CN112946283B

CN112946283B - Application of quantitative A2M product in preparation of preeclampsia diagnosis tool

Info

Publication number: CN112946283B
Application number: CN202110134187.4A
Authority: CN
Inventors: 李瑞满; 王广; 王静云
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2021-01-29
Filing date: 2021-01-29
Publication date: 2022-11-29
Anticipated expiration: 2041-01-29
Also published as: CN112946283A

Abstract

The invention discloses application of a quantitative A2M product in preparation of a preeclampsia diagnosis tool. The invention discovers that the expression level of A2M in the placenta and the plasma of a PE pregnant woman is obviously increased for the first time. The invention provides application of A2M serving as a novel biomarker of PE in preparation of a preeclampsia diagnosis tool, and PE can be prompted when the A2M level is detected to rise. Reagents for quantifying A2M levels for clinical diagnosis of PE are a supplement to existing studies. The invention also provides application of at least one of sFlt-1 and PIGF and A2M as a biomarker composition of PE in preparation of a pre-eclampsia diagnostic tool. The invention not only provides a new idea for the research of PE prediction or diagnosis, but also is beneficial to further research and development of new drugs for preventing or treating PE.

Description

Application of quantitative A2M product in preparation of preeclampsia diagnosis tool

Technical Field

The invention belongs to the field of biological medicine, and particularly relates to application of a quantitative A2M product in preparation of a preeclampsia diagnosis tool.

Background

Preeclampsia (PE) is a pregnancy idiopathic disease occurring after 20 weeks of pregnancy, affects 2% -8% of pregnancies worldwide, seriously threatens the safety of mothers and infants, and has attracted attention of numerous scholars at home and abroad at present. The prevalence of PE is reported to be about 3% -7% worldwide, and much higher in china than the world's average prevalence of about 9% -10%. The disease seriously affects the health of the mother and the child and is one of the main causes of the increase of the death rate of the pregnant and lying-in women and the childbirth. The clinical manifestations include the increase of blood pressure, edema, proteinuria and the like of pregnant women, and the continuous deterioration of the disease conditions may cause eclampsia, HELLP syndrome, fetal Growth Restriction (FGR), intrauterine death and the like. The cause of PE is not completely clarified so far, and the PE is considered to be a placenta disease at present and is related to insufficient recasting of spiral arterioles of the uterus, excessive activation of inflammatory immunity, dysfunction of vascular endothelium, genetic factors and the like. The current common biomarkers of PE include Soluble vascular endothelial growth factor receptor-1 (Soluble fms-like tyrosine kinase 1, sflt-1), placental growth factor (PIGF), etc., but their specificity and sensitivity are uncertain, and it is still uncertain to be widely applied clinically in diagnosis and prediction of PE, to find biomarkers related to PE, or to combine multiple protein markers as a challenge for scholars both at home and abroad.

Alpha 2 macroglobulin (A2M), a macromolecular glycoprotein abundant in plasma, has a relative molecular mass of 725000 and is composed of four identical subunits 180000 in size, each of which contains 5 functional regions, namely a decoy region, an internal thioester bond, a receptor binding site, a transglutaminase reaction site and a zinc ion binding site. It was first recognized as a broad-spectrum protease inhibitor and is a pan-protease inhibitor that can mechanically inhibit all protease families. It plays an important role in the clearance of proteases in the circulation, regulation of fibrinolysis, coagulation and complement activation. Unlike other protease inhibitors that act by blocking the active site of the protease, A2M acts by binding to the protease molecule through a special "trapping" mechanism, isolating it from the substrate. The molecular mechanism is as follows: when A2M binds to a protease, the bait region of its molecule is first exposed to the protease, which breaks internal thioester bonds and initiates a change in its molecular conformation, forming a cage structure and encapsulating the protease within, hindering access to the protein substrate. Allosterically, A2M reacts with proteases, resulting in its exposed C-terminal receptor binding domain that is recognized by the CD91/LRP receptor on the surface of some cells (e.g., hepatocytes, macrophages, etc.) and mediates endocytosis, which is then rapidly cleared by the intracellular lysosomal pathway. In addition, A2M binds to many cytokines (e.g., TGF β, IL1 β, TNF α, etc.), growth factors (e.g., VEGF, PDGF, NGF, etc.), hormones, etc., and has a different affinity for A2M in different conformations. TGF β, IL1 β, TNF α, etc. have a strong affinity for activated A2M, whereas PDGF, NGF, EGF, etc. preferentially bind to A2M in its original conformation. In addition, after the A2M is oxidized by oxygen free Radicals (ROS), the property of binding cytokines of the A2M is changed, for example, the binding force to proinflammatory cytokines such as TNF alpha, IL-6, IL1 beta and the like is obviously enhanced, the activity of the proinflammatory cytokines is inhibited, and the affinity of the proinflammatory cytokines to growth factors such as TGF beta, bFGF, NGF, PDGF-BB and the like is greatly reduced. Thus, A2M specifically regulates the distribution, half-life and biological activity of different cytokines in vivo, exerting a wide and diverse range of biological functions.

In recent years, researches show that the A2M has good effects on treating radioactive skin mucosal ulcer injury, radioactive pneumonia, radioactive cystitis, keratitis and corneal ulcer. A2M can also be used as a biomarker for judging the stage of hepatic fibrosis and the severity of pancreatitis. However, no correlation between A2M and PE has been found.

In view of the above, there is a great clinical need to select more valuable biomarkers to provide a more feasible, efficient and economical reference for the diagnosis of PE.

Disclosure of Invention

In view of the shortcomings and drawbacks of the prior art, it is an object of the present invention to provide a method for quantifying the amount of A2M in the preparation of a pre-eclampsia diagnostic tool.

The purpose of the invention is realized by the following technical scheme:

use of a product for quantifying A2M in the preparation of a pre-eclampsia diagnostic tool.

Further, the products include products for diagnosing preeclampsia by quantifying A2M levels by immunoblotting, immunohistochemistry, immunofluorescence, enzyme-linked immunosorbent assays, and any suitable biological detection method.

Still further, the product comprises an antibody specific for A2M.

Still further, the specific antibody includes a monoclonal antibody or a polyclonal antibody.

Further, the sample of quantitative A2M is placental tissue of a pregnant woman or peripheral venous blood.

Furthermore, the pregnant women are pregnant women in middle and late pregnancy.

Application of A2M in preparing products for regulating expression of sFlt-1 and/or PIGF.

Use of a product for quantifying a protein composition comprising at least one of sFlt-1 and PIGF and A2M in the manufacture of a pre-eclampsia diagnostic tool.

Further, the products include products for diagnosing preeclampsia by quantifying protein levels by immunoblotting, immunohistochemistry, immunofluorescence, enzyme-linked immunosorbent assays, and any suitable biological detection method.

Compared with the prior art, the invention has the following beneficial effects:

the invention adopts quantitative proteomics analysis and carries out laboratory verification, and the result shows that A2M in the placenta and the blood plasma of a PE pregnant woman is remarkably increased:

(1) Enzyme-linked immunosorbent assay (Elisa) indicates that the concentrations of A2M and sFlt-1 in PE pregnant women and plasma of later pregnancy are increased compared with normal pregnant women; PIGF is reduced in the late pregnancy compared with normal pregnant women;

(2) Immunofluorescence experiments (IF) suggest that A2M expression in placental arterial smooth muscle of PE pregnant women is increased compared to normal pregnant women;

(3) Immunoblotting (Western Blot, WB) suggested that the expression of A2M in the placenta of PE pregnant women was increased compared to normal pregnant women;

(4) The blood pressure of the SD pregnant mice with the systemic overexpression of A2M in the late pregnancy is obviously increased;

(5) The urinary protein of SD pregnant mice systemically over-expressing A2M in the late pregnancy period of 24 hours is increased;

(6) The weight of the embryo and the placenta of the SD pregnant mouse with the A2M over-expressed on the whole body is reduced;

(7) The concentration of sFlt-1 and PIGF in the plasma of SD pregnant mice overexpressing A2M systemically was varied.

The invention firstly provides that A2M can become a novel biomarker of PE, and provides a certain theoretical basis and research reference for clinical prediction, prevention and drug research and development of PE.

The current research on PE biomarkers focuses on sFlt-1 and PIGF, but the two cannot be clinically used generally, and the specificity and sensitivity of the two are still to be further verified. The present invention proposes a novel biomarker composition that is A2M binding to sFlt-1 and/or PIGF, and is undoubtedly a complement to existing research.

The marker provided by the invention not only provides a new thought for the research of PE prediction or diagnosis, but also is beneficial to further research and development of new drugs for preventing or treating PE.

Drawings

FIG. 1 is a graph showing the results of Elisa testing the concentrations of A2M, sFlt-1 and PIGF in the plasma of normal pregnant women and PE pregnant women during different pregnancy periods; wherein, the A2M detection result, the B sFlt-1 detection result and the C PIGF detection result are shown in a picture A.

FIG. 2 is a graph showing the results of immunohistochemical and immunofluorescence assays detecting the expression level of A2M in placental arterial smooth muscle of PE pregnant women; wherein, the graph A is the immunohistochemical detection result, the graph B is the immunofluorescence detection result, and the graph C is the statistical result of the expression of A2M in the smooth muscle of the middle, small and arterioles.

FIG. 3 is a graph showing the results of Western Blot detecting the expression level of A2M in placenta tissues of PE pregnant women.

FIG. 4 is a graph showing the result of blood pressure test of SD pregnant mice with A2M over-expressed systemically in the late pregnancy period.

FIG. 5 is a graph showing the result of 24-hour urine protein detection in late pregnancy of SD pregnant mice with systemically over-expressed A2M.

FIG. 6 is a diagram of the results of the weight measurements of SD mouse-pregnant embryos and placentas overexpressing A2M systemically.

FIG. 7 is a graph showing the results of detecting the concentrations of sFlt-1 and PIGF in the plasma of SD pregnant mice systemically overexpressing A2M; wherein, the A2M detection result, the B sFlt-1 detection result and the C PIGF detection result are shown in the figure.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

The study selects preeclamptic pregnant women in a first hospital affiliated to river university as preeclampsia groups; in addition, the normal pregnant women in the same period are randomly selected as a normal group. All pregnant women are single birth and early childbirth, have no bad hobbies such as smoke and wine, and the exclusion criteria comprise multiple deliveries, the age of a delivery person is less than 20 years old or more than 40 years old, diabetes, pre-pregnancy hypertension, chronic liver and kidney diseases, thyroid and other endocrine diseases, and the like. The PE diagnosis standard refers to the pregnancy-induced hypertension disease published in 2018 by the Chinese medical society, department of obstetrics and gynecology (ISSHP): ISSHP Classification, diagnosis and management guidelines. The medical ethical committee approves the method, and the pregnant women and the family members agree and sign informed consent.

Collecting placenta tissues: within 10min after cesarean delivery of pregnant women, placenta tissue with umbilical cord as center in placenta central region is taken to avoid infarction, calcification and blood vessel. The collected placenta tissue is fixed in 4% paraformaldehyde and stored at-80 deg.C for use.

Collecting blood plasma: peripheral venous blood of pregnant women is collected by an anticoagulation tube, and after centrifugation (3000 g, 4 ℃ for 10 minutes), supernatant is taken to obtain blood plasma for standby.

Example 1 concentrations of A2M and sFlt-1 in plasma of PE pregnant women and late pregnancy were increased compared to normal pregnant women; PIGF is reduced in pregnant women

To study the expression of A2M, sFlt-1 and PIGF in maternal plasma, we first collected 42 peripheral venous blood of PE pregnant women and 50 peripheral venous blood of normal pregnant women in different pregnancy using an anticoagulation tube and centrifuged (3000 g, 4 ℃, 10 min) to obtain supernatant as plasma. The concentrations of A2M, sFlt-1 and PIGF in plasma of two groups of pregnant women during different pregnancy were then determined using Elisa kit (MB-1240A, MB-0099A, MB-3845A, jiangsu enzyme-labeled Biotechnology Co., ltd.) according to the instruction procedures, and the results are shown in FIG. 1. The results show that the concentrations of A2M and sFlt-1 in plasma of PE pregnant women and late pregnancy are increased compared with normal pregnant women; PIGF is reduced in late pregnancy compared with normal pregnant women.

Experiments prove that the concentrations of A2M and sFlt-1 in plasma of PE pregnant women and late pregnancy are increased compared with normal pregnant women; PIGF is reduced in late pregnancy compared with normal pregnant women.

Example 2A2M expression in placental arterial smooth muscle of PE pregnant women is increased compared to normal pregnant women

We used immunohistochemistry and immunofluorescence techniques to detect localization of A2M in placental tissues (26 in the preeclamptic group, 24 in the normal group) and their expression. The immunohistochemical procedure was as follows: slicing and dewaxing to water conventionally; antigen retrieval; washing with buffer solution for 5min/3 times; incubating for 10-15 minutes with hydrogen peroxide; washing with buffer solution for 5min/2 times; adding 5% BSA blocking solution dropwise, wiping off the blocking solution with filter paper, and not washing; dropwise adding a primary anti-working solution (Abcam, ab58730,1, 1000) at 4 ℃ for overnight and then rewarming; washing with buffer solution for 5min/2 times; adding a secondary antibody dropwise, and incubating for 2 hours at room temperature; washing with buffer solution for 5min/2 times; adding DAB staining solution, and staining cell nucleus with hematoxylin; the plate was washed with PBS buffer, wiped off with filter paper, and mounted on neutral resin, and immediately observed under a fluorescent microscope. As shown in fig. 2A, A2M is localized primarily to the placental smooth muscle layer. The immunofluorescence procedure was as follows: slicing paraffin, baking, dewaxing, gradient alcohol rehydration, antigen retrieval, adding 5% BSA blocking solution dropwise, wiping off the blocking solution with filter paper, and not washing; dropwise adding a proper amount of primary antibody (Abcam, ab58730, 1; washing with PBS buffer solution, adding a second antibody dropwise, and incubating in dark; washing with PBS buffer solution, and dropwise adding Hochest to incubate in dark; the sample was washed with PBS buffer, wiped off with filter paper, and then mounted with glycerol and immediately observed under a fluorescence microscope. The results are shown in FIG. 2B, comparing the expression difference of the two groups from the middle, small and arterioles, respectively. Blood vessels are shown in the dotted line, and it can be seen that the expression of A2M in the smooth muscle of the placental arterioles and arterioles of PE pregnant women is increased as compared with that of normal pregnant women.

Experiments prove that the expression of A2M in placental arterial smooth muscle of PE pregnant women is increased compared with that of normal pregnant women.

Example 3WB validation that expression of A2M in placenta of PE pregnant women is increased compared to normal pregnant women

WB is a conventional and commonly used experimental technique for qualitative detection of protein expression or semi-quantitative analysis of protein expression. In order to further verify whether the expression of A2M in the placental tissues of PE pregnant women and normal pregnant women is different, 50 placental tissues (26 in a preeclampsia group and 24 in a normal group) from different sources are collected according to the method, and the expression level of A2M is detected by adopting a WB method, and the method comprises the following steps: after extracting proteins from placenta tissues using a protein extractant (Millipore, USA), protein separation was performed by 10-% SDS-polyacrylamide gel electrophoresis, and then transferred to a polyvinylidene fluoride membrane (BioRad, USA). After blocking the membrane for 1 hour using TBS buffer containing 5% skim milk and 0.1% tween 20, it was incubated with primary antibody (Abcam, ab58730,1 1000) overnight at 4 ℃. Then incubated for 2 hours at room temperature with a secondary antibody (CST, 7074S, 1. The results showed that A2M expression in the placenta of PE pregnant women was increased compared to normal pregnant women (fig. 3).

Experiments prove that the expression of A2M in placenta tissues of PE pregnant women is increased compared with that of normal pregnant women.

Example 4 SD pregnant mice overexpressing A2M systemically had significantly elevated blood pressure in late pregnancy

To confirm that high expression of A2M is responsible for eclampsia, we injected pregnant SD rats (purchased from beijing wilkinsoniwa laboratory animal technology limited, cat #: viton wayside-101-SD rats-018,8 weeks in cages) were injected with viral empty vector (purchased from and biotechnology (shanghai) gmbh, cat # H17668) and A2M adenovirus (purchased from and biotechnology (shanghai) gmbh, cat # control H221) in GD (pregnancy days) 11.5 via tail vein, respectively (8 injections of viral empty vector, 9 injections of A2M adenovirus, method reference "Karumanchi S a, stillman I e.in vivo rat model of precampsia. [ J. The method is operated according to the instrument instruction, and comprises the following specific steps:

(1) Preheating the heating cabin 20 minutes in advance, adjusting the heating gear to be between 5 and 6 to ensure that the temperature in the cabin is between 36.5 and 38 ℃, and putting the rat fixer into the cabin for preheating;

(2) And the VPR and the O-Cuff sensors are respectively connected with corresponding interfaces of the host according to colors. And connecting the USB interface with a computer, and setting parameters after opening the Coda4.1 pressure measurement software. Firstly, the accuracy of the VPR and O-Cuff sensors is detected, and the pressure measurement is started after the detection is normal. Fixing the rat in a proper fixer, sleeving the VPR and O-Cuff sensors at the root of the rat tail as much as possible, putting the rat into a preheated heating chamber, measuring the pressure after adapting for 3-5 minutes, and finishing 15 inflation and deflation cycles for each measurement.

(3) Observing the blood pressure waveform after 15 cycles, wherein the result that the waveform stability is similar to the standard manometry pattern is a qualified result, and if the waveform stability is more than 8, taking the average value as the current blood pressure value of the rat; if the number of the blood pressure measurement is less than 8, the blood pressure measurement is recorded as invalid, the rat is put back into a mouse cage, and the measurement is carried out again every other day by the method.

(4) Note that: the time point for each blood pressure measurement was fixed to exclude the effect of different times on the blood pressure of rats: the pressure measuring environment should be relatively quiet, so that the stress of the rat caused by noise is prevented from influencing the blood pressure: after the adaptive feeding and before the pressure measurement, the rat is adaptive to the fixator and the tail pressure sensor for 2-3 days, and a fixing device with a proper size is selected; the pressure measurement should be carried out in a constant temperature environment, and the temperature of a rat room and the pressure measurement room are both 23 ℃ soil and 1 ℃; all operations on the rat should be gentle in the pressure measuring process to prevent the rat from being stressed; the rat is not subjected to any operation before the pressure measurement, and is subjected to blood pressure measurement after all the operations are finished, namely the rat after the pressure measurement is finished is not put back to the original mouse cage temporarily, so that other rats to be measured in the mouse cage are prevented from being influenced, and the rat is put back to the original position when the pressure measurement of the rats in the same mouse cage is finished or the rat to be measured in the same day is not available in the mouse cage.

As a result, it was found that the blood pressure of pregnant mice after A2M administration began to rise at GD13.5 and continued to GD19.5, which was significantly higher than that of the control group (fig. 4). This suggests that overexpression of A2M causes an increase in blood pressure in pregnant rats, mimicking the symptoms of an increase in blood pressure in preeclampsia.

Experiments prove that the blood pressure of SD pregnant mice with systemic overexpression of A2M in the late pregnancy period is obviously increased.

Example 5 Total A2M overexpressing SD pregnant mice late pregnancy 24 hours urine protein elevation

Respectively injecting a virus empty carrier and an A2M adenovirus into a GD11.5 in a pregnant SD rat body through a tail vein, respectively placing the pregnant rat into a relatively sterile metabolism cage at GD8 and GD20 to ensure that the rat can eat freely, sealing the joint of a urine cup and the metabolism cage by using a sealing film to reduce urine evaporation, transferring urine collected in the urine cup into an autoclave centrifuge tube after 24 hours, transferring supernatant into a new autoclave centrifuge tube after centrifugation (3000 g, 4 ℃ and 10 minutes), recording total urine volume and marking. 24-hour urine protein was measured by the BCA method. As a result, it was found that the urine protein at 24 hours in SD pregnant mice GD20, which systemically over-expressed A2M, was significantly higher than that in the control group (FIG. 5).

Experiments prove that the urine protein of SD pregnant mice with over-expression A2M systemically increases in the late pregnancy period of 24 hours.

Example 6 reduction of embryo and placenta weight in SD pregnant mice overexpressing A2M systemically;

we injected viral empty vector and A2M adenovirus into the pregnant SD rats via tail vein at GD11.5, and collected two groups of pregnant mouse embryos and placenta at GD 20. Studies have shown that adenovirus cannot cross the placental barrier, and in addition, PE causes restricted embryonic development and growth. Therefore, the placenta of the pregnant mouse and the top buttock length and the weight of the embryo of the pregnant mouse are detected; diameter, weight, etc. of placenta (fig. 6), and it was found that the increase of A2M in SD pregnant mice caused the growth restriction of embryonic development.

Experiments prove that the weight of the embryo and the placenta of the SD pregnant mouse with the over-expression of A2M systemically is reduced.

Example 7 the plasma concentrations of sFlt-1 and PIGF in SD pregnant mice overexpressing A2M systemically

Viral empty vectors and A2M adenovirus are respectively injected into SD pregnant mice through tail veins at GD11.5, peripheral venous blood of two groups of pregnant mice is respectively collected at GD8 and GD20, and supernatant, namely plasma, is taken after centrifugation (3000 g, 4 ℃ and 10 minutes). The concentrations of A2M, sFlt-1 and PIGF in plasma of two groups of pregnant mice at different pregnancy periods are respectively detected by using an Elisa kit (MB-7300B, MB-1659B, MB6627B, jiangsu enzyme labeling Biotech limited company) according to the instruction steps, and the result shows that the A2M is unchanged at GD8, and the A2M group is obviously higher than the control group at GD 20; sFlt-1 was unchanged at GD8, with the A2M group at GD20 being significantly higher than the control group; PIGF was unchanged at GD8, with A2M group significantly lower than the control at GD20 (fig. 5). The results of animal experiments indicate that the concentrations of sFlt-1 and PIGF in the plasma of SD pregnant mice which systemically over-express A2M change along with the change, and the SD pregnant mice have the change trend of PE. This indicates that the change of A2M can affect the change of sFlt-1 and PIGF in plasma of pregnant mice.

Experiments prove that the concentrations of sFlt-1 and PIGF in the plasma of SD pregnant mice with systemically over-expressed A2M have the variation trend of PE.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such modifications are intended to be included in the scope of the present invention.

Claims

1. Application of a quantitative A2M product in preparation of a pre-eclampsia diagnostic tool is characterized in that: the sample used for quantifying A2M is plasma of preeclamptic patients in middle and late pregnancy, and the expression quantity of A2M in preeclamptic patients is increased compared with that of normal pregnant women.

2. Use according to claim 1, characterized in that: the product comprises a product for diagnosing preeclampsia by quantifying A2M level through an immunoblotting method, an immunohistochemistry method, an immunofluorescence method and an enzyme-linked immunosorbent assay.

3. Use according to claim 2, characterized in that: the product comprises antibodies specific for A2M.

4. Use according to claim 3, characterized in that: the specific antibody comprises a monoclonal antibody or a polyclonal antibody.