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Application of exosomes derived from decidual NK cells and cell subgroups in the preparation of infertility-related disease drugs and adjuvant therapeutic agents

A technology of NK cells and infertility, which is applied in the field of biomedicine and can solve problems such as no disclosure or prompting

Active Publication Date: 2021-07-06
PHARCHOICE THERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The prior art does not disclose or suggest a technical solution for preparing products from exosomes derived from decidual NK cell subsets expressing specific markers for disease treatment

Method used

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  • Application of exosomes derived from decidual NK cells and cell subgroups in the preparation of infertility-related disease drugs and adjuvant therapeutic agents
  • Application of exosomes derived from decidual NK cells and cell subgroups in the preparation of infertility-related disease drugs and adjuvant therapeutic agents
  • Application of exosomes derived from decidual NK cells and cell subgroups in the preparation of infertility-related disease drugs and adjuvant therapeutic agents

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Screening of uterine decidua NK cell candidate markers

[0039] Statistical calculations in this example and the following examples are based on different statistical modes of the software SPSS 22.0 version for two-group comparison, multi-group comparison, and rate comparison to calculate p values. A P value of less than 0.05 was considered statistically significant.

[0040] Firstly, NK cells were isolated from the decidual tissues of 10 cases of healthy non-medical termination of early pregnancy (normal group) and 5 cases of spontaneous abortion early pregnancy decidual tissues (abortion group). For the isolation and flow cytometry of NK cells, please refer to [ Fu B, et al. Immunity, 2017, 47(6):1100-1113.e6.]. An example is as follows: Lymphocytes were digested with 1 mg / mL collagenase IV (Sigma-Aldrich) and 0.01 mg / mL DNase I (Shanghai Sangon) for 1 h, and then obtained by Percoll (GE Healthcare) density gradient centrifugation. Stromal cells and macrop...

Embodiment 2

[0041] Example 2 Preparation of uterine decidua NK cells and cell subsets

[0042] Utilize healthy non-medical reasons to terminate early pregnancy decidual tissue to prepare decidual NK cells, implement according to the NK cell separation method described in Example 1, briefly as follows: After 1 mg / mL collagenase IV (Sigma-Aldrich) and 0.01 mg / mL Lymphocytes were obtained by digesting with DNase Ⅰ (Shanghai Sangon) for 1 h, and then centrifuging with Percoll (GE Healthcare) density gradient. Culture dishes were cultured at 37°C for 2 hours to remove stromal cells and macrophages, and then NK cells were separated by flow cytometry. The resulting phenotype is CD56 bright CD16 - CD49a + decidua NK cells. Further use of antibody magnetic beads to sort CD56 bright CD16 - CD49a + CD39 + Decidual NK cell population, CD56 bright CD16 - CD49a + CD27 + Decidual NK cell population, CD56 bright CD16 - CD49a + CD160 + Decidual NK cell population, CD56 bright CD16 - CD49...

Embodiment 3

[0043] Example 3 Preparation of exosomes derived from uterine decidua NK cells and cell subsets

[0044] The freshly isolated decidual NK cells, decidual NK cell subsets and control NK cells described in Example 2 were cultured in serum-free 1640 medium for 24 hours. Centrifuge to remove cells, filter the medium supernatant with a 0.45 μm filter membrane, centrifuge at 1000 g at 4 °C for 10 min, and collect the supernatant; collect the supernatant at 4 °C, centrifuge at 2000 g for 20 min, collect the supernatant; collect the supernatant at 4 °C , centrifuge at 10000g for 30min, collect the supernatant; centrifuge the collected supernatant at 110000g for 100min, discard the supernatant, and resuspend the pellet with phosphate buffer; Filter with a 0.45 μm filter membrane to obtain exosomes. The total protein content of exosomes was detected by Bradford method (Bio-Rad Protein Assay Reagent). Exosomes were lyophilized and stored at -80°C. That is, the following six exosomes w...

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Abstract

The invention provides the use of exosomes derived from decidual NK cells and cell subgroups in the preparation of infertility-related disease medicines and auxiliary therapeutic agents. It has been confirmed by experiments that the exosomes treat the endometrium by promoting the increase of endometrial thickness, enhancing endometrial cell viability, reducing endometrial cell damage, promoting VEGF expression, maintaining endometrial stromal cell stemness and stimulating proliferation. Growth disorders, which increase the pregnancy success rate of endometrial injury model mice from 20% to 50-70%; treat maternal-fetal immunity by exerting immune tolerance, reducing spontaneous abortion rate, and increasing the level of T helper lymphocytes Tolerance related disorders. In addition, the exosomes described in the present invention can effectively increase the development rate of fertilized eggs fertilized in vivo or fertilized eggs in vitro, and at the same time improve the implantation rate and litter rate of in vitro fertilization rate transplantation. It plays a positive auxiliary role in the treatment of infertility.

Description

technical field [0001] The invention belongs to the field of biomedicine, and relates to the use of exosomes derived from decidual NK cells and cell subgroups in the preparation of infertility-related disease drugs and auxiliary therapeutic agents, the preparation method of the exosomes and the exosomes containing the exosomes. body pharmaceutical composition. Background technique [0002] The endometrial injury is mainly the injury of the endometrial basal layer, which is mainly related to curettage during pregnancy. Compared with the normal endometrium, the endometrial basal layer during pregnancy is loose and more susceptible to damage. Based on my country's national conditions and the increasing rate of induced abortion, the occurrence of endometrial damage cannot be ignored. The endometrial basal layer is damaged, which may lead to damage or loss of endometrial stem cells; at the same time, local infection and aseptic inflammation of the injured endometrium can destroy...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0783A61P15/08A61K35/17
CPCA61K35/17A61P15/08C12N5/0646C12N2509/00C12N2501/165
Inventor 胡适丁敏
Owner PHARCHOICE THERAPEUTICS INC
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