Use of peripheral blood NK cells expressing tim-3 in the preparation of biomarkers of spontaneous abortion

A biomarker, NK cell technology, applied in the biological field, can solve the problems of lack of early diagnosis and prevention strategies, difficulty in obtaining decidual tissue, and inability to overcome pregnancy failure.

Active Publication Date: 2021-03-09
THE OBSTETRICS & GYNECOLOGY HOSPITAL OF FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

According to statistics, at present, the incidence of spontaneous abortion is as high as 15-40%. Even with the rapid development of assisted reproductive technology today, it is still unable to overcome the pregnancy failure caused by the disorder of maternal-fetal immune regulation. Lack of effective early diagnosis and prevention strategies
The occurrence of spontaneous abortion is mostly attributed to genetics, infection, endocrine, reproductive tract abnormalities, etc., but there are still more than 50% of clinical abortions, especially repeated spontaneous abortions, whose etiology is unknown. Among them, the imbalance of maternal-fetal immune regulation is considered to be an important reason One; studies have shown that immune cells are the executors and effectors of maternal-fetal immune tolerance, in which NK cells play a crucial regulatory role in maternal-fetal tolerance; the previous study of this application found that decidua NK cells are crucial to the establishment and maintenance of maternal-fetal tolerance. During pregnancy, decidual NK cells maintain maternal-fetal tolerance and normal pregnancy through the action of cell surface molecules and various intracellular factors. It is difficult to obtain decidual tissue in the early stage, and it is almost impossible to use it as a target for clinical diagnosis and prevention

Method used

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  • Use of peripheral blood NK cells expressing tim-3 in the preparation of biomarkers of spontaneous abortion
  • Use of peripheral blood NK cells expressing tim-3 in the preparation of biomarkers of spontaneous abortion
  • Use of peripheral blood NK cells expressing tim-3 in the preparation of biomarkers of spontaneous abortion

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Peripheral blood Tim-3 in different pregnancy states + NK cell percentage comparison and differential analysis / analysis of the expression characteristics and regulation of Tim-3 in peripheral blood NK cells in different pregnancy states

[0027] 1) Analyze the expression characteristics of Tim-3 in peripheral blood NK cells in different pregnancy states

[0028] Firstly, the peripheral blood of normal non-pregnant, early pregnant and mid-pregnant women was separated, lymphocytes were separated by Ficoll, and Tim-3 was analyzed by FCM in CD4 + T cells, CD8 + Expression of T cells, NK cells, NKT cells, DC cells and monocytes (such as figure 1 Shown in A), the results showed that NK cells, DC cells and monocytes highly expressed Tim-3 (respectively 80%, 80%, 90%), and the expression level of Tim-3 in NK cells was significantly different in different pregnancy periods, figure 1 As shown in B and C, the expression of Tim-3 in peripheral blood NK cells was signi...

Embodiment 2

[0033] Example 2: Experiment of Galectin-9 / Tim-3 Signal Mediating Peripheral NK Cell Immune Tolerance in Early Pregnancy

[0034] 1) Phenotype comparison of peripheral blood Tim-3+NK and Tim3-NK cells

[0035] In order to explore whether the expression of Tim-3 on NK cells is related to the function of NK cells, the isolated peripheral mononuclear cells were further sorted out Tim-3 by magnetic beads. + NK and Tim3 - NK cells, identified and sorted with a purity of 92%, meeting the experimental requirements, extracting total RNA and performing genome-wide microarray analysis; such as image 3 As shown in A and B, Tim-3+ and Tim-3-NK cells are two groups of NK cell subsets with differential gene expression, Tim-3 + In NK cells, 865 genes are up-regulated and 100 genes are down-regulated, and most of the up-regulated genes are related to Th2 type immune response and immune tolerance; in this experiment, PMA, ionomycin and BFA were stimulated for 4 to 6 hours before further inv...

Embodiment 3

[0045] Example 3: Abnormal Galectin-9 / Tim-3 signaling of peripheral NK cells in early pregnancy caused NK dysfunction and pregnancy failure experiment

[0046] 1) The expression level of Tim-3 in the peripheral NK cells of patients with spontaneous abortion decreased, the production level of Gal-9 decreased, and the expression level of NK cells decreased.

[0047] The level of cytokine secretion is disordered, and the killing activity is enhanced

[0048] In order to study the abnormality of the number and function of peripheral NK cells is closely related to the occurrence of spontaneous abortion, and further confirm whether the expression of Tim-3 in peripheral blood NK cells of patients with normal pregnancy and spontaneous abortion is different, and whether the content of Gal-9 in the blood is changed , Does the function of NK cells change accordingly? In this experiment, the peripheral blood of patients with early normal pregnancy and unexplained spontaneous abortion was...

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Abstract

The invention belongs to the field of biotechnology, and relates to the use of peripheral blood NK cells expressing Tim-3 in the preparation of spontaneous abortion biomarkers. The NK cells of the present invention have a tolerant phenotype and low cytotoxicity. It has been proved by experiments that the peripheral blood NK cells expressing Tim-3 are highly expressed in the peripheral blood of normal pregnancy, and the number of such cells in patients with spontaneous abortion Decline, dysfunction; Antagonizing Tim-3 of NK cells leads to pregnancy failure, and adoptive transfer of this NK cell subset can rescue immune spontaneous abortion model and pregnancy failure caused by NK knockout; the peripheral tumor expressing Tim-3 Blood NK cells can be used to prepare biomarkers of spontaneous abortion, and further serve as a new target for the prediction and prevention of spontaneous abortion of unknown clinical reasons.

Description

technical field [0001] The invention belongs to the field of biotechnology. It relates to biomarkers for predicting and preventing spontaneous abortion, and specifically relates to the use of peripheral blood NK cells expressing Tim-3 in preparing biomarkers for spontaneous abortion. The biomarker of spontaneous abortion can be used as a target for prediction and prevention of early pregnancy failure. Background technique [0002] The prior art discloses that the prediction method of pregnancy outcome is mainly based on the peripheral blood HCG-β and progesterone levels of pregnant women, but a large amount of evidence in practice shows that the relationship between pregnant women’s peripheral blood P levels and pregnancy outcomes is not completely consistent; although HCG-β β can truly reflect the pregnancy prognosis of pregnant women, but more and more clinical data show that once the peripheral blood HCG of pregnant women decreases (except for the physiological decline o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/02
CPCG01N33/5091
Inventor 杜美蓉李彦虹张迪常兴李大金
Owner THE OBSTETRICS & GYNECOLOGY HOSPITAL OF FUDAN UNIV
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