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Application of decidua NK cells and decidua NK cell exosomes from cell subsets in preparing drugs and auxiliary therapeutic agents for treating diseases related to infertility

A technology of NK cells and cell subsets, applied in the field of biomedicine, can solve the problems of no disclosure or prompt, and achieve the effect of improving the development rate, the implantation rate and the farrowing rate

Active Publication Date: 2020-01-24
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 decidua NK cells and decidua NK cell exosomes from cell subsets in preparing drugs and auxiliary therapeutic agents for treating diseases related to infertility
  • Application of decidua NK cells and decidua NK cell exosomes from cell subsets in preparing drugs and auxiliary therapeutic agents for treating diseases related to infertility
  • Application of decidua NK cells and decidua NK cell exosomes from cell subsets in preparing drugs and auxiliary therapeutic agents for treating diseases related to infertility

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 application of decidua NK cells and decidua NK cell exosomes from cell subsets in preparing drugs and auxiliary therapeutic agents for treating diseases related to infertility.Experiments prove that the exosomes treat endometrium growth disorders by promoting endometrial thickness increasing, improving endometrial cell activity, reducing endometrial cell damage, promoting VEGF expression, maintaining stem features of endometrium matrix cells and stimulating proliferation, so that the pregnancy success rate of endometrium damage model mice is increased to 50-70% from 20%; and the exosomes treat diseases related to maternal-fetal immunologic tolerance disorders by playing a role of immunologic tolerance, reducing a spontaneous abortion rate and improving a help T lymphocyte level. Besides, the exosomes can effectively increase the developmental rate of zygotes fertilized no matter in vitro or in vivo in a multiple mode, meanwhile, an in-vitro fertilization rate, an implantation rate of implantation and a birth rate are also increased, and positive assisting effects on infertility treatment are achieved.

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