Application of Nrf2 in the Auxiliary Diagnosis of Lung Developmental Retardation in Fetal Growth Restriction

Abstract

The present invention discloses the application of Nrf2 in the auxiliary diagnosis of fetal lung hypoplasia caused by intrauterine growth restriction, belonging to the field of biotechnology. This study found that the knockout of the Nrf2 gene in mice with intrauterine growth restriction led to a decrease in the live birth rate compared with wild-type mice, a decrease in the birth weight of fetal mice and the appearance of catch-up growth, an exacerbation of asymmetric development, and an obstruction in the development of pulmonary blood vessels; the oxidative stress index in lung tissue was aggravated; the pyroptosis index GSDMD in lung tissue increased. Overexpression of Nrf2 can effectively inhibit the expression of GSDMD and inhibit cell pyroptosis. The above results indicate that Nrf2 plays an important protective role in the lung hypoplasia of IUGR. Overexpression of Nrf2 can play a role in preventing and / or alleviating fetal lung hypoplasia caused by intrauterine growth restriction, and detecting the content of Nrf2 can predict the development of the fetal lung.

Description

Technical Field

[0001] The present invention relates to the application of Nrf2 in the auxiliary diagnosis of fetal lung hypoplasia with intrauterine growth restriction, and belongs to the field of biotechnology. Background Art

[0002] Intrauterine growth restriction (IUGR) of the fetus generally refers to the fetal birth weight being lower than two standard deviations or the tenth percentile of the average weight of fetuses of the same gestational age, and it is a major cause of morbidity and mortality of fetuses and neonates. Placental insufficiency generally occurs in the second half of pregnancy, which is the development period of alveoli and alveolar sacs. Therefore, the distal lungs are most vulnerable to the effects of IUGR. Long-term restriction of nutrition or oxygen in the later stage of pregnancy can lead to abnormal development of the airways and lungs in the offspring, including alveolar simplification, thickening of the intermediate septum and basement membrane. The structural abnormalities and lung function damage that appear shortly after the fetus is born persist and even develop with age. Currently, there are several common methods for diagnosing IUGR during pregnancy. One is to measure the distance from the upper edge of the symphysis pubis of the pregnant woman to the top of the uterine fundus, and the other is to detect the fetus by ultrasound. Standard fetal biometry includes assessing the head circumference, biparietal diameter, abdominal circumference and femur length, and estimating the fetal weight based on these four indicators. These methods not only have large errors, but also have low fineness and cannot accurately evaluate the development of the fetal lungs. By the time obvious morphological changes in the fetal lungs are found, the best intervention time has often been missed. Therefore, it is of great significance to develop a method for auxiliary diagnosis of IUGR lung hypoplasia.

[0003] Inflammation and oxidative stress are recognized pathogenic mechanisms of IUGR. Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a basic leucine zipper redox-sensitive transcription factor and is a pleiotropic protein that regulates the basal and inducible expression of a series of antioxidant and other cytoprotective genes by binding to cis-acting elements. The enhancer sequence is called the antioxidant response element. Nrf2 is an upstream regulator of cytokine production and inhibits the transcriptional upregulation of pro-inflammatory cytokine genes, and can regulate the inflammatory response of macrophages. Summary of the Invention

[0004] Based on the technical problems existing in the background art, the present invention proposes an application of Nrf2 in the diagnosis of fetal lung hypoplasia with intrauterine growth restriction.

[0005] The first object of the present invention is to provide the application of Nrf2 protein as a diagnostic marker for fetal lung hypoplasia with intrauterine growth restriction.

[0006] In one embodiment of the present invention, the application of Nrf2 protein or a reagent for quantitatively detecting the expression of Nrf2 protein in the preparation of a diagnostic product for fetal lung hypoplasia caused by intrauterine growth restriction.

[0007] In one embodiment of the present invention, the detection product includes, but is not limited to, a reagent or a kit.

[0008] The second object of the present invention is to provide a diagnostic kit for fetal lung hypoplasia caused by intrauterine growth restriction, which contains a detection reagent for Nrf2 protein.

[0009] In one embodiment of the present invention, the detection reagent for Nrf2 protein is used for the quantitative detection of Nrf2 protein or Nrf2 gene.

[0010] In one embodiment of the present invention, the amino acid sequence of the Nrf2 protein is as shown in SEQ ID NO: 1.

[0011] In one embodiment of the present invention, the detection reagent detects the expression of Nrf2 protein in a sample by fluorescence quantitative PCR, Southern hybridization, Northern hybridization, fluorescence in situ hybridization, DNA microarray, high-throughput sequencing method or immunoassay.

[0012] In one embodiment of the present invention, the immunoassay includes ELISA method, radioimmunoassay, immunohistochemistry method, Western blotting method.

[0013] In one embodiment of the present invention, the detection product contains primers and / or probes capable of specifically amplifying the Nrf2 gene; or antibodies capable of specifically binding to the Nrf2 protein.

[0014] In one embodiment of the present invention, the detection reagent for Nrf2 protein is an antibody against Nrf2.

[0015] In one embodiment of the present invention, the antibody against Nrf2 is a monoclonal antibody.

[0016] In one embodiment of the present invention, the sample is lung tissue.

[0017] In one embodiment, the kit further includes a detection label; the detection label includes, but is not limited to, a fluorophore, an enzyme, a metal ion, or an isotope.

[0018] In one embodiment, if the expression level of the Nrf2 protein in the lung tissue is more than 1.2 times higher than that of the control, it is regarded as having a risk of fetal lung hypoplasia caused by intrauterine growth restriction.

[0019] The third object of the present invention is to provide a pharmaceutical composition, which comprises a drug for enhancing Nrf2 expression and a pharmaceutically acceptable excipient.

[0020] In one embodiment of the present invention, the excipient includes any one or a combination of at least two of a carrier, a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an emulsifier, a cosolvent, a solubilizer, an osmotic pressure regulator, a surfactant, a coating material, a colorant, a pH regulator, an antioxidant, an antibacterial agent or a buffer.

[0021] In one embodiment of the present invention, the combination of the at least two, for example, the combination of a diluent and an excipient, the combination of a binder and a wetting agent, the combination of an emulsifier and a cosolvent, etc., any other combination method can be selected and will not be elaborated one by one here.

[0022] The present invention also provides the use of the pharmaceutical composition in the preparation of a drug for relieving and / or treating fetal lung hypoplasia caused by intrauterine growth restriction.

[0023] The present invention also provides the use of the pharmaceutical composition in the preparation of a drug having at least one of the following functions:

[0024] (1) Relieving oxidative stress in lung hypoplasia caused by intrauterine growth restriction;

[0025] (2) Pyroptosis in lung hypoplasia caused by intrauterine growth restriction.

[0026] Beneficial effects:

[0027] During the invention process, the inventors found that after pregnant mice were given 10.5% O 2 After hypoxia, the live birth rate of Nrf2 gene knockout mice was lower than that of wild-type mice, the birth weight of fetal mice decreased and showed catch-up growth, and the asymmetric development was aggravated; the morphological changes of lung tissue, thickening of alveolar walls, inflammatory infiltration, and the development of pulmonary blood vessels were blocked; the oxidative stress indexes (SOD1, SOD2, Gclm and Txn) in lung tissue were aggravated; the pyroptosis indexes GSDMD, caspase-1 and IL-18 in lung tissue were increased; the inventors of the present invention found that overexpression of Nrf2 in 293T cells could significantly inhibit the transcription of GSDMD and further reduce cell pyroptosis. Description of the drawings

[0028] Figure 1 : Nrf2 expression level in mouse lung tissue; A: Immunoblotting experiment, B: Gray value.

[0029] Figure 2: Effects of Nrf2 knockout on the live birth rate, birth and postnatal body weight of mice with intrauterine growth restriction caused by hypoxia, and the brain / liver ratio at 4 weeks of age; A: Live birth rate of mice, B: Birth weight of mice, C: Postnatal body weight of mice, D: Body weight change rate, E: Brain / liver weight ratio of 2-week-old mice, F: Brain / liver weight ratio of 4-week-old mice.

[0030] Figure 3 : Effects of Nrf2 knockout on the lung tissue morphology of 2-week-old mice; A: H&E staining, B: Immunohistochemistry.

[0031] Figure 4 : Effects of Nrf2 knockout on the oxidative stress indexes of lung tissue in 4-week-old mice; A: Relative expression level of SOD1 mRNA, B: Relative expression level of SOD2 mRNA, C: Relative expression level of Gclm mRNA, D: Relative expression level of Txn mRNA.

[0032] Figure 5 : Effects of Nrf2 on the pyroptosis indexes GSDMD, caspase-1 and IL-18 in lung tissue; A: Expression level of GSDMD protein, B: Gray value of GSDMD protein, C: Detection of GSDMD expression level by immunofluorescence, D: Relative expression level of NLRP3 mRNA, E: Relative expression level of caspase-1 mRNA, F: Relative expression level of GSDMD mRNA, G: Relative expression level of IL-18 mRNA.

[0033] Figure 6 : Nrf2 plasmid construction map; A: Empty plasmid map. B: Nrf2 plasmid map.

[0034] Figure 7 : GSDMD promoter luciferase plasmid construction map; A: Empty plasmid map. B: GSDMD promoter luciferase plasmid map.

[0035] Figure 8 : Inhibitory effect of Nrf2 on pyroptosis; A: Detection of relative fluorescence activity of GSDMD by luciferase reporter gene, B: Inhibitory effect of overexpressed Nrf2 on GSDMD, C: Detection of gray value of Nrf2 protein expression, D: Detection of gray value of GSDMD protein expression, E: Detection of gray value of GSDMD protein expression. Detailed implementation mode

[0036] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments. However, the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

[0037] Expression of Nrf2 in the lung tissue of IUGR mice in Example 1

[0038] (1) Establishment of intrauterine growth restriction (IUGR) model

[0039] Wild-type C57BL / 6 mice aged 8 - 10 weeks were randomly divided into two groups. One group served as the normoxia control group, and the other group was the IUGR model group.

[0040] Method for establishing IUGR model: Mice were caged according to the ratio of female:male = 2:1. The vaginal plugs of female mice were checked the morning after cohabitation as the standard for successful pregnancy. Pregnant mice were placed in a hypoxic environment (10.5% O 2 ) from gestational day 11.5 to 17.5 (pseudoglandular - tubule stage of mouse lung development), and then returned to the normal environment (21% O 2 ) and continued to be raised until birth.

[0041] 1) Normal: Wild-type mice were placed in a normoxic environment;

[0042] 2) IUGR: Wild-type mice were subjected to IUGR model establishment;

[0043] (2) Immunoblotting assay

[0044] Performed according to the method established in our laboratory. The main steps were as follows: Total protein of lung tissue samples of 14-day-old mice after birth was extracted using a kit, quantified by BCA method, and the samples were routinely processed, electrophoresed, and electrotransferred to membranes. The NC membranes were blocked in 5% non-fat milk for 2 hours, then primary antibodies were added and incubated overnight on a shaker at 4°C. After washing three times with TBST solution, corresponding secondary antibodies were added and incubated at room temperature for 1 h. After washing the membranes with TBST, the developer was added and exposed to develop the image.

[0045] The results were as Figure 1 shown. The expression of Nrf2 in the lung tissue of the IUGR group mice increased, which was 1.221 times that of the Normal group.

[0046] Example 2 Effects of Nrf2 knockout on fetal mice

[0047] (1) Establishment of intrauterine growth restriction (IUGR) model

[0048] Wild-type C57BL / 6 mice and Nrf2 knockout mice aged 8 - 10 weeks were randomly divided into two groups. One group served as the normoxia control group, and the other group was the IUGR model group.

[0049] IUGR modeling method: Mice were caged together at a ratio of female: male = 2:1. The vaginal plugs of female mice were checked the morning after cohabitation as the criterion for successful pregnancy. Pregnant mice were placed in a hypoxic environment (10.5% O 2 ) from gestational day 11.5 to 17.5 (pseudoglandular-tubular stage of mouse lung development), and then returned to a normal environment (21% O 2 ) and continued to be raised until birth.

[0050] The experimental groups were as follows:

[0051] 1) WTNormal: Wild-type mice were placed in a normoxic environment;

[0052] 2) WT IUGR: Wild-type mice were modeled with IUGR;

[0053] 3) Nrf2 - / - Normal: Nrf2 knockout mice were placed in a normoxic environment;

[0054] 4) Nrf2 - / - IUGR: Nrf2 knockout mice were modeled with IUGR.

[0055] All data were expressed as mean ± standard deviation (mean ± SD), and statistical analysis was performed using GraphPad Prism 7.0 statistical software. One-way or multi-way ANOVA was used for multiple comparisons. P < 0.05 represented significant differences and statistical significance (*P < 0.05; **P < 0.01; ***P < 0.001).

[0056] (2) Experimental results

[0057] 1) Organ-to-body weight ratio

[0058] At 14 days and 28 days after birth of fetal mice, they were anesthetized with sodium pentobarbital, and their body weights were measured. After disinfecting the skin from the neck to the abdomen with povidone-iodine, the skin was cut along the midline, the glands and muscles were bluntly separated, and the thoracic and rib bones were cut open to remove the brain, liver, and lung tissues. The tissues were rinsed with PBS placed on ice, and the water was blotted off with dust-free paper and then weighed.

[0059] The results were as Figure 2 shown. Compared with wild-type mice, in a hypoxic environment, Nrf2 knockout significantly reduced the live fetal rate ( Figure 2 A), birth weight, and later body weight ( Figure 2 B - D) of mice, and exacerbated asymmetric development ( Figure 2 E - F). Thus, it can be seen that the hypoxic environment significantly increased the incidence of IUGR in Nrf2 knockout mice.

[0060] 2) H&E staining

[0061] The lung tissues of 14-day-old postnatal mice were immersed in 10% neutral formaldehyde solution for fixation for about 48 h, then dehydrated step by step, cleared with xylene, embedded in paraffin, sectioned, and after the sections were placed at room temperature overnight, they were stored at 4°C for a long time; the tissue sections were baked at 62°C for 2 h → dewaxed in xylene I for 15 min → dewaxed in xylene II for 15 min → soaked in absolute ethanol for 5 min → soaked in 95% ethanol for 5 min → soaked in 90% ethanol for 5 min → soaked in 80% ethanol for 5 min → soaked in 70% ethanol for 5 min → rinsed with double-distilled water for 5 min × 3 times → stained with hematoxylin for 2 min → rinsed with tap water for 1 min → differentiated with 1% hydrochloric acid ethanol for 1 - 3 s → rinsed with tap water for 1 min → stained with eosin for 3 min → washed with tap water for 40 s to remove floating color → dehydrated with 85% ethanol for 20 s → dehydrated with 90% ethanol for 30 s → dehydrated with 95% ethanol I for 1 min → dehydrated with 95% ethanol II for 1 min → dehydrated with absolute ethanol I for 4 min → soaked and dehydrated with absolute ethanol II for 4 min → cleared in xylene I for 2 min → cleared in xylene II for 4 min → cleared in xylene III for 4 min → sealed with neutral gum → observed under a light microscope.

[0062] The results are as Figure 3 shown in A. Compared with wild-type mice, under hypoxic conditions, Nrf2 knockout caused morphological changes in lung tissues, thickening of alveolar walls, and infiltration of inflammatory cells.

[0063] 3) Immunohistochemistry

[0064] The lung tissue sections of 14-day-old postnatal mice were baked in an oven at 60°C for 1 h, dewaxed with xylene for 10 min, repeated 3 times, rehydrated with 100% - 70% gradient alcohol, 2 min each time, and washed twice with PBS for 5 min each.

[0065] The sections were subjected to antigen retrieval using a sodium citrate solution (Solarbio, Beijing, China). The sections were blocked with avidin and biotin in sequence, and then incubated with a 1 / 100 diluted anti-CD31 antibody (santa) at 4°C overnight. After washing away the primary antibody with PBS, the sections were incubated with biotinylated IgG at 37°C for 30 minutes. Staining was performed using the SABC (streptavidin-biotin complex) method, and diaminobenzidine was used as the staining substrate, and photographs were taken by microscopic observation.

[0066] The results are as Figure 3 shown in B. Compared with wild-type mice, under hypoxic conditions, Nrf2 knockout inhibited the development of lung blood vessels ( Figure 2 B).

[0067] 4) Real-time fluorescence quantitative PCR

[0068] Extract the RNA of lung tissue samples from 28-day-old mice after birth according to the kit. After measuring its purity and concentration, quantitatively reverse transcribe it into cDNA, and perform real-time fluorescence quantitative PCR detection using SYBR green fluorescent dye according to the kit instructions. Use the 2 -ΔΔCt method to relatively quantify the expression levels of genes such as superoxide dismutase 1 (SOD1), superoxide dismutase 2 (SOD2), glutamate-cysteine ligase (Gclm), and thioredoxin (Txn), and calculate the expression fold of each related gene.

[0069] The results are as Figure 4 shown. Compared with wild-type mice, in a hypoxic environment, the mRNA levels of SOD1, SOD2, Gclm, and Txn in Nrf2 knockout mice were significantly decreased, indicating that Nrf2 deficiency would lead to aggravated oxidative stress in mice.

[0070] 5) Immunoblotting experiment

[0071] Perform according to the method established in this laboratory. The main steps are as follows: Extract the total protein of lung tissue samples from 14-day-old mice after birth using the kit, quantify by BCA method, and routinely process the samples, perform electrophoresis, and electrotransfer the membrane. Block the NC membrane in 5% non-fat milk powder for 2 hours, then add the primary antibody and incubate overnight on a shaker at 4°C. After washing three times with TBST solution, add the corresponding secondary antibody and incubate at room temperature for 1 h. After washing the membrane with TBST, add the developer and expose and develop the image.

[0072] The results are as Figure 5 shown. Compared with wild-type mice, in a hypoxic environment, Nrf2 knockout led to a significant increase in the protein level (5A-B) and mRNA level (5F) of the key pyroptosis molecule GSDMD, and a significant increase in the mRNA levels of caspase-1 and IL-18 (5E, 5G).

[0073] 6) Immunofluorescence

[0074] Lung tissue sections of 14-day-old mice after birth were baked in an oven at 60 °C for 1 h, dewaxed with xylene for 10 min, repeated 3 times, rehydrated with 100%-70% gradient ethanol, 2 min each time; 2. Washed twice with PBS for 5 min each. Antigen retrieval was performed in 0.01 M sodium citrate buffer (pH 6.0) at 95 °C for 15 min. After the buffer cooled, the sections were taken out and washed with PBS for 5 min. Incubated with 1% Triton X-100 at room temperature for 10-20 min to permeabilize the membrane, washed with PBS 3 times, 5 minutes each time. Added 5% normal goat serum and blocked at room temperature for 30 min, then drained off the excess liquid. Added the primary antibody and incubated overnight at 4 °C. After incubation overnight at 4 °C, the samples were rewarmed at 37 °C for 45 min and washed with PBS 3 times, 5 min each time. Added the secondary antibody in the order of the primary antibody (secondary antibody diluted 1:300 with PBS), incubated at room temperature for 1 h, washed with PBS for 5 min, protected from light. Stained the nuclei with DAPI (diluted 1:100 with PBS) for 10 min, rinsed with PBS for 5 minutes, and added an anti-quenching agent to mount the slides. Observed using a Zeiss upright fluorescence microscope.

[0075] The results are as Figure 5 shown in C. The immunofluorescence results showed that knockout of Nrf2 led to a significant increase in the expression level of GSDMD, a key molecule in pyroptosis.

[0076] Example 3 Effect of Overexpressing Nrf2 on Lung Development with Intrauterine Growth Restriction

[0077] (1) Plasmid construction

[0078] Plasmid Nrf2 OE( Figure 6 ):

[0079] Table 1 Information on Nrf2 plasmid construction

[0080]

[0081] GSDMD promoter plasmid( Figure 7 ):

[0082] Table 2 Information on GSDMD luciferase plasmid construction

[0083]

[0084] (2) Cell transfection

[0085] When the 293t cells reached 90% confluence, replace the medium with 10% FBS DMEM without antibiotics. After 2 hours, dilute the Nrf2 overexpression plasmid (Nrf2 OE) and the negative control plasmid (pcDNA3.1(+)-3×FLAG-P2A-EGFP) with DMEM respectively (this is solution A). Dilute the liposome transfection reagent with DMEM and gently pipette to mix evenly (this is solution B). After standing at room temperature for 5 minutes, mix solution A and B, and gently pipette to mix evenly. After standing at room temperature for 20 minutes, add the mixture to the corresponding culture dishes according to the experimental groups, gently mix and place in an incubator at 37°C and 5% CO 2 and incubate. After 6 hours, replace the medium with 10% FBS DMEM without antibiotics and continue to incubate. After 24 hours of transfection, perform subsequent experiments.

[0086] (3) Dual luciferase reporter gene

[0087] After transfecting 293t cells with Nrf2 overexpression plasmid (Nrf2 OE), negative control plasmid (pcDNA3.1(+)-3×FLAG-P2A-EGFP), GSDMD promoter plasmid (pGL4.10-GSDMD Promoter (Wt)), empty plasmid (pGL4.10), and sea cucumber internal reference plasmid (pRL.CWV) for 24 hours, use the Dual Luciferase Reporter Assay Kit to detect the activity of the luciferase reporter gene. Take out the kit in advance to balance its temperature to room temperature. Aspirate the cell culture medium, wash twice with PBS, add an appropriate amount of 1×Cell Lysis Buffer, shake and lyse for 5 minutes, pipette and aspirate the cell lysate into a 1.5 mL centrifuge tube, centrifuge at 12,000 g at room temperature for 2 minutes, and take the supernatant for use. Add 100 μl of Luciferase Substrate balanced to room temperature to the microplate, carefully aspirate 20 μL of the cell lysate supernatant into the microplate, quickly mix and immediately detect the activity of the Firefly luciferase reporter gene in the microplate reader. Subsequently, add 100 μl of freshly prepared Renilla substrate working solution to the above reaction solution, quickly mix and immediately detect the activity of the Renilla luciferase reporter gene in the microplate reader.

[0088] The results are as Figure 8 shown in A. The relative fluorescence value of the GSDMD promoter in 293t cells overexpressing Nrf2 decreased significantly, indicating that overexpression of Nrf2 can inhibit the activity of the GSDMD promoter and inhibit the expression of GSDMD protein in a gradient manner ( Figure 8B-E). It can be seen that overexpression of Nrf2 can significantly inhibit pyroptosis, indicating that Nrf2 has a protective effect on the lung development of fetuses with intrauterine growth restriction.

[0089] Although the present invention has been disclosed above in preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims. SEQUENCE LISTING <110> Jiangnan University <120> Application of Nrf2 in the Auxiliary Diagnosis of Lung Development Retardation in Fetuses with Intrauterine Growth Restriction <130> BAA220459A <160> 1 <170> PatentIn version 3.3 <210> 1 <211> 605 <212> PRT <213> Artificial Sequence <400> 1 Met Met Asp Leu Glu Leu Pro Pro Pro Gly Leu Pro Ser Gln Gln Asp 1 5 10 15 Met Asp Leu Ile Asp Ile Leu Trp Arg Gln Asp Ile Asp Leu Gly Val 20 25 30 Ser Arg Glu Val Phe Asp Phe Ser Gln Arg Arg Lys Glu Tyr Glu Leu 35 40 45 Glu Lys Gln Lys Lys Leu Glu Lys Glu Arg Gln Glu Gln Leu Gln Lys 50 55 60 Glu Gln Glu Lys Ala Phe Phe Ala Gln Leu Gln Leu Asp Glu Glu Thr 65 70 75 80 Gly Glu Phe Leu Pro Ile Gln Pro Ala Gln His Ile Gln Ser Glu Thr 85 90 95 Ser Gly Ser Ala Asn Tyr Ser Gln Val Ala His Ile Pro Lys Ser Asp 100 105 110 Ala Leu Tyr Phe Asp Asp Cys Met Gln Leu Leu Ala Gln Thr Phe Pro 115 120 125 Phe Val Asp Asp Asn Glu Val Ser Ser Ala Thr Phe Gln Ser Leu Val 130 135 140 Pro Asp Ile Pro Gly His Ile Glu Ser Pro Val Phe Ile Ala Thr Asn 145 150 155 160 Gln Ala Gln Ser Pro Glu Thr Ser Val Ala Gln Val Ala Pro Val Asp 165 170 175 Leu Asp Gly Met Gln Gln Asp Ile Glu Gln Val Trp Glu Glu Leu Leu 180 185 190 Ser Ile Pro Glu Leu Gln Cys Leu Asn Ile Glu Asn Asp Lys Leu Val 195 200 205 Glu Thr Thr Met Val Pro Ser Pro Glu Ala Lys Leu Thr Glu Val Asp 210 215 220 Asn Tyr His Phe Tyr Ser Ser Ile Pro Ser Met Glu Lys Glu Val Gly 225 230 235 240 Asn Cys Ser Pro His Phe Leu Asn Ala Phe Glu Asp Ser Phe Ser Ser 245 250 255 Ile Leu Ser Thr Glu Asp Pro Asn Gln Leu Thr Val Asn Ser Leu Asn 260 265 270 Ser Asp Ala Thr Val Asn Thr Asp Phe Gly Asp Glu Phe Tyr Ser Ala 275 280 285 Phe Ile Ala Glu Pro Ser Ile Ser Asn Ser Met Pro Ser Pro Ala Thr 290 295 300 Leu Ser His Ser Leu Ser Glu Leu Leu Asn Gly Pro Ile Asp Val Ser 305 310 315 320 Asp Leu Ser Leu Cys Lys Ala Phe Asn Gln Asn His Pro Glu Ser Thr 325 330 335 Ala Glu Phe Asn Asp Ser Asp Ser Gly Ile Ser Leu Asn Thr Ser Pro 340 345 350 Ser Val Ala Ser Pro Glu His Ser Val Glu Ser Ser Ser Tyr Gly Asp 355 360 365 Thr Leu Leu Gly Leu Ser Asp Ser Glu Val Glu Glu Leu Asp Ser Ala 370 375 380 Pro Gly Ser Val Lys Gln Asn Gly Pro Lys Thr Pro Val His Ser Ser 385 390 395 400 Gly Asp Met Val Gln Pro Leu Ser Pro Ser Gln Gly Gln Ser Thr His 405 410 415 Val His Asp Ala Gln Cys Glu Asn Thr Pro Glu Lys Glu Leu Pro Val 420 425 430 Ser Pro Gly His Arg Lys Thr Pro Phe Thr Lys Asp Lys His Ser Ser 435 440 445 Arg Leu Glu Ala His Leu Thr Arg Asp Glu Leu Arg Ala Lys Ala Leu 450 455 460 His Ile Pro Phe Pro Val Glu Lys Ile Ile Asn Leu Pro Val Val Asp 465 470 475 480 Phe Asn Glu Met Met Ser Lys Glu Gln Phe Asn Glu Ala Gln Leu Ala 485 490 495 Leu Ile Arg Asp Ile Arg Arg Arg Gly Lys Asn Lys Val Ala Ala Gln 500 505 510 Asn Cys Arg Lys Arg Lys Leu Glu Asn Ile Val Glu Leu Glu Gln Asp 515 520 525 Leu Asp His Leu Lys Asp Glu Lys Glu Lys Leu Leu Lys Glu Lys Gly 530 535 540 Glu Asn Asp Lys Ser Leu His Leu Leu Lys Lys Gln Leu Ser Thr Leu 545 550 555 560 Tyr Leu Glu Val Phe Ser Met Leu Arg Asp Glu Asp Gly Lys Pro Tyr 565 570 575 Ser Pro Ser Glu Tyr Ser Leu Gln Gln Thr Arg Asp Gly Asn Val Phe 580 585 590 Leu Val Pro Lys Ser Lys Lys Pro Asp Val Lys Lys Asn 595 600 605

Claims

1. Use of a reagent for quantitatively detecting the expression of Nrf2 protein in the preparation of a diagnostic product for fetal lung hypoplasia with intrauterine growth restriction; It is characterized in that the amino acid sequence of the Nrf2 protein is as shown in SEQ ID NO:

1.

2. The use according to claim 1, It is characterized in that the diagnostic product is a diagnostic kit, and the diagnostic kit contains a detection reagent for Nrf2 protein.

3. The use according to claim 2, It is characterized in that the detection reagent for Nrf2 protein is used for quantitative detection of Nrf2 protein.

4. The use according to claim 2 or 3, It is characterized in that the detection reagent detects the expression of Nrf2 protein in a sample by fluorescence quantitative PCR, Southern hybridization, Northern hybridization, fluorescence in situ hybridization, DNA microarray, high-throughput sequencing method or immunoassay.

5. The use according to claim 4, It is characterized in that the immunoassay includes ELISA method, radioimmunoassay, immunohistochemistry method, Western blotting method.

6. The use according to claim 2 or 3, It is characterized in that if the expression level of the Nrf2 protein in lung tissue is more than 1.2 times higher than that of the control, it is regarded as a risk of fetal lung hypoplasia with intrauterine growth restriction.

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