Application of decidua NK cells and cell subsets thereof in preparation of medicines for treating infertility related diseases

A technology of NK cells and cell subsets, applied in the field of biomedicine, can solve problems such as no disclosure or prompt, and achieve the effects of promoting VEGF expression, reducing endometrial cell damage, and reducing spontaneous abortion rate

Active Publication Date: 2020-03-06
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 produ

Method used

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  • Application of decidua NK cells and cell subsets thereof in preparation of medicines for treating infertility related diseases
  • Application of decidua NK cells and cell subsets thereof in preparation of medicines for treating infertility related diseases
  • Application of decidua NK cells and cell subsets thereof in preparation of medicines for treating infertility related diseases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Screening of uterine decidua NK cell candidate markers

[0027] 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.

[0028] 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 obtained by digesting with 1 mg / mL collagenase IV (Sigma-Aldrich) and 0.01 mg / mL DNase I (Shanghai Sangon) for 1 h, and then centrifuging with Percoll (GE Healthcare) density gradient. Culture dishes were ...

Embodiment 2

[0029] Example 2 Preparation of uterine decidua NK cells and decidua NK cell subsets

[0030] 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 - C D49a + 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...

Embodiment 3

[0031] Example 3 Decidual NK cells and decidual NK cell subsets enhance endometrial cell viability, reduce endometrial cell damage, and increase VEGF expression

[0032] After culturing non-pathological endometrial stromal cells (stroma cells) for 24 hours, each group of NK cells described in Example 2 was added, and the ratios of stromal cells: NK cells were 10:1 and 5:1, respectively. The control group used the control NK cells described in Example 2. NK cells were cleared 48 hours after treatment, and stromal cells and media were sampled for analysis.

[0033] Cell viability of stromal cells was detected by the PrestoBlue method (Thermo Fisher Scientific), measured 48 hours after treatment, and values ​​are expressed as mean % normalized to control (Table 1 and Table 2).

[0034] Table 1 Relative cell viability of stromal cells under 10:1 culture condition

[0035] average value SD p-value Blank (cultured alone, no NK cells) 100 6.72 control c...

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Abstract

The invention provides an application of decidua NK cells and cell subsets thereof in preparation of medicines for treating infertility related diseases. Through experimental verification, according to the decidua NK cell and the cell subset thereof, endometrial growth disorder diseases are treated by promoting endometrial thickness increase, enhancing endometrial cell viability, reducing endometrial cell damage, promoting VEGF expression, maintaining endometrial stromal cell dryness and stimulating proliferation; the pregnancy success rate of endometrial injury model mice is increased from 30% to 60-70%; by exerting the immune tolerance effect, reducing the spontaneous abortion rate and improving the T helper lymphocyte level to treat maternal fetal immune tolerance disorder related diseases, a new way is provided for treatment of infertility.

Description

technical field [0001] The invention belongs to the field of biomedicine, and relates to the use of decidual NK cells and cell subgroups in preparing medicines for treating infertility-related diseases, a method for separating and culturing cells, and a pharmaceutical composition containing the same. 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 the niche microenvironment of stem cells, resulting in regene...

Claims

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Application Information

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IPC IPC(8): C12N5/0783A61K35/17A61P15/08
CPCA61K35/17A61P15/08C12N5/0646C12N2509/00
Inventor 胡适
Owner PHARCHOICE THERAPEUTICS INC
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