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Application of miR-663a or target gene CKDN2A thereof in thin endometrium

A technology of mir-663a and endometrium, applied in the field of biomedicine, can solve problems such as decrease, increase, and unclear molecular mechanism

Pending Publication Date: 2022-06-24
PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The technical effect of this patented method involves identifying small molecules called miR-36 which are involved with cellular functions such as DNA synthesis or regulation during menstrual cycles. These compounds were found to reduce damage from oxygen deficiency caused by low blood flow after childbirths. This discovery could lead to treating women who have had their uterus removed due to these injuries.

Problems solved by technology

This patented technical solution involves studying how small placenta can help reduce pain from anemia associated with endometritis while also promoting regrowth of new vessels into the womb space around them through vasculature.

Method used

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  • Application of miR-663a or target gene CKDN2A thereof in thin endometrium
  • Application of miR-663a or target gene CKDN2A thereof in thin endometrium
  • Application of miR-663a or target gene CKDN2A thereof in thin endometrium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Extraction and identification of HucMSC-EX

[0050] Extraction of exosomes from human umbilical cord mesenchymal stem cells (HucMSC-EX): clean bench operation, take the cell culture medium, divide into sterilized centrifuge bottles or centrifuge tubes, centrifuge at 4°C, 1000 x g for 10 min, remove Cell culture supernatant, debris and dead cells in culture medium;

[0051] Then, operate on an ultra-clean bench, filter the supernatant through a 0.2mm filter, and divide into sterilized centrifuge bottles; centrifuge the supernatant culture medium in an ultra-high-speed centrifuge at 4°C, 100,000 x g for 3 hours; discard the supernatant and add 10ml PBS, ultracentrifuge, 4°C, 100,000x g for 90min; discard the PBS, resuspend the exosome-containing particles at the bottom of the tube with 50μl PBS, transfer to a sterile imported 1.5ml EP tube, seal with parafilm, and transfer to Store at -80°C.

[0052] Identification of HucMSC-EX: HucMSC-EX were identified by We...

Embodiment 2

[0054] Example 2 Construction of EEC hypoxia model

[0055] (1) Human endometrial glandular epithelial cells (EEC) were cultured under normoxic conditions, and the cells were grown to a density / plate rate of about 60-70%.

[0056] (2) During hypoxia treatment, cells were exposed to humidified hypoxic air (1% O 2 ,94%N 2 ,5%CO 2 ), placed in an incubator (Thermo), and cultured at 37°C for 1 h, 4 h, 8 h, 16 h and 24 h; EECs cultured under normoxia were used as the control group.

[0057] (3) Western blot was used to detect the expressions of VEGF (Abcam ab214424), avβ3 (Abcam ab179473) and E-Cadherin (Abcam ab40772) to identify hypoxic injury.

[0058] The result is as image 3 The results of Western Blot showed that after 4 hours of hypoxia, the hypoxia-related indicators VEGF and avβ3 were significantly up-regulated, and the expression of epithelial cell marker molecule E-Cadherin was also significantly down-regulated. Co-culture experiments were carried out.

Embodiment 3

[0059] Example 3 Hypoxic EEC to HucMSC exosome uptake experiment

[0060] HucMSC-Ex were co-cultured with EECs injured by hypoxia for 4 h and EECs treated with non-hypoxia, respectively, and then the two groups of cells were subjected to RKH67 fluorescence staining and observed under a 400-fold lens.

[0061] The specific steps are as follows. Exosome exosomes are labeled with PKH67 (company: Sigma-Aldrich, product number: MINI67-1KT). PKH67-labeled exosomes were co-cultured and incubated with HucMSCs for 24 hours; cells were fixed with paraformaldehyde (4%) for 15 minutes at room temperature; cells were punched with TBST in 0.5% Triton X-100 for 15 minutes at room temperature; labeled with DAPI staining Nuclei (4′,6-diamidino-2-phenylindole (DAPI) solution (Selleck S9980)); cellular uptake of labeled exosomes was detected using a laser scanning confocal microscope (OLYMPUS FLUOVIEW FV3000).

[0062] The result is as Figure 4 As shown, the results showed that the ability of...

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Abstract

The invention relates to the technical field of biological medicines, in particular to application of miR-663a or a target gene CKDN2A thereof in thin endometrium. The invention discloses application of a reagent for detecting the expression level of miR-663a or a target gene CKDN2A of the miR-663a in preparation of a product for detecting thin endometrium. Furthermore, the invention further discloses a biological agent for relieving or repairing the hypoxia injury EEC and application of the biological agent. According to the application, the miR-663 is screened out through a transcriptome sequencing technology, a cell function experiment and a Western blot experiment show that the miR-663 can obviously inhibit the occurrence of EEC hypoxia injury, the inhibition can be realized through a target gene CDKN2A, and a new thought is provided for the treatment of thin endometrium.

Description

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Claims

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Application Information

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Owner PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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