Application of biomarker in preparation of Farotetrakis diagnosis product

Abstract

The invention belongs to the technical field of biological medicine and medical diagnosis, and particularly relates to application of a biomarker in preparation of a diagnostic product for Fallotetrakis. The invention provides application of a biomarker in preparation of a diagnostic product for Fallotetrakis. The biomarker is HSPA6 protein or mRNA thereof. Through comparative study on expression levels of the HSPA6 protein and mRNA of a Fallotetrakis patient sample and a normal individual sample, it is found that compared with the normal individual sample, the expression levels of the HSPA6 protein and mRNA of the Fallotetrakis patient are remarkably reduced, and the difference has statistical significance. Therefore, it is prompted that the HSPA6 expression level has a diagnostic value for diagnosis of the Fallotetrakis, the HSPA6 protein or mRNA of the HSPA6 protein can be used as a basis for early clinical diagnosis of the Fallotetrakis, and a new direction can also be provided for development of diagnostic products of the Fallotetrakis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and medical diagnosis technology, and in particular, relates to the application of biomarkers in the preparation of diagnostic products for tetralogy of Fallot. Background Art

[0002] Tetralogy of Fallot (TOF) is a common congenital heart and vascular malformation. Its basic pathology includes pulmonary stenosis, ventricular septal defect, right aorta (riding over the defective ventricular septum), and right ventricular hypertrophy. Among them, ventricular septal defect and pulmonary stenosis are more important. Patients with only ventricular septal defect, pulmonary stenosis, and right ventricular hypertrophy without aortic riding are called atypical tetralogy of Fallot. TOF is the most common cyanotic congenital heart disease, accounting for about 7% to 10% of congenital heart defects, which seriously affects the health of children.

[0003] At present, the cause of TOF is still unclear. Epidemiological surveys have shown that genetic abnormalities are its main pathogenic factors. Some gene mutations are closely related to the occurrence of tetralogy of Fallot, among which NKX2.5, GATA4, TBX5 and TBX20 have been confirmed to be highly correlated with tetralogy of Fallot. However, the genetic cause of most patients with tetralogy of Fallot, especially sporadic patients, is still unclear. Therefore, revealing the molecular mechanism of the occurrence and development of tetralogy of Fallot and exploring new biomarkers are of great significance for the early diagnosis and early treatment of tetralogy of Fallot, as well as the later design of drugs and the realization of clinical transformation.

[0004] Heat shock protein 70-kDaA6 (heat shock 70-kDa protein 6, HSPA6) is a member of the HSP70 family. Its gene is located on human chromosome 1q23.3, and its expression is mainly located in the cytoplasm and exosomes. HSPA6 plays an important role in a variety of physiological and pathological processes. Cardiovascular diseases are often associated with vascular endothelial cell damage. After vascular endothelial damage, the middle smooth muscle cells (SMC) will migrate to the intima layer of the vascular wall and proliferate there, eventually leading to vascular fibrosis and vascular stenosis. Studies have found that after SMCs were infected with adenovirus carrying the HSPA6 gene, serum stimulation could inhibit SMC proliferation at 3d and 6d, indicating that HSPA6 can inhibit SMC proliferation and promote the development of vascular repair. In addition, early studies have confirmed that abnormal aggregation and abnormal folding of proteins can lead to the occurrence of neurodegenerative diseases. Heat shock proteins are involved in the repair process of this cell damage. HSPA6 also helps neuronal cells to avoid cell stress damage.

[0005] However, there are currently no studies on the association between HSPA6 and tetralogy of Fallot, nor any reports on the molecular mechanism of action between the two. Summary of the invention

[0006] In order to overcome the deficiency of the lack of diagnostic markers for tetralogy of Fallot in the prior art, the purpose of the present invention is to provide the use of a biomarker in the preparation of a diagnostic product for tetralogy of Fallot, wherein the biomarker is HSPA6 protein or its mRNA. The present invention has found in experiments that HSPA6 protein and its mRNA in the blood or myocardial tissue of TOF patients are significantly low in expression, so HSPA6 can be used as a biomarker, and by detecting the expression level of HSPA6 protein or its mRNA, a basis can be provided for the early diagnosis of tetralogy of Fallot.

[0007] To achieve the above object, the technical solution adopted by the present invention is: A use of a biomarker in preparing a diagnostic product for tetralogy of Fallot, wherein the biomarker is HSPA6 protein or its mRNA.

[0008] As a preferred embodiment, the diagnostic product is used to detect the expression level of the biomarker in a biological sample.

[0009] As a further preferred embodiment, the biological sample is blood or myocardial tissue of the subject to be detected.

[0010] As a preferred solution, the diagnostic product is a kit.

[0011] As a further preferred solution, the kit includes: a reagent for detecting the expression level of the biomarker and a diagnostically acceptable auxiliary reagent.

[0012] As a further preferred solution, the reagent used to detect the expression level of the biomarker is an enzyme-linked immunosorbent assay reagent. The enzyme-linked immunosorbent assay reagent can be used to detect HSPA6 protein, and tetralogy of Fallot can be diagnosed based on the expression level of HSPA6 protein.

[0013] As a further preferred solution, the reagent used to detect the expression level of the biomarker is a real-time fluorescence quantitative reagent. The real-time fluorescence quantitative reagent can be used to detect HSPA6 mRNA, and tetralogy of Fallot can be diagnosed based on the expression level of HSPA6 mRNA.

[0014] The technical solution of the present invention has the following advantages and beneficial effects: The present invention provides the use of a biomarker in the preparation of a diagnostic product for tetralogy of Fallot, wherein the biomarker is HSPA6 protein or its mRNA. The present invention compares the expression levels of HSPA6 protein in samples of patients with tetralogy of Fallot and samples of normal individuals, and finds that compared with samples of normal individuals, the expression level of HSPA6 protein in patients with tetralogy of Fallot is significantly reduced, and the difference is statistically significant. Further, by detecting the expression level of HSPA6 mRNA, it is found that the expression level of HSPA6 mRNA in patients with tetralogy of Fallot is also significantly reduced, and the difference is statistically significant.

[0015] Therefore, the above results suggest that the expression level of HSPA6 has diagnostic value for the diagnosis of tetralogy of Fallot, and HSPA6 protein or its mRNA can be used as the basis for the early clinical diagnosis of tetralogy of Fallot.

[0016] The present invention provides a new biomarker HSPA6 protein or its mRNA for the early diagnosis of tetralogy of Fallot by revealing the close correlation between tetralogy of Fallot and HSPA6. By detecting the expression of HSPA6 protein in the blood of the subject, it can be determined whether the subject suffers from tetralogy of Fallot or whether the subject is at risk of suffering from tetralogy of Fallot, thereby providing strong technical support for the early diagnosis and intervention treatment of tetralogy of Fallot, and also providing a new direction for the development of diagnostic products for tetralogy of Fallot. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The results of the present invention using the HSPA6 enzyme-linked immunosorbent assay kit to detect the HSPA6 protein expression levels of normal blood samples and tetralogy of Fallot blood samples respectively; Figure 2 The results of the present invention using real-time fluorescence quantitative technology to detect the expression of HSPA6 mRNA in normal myocardial tissue and myocardial tissue of tetralogy of Fallot; Figure 3 This is the ROC curve analysis result of the clinical diagnosis of tetralogy of Fallot using HSPA6 protein content in the present invention. DETAILED DESCRIPTION

[0018] The technical scheme and technical effects of the present invention will be clearly and completely described below in conjunction with specific embodiments and test examples. However, it should be understood by those skilled in the art that the embodiments are only used to illustrate the technical scheme of the present invention and should not be regarded as limiting the scope of protection of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the raw materials used are all commonly used in the art, available to the public or commercially available, unless otherwise specified.

[0019] Example 1

[0020] This embodiment provides an application of a biomarker in preparing a diagnostic product for tetralogy of Fallot, wherein the biomarker is HSPA6 protein. The diagnostic product is used to detect the expression level of HSPA6 protein in a biological sample. The biological sample is the blood of a subject to be detected.

[0021] Example 2

[0022] This embodiment provides an application of a biomarker in preparing a diagnostic product for tetralogy of Fallot, wherein the biomarker is HSPA6 mRNA. The diagnostic product is used to detect the expression level of HSPA6 mRNA in a biological sample. The biological sample is myocardial tissue of a subject to be detected.

[0023] Test example The data involved in the following test examples of the present invention were statistically analyzed using the statistical analysis software Prism 9. P <0.05 indicates that the difference is statistically significant. * P <0.05,** P <0.01,*** P <0.001, **** P <0.0001 indicates different degrees of difference.

[0024] 1. Detection of HSPA6 protein expression level Blood samples from 10 patients with tetralogy of Fallot from Qingdao Women and Children's Hospital were selected, and blood samples from 10 healthy subjects were selected as controls. Among them, the patients with tetralogy of Fallot included were diagnosed with tetralogy of Fallot based on their pathological reports. The implementation of the present invention was approved by the ethics committee of Qingdao Women and Children's Hospital through ethical voting standards and supervision.

[0025] (1) Separation of plasma Collect whole blood samples from patients with tetralogy of Fallot or healthy subjects, gently invert and mix in an EDTA anticoagulant tube, precool the centrifuge to 4°C, centrifuge at 1600g for 10 min, collect the supernatant into a new 1.5mL EP tube, centrifuge at 16000g for 10 min to remove cell debris, and dispense the plasma into new centrifuge tubes, mark them for subsequent testing or freeze them in a -80°C ultra-low temperature refrigerator for later use.

[0026] (2) Detection of human HSPA6 protein content in plasma (a) Dilution of standard: The HSPA6 protein content was detected using the human HSPA6 ELISA kit (model MM-1823H1, Jiangsu, China). The kit provided one original standard, which was diluted in a small test tube according to the ratio shown in the chart in the kit manual; (b) Sample addition: Set up blank wells (blank control wells do not add samples and enzyme-labeled reagents, and the rest of the steps are the same), standard wells, and test sample wells. Accurately add 50uL of the standard sample to the enzyme-labeled plate, first add 40uL of the sample diluent to the test sample well, and then add 10uL of the test plasma sample obtained by the above treatment (the final sample dilution is 5 times). Add the sample to the bottom of the well of the enzyme-labeled plate, try not to touch the well wall, and gently shake to mix; (c) Incubation: Seal the plate with a sealing film and incubate at 37°C for 30 min. (d) Solution preparation: dilute the 30-fold concentrated washing solution with 30-fold distilled water for later use; (e) Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and then discard, repeat this 5 times, and pat dry; (f) Add enzyme: add 50uL of enzyme-labeled reagent to each well, except for the blank well; then incubate at 37℃ for 30min, and repeat the washing operation in the previous step; then develop the color, first add 50uL of color developer A to each well, then add 50uL of color developer B, gently shake and mix, and develop the color at 37℃ in the dark for 15min; finally stop: add 50uL of stop solution to each well to stop the reaction (the blue color will turn yellow immediately); (g) Determination: Use blank as zero and measure the absorbance (OD value) of each well in sequence at a wavelength of 450 nm. The determination should be carried out within 15 minutes after adding the stop solution.

[0027] (3) Data analysis The OD450 value of HSPA6 of each sample measured by the microplate reader was used to calculate the content of HSPA6 in the plasma sample according to the standard curve. Each sample was tested in parallel three times. Figure 1 shown.

[0028] In this experiment, in order to verify the content of HSPA6 protein in the plasma of TOF patients and normal people, the present invention uses a human HSPA6 enzyme-linked immunosorbent assay kit to detect the content of HSPA6. Figure 1 The results showed that compared with the normal control group, the HSPA6 content in the plasma of TOF patients was significantly reduced, and there was a significant difference between the two.

[0029] 2. Detection of HSPA6 mRNA expression level The test samples were from Qingdao Women and Children's Hospital. The myocardial tissue of TOF patients used to detect the expression level of HSPA6 mRNA was collected during the tetralogy of Fallot surgery, and the corresponding normal myocardial tissue was from volunteers who donated organs with the consent of their families and did not suffer from tetralogy of Fallot. The included tetralogy of Fallot patients were diagnosed with tetralogy of Fallot based on their pathological reports. The implementation of the present invention was approved by the ethics committee of Qingdao Women and Children's Hospital through ethical voting standards and supervision.

[0030] (1) Extraction of mRNA from normal myocardial tissue and myocardial tissue of TOF patients The corresponding myocardial tissue was taken out from the liquid nitrogen tank, 100 mg of myocardial tissue was weighed and thawed in a 1.5 mL EP tube, 500 uL of Trizol was added and the tissue was broken with a special tissue crusher, 100 uL of chloroform was added and vortexed for 15 seconds, then ice-bathed for 5 minutes, centrifuged at 12000 rpm for 15 minutes, RNA was separated from the aqueous phase and an equal volume of isopropanol was added, inverted and ice-bathed for 30 minutes, and then centrifuged at 12000 rpm for 10 minutes; after discarding the supernatant, freshly prepared 75% ethanol was added for washing up and down, and the supernatant was removed by centrifugation, 50 uL of DEPC was added to dissolve the precipitate to obtain myocardial tissue mRNA, which was reversed into cDNA for subsequent detection.

[0031] (2) Obtaining cDNA from normal myocardial tissue and myocardial tissue of TOF patients The mRNA of myocardial tissue extracted in the previous step was reverse transcribed using a reverse transcription kit (Vazyme, China) according to the instructions, thereby converting the unstable and difficult to preserve mRNA into stable cDNA that can be used for subsequent detection of raw materials.

[0032] (3) Design and synthesis of HSPA6 primers According to the different detection indicators, the CDS sequence of HSPA6 gene was obtained from the NCBI website, and the primers were designed using Primer5.0 software. The primer information was sent to Shanghai Bioengineering Company for synthesis and used for subsequent detection. The specific primer sequences are as follows: F: 5'-CTGGTGTCTTTGAGGTGAAAGCC-3', R: 5'-GCGCAGGGCACGCTTGTT-3'.

[0033] (4) Detection of HSPA6 mRNA expression levels in normal myocardial tissue and myocardial tissue of TOF patients The cDNA samples were mixed according to the following system and tested on the machine: cDNA Template: 2.0uL; 2×SYBRGreen qPCR Mix: 10.0uL; 10μM Primer Forward: 0.4uL; 10μM Primer Reverse: 0.4uL; DEPC water: 7.2uL; Total volume: 20uL; Place the PCR tube in a thermal cycler and perform the PCR reaction. The reaction conditions are set as follows: 95°C, 10 min; 45 PCR cycles (95°C, 20 s; 55°C, 30 s; 72°C, 20 s). To establish the melting curve of the PCR product, after the amplification reaction, 95°C, 5 s; 65°C, 60 s; 97°C, 15 s; and cooling at 40°C, 10 s.

[0034] (5) Data analysis Adoption 2 －△△Ct Method calculation, RQ = 2 －△△Ct Three replicate wells were set up for each sample's internal reference gene and target gene for independent enrichment and repeated verification. Figure 2 shown.

[0035] Depend on Figure 2 It can be seen that the present invention collected normal human myocardial tissue and TOF myocardial tissue, and used real-time fluorescence quantitative PCR technology to detect the expression level of HSPA6 mRNA in the two batches of tissues. The results showed that HSPA6 mRNA was significantly lowly expressed in the myocardial tissue of TOF patients, which was significantly different from that in normal myocardial tissue.

[0036] 3. Sensitivity and specificity analysis The above experiment confirmed that HSPA6 was significantly underexpressed in the blood of TOF patients, but the reliability of this indicator for TOF disease diagnosis is still unclear. The present invention further analyzed the relationship between the content of the above HSPA6 protein and the occurrence of TOF disease, and performed ROC curve analysis for clinical diagnosis. Figure 3 shown.

[0037] Depend on Figure 3 It can be seen that the present invention uses the content of HSPA6 protein as an evaluation index, and its sensitivity for clinical diagnosis of TOF disease is 0.8, specificity is 0.9, accuracy is 0.85, the corresponding cut-off value is 80.754, and the area under the curve is 0.791. This result shows that the HSPA6 content in the plasma of TOF patients is lower than that of normal people, and can be effectively used to diagnose whether they have TOF.

[0038] In summary, the present invention provides a new biomarker HSPA6 protein or its mRNA for the early diagnosis of tetralogy of Fallot by revealing the close correlation between tetralogy of Fallot and HSPA6. By detecting the expression of HSPA6 protein in the blood of the subject, it can be determined whether the subject suffers from tetralogy of Fallot or whether the subject is at risk of suffering from tetralogy of Fallot, thereby providing strong technical support for the early diagnosis and intervention treatment of tetralogy of Fallot, and also providing a new direction for the development of diagnostic products for tetralogy of Fallot.

[0039] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. The use of a biomarker in the preparation of a diagnostic product for tetralogy of Fallot, characterized in that: The biomarker is HSPA6 protein or its mRNA.

2. Use of the biomarker according to claim 1 in preparing a diagnostic product for tetralogy of Fallot, characterized in that: The diagnostic product is used to detect the expression level of the biomarker in a biological sample.

3. Use of the biomarker according to claim 2 in preparing a diagnostic product for tetralogy of Fallot, characterized in that: The biological sample is the blood or myocardial tissue of the subject to be detected.

4. Use of the biomarker according to claim 1 in preparing a diagnostic product for tetralogy of Fallot, characterized in that: The diagnostic product is a kit.

5. Use of the biomarker according to claim 4 in preparing a diagnostic product for tetralogy of Fallot, characterized in that: The kit includes: reagents for detecting the expression level of the biomarkers and diagnostically acceptable auxiliary reagents.

6. Use of the biomarker according to claim 5 in preparing a diagnostic product for tetralogy of Fallot, characterized in that: The reagent used to detect the expression level of the biomarker is an enzyme-linked immunosorbent assay reagent.

7. Use of the biomarker according to claim 5 in preparing a diagnostic product for tetralogy of Fallot, characterized in that: The reagent used to detect the expression level of the biomarker is a real-time fluorescence quantitative reagent.

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