Marker group for immune-related poor pregnancy and application of marker group

Abstract

The invention provides a marker group for immune-related poor pregnancy and application of the marker group, and belongs to the technical field of poor pregnancy diagnosis. According to the invention, a marker of immune-related poor pregnancy is screened, and compared with normal immune-related poor pregnancy, the expression levels of TNF alpha and IL-6 in peripheral blood plasma of immune-related poor pregnancy are obviously improved; the expression of CD3 cells in the spleen and the drainage lymph node is reduced, the activation level of CD4 + T cells to effector memory T cells (Tems): CD44 + CD62L-and central memory T cells (Tcm): CD44 + CD62L + is increased, the proportion of monocytes, neutrophils and M1 type macrophages is increased, the proportion of dendritic cells and M2 type macrophages is reduced, and the proportion of Tc1 and NK + CD107A cells is increased; and the proportion of NKT, NK, CD11B and CD4 + T cells in the placenta is increased.

Description

Technical Field

[0001] The present invention relates to the technical field of adverse pregnancy diagnosis, and in particular to a marker group for immune-related adverse pregnancy and applications thereof. Background Art

[0002] Pregnancy is a common problem in women with rheumatoid diseases (RD). Women with RD are at increased risk for adverse pregnancy and offspring outcomes, with rheumatoid arthritis (RA) also having a significant impact on adverse pregnancy outcomes. RA is a chronic inflammatory disease that can affect women of childbearing age. Women with RA are prone to infertility, with 42% of women taking more than 12 months to conceive. High disease activity and high-dose glucocorticoids and nonsteroidal anti-inflammatory drugs (NSAIDs) in RA patients are associated with infertility and adverse pregnancy outcomes. Some RA treatment drugs, such as methotrexate, are associated with teratogenicity.

[0003] While 48-65% of women with RA experience improvement in RA disease activity during pregnancy, approximately half of these women experience worsening of their disease postpartum. This pattern is associated with immune changes that occur during pregnancy to accommodate the developing fetus. During early pregnancy, paternal antigens (such as sperm, transforming growth factor, prostaglandins, and soluble human leukocyte antigen) are present, increasing the risk of adverse obstetric and delivery outcomes. Among autoimmune diseases, SLE carries the greatest risk of poor prognosis, and these risks are well described and include intrauterine growth restriction, low birth weight, and increased cesarean sections.

[0004] It is now widely recognized that the presence of immune-related diseases during pregnancy increases the risk of low birth weight and preterm birth due to placental insufficiency. However, the association between active RA and hypertension during pregnancy remains controversial.

[0005] Elevated serum cytokine levels are a hallmark of active rheumatoid arthritis. Although not typically seen in healthy pregnant women, cytokines nonetheless play a crucial role in driving disease activity in RA. Excessive maternal cytokine levels initiate and accelerate the inflammatory cytokine cascade that occurs in healthy pregnancies, potentially leading to placental malformation and spontaneous abortion, intrauterine growth restriction (IUGR), or preterm birth. Several perinatal complications, including preeclampsia, preterm birth, threatened miscarriage, and altered maternal cytokine levels, have been associated with these abnormalities.

[0006] RA impairs fertility, and compared with the general population, women with RA have less favorable pregnancy outcomes, especially those with high disease activity. Several studies have reported an association between RA and adverse pregnancy outcomes. A meta-analysis reported that maternal RA increased the risk of autism spectrum disorder in offspring. However, this meta-analysis did not report maternal outcomes or other fetal outcomes. A meta-analysis conducted by Huang et al. showed that maternal RA was significantly associated with an increased risk of adverse maternal and fetal outcomes; however, their meta-analysis did not explore the association between disease activity and pregnancy outcomes. Existing studies have shown that higher RA disease activity is associated with a higher risk of adverse pregnancy outcomes. A study by Langen et al. showed no association between disease activity and pregnancy outcomes in women with RA, but they found that discontinuation of medication increased the chance of adverse pregnancy outcomes at delivery.

[0007] Pregnancy and childbirth can be significant challenges for women with RA, as altered medication regimens, disease flares, and immune dysregulation can adversely affect maternal and fetal outcomes. Over the past decade, optimal use of antirheumatic drugs and the development of novel biologics have led to significant advances in the treatment of RA. A growing number of studies have demonstrated that RA adversely affects pregnancy; however, evidence regarding specific pregnancy outcomes is limited and conflicting. Furthermore, several limitations have been observed in these studies, including insufficient sample sizes, predominantly regional studies, and limited pregnancy outcomes. Current evidence in this area does not include data syntheses or meta-analyses to guide practice. With the advent of these new therapies, ensuring a healthy and uncomplicated pregnancy has become a top priority in the management of pregnant women with RA. Therefore, biomarkers that can detect immune-related adverse pregnancy events are urgently needed to facilitate early diagnosis and predict pregnancy outcomes. Summary of the Invention

[0008] The purpose of the present invention is to provide a marker group for immune-related adverse pregnancy and its application. The marker group can realize the early diagnosis of immune-related adverse pregnancy, provide support for clinicians to accurately predict the pregnancy outcomes of pregnant women, and timely adopt more personalized prevention and treatment plans, thereby minimizing the incidence of immune-related adverse pregnancy and reducing adverse pregnancy outcomes.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0010] The present invention provides a marker panel for immune-related adverse pregnancy, comprising one or more of the following marker groups:

[0011] (1) Expression levels of TNFα and IL-6 in peripheral blood plasma;

[0012] (2) the proportion of CD3 cells in the spleen or draining lymph nodes;

[0013] (3) the activation level of CD4 + T cells to effector memory T cells CD44 + CD62L − in the spleen or draining lymph nodes;

[0014] (4) the activation level of CD4 + T cells in the spleen or draining lymph nodes to central memory T cells CD44 + CD62L + ;

[0015] (5) the proportion of monocytes, neutrophils, or M1 macrophages in the spleen or draining lymph nodes;

[0016] (6) the proportion of dendritic cells or M2 macrophages in the spleen or draining lymph nodes;

[0017] (7) the proportion of Tc1 or NK + CD107A cells in the spleen or draining lymph nodes;

[0018] (8) The proportion of NKT, NK, CD11B or CD4+T cells in the placenta.

[0019] Preferably, among the markers in group (1), the expression levels of TNFα and IL-6 are elevated in patients with immune-related adverse pregnancy.

[0020] Preferably, among the markers in group (2), the CD3 cell expression is decreased in patients with immune-related adverse pregnancy.

[0021] Preferably, in group (3) of markers, the activation level of the CD4+T to effector memory T cells CD44+CD62L- is increased;

[0022] Preferably, in group (4) of markers, the activation level of the CD4+T cells to central memory T cells CD44+CD62L+ is increased.

[0023] Preferably, in group (5) of markers, the proportion of monocytes, neutrophils or M1 macrophages is increased in patients with immune-related adverse pregnancy.

[0024] Preferably, in group (6) of markers, the proportion of dendritic cells or M2 macrophages is reduced in patients with immune-related adverse pregnancy.

[0025] Preferably, in group (7) of markers, the proportion of Tc1 or NK+CD107A cells is increased in patients with immune-related adverse pregnancy.

[0026] Preferably, in group (8) of markers, the proportion of NKT, NK, CD11B or CD4+T cells is increased in patients with immune-related adverse pregnancy.

[0027] The present invention also provides an application of an immune-related adverse pregnancy marker panel in the preparation of an immune-related adverse pregnancy detection product.

[0028] The beneficial effects of the present invention compared with the prior art are:

[0029] (1) The present invention screens markers of immune-related adverse pregnancy by constructing an RA pregnancy animal model, studies the application prospects of these plasma / cell markers in the early diagnosis of immune-related adverse pregnancy, and reveals their screening and early diagnostic value. The results show that compared with normal, the expression levels of TNFα and IL-6 in peripheral blood plasma of immune-related adverse pregnancy are significantly increased; the expression of CD3 cells in the spleen and draining lymph nodes is reduced, the activation level of CD4+T effector memory T cells (Tem): CD44+CD62L- and central memory T cells (Tcm): CD44+CD62L+ is increased, the proportion of monocytes, neutrophils, macrophages, and M1 macrophages is increased, the proportion of dendritic cells and M2 macrophages is reduced, and the proportion of Tc1 and NK+CD107A cells is increased; in the placenta, the proportion of NKT, NK, CD11B, and CD4+T cells is increased.

[0030] (2) The present invention has screened a marker group that can be used to detect immune-related adverse pregnancy. By measuring this marker group, early diagnosis of immune-related adverse pregnancy can be achieved, providing support for clinicians to accurately predict the pregnancy outcomes of pregnant women and to promptly adopt more personalized prevention and treatment plans, thereby minimizing the incidence of immune-related adverse pregnancy, providing guidance for the monitoring and management of pregnant women with RA, and reducing adverse pregnancy outcomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0032] Figure 1 Figure 1 shows RA-like changes in C57BL / 6J-SKG female mice after intraperitoneal injection of mannan in Example 1 of the present invention, wherein A is a photo of the joints of C57BL / 6J female mice after intraperitoneal injection of PBS and C57BL / 6J-SKG female mice after intraperitoneal injection of mannan; B is the joint scores of C57BL / 6J female mice after intraperitoneal injection of PBS and C57BL / 6J-SKG female mice at different time points; C is the percentage of anti-CCP antibodies detected by ELISA after blood was collected from the anterior eyeballs of mice after dissection, ***: P < 0.001;

[0033] Figure 2 Figure 1 shows the results of mouse pregnancy in Example 1 of the present invention, wherein A is a mouse vaginal plug image; B is a representative image of the fetus-umbilical cord-placenta connection; C is a uterine morphology image of the pregnant mouse model, wherein the left side is a representative image of the uterine morphology of the normal mouse control group on the 15th day of pregnancy, the mouse has a bicornuate uterus and multiple pregnancies, no dead fetuses or miscarriage products, the fetuses are uniform in size, and the placenta has good blood supply; the right side is a representative image of the uterine morphology of the RA mouse model group on the 15th day of pregnancy, as indicated by the arrow, miscarriage and dead fetuses wrapped by fat tissue were observed in the RA mouse model; D is the offspring of the pregnant mouse model, wherein the left side is a representative image of the control group and the model Representative pictures of the morphology of offspring fetuses and placentas born to pregnant mice in the type group, the mice had a bicornuate uterus and multiple pregnancies, and the arrows indicated growth-restricted fetuses and placental hematomas born to pregnant mice in the model group; on the right is a comparison chart of the weights of fetal offspring born to pregnant mice in the control group and the model group; E is a comparison chart of the weights of placentas born to pregnant mice in the control group and the model group; F is a comparison chart of the weights of embryos born to pregnant mice in the control group and the model group; G is a statistical chart of the number of vaginal plugs detected in female mice in each group; H is a pie chart of the pregnancy status of the control group and the model group in Example 1 of the present invention; I is a statistical chart of the embryo absorption rate of pregnant mice in the control group and the model group; J is a statistical chart of the pregnancy success rate of the control group and the model group;

[0034] Figure 3 Schematic diagram of the concentration of the standard sample in Example 1 of the present invention;

[0035] Figure 4 The cytokine expression levels of the peripheral blood control group and the model group in Example 1 of the present invention, wherein A is a flow cytometry representative graph of the cytokine expression level in the control group, and B is a flow cytometry representative graph of the cytokine expression level in the model group; CN is the expression level of different cytokines between the two groups (*P<0.05);

[0036] Figure 5 Schematic diagram of the serial dilution of the standard substance in Example 1 of the present invention;

[0037] Figure 6 This is the flow cytometry gating strategy for various cell subsets in the spleen and draining lymph nodes in Example 1 of the present invention;

[0038] Figure 7 Statistical graphs of NK, CD11B, CD19, CD3+T, CD4+ / CD3+T cell ratios and activation levels of CD4+T effector memory T cells (Tem): CD44+CD62L- and central memory T cells (Tcm): CD44+CD62L+ in the spleen of the control group and the model group in Example 1 of the present invention;

[0039] Figure 8This is the flow cytometry gating strategy for myeloid cell subsets in the spleen and draining lymph nodes in Example 1 of the present invention;

[0040] Figure 9 Statistical graph of the proportions of dendritic cells (CD11B+CD11C+MHCII+), monocytes (CD11B+Ly6G+Ly6C+), neutrophils (CD11B+Ly6G+), macrophages (CD11B+F4 / 80+), M1 macrophages (CD11B+F4 / 80+CD80+), and M2 macrophages (CD11B+F4 / 80+CD206+) in the spleen of the control group and the model group in Example 1 of the present invention;

[0041] Figure 10 Tc1 (CD8 + IFNγ + / CD8), NK+CD107A cell ratio statistics;

[0042] Figure 11 The various cell subsets in the placental tissue in Example 1 of the present invention are NKT, NK, CD11B, CD3+T, CD4+T, CD8+T, CD69+CD3, CD11b+CD27+NK, CD11b+CD27 - NK cell ratio statistics. DETAILED DESCRIPTION

[0043] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0044] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0045] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0046] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0047] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0048] Example 1

[0049] Example 1 of the present invention provides a method for analyzing a panel of immune-related adverse pregnancy markers, and the specific steps are as follows:

[0050] 1. Experimental Animals

[0051] All experimental animals used in this study were of the C57BL / 6J background. Wild-type mice were obtained from Jiangsu Jicui Biotechnology Co., Ltd. ZAP-70W163 (SKG) mice of the C57BL / 6J background were generated by our laboratory at Shanghai Nanmo Biotechnology Co., Ltd. and verified by DNA sequencing. Adult mice aged 8-12 weeks were housed in a specific pathogen-free (SPF) environment in the research building of Tongji Hospital. This study was approved by the Animal Ethics Committee of Tongji Hospital (approval number: S-106-24-12-2J).

[0052] 2. Construction of RA pregnancy animal model

[0053] (1) Construction of RA animal model

[0054] (A) In a biosafety cabinet, 500 mg of mannan was weighed and dissolved in 5 mL of sterile PBS to a final concentration of 100 mg / mL.

[0055] (B) All experimental animals were housed in a SPF barrier environment. 8-12 week old C57BL / 6J-SKG mice were intraperitoneally injected with 20 mg (200 μL) of mannan (experimental group) or 200 μL of sterile PBS (SKG control group). Age- and sex-matched C57BL / 6J wild-type mice were also intraperitoneally injected with 200 μL of sterile PBS as WT controls.

[0056] (C) After intraperitoneal injection of mannan / sterile PBS, mice were weighed and their joints were scored weekly. Joint scoring criteria were: 0, no joint swelling; 0.1, swelling of one finger or toe; 0.5, mild swelling of the wrist or ankle; and 1.0, severe swelling of the wrist or ankle. The scores for all fingers (or toes), wrists, and ankles of each mouse's palms and soles were summed to obtain a total score for each mouse.

[0057] like Figure 1 As shown, C57BL / 6J-SKG female mice showed obvious joint swelling 3 days after intraperitoneal injection of mannan, while mice in the PBS injection group did not show joint swelling ( Figure 1 Middle A). Closely monitor the joint swelling of mice and record the joint score according to the scoring criteria (P<0.001), indicating that the RA model was successfully established ( Figure 1 Middle B). The expression level of Anti-CCP antibodies in the peripheral blood plasma of mice was detected by ELISA. It was found that the expression level of Anti-CCP antibodies in the model group was increased compared with the control group (P<0.001) ( Figure 1 Middle C).

[0058] (2) Construction of RA pregnancy model

[0059] (A) Fourteen days after intraperitoneal injection of mannan or PBS, female mice were co-housed with C57BL / 6 male mice, with one male mouse per two female mice, to establish a pregnancy model. Co-housing was generally performed between 5 and 6 PM. Starting the next day, vaginal plugs were checked at 8:30 AM and 3:00 PM daily. The day of detection of a vaginal plug was designated as pregnancy day 0 (P0).

[0060] (B) When female C57BL / 6J-SKG mice develop vaginal plugs ( Figure 2 Male and female mice were housed in separate cages. Mice were sacrificed on gestational day 15 (P15). Thromboembolism detection in the control and model groups was continuously observed, and the number of mice successfully detected in each group was recorded daily.

[0061] 3. Animal materials

[0062] 1) Weighing: Weigh the pregnant mice on day 15 of gestation and record the values.

[0063] 2) Anesthesia: Anesthetize the mouse with isoflurane and proceed with subsequent procedures after the mouse stops struggling.

[0064] 3) Blood collection: Peripheral blood was collected from each group of pregnant mice by enucleation on a small animal dissection table (using EP tubes containing EDTA anticoagulant, and taking care not to shake violently to avoid hemolysis).

[0065] 4) Fixation: Pregnant mice were sacrificed by cervical dislocation, and the mice were immersed in 75% alcohol for 2 minutes for disinfection. The limbs of the mice were fixed with pins.

[0066] 5) Sampling: Expose the mouse pelvic and abdominal cavities by incising the skin from above the anus to the xiphoid process. Separate and remove the uterus. Remove the spleen and weigh and record the values. Also, remove the mesenteric, axillary, and inguinal draining lymph nodes and their surrounding tissues (fascia, adipose tissue).

[0067] 6) Separation of placenta and fetus: Use ophthalmic scissors to completely cut off and remove the uterus, observe and record the number of normal surviving embryos (tender red in color, embryos have formed, and the attached amniotic sac and placenta are relatively intact) and abnormal embryos, bluntly tear the uterine wall from the uterine horn, select the normal surviving embryos adjacent to the absorbed embryo, peel the embryo-placenta unit from the uterine wall, ensure the integrity of the basal decidua, bluntly separate the embryo and placenta, remove the fetus and placenta, weigh them, and test their pregnancy rate. The results are as follows: Figure 2 shown.

[0068] 7) Calculate embryo absorption rate

[0069] The mouse embryo absorption rate was calculated according to "embryo absorption rate = number of absorbed embryos / (number of surviving embryos + number of absorbed embryos) x 100%".

[0070] Figure 2 Middle B and C show that the uterine placental blood supply of pregnant mice in the control group was better than that in the model group.

[0071] Figure 2 Figures D, E, and F show that the control group had fewer adverse pregnancy outcomes such as miscarriage, stillbirth, growth-restricted fetuses, and placental hematoma, and there was no significant difference in the weight of fetuses and placentas between the two groups.

[0072] Figure 2 Middle G shows that the control group had completed all the procedures after 5 days of cage placement. At this time, the number of mice in the control group (n=12) with successful thrombus detection was higher than that in the model group (n=11), and the success rate of thrombus detection in the control group (100%) was also significantly higher than that in the model group (45.83%).

[0073] Figure 2 In the H, I, and J control groups, 9 out of 12 female mice successfully tested for thrombus formation became pregnant, with an embryo absorption rate of 5.33% (4 / 75). In the model group, 9 out of 13 24 female mice successfully tested for thrombus formation became pregnant, with an embryo absorption rate of 13.79% (4 / 29). The pregnancy success rates of each group were 75% (9 / 12) and 37.5% (4 / 24), respectively.

[0074] 8) Separation of limb joints: Use ophthalmic scissors and forceps to peel off the skin on the limb joints. After separation, place the upper and lower limbs on the same side in 4% paraformaldehyde for fixation. Place the upper and lower limbs on the other side in cryotubes and quickly place them in a liquid nitrogen tank (note that the entire process should be as rapid as possible). After 30 minutes, transfer the cryotubes to an ultra-low temperature freezer.

[0075] 9) Tissue Placement: Place one-half of the placenta in PBS on ice for subsequent preparation of a single-cell suspension for flow cytometry. Place one-quarter of the placenta in 4% paraformaldehyde for pathological analysis. Place one-quarter of the placenta in a cryovial and quickly place it in a liquid nitrogen tank (note that the entire process should be as rapid as possible). After 30 minutes, transfer the cryovial to an ultra-low temperature freezer.

[0076] 4. Flow cytometry staining method

[0077] (1) Cell surface molecule staining

[0078] Lymphocyte subset color scheme: FVS-AF700, CD45-BV785, CD3-BV605, CD4-BB700, CD8-ECD, NK1.1-BV605, CD11b-PE / CY7, CD25-PE, CD44-BV421, CD62L-BV510, CD69-APC

[0079] (A) Cell counting: Take 10 5 The liquid of 100 μL of cells was placed in a flow cytometry tube and centrifuged at 300 g for 5 min at 4°C. After centrifugation, the supernatant was discarded.

[0080] (B) Cell viability staining: Wash once with PBS to remove serum (serum will inactivate the viability stain), then add 30 μL of Fixable Viability Stain 700 at a 1:1000 dilution to each tube, vortex to mix, incubate at 4°C in the dark for 15 min, add 1 mL of PBS, centrifuge at 300 g for 5 min at 4°C, and discard the supernatant.

[0081] (C) Surface staining: Resuspend cells in 30 μL PBS and add BrilliantViolet605 at a ratio of 1:100-1:200. TM anti-mouse CD3, BB700 RatAnti-Mouse CD4(RM4-5), PE-CF594 Rat Anti-Mouse CD8a, Brilliant Viole605 TM anti-mouse NK1.1, PE / Cyanine7anti-mouse / human CD11b, APC-Cy TM7Rat Anti-Mouse CD19, FITC anti-mouse / human / rat CD27, PE anti-mouse CD26 (DPP-4) Antibody, Brilliant Violet 421 TM Incubate with anti-mouse CD44, APC RatAnti-Mouse CD62L, and APC anti-mouse CD69 flow cytometry antibodies on ice in the dark for 30 minutes.

[0082] (D) After incubation, add 1 mL of PBS, mix thoroughly, and centrifuge at 300 g for 5 min at 4°C. Discard the supernatant.

[0083] (E) Add appropriate amount of PBS to resuspend the cells and store in the dark at 4°C until analysis by flow cytometry.

[0084] (2) Cell surface molecule staining

[0085] Myeloid staining: FVS-AF700, CD45-BV785, CD80-FITC, MHCII-BV65, CD11b-PE / CY7, CD206-PE, F4 / 80-BV421, Ly6G-BV510, Ly6C-APC

[0086] (A) Cell counting: Take 10 5 The liquid of 100 μL of cells was placed in a flow cytometry tube and centrifuged at 300 g for 5 min at 4°C. After centrifugation, the supernatant was discarded.

[0087] (B) Cell viability staining: Wash once with PBS to remove serum (serum will inactivate the viability stain), then add 30 μL of Fixable Viability Stain 700 at a 1:1000 dilution to each tube, vortex to mix, incubate at 4°C in the dark for 15 min, add 1 mL of PBS, centrifuge at 300 g for 5 min at 4°C, and discard the supernatant.

[0088] (C) Fc blocking: Resuspend the centrifuged sample in 50 μL PBS (to a final volume of 100 μL). Add 2 μL of Fc receptor blocker to the sample tube and mix thoroughly. (If there are a lot of cells and the cell is turbid and white, increase the resuspension volume and add more blocker in proportion. For example, add 4 μL of blocker to resuspend the cells in 200 μL PBS.) Incubate at 4°C in the dark for 15 min. Add 1 mL of PBS and centrifuge at 300 g to remove the supernatant.

[0089] (D) Surface staining: Resuspend cells in 30 μL PBS and add FITC anti-mouse CD80 and Brilliant Violet 650 at a dilution of 1:100-1:200. TM Anti-Mouse IA / IE, PE / Cyanine7anti-mouse / human CD11b, APCanti-mouse Ly6C, Brilliant Violet 510 TM anti-mouse Ly6G, APC-Cy TM 7RatAnti-MouseCD11c、Brilliant Violet 421 TM Anti-mouse F4 / 80 and PE anti-mouse CD206 flow cytometry antibodies were incubated on ice in the dark for 30 minutes.

[0090] (E) After incubation, add 1 mL of PBS, mix thoroughly, and centrifuge at 300 g for 5 min at 4°C. Discard the supernatant.

[0091] (F) Add appropriate amount of PBS to resuspend the cells and store in the dark at 4°C until analysis by flow cytometry.

[0092] (3) Intracellular cytokine staining

[0093] Intracellular staining scheme: FVS-AF700, CD3-BV605, CD4-BB700, CD8-ECD, NK1.1-BV605, CD26-PE / CY7, IL-17A-APC, IFN-γ-BV480, CD107a-APC / CY7

[0094] (A) Cell counting: Take 10 6 The liquid of 100 μL of cells was placed in a flow cytometry tube and centrifuged at 300 g for 5 min at 4°C. After centrifugation, the supernatant was discarded.

[0095] (B) Resuspend the cells in 500 μL of RPMI 1640 complete medium containing 10% FBS, add 1 μL of cell stimulator (plus protein transport inhibitor), mix well, and incubate in a 37°C incubator for 4.5 h.

[0096] (C) After incubation for 4.5 h, remove the cells and centrifuge at 300 g for 5 min at 4°C in a pre-cooled centrifuge. Discard the supernatant.

[0097] (D) Cell viability staining: Wash once with PBS to remove serum (serum will inactivate the viability stain), then prepare Fixable Viability Stain 700 at a 1:1000 dilution, add 50 μL to each tube, vortex to mix, incubate at 4°C in the dark for 15 min, add 1 ml of PBS, centrifuge at 300 g for 5 min at 4°C, and discard the supernatant.

[0098] (E) Resuspend cells in 50 μL PBS and add BrilliantViolet 605 at a dilution of 1:100-1:200. TM anti-mouseCD3, BB700 Rat Anti-Mouse CD4(RM4-5), PE-CF594 Rat Anti-Mouse CD8a, BrilliantViolet 605 TM Anti-mouse NK1.1, PE anti-mouse CD25, PE / Cyanine7 anti-mouse CD26 (DPP-4) flow cytometry antibodies were incubated on ice in the dark for 30 minutes.

[0099] (F) After incubation, add 1 mL of PBS, mix thoroughly, and centrifuge at 300 g for 5 min at 4°C. Discard the supernatant.

[0100] (G) Add 100 μL of fixative, mix well, and incubate on ice in the dark for 50-60 min.

[0101] (H) After the incubation, add 1 mL of 1× permeabilization reagent (10× permeabilization reagent is diluted to 1× with ddH2O), mix thoroughly, and centrifuge at 600g for 5 min at 4°C. Discard the supernatant.

[0102] (I) Resuspend the cells in 50 μL 1× permeabilization reagent and add APC anti-mouse IL-17A Antibody and Brilliant Violet 480 at a dilution of 1:100. TM Anti-mouse IFN-γ, APC / Cyanine7 anti-mouse CD107a (LAMP-1). Incubate on ice in the dark.

[0103] (J) After incubation, add 1 mL of PBS, mix thoroughly, and centrifuge at 600 g for 5 min at 4°C. Discard the supernatant.

[0104] (K) Add appropriate amount of PBS to resuspend the cells and store in the dark at 4°C until analysis by flow cytometry.

[0105] 5. Enzyme-linked immunosorbent assay (ELISA)

[0106] (1) Preparation of items

[0107] 1) Bring all reagents to room temperature (18-25°C) and equilibrate for at least 30 minutes. Prepare the reagents as described above and set aside.

[0108] (2) Sample preparation

[0109] 1) Take the aliquoted plasma out of the -80°C freezer and thaw on ice.

[0110] 2) Cool down the low-temperature high-speed centrifuge in advance.

[0111] 3) Place the thawed plasma in a low-temperature high-speed centrifuge, centrifuge at 1000g, 4°C, for 10 min.

[0112] 4) Carefully take out the Eppendorf tube and place it on the EP tube. Let it stand on ice until the sample is added.

[0113] (3) Configuration of standard products

[0114] 1) Remove one standard from the kit and centrifuge at 6,000-10,000 rpm for 30 seconds. Dissolve in 1 ml of sample diluent and repeatedly pipette five times toward the bottom of the cryotube to aid dissolution. Mix thoroughly to obtain standard S7 and set aside.

[0115] 2) Arrange seven 1.5ml centrifuge tubes (S0-S6) in sequence and add 250μl of sample diluent to each. Pipette 250μl of standard S7 into the first centrifuge tube (S6) and gently pipette to mix. Pipette 250μl from S6 into the second EP tube (S5) and gently pipette to mix. Repeat this process to dilute the standard sample in multiples. S0 is the sample diluent, e.g. Figure 3 , as shown in Table 1.

[0116] Table 1 Standard concentrations

[0117] serial number S7 S6 S5 S4 S3 S2 S1 S0 U / mL 400 200 100 50 25 12.5 6.25 0

[0118] (4) Add standards and samples

[0119] 1) Open the biochemical incubator in advance and adjust the temperature to 37°C.

[0120] 2) Sample Addition: Set up separate wells for standards and samples. Add 100 μL of standard or sample to each well, gently shake to mix, cover with a plate sticker, and incubate at 37°C for 2 hours.

[0121] 3) Key points of operation: Both standards and samples are set up for double-well determination. When adding samples, please use disposable clean pipette tips to avoid cross contamination. When adding samples, be as gentle as possible to avoid foaming. Add the samples to the bottom of the ELISA plate wells and do not add samples along the well walls. The time for one addition is best controlled within 10 minutes. If there are a large number of samples, it is recommended to use a discharge gun for addition. To prevent sample evaporation, the ELISA plate must be covered with a plate sticker during the incubation process, and the ELISA plate should not be left in an open and dry state during the experiment. During the incubation process, the incubator temperature should be observed at any time to ensure that it is constant at 37°C and adjusted in time. During the incubation reaction, the incubator should not be opened too many times to avoid affecting the temperature balance.

[0122] (5) Add biotin marker working solution

[0123] 1) Prepare the biotin marker working solution 10 minutes in advance. Dilute the biotin marker with biotin marker diluent at a ratio of 1:100. Prepare the total volume required for each experiment based on the pre-calculated amount. For each experiment, prepare an extra 0.1-0.2 mL and mix gently.

[0124] 2) After incubation, carefully remove the sealing film, discard the liquid, and spin dry without washing.

[0125] 3) Add 100 μL of biotin marker working solution to each well, cover with a new plate sticker, and incubate at 37°C for 1 hour.

[0126] (6) Add horseradish peroxidase-labeled avidin

[0127] 1) Prepare concentrated wash solution 10 minutes in advance and dilute it 1:25 with deionized water. Store the concentrated wash solution at low temperatures, as salt precipitation may occur. Warm the solution in a water bath to aid dissolution during dilution. Prepare the solution based on the pre-calculated total volume required for each experiment. Prepare an extra 0.1-0.2 mL and mix gently.

[0128] 2) Prepare the horseradish peroxidase-labeled avidin working solution 10 minutes in advance. Dilute the horseradish peroxidase-labeled avidin with the biotin label diluent at a ratio of 1:100. Prepare the total volume required for each experiment based on the pre-calculated amount. For each experiment, prepare an extra 0.1-0.2 mL and mix gently.

[0129] 3) After incubation, carefully remove the sealing film, discard the liquid in the wells, spin dry, and wash the plate three times, soaking for 2 minutes each time, 200 μL / well, and spin dry.

[0130] 4) Key Points: The washing process is crucial; inadequate washing can easily result in false positives. During each wash, completely drain the liquid from the wells and pat dry on absorbent paper. Never place absorbent paper directly into the wells to absorb water, or use a pipette to remove liquid from the wells. If using a pipette to remove liquid from the wells, avoid touching the plate walls.

[0131] (7) Add substrate solution

[0132] 1) After incubation, carefully remove the sealant, discard the liquid in the wells, spin dry, and wash the plate five times. Soak for 2 minutes each time, 200 μL / well, and spin dry.

[0133] 2) Use a pipette to add 90 μL of substrate solution to each well in sequence and incubate at 37°C in the dark for 15-30 minutes.

[0134] (8) Add stop solution

[0135] 1) Use a pipette to add 50 μL of stop solution to each well to terminate the reaction.

[0136] 2) Key points: To ensure the accuracy of experimental results, the stop solution should be added as soon as the substrate reaction time expires. After adding the substrate solution, observe the color development at regular intervals (e.g., every 10 minutes) to control the reaction time. When a clear blue gradient is visible in the first 3-4 wells of the standard sample and the color development is less obvious in the last 3-4 wells, add the stop solution to terminate the reaction. The blue color will immediately turn yellow. The stop solution should be added in the same order as the substrate solution as much as possible.

[0137] (9) Reading detection

[0138] 1) Open the microplate reader in advance and set the detection parameters.

[0139] 2) Within 5 minutes after the reaction is terminated, the optical density (OD value) of each well is measured in sequence at a wavelength of 450 nm using a microplate reader.

[0140] (10) Data Analysis

[0141] Draw a standard curve on logarithmic paper, plotting the standard concentration on the vertical axis (logarithmic scale) and the OD value on the horizontal axis (logarithmic scale). We recommend using professional curve analysis software. Use "Curve Expert" and follow the prompts to create a standard curve. Based on the sample OD value, use the standard curve to determine the corresponding concentration. Alternatively, calculate the regression equation for the standard curve using the standard concentration and OD value. Substitute the sample OD value into the equation to calculate the sample concentration. If the sample is diluted before testing, multiply by the dilution factor in the final calculation to obtain the actual sample concentration.

[0142] 7. Separation of plasma

[0143] (1) After obtaining peripheral blood from pregnant mice, place it at room temperature for half an hour, pre-cool the centrifuge, centrifuge (1000g, 4℃, 10min), carefully divide the upper pale yellow plasma into 1.5ml Eppendorf tubes, store in a -80℃ refrigerator, and test relevant indicators using CBA kits. The results are as follows: Figure 4 shown.

[0144] (2) Cytokine CBA detection

[0145] ① Preparation of cytokine standards

[0146] 1) Transfer one vial of lyophilized cytokine standard pellets to a 15 mL conical centrifuge tube and label it as the highest concentration standard (5000 pg / mL).

[0147] 2) Dilute the standard with 2 mL of Assay Diluent and equilibrate at room temperature for at least 15 minutes;

[0148] 3) Gently mix the standard with a pipette tip; do not vortex or shake vigorously.

[0149] 4) Take nine 12x75mm flow cytometry tubes and label them with the gradient dilution ratios of 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, and 1:256;

[0150] 5) Add 300 μL Assay Diluent to each tube;

[0151] 6) Starting from the highest concentration standard, add 300 μL of dilution buffer one by one to dilute the standard sample in a gradient manner, e.g. Figure 5 As shown: (take 300 μL of the solution from the highest concentration standard tube to the 1:2 tube and mix thoroughly by pipetting. Then take 300 μL of the solution from the 1:2 tube to the 1:4 tube and mix thoroughly by pipetting. And so on, until the 1:256 tube is reached).

[0152] 7) Finally, take a 12×75 mm flow cytometry sample tube and add 300 μL Assay Diluent as a negative control tube (0 pg / mL).

[0153] ② Preparation of mixed cytokine capture microspheres

[0154] 1) Determine the number of experimental samples (including all standards, negative controls, and test samples).

[0155] 2) Each capture microsphere needs to be fully vortexed for 5-15 seconds before mixing (this step is critical to the entire experiment. To ensure sufficient microspheres, it is recommended to fully mix the microsphere solution before aspirating each microsphere).

[0156] 3) Pipette an appropriate amount of capture microspheres according to 10 μL / sample, and mix all microspheres in a flow tube, marked as "mixed microspheres".

[0157] ③ Sample incubation

[0158] 1) Incubate at room temperature in the dark for 3 hours; thoroughly vortex and resuspend the "mixed microspheres" and add 50 μL to each experimental tube;

[0159] 2) Standards and test samples:

[0160] a) Add 50 μL of the serially diluted standard to each standard tube. The concentrations of the standard are shown in Table 2:

[0161] Table 2 Dilution multiples

[0162] pipe number Concentration (pg / mL) Dilution multiple 1 0 AssayDiluent 2 20 1：256 3 40 1：128 4 80 1：64 5 156 1：32 6 312.5 1：16 7 625 1：8 8 1250 1：4 9 2500 1：2 10 5000 Highest concentration standard

[0163] b) Add 50 μL of the sample to be tested to each sample tube;

[0164] c) Add 50 μL of PE-labeled cytokine detection antibody to all experimental tubes;

[0165] d) Incubate at room temperature in the dark for 3 hours;

[0166] ④On-machine testing

[0167] 1) Add 1 mL of washing solution to each tube and centrifuge at 300 g for 5 minutes at 4°C.

[0168] 2) Carefully aspirate or gently pour off the supernatant and resuspend the cells by adding 300 μL of wash buffer to each tube;

[0169] 3) After the instrument is calibrated, start testing as soon as possible (to avoid degradation of cytokines in the standards and test samples, start testing as soon as possible. If there is any delay, place the samples on ice, but not overnight).

[0170] ⑤Data analysis

[0171] After the samples were collected (FCS2.0 format), the CBA-specific analysis software FCAPArray v1.0 was used to draw the standard curve and perform data analysis.

[0172] Figure 4 The results showed that compared with the control group, the expression levels of TNFα and IL-6 in the model group were significantly increased, and there was a statistical difference between the two groups (P<0.05); while the expression levels of IFNγ, IL-17, IL-4, and IL-2 relative to TNFα and IL-6 did not change significantly, and there was no statistical difference.

[0173] 8. Mouse spleen and draining lymph node detection

[0174] (1) Preparation of mouse spleen single cell suspension

[0175] (A) The spleen is placed in a culture dish or plate, an appropriate amount of PBS is added, and the spleen is gently ground with a pestle. During the grinding process, single spleen cells will be seen flowing out. After the grinding is completed, only the spleen capsule remains.

[0176] (B) Rinse the culture dish or plate with PBS, remove as many of the ground cells as possible, and transfer them to a labeled EP tube.

[0177] (C) Place in a pre-cooled centrifuge at 300g for 5 minutes.

[0178] (D) After centrifugation, discard the supernatant, add 1-2 mL of red blood cell lysis buffer, mix well, and let it stand for 5 minutes.

[0179] (E) Add an equal amount of PBS to stop the red lysis, filter through a 300-mesh filter cloth, and centrifuge at 300 g for 5 min at 4°C.

[0180] (F) Discard the supernatant and resuspend the cells in PBS for subsequent flow cytometry analysis.

[0181] (2) Preparation of single cell suspension of mesenteric draining lymph nodes

[0182] (A) Place the mesenteric draining lymph node in a culture dish or plate, add 500 μL of PBS, and gently grind with a pestle. During the grinding process, single cells from the mesenteric draining lymph node will be seen to flow out. After grinding, only the surrounding connective tissue or adipose tissue remains.

[0183] (B) Rinse the culture dish or plate with PBS, remove as many of the ground cells as possible, and transfer them to a labeled EP tube.

[0184] (C) Filter through a 300-mesh filter cloth and centrifuge at 300 g for 10 min at 4°C.

[0185] (D) Discard the supernatant and resuspend the cells in PBS for subsequent flow cytometry analysis.

[0186] (3) Preparation of single cell suspension of axillary draining lymph nodes

[0187] (A) The removed axillary draining lymph node is placed in a culture dish or plate, 500 μL of PBS is added, and the lymph node is gently ground with a pestle. During the grinding process, single cells from the axillary draining lymph node are observed to flow out. After grinding, only the surrounding connective tissue or adipose tissue remains.

[0188] (B) Rinse the culture dish or plate with PBS, remove as many of the ground cells as possible, and transfer them to a labeled EP tube.

[0189] (C) Filter through a 300-mesh filter cloth and centrifuge at 300 g for 10 min at 4°C.

[0190] (D) Discard the supernatant and resuspend the cells in PBS for subsequent flow cytometry analysis.

[0191] (4) Preparation of single cell suspension of inguinal draining lymph nodes

[0192] (A) The removed inguinal draining lymph node is placed in a culture dish or plate, 500 μL of PBS is added, and the lymph node is gently ground with a pestle. During the grinding process, single cells are observed to flow out of the inguinal draining lymph node. After grinding, only the surrounding connective tissue or adipose tissue remains.

[0193] (B) Rinse the culture dish or plate with PBS, remove as many of the ground cells as possible, and transfer them to a labeled EP tube.

[0194] (C) Filter through a 300-mesh filter cloth and centrifuge at 300 g for 10 min at 4°C.

[0195] (D) Discard the supernatant and resuspend the cells in PBS for subsequent flow cytometry analysis.

[0196] The flow gate strategy for the above flow detection is as follows Figure 6 、 8 As shown, the results are Figure 7 、 9 , as shown in 10.

[0197] Figure 7 Results showed that compared with the control group, the expression of NK% and CD11B% cells in the spleen and draining lymph nodes of mice in the C57BL / 6J-SKG model group was increased, while the expression of CD3% cells was decreased. Furthermore, the activation levels of CD4+ T cells in the spleen and draining lymph nodes of mice in the C57BL / 6J-SKG model group to effector memory T cells (Tem): CD44+CD62L- and central memory T cells (Tcm): CD44+CD62L+ were increased.

[0198] Figure 9 The results showed that the proportions of monocytes, neutrophils, and M1 macrophages in the spleen and draining lymph nodes of mice in the C57BL / 6J-SKG model group were increased, while the proportions of dendritic cells and M2 macrophages were decreased.

[0199] Figure 10 The results showed that Tc1(CD8 + IFNγ + / CD8) and NK+CD107A cell ratios increased.

[0200] 7. Preparation of placental single cell suspension

[0201] (A) Place the removed 1 / 2 placenta into a 2 ml EP tube containing 0.5 ml PBS and place on ice for subsequent mincing and digestion.

[0202] (B) Cut the placenta into pieces on ice, being careful to be gentle.

[0203] (C) Pre-cool the centrifuge and centrifuge at 400 g, 4°C, for 5 min.

[0204] (D) Add 1 ml of digestion solution.

[0205] (E) Seal the EP tube cap with parafilm, turn the EP tube upside down to fully resuspend the placental tissue in the digestion solution, and place the EP tube in a preheated 37°C constant temperature shaking water bath for 30 minutes.

[0206] (F) After digestion, quickly add 1 ml of complete culture medium to the EP tube to terminate the digestion. Invert the EP tube upside down. The entire digestion process should be performed as quickly as possible.

[0207] (G) The mixed liquid was aspirated with a Pasteur pipette, and the placental single cell suspension was filtered through a 70 μm filter into an EP tube. The collected filtrate was centrifuged at 400 g and 4°C for 5 min.

[0208] (H) Discard the supernatant and add 500 μL of red blood cell lysis buffer to the pellet. Mix thoroughly and let it stand at room temperature for 5 min.

[0209] (I) After red cell lysis, add 1 ml of 1× PBS to terminate the reaction.

[0210] (J) Centrifugation, 400 g, 4°C, 5 min.

[0211] (E) Discard the supernatant and resuspend the cells in PBS for subsequent flow cytometry analysis.

[0212] The results of flow cytometry testing are as follows Figure 11 shown.

[0213] Figure 11 It was shown that the proportions of NKT, NK, CD11B, and CD4+ / CD3+T cells in the placenta of mice in the C57BL / 6J-SKG model group were increased.

[0214] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A marker panel for immune-related adverse pregnancy, characterized in that: Include one or more of the following groups of markers: (1) Expression levels of TNFα and IL-6 in peripheral blood plasma; (2) the proportion of CD3 cells in the spleen or draining lymph nodes; (3) the activation level of CD4+ T cells to effector memory T cells CD44+CD62L- in the spleen or draining lymph nodes; (4) the activation level of CD4+ T cells in the spleen or draining lymph nodes to central memory T cells CD44+CD62L+; (5) the proportion of monocytes, neutrophils, or M1 macrophages in the spleen or draining lymph nodes; (6) the proportion of dendritic cells or M2 macrophages in the spleen or draining lymph nodes; (7) the proportion of Tc1 or NK+CD107A cells in the spleen or draining lymph nodes; (8) The proportion of NKT, NK, CD11B or CD4+T cells in the placenta.

2. The immune-related adverse pregnancy marker panel according to claim 1, characterized in that: Among the markers in group (1), the expression levels of TNFα and IL-6 were elevated in patients with immune-related adverse pregnancy.

3. The immune-related adverse pregnancy marker panel according to claim 1, characterized in that: Among the markers in group (2), the CD3 cell expression was decreased in patients with immune-related adverse pregnancy.

4. The immune-related adverse pregnancy marker panel according to claim 1, characterized in that: In the third group of markers, the activation level of the CD4+T to effector memory T cells CD44+CD62L- is increased.

5. The immune-related adverse pregnancy marker panel according to claim 1, characterized in that: In the fourth group of markers, the activation level of the CD4+T to central memory T cells CD44+CD62L+ is increased.

6. The immune-related adverse pregnancy marker panel according to claim 1, characterized in that: In group (5) of markers, the proportion of monocytes, neutrophils or M1 macrophages is increased in patients with immune-related adverse pregnancy.

7. The immune-related adverse pregnancy marker panel according to claim 1, characterized in that: In group (6) markers, the proportion of dendritic cells or M2 macrophages was decreased in patients with immune-related adverse pregnancy.

8. The immune-related adverse pregnancy marker panel according to claim 1, characterized in that: In group (7) of markers, the proportion of Tc1 or NK+CD107A cells was increased in patients with immune-related adverse pregnancy.

9. The immune-related adverse pregnancy marker panel according to claim 1, characterized in that: In the group (8) of markers, the proportion of NKT, NK, CD11B or CD4+T cells is increased in patients with immune-related adverse pregnancy.

10. Use of the immune-related adverse pregnancy marker panel according to any one of claims 1 to 9 in preparing a product for detecting immune-related adverse pregnancy.

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