CN114217074A - Biomarker for assisting in diagnosing recurrent abortion and application thereof - Google Patents
Biomarker for assisting in diagnosing recurrent abortion and application thereof Download PDFInfo
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- CN114217074A CN114217074A CN202111506638.9A CN202111506638A CN114217074A CN 114217074 A CN114217074 A CN 114217074A CN 202111506638 A CN202111506638 A CN 202111506638A CN 114217074 A CN114217074 A CN 114217074A
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Abstract
The invention discloses a biomarker for assisting in diagnosing recurrent abortion and application thereof, and belongs to the technical field of biology. The invention discovers that the change of LeY oligosaccharide on MEST can change the migration invasion and proliferation capacity of trophoblast cells, and the LeY oligosaccharide on MEST and MEST can be used as a biological marker for auxiliary diagnosis of recurrent abortion, which is helpful for the prediction and diagnosis of recurrent abortion, deeply understands the embryo implantation mechanism and also provides a new theoretical basis for the clinical treatment of recurrent abortion with glycoprotein as a target.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a biomarker for assisting in diagnosing recurrent abortion and application thereof.
Background
Successful pregnancy requires functional synchronization of both the embryo and the endometrium. The mature embryo is positioned, adhered and invaded into the endometrium to complete the embryo implantation. The embryo consists of an inner cell mass and outer trophoblast cells, which act as "drivers" and "feeders" for the embryo. However, dysfunction of trophoblast cells can cause abortion. In early pregnancy (< 20 weeks) of women, the natural loss of less than 500g of pregnancy is called Spontaneous Abortion (spirochetous Abortion), which is a common bad outcome in obstetrics and gynecology. However, spontaneous abortion (RSA) occurring twice or more continuously in early pregnancy is recurrent abortion. The etiology of recurrent abortion is complex, and immune factors, endocrine factors, genetic factors, thrombotic diseases, infection and the like are common. Currently, there is no clinical method for diagnosing and preventing RSA, so that the search for possible pathogenesis of recurrent abortion in a new field is one of the hot spots of reproduction.
Although much research is currently being conducted to elucidate the causes of RSA and to predict, or even prevent, the disease before RSA appears. The pathogenesis of RSA is not clear. Currently, the RSA can only diagnose by elimination methods including: inquiring about related medical history, perfecting physical examination and related auxiliary examination to find out etiology. Nevertheless, more than 50% of RSA patients have unknown etiology. This group of patients with recurrent abortion of unknown origin can only be treated by active immunization and passive immunization. Therefore, research on pathogenesis and diagnostic indexes of RSA is particularly important.
Disclosure of Invention
In view of the above, the present invention provides a biomarker for assisting in diagnosing recurrent abortion and applications thereof.
The purpose of the invention is realized by the following modes:
mesoderm-specific transcription homolog protein (MEST) plays an important role in embryonic development and migration invasion, dysfunction of the Mesoderm-specific transcription homolog protein can cause early spontaneous abortion, dead fetuses and embryonic tumors, and imprinted gene MEST is highly expressed in human placenta tissues, mainly expressed in choriotrophoblast cells and vascular endothelial cells and is an essential gene for regulating the growth and development of embryos and placentas, and a glycosylation site Asn163 is arranged on the MEST according to the prediction of the glycosylation site. During the pregnancy process, both the embryonic trophoblast and endometrial epithelial cell surfaces are highly glycosylated.
The invention provides a biomarker for assisting in diagnosing recurrent abortion, wherein the biomarker is LeY oligosaccharide [ Fuc1 → 2Gal beta 1 → 4(Fuc1 → 3) GlcNAc beta 1 → R ] on MEST.
Further, the attachment site of the LeY oligosaccharide is Asn163 on MEST.
In another aspect, the invention provides the use of LeY oligosaccharide on MEST in the preparation of a reagent or a kit for the auxiliary diagnosis of recurrent spontaneous abortion.
Further, the detection method of the reagent or the kit comprises an immunoprecipitation method and an immunofluorescence method.
Further, the detected pathological sample is villus tissue.
Further, the detected pathological sample is trophoblast cells.
Further, when LeY oligosaccharide synthesis on MEST is reduced, the proliferation and migratory invasion abilities of trophoblast cells are inhibited, resulting in recurrent abortion.
Further, when LeY oligosaccharide synthesis on MEST is decreased, binding to the translation initiation inhibitor eIF4E2 is decreased, thereby inhibiting translation of proteins associated with migratory invasion and proliferative capacity, resulting in recurrent abortion.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention discovers that the change of LeY oligosaccharide on MEST can change the migration, invasion and proliferation capacity of trophoblast cells for the first time, and LeY oligosaccharide on MEST and MEST can be used as a biological marker for the auxiliary diagnosis of recurrent abortion, which is helpful for the diagnosis of recurrent abortion, deeply understands the action mechanism of LeY oligosaccharide on MEST and provides an auxiliary diagnosis target for recurrent abortion.
2. The invention discovers that the glycosylation site mutation of MEST can inhibit the synthesis of LeY oligosaccharide, inhibit the proliferation capacity and the migration invasion capacity of trophoblast cells and reduce the combination with a translation initiation inhibitor eIF4E2, thereby inhibiting the translation of proteins related to the migration invasion and proliferation capacity.
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 shows the detection of the change of MEST and the presence of LeY oligosaccharide thereon and of LeY oligosaccharide after the mutation of MEST in the embryonic trophoblast cells of different experimental groups (empty vector group, transfected MEST cDNA group and MEST mutant group) by immunoprecipitation.
FIG. 2 shows cellular immunofluorescence (A), EdU proliferation (B), Transwell migration invasion (C) and cell scratch (D) of the embryonic trophoblasts of different experimental groups (empty vector, MEST-transfected cDNA and MEST mutant).
FIG. 3 shows the confirmation of the interaction of eST with eIF4E2 in fetal trophoblast cells from different experimental groups (empty vector, transfected MEST cDNA and MEST mutant) by immunoprecipitation (A) and confocal cells (B).
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: the presence of LeY oligosaccharide on MEST of embryo trophoblast cells of different experimental groups (empty vector group, transfection MEST cDNA group and MEST mutation group) and the change of the LeY oligosaccharide after MEST mutation are detected.
1. Cells in the logarithmic growth phase are taken and observed for contamination, and are inoculated in a 3.5cm cell culture dish one day before transfection, and the cell density should reach 60-80% coverage on the day of transfection. Before transfection, the serum-containing medium is aspirated, a serum-free medium is added, and the medium is placed in an incubator for 2 hours and then taken out to achieve the aim of starvation.
(1) Solution A: 4ug vector, 4ug MEST-His-cDNA and 4ug MEST-His-MUT (Asn163) were dissolved in 250. mu.L of DMEM/F12 medium (no serum and double antibody), the tube bottom was flicked with a finger, and placed on a clean bench and allowed to stand for 5 minutes.
(2) And B, liquid B: mu.L of Lipo2000 was dissolved in 250. mu.L of DMEM/F12 medium (no serum or double antibody), the bottom of the tube was flicked with a finger, and the tube was left on a clean bench for 5 minutes.
(3) Slowly dropping the solution A in the step (2) into the solution B by using a pipette, gently mixing the solution A and the solution B uniformly, and standing the mixture at room temperature for 20 minutes to form a liposome-encapsulated DNA complex.
(4) The cells to be transfected were gently washed once with PBS and 500. mu.L of the complex in step (3) was added.
(5) After the cell culture dish is sprayed with alcohol and is gently placed back into the incubator for 5-6 hours, the cell culture dish is replaced by a complete culture medium containing 10% serum, and the cell culture dish is cultured at 37 ℃ and 5% CO2And after continuously culturing for 48-72 hours under the condition of 90% humidity, detecting the transfection level and carrying out subsequent tests.
2. Immunoprecipitation (IP)
(1) Protein extraction: the embryonic trophoblast cells in the logarithmic growth phase were plated in 10cm dishes until they had grown to 60% -80% confluence and were in good condition, and after 48 hours of cell transfection, the cells were trypsinized, collected and pelleted in 1.5mL EP tubes, lysed with 1mL of an IP lysate (containing 10. mu.L of protease inhibitor) on ice for 30 minutes, then centrifuged at 12000rpm for 10 minutes, the supernatant was removed, transferred to a new EP tube, and the pelleted cells were discarded. Taking out a small amount of total protein lysate, quantifying by using BCA protein, and calculating the total protein with the same quality to perform subsequent tests.
(2) Binding of magnetic beads to antibodies
Taking out the magnetic beads from 4 ℃, turning the magnetic beads upside down and mixing the magnetic beads evenly, extracting 50 mu L of magnetic bead suspension into a 1.5mL EP tube, placing the EP tube on a magnetic frame to remove supernatant, taking the 1.5mL EP tube off the magnetic frame, using 200 mu L binding buffer to resuspend and wash the magnetic beads, placing the EP tube on the magnetic frame to suck the liquid, repeating the operation for 2 times, and finally only remaining the magnetic beads.
② calculate the volume of Antibody 6 His, His-Tag Monoclonal Antibody (Proteintech, Wuhan, China, 1-10. mu.g), add the corresponding Antibody volume in 200. mu.L Ab Binding & Washing Buffer, resuspend the beads with shaker.
Thirdly, placing the mixed solution of the antibody and the magnetic beads on a shaking bed, incubating for 10-30 minutes (the incubation time is properly adjusted according to the experimental result), then taking off the EP tube, placing the EP tube on a magnetic frame, standing for 30 seconds, and slightly absorbing and removing the supernatant by using a pipette. The EP tube was removed and placed on an EP tube stand.
And fourthly, 200 mu L of Ab Binding & Washing Buffer is added into an EP tube, the mixture of the magnetic beads and the antibodies is gently resuspended by a pipette gun for at least 2 times, the gentle resuspension is cut off, and finally the Ab Binding & Washing Buffer is removed on a magnetic frame.
(3) Antigen co-immunoprecipitation
Putting the EP tube obtained in the step (2) on an EP tube frame, adding 1000 mu L of the protein lysate sample containing the antigen (MEST, LeY oligosaccharide) obtained in the step (1), and gently resuspending the magnetic bead-antigen-antibody mixture by using a pipette gun.
② placing the antigen-antibody-magnetic bead mixed solution on a shaking table, incubating for 10-60 minutes (properly adjusting incubation time according to experimental results), then placing the EP tube on a magnetic frame, and absorbing supernatant.
③ Place the EP tube on the EP tube holder, add 200. mu.L of Washing Buffer to the EP tube, gently wash the antigen-antibody-magnetic bead mixture, and then add 100. mu.L of Washing Buffer to the EP tube. After mixing with a pipette, the mixture was aspirated and placed in another EP tube.
And fourthly, placing the EP tube on a magnetic frame, removing the supernatant, adding 30 mu L of precipitation Buffer into the tube to dissolve the antigen-antibody-magnetic bead mixed solution, then adding 30 mu L of Loading Buffer into the tube, mixing the mixture evenly, incubating the mixture in a water bath kettle at 70 ℃ for 10 minutes, and detecting the immunoprecipitation result by using MEST (Proteitech, Wuhan, China) and LeY (Abcam, USA) through a Western blot method. Note that Western blot detection is carried out on the best day, and the storage time at-20 ℃ is not suitable for too long.
As can be seen from FIG. 1, the embryonic trophoblast cells of the empty vector group expressed only a trace amount of MEST and LeY oligosaccharides, the embryonic trophoblast cells of the transfected MEST cDNA group expressed a large amount of MEST and LeY oligosaccharides, and the embryonic trophoblast cells of the MEST mutant group expressed MEST but did not express LeY oligosaccharides, and the MEST N-glycosylation site mutation inhibited the synthesis of LeY oligosaccharides on MEST.
Example 2: detection of MEST expression and functional Change in MEST in trophoblast cells after overexpression of MEST and MEST mutation
1. Cellular immunofluorescence
Climbing sheets: after trypsinization, the 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 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 slides were removed from the blocking solution, blotted dry on filter paper, and incubated with primary antibody overnight at 4 deg.C (rabbit anti-human MEST1: 150).
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.
A fluorescently-labeled secondary antibody (FITC-labeled goat anti-rabbit IgG) is added dropwise, and the mixture is incubated at room temperature for 1h and washed 6 times with PBS for 5min each time.
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.
2. EdU proliferation assay
(1) Cell culture: taking cells in logarithmic growth phase, 5X 10 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.
3. 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 the cells to be treated in the incubator, digesting with pancreatin, centrifuging at 800rpm for 4min, resuspending with serum-free medium, counting for 3 times with counting plate, and adjusting 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 24-well plate in a Transwell chamber, sucking 200. mu.L of cell suspension from the cell suspension, adding the cell suspension to an upper chamber, placing the upper chamber in an incubator at 37 ℃ and continuing culturing for a plurality of hours
(4) Dyeing with a live cell dye: adding a living Cell dye (Cell-Tracker Green CMGDA living Cell tracing probe) (1: 1000) into the chamber, fixing with methanol for 15min, and rinsing the chamber with PBS for 5min for 2 times; 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.
4. Cell scratch test
(1) Preparation work: all instruments capable of sterilization are sterilized, and the ruler and marker pen are irradiated by ultraviolet for 30min before operation (in a super clean bench)
(2) A marker pen is firstly used at the back of the 6-hole plate, a straight ruler is used for comparison, transverse lines are uniformly drawn, and the transverse lines cross the through holes approximately every 0.5-1 cm. Each hole passes through at least 5 lines.
(3) About 5X 10 addition to the well plate5The specific number of each cell is different from cell to cell, and the cells can be fully filled overnight.
(4) The gun head is used on the next day, the ruler is compared, the scratch is vertical to the transverse line at the back as much as possible, and the gun head is vertical and does not incline.
(5) Cells were washed 3 times with PBS, scraped cells were removed, and serum-free medium was added.
(6) Put in 5% CO at 37 DEG C2Culturing in an incubator. Samples were taken at 0, 6, 12, 24 hours and photographed.
FIG. 2A further verifies the results of example 1, and from FIG. 2B, it can be seen that the glycosylation site mutation of MEST inhibits the proliferative capacity of trophoblast cells; as can be seen from FIGS. 2C and 2D, the glycosylation site mutation of MEST inhibits the migratory invasion capacity of trophoblast cells.
Example 3: immunoprecipitation (IP) and confocal cell confirmation of MEST interaction with eIF4E2
Immunoprecipitation (IP)
(1) Protein extraction: the embryonic trophoblast cells in the logarithmic growth phase were plated in 10cm dishes until they had grown to 60% -80% confluence and were in good condition, and after 48 hours of cell transfection, the cells were trypsinized, collected and pelleted in 1.5mL EP tubes, lysed with 1mL of an IP lysate (containing 10. mu.L of protease inhibitor) on ice for 30 minutes, then centrifuged at 12000rpm for 10 minutes, the supernatant was removed, transferred to a new EP tube, and the pelleted cells were discarded. Taking out a small amount of total protein lysate, quantifying by using BCA protein, and calculating the total protein with the same quality to perform subsequent tests.
(2) Binding of magnetic beads to antibodies
Taking out the magnetic beads from 4 ℃, turning the magnetic beads upside down and mixing the magnetic beads evenly, extracting 50 mu L of magnetic bead suspension into a 1.5mL EP tube, placing the EP tube on a magnetic frame to remove supernatant, taking the 1.5mL EP tube off the magnetic frame, using 200 mu L binding buffer to resuspend and wash the magnetic beads, placing the EP tube on the magnetic frame to suck the liquid, repeating the operation for 2 times, and finally only remaining the magnetic beads.
② calculate the volume of Antibody 6 His, His-Tag Monoclonal Antibody (1-10. mu.g), add the corresponding Antibody volume to 200. mu.L Ab Binding & Washing Buffer, and resuspend the beads with shaker.
Thirdly, placing the mixed solution of the antibody and the magnetic beads on a shaking bed, incubating for 10-30 minutes (the incubation time is properly adjusted according to the experimental result), then taking off the EP tube, placing the EP tube on a magnetic frame, standing for 30 seconds, and slightly absorbing and removing the supernatant by using a pipette. The EP tube was removed and placed on an EP tube stand.
And fourthly, 200 mu L of Ab Binding & Washing Buffer is added into an EP tube, the mixture of the magnetic beads and the antibodies is gently resuspended by a pipette gun for at least 2 times, the gentle resuspension is cut off, and finally the Ab Binding & Washing Buffer is removed on a magnetic frame.
(3) Antigen co-immunoprecipitation
Putting the EP tube obtained in the step (2) on an EP tube frame, adding 1000 mu L of the protein lysate sample containing the antigen (LeY oligosaccharide) obtained in the step (1), and gently resuspending the mixed solution of the magnetic beads, the antigen and the antibody by using a pipette gun.
② placing the antigen-antibody-magnetic bead mixed solution on a shaking table, incubating for 10-60 minutes (properly adjusting incubation time according to experimental results), then placing the EP tube on a magnetic frame, and absorbing supernatant.
③ Place the EP tube on the EP tube holder, add 200. mu.L of Washing Buffer to the EP tube, gently wash the antigen-antibody-magnetic bead mixture, and then add 100. mu.L of Washing Buffer to the EP tube. After mixing with a pipette, the mixture was aspirated and placed in another EP tube.
And fourthly, placing the EP tube on a magnetic frame, removing the supernatant, adding 30 mu L of precipitation Buffer into the tube to dissolve the antigen-antibody-magnetic bead mixed solution, then adding 30 mu L of Loading Buffer into the tube, uniformly mixing, incubating the mixture in a water bath kettle at 70 ℃ for 10 minutes, and detecting the immunoprecipitation result by using eIF4E2 (Proteitech, Wuhan, China) through a Western blot method. Note that Western blot detection is carried out on the best day, and the storage time at-20 ℃ is not suitable for too long.
Cellular confocal imaging
Climbing sheets: after trypsinization, the 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 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 slides were removed from the blocking solution, blotted dry with filter paper, and incubated with primary antibody overnight at 4 deg.C (goat anti-mouse MEST1:150, goat anti-rabbit EIF4E 21: 150).
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.
Adding fluorescently-labeled secondary antibody (FITC-labeled goat anti-rabbit IgG and TRITC-labeled goat anti-mouse IgG), incubating at room temperature for 1h, washing with PBS for 5min each time 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 a confocal fluorescence microscope.
As can be seen from fig. 3A and 3B, the MEST N-glycosylation site mutation inhibits the synthesis of LeY oligosaccharide, reduces the binding to the translation initiation inhibitor eIF4E2, and thereby inhibits the translation of proteins associated with migratory invasion and proliferative capacity.
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 biomarker for assisting in the diagnosis of recurrent abortion, wherein the biomarker is LeY oligosaccharide on MEST, and the LeY oligosaccharide is Fuc1 → 2Gal β 1 → 4(Fuc1 → 3) GlcNAc β 1 → R.
2. Use of LeY oligosaccharides on MEST of claim 1 in the preparation of a reagent or kit for the adjuvant diagnosis of recurrent miscarriage.
3. The use according to claim 2, wherein the method of detection of the reagent or kit comprises immunoprecipitation, immunofluorescence.
4. Use according to claim 3, characterized in that the pathological sample to be examined is villous tissue.
5. The use according to claim 4, wherein the pathological sample to be tested is trophoblast cells.
6. The use of claim 2, wherein reduced LeY oligosaccharide synthesis on MEST inhibits the proliferative and migratory invasive capacity of trophoblast cells, resulting in recurrent abortion.
7. The use of claim 2, wherein the reduction in LeY oligosaccharide synthesis on MEST reduces binding to the translation initiation inhibitor eIF4E2, thereby inhibiting translation of proteins associated with migratory invasion and proliferative capacity, resulting in recurrent abortion.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108387722A (en) * | 2018-03-19 | 2018-08-10 | 复旦大学附属妇产科医院 | Application of the 1,6- diphosphofructoses in preparing spontaneous abortion anti-abortion medicine |
| CN109925509A (en) * | 2019-03-19 | 2019-06-25 | 大连医科大学 | A kind of drug for treating choriocarcinoma |
| CN110951880A (en) * | 2019-12-30 | 2020-04-03 | 西安交通大学医学院第二附属医院 | Application of reagent for detecting lncRNA marker of hypopharynx cancer in preparation of product for diagnosing hypopharynx cancer |
| CN112322734A (en) * | 2020-11-11 | 2021-02-05 | 江苏省肿瘤医院 | Lung cancer related diagnostic marker and application thereof |
| CN112961913A (en) * | 2021-02-05 | 2021-06-15 | 山东大学第二医院 | Application of lncRNA in diagnosis and treatment of recurrent abortion |
| CN113406326A (en) * | 2021-06-01 | 2021-09-17 | 大连医科大学 | Biological marker for predicting preeclampsia and application thereof |
| CN113755570A (en) * | 2021-08-31 | 2021-12-07 | 中国人民解放军空军军医大学 | Biomarker for predicting recurrent abortion with unknown cause and application thereof |
-
2021
- 2021-12-10 CN CN202111506638.9A patent/CN114217074A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108387722A (en) * | 2018-03-19 | 2018-08-10 | 复旦大学附属妇产科医院 | Application of the 1,6- diphosphofructoses in preparing spontaneous abortion anti-abortion medicine |
| CN109925509A (en) * | 2019-03-19 | 2019-06-25 | 大连医科大学 | A kind of drug for treating choriocarcinoma |
| CN110951880A (en) * | 2019-12-30 | 2020-04-03 | 西安交通大学医学院第二附属医院 | Application of reagent for detecting lncRNA marker of hypopharynx cancer in preparation of product for diagnosing hypopharynx cancer |
| CN112322734A (en) * | 2020-11-11 | 2021-02-05 | 江苏省肿瘤医院 | Lung cancer related diagnostic marker and application thereof |
| CN112961913A (en) * | 2021-02-05 | 2021-06-15 | 山东大学第二医院 | Application of lncRNA in diagnosis and treatment of recurrent abortion |
| CN113406326A (en) * | 2021-06-01 | 2021-09-17 | 大连医科大学 | Biological marker for predicting preeclampsia and application thereof |
| CN113755570A (en) * | 2021-08-31 | 2021-12-07 | 中国人民解放军空军军医大学 | Biomarker for predicting recurrent abortion with unknown cause and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 刘帅: "FUT4 过表达促进胚胎细胞与子宫内膜细胞的黏附", 中国生物化学与分子生物学报, vol. 10, no. 27, 31 October 2011 (2011-10-31), pages 968 * |
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