Use of serum microvesicle-asparagine endopeptidase pseudogene 1 in the diagnosis and recurrence prediction of endometriosis

A technology of asparagine and microvesicles is applied in the field of medical detection to achieve the effect of obvious technical effect

Active Publication Date: 2022-07-22
THE OBSTETRICS & GYNECOLOGY HOSPITAL OF FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The object of the present invention is to provide the purposes of serum microvesicle-asparagine endopeptidase pseudogene 1 (EV-LGMNP1), and described this circulation serum microvesicle-asparagine endopeptida

Method used

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  • Use of serum microvesicle-asparagine endopeptidase pseudogene 1 in the diagnosis and recurrence prediction of endometriosis
  • Use of serum microvesicle-asparagine endopeptidase pseudogene 1 in the diagnosis and recurrence prediction of endometriosis
  • Use of serum microvesicle-asparagine endopeptidase pseudogene 1 in the diagnosis and recurrence prediction of endometriosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1 Extraction and identification of microvesicles (EV / Ecto-ESCs) in the supernatant of ectopic endometrial stromal cells (Ecto-ESCs)

[0017] Cell supernatant microvesicle extraction and identification process:

[0018] 1) Primary culture of ectopic endometrial stromal cells (1# and 2# Ecto-ESCs)

[0019] 2) Ecto-ESCs were identified by immunohistochemical method and specific markers such as vimentin (vimentin), cytokeratin (keratin), CD45 and CD10.

[0020] 3) The microvesicles in the supernatant of the above cells were extracted with a microvesicle extraction kit ExoQuick EV precipitation solution kit (catalogueno. EXOTC50A-1; System Biosciences, Palo Alto, California).

[0021] 4) Transmission electron microscope (TEM) identification of microvesicles: Take 20ul of the microvesicle solution obtained above, add 20uL of PBS to dilute it, and then drop it onto a copper wire mesh specially designed for microscopy, place at room temperature for 3 minutes, and let i...

Embodiment 2

[0025] Example 2 Microvesicles (EV / Ecto-ESCs) secreted by ectopic endometrial stromal cells are endocytosed by recipient cells THP-1 and induce THP-1 polarization to M2 macrophages

[0026] Microvesicle labeling and endocytosis experiments:

[0027] 1) The microvesicles were labeled with the green dye PKH67 (green dye, Sigma-Aldrich, St. Louis, MO, USA).

[0028] 2) Microvesicles (EV / Ecto-ESCs) secreted by ectopic endometrial stromal cells were co-cultured with the receptor THP-1, and the endocytosis of microvesicles was observed by fluorescence microscope after 16 hours.

[0029] Validation of receptor cell THP-1 polarization towards M2 macrophages:

[0030]The expression of specific M1-type molecule CD86 and M2-type molecule CD206 in THP-1 cells was identified by real-time quantitative PCR and Western blot. GAPDH was used as an internal reference in qRT-PCR, and the primer sequences were as follows.

[0031] Table 1 CD86, CD206 primer sequences

[0032] Target G...

Embodiment 3

[0038] Example 3 Overexpressed EV-LGMNP1 secreted by donor Ecto-ESCs can promote the polarization of recipient cells THP-1 to M2 macrophages and upregulate the downstream target gene LGMN

[0039] Construction of overexpression lentiviral vector and cell transfection experiment

[0040] 1) Construction of lentiviral vector: select lentiviral vector, select specific primers according to the primer design principle, and specifically amplify the LGMNP1 gene fragment (from: >NC_000013.11:c64959518-64957561Homo sapienschromosome 13,GRCh38.p13 Primary Assembly, such as SEQ ID NO.1). The gene fragment LGMNP1 was connected to the digested lentiviral vector through the restriction sites at both ends, and then transferred into the prepared competent bacteria. After cultivation, the monoclonal colonies were identified by PCR, and then sequenced, compared and selected. A lentiviral vector overexpressing LGMNP1 was successfully expressed.

[0041] 2) Lentiviral packaging and assay: Use t...

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Abstract

Use of serum microvesicle-asparagine endopeptidase pseudogene 1 in the preparation of a biomarker for diagnosing endometriosis or predicting the recurrence of endometriosis, serum microvesicle-asparagine endopeptide The gene sequence of the enzyme pseudogene 1 is shown in SEQ ID NO.1. Also provided is a kit for diagnosing endometriosis or predicting the recurrence of endometriosis, containing reagents for detecting EV-LGMNP1, including upstream primer sequences, downstream primer sequences and probes, the sequences of which are as shown in SEQ ID NO.5-7. It was found that EV-LGMNP1 could be detected in the serum of patients with endometriosis, and its expression level was significantly higher than that of women without endometriosis, and its expression was significantly increased in patients with recurrent endometriosis. Novel non-invasive biomarkers for the diagnosis of endometriosis, prediction of endometriosis recurrence, and condition monitoring.

Description

technical field [0001] The invention belongs to the field of medical detection, and relates to a diagnostic marker, in particular to the preparation of circulating microvesicle-asparagine endopeptidase pseudogene 1 (EV-LGMNP1) as a diagnostic marker for endometriosis or prediction of uterine Use of markers and diagnostic kits for endometriosis recurrence. Background technique [0002] Endometriosis (EMs / EMT, referred to as "endometriosis") is an aggressive, estrogen-dependent disease that exists in parts other than the uterus in the form of glands and stroma of the endometrium. as characteristic. Mild lesions of endometriosis may have tiny lesions on the surface of organs, and severe lesions may lead to extensive adhesions of pelvic organs, bowel, bladder, and ureter. The main clinical manifestations of endometriosis are pain and infertility, affecting 6%-10% of women of reproductive age worldwide, seriously affecting women's quality of life. At the same time, although en...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/158C12Q2600/118
Inventor 邱君君华克勤孙淑根郭靖靖郭晨妍瞿欣瑜
Owner THE OBSTETRICS & GYNECOLOGY HOSPITAL OF FUDAN UNIV
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