to differentiate into EVT or STB cells. This will help in the diagnosis and prognosis of threatened abortion, and will provide a new theoretical basis for the deep knowledge of embryo implantation mechanism and for the clinical diagnosis and treatment of abortions with sugar as target.
2. The invention discovers that ALG3 changes alpha 1,3 mannosylation and affects the self-renewal capacity and differentiation capacity of trophoblast cells; ALG3 siRNA reduces synthesis of manα1,3Man, thereby inhibiting the self-renewing ability of trophoblasts and promoting the ability of trophoblasts 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.
FIG. 1 is a graph showing the comparison of Manα1,3Man and ALG3 expression in the villus tissues of normal women and patients with inevitable abortion, wherein NP represents the villus tissue of a female of a normal premature pregnant woman, and MIS represents the villus tissue of a patient with abortion, by Lectin Histochemistry (A), lectin blot (B), immunohistochemistry (C) and Western immunoblotting (D).
FIG. 2 is a graph comparing the expression of Manα1,3Man and ALG3 in different experimental groups (Scramble control group, transfected ALG3 siRNA group, empty vector group and transfected ALG3 cDNA group) by detecting human villous trophoblast cells (HTR 8/SVneo) by means of Lectin blot and Western blot (A) and cellular immunofluorescence (B and C).
FIG. 3 is a graph showing the expression of proteins involved in verifying the ability of transfected ALG3 siRNA to inhibit self-renewal in trophoblasts by Western blot (A and B) and spheronization experiments (C and D), whereas transfected ALG3 cDNA promotes self-renewal in trophoblasts.
FIG. 4 shows the differentiation capacity of trophoblast cells in the various experimental groups (Scramble control group, transfected ALG3 siRNA group, empty vector group and transfected ALG3 cDNA group), namely STB cell integration experiments (A and B) and EVT cell migration invasion experiments (C).
Detailed Description
The following detailed description of the invention is provided in connection with examples, but the implementation of the invention is not limited thereto, and it is obvious that the examples described below are only some examples of the invention, and that it is within the scope of protection of the invention to those skilled in the art to obtain other similar examples without inventive faculty.
Example 1
Clinical specimens (villus tissue) were from both patients with inevitable abortion and healthy women with normal pregnancy, paraffin embedded.
Expression of Manα1,3Man and ALG3 in villous tissues was examined.
1. Immunohistochemical detection of localization and expression of Manα1,3Man and ALG3 in villus tissues
(1) Dewaxing: the prepared paraffin sections were placed in the following solutions in order: xylene I10 min, xylene II 10min,100% ethanol I5 min,100% ethanol II 5min,95% ethanol 5min,85% ethanol 5min,70% ethanol 5min; then flushing with slow water flow for 10min. PBS was used for 3 washes of 5min each.
(2) Antigen retrieval: the slices were placed in citrate buffer and thawed in a microwave oven for 20min. Taking out and naturally cooling to room temperature.
(3) Soaking and washing with PBS for 3 times, each time for 5min; the sections were dried and placed in a brown box, 3% hydrogen peroxide was added dropwise, and incubated at room temperature for 20min in the dark.
(4) Goat serum blocking: soaking and washing with PBS for 3 times, each time for 5min; goat serum was added dropwise for 1h of blocking.
(5) Incubation resistance: the filter paper was blotted dry of excess liquid, primary antibody overnight at 4 ℃ (rabbit anti-human ALG 3:150; lectin GNA 1:300).
(6) And (3) rewarming: the next day was taken out and left at room temperature for 1h.
(7) Washing with PBS for 3 times, each time for 10min, sucking the liquid, and adding the biotinylated secondary antibody for incubation for 1h at room temperature.
(8) Washing with PBS for 5min 3 times, adding horseradish enzyme labeled streptavidin, and incubating at room temperature for 1h.
(9) PBS was washed 3 times for 10min each time, DAB developed, and observed under a microscope.
(10) Washing with running water for 10min, and staining with hematoxylin.
(11) Dewatering is started after washing for 10min by running water: 70% ethanol (2 min), 85% ethanol (2 min), 95% ethanol (2 min), absolute ethanol I (2 min), absolute ethanol II (2 min), xylene I (2 min).
(12) Sealing piece: the neutral gum was covered on a glass slide to confirm that the sample was bubble free. And (5) after airing, observing and photographing by a normal microscope.
From FIGS. 1A and 1C, it can be seen that α1,3mannosylation (α1,3mannosylation, manα1,3 Man) and α1,3 mannosyltransferase (ALG 3) are abnormally highly expressed in tissues of patients suffering from unavoidable abortion.
2. Western blot and Lectin blot for detecting expression of Manalpha 1,3Man and ALG3
(1) Extraction of cell total protein
The cells were removed from the incubator, the supernatant was discarded, washed 2 times with PBS, cell lysate was added, and incubated on ice for 10 minutes. The cells were scraped with protein and placed in a 1.5mL EP tube, sealed with a sealing film, and boiled in boiling water for 15 minutes. Preserving at-20 ℃ for standby.
(2) Extraction of villus tissue proteins
Taking out tissue from a refrigerator at-80 ℃, placing the tissue on ice, shearing the proper tissue, placing the tissue into an EP tube, adding a tissue protein lysate, and mixing the tissue protein lysate with 100:1 adding protease inhibitor, and grinding on ice. Incubation is carried out for 4-6h at 4 ℃ and vortex oscillator oscillation is carried out for 15 seconds every 30 min. The tissue protein lysate was centrifuged at 10000g at 4℃for 10min, the supernatant was collected in a fresh EP tube, protein was quantified using BCA protein quantification kit, and loading buffer was added for denaturation by boiling for 15min.
(3) SDS-PAGE electrophoresis
Acrylamide gel was prepared and protein samples were applied, and the position of the protein Maker was observed in real time, and electrophoresis was terminated. Transfer was performed using a cellulose acetate film (NC film) at a voltage of 150V and a current of 250mA for a suitable time. Incubation resistance: preparing primary antibody (rabbit anti-human ALG 3:1000; rabbit anti-human GAPDH 1:2000; lectin GNA 1:2000), incubating overnight at 4deg.C, taking out the primary antibody the next day, and washing the membrane with TBST for 4 times each for 10min. Secondary antibody incubation: HRP-labeled secondary antibody (goat anti-rabbit IgG 1:3000; streptavidin 1:2000) was incubated for 1h at room temperature. TBST membrane washing is carried out for 4 times and 10 minutes each time. Placing the mixture into a luminous solution, placing the luminous solution into a Bio-Rad imager for color development, and analyzing the strips by using an Image Lab.
As can be seen from FIGS. 1B and 1D, only trace amounts of Manα1,3Man and ALG3 are present in the villus tissues of the control group, and the expression levels of Manα1,3Man and ALG3 in the villus tissues of the patients suffering from the inevitable abortion are obviously increased, i.e., the expression levels of Manα1,3Man and ALG3 in the villus tissues of the patients suffering from the inevitable abortion are obviously increased compared with the normal control.
Example 2: LECTIN STAINING and cellular immunofluorescence detection of Manα1,3Man expression in trophoblast cells
Climbing piece: after digestion of the trophoblast cells with pancreatin, centrifugation at 800rpm for 4min, fresh medium was resuspended and the cell suspension was placed in a petri dish with a slide.
And (3) collecting: the medium in the dish was removed and washed 3 times with PBS for 3min each.
Fixing: 4% paraformaldehyde was added to the dish and the mixture was fixed for 20min.
The paraformaldehyde was discarded, and washed 3 times with PBS for 3min each.
Closing: the immunostaining blocking solution was blocked for 1h.
Incubation resistance: overnight at 4 ℃ (rabbit anti-human ALG 3:150; lectin GNA 1:300). PBS was washed 6 times for 5min each.
The fluorescent-labeled secondary antibody was added dropwise, incubated at room temperature for 1h, and washed with PBS 6 times for 5min each time.
DAPI nuclear staining: DAPI was added dropwise to the slide and stained for 10min at room temperature (DAPI 1:4000).
Sealing piece: PBS is washed for 3 times and 3min each time; and (5) sealing the anti-fluorescence quenching agent, and observing and photographing under an inverted fluorescence microscope.
As can be seen from FIG. 2, transfected ALG3 siRNA inhibited expression of Manα1,3Man in trophoblasts, while transfected ALG3 cDNA promoted synthesis of Manα1,3 Man.
Example 3: balling experiments to detect trophoblast self-renewal
(1) Trophoblast cells with good growth state are digested and centrifuged, the serum-containing medium is removed, and washed twice with PBS.
(2) Preparation of Stem cell Medium resuspended cells (10 4 cells): DMEM/F12+1xB27+20 ng/mL bFGF+20ng/mL EGF.
(3) Cell plating: a low adhesion 6-well plate was selected with 4 ml of medium per well. Culturing is completed about 10 days, and the balling state is observed.
As can be seen from fig. 3, transfected ALG3 siRNA inhibited the self-renewal capacity of trophoblast cells and the stem properties of trophoblast cells; while ALG3 cDNA promotes the self-renewal capacity of trophoblasts and the stem nature of trophoblasts.
Example 4: transwell experiments to test the migratory invasive Capacity of trophoblasts
(1) Spreading glue in a small chamber: melting matrigel in a refrigerator at 4deg.C, diluting matrigel with serum-free culture medium DMEM/F-12 at 1:9, gently blowing and mixing, adding 50μ L MATRIGEL into a Transwell chamber, avoiding bubble generation as much as possible, placing the chamber in a 24-well plate, and placing in a 37 deg.C incubator.
(2) Cell count: trophoblast cells of the Scramble and ALG3 siRNA, vector, ALG cDNA set were removed from the incubator, digested with pancreatin, centrifuged at 800rpm for 4min, resuspended in serum-free medium, counted 3 times in a counter plate, and the cell concentration adjusted to 8X10 4 cells/mL.
(3) After 700. Mu.L of DMEM/F-12 medium containing 10% FBS was added to the 24-well plate, the mixture was gently placed in a Transwell chamber, 200. Mu.L of the cell suspension was aspirated from the cell suspension, and the mixture was placed in an upper chamber and cultured at 37℃for several hours.
(4) Crystal violet staining: taking out the cell, and slightly rinsing the cell by PBS; methanol is fixed for 15min; dyeing with 0.5% crystal violet dye solution for 15min, and rinsing the cell with PBS for 2 times for 1min each time; gently wiping off cells inside the cells with a cotton swab; randomly selecting 3 different visual field photographs under an inverted microscope and calculating the cell number; the experiment was repeated three times and the results were counted.
From fig. 4C, transwell migration invasion experiments demonstrate the ability of transfected ALG3 siRNA to promote differentiation of trophoblasts into EVT cells and promote migration invasion; while ALG3 cDNA inhibits trophoblast differentiation into EVT cells and reduces migration invasion.
Example 5: cell fusion assay to test the ability of trophoblast cells to differentiate into STB cells
(1) Trophoblast cells are typically dosed with an adenylate cyclase activator (Forskolin) at a confluence of about 20% at a concentration of typically 50-100nM and after 48 hours of treatment the cells spontaneously fuse.
(2) Identification of syncytia: and detecting the expression condition of the CGB through a Western blot experiment.
As can be seen from FIG. 4A, transfected ALG3 siRNA promoted the ability of trophoblast cells to be integrated, i.e., transformed into STB cells, whereas transfected ALG3 cDNA inhibited the ability of trophoblast cells to differentiate into STB cells.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (7)
1. A biological marker for diagnosing abortion, characterized in that said biological marker is α1,3 mannose (manα1,3 Man) or/and α1,3 mannosyltransferase (ALG 3).
2. Use of α1,3 mannose (manα1,3 Man) or/and α1,3 mannosyltransferase (ALG 3) as claimed in claim 1 for the preparation of a reagent or kit for diagnosing abortion.
3. The use according to claim 2, wherein the method of detection of the reagent or kit comprises immunohistochemical experiments, lectin-imprinting experiments, western-immunoblotting experiments and ELISA experiments.
4. Use according to claim 3, characterized in that α1,3 mannose (manα1,3 Man) is detected by a lectin blotting assay.
5. The use according to claim 3, wherein the alpha 1,3 mannosyltransferase (ALG 3) is detected by immunohistochemical, western immunoblotting 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 detected is villus tissue or serum detection.
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