MiRNA marker for diagnosing early abortion and application thereof

A technology of early miscarriage and markers, applied in the field of miRNA biomarkers, can solve the problems of early miscarriage without molecular markers and unsatisfactory treatment

Active Publication Date: 2022-05-06
ATTACHED OBSTETRICS & GYNECOLOGY OSPITAL MEDICALCOLLEGE ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the role of miR-29a affecting the proliferation and decidualization of ESCs and its potential application as a molecular marker of early abortion remain to be elucidated
[0006] At present, there is no effective molecular marker for early miscarriage, and the treatment after miscarriage is unsatisfactory due to the inadequ

Method used

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  • MiRNA marker for diagnosing early abortion and application thereof
  • MiRNA marker for diagnosing early abortion and application thereof
  • MiRNA marker for diagnosing early abortion and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: qRT-PCR detection of miR-29a expression levels in decidual tissues of endometrium and early abortion patients in different pregnancy cycles

[0025] (1) RNA extraction:

[0026] Part of the tissue was cut with surgical scissors and placed in a homogenization tube containing 1 mL of Trizol, thoroughly ground using an electric homogenizer (operated on ice), incubated for 5 min, 12,000 rpm, and centrifuged for 10 min. Aspirate the supernatant into a new 1.5mL centrifuge tube, add 200μL of chloroform, shake well, let stand at room temperature for 2min, centrifuge at 12000rpm at 4°C for 10min. Pipette the supernatant into a new 1.5mL centrifuge tube, add 600μL of isopropanol, mix well, let stand at room temperature for 15min, centrifuge at 4°C, 12000rpm for 15min, and discard the supernatant. Add 1 mL of 75% absolute ethanol (750 μL of absolute ethanol and 250 μL of DEPC water) to rinse the precipitate, centrifuge at 12,000 rpm for 5 min at 4° C., and discard the...

Embodiment 2

[0037] Example 2: Isolation and culture of human primary ESCs and induction of decidualization model

[0038] Take human uterine tissue, move it to an ultra-clean workbench, and put it into PBS containing double antibodies; carefully clean up excess peripheral tissue, clean blood, gently scrape off the surface mucosa, separate endometrial tissue, and cut the tissue into about 1 mm3 pieces. Wash the tissue once with PBS, collect the tissue in a centrifuge tube, add the digestive solution and digest it in a water bath shaker at 37°C, add complete medium after 40 minutes to stop the digestion, blow the digestive solution repeatedly with a straw, centrifuge the digestive solution at 300g for 5 minutes, discard the supernatant, and keep Cell pellet.

[0039] (1) Cell culture:

[0040] Resuspend the cell pellet with the complete human ESCs medium, inoculate it on a polylysine pre-coated culture dish, and culture it statically in a 5% CO2 constant temperature incubator at 37°C, cha...

Embodiment 3

[0043] Example 3: CCK-8 detects the effect of miR-29a inhibitors on decidualization-induced ESCs cell proliferation

[0044] (1) Cell culture:

[0045]The cells were placed in human ESCs complete medium containing 10% fetal bovine serum and 1% double antibody (100U / ml penicillin and streptomycin), and cultured in an incubator at 37°C, containing 5% CO2 and saturated humidity. The medium was replaced once every 2-3 days, and digested and passaged with 0.25% trypsin on 5-6 days.

[0046] (2) Experimental grouping:

[0047] ① Control group;

[0048] ② Decidualization group;

[0049] ③ Decidualization + inhibitor control group;

[0050] ④ Decidualization + miR-29a inhibitor group.

[0051] When the ESCs grew to about 60%, decidualization in vitro was performed. Primary human endometrial stromal cells were cultured in medium containing 2% FBS, estradiol E2 (10nM), medroxyprogesterone P4 (1μM), cAMP (0.5mM) for 5 days to induce decidualization, miR-29a inhibitor Treatment was...

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Abstract

The invention discloses a novel biomarker for early abortion diagnosis, and particularly relates to a method for diagnosing, predicting and/or monitoring early abortion by hsa-miR-29a-3p and application of hsa-miR-29a-3p. According to the invention, hsa-miR-29a-3p is proposed as a marker for early pregnancy embryo implantation and bad pregnancy outcome for the first time, and then the regulation effect of hsa-miR-29a-3p on decidualization is proved by using an in-vitro endometrial stromal cell decidualization model. The temporal-spatial expression specificity based on the marker has wide clinical diagnosis and prediction application prospects. According to the specific expression of the hsa-miR-29a-3p in different pregnancy periods and in decidua tissues of early abortion, the early abortion of pregnancy can be effectively predicted and diagnosed.

Description

[0001] Technical field [0002] The invention belongs to the field of biological detection and diagnosis, and in particular relates to a miRNA biomarker for early pregnancy abortion diagnosis and application thereof. Background technique [0003] Miscarriage is a common problem, affecting 25% of pregnant women under the age of 39. Miscarriage affects approximately one-third of pregnant women and occurs most often during the second and first trimesters. In recent years, the incidence of early miscarriage has been increasing year by year. Although the development of early intervention methods and drug treatment has achieved certain effects on the prevention of early miscarriage, the lack of early miscarriage prediction and diagnosis methods makes it difficult to effectively prevent and control this pregnancy disease. [0004] Normal communication between mother and fetus is fundamental to maintaining a successful and healthy pregnancy. Endometrial stromal cells (ESCs) continue...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12N15/113C12N15/11
CPCC12Q1/6883C12Q2600/158C12Q2600/178
Inventor 刘爱霞王迪民
Owner ATTACHED OBSTETRICS & GYNECOLOGY OSPITAL MEDICALCOLLEGE ZHEJIANG UNIV
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