CN112414925A - Application of peripheral blood NK (Natural killer) cells expressing NKG2C as natural abortion biomarker - Google Patents
Application of peripheral blood NK (Natural killer) cells expressing NKG2C as natural abortion biomarker Download PDFInfo
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Abstract
The invention discloses application of peripheral blood NK cells expressing an activating receptor NKG2C in preparation of natural abortion biomarkers, application in preparation of products for predicting early pregnancy failure and application in preparation of products for preventing and treating early pregnancy failure. Experimental research shows that in peripheral blood of an RSA patient suffering from spontaneous abortion, the proportion of peripheral blood NK cells expressing an activating receptor NKG2C in total NK cells of the peripheral blood is remarkably higher than that of normal pregnant women and also remarkably higher than that of RSA patients who succeed in pregnancy, so that the peripheral blood NK cells expressing the activating receptor NKG2C can be used as biomarkers of spontaneous abortion and used for prediction and prevention and treatment of spontaneous abortion.
Description
Technical Field
The invention relates to application of peripheral blood NK cells expressing an activating receptor NKG2C as a natural abortion biomarker in preparation of products for predicting early pregnancy failure and products for preventing and treating early pregnancy failure, belonging to the technical field of biology.
Background
Currently, methods for monitoring serum HCG, progesterone and ultrasonic diagnosis are mostly adopted in clinical practice to predict pregnancy outcome. However, studies have shown that early pregnancy diagnosis using serum HCG and ultrasound lacks specificity and sensitivity. The HCG level has individual difference in the early pregnancy, the stability is low, and continuous dynamic monitoring is required; and there have been a number of studies showing that embryos have failed to be saved when a drop in peripheral blood HCG levels is found. Progesterone generally begins to stabilize gradually after 5 weeks of gestation, with normal levels of progesterone > 78nmol/L, and patients with progesterone < 15.6nmol/L are considered to be poorly pregnant; in the early gestation period, the progesterone level of a patient is mostly less than 62.4nmol/L, and if the pregnancy outcome is predicted, the pregnancy outcome cannot be accurately judged only by the progesterone level. Therefore, the index for predicting pregnancy outcome still has certain defects at present, so that an early prediction and prevention index which is simple and convenient and can reflect the pregnancy state in the early pregnancy period is urgently needed.
It has been found that the fetal-placental unit contains both paternal and maternal genetic genes and can be considered a maternal allo-hapten, which is not rejected by the mother throughout pregnancy and depends on the maternal immune tolerance status. The immune tolerance starts from contacting a parent system antigen carried by sperms with a mother body, and is presented to decidua immune cells through dendritic cells, so that the antigen-specific regulatory T cells proliferate and differentiate, and the immune tolerance and various immune cells jointly act to create a tolerance microenvironment for embryo implantation; at the same time, immune cells also provide nutritional assurance for embryo implantation by promoting the generation and remodeling of uterine vessels. If the immune tolerance environment is disturbed or the blood supply is insufficient, implantation failure and miscarriage and other poor pregnancy outcomes can occur. With the continuous and deep research of reproductive immunology, new understanding is clinically provided for many poor pregnancy outcomes such as RSA (recurrent abortion), and about 50% of RSA is caused by immune disorder, and 80% of abortion caused by unknown reasons is related to immune tolerance imbalance.
Immune cells play an important role in maternal-fetal interface immune tolerance. Decidua NK cells are the main immune cells of the early maternal-fetal interface, accounting for about 70%, mainly CD56brightCD16-Phenotype, low cytotoxicity, high secretory activity, plays a role in uterine spiral artery recasting and trophoblast invasion; the main phenotype of NK cells in peripheral blood is CD56dimCD16+Mainly plays a role in killing and toxicity of cells. The changes in NK cell number, function and expression of surface receptors are thought to be closely related to the occurrence of spontaneous abortion. Since decidua tissue is difficult to obtain and is often difficult to be used as an index for prediction and prevention, a simple and convenient method for evaluating the pregnancy state of a pregnant woman in the early pregnancy stage is urgently needed in clinical practice, so that early identification and early prevention and treatment are realized.
Disclosure of Invention
Aiming at the prior art, the invention discovers a natural abortion biomarker, namely peripheral blood NK (natural blood NK) cells expressing an activating receptor NKG2C, and the biomarker has important significance in predicting and preventing natural abortion.
The invention is realized by the following technical scheme:
a natural abortion biomarker, namely peripheral blood NK cells expressing an activating receptor NKG 2C. Experimental research shows that the proportion of peripheral blood NK cells expressing an activating receptor NKG2C (the proportion of the peripheral blood NK cells in the peripheral blood of an RSA patient suffering from spontaneous abortion) is remarkably higher than that of a normal pregnant woman and that of an RSA patient suffering from successful pregnancy, so that the peripheral blood NK cells expressing the activating receptor NKG2C can be used as biomarkers of spontaneous abortion.
Use of peripheral blood NK cells expressing the activating receptor NKG2C for the preparation of a product for the prediction of early pregnancy failure.
Further, in specific application, the proportion of peripheral blood NK cells expressing an activating receptor NKG2C in peripheral blood of a detected person to the total NK cells in the peripheral blood is detected, and the detected result is compared with the detection result of a normal pregnant woman or/and an RSA patient with successful pregnancy, and the comparison result can be used as a reference index to assist a doctor in early pregnancy failure prediction or risk assessment. If the proportion of peripheral blood NK cells expressing an activating receptor NKG2C in the peripheral blood of a subject to the total NK cells of the peripheral blood is obviously higher than that of a normal pregnant woman or/and an RSA patient who succeeds in pregnancy, the risk of early pregnancy failure of the subject is indicated to be extremely high, and related measures are suggested to be taken as early as possible to achieve a positive effect.
Further, the product for predicting early pregnancy failure is used for detecting the proportion of peripheral blood NK cells expressing the activating receptor NKG2C in peripheral blood to total peripheral blood NK cells.
The application of peripheral blood NK cells expressing an activating receptor NKG2C as a prevention and treatment target in preparing products for preventing and treating early pregnancy failure.
The invention carries out related experimental research, selects the crowds with different pregnancy states, divides the crowds into an RSA group and a normal pregnancy group, further divides the RSA group into an RSA abortion group and an RSA pregnancy group according to the pregnancy outcome, carries out a percentage comparison experiment of the total NK cells of the peripheral blood on the three groups, and analyzes the expression characteristics of the total NK cells of the peripheral blood in different pregnancy states. The result shows that the percentage of the total NK cells of the peripheral blood of the RSA patient who has spontaneous abortion in the pregnancy is obviously higher than that of the RSA patient who succeeds in the pregnancy and the pregnant woman in the normal early pregnancy; meanwhile, the RSA patient is found that if spontaneous abortion does not occur in the early pregnancy, the peripheral blood NK cell number of the RSA patient can be kept at a lower level and even slightly lower than that of a pregnant woman in the normal early pregnancy. The invention also aims at the peripheral blood NK cell CD56 in different pregnancy statesdimCD16+And CD56brightCD16-Carrying out percentage comparison experiments on the cell subsets, and analyzing the expression characteristics of different pregnancy states of peripheral blood NK cells of different subsets; the results showed that the peripheral blood NK cells CD56brightCD16-Subgroup in this pregnancyThe middle-aged and lower-aged RSA patients who suffer from natural abortion is lower than the normal RSA patients and normal pregnant women who suffer from the pregnancy. In order to further discuss the relation between the NK cell activating receptor NKG2C and RSA, percentage comparison experiments are also carried out on the three groups of activating receptors NKG2C of peripheral blood NK cells, and the expression levels of the activating receptor NKG2C in the peripheral blood NK cells in different pregnancy states are analyzed; the result shows that the peripheral blood of the RSA patient with spontaneous abortion in the pregnancy is obviously higher than that of the RSA patient and the normal pregnant woman who succeed in the pregnancy by the activating receptor NKG 2C.
The experiments prove that the expression ratio of the activating receptor NKG2C in the peripheral blood NK cells of the RSA patient who has spontaneous abortion is higher than that of the patient who continues to be pregnant. Therefore, the peripheral blood NK cells expressing the activating receptor NKG2C can be used as biomarkers of spontaneous abortion, as an index for predicting early pregnancy failure of RSA patients, and as a prediction and prevention target spot of clinical spontaneous abortion.
When the biomarker is applied, peripheral blood is used as a detected object, and compared with decidua tissue in gestation, the decidua tissue in gestation is not easy to obtain and is difficult to be used as a target spot for prediction and prevention and treatment of spontaneous abortion; while NKG2C+NK cells exist in peripheral blood and are easy to obtain, the peripheral blood NK cells can reflect the level change of decidua NK cells, only a small amount of peripheral blood needs to be extracted, and the peripheral blood NK cell expression condition can be monitored through flow cytometry analysis, so that the method is used for predicting the occurrence condition of spontaneous abortion.
The various terms and phrases used herein have the ordinary meaning as is well known to those skilled in the art. To the extent that the terms and phrases are not inconsistent with known meanings, the meaning of the present invention will prevail.
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FIG. 1: distribution of different subsets of peripheral blood NK cells.
Detailed Description
The present invention will be further described with reference to the following examples. However, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention.
The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible.
The instruments, reagents, materials and the like used in the following examples are conventional instruments, reagents, materials and the like in the prior art and are commercially available in a normal manner unless otherwise specified. Unless otherwise specified, the experimental methods, detection methods, and the like described in the following examples are conventional experimental methods, detection methods, and the like in the prior art.
Example 1: peripheral blood NK cells and NKG2C in different pregnancy states+NK cell percentage comparison/analysis of expression characteristics of different cell subsets and activation receptor NKG2C in peripheral blood NK cells of different pregnancy states
Experimental method (peripheral blood flow cytometry detection method): collecting peripheral blood of an experimental object by using an EDTA anticoagulation tube, temporarily storing the peripheral blood in a refrigerator at 4 ℃ for detection, and adding a mixed monoclonal fluorescent antibody into a flow tube comprises the following steps: PerCP-Cy5.5-CD3 antibody, FITC-CD56 antibody, PE-CY7-CD16 antibody and APC-NKG2C antibody, 100ul of peripheral blood in an anticoagulation tube is sucked, added into a flow tube, oscillated and mixed uniformly, applied for 15min in a dark place, added with 1ml of erythrocyte lysate, oscillated, mixed uniformly and cracked for 10min, washed for 2 times by PBS, added with 300 mu L of PBS solution, resuspended and oscillated, and detected on a machine by adopting a FACS Calibur flow cytometer.
Circle gate strategy for lymphocyte subpopulations: performing a circulant experiment on peripheral blood NK cells by using CD3, CD56 and CD16, determining lymphocyte regions according to an anterior angle and a lateral angle, and determining lymphocyte regions according to CD3-CD56+Determining the approximate region of NK cells, classifying the NK cells into CD56 according to the expression intensity of CD56 and CD16brightCD16-And CD56dimCD16+Subpopulations.
(1) Analysis of expression characteristics of Total peripheral blood NK cells in different pregnancy states
Firstly, selecting proper people according to strict inclusion and exclusion standards, and dividing the proper people into two groups of RSA and normal pregnant women; the pregnancy is divided into RSA abortion group and RSA pregnancy group according to whether the pregnancy has abortion, elbow venous blood of three groups of people in early pregnancy is collected, and the percentage of total NK cells of peripheral blood in different pregnancy states is detected by flow cytometry (as shown in table 1-row 1). The result shows that the percentage of NK cells in peripheral blood of RSA patients in spontaneous abortion of the pregnancy is higher than that of the RSA patients in successful pregnancy and pregnant women in normal early pregnancy. The research also finds that if the RSA patient does not have spontaneous abortion in the early pregnancy, the peripheral blood NK cell number of the RSA patient can be kept lower or even slightly lower than that of the pregnant woman in the normal early pregnancy. The occurrence of spontaneous abortion of RSA patients is closely related to the increase of the number of NK cells, and the continuous spontaneous abortion of the RSA patients with NK cell proliferation can be avoided by changing the number of the NK cells.
TABLE 1
Comparison of proportion of 3 groups of NK cells to NKG2C + NK cells (%)
NK cells and NKG2C+NK cell ratios of three groups(％)
1)P＜0.05 vs RSA pregnancy group；2)P＜0.05 vs normal early pregnancy group.
(2) Analyzing the expression characteristics of different sub-population peripheral blood NK cells in different pregnancy states
In the study, CD3, CD56 and CD16 are used for performing a gate experiment of peripheral blood NK cells, lymphocyte regions are determined according to an anterior angle and a lateral angle, then approximate NK cell regions are determined according to CD3-CD56+, and the NK cells are divided into CD56bright CD 16-and CD56dim CD16+ subgroups according to CD56 expression strength and CD16, and the results are shown in figure 1.
Flow cytometry detection of peripheral blood NK (natural killer) cell CD56 in different pregnancy statesdimCD16+And CD56brightCD16-Percentage of cell subpopulations (as shown in table 1-rows 2, 3). The result shows that the RSA patient peripheral blood CD56 of the spontaneous abortion of the pregnancydimCD16+The NK cell proportion is higher than that of RSA patients who succeed in the pregnancy and pregnant women in normal early pregnancyBut the difference is not statistically significant. CD56dimCD16+NK cells play a cytotoxic role in peripheral blood, and the increase of the proportion indicates that the NK cell killing function in peripheral blood is probably an important factor causing spontaneous abortion of RSA patients. The results also show that the successful RSA patients in the current pregnancy have peripheral blood CD56brightCD16-The proportion of NK cells is obviously higher than that of the pregnant women in the normal early pregnancy, and the suggestion is that the CD56 in the peripheral bloodbrightCD16-NK cells play an active role in pregnancy.
(3) Analysis of expression characteristics of the activating receptor NKG2C in peripheral blood NK cells under different gestational states
The ratio of expression of the activating receptor NKG2C in peripheral blood NK cells at different gestational states was examined by flow cytometry (as shown in table 1-line 3). The results show that NKG2C in peripheral blood of RSA patients with spontaneous abortion+The NK cell proportion is obviously higher than that of an RSA patient who succeeds in the pregnancy and a pregnant woman in the normal early pregnancy, and NKG2C in peripheral blood is prompted+NK cells are a negative factor for successful pregnancy (the inventors hypothesize that increased expression of the activating receptor NKG2C in NK cells may lead to increased killing of NK cells, thereby affecting the infiltration capacity of trophoblasts and consequently pregnancy outcome). The inventors predict that by reducing the number or proportion of the activating receptor NKG2C in NK cells, it may be possible to avoid RSA patients from continuing to have spontaneous abortion events.
The above examples are provided to those of ordinary skill in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications apparent to those skilled in the art are intended to be within the scope of the appended claims.
Claims (7)
1. Use of peripheral blood NK cells expressing the activating receptor NKG2C as/for the preparation of biomarkers for spontaneous abortion.
2. Use of peripheral blood NK cells expressing the activating receptor NKG2C for the preparation of a product for the prediction of early pregnancy failure.
3. Use according to claim 2, characterized in that: in specific application, the proportion of peripheral blood NK cells expressing an activating receptor NKG2C in peripheral blood of a detected person to the total NK cells in the peripheral blood is detected, and the detected result is compared with the detection result of a normal pregnant woman or/and an RSA patient who succeeds in pregnancy.
4. Use according to claim 3, characterized in that: if the proportion of the peripheral blood NK cells expressing the activating receptor NKG2C in the peripheral blood of the subject to the total NK cells in the peripheral blood is obviously higher than that of normal pregnant women or/and RSA patients with successful pregnancy, the risk of early pregnancy failure of the subject is indicated to be extremely high.
5. Use according to claim 2, characterized in that: the product for predicting early pregnancy failure is used for detecting the proportion of peripheral blood NK cells expressing an activating receptor NKG2C in peripheral blood to total peripheral blood NK cells.
6. The application of peripheral blood NK cells expressing an activating receptor NKG2C in preparing products for preventing and treating early pregnancy failure.
7. Use according to claim 6, characterized in that: the product for preventing and treating early pregnancy failure takes peripheral blood NK cells expressing an activating receptor NKG2C as a prevention and treatment target.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107287273A (en) * | 2016-03-31 | 2017-10-24 | 复旦大学附属妇产科医院 | Express purposes of the Tim-3 peripheral blood NK cell in spontaneous abortion biomarker is prepared |
| CN108048559A (en) * | 2018-01-24 | 2018-05-18 | 上海市第妇婴保健院 | Applications of the miR-184 in spontaneous abortion forewarning function |
| US20190314445A1 (en) * | 2018-03-16 | 2019-10-17 | Deutsches Rheuma-Forschungszentrum Berlin | Activation and expansion of nkg2c+ nk cells |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107287273A (en) * | 2016-03-31 | 2017-10-24 | 复旦大学附属妇产科医院 | Express purposes of the Tim-3 peripheral blood NK cell in spontaneous abortion biomarker is prepared |
| CN108048559A (en) * | 2018-01-24 | 2018-05-18 | 上海市第妇婴保健院 | Applications of the miR-184 in spontaneous abortion forewarning function |
| US20190314445A1 (en) * | 2018-03-16 | 2019-10-17 | Deutsches Rheuma-Forschungszentrum Berlin | Activation and expansion of nkg2c+ nk cells |
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| 余琴梅等: "白介素10可保持蜕膜NKG2D＋TGF-β－NK 细胞低水平并有利 于妊娠成功", 现代免疫学, vol. 31, no. 2, pages 139 - 142 * |
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