阅读说明：本技术 一组反复妊娠丢失疾病诊疗免疫标志物及应用 (Diagnosis and treatment immune marker for recurrent pregnancy loss diseases and application thereof ) 是由 廖爱华 黄晓博 刘红 蔺新秀 尹芝南 于 2020-11-25 设计创作，主要内容包括：本发明属于生物医学检测技术领域,尤其涉及一组反复妊娠丢失疾病诊疗免疫标志物及应用。免疫标志物包括：初始CD4～+T细胞、中心记忆CD4～+T细胞、效应记忆CD4～+T细胞、终末分化CD4～+T细胞、Treg细胞、外周辅助T细胞、中心CD8～+T细胞、终末分化CD8～+T细胞、非活跃期特异性的终末分化CD8～+T细胞、耗竭型CD8～+T细胞、T NK细胞、成熟NK细胞、未成熟NK细胞、未成熟NK/成熟NK比值、γδT细胞中的至少一种。这15种与反复妊娠丢失有关的免疫细胞亚型提示反复妊娠丢失妇女免疫系统处于免疫耐受不足的状态,从而不能够容受携带父系抗原的胎儿导致流产的发生。(The invention belongs to the technical field of biomedical detection, and particularly relates to a group of diagnosis and treatment immune markers for diseases caused by repeated pregnancy loss and application thereof. The immune markers include: initial CD4 + T cell, central memory CD4 + T cell, effector memory CD4 + T cell, terminally differentiated CD4 + T cells, Treg cells, peripheral helper T cells, central CD8 + T cell, terminally differentiated CD8 + T-cell, non-active phase specific terminally differentiated CD8 + T cell, depleted CD8 + At least one of T cells, T NK cells, mature NK cells, immature NK/mature NK ratio, γ δ T cells. These 15 subtypes of immune cells associated with recurrent pregnancy loss suggest that the immune system of women with recurrent pregnancy loss is under-tolerated and therefore unable to tolerate foetuses carrying paternal antigens leading to the development of miscarriage.)

1. A group of immune markers for diagnosing and treating recurrent pregnancy loss diseases, which is characterized in that the markers comprise primary CD4⁺T cell, central memory CD4⁺T cell, effector memory CD4⁺T cell, terminally differentiated CD4⁺T cells, Treg cells, peripheral helper T cells, central memory CD8⁺T cell, terminally differentiated CD8⁺T-cell, non-active phase specific terminally differentiated CD8⁺T cell, depleted CD8⁺At least one of T cells, T NK cells, mature NK cells, immature NK/mature NK ratio, γ δ T cells.

2. Use of a set of the biomarkers of recurrent pregnancy loss disease diagnosis and treatment according to claim 1 in the preparation of a reagent or a kit for diagnosing recurrent pregnancy loss disease.

3. A reagent or kit for diagnosing recurrent pregnancy loss disease, comprising a reagent for detecting an immune-related molecule expressed by the immune marker of claim 1.

4. The reagent or the kit for diagnosing and treating recurrent pregnancy loss diseases according to claim 3, wherein: the immune related molecules comprise CD3, CD4, CD8, CD56, CD45RA, CCR7, CD127, CD28, gamma delta T, CXCR5 and PD-1.

5. Use of peripheral helper T cells as an immune marker for the prediction of normal pregnancy.

Technical Field

The invention belongs to the technical field of biomedical detection, and particularly relates to a group of diagnosis and treatment immune markers for diseases caused by repeated pregnancy loss and application thereof.

Background

Recurrent pregnancy loss refers to the loss of pregnancy occurring 2 or more times before 20 weeks of gestation, the incidence rate of which in women of childbearing age is 1% -5%, and the risk of miscarriage of patients of this type of pregnancy increases with the number of miscarriages. The causes of the disease mainly include chromosome abnormality, anatomical structure abnormality, endocrine factors, autoimmune factors, thrombosis liability, infection, environmental factors, psychology and other factors. And bothers clinicians and patients: about 50% of women still do not find the cause of recurrent pregnancy loss after excluding the above known causes, which is also called recurrent pregnancy loss of unknown cause. More and more studies are currently underway to show that maternal immune imbalance may be the major cause of recurrent pregnancy loss of unknown origin, accounting for over 60%.

During the menstrual cycle and gestation, immune cells (NK cells, T cells and γ δ T cells) are dynamically changed as the hormone levels change. It has been shown that the abnormal number and/or function of a single immune cell or a certain kind of immune cell subsets in the peripheral blood of a mother are closely related to the occurrence of recurrent pregnancy loss, but the systematic and accurate detection and screening of multiple immune cell subsets of the mother are lacked. Thus, there is a lack of assessment of the number and function of maternal whole immune cell subsets.

The current examinations for patients with recurrent pregnancy loss of unknown origin mainly include basic physical examination, ultrasound examination and invasive endometrial pathology, which cannot assess the immune status of the mother and lack specific immune markers. Nevertheless, patients with recurrent pregnancy loss still have a strong desire to detect an indicator of immunity or monitor an assessment of an indicator of immunotherapy effectiveness.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a group of diagnosis and treatment immune markers for recurrent pregnancy loss diseases and application thereof, and aims to solve part of problems in the prior art or at least alleviate part of problems in the prior art.

The invention mainly adopts multi-parameter flow cytometry to simultaneously detect 63 immune cell subtypes in peripheral blood of non-pregnant women, born women and women with recurrent pregnancy loss (50 cases in each group), and through multi-group unpaired t test, immune indexes of the non-pregnant women and the women with recurrent pregnancy loss and the born women and the women with recurrent pregnancy loss which are simultaneously different are selected as immune cell subtypes related to recurrent pregnancy loss.

The invention is realized by that a group of immune markers for diagnosing and treating recurrent pregnancy loss diseases comprises initial CD4⁺T cell, central memory CD4⁺T cell, effector memory CD4⁺T cell, terminally differentiated CD4⁺T cells, Treg cells, peripheral helper T cells, central memory CD8⁺T cell, terminally differentiated CD8⁺T-cell, non-active phase specific terminally differentiated CD8⁺T cell, depleted CD8⁺At least one of T cells, T NK cells, mature NK cells, immature NK/mature NK ratio, γ δ T cells.

The invention also discloses application of the group of diagnosis and treatment immune markers for the recurrent pregnancy loss diseases in preparation of a reagent or a kit for diagnosing the recurrent pregnancy loss diseases.

The present invention also discloses a reagent or a kit for diagnosing and treating recurrent pregnancy loss diseases, which comprises a reagent for detecting an immune-related molecule expressed by the immune marker of claim 1.

Further, the immune-related molecule comprises at least one of CD3, CD4, CD8, CD56, CD45RA, CCR7, CD127, CD28, γ δ T, CXCR5, PD-1.

The invention also provides the application of the peripheral helper T cells as immune markers for normal pregnancy prediction.

In summary, the advantages and positive effects of the invention are:

the invention adopts multiple parametersFlow cytometry simultaneously detected 63 immune cell subtypes in peripheral blood of infertile women, born women and women with recurrent pregnancy loss (50 cases each group); through a plurality of groups of unpaired t tests, screening immune indexes with difference between a non-bearing woman group and a recurrent pregnancy loss group and between a bearing woman group and a recurrent pregnancy loss group as immune cell subtypes related to recurrent pregnancy loss, wherein the immune function indexes comprise: initial CD4⁺T cell, central memory CD4⁺T cell, effector memory CD4⁺T cell, terminally differentiated CD4⁺T cells, Treg cells, peripheral helper T cells, central CD8⁺T cell, terminally differentiated CD8⁺T-cell, non-active phase specific terminally differentiated CD8⁺T cell, depleted CD8⁺T cells, T NK cells, mature NK cells, immature NK/mature NK ratio, γ δ T cells.

The invention provides immune markers for detecting recurrent pregnancy loss of unknown reasons, and the immune markers comprise 15 cell subtypes. These subtypes of immune cells associated with recurrent pregnancy loss suggest that the immune system of a woman with recurrent pregnancy loss is under-tolerated and therefore unable to tolerate a fetus carrying a paternal antigen leading to the development of an abortion.

Drawings

FIG. 1 is a graph of data from an analysis of the detection of immune cell subtypes closely associated with recurrent pregnancy loss.

FIG. 2 is a ROC graph of 9 immune indexes with potential clinical application value;

FIG. 3 shows the results of ROC analysis of nine differential indicators in the born vs. non-born group and the recurrent pregnancy loss vs. non-born group.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the equipment and reagents used in the examples and test examples are commercially available without specific reference. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.

Various modifications to the precise description of the invention will be readily apparent to those skilled in the art from the information contained herein without departing from the spirit and scope of the appended claims. It is to be understood that the scope of the invention is not limited to the procedures, properties, or components defined, as these embodiments, as well as others described, are intended to be merely illustrative of particular aspects of the invention. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in the art or related fields are intended to be covered by the scope of the appended claims.

For a better understanding of the invention, and not as a limitation on the scope thereof, all numbers expressing quantities, percentages, and other numerical values used in this application are to be understood as being modified in all instances by the term "about". Accordingly, unless expressly indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. In the present invention, "about" means within 10%, preferably within 5% of a given value or range.

The normal temperature in the following embodiments of the present invention refers to a natural room temperature condition in four seasons, and is not subjected to additional cooling or heating treatment, and is generally controlled at 10 to 30 ℃, preferably 15 to 25 ℃.

The invention discloses a group of immune markers for diagnosis and treatment of recurrent pregnancy loss diseases and application thereof, and particularly relates to the following embodiments.

Example 1 screening of significantly different subtypes of immune cells in peripheral blood

The examples mainly include three groups of people: 50 non-pregnant women (non-pregnant group), 50 pregnant women (pregnant group) and 50 women with recurrent pregnancy loss (recurrent pregnancy loss group). The menstrual cycle of women in the non-bearing group and the bearing group is normal, and no adverse life history exists; women in the fertile group all delivered a normal child and had no history of poor fertility between 1 and 2 years after delivery. Women in the recurrent pregnancy loss group have had a history of spontaneous abortion or embryo abortion of two or more times before 20 weeks gestation, and have excluded chromosomal abnormalities, uterine anatomical abnormalities, prothrombotic states, endocrine factors, infections, and autoimmune diseases.

Each person in the examples 5ml of peripheral blood was drawn into heparin sodium treated blood collection tubes and tested within 24h after sampling.

After the peripheral blood was allowed to stand at room temperature, the upper plasma was aspirated, and the plasma was diluted with PBS buffer at a ratio of 1: 1, diluting, uniformly mixing, paving on the peripheral blood lymphocyte separation liquid, and performing density gradient centrifugation. The cells were collected and resuspended in RPMI1640 medium containing 10% fetal bovine serum, the flow antibody required for detection was added according to the instructions, incubated at 4 ℃ in the dark for 30min, washed twice with PBS, resuspended in separation buffer, and subjected to multiparameter flow cytometry detection using BD LSRFortessa X-20, and the results were analyzed using FlowJo V10 software.

63 of multiparameter flow cytometric analyses include: CD4 and CD8 of 41T cell subtypes, NK cell 10 subtypes and gamma delta T cell 12 subtypes.

The data distribution type is tested by Shapiro-Wilk, the normal distribution type is the mean and the standard deviation, and the skewed distribution data is represented by the median and the range; data differences were statistically analyzed using analysis of variance for multiple comparisons.

FIG. 1 shows 15 subtypes of immune cells closely related to recurrent pregnancy loss and successful pregnancy. Wherein, A-O: 15 subtypes of immune cells associated with recurrent pregnancy loss, including: initial CD4⁺T cell, central memory CD4⁺T cell, effector memory CD4⁺T cell, terminally differentiated CD4⁺T cells, Treg cells, peripheral helper T cells, central memory CD8⁺T cell, terminally differentiated CD8⁺T-cell, non-active phase specific terminally differentiated CD8⁺T cell, depleted CD8⁺T cells, T NK cells, mature NK cells, immature NK/mature NK ratio, γ δ T cells. A-D and L-M are immune indexes with statistical difference in comparison of a repeated pregnancy loss group vs. a born group and a repeated pregnancy loss group vs. a non-born group; f is simultaneously atThe immunity indexes of the recurrent pregnancy loss group vs. the non-bearing group and the born group vs. the non-bearing group are statistically different; e and H-K are immune indicators with statistical differences in repeated pregnancy loss group vs. born group comparison; g and N-O are immune indexes with statistical difference in comparison of repeated pregnancy loss group vs. non-bearing group; normal distribution data is represented by mean + -standard deviation, and skewed distribution data is represented by median + -extreme value. Data differences were statistically analyzed using analysis of variance for multiple comparisons. The difference level: p<0.05(*)；P<0.01(**)；P<0.001(***)；and P<0.0001(****). ns indicates that the difference is not statistically significant.

Example 2 clinical application value of the selected differential index

Three groups of people were included in the examples, 50 non-pregnant women (non-pregnant group), 50 pregnant women (pregnant group) and 50 women with recurrent pregnancy loss (recurrent pregnancy loss group). The menstrual cycle of women in the non-bearing group and the bearing group is normal, and no adverse life history exists; women in the fertile group all delivered a normal child and had no history of poor fertility between 1 and 2 years after delivery. Women in the recurrent pregnancy loss group have had a history of spontaneous abortion or embryo abortion of two or more times before 20 weeks gestation, and have excluded chromosomal abnormalities, uterine anatomical abnormalities, prothrombotic states, endocrine factors, infections, and autoimmune diseases.

The group of non-pregnant women, as represented by the group of non-pregnant women, was taken as the baseline pre-pregnant state for women of childbearing age. The 95% confidence intervals for the proportion of 15 immune cell subtypes in the peripheral blood of non-pregnant women, and the 95% confidence intervals for the same indices in normal pregnancy (born group) and recurrent pregnancy loss groups are shown in Table 1. The comparison shows that the confidence interval range of the 15 different immunity indexes is smaller in the group without fertility and the group with fertility, and is larger in the group with repeated pregnancy loss. Among them, the original CD4⁺T cells, central memory CD8+ T cells, mature NK cells and central memory CD4⁺The confidence interval of T cells was completely higher in the recurrent pregnancy loss group than in the non-fertile group, while effector memory CD4⁺T cells, immature NK cells, mature NK/immature NK ratio, peripheral helper T cellsAnd terminally differentiated CD4⁺The confidence interval for T cells was lower in the recurrent pregnancy loss group than in the non-fertile group; the 9 immune indexes are prompted to provide critical value judgment for prediction of repeated pregnancy loss diseases, and the critical value judgment has important reference value. The more the index detected by the peripheral blood immune function of the patient with recurrent pregnancy loss coincides with the index in the present embodiment, the higher the risk of recurrent pregnancy loss of the patient is suggested.

Table 1 summary of differential immune index function and its confidence intervals in each group

In addition, the 9 indexes of difference are subjected to receiver operating characteristic curve (ROC curve for short) (figure 2) and area under the curve (AUC) (figure 3) to analyze the critical value, sensitivity and specificity of disease prediction. Peripheral helper T cells were found to be of significant value in predicting normal pregnancy with a cutoff of 61.1% (specificity up to 88%, sensitivity 54%) in comparison between the fertile and non-fertile groups. In comparison between the recurrent pregnancy loss group and the non-bearing group, the p values of the 9 indexes are less than 0.01, which indicates that the method has important value for predicting recurrent pregnancy loss diseases, and the sensitivity and specificity of the method for diagnosing recurrent pregnancy loss are shown in figure 3 (recurrent pregnancy loss group vs. non-bearing group).

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.