Biomarker for auxiliary diagnosis of recurrent abortion and application thereof

A recurrent miscarriage and biomarker technology, applied in disease diagnosis, biological testing, biomaterial analysis, etc., can solve problems such as unclear pathogenesis and achieve the effect of inhibiting protein translation

Pending Publication Date: 2022-03-22
DALIAN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the pathogenesis of RSA is still unclear
At present, RSA can only be diagnosed by exclusion method, including: asking relevant medical history, improving physical examination and related auxiliary examinations to find the cause

Method used

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  • Biomarker for auxiliary diagnosis of recurrent abortion and application thereof
  • Biomarker for auxiliary diagnosis of recurrent abortion and application thereof
  • Biomarker for auxiliary diagnosis of recurrent abortion and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Detection of the presence of LeY oligosaccharides on MEST of embryonic trophoblast cells in different experimental groups (empty vector group, MEST cDNA transfection group and MEST mutation group) and the changes of LeY oligosaccharides after MEST mutation.

[0024] 1. Take the cells in the logarithmic growth phase and observe whether there is contamination. The day before transfection, inoculate in a 3.5cm cell culture dish, and the cell density should reach 60% to 80% coverage on the day of transfection. Before transfection, remove the serum-containing medium, add serum-free medium, put it in the incubator for 2 hours, and take it out to achieve starvation.

[0025] (1) Solution A: Dissolve 4ug vector, 4ug MEST-His-cDNA and 4ug MEST-His-MUT(Asn163) in 250μL of DMEM / F12 medium (no serum and no double antibody), flick the bottom of the tube with your fingers, Put it on the ultra-clean table and let it stand for 5 minutes.

[0026] (2) Solution B: Dissolve 2....

Embodiment 2

[0043] Example 2: Detection of the expression and functional changes of MEST in trophoblast cells after overexpressing MEST and MEST mutation

[0044] 1. Immunofluorescence of cells

[0045] Slide: After the cells are digested with trypsin, centrifuge at 800 rpm for 4 minutes, resuspend in fresh medium, put the cell suspension into a petri dish with slide, and let the cells grow on the slide.

[0046] Collection: remove the culture medium in the culture dish, wash with PBS 3 times, 3 minutes each time.

[0047] Fixation: add 4% paraformaldehyde to the petri dish obliquely, and fix it for 20 minutes.

[0048] Paraformaldehyde was discarded, washed 3 times with PBS, 3 min each time.

[0049] Sealing: Select slides and place them in a wet box, add immunostaining blocking solution to seal for 1 hour.

[0050] Primary antibody incubation: Clip the slices from the blocking solution, blot dry on filter paper, incubate with primary antibody, overnight at 4°C (rabbit anti-human MEST...

Embodiment 3

[0074] Example 3: Immunoprecipitation (IP) and cell confocal verification of MEST interaction with eIF4E2

[0075] Immunoprecipitation (IP)

[0076] (1) Protein extraction: spread the embryonic trophoblast cells in the logarithmic growth phase in a 10cm culture dish, and when they grow to 60%-80% confluence, and the cells are in good condition, after 48 hours of cell transfection, use pancreatic Digest the cells with enzymes, collect the cell pellet in a 1.5mL EP tube, lyse with 1mL IP lysis solution (containing 10μL protease inhibitor) on ice for 30 minutes, then centrifuge at 12000rpm for 10 minutes, take the supernatant, and transfer it to a new EP tube. Discard the cell pellet. A small amount of total protein lysate was taken out and quantified with BCA protein, and the total protein of the same mass was calculated for subsequent experiments.

[0077] (2) Binding of magnetic beads and antibodies

[0078] ①Take out the magnetic beads from 4°C, mix them upside down, draw ...

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Abstract

The invention discloses a biomarker for auxiliary diagnosis of recurrent abortion and application thereof, and belongs to the technical field of biology. It is found that the migration, invasion and proliferation capacities of trophoblast cells can be changed by changing the LeY oligosaccharide on the MEST, and the MEST and the LeY oligosaccharide on the MEST can serve as biological markers for auxiliary diagnosis of the recurrent abortion, so that prediction and diagnosis of the recurrent abortion are facilitated, the embryo implantation mechanism is deeply understood, and the development of the recurrent abortion is promoted. And a new theoretical basis can be provided for clinical treatment of recurrent abortion taking glycoprotein as a target spot.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a biomarker for auxiliary diagnosis of recurrent miscarriage and its application. Background technique [0002] A successful pregnancy requires functional synchronization of both the embryo and the endometrium. The mature embryo settles, adheres, and invades the endometrium, completing embryo implantation. The embryo is composed of the inner cell mass and the outer trophoblast cells, in which the trophoblast cells play the role of "driver" and "feeder" for the embryo. However, abnormal function of trophoblast cells can cause miscarriage. In women's early pregnancy (≤20 weeks), the natural loss of less than 500g of pregnancy is called spontaneous abortion (Spontaneous Abortion), which is a common adverse outcome in obstetrics and gynecology. However, two or more consecutive spontaneous abortions in early pregnancy are recurrent spontaneous abortions (RSA). The etiology...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/6893G01N2800/368G01N2333/47G01N2400/02
Inventor 刘帅燕秋王浩崔馨元王录尧
Owner DALIAN MEDICAL UNIVERSITY
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