Use of csrp2bp gene as a marker for diagnosing preeclampsia

Abstract

The application provides a marker for diagnosing preeclampsia, use of a reagent for detecting a CSRP2BP gene expression level in preparation of a kit, use of a preparation for improving the CSRP2BP gene expression level in preparation of a medicine, a non-treatment method for regulating migration ability and / or proliferation ability of trophoblasts, and a system for diagnosing preeclampsia, wherein the marker comprises a CSRP2BP gene. The CSRP2BP gene is used as the marker, the expression level of the CSRP2BP gene is detected, whether preeclampsia is suffered can be accurately judged, the diagnosis of preeclampsia is facilitated, and the application prospect is good.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine. Specifically, the present application relates to the use of CSRP2BP gene as a marker for diagnosing preeclampsia. BACKGROUND

[0002] Preeclampsia is a disease unique to pregnancy and is one of the main causes of morbidity and mortality of pregnant women and perinatal infants. Insufficient invasion of trophoblasts in early pregnancy causes "placental shallow implantation" and placental vascular formation disorder, which are important links leading to placental ischemia and hypoxia and further causing the occurrence and development of preeclampsia. At present, preeclampsia can only be clinically diagnosed in the middle and late stages of pregnancy, and there is a lack of effective treatment, so if the molecular mechanism of early onset of preeclampsia can be elucidated and a new diagnostic marker can be found, it will have important significance for the early prediction and intervention of preeclampsia.

[0003] The placenta is a bridge connecting the embryo and the maternal uterine artery, ensuring the supply of nutrients to the embryo and maintaining normal pregnancy, and is the guarantee of fetal development. The differentiation and invasion of trophoblasts are the basis for ensuring the normal development of the placenta and maintaining the development of pregnancy. The invasion function of placental trophoblasts mainly refers to the infiltration of extravillous trophoblasts differentiated from cytotrophoblasts into the endometrial stroma, and into the lumen of uterine spiral arterioles and gradually replacing vascular smooth muscle cells and endothelial cells, so that the artery changes from a high-resistance low-flow vessel to a low-resistance high-flow vessel, thereby increasing placental blood flow and ensuring maternal-fetal nutrient exchange.

[0004] If the invasion ability of placental trophoblasts decreases, the trophoblasts cannot enter the uterine muscle layer, and the vascular smooth muscle is not completely replaced, resulting in "vascular remodeling" disorder of uterine spiral arterioles, causing "placental shallow implantation", vascular lumen stenosis, and increased vascular resistance, leading to reduced placental perfusion, thereby triggering a series of symptoms of preeclampsia. Therefore, studying the regulatory mechanism of placental trophoblasts in preeclampsia is conducive to elucidating the pathogenesis of preeclampsia and finding new markers for the occurrence and development of preeclampsia. SUMMARY

[0005] The present application aims to at least partially solve at least one of the technical problems existing in the prior art. To this end, the present application proposes a marker for diagnosing preeclampsia, the use of a reagent for detecting the expression level of CSRP2BP gene in the preparation of a kit, the use of a preparation for increasing the expression level of CSRP2BP gene in the preparation of a medicine, a non-therapeutic method for regulating the migration and proliferation abilities of trophoblasts, and a system for diagnosing preeclampsia. By taking CSRP2BP gene as a marker and detecting its expression level, it can be accurately judged whether preeclampsia is present, which is helpful for the diagnosis of preeclampsia and has a good application prospect.

[0006] In one aspect of the present application, the present application provides a marker for diagnosing preeclampsia. According to an embodiment of the present application, the marker comprises: a CSRP2BP gene. By detecting the expression level of the CSRP2BP gene, it can be accurately determined whether preeclampsia is present, which is helpful for the diagnosis of preeclampsia and has a good application prospect.

[0007] In another aspect of the present application, the present application provides use of a reagent for detecting the expression level of a CSRP2BP gene in the preparation of a kit. According to an embodiment of the present application, the kit is used for diagnosing preeclampsia. By detecting the expression level of the CSRP2BP gene, it can be accurately determined whether preeclampsia is present, which is helpful for the diagnosis of preeclampsia.

[0008] The term "gene expression level" should be interpreted broadly. It can be a numerical value obtained by direct detection, such as an absolute expression value or a relative expression value obtained by fluorescence quantitative PCR detection. It can also be a non-numerical value, such as the brightness of an electrophoresis band. It can also be a parameter obtained by indirect detection, such as the expression level of a protein encoded by a gene, specifically a protein level expression score calculated by immunohistochemical experiment. The present application does not make strict limitations thereon, and the selection can be flexible according to actual conditions.

[0009] It should be noted that the present application does not make strict limitations on the type of reagent for detecting the expression level of the CSRP2BP gene. As long as the expression level of the CSRP2BP gene can be detected, the reagent can be, for example, a reagent used in Real-time PCR, Western Blot, such as a primer for amplifying the CSRP2BP gene, dNTP, DNA polymerase, Buffer, etc. The selection can be flexible according to actual needs.

[0010] According to an embodiment of the present application, the expression level of the CSRP2BP gene is lower than the normal level, which is an indication of preeclampsia; and the expression level of the CSRP2BP gene is not lower than the normal level, which is an indication of not having preeclampsia.

[0011] It should be noted that the "normal level" described in the present application refers to the expression level of the CSRP2BP gene in a population not suffering from preeclampsia.

[0012] According to an embodiment of the present application, the kit is used for diagnosing whether preeclampsia is present or the severity of preeclampsia. By comparing the expression level of the CSRP2BP gene of the subject to be tested with the normal level, if it is lower than the normal level, it indicates that the subject is suffering from preeclampsia; if it is not lower than the normal level, it indicates that the subject is not suffering from preeclampsia. When it is diagnosed that the subject is suffering from preeclampsia, the greater the difference between the expression level of the CSRP2BP gene of the subject to be tested and the normal level, the more severe the preeclampsia.

[0013] According to an embodiment of the present application, the kit is used for predicting the migration ability, invasion ability and / or proliferation ability of the trophoblasts. The inventors have found that the migration ability, invasion ability and / or proliferation ability of the trophoblasts can be changed, such as increased or decreased, by silencing the CSRP2BP gene or overexpressing the CSRP2BP gene, thereby affecting the occurrence and development of preeclampsia. Specifically, the "migration ability" is characterized by a migration rate, the "invasion ability" is characterized by a relative invasion number of the trophoblasts, and the "proliferation ability" is characterized by a proliferation rate of the trophoblasts.

[0014] According to an embodiment of the present application, the expression level of the CSRP2BP gene being lower than a normal level is an indication of a decreased migration ability, invasion ability and / or proliferation ability of the trophoblasts, and the expression level of the CSRP2BP gene being higher than the normal level is an indication of an increased migration ability, invasion ability and / or proliferation ability of the trophoblasts.

[0015] In yet another aspect of the present application, the present application provides a use of a preparation for increasing the expression level of the CSRP2BP gene in the preparation of a drug. According to an embodiment of the present application, the drug is used for preventing or treating preeclampsia. The inventors have found that the expression level of the CSRP2BP gene of a population suffering from preeclampsia is lower than a normal level, and thus, by administering a drug for increasing the expression level of the CSRP2BP gene to the population, preeclampsia can be effectively prevented or treated.

[0016] According to an embodiment of the present application, the drug is used for increasing the migration ability, invasion ability and / or proliferation ability of the trophoblasts. The inventors have found that the migration ability, invasion ability and / or proliferation ability of the trophoblasts is decreased when the expression level of the CSRP2BP gene is lower than a normal level, and thus, by administering a drug for increasing the expression level of the CSRP2BP gene, the migration ability, invasion ability and / or proliferation ability of the trophoblasts can be effectively increased.

[0017] In yet another aspect of the present application, the present application provides a non-therapeutic method for regulating the migration ability, invasion ability and proliferation ability of the trophoblasts. According to an embodiment of the present application, the method comprises: regulating the expression level of the CSRP2BP gene of the trophoblasts.

[0018] The inventors have found that the migration ability, invasion ability and proliferation ability of the trophoblasts can be changed, such as increased or decreased, by silencing the CSRP2BP gene or overexpressing the CSRP2BP gene, thereby helping to better scientifically study the biological functions of the trophoblasts. Furthermore, based on the biological functions of the trophoblasts, the pathogenesis and causes of preeclampsia can be further theoretically studied, thereby laying a theoretical foundation for the diagnosis of preeclampsia.

[0019] According to an embodiment of the present application, the method comprises inhibiting the expression of the CSRP2BP gene of the trophoblast cells so as to reduce the migration ability, invasion ability and / or proliferation ability of the trophoblast cells.

[0020] According to an embodiment of the present application, the method comprises promoting the expression of the CSRP2BP gene of the trophoblast cells so as to increase the migration, invasion and / or proliferation ability of the trophoblast cells.

[0021] According to an embodiment of the present application, the trophoblast cells described above are selected from human trophoblast cells HTR-8.

[0022] In yet another aspect of the present application, the present application provides a system for diagnosing preeclampsia. According to an embodiment of the present application, the system comprises a detecting device for detecting the expression level of the CSRP2BP gene in a biological sample; and an analyzing device connected to the detecting device for determining whether the biological sample has preeclampsia based on the expression level of the CSRP2BP gene. Thus, the system according to an embodiment of the present application can accurately determine whether the biological sample has preeclampsia.

[0023] According to an embodiment of the present application, the expression level of the CSRP2BP gene lower than a normal level is an indication that the biological sample has preeclampsia; and the expression level of the CSRP2BP gene not lower than a normal level is an indication that the biological sample does not have preeclampsia.

[0024] Additional aspects and advantages of the present application will be given in part in the following description, become apparent from the following description, or be learned by practice of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0025] The above and / or additional aspects and advantages of the present application will become apparent and be readily understood from the following description, taken in conjunction with the accompanying drawings, in which:

[0026] Figure 1 and Figure 2 respectively show the experimental flowchart according to an embodiment of the present application;

[0027] Figure 3 show the analysis diagram of the plate clone experiment results according to an embodiment of the present application, wherein shNC: HTR8 CSRP2BP silencing control group cells; shCSRP2BP: HTR8 CSRP2BP silencing cells; NC: HTR8 CSRP2BP overexpression control group cells; CSRP2BP: HTR8 CSRP2BP overexpression cells;

[0028] Figure 4A schematic diagram showing analysis of EdU experimental results according to one embodiment of the present application is shown.

[0029] Figure 5 A flow chart of an experiment according to one embodiment of the present application is shown.

[0030] Figure 6 A schematic diagram showing analysis of scratch experimental results according to one embodiment of the present application is shown.

[0031] Figure 7 A schematic diagram showing analysis of Transwell chamber experimental results according to one embodiment of the present application is shown.

[0032] Figure 8 A schematic diagram showing analysis of ROC curve results according to one embodiment of the present application is shown, in which the curve represents the expression amount of CSRP2BP in placental tissue, and the diagonal line represents AUC of 0.5. DETAILED DESCRIPTION

[0033] The present application relates to a marker for diagnosing preeclampsia and the effect of the marker on the biological behavior of trophoblasts, including proliferation, migration and invasion. The present application constructs a CSRP2BP silencing and overexpression lentiviral expression plasmid, knocks down and overexpresses the expression of CSRP2BP gene in trophoblast HTR-8 / SVneo cells, verifies the effect of the molecular marker on the biological behavior of trophoblasts through cloning formation experiment, EdU experiment, scratch experiment, Transwell invasion experiment and other experimental methods, and further contributes to the diagnosis of preeclampsia. The technical solution is expected to be used for the diagnosis of preeclampsia, and is conducive to early diagnosis and early treatment of the disease of the patient.

[0034] The technical solution of the present application will be explained below in conjunction with the embodiments. Those skilled in the art will understand that the following embodiments are only used to illustrate the present application, and should not be regarded as limiting the scope of the present application. If the specific technology or condition is not specified in the embodiments, the technology or condition described in the literature in the art or according to the product manual is used. If the reagent or instrument is not specified by the manufacturer, it is a conventional product that can be obtained by purchase.

[0035] Example 1

[0036] The human trophoblast HTR-8 cells were used as the object to establish CSRP2BP silencing and overexpression cell strains, and the effect of the strains on the proliferation, migration and invasion of trophoblasts was studied.

[0037] 1. Construction of HTR-8 cell strains stably silencing and overexpressing CSRP2BP

[0038] Reference Figure 1, construct the lentivirus expression plasmid of silencing CSRP2BP (GV248-shRNA-CSRP2BP-EGFP-puro) and overexpressing CSRP2BP (plv-hCSRP2BP-EGFP-puro), transfect 293T cells, harvest virus, establish stable HTR-8 cell strain of silencing and overexpressing CSRP2BP, and verify the expression of CSRP2BP by Real-time PCR.

[0039] The Real-time PCR is operated as follows:

[0040] (1) Extraction of RNA in cell sample

[0041] 1) Remove cell culture medium, add 1 ml Trizol in 10 cm dish, collect cell suspension in EP tube, and lyse on ice.

[0042] 2) Add 200 ul chloroform according to the volume ratio of chloroform: Trizol = 1:5, mix well by oscillation for 10 s, avoid vortex oscillation, and destroy the structure of RNA. Stand on ice for 2 min, and repeat oscillation and standing for 3 times, centrifuge at 4℃, 12000 rpm / min for 15 min.

[0043] 3) When the centrifugation is almost finished, prepare a new EP tube, and add equal volume of pre-cooled isopropanol, about 400 ul.

[0044] 4) Take out the sample after centrifugation, and gently suck 400 ul supernatant and add to the above isopropanol centrifuge tube, avoid sucking the lower precipitate and contaminating RNA, and mix well for 10 times.

[0045] 5) Centrifuge at 4℃, 12000 rpm / min for 10 min, and the white precipitate can be seen at the bottom of the tube after taking out, and the supernatant is removed as much as possible.

[0046] 6) Add 750 ul of 75% ethanol to the tube, centrifuge at 4℃, 12000 rpm / min for 5 min.

[0047] 7) The white precipitate can be seen at the bottom of the tube after taking out, and the supernatant is gently removed, and then centrifuge at 4℃, 12000 rpm / min for 2 min, and remove the liquid drops on the tube wall.

[0048] 8) Use 10 ul of gun head to remove the liquid drops, and dry the precipitate in the safety cabinet for about 5-10 min. The EP tube is placed on one side during drying to prevent RNAase in the air from contaminating and degrading RNA.

[0049] 9) Dissolve the RNA with appropriate amount of DEPC water.

[0050] (2) Reverse transcription of RNA to cDNA

[0051] Reverse transcription was performed by two-step method, divided into two systems, respectively:

[0052] 1) System 1: total volume 10ul

[0053]

[0054] Machine program: 42℃, 2min.

[0055] 2) System 2: total volume 20ul

[0056]

[0057]

[0058] Machine program: 42℃, 15min, 85℃, 5s, 4℃ preservation.

[0059] 3) After the end of the program, take out the cDNA, DEPC water dilution 10 times after used for RT-PCR experiment to detect.

[0060] (3) Real-time PCR detection of CSRP2BP expression

[0061] 1) Reaction system as follows:

[0062]

[0063] Ice sample mixing, Agilent Stratagene Mx3000P set as follows: 50℃ 2min, 95℃ 10min, 95℃ 10s, 60℃ 20s 40-45 cycles, 72℃ 30s, 72℃ 7min.

[0064] 2) Each sample 3 complex hole, Agilent Stratagene Mx3000P software analysis, GraphPad software mapping.

[0065] 3) 2 －△△Ct Method analysis results, ΔΔCt calculation: sample ΔCT value minus the ΔCt value of the control group, the ΔΔCT value of each sample is calculated.

[0066] 4) Into the formula Relative Expression Ratio(sample) = 2 –ΔΔCt (sample).

[0067] 2, study in the case of silence and overexpression of CSRP2BP, HTR-8 cell growth, proliferation changes

[0068] Reference Figure 2 , using HTR-8 cells with silenced and overexpressed CSRP2BP, plate clone formation experiments, EdU experiments were carried out to study the changes in cell growth and proliferation of HTR-8 cells under the condition of silencing or overexpressing CSRP2BP.

[0069] As shown in Figure 3 , the plate clone experiment results show that silencing CSRP2BP inhibits the proliferation of trophoblast cells, and overexpressing CSRP2BP promotes the proliferation of trophoblast cells.

[0070] As shown in Figure 4 , the EdU results show that silencing CSRP2BP inhibits cell proliferation, and overexpressing CSRP2BP promotes the proliferation of trophoblast cells.

[0071] 3. Study the changes in migration and invasion ability of HTR-8 cells under the condition of silencing and overexpressing CSRP2BP

[0072] Reference Figure 5 , using HTR-8 cells with silenced and overexpressed CSRP2BP, scratch test and Transwell test were carried out to study the effect of silencing and overexpressing CSRP2BP on the migration and invasion of HTR-8 cells.

[0073] As shown in Figure 6 , the scratch test results show that silencing CSRP2BP inhibits the migration of trophoblast cells, and overexpressing CSRP2BP promotes the migration of trophoblast cells.

[0074] As shown in Figure 7 , the Transwell chamber experiment results show that silencing CSRP2BP inhibits the invasion of trophoblast cells, and overexpressing CSRP2BP promotes the invasion of trophoblast cells.

[0075] In summary, the expression level of CSRP2BP gene not only affects the migration, invasion and proliferation ability of trophoblast cells, but also has a significant correlation with whether or not to have preeclampsia. Further, by detecting the expression level of CSRP2BP gene, whether or not to have preeclampsia can be accurately determined.

[0076] Example 2

[0077] In this example, the efficiency of diagnosing the occurrence of preeclampsia by the expression level of CSRP2BP in placental tissue was studied, and the specific steps were as follows:

[0078] (1) Collect 29 cases of normal pregnant women and 55 cases of preeclampsia patients discarded after delivery, and prepare paraffin-embedded sections by fixing the placental tissue, dehydrating, transparent, wax embedding and sectioning;

[0079] (2) The expression level of CSRP2BP in placenta sections was detected by immunohistochemical experiment, and the specific steps were as follows:

[0080] Under the electron microscope, two experienced pathologists and clinicians scored according to the scoring criteria by double-blind method. The placental trophoblast immunohistochemical staining score: positive marker immune characteristics: according to the staining degree, it can be divided into no staining (no color), weak staining (light yellow), moderate staining (brown yellow) and strong staining (dark brown), which are scored as 0, 1, 2 and 3 points respectively. According to the number of positive cells, it can be divided into 0, <10%, 10%-50%, 50%-80% and >80%, which are scored as 0, 1, 2, 3 and 4 points respectively. The calculation formula is protein level expression score = staining degree score x positive cell number score.

[0081] (3) According to the protein level expression score, the relationship between the expression of CSRP2BP in placental tissue and preeclampsia was analyzed by ROC curve.

[0082] (4) The ROC curve analysis result showed that the expression level of CSRP2BP was significantly decreased in preeclampsia, and the area under the curve (AUC) of CSRP2BP in diagnosing preeclampsia was 0.817 (95% CI: 0.719-0.915, P=0.000). Figure 8 ) Among them, the ROC cutoff value was 7.75, and when the expression of CSRP2BP was less than 7.75, the sensitivity for diagnosing preeclampsia was 82.8%, and the specificity was 76%.

[0083] The results showed that by determining the expression level of CSRP2BP, whether preeclampsia was accurately determined.

[0084] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms is not necessarily directed to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. Furthermore, the different embodiments or examples described in the present specification and the features of the different embodiments or examples can be combined and modified by those skilled in the art without contradiction.

[0085] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above embodiments within the scope of the present application.

Claims

1. Use of a reagent for detecting the expression level of the CSRP2BP gene in the manufacture of a kit, characterized in that, The kit is used for diagnosing preeclampsia.

2. Use according to claim 1, characterized in that, The expression level of the CSRP2BP gene is lower than the normal level, which is an indication of preeclampsia; and the expression level of the CSRP2BP gene is not lower than the normal level, which is an indication of not suffering from preeclampsia.

3. A system for diagnosing pre-eclampsia, characterized by The kit comprises: a detection device for detecting the expression level of the CSRP2BP gene in a biological sample; an analysis device connected to the detection device, for judging whether the biological sample suffers from preeclampsia based on the expression level of the CSRP2BP gene, wherein the expression level of the CSRP2BP gene is lower than the normal level, which is an indication of preeclampsia; and the expression level of the CSRP2BP gene is not lower than the normal level, which is an indication of not suffering from preeclampsia.

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