OAB biomarker caused by vitamin D deficiency and application thereof

Abstract

The invention discloses a biomarker for OAB caused by deficiency of vitamin D and application thereof, specifically, human myelin basic protein and human trans-2, 3-enoyl-CoA reductase-like protein are used as biomarkers for children OAB, especially for OAB caused by deficiency of vitamin D for the first time, and the biomarker has relatively important guiding significance for clinical prognosis of OAB, especially for children OAB. And clinical treatment and prognosis judgment can be better guided. The biomarker disclosed by the invention can be realized only by collecting urine, is non-invasive, low in cost and rapid and convenient in diagnosis, improves the working efficiency, is beneficial to diagnosis and treatment of OAB in children, and has good clinical diagnosis potential and popularization value.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and provides OAB biomarkers caused by vitamin D deficiency and applications thereof. Background Art

[0002] Overactive bladder syndrome (OAB) is a concept formally proposed by the International Continence Society in 2002. Its main manifestations are urinary urgency, with or without urge incontinence, usually accompanied by frequent urination and nocturia, without urinary tract infection or other definite lesions. The cause of OAB is still unclear. The current mainstream view is that factors such as bladder detrusor instability, bladder hyperesthesia, urethral and pelvic floor muscle dysfunction, mental and behavioral abnormalities, and abnormal hormone metabolism are jointly involved in the pathogenesis of OAB.

[0003] As the research on vitamin D gradually deepens, the biological effects of vitamin D, known as the "anti-rickets vitamin", have gradually expanded to other extraskeletal systems such as autoimmune, endocrine, and cardiovascular systems. In recent years, as the expression of VDR in the bladder detrusor and pelvic muscles has been confirmed, the role and mechanism of vitamin D in lower urinary tract symptoms such as frequent urination, urgency, and urinary incontinence have gradually become a hot topic in basic and clinical research. A meta-analysis of approximately 4,000 adult patients suggests that compared with people without lower urinary tract symptoms, people with lower urinary tract symptoms have significantly lower serum vitamin D levels. In addition, studies targeting adults have shown that the risk of lower urinary tract symptoms in people with vitamin deficiency is more than three times that of people with sufficient vitamins. A randomized controlled trial designed by Markland found that exogenous supplementation of vitamin D (5000 IU / week) can significantly reduce the symptoms of urgency and urinary incontinence in women of a specific ethnic group. A randomized controlled trial designed by Oberg et al. found that high-dose vitamin D supplementation can reduce the severity of urinary incontinence in postmenopausal women. The study by Digesu et al. showed that vitamin D analogs relieve lower urinary tract symptoms by inhibiting the overactivity of the bladder detrusor. In basic research, some researchers have found that the polymorphism of VDR is closely related to the preventive and protective effect of vitamin D on male lower urinary tract symptoms. Studies have also shown that vitamin D deficiency may lead to an imbalance in cellular calcium homeostasis and thus abnormal contraction activity of the bladder detrusor, while vitamin D receptor agonists can inhibit bladder contraction activity by regulating calcium channel activity and the RhoA / ROCK signaling pathway. Another study at the genetic level found that the rs731236 variant of the gene encoding the vitamin D receptor can reduce the risk of lower urinary tract symptoms such as frequent urination and urgency. Recent studies have shown that most children with OAB have insufficient or insufficient vitamin D levels, and short-term, high-dose vitamin D supplementation can cure or effectively relieve their lower urinary tract symptoms, which is far better than the currently widely used anticholinergic drugs, and this treatment strategy is well tolerated in children. The above research progress shows that vitamin D deficiency may be the main cause of overactive bladder in children, suggesting that OAB caused by vitamin D deficiency may be the main cause of OAB in children.

[0004] Recently, based on a prospective randomized controlled study, the applicant conducted a vitamin D test and intervention control study on more than 300 children with OAB. The results showed that OAB children generally lack vitamin D, and short-term, high-dose vitamin D supplementation can cure or effectively relieve their lower urinary tract symptoms. Its efficacy and safety are far superior to the currently widely used anticholinergic drugs, and the children have good compliance. Therefore, the applicant proposed the concept of OAB caused by vitamin D deficiency, and related research has been published in authoritative journals in the field. However, the diagnosis of OAB related to vitamin D deficiency in children depends on venous blood sampling to detect vitamin D content and the therapeutic effect after vitamin D supplementation. There is a lack of non-invasive monitoring indicators, making it difficult to implement symptom assessment and follow-up effect evaluation of the disease. Urine has the advantages of non-invasiveness, low background, relative stability, and large collection volume. As a source of OAB biomarkers to assist clinical decision-making, it has great research and application value. In recent years, proteomics technology has made rapid progress, laying the foundation for the establishment and quantitative analysis of urine proteomics maps. Based on proteomics technology, there is a very broad prospect in finding reliable and non-invasive OAB urine protein biomarkers suitable for children to assist in clinical diagnosis and treatment decision-making and follow-up monitoring. Summary of the invention

[0005] In view of this, the object of the present invention is to provide OAB biomarkers caused by vitamin D deficiency and applications thereof.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a biomarker for OAB caused by vitamin D deficiency, selected from any one or a combination of the following:

[0008] (a) human myelin basic protein (MBP), the amino acid sequence of which is shown in SEQ ID NO.1;

[0009] (b) Human trans-2,3-enoyl-CoA reductase-like protein (TECRL), the amino acid sequence of which is shown in SEQ ID NO.2.

[0010] The present invention also provides the use of the aforementioned biomarkers in preparing a detection kit for OAB caused by vitamin D deficiency.

[0011] The present invention also provides a kit comprising the detection reagent of the above-mentioned biomarker.

[0012] As one of the preferred technical solutions, the kit detects OAB caused by vitamin D deficiency by detecting the expression level of biomarkers.

[0013] As one of the preferred technical solutions, the test sample of the kit is urine.

[0014] As one of the preferred technical solutions, the kit adopts mass spectrometry or ELISA.

[0015] The beneficial effects of the present invention are:

[0016] The present invention is the first to use human myelin basic protein and human trans-2,3-enoyl-CoA reductase-like protein as biomarkers for childhood OAB, especially OAB caused by vitamin D deficiency, which has important guiding significance for the clinical prognosis of OAB, especially childhood OAB, and can better guide clinical treatment and judge prognosis.

[0017] The present invention assists in the diagnosis of childhood OAB caused by vitamin D deficiency by detecting the expression levels of human myelin basic protein and human trans-2,3-enoyl-CoA reductase-like protein in the urine of children with OAB, thereby achieving a more accurate selection of treatment options. The present invention proposes for the first time to use the above two proteins as biomarkers for childhood OAB, especially OAB caused by vitamin D deficiency, which has high specificity and sensitivity, and provides a theoretical basis for the formulation of new standards for the etiology of OAB. This method has important guiding significance for the clinical treatment of childhood OAB, especially OAB caused by vitamin D deficiency, and helps to deeply understand its pathogenesis.

[0018] The biomarkers of the present invention can be achieved simply by collecting urine, are non-invasive, low-cost, and enable rapid and convenient diagnosis, thereby improving work efficiency, being beneficial to the diagnosis and treatment of OAB in children, and having good clinical diagnostic potential and promotion value.

[0019] Other advantages, objectives and features of the present invention will be described in the following description to some extent, and to some extent, will be obvious to those skilled in the art based on the following examination and study, or can be taught from the practice of the present invention. The objectives and other advantages of the present invention can be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be described in detail below in conjunction with the accompanying drawings, wherein:

[0021] Figure 1 This is the PCA score diagram (2D) of urine samples of children with OAB based on UPLC G2-Si HDMS; PC1 represents the first principal component, PC2 represents the second principal component, the percentage represents the variance explanation rate of the principal component for the data set, each point in the figure represents a sample, samples in the same group are represented by the same color, and Group represents the grouping.

[0022] Figure 2 Figure 3 is the PCA score diagram of urine samples of children with OAB based on UPLC G2-Si HDMS (3D); PC1 represents the first principal component, PC2 represents the second principal component, and PC3 represents the third principal component.

[0023] Figure 3 This is a heat map of differential protein clustering in urine between healthy children and children with OAB associated with vitamin D deficiency.

[0024] Figure 4 Volcano plot of differential proteins in urine between healthy children and children with OAB associated with vitamin D deficiency.

[0025] Figure 5 Box plots comparing the urinary MBP and TECRL protein levels in healthy children and children with OAB associated with vitamin D deficiency. DETAILED DESCRIPTION

[0026] The present invention will be further described below in conjunction with specific implementation modes.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0028] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0029] Embodiment 1:

[0030] Initial screening of urine markers for OAB in children

[0031] (1) Case selection: 12 children with OAB caused by vitamin D deficiency diagnosed in the outpatient department of the Children's Hospital Affiliated to Chongqing Medical University from August 2022 to November 2024 were selected, and 8 healthy children (vitamin D more than 35 pg / ml) were also included. All patients met the corresponding inclusion and exclusion criteria.

[0032] (2) Sample pretreatment: Fasting morning urine (midstream urine) was collected from children with OAB caused by vitamin D deficiency and healthy children, centrifuged at 4°C and 5000 rpm for 10 min, and the supernatant was collected and stored in a -80°C refrigerator;

[0033] (3) Protein extraction:

[0034] First, take out the sample from -80℃ and thaw it on ice. Then, add PMSF (phenylmethylsulfonyl fluoride) with a final concentration of 1mM to the sample and mix well. Centrifuge at 4500g for 10min at 4℃ and collect the supernatant; finally, determine the protein concentration using a BCA kit (Shanghai Biotech Co., Ltd.). If the sample concentration is too low, transfer the sample to a 10Kd ultrafiltration tube (Beckman Coulter Inc., Brea, CA), repeat ultrafiltration and concentration, collect the protein solution in the ultrafiltration tube, and determine the protein concentration using a BCA kit (Shanghai Biotech Co., Ltd.).

[0035] (4) Protein enzyme desalination

[0036] First, according to the protein concentration, 100 μg of protein solution was taken, and the volume was filled to 200 μl with 8M urea, and then DTT (dithiothreitol, final concentration 5mM) was added to reduce at 37°C for 45 minutes, and iodoacetamide (final concentration 11mM) was used for alkylation for 15 minutes in a dark room at room temperature. Then, 800 μl of 25mM ammonium bicarbonate solution and 2 μl of trypsin (Promega, V5280) were added, and digested at 37°C overnight. The enzymatic peptides were adjusted to pH 2-3 with a mass concentration of 20% TFA (trifluoroacetic acid) aqueous solution, and desalted with C18 (Millipore, Billerica, MA) column material. Finally, Pierce TM The peptide concentration was determined using a quantitative peptide detection reagent and standard kit (Thermo Fisher).

[0037] (5) Mass spectrometry: The urine samples were subjected to LC-MS / MS detection. 1) Nanoliter liquid chromatography detection: The samples were separated using a Vanquish Neo UHPLC nanoliter liquid phase system. Mobile phase A was a 0.1% formic acid aqueous solution, and mobile phase B was a 0.1% formic acid acetonitrile solution (100% acetonitrile). The injection mode was a capture-analysis dual-column method, in which the trap column was a PepMap Neo Trap Cartridge (300μm*5mm, 5μm), and the analytical column was an Easy-Spray TM PepMap TMNeo UHPLC column (150μm x 15cm, 2um). The temperature of the analytical column was controlled at 55°C by an integrated column oven, the sample load was 200pg, the flow rate was 2.5μl / min, the effective gradient was 22 minutes, and the total time was 24 minutes. 2) Orbitrap Astral mass spectrometer detection: DIA analysis used a nanoliter-speed Vanquish Neo system (Thermo Fisher) for chromatographic separation, and the samples separated by nanoliter HPLC were analyzed by DIA (data-independent) mass spectrometry using an Orbitrap Astral high-resolution mass spectrometer (ThermoScientific). Detection mode: positive ion, parent ion scanning range of 380-980m / z, primary mass spectrometry resolution of 240000at 200m / z, Normalized AGC Target of 500%, and MaximumIT of 5ms. MS2 uses DIA data acquisition mode, sets 299 scanning windows, Isolation Window is 2Th, HCDCollision Energy is 25%, Normalized AGC Target is 500%, and Maximum IT is 3ms. Based on the OPLS-DA model, proteins with differences between different varieties or tissues were preliminarily screened.

[0038] ( Figures 1 to 3 )

[0039] (6) Marker screening: According to the above steps, a total of 68 potential urine biomarkers were obtained (satisfying FC>1.5 and P-value≤0.05). The two biomarkers with the highest difference fold were selected as candidate biomarkers: human trans-2,3-enoyl-CoA reductase-like protein (TECRL) and human myelin basic protein (MBP) ( Figure 4 ).

[0040] Embodiment 2:

[0041] Validation of urine markers for OAB in children

[0042] The urine samples of 40 children with OAB and 40 healthy children were quantitatively analyzed by enzyme-linked immunosorbent assay (ELISA) (refer to the instructions of relevant commercial kits), and the urine creatinine was used for standardization correction (the target protein content in the sample was divided by the creatinine content in the sample). The specific reference is "Percy AK, Lane JB, Goodwin J, Kachelhofer RD, Whitaker JN (1998) Age-related chapges in the level of urinarymyelin basic protein-like material in childhood. Neurology 51(5):1339-1341".

[0043] Enzyme-linked immunosorbent assay (ELISA) for determination of MBP and TECRL:

[0044] 1. Sample processing

[0045] 1.1 Urine sample collection and storage: Collect fasting morning urine (midstream urine) from children with OAB caused by vitamin D deficiency and healthy children, centrifuge at 4°C and 5000 rpm for 10 min, take the supernatant, and store it in a -80°C refrigerator;

[0046] 1.2 Urine sample concentration: Take 10mL urine sample and add PMSF with a final concentration of 1mM and mix well. Add the sample to a 3Kd / 10Kd ultrafiltration tube and centrifuge in a 4℃ low-temperature centrifuge with a centrifugal force of 4000g for 45min. If the concentration multiple is low, ultrafiltration and concentration can be repeated. Collect the protein solution in the ultrafiltration tube for subsequent experiments.

[0047] 2. Reagent Preparation

[0048] 2.1 Take out the test kit from the refrigerator 20 minutes in advance and equilibrate to room temperature (18-25°C).

[0049] 2.2 Washing solution: Dilute the concentrated washing solution at a ratio of 1:24 and dissolve possible crystals in a 40°C water bath.

[0050] 2.3 Standard working solution: Add 1 mL of diluent to the lyophilized standard, let it stand for 10 minutes to fully dissolve, then prepare a 1000 pg / mL working solution, and dilute it in multiples to concentrations of 1000, 500, 250, etc.

[0051] 2.4 Biotinylated antibody working solution: Before the experiment, calculate the dosage at 100 μL / well and dilute the 100× concentrate to 1× working concentration.

[0052] 2.5 HRP enzyme conjugate working solution: Calculate the dosage based on 100 μL / well and dilute the 100× concentrate to 1× working concentration.

[0053] 3. Operation steps

[0054] 3.1 Sample addition and incubation

[0055] Set up standard wells, blank wells, and sample wells: Standard wells: Add 100 μL of the diluted standard. Blank wells: Add 100 μL of diluent. Sample wells: Add 100 μL of diluted sample. Cover with film and incubate at 37°C for 90 minutes.

[0056] 3.2 Add biotinylated antibody

[0057] Shake off the liquid in the wells without washing. Add 100 μL of biotinylated antibody working solution to each well, cover with membrane, and incubate at 37°C for 1 hour.

[0058] 3.3 Washing plate

[0059] Add 350 μL of washing solution to each well, soak for 1 minute, remove the liquid and pat dry. Repeat washing 3 times.

[0060] 3.4 Add HRP enzyme conjugate

[0061] Add 100 μL HRP enzyme conjugate working solution to each well, cover with membrane, and incubate at 37°C for 30 minutes. Wash the plate 5 times using the same method as above.

[0062] 3.5 Color reaction

[0063] Add 90 μL of substrate solution (TMB) to each well, cover with membrane, and incubate at 37°C in the dark for 15 minutes (adjust the time according to the color development, but not more than 30 minutes).

[0064] 3.6 Termination of the reaction

[0065] Add 50 μL of stop solution to each well, and immediately measure the optical density (OD value) of each well at a wavelength of 450 nm using an enzyme reader.

[0066] 4. Calculation of results

[0067] 4.1 Calculate the average OD value of the standard and sample duplicate wells, and subtract the OD value of the blank well as the correction value.

[0068] 4.2 Draw a standard curve (concentration as the horizontal axis, OD value as the vertical axis) and fit a four-parameter logistic function.

[0069] 4.3 Calculate the concentration of target protein in the sample based on the standard curve and dilution factor.

[0070] The results show that:

[0071] 1. The level of human myelin basic protein (MBP) in urine of healthy children was 91.94±57.85 pg / mg Cr, while that of children with OAB increased to 918.80±479.13 pg / mg Cr (P<0.001).

[0072] 2. The level of human trans-2,3-enoyl-CoA reductase-like (TECRL) in the urine of healthy children was 39.77±27.77 pg / mg Cr, while the level in children with OAB was significantly increased to 318.38±137.50 pg / mg Cr (P<0.001).

[0073] The above differences were statistically significant ( Figure 5 ).

[0074] Standardized (by urine creatinine level) human trans-2,3-enoyl-CoA reductase-like protein (TECRL) and human myelin basic protein (MBP) were significantly increased in the urine of children with OAB, increasing by about 10 times and 8 times, respectively, with good discriminatory effect. (Table 1)

[0075] Table 1

[0076]

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution, which should be included in the scope of the claims of the present invention.

Claims

1. A biomarker for OAB caused by vitamin D deficiency, characterized in that: Select from any one or a combination of the following: (a) human myelin basic protein, the amino acid sequence of which is shown in SEQ ID NO.1; (b) Human trans-2,3-enoyl-CoA reductase-like protein, the amino acid sequence of which is shown in SEQ ID NO.

2.

2. Use of the biomarker according to claim 1 in preparing a detection kit for OAB caused by vitamin D deficiency.

3. A kit, characterized in that: A detection reagent comprising the biomarker according to claim 1.

4. A kit according to claim 3, characterized in that, The kit detects OAB caused by vitamin D deficiency by detecting the expression level of biomarkers.

5. A kit according to claim 3, characterized in that: The test sample of the kit is urine.

6. A kit according to claim 3, characterized in that, The kit adopts mass spectrometry or ELISA method.

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