CN112924696A - Method for evaluating maternal-fetal immune tolerance by detecting human choriotrophoblast exosome HLA-E level - Google Patents

Abstract

The invention discloses a method for detecting the level of human choriotrophoblast exosome HLA-E and evaluating maternal and fetal immune tolerance. The method of the invention needs less sample amount, is convenient to obtain and is safe for patients; the invention adopts the flow cytometer for detection, which is convenient and fast; the level of exosome HLA-E can accurately reflect the degree of maternal-fetal immune tolerance.

Description

Method for evaluating maternal-fetal immune tolerance by detecting human choriotrophoblast exosome HLA-E level

Technical Field

The invention particularly relates to a method for evaluating maternal-fetal immune tolerance by detecting the level of human choriotrophoblast exosome HLA-E.

Background

During normal pregnancy, the maternal immune system makes a series of immune regulation mechanisms at the maternal-fetal interface, so that the maternal immune tolerance is generated on the fetus of the hemiallograft, and spontaneous abortion can occur if the immune regulation function of the maternal-fetal interface is unbalanced. Spontaneous abortion occurring 2 or more times in succession with the same partner is called recurrent abortion (RSA), which seriously affects the physical and mental health of women of childbearing age. In recent years, the incidence of spontaneous abortion has shown an increasing trend in the population. The causes of RSA are diverse and include: anatomical abnormalities of genital tract, chromosomal abnormalities of partners, maternal thrombotic disorders, maternal immune dysfunction, and various endocrine disorders. However, 40% -50% of RSA patients still do not find a clear cause, called recurrent abortion of unknown origin (URSA).

HLA-E is a non-classical HLA (human histocompatibility leucocyte antigen) class I molecule that is distributed among early placental trophoblasts. HLA-E can bind to NK cells at maternal-fetal interface, NKG2A and NKG2C receptors on T cells, and HLA-E has 6-fold higher affinity for NKG2A than NKG 2C. NKG2A is an inhibitory receptor on the cell surface, and can inhibit attack of NK cells and T cells on fetus, and induce maternal-fetal immune tolerance. HLA-E binding to receptors is dependent on HLA-E expression on the cell surface, however previous studies have shown that: HLA-E is present primarily in the cytoplasm of the villous trophoblasts rather than on the surface of the cell membrane and is therefore difficult to work with by direct contact with receptors on cells in the maternal-fetal interface. Literature reports show that trophoblast-derived exosomes play a role in cellular communication in pregnancy to maintain maternal-fetal immune tolerance.

The methods for assessing maternal-fetal immune tolerance reported in the literature are mainly: measuring cytokine or estrogenic progestogen levels in peripheral blood, comparing changes in the proportion of the population of immune cells in peripheral blood, and comparing changes in the proportion of the population of immune cells in the decidua.

The main steps for measuring the cytokine or estrogen progestogen level in peripheral blood are: appropriate amount of peripheral blood was drawn from the patient, and plasma was collected and measured for cytokine or estrogen progestogen levels by ELISA. ELISA is an experimental method which connects a substance to be detected with enzyme by utilizing the specific reaction of antigen and antibody, then generates color reaction between the enzyme and a substrate, and then carries out quantitative determination. It has significant disadvantages: the ELSA experiment has various steps, so that a plurality of interference factors are caused, and the experiment repeatability is poor; 2, a plurality of steps of ELISA all need manual operation, and only one factor or hormone expression can be measured in one experiment, so that the method is time-consuming and labor-consuming; 3, the factors influencing the cytokine level are more, and the immune tolerance level of the maternal and fetal cannot be accurately reflected.

The steps by which changes in the immune cell population in peripheral blood are compared are mainly: after a proper amount of peripheral blood of a patient is extracted and required immune cells are obtained through separation, the proportion of different immune cells is compared through a flow analysis method, and the immune tolerance function of a maternal fetus is measured by mostly comparing the change of the proportion of NK cell populations or marrow-derived inhibitory cells or T cells. However, this method only indirectly reflects the immune tolerance of maternal-fetal interfaces, and changes in immune cells in peripheral blood are susceptible to other diseases: for example, an increased proportion of myeloid-derived suppressor cells in the peripheral blood of an acutely infected patient; the asthma patients have imbalance of Th1/Th2 in peripheral blood and the proportion of Tregs is increased; in some hypersensitivity and innate immune responses, the proportion of NK cells in patients also increases.

The main steps by measuring the immune cell population changes in the decidua are: collecting decidua abortus, separating out required immune cells which are mostly NK cells or myeloid-derived suppressor cells or T cells, and analyzing the change of the proportion of the immune cells by a flow technology to measure the immune tolerance function of a maternal fetus. Although theoretically, the method can intuitively embody the immune tolerance function of the maternal-fetal interface, the method has uncertainty because immune cells are unevenly distributed in the decidua, namely the immune cells in the basal decidua are obviously higher than the decidua subcontrata, and the uterus cleaning cannot scrape all decidua tissues. In addition, in order to protect the endometrium of a URSA patient and not influence the subsequent fertility demand, a method for promoting the discharge of the decidua and the gestational sac by mifepristone is advocated by various hospitals at present, and the decidua tissue cannot be discharged at one time, so that the collection of the decidua is difficult.

Disclosure of Invention

In view of the above situation, the present invention provides a method for detecting HLA-E level of human villous trophoblast exosome to evaluate maternal-fetal immune tolerance.

In order to achieve the purpose, the invention provides the following technical scheme:

a method for assessing maternal-fetal immune tolerance by detecting human choriotrophoblast exosome HLA-E levels, comprising the steps of:

1) extracting exosomes of the villous trophoblasts of the patient;

2) combining the exosomes obtained in step 1) with latex particles;

3) HLA-E flow antibodies are labeled and the level of HLA-E in exosomes is analyzed on a flow cytometric analyzer.

Further, the step 1) is specifically as follows:

1.1) on the day of uterine curettage of patients with unexplained recurrent abortion (URSA), the patient's villi are collected and rinsed with sterile phosphate buffer at pH 7.4;

1.2) cutting down the villus tissue obtained in the step 1.1) into small pieces with the size of 1 cubic millimeter, re-suspending the small pieces with PBS to obtain a tissue mixed solution, and then digesting the tissue mixed solution with 1mg/ml collagenase IV and 0.01mg/ml DNase on a shaking bed at 37 ℃ for 1 hour;

1.3) filtering the tissue mixed solution obtained in the step 1.2) by using a sterile filter with the diameter of 70 mu m, centrifuging the filtered cell suspension by using a centrifuge at the rotating speed of 1,000 Xg for 5 minutes, and taking out supernatant after the centrifugation is finished;

1.4) filtering the supernatant obtained in the step 1.3) by using a sterile filter with the diameter of 0.22 mu m, filling the filtered liquid into an ultracentrifuge tube, centrifuging the liquid for 1 hour at the temperature of 4 ℃ and at the speed of 100,000 Xg in the ultracentrifuge, removing the supernatant, and precipitating the supernatant into exosomes;

1.5) resuspending the exosome by PBS, centrifuging for 1 hour at 4 ℃ and 100,000 Xg in an ultracentrifuge to obtain a precipitate, namely the cleaned exosome;

further, the step 2) is specifically as follows:

after the exosomes were resuspended in 200. mu.l PBS, the exosome concentration was quantitatively analyzed by BCA method, according to the BCA result, 20. mu.g of exosomes were taken out from the obtained exosome suspension, 10. mu.l of latex particles diluted 100 times was added to mix well, after being left at room temperature for 15 minutes, 1ml of PBS was added, then shaking was carried out on a shaker at 4 ℃ for 2 hours, then the mixture of latex particles and exosomes was centrifuged for 5min at 3,000 Xg, the supernatant was removed, washed once with PBS, and the precipitate was resuspended in 100. mu.l PBS.

Further, the step 3) is specifically as follows:

3.1) Add fluorescently labeled flow antibody to 100. mu.l of the resuspension: HLA-E-APC, mixing, incubating at room temperature in dark for 30 min;

3.2) after the incubation in the dark, adding 1ml PBS and 3000 Xg, centrifuging for 5min to remove the supernatant, finally adding 200 μ l PBS into the precipitate, and analyzing the proportion of the HLA-E positive particles.

The invention has the beneficial effects that:

(1) HLA-E is a ligand of an inhibitory receptor NKG2A, plays an immunosuppressive effect after being combined with NKG2A on T cells and NK cells, and is required to be secreted to the extracellular space through exosomes to play a role because most of the HLA-E is expressed in cytoplasm of trophoblasts, so that the level of the HLA-E of the exosomes can reflect the level of maternal-fetal immune tolerance.

(2) The invention firstly extracts the human primary villus trophoblast exosome, and then detects and analyzes the level of HLA-E in the exosome through flow cytometry. The detection method is simple, convenient and quick to operate, and time-saving and labor-saving.

(3) The detection method of the invention needs less villus tissue amount, is easy to obtain, does not increase additional damage to patients, and avoids the waste of manpower and material resources caused by the small tissue amount and the failure of detection results.

Drawings

FIG. 1 is a flow chart of the detection method of the present invention.

Figure 2 is a schematic of a Beckman flow cytometer employed in the present invention.

FIG. 3 is a graph showing the identification of the obtained super-dissociated exosomes, wherein FIG. 3(a) is a size and morphology graph of human choriotrophoblast exosomes identified by cryo-TEM, FIG. 3(b) is a particle size distribution graph of human choriotrophoblast exosomes identified by exosome concentration particle size (NTA), FIG. 3(c) is a marker-specific molecular map of human choriotrophoblast exosomes identified by Western blot, and FIG. 3(d) is a graph showing the immune cell surface molecular carriage in human choriotrophoblast exosomes identified by flow cytometry.

FIG. 4 is a data statistical chart showing the HLA-E positive ratio in villus trophoblast exosomes of normal women, patients with recurrent abortion due to embryonic chromosomal abnormality, and patients with unexplained recurrent abortion.

Detailed Description

The technical solutions of the present invention are further described in detail below with reference to the accompanying drawings, and it should be noted that the detailed description is only for describing the present invention, and should not be construed as limiting the present invention. Reagents, kits, instruments, and the like used in the following examples are commercially available; wherein 1M is 1 mol/L; PBS refers to sterile phosphate buffer at pH 7.4.

Example 1

A method of detecting HLA-E levels in human villous trophoblast exosomes comprising the steps of:

(1) extracting exosomes of the villous trophoblasts of the patient:

1.1) on the day of uterine curettage or villus discharge in patients with unexplained recurrent abortion (URSA), collecting villus tissue, rinsing with sterile Phosphate Buffered Saline (PBS) pH 7.4 for 2 times;

1.2) the villus tissue is cut into small pieces of 1 cubic millimeter with sterilized scissors, resuspended in PBS to a tissue mixture, and digested with 1mg/ml collagenase IV (Sigma-Aldrich) and 0.01mg/ml DNase (purchased from Sigma-Aldrich) on a shaker at 37 ℃ for 1 hour;

1.3) filtering the digested tissue mixed solution by using a sterile filter with the diameter of 70 mu m, then centrifuging the filtered cell suspension by using a centrifuge at the rotating speed of 1,000 Xg for 5 minutes, wherein the centrifuged precipitate is cells and cell debris, exosome exists in supernatant, and taking out the supernatant;

1.4) filtering the supernatant by using a sterile filter of 0.22 mu m to further achieve the aim of removing fine cell debris, putting the filtered liquid into an ultracentrifuge tube, weighing the total mass of the liquid and the ultracentrifuge tube by using an electronic balance to be accurate to 0.01g, wherein the mass of two ultracentrifuge tubes filled with the liquid on the diagonal is consistent, putting the ultracentrifuge tube into a fixed-angle rotor in a diagonal symmetry mode, centrifuging for 1 hour at 4 ℃ and 100,000 Xg in the ultracentrifuge, pouring out the supernatant, and precipitating to be an exosome;

1.5) resuspending the exosome with 25ml of PBS to obtain an exosome solution, weighing the total mass of the exosome solution and an ultra-centrifugal tube by an electronic balance to be accurate to 0.01g, enabling the mass of two ultra-centrifugal tubes filled with the exosome solution on a diagonal line to be consistent, then placing the ultra-centrifugal tubes into a fixed-angle rotor in a diagonal line symmetry mode, centrifuging the ultra-centrifugal tubes for 1 hour at 4 ℃ for cleaning once at 100,000 Xg in an ultra-centrifugal machine, and finally obtaining a precipitate which is the cleaned exosome.

FIG. 3 is a diagram showing the identification of the obtained Exosomes, in which the human choriotrophoblast Exosomes were identified (NP: Normal suppression, a healthy female; AK-RSA: Current specific ability abortions with aberration karyotype, a Recurrent abortion patient with an embryonic chromosomal abnormality; URSA: Unexplained Recurrent abortion patients; TC-EXO: Trophoblast cell derived Exosomes, a Trophoblast-derived exosome; TC-Lysate, Trophoblast cell Lysate, a Trophoblast Lysate).

FIG. 3a is a graph of exosomes of human trophoblast cells identified by cryo-transmission electron microscopy, FIG. 3b is a distribution graph of exosome particle size of human trophoblast cells identified by exosome concentration particle size (NTA), with the abscissa being the size of the exosome particle size and the ordinate being the concentration of exosomes, and FIG. 3b shows that the particle sizes of three groups of exosomes were measured as 154.7, 151.9 and 145.8nm, respectively; FIGS. 3a and 3b illustrate that the extracted material is exosome, conforming to the morphology and size of exosomes. FIG. 3c is a marker-specific molecular diagram of human trophoblast exosomes identified by Western blot, FIG. 3c illustrates that the extracted exosomes carry exosome-specific molecules Alix, CD63, TSG101, placental trophoblast-specific molecules PLAP and HLA-G, and do not carry mesenchymal cell-specific molecules Vimentin and endoplasmic reticulum protein GRP94 in villous tissues; FIG. 3d is a graph showing the identification of the molecular carriage of the surface of immune cells in human villous trophoblast exosomes by flow cytometry. FIG. 3d illustrates that the extracted exosomes do not carry immune cell surface molecules, are not of lymphocyte origin, but of human villous trophoblast origin.

(2) The obtained exosome of the patient villus trophoblast is combined with the latex particle, and the exosome has smaller diameter and cannot be detected by a flow cytometry analyzer, so that the exosome is adsorbed on the latex particle to be better detected by the flow analyzer.

2.1) measuring the concentration of the exosome according to the BCA method, taking out 20 mu g of exosome to a 1.5ml ep tube, adding 10 mu l of aldehyde/sulfate latex particles (Invitrogen, New York, NY, USA) diluted by 100 times, uniformly mixing, and standing for 15 minutes at room temperature; the concentration of the 100-fold diluted aldehyde/sulfate latex particles was 410. mu.g/. mu.l, specifically diluted as described in the specification. The present invention does not improve on this. In this example, the BCA method was used to determine protein concentration, which is a conventional procedure in the prior art and is not modified by the present invention.

2.2) then adding 1ml of PBS and mixing evenly, and slowly shaking evenly for 2 hours on a shaking table at 4 ℃ to ensure that the exosome is fully combined with the latex particles;

2.3) the latex particles and exosomes mixture was centrifuged at 3,000 Xg for 5min, the supernatant was discarded, the pellet was washed once with 1ml PBS,3,000 Xg, centrifuged for 5min, the supernatant was discarded, and the pellet was mixed well with 100. mu.l PBS.

(3) Circling a population of trophoblast-derived exosomes on a flow cytometric analyzer, analyzing the expression of HLA-E in the population:

3.1) Add 5. mu.l of fluorescently labeled flow antibody to 100. mu.l of exosome solution (i.e. solution from step 2.3): HLA-E-APC (Cat No. 130-;

3.2) adding 1ml PBS 3,000 Xg after the lightproof incubation is finished, centrifuging for 5min, removing supernatant, adding 1ml PBS for centrifugal cleaning, adding 200 mul PBS in the final precipitate, and analyzing the proportion of the HLA-E positive particles on a machine.

The present study shows that the alteration of villous trophoblast exosome content is mainly reflected in the reduction of HLA-E expression, and therefore, the detection of HLA-E levels in human villous trophoblast exosomes may be used to assess maternal-fetal immune tolerance.

The collected HLA-E level of decidua human choriotrophoblast exosomes is shown in FIG. 4, wherein in FIG. 4, a is a flow cytometric analysis chart, and the abscissa is fluorescence intensity which represents the expression intensity of HLA-E; b is a statistical plot of the proportion of HLA-E positive particles;

human choriotrophoblast Exosomes HLA-E positive rate (NP: Normal differentiation, healthy female; AK-RSA: current specific metabolic abundance with abnormal chromosomal type, Recurrent abortion patient with embryonic chromosomal abnormality; URSA: Unexplained Recurrent abortion patient; TC-EXO: Trophoblast cell derived Exosomes) was flow-detected, and the ordinate in FIG. 4b is the proportion of HLA-E positive in human choriotrophoblast Exosomes.

As can be seen from FIG. 4, compared with healthy women and RSA patients with embryonic chromosomal abnormalities, the URSA patients have the smallest proportion of HLA-E positivity in the exosomes of the villous trophoblasts (i.e. the HLA-E in the exosomes is positive when expressed); the average value M of the positive proportion of HLA-E in exosomes of healthy villous trophoblasts is: 37.02% and a standard deviation SD of 15.83%, so that when the human maternal and fetal immune tolerance function is deduced, the cutoff value of HLA-E positive proportion in villous trophoblast exosomes is: m-2SD ═ 5.36%; that is, when the flow cytometry method is used to measure the level of HLA-E in human villus trophoblast exosomes, if the positive proportion of HLA-E in villus trophoblast exosomes is less than 5.36%, the maternal-fetal immune tolerance function may be impaired.

As is clear from fig. 4, since the HLA-E level in the exosomes of the villus trophoblasts is significantly lower in the URSA patients than in normal women, the human villus trophoblasts secrete exosomes, and bind the HLA-E in the exosomes to NKG2A on immune cells such as NK and T cells at the maternal-fetal interface, thereby suppressing the toxicity of NK cells and T cells to the fetus and maintaining maternal-fetal immune tolerance. The HLA-E level of the villus trophoblast exosome of the URSA patient is reduced, so that the immune tolerance function is damaged, and the detection of the HLA-E level of the human villus trophoblast exosome can be used for evaluating the immune tolerance function of the maternal fetus.

It is to be understood that the described embodiments are merely a few embodiments of the invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Claims (4)

1. A method for detecting the level of human choriotrophoblast exosome HLA-E to evaluate maternal-fetal immune tolerance, which is characterized by comprising the following steps:

1) extracting exosomes of villus trophoblasts of patients with recurrent abortion of unknown reasons;

2) combining the exosomes obtained in step 1) with latex particles;

3) HLA-E flow antibodies are labeled and the level of HLA-E in exosomes is analyzed on a flow cytometric analyzer.

2. The method for detecting the level of human choriotrophoblast exosome HLA-E to assess maternal-fetal immune tolerance according to claim 1, wherein the step 1) comprises the following steps:

1.1) on the day of uterus cleaning of patients with unexplained recurrent abortion, collecting villi of the patients, and rinsing the villi with a sterile phosphate buffer solution with the pH value of 7.4;

1.2) cutting down the villus tissue obtained in the step 1.1) into small pieces with the size of 1 cubic millimeter, re-suspending the small pieces with PBS to obtain a tissue mixed solution, and then digesting the tissue mixed solution with 1mg/ml collagenase IV and 0.01mg/ml DNase on a shaking bed at 37 ℃ for 1 hour;

1.3) filtering the tissue mixed solution obtained in the step 1.2) by using a sterile filter with the diameter of 70 mu m, centrifuging the filtered cell suspension by using a centrifuge at the rotating speed of 1,000 Xg for 5 minutes, and taking out supernatant after the centrifugation is finished;

1.4) filtering the supernatant obtained in the step 1.3) by using a sterile filter with the diameter of 0.22 mu m, filling the filtered liquid into an ultracentrifuge tube, centrifuging the liquid in the ultracentrifuge tube for 1 hour at the temperature of 4 ℃ and at the speed of 100,000 Xg, discarding the supernatant, precipitating the supernatant into an exosome,

1.5) resuspending the exosome with PBS, centrifuging for 1 hour at 4 ℃ and 100,000 Xg in an ultracentrifuge to obtain a precipitate, namely the cleaned exosome.

3. The method for detecting the level of human choriotrophoblast exosome HLA-E to assess maternal-fetal immune tolerance according to claim 2, wherein the step 2) comprises:

after resuspending the exosomes again with 200. mu.l of PBS, quantitatively analyzing the exosome concentration by BCA method, according to the BCA result, taking out 20. mu.g of exosomes from the obtained exosome suspension, adding 10. mu.l of latex particles diluted by 100 times, uniformly mixing, standing at room temperature for 15 minutes, adding 1ml of PBS, then shaking on a shaker at 4 ℃ for 2 hours, then centrifuging the mixture of latex particles and exosomes for 5 minutes at 3,000 Xg, removing supernatant, washing once with PBS, and adding the precipitate into 100. mu.l of PBS for resuspension.

4. The method for detecting the level of human choriotrophoblast exosome HLA-E to assess maternal-fetal immune tolerance according to claim 3, wherein the step 3) comprises:

3.1) Add fluorescently labeled flow antibody to 100. mu.l of the resuspension: HLA-E-APC, mixing, incubating at room temperature in dark for 30 min;

3.2) after the incubation in the dark, 1ml PBS and 3000 Xg are added, the supernatant is removed by centrifugation for 5min, and finally 200. mu.l PBS is added into the precipitate to be loaded on a machine, and then the proportion of the HLA-E positive particles is analyzed.

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