Application of TCTP protein in preparation of noninvasive premature delivery diagnostic product

Abstract

The invention discloses application of TCTP protein in preparation of a noninvasive premature delivery diagnosis product, and relates to the technical field of biological medicine. According to the method, the premature delivery risk can be estimated by detecting the TCTP protein content or TCTP gene expression quantity in the peripheral blood of the pregnant woman, only 0.5 ml of peripheral blood of the mother is needed, the method is safe to the mother and the infant, and the method is simple, high in sensitivity, low in cost and easy to operate. In 126 samples in the embodiment of the invention, the premature delivery detection rate detected by the method is 91%, the false positive rate in a control group is 8.6% (5 samples / 58 samples), and the method has high accuracy.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and more specifically, to the application of TCTP protein in the preparation of non-invasive preterm birth diagnostic products. Background Art

[0002] Preterm birth accounts for up to 35.4% of the causes of neonatal death globally, being the leading cause of neonatal death and birth defects, and also a major cause of death among children under five years old. Preterm birth is essentially related to the abnormal initiation of labor, so it is crucial to analyze the motivation and mechanism of labor initiation. Predicting preterm birth can strengthen prenatal care, reduce the incidence of preterm birth, and improve the growth and development of preterm infants.

[0003] Currently, the methods for evaluating the risk of preterm birth mainly include: imaging examinations, fetal fibronectin (fFN) detection, and inflammatory marker detection, etc. Among them, imaging examinations mainly measure the cervical length through transvaginal ultrasound to evaluate the cervical function status for assessing the risk of preterm birth, but the positive rate of predicting preterm birth by this method is relatively low. Biochemical marker detections, such as fetal fibronectin (fFN) detection and inflammatory marker detection, are insufficient in sensitivity and specificity.

[0004] Therefore, there is an urgent clinical need to find non-invasive biomarker detections for predicting and early diagnosing preterm birth. For example, looking for molecular markers from proteins, cfRNA, cfDNA, and exosomal RNA in maternal peripheral blood to reflect the pregnancy status, so as to determine the gestational age and estimate the risk of preterm birth, etc. Summary of the Invention

[0005] In view of this, the present invention provides the application of TCTP protein in the preparation of non-invasive preterm birth diagnostic products.

[0006] To achieve the above object, the present invention adopts the following technical scheme:

[0007] The application of a product for detecting TCTP protein in the preparation of a non-invasive preterm birth diagnostic reagent, wherein the amino acid sequence of the TCTP protein is as shown in SEQ ID NO.3.

[0008] Preferably, the product is a kit.

[0009] Another object of the present invention is to provide the application of a product for detecting the expression level of TCTP gene in the preparation of a non-invasive preterm birth diagnostic reagent, wherein the amino acid sequence encoded by the TCTP gene is as shown in SEQ ID NO.3.

[0010] Preferably, the product is a probe set or a kit.

[0011] More preferably, the kit further comprises reagents for extracting total RNA from peripheral blood, and / or reagents for reverse-transcribing total RNA into cDNA using the total RNA as a template, and / or reagents for quantitative PCR of cDNA.

[0012] Advantageous effects:

[0013] 1. By detecting the content of TCTP protein or the expression level of TCTP gene in the peripheral blood of pregnant women, the present invention can estimate the risk of preterm birth. This method only requires 0.5 ml of peripheral blood from the mother, is safe for the mother and baby, and has the advantages of simple method, high sensitivity, low cost and simple operation.

[0014] 2. In 126 samples of the embodiment of the present invention, the preterm birth detection rate detected by this method is 91%, while the false positive rate in the control group is 8.6% (5 cases / 58 cases), and this method has a high accuracy rate. Description of the drawings

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to the provided drawings.

[0016] Figure 1 Cell subtype identification of lung tissues of mice at different gestational ages and adult mice; Epi-AT1, type I alveolar epithelial cells; Epi-AT2, type II alveolar epithelial cells; Cilited cells, ciliated epithelial cells; Club cells, ciliated cells; Differentiation cells, differentiated cells; Fibroblast, fibroblasts; Endothelial cell, endothelial cells; Lymphocyte, lymphocytes; Myeloid cell, myeloid cells.

[0017] Figure 2 Molecular markers for identifying cell subtypes of mouse lung tissues.

[0018] Figure 3 Expression and distribution of Epi-AT1-Hopx cells in mouse lung tissues at different gestational ages and adult mice; where E15.5: fetal lung at 15.5 days of gestation; E18.5: fetal lung at 18.5 days of gestation; labor: fetal lung at delivery; Adult-8w: adult lung tissue. DAPI (blue) is a nuclear marker, and PDPN (green) and Hopx (red) are markers for type I lung epithelial cells. +

[0019] Figure 4 Alveolar epithelial cell subsets in different pregnancy trimesters, neonatal mice, and adult mice.

[0020] Figure 5 Proportion distributions of each alveolar epithelial cell subset at different developmental stages (E12.5 is the fetal mouse at 12.5 days of pregnancy, E18.5 is the fetal mouse at 18.5 days of pregnancy, Neonata is the neonatal mouse within 0.5 days after birth, Adult is the adult mouse at 8 weeks after birth). The left side of the figure is the percentage ratio, and the right side of the figure is the actual ratio.

[0021] Figure 6 Distribution of upregulated genes in the fetal lung epithelium of E18.5 mice.

[0022] Figure 7 Expression distribution of the TCTP gene in alveolar epithelial cell subsets at different developmental stages.

[0023] Figure 8 Transcription level and protein expression level of the TCTP gene in the lung tissues of fetal mice at different pregnancy trimesters.

[0024] Figure 9 Expression levels of mRNA and encoded protein of the TCTP gene in the fetal lungs of preterm mice (RU-PTL) and control mice (TM-PTNL) at the same pregnancy trimester.

[0025] Figure 10 Standard curve for TCTP protein determination (r > 0.99).

[0026] Figure 11 TCTP protein was significantly increased in the maternal serum of the preterm group, P < 0.001. Detailed implementation manners

[0027] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0028] Embodiment 1

[0029] 1. Analyze the single-cell transcriptome sequencing results of lung tissues from mid-gestation (E12.5) (GSM4504959), late-gestation (E18.5) (GSM5343154, GSM5343155), neonatal mice (Neonatal) (GSM4647247, GSM4647248), and adult mice (Adult) (GSM4647254, GSM4647255) in the public database using the Seurat package in R software. The analysis process includes data normalization, identification of differential genes, and dimensionality reduction and clustering. Visualize cell types using the DimPlot function in the Seurat package (see Appendix Figure 1 ), and visualize the molecular markers for identifying cell types using the ggplot2 software (see Appendix Figure 2 ). The comprehensive results show that in the fetal lung tissues at late gestation (E18.5), there is specifically a subset of type I alveolar epithelial cells, Epi-AT1-Hopx.

[0030] 2. Perform immunofluorescence staining on the lungs of pregnant mice at E15.5, E18.5, at parturition, and adult mice respectively. The specific method is as follows:

[0031] (1) Sample collection and paraffin sectioning: Place the lungs in 4% paraformaldehyde for 24 hours, then rinse with PBS for 10 minutes and embed in paraffin. After the paraffin solidifies overnight, use a microtome to section the samples and select suitable fields of view under the microscope.

[0032] (2) Dewaxing: Place the paraffin sections on the slide rack in the same orientation, and bake the sections in an incubator at 55°C for 30 minutes. At the same time, place the first dewaxing solution in an incubator at 55°C. Place the paraffin sections together with the slide rack into the first dewaxing solution, then take them out of the incubator and let them cool to room temperature. After 5 minutes, take out the sections and immerse them in the second dewaxing solution at room temperature. Then, place the paraffin sections into the second dewaxing solution, the third dewaxing solution, absolute ethanol 1, absolute ethanol 2, and absolute ethanol 3 in sequence (you can also choose gradient-concentration ethanol), 5 minutes for each solution. Wash the sections with running water for 5 minutes;

[0033] (3) Staining: Completely cover the sample with 5% blank goat serum. The sections need to be placed in a humid chamber, and the cell well plates can be directly sealed and incubated in a 37°C constant temperature and humidity incubator for 30 min; Dilute the primary antibody at 1:1000, place the sections in the primary antibody dilution solution and incubate overnight at 4°C; Recover the primary antibody, wash once with TBST for 5 minutes. After diluting the secondary antibody at 1:5000; Incubate at room temperature in the dark for 1 h, wash once with buffer TBST for 5 minutes; Drop the DAPI working solution on the sample, incubate in the dark at room temperature for 10 minutes; Remove the DAPI working solution, wash once with buffer TBST for 5 minutes; Wash 3 times with buffer TBS, 5 minutes each time; After adding the anti-fluorescence quenching mounting medium, observe and collect images under a fluorescence microscope (see attachment Figure 3 ).

[0034] Immunofluorescence staining showed that the content of Epi-AT1-Hopx cells in the fetal lungs of late pregnancy (18.5 dpc) was significantly increased, reaching the peak at parturition, while in the lung tissues of adult fetal mice, this subset of cells almost completely disappeared. This result once again suggested that this subset of cells showed drastic dynamic changes during fetal lung development and might be related to the initiation of parturition.

[0035] 3. For the lung epithelial cells in attachment Figure 1 , further clustering analysis was performed using the seurat software package, which further confirmed the specific distribution of the Epi-AT1-Hopx cell subset in the fetal lungs of late pregnancy (see attachment Figures 4 - 5 ). To analyze the fate process of this cell subset, we performed differential gene analysis on the sequencing results of mouse fetal lungs at each stage and found that in the fetal lungs of E18.5, a series of genes related to protein translation, synthesis, etc. were significantly upregulated (see attachment Figure 6 ). Among them, the TCTP gene was specifically highly expressed in the Epi-AT1-Hopx cell subset of the fetal lungs of E18.5 (see attachment Figure 7 ).

[0036] 4. Collect the mouse lungs at E12.5, E15.5, E18.5 of pregnancy and the initiation of parturition, and analyze the mRNA expression of the TCTP gene and the expression level of the encoded TCTP protein in the mouse fetal lung tissues by Realtime qPCR and Western blot experiments.

[0037] qPCR: (1) Total RNA extraction: Collect the tissues, grind them in liquid nitrogen and then add 1 ml of TRIzol (Invitrogen), and perform total RMA extraction according to the standard procedure. (2) After reverse transcribing cDNA using the PrimeScript RT Reagent Kit, use qPCR amplification was performed using qPCR SYBR Green Master Mix (No Rox), and the primers were: forward primer GGCAAACTTGAAGAGCAGAAACC (SEQ ID NO.1) and reverse primer TCACGGTAGTCCAGGAGAGCAA (SEQ ID NO.2).

[0038] WB: (1) Gel preparation: Take equal volumes (2 mL each) of the lower gel solution and the lower gel buffer, and mix them; add 40 μL of the modified coagulant, mix well, and pour it into the gel-making glass plate. After the lower gel solidifies (about 15 min), pour off the upper water or alcohol; after solidification, take equal volumes (0.5 mL each) of the upper gel solution and the colored upper gel buffer, mix well, add 10 μL of the modified coagulant, mix well, pour it into the glass plate, and insert the comb; remove the comb after the upper gel solidifies. (2) Electrophoresis: Mix the protein sample to be tested with the loading buffer and add it to the sample wells in the gel. At the same time, add the molecular weight marker to the sample wells; (3) Membrane transfer: Soak the PVDF membrane in absolute methanol in advance; prepare the membrane transfer device. Then place the membrane transfer tank with the membrane transfer device in an ice box and transfer the membrane at a constant current of 300 mA for 100 min. (4) Blocking: Block with 5% BSA / TBST for 1 - 2 h; (5) Primary antibody incubation: Dilute the primary antibody (TCTP protein, Cell Signaling Technology, #8441) to 1:1000 dilution with 5% BSA / TBST, place the cut PVDF membrane into the primary antibody, incubate overnight on a shaker at 4°C; then take out the membrane and equilibrate at room temperature for 30 min; wash the membrane 3 times with 1×TBST, 10 min each time; (6) Secondary antibody incubation: Incubate with the secondary antibody (proteintech, SA00001 - 2) diluted with 1×TBST for 1 - 2 h; wash the membrane 3 times with 1×TBST, 10 min each time; (7) Prepare the chemiluminescent solution for development and exposure.

[0039] It was confirmed by Realtime qPCR and Western blot experiments that as pregnancy progresses, the mRNA of the TCTP gene and the encoded TCTP protein expression in the fetal lung tissues of mice gradually increase and reach the peak at parturition (see Appendix Figure 8 ).

[0040] Example 2

[0041] To further clarify the correlation between the TCTP gene and the onset of labor and preterm birth, a mouse model of preterm birth induced by mifepristone (RU486) was constructed. The construction method was to subcutaneously inject mifepristone (RU486; 50 μg / 100 μL propylene glycol) or propylene glycol control into the nape of the neck of 8-week-old female mice 8 days after pregnancy. At 15.5 days of pregnancy, the fetal lungs were taken by cesarean section, and qPCR experiments and WB experiments were carried out using the same methods as in Example 1.

[0042] The experimental results showed that the mRNA and protein expression levels of TCTP in the fetal lungs of preterm mice (RU-PTL) were significantly higher than those in the fetal lungs of control mice (injected with DMSO, P15.5d cesarean section) at the same gestational age (see attached Figure 9 ). The above results suggest that the expression level of TCTP in the fetal lungs is closely related to the onset of labor.

[0043] Example 3

[0044] Collect 0.5 ml of peripheral blood from 126 pregnant women, including 0.5 ml of peripheral blood from 68 preterm pregnant women who gave birth before 37 weeks and 58 term pregnant women who had not yet gone into labor as controls, and detect the content of TCTP protein (the amino acid sequence is: miiyrdlishdemfsdiykireiadglclevegkmvsrtegniddsliggnasaegpegegtestvitgvdivm nhhlqetsftkeaykkyikdymksikgkleeqrpervkpfmtgaaeqikhilanfknyqffigenmnpdgmvalldyredgvtpymiffkdglkmekc, SEQ ID NO.3). The specific detection method is as follows:

[0045] (1) Place the whole blood in a tube without anticoagulant, let it coagulate naturally at room temperature for 30 - 60 min until the blood clots; centrifuge at a speed of 2000 - 3000 rpm for 5 - 10 min, and take 250 μl of the supernatant as serum.

[0046] (2) Detect the content of TCTP protein in the serum by ELISA kit (CUSABIO, product number: CSB-EL024134HU). The specific method includes making a standard curve and detecting the sample to be tested.

[0047] ① Making the standard curve: Add 1 ml of sample diluent to the TCTP recombinant protein standard. Take 7 centrifuge tubes and add 250 μl of sample diluent to each. Then, dilute them in sequence according to the instructions and take 100 μL for detection;

[0048] ②Take 100 μl of the serum sample to be tested for detection. Add the standard product and the sample to be tested into a 96-well plate pre-coated with TCTP antigen respectively. After attaching the plate sticker, incubate at 37°C for 2 hours;

[0049] ③Discard the liquid, shake dry, add 100 μl of the working solution of the biotinylated antibody to each well, cover with a new plate sticker, and incubate at 37°C for 1 hour;

[0050] ④Discard the liquid in the wells, wash the plate 3 times, soak for 2 minutes each time, and shake dry;

[0051] ⑤Add 100 μl of the working solution of horseradish peroxidase-labeled avidin to each well, attach the plate sticker, incubate at 37°C for 1 hour, discard the liquid in the wells, shake dry, wash the plate 5 times, soak for 2 minutes each time, and shake dry;

[0052] ⑥Sequentially add 90 μl of the substrate solution and develop color at 37°C in the dark for 30 minutes;

[0053] ⑦Add 50 μl of the termination solution to terminate the reaction. Then, use an enzyme-linked immunosorbent assay (ELISA) reader to sequentially measure the optical density (OD value) of each well at a wavelength of 450 nm.

[0054] ⑧Use Curve Expert 1.4.zip software (https: / / www.cusabio.cn / statics / 200822010756694.rar) to draw a standard curve (see Appendix Figure 10 ) and calculate the content of TCTP protein in the sample to be tested.

[0055] The results show that the TCTP protein is significantly increased in the preterm birth group (see Appendix Figure 11 ). Among them, the first quartile and the third quartile of the TCTP protein level in the term birth group are 41 pg / ml and 63 pg / ml respectively; while the first quartile and the third quartile of the TCTP protein level in the preterm birth group are 77 pg / ml and 94 pg / ml respectively. Therefore, we suggest using a TCTP serum content greater than 70 pg / ml as the detection standard for high-risk preterm birth. Among 126 samples, the detection rate of preterm birth by this method is 91%, and the false positive rate among 58 term births is 8.6% (5 cases).

[0056] In this specification, each embodiment is described in a progressive manner. The key point of each embodiment is to illustrate the differences from other embodiments. For the same or similar parts among the embodiments, reference can be made to each other.

[0057] The foregoing description of the disclosed embodiments enables those skilled in the art to practice or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The use of a product for detecting TCTP protein in the preparation of a non-invasive premature birth diagnostic reagent, characterized in that: The amino acid sequence of the TCTP protein is shown in SEQ ID NO.

3.

2. The use according to claim 1, characterized in that: The product is a test kit.

3. The use of a product for detecting TCTP gene expression in the preparation of a non-invasive premature birth diagnostic reagent, characterized in that: The amino acid sequence encoded by the TCTP gene is shown in SEQ ID NO.

3.

4. The use according to claim 3, characterized in that: The product is a probe set or a kit.

5. The use according to claim 4, characterized in that: The kit also includes reagents for extracting total RNA from peripheral blood, and / or reagents for reverse transcribing total RNA as a template into cDNA, and / or reagents for quantitative PCR of cDNA.

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