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Usage of circulating exosome long-chain non-coding RNA-TC0101441 as marker for diagnosing endometriosis

An endometrium, non-coding technology, applied in the field of medical detection, can solve problems such as the difficulty of early diagnosis of endometriosis, achieve the effect of promoting invasion and metastasis, and the technical effect is obvious

Active Publication Date: 2018-10-19
THE OBSTETRICS & GYNECOLOGY HOSPITAL OF FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide circulating exosome long-chain non-coding RNA-TC0101441 as a biomarker for the diagnosis of endometriosis, and the use of the circulating exosome long-chain non-coding RNA-TC0101441 Solve the technical problems in the prior art that are difficult for early diagnosis of endometriosis

Method used

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  • Usage of circulating exosome long-chain non-coding RNA-TC0101441 as marker for diagnosing endometriosis
  • Usage of circulating exosome long-chain non-coding RNA-TC0101441 as marker for diagnosing endometriosis
  • Usage of circulating exosome long-chain non-coding RNA-TC0101441 as marker for diagnosing endometriosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1 Expression of metastasis-promoting lncRNA-TC0101441 in ectopic, eutopic, and normal endometrial tissues in patients with endometriosis

[0016] Fluorescence in situ hybridization (FISH):

[0017] 1) Fix and dehydrate the ectopic, eutopic and normal endometrial tissue samples in endometriosis;

[0018] 2) The lncRNA-TC0101441 probe was added to the hybridization buffer, and the in situ hybridization kit (Biochain, Hayward, CA, USA) was used for in situ hybridization.

[0019] 3) After the nuclei were stained with DAPI, they were observed with a fluorescence microscope.

[0020] Expression of metastasis-promoting lncRNA-TC0101441 in ectopic, eutopic, and normal endometrial tissues of patients with endometriosis detected by real-time fluorescent quantitative PCR (qRT-PCR)

[0021] Detection process:

[0022] Real-time fluorescence quantitative PCR method:

[0023] The primer sequence is as follows: lncRNA-TC0101441:

[0024] Upstream primer 5'- caaggcaggtgag...

Embodiment 2

[0034] Example 2 Extraction and identification of exosomes from the supernatant of ectopic endometrial stromal cells (ECSCs)

[0035] Cell supernatant exosome extraction and identification process:

[0036] 1) Primary culture of ectopic endometrial stromal cells (3# and 5# ECSCs) highly expressing TC0101441, and culture ECSCs in serum-free medium for 3 days;

[0037] 2) The expression of TC0101441 in 3# and 5# ECSCs was knocked down by siRNA and lentivirus knockdown (knockdown, KD) technology, and TC0101441 knockdown and control ECSCs were obtained.

[0038] 3) Use exosome extraction kit #44578259 (Thermo Fisher Scientific, CA, USA) to extract exosomes in the above cell supernatant.

[0039] 4) Transmission electron microscope (TEM) identification of exosomes: Take 20 ul of the exosome solution obtained above, add 20 ul of PBS to dilute it, and drop it on the sample-loading copper grid dedicated to the microscope, place it at room temperature for 3 minutes, and dry it in the ...

Embodiment 3

[0042] Example 3 Exosomes secreted by donor ectopic endometrial stromal cells (ECSCs) supernatant were endocytosed by recipient cells

[0043] Exosome labeling and endocytosis experiments:

[0044] 1) Exosomes were labeled with green dye PKH67 (green dye, Sigma-Aldrich, St. Louis, MO, USA),

[0045] 2) The exosomes secreted by the supernatant of donor ectopic endometrial stromal cells (ECSCs) were co-cultured with recipient ECSCs, and the endocytosis of exosomes was observed by fluorescence microscope after 4 hours.

[0046] We found that: if image 3 As shown, exosomes released from the supernatant of TC0101441 knockdown and control ECSCs can be endocytosed by recipient cells, suggesting that exosomes and exosomes TC0101441 can be transported from donor ectopic endometrial stromal cells (ECSCs) to recipient cells.

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Abstract

The invention discloses a usage of circulating exosome long-chain non-coding RNA-TC0101441 as a marker for diagnosing endometriosis. A gene sequence of the circulating exosome long-chain non-coding RNA-TC0101441 is as shown in SEQ ID NO. 1. A kit for diagnosing the endometriosis is provided, the kit contains a reagent for detecting the circulating exosome long-chain non-coding RNA-TC0101441, the reagent is prepared from an upstream primer sequence and a downstream primer sequence, wherein the upstream primer sequence is as shown in SEQ ID NO. 2, and the downstream primer sequence is as shown in SEQ ID NO. 3. Condition that exosome TC0101441 can be detected in serum of an endometriosis patient is discovered, and the expression level of the exosome TC0101441 is apparently higher than that ina non-endometriosis female, so the circulating exosome TC0101441 can be used as a novel non-invasive biomarker for the early diagnosis of the endometriosis and illness monitoring.

Description

technical field [0001] The invention belongs to the field of medical detection, and relates to a serum and cell supernatant exosome lncRNA-TC0101441, specifically circulating exosome long-chain non-coding RNA-TC0101441 as a biomarker for diagnosing endometriosis and use in diagnostic reagents. Background technique [0002] Endometriosis (EMs, referred to as "endometriosis") is an aggressive, estrogen-dependent disease characterized by the presence of endometrial glands and stroma in other parts of the uterus . The main clinical manifestations of endometriosis are pain and infertility, which seriously affect the quality of life of women. Endometriosis affects 10%-15% of reproductive-age women worldwide and more than 70 million people. Mild lesions of endometriosis may have tiny lesions on the surface of the organs, and severe lesions may lead to extensive adhesions of the bowel, bladder, and ureter in the pelvic organs. [0003] Laparoscopy is not practical as an invasive...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/158C12Q2600/178
Inventor 邱君君华克勤唐晓燕林晓静郑婷婷
Owner THE OBSTETRICS & GYNECOLOGY HOSPITAL OF FUDAN UNIV
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