Non-invasive predictive kit and method for the progression of benign prostatic hyperplasia based on prostatic fluid.

Abstract

This invention relates to a non-invasive predictive kit and method for the progression of benign prostatic hyperplasia (BPH) based on prostatic fluid. The invention provides the application of FGF9 as a non-invasive predictive biomarker for the progression of BPH based on prostatic fluid, wherein the concentration of FGF9 in prostatic fluid serves as an indicator of BPH progression. The invention also provides a method for detecting the concentration of FGF9 in prostatic fluid for non-disease diagnostic purposes. Compared with existing technologies, the non-invasive BPH detection method based on prostatic fluid of this invention has advantages such as non-invasiveness, accuracy, simplicity, practicality, and scalability, and is expected to be widely used in the clinical diagnosis and treatment of prostate diseases.

Description

Technical Field

[0001] This invention belongs to the field of prostate disease detection reagent technology, and in particular relates to a non-invasive predictive reagent kit and method for the progression of benign prostatic hyperplasia based on prostate fluid. Background Technology

[0002] Benign prostatic hyperplasia (BPH) is a major cause of bladder outlet obstruction in middle-aged and elderly men, leading to lower urinary tract symptoms (LUTS) and even urinary retention.

[0003] According to data from the MTOPS, PLESS studies and their follow-up studies, the annual incidence of urinary retention in men aged 50 and older in the general population is 0.5% to 2.5%. However, the incidence of urinary retention is a cumulative risk that increases rapidly with age. That is, assuming a 60-year-old man survives to age 80, with clinical progression of benign prostatic hyperplasia, the cumulative risk of experiencing difficulty urinating or mild urinary retention symptoms is approximately 23%; the risk of developing acute urinary retention requiring surgical intervention is approximately 8-20%.

[0004] Patients with moderate to severe urinary symptoms of benign prostatic hyperplasia (BPH) often experience repeated medication and progressively worsening lower urinary tract symptoms before undergoing invasive surgical treatment. The socioeconomic and healthcare burden caused by the clinical progression of BPH is expected to continue to increase in the foreseeable future.

[0005] Although research on benign prostatic hyperplasia (BPH) from a molecular biological perspective is still insufficient, it is certain that the androgen signaling pathway plays a crucial role. Based on this, classic drugs that can block dihydrotestosterone synthesis—5-alpha reductase inhibitors—were developed. Simultaneously, due to the presence of prostatic stroma and urethral smooth muscle, alpha-receptor blockers were introduced into BPH treatment. The development of these two drugs spurred large-scale clinical trials such as MTOPS and CombAT, thus laying the foundation for the classic two-drug combination therapy for BPH. However, clinical data shows that approximately 5% of patients still do not respond well to medication, failing to halt the clinical progression of BPH and necessitating surgical intervention. This group of patients is currently the focus of basic, clinical, and translational research in the field of BPH. Existing research on benign prostatic hyperplasia does not adequately elucidate the mechanisms of drug ineffectiveness and clinical progression of BPH from a molecular biological perspective. Identifying key molecules responsible for the clinical progression of BPH can help predict patients with progressive benign prostatic hyperplasia and allow for earlier surgical intervention.

[0006] Meanwhile, current diagnostic methods for the progression of benign prostatic hyperplasia are mainly based on invasive diagnostic methods (which require surgery or catheterization to make a diagnosis), which puts pressure on patients both physiologically and economically. Summary of the Invention

[0007] Given that current diagnostic methods for benign prostatic hyperplasia (BPH) progression are primarily invasive, this invention provides a non-invasive predictive kit and method for BPH progression based on prostatic fluid to enrich diagnostic tools for BPH progression.

[0008] The objective of this invention can be achieved through the following technical solutions:

[0009] This invention first provides the application of FGF9 as a non-invasive predictive biomarker for the progression of benign prostatic hyperplasia (BPH) based on prostatic fluid, wherein the concentration of FGF9 in prostatic fluid serves as an indicator of whether a patient already diagnosed with BPH will experience symptom progression in the future.

[0010] This invention provides the application of FGF9 as a biomarker to predict whether patients diagnosed with benign prostatic hyperplasia will experience symptom progression in the future.

[0011] The present invention also provides the use of FGF9, or reagents for detecting FGF9 protein levels, in the preparation of kits for predicting whether patients with benign prostatic hyperplasia will experience symptom progression in the future.

[0012] The present invention also provides the use of FGF9, or reagents for detecting FGF9 protein content, in the preparation of kits for predicting whether patients with benign prostatic hyperplasia will experience clinical progression after failure of drug treatment for benign prostatic hyperplasia.

[0013] This invention provides a non-invasive predictive kit for the progression of benign prostatic hyperplasia based on prostatic fluid, including reagents for detecting the concentration of FGF9 in prostatic fluid.

[0014] In one embodiment of the present invention, the reagents include an FGF9 antibody solution and an ELISA reagent.

[0015] In one embodiment of the present invention, the ELISA reagent comprises a horseradish peroxidase (HRP)-labeled avidin solution at a concentration of 0.01-0.05 μg / mL, a TMB chromogenic solution, and a stop solution.

[0016] The TMB chromogenic solution refers to tetramethylbenzidine chromogenic solution, which is the substrate for ELISA color development. The reaction occurs under the catalysis of HRP. Specific components: Solution A: Sodium acetate 13.6g, citric acid 1.6g, 30% hydrogen peroxide 0.3ml, distilled water to 500ml. Solution B: Disodium EDTA 0.2g, citric acid 0.95g, glycerol 50ml, TMB 0.15g, distilled water to 500ml. Mix solutions A and B in a 1:1 ratio before use.

[0017] In one embodiment of the present invention, the terminating solution is 2 mol / L H2SO4.

[0018] This invention also provides a non-invasive method for predicting the progression of benign prostatic hyperplasia (BPH) based on prostatic fluid. This method can predict whether patients already diagnosed with BPH will experience symptom progression in the future. It should be noted that this method is not for diagnosing the disease, but rather a predictive method, and includes the following steps:

[0019] The concentration of FGF9 in the prostatic fluid of patients diagnosed with benign prostatic hyperplasia (BPH) was detected, and the probability of symptom progression in future patients diagnosed with BPH was predicted based on the FGF9 concentration in the prostatic fluid.

[0020] In one embodiment of the present invention, the concentration of FGF9 in the patient's prostatic fluid is compared with a set value. When the concentration of FGF9 in the patient's prostatic fluid is higher than the set value, it is predicted that the patient is positive for BPH progression. The set value is 100 pg / ml.

[0021] This invention also provides a method for detecting the concentration of FGF9 in prostatic fluid for non-disease diagnostic purposes, comprising the following steps:

[0022] 1) Prepare a 500 pg / ml FGF9 standard solution. Perform serial dilutions of the standard. Set up 7 standard wells and add the serially diluted standard solutions at concentrations of 500 pg / ml, 250 pg / ml, 125 pg / ml, 62.5 pg / ml, 31.25 pg / ml, 15.625 pg / ml, and 7.8125 pg / ml. Cover the microplate with a membrane and incubate at 37°C for 1 hour.

[0023] 2) Discard the liquid, spin dry, no washing required;

[0024] 3) Add 100 μL of FGF9 antibody solution to each well, cover the microplate with a membrane, and incubate at 37°C for 1 hour;

[0025] 4) Discard the liquid in the wells. Wash each well with 350 μL of PBS, soaking for 1-2 minutes. Gently tap the plate on absorbent paper to remove all liquid from the wells. Repeat the washing process 3 times. After the final wash, aspirate or pour out the remaining PBS, invert the plate onto absorbent paper, and blot away any remaining liquid in the wells.

[0026] 5) Add 100 μL of horseradish peroxidase-labeled avidin solution to each well, cover the microplate with a membrane, and incubate at 37°C for 30 minutes.

[0027] 6) Discard the liquid in the wells, wash each well with 350 μL of PBS, soak for 1-2 minutes, gently tap the microplate on absorbent paper to remove all the liquid in the wells, shake dry, repeat the washing 5 times, after the last wash, aspirate or pour out the remaining PBS, invert the microplate on absorbent paper, and absorb all the liquid remaining in the wells.

[0028] 7) Add 90 μL of TMB chromogenic solution to each well, cover the microplate with a membrane, and incubate at 37°C in the dark.

[0029] 8) Add 50 μL of 2 mol / L H2SO4 to each well to terminate the reaction;

[0030] 9) After ensuring that there are no water droplets at the bottom of the ELISA plate and no air bubbles in the wells, immediately use an ELISA reader to measure the optical density value (OD value) of each well at a wavelength of 450 nm. Based on the OD values ​​of different standards in the standard wells and the concentration of FGF9 in different standards, calculate the relationship between the OD value and the FGF9 concentration to obtain the standard curve and the linear formula.

[0031] 10) For the sample to be tested, the OD value of the sample is detected using the same method as the standard. Then, based on the linear formula obtained in step 9) and the OD value of the prostatic fluid of the patient to be tested in the sample well, the concentration of FGF9 in the sample is calculated.

[0032] The sample to be tested is the prostatic fluid of the subject. The prostatic fluid is collected using a prostate massage method. Specifically, the subject needs to lie on their side with an empty bladder after urination, while medical personnel perform prostate massage to collect the prostatic fluid into a container.

[0033] In this invention, the amino acid sequence of FGF9 is as shown in SEQ ID NO.1, specifically: MPLGEVGNYFGVQDAVPFGN VPVLPVDSPV LLSDHLGQSE AGGLPRGPAV TDLDHLKGIL RRRQLYCRTG FHLEIFPNGTIQGTRKDHSR FGILEFISIA VGLVSIRGVD SGLYLGMNEK GELYGSEKLT QECVFREQFE ENWYNTYSSNLYKHVDTGRR YYVALNKDGT PREGTRTKRH QKFTHFLPRP VDPDKVPELY KDILSQS.

[0034] ELISA (Enzyme-Linked Immunosorbent Assay) is a commonly used immunological assay used to detect the presence and concentration of specific biomolecules such as proteins or antibodies in a sample. Its main principle is to utilize specific antibodies and enzyme-labeled secondary antibodies or substrates to detect target molecules. ELISA is a highly sensitive assay based on immunological reactions, combining the specific reaction of antigens and antibodies with the highly efficient catalytic action of enzymes on substrates.

[0035] Because the antigen-antibody reaction takes place in the wells of a solid-phase carrier—a polystyrene microtiter plate—excess free reactants can be removed by washing after each reagent is added and incubated, thus ensuring the specificity and stability of the test results. In practical applications, various methods and steps can be used depending on the design. These include indirect methods for detecting antibodies, double-antibody sandwich methods for detecting antigens, and antigen competition methods for detecting small molecule antigens or haptens, etc. The most commonly used are the double-antibody sandwich method and the indirect method of ELISA.

[0036] This invention uses the ELISA method to detect the corresponding OD value in the sample, and then calculates the concentration of FGF9 in the sample based on the linear formula obtained from the standard and the OD value of the prostatic fluid of the patient to be tested in the sample well.

[0037] Human FGF9 is a protein commonly used in laboratory studies as a biological agent to activate or inhibit certain biological processes. FGF9 is a member of the fibroblast growth factor (FGF) family and is involved in the regulation of many biological processes, such as embryonic development, tissue repair, and tumorigenesis. In laboratory studies, human FGF9 is commonly used for:

[0038] Inducing cell proliferation and growth: FGF9 can promote the growth and proliferation of certain cell types, so FGF9 can be added in some cell culture experiments to promote cell proliferation and differentiation.

[0039] Cell signal transduction research: FGF9 can bind to Fibroblast Growth Factor Receptor (FGFR) on the cell surface and activate signal transduction pathways, thus it can be used to study cell signal transduction mechanisms.

[0040] In vitro fertilization (IVF): FGF9 is involved in the development and maturation of the reproductive system, and therefore can be used in IVF, such as to promote ovarian cell proliferation and sperm maturation.

[0041] In this invention, human FGF9 is used as a non-invasive predictive biomarker for the progression of benign prostatic hyperplasia (BPH) based on prostatic fluid, and the concentration of FGF9 in prostatic fluid is used as an indicator for judging the progression of BPH.

[0042] This invention provides a highly efficient, non-invasive diagnostic method for benign prostatic hyperplasia (BPH). By comparing prostatic fluid samples with the FGF9 standard group and performing calculations, BPH progression can be accurately predicted non-invasively, reducing patient suffering and financial burden.

[0043] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0044] 1. Non-invasive: The kits and methods provided by this invention do not require invasive procedures such as prostate biopsy, reducing detection risks and discomfort.

[0045] 2. Precision: The concentration of FGF9 in prostatic fluid is related to the progression of BPH, but there is no method in the prior art to detect the concentration of FGF9 in prostatic fluid. This invention can predict whether BPH patients will experience clinical symptom progression by detecting the content of the specific protein FGF9 in prostatic fluid, thus improving the accuracy and reliability of the prediction.

[0046] 3. Simplicity: This method is simple to operate, requiring only the collection of prostate fluid and ELISA testing. It is easy for medical staff to master and does not require complicated equipment or techniques.

[0047] 4. Practicality: This method is suitable for patients with mild to moderate symptoms of benign prostatic hyperplasia (IPSS questionnaire <20). It can quickly and accurately detect BPH and is expected to play an important role in clinical applications.

[0048] 5. Scalability: This method can also be used to detect other BPH-related proteins and diagnose other prostate diseases, demonstrating strong scalability and application prospects.

[0049] Therefore, this invention provides a non-invasive BPH detection method based on prostatic fluid, which has the advantages of non-invasiveness, accuracy, simplicity, practicality and scalability, and is expected to be widely used in the clinical diagnosis and treatment of prostate diseases. Attached Figure Description

[0050] Figure 1 The difference in FGF9 concentration in prostatic fluid between patients with progressive benign prostatic hyperplasia and those with non-progressive benign prostatic hyperplasia was investigated.

[0051] Figure 2The improvement of symptoms after treatment with finasteride + tamsulosin in patients with benign prostatic hyperplasia whose prostatic fluid concentration was <25 pg / ml and ≥25 pg / ml.

[0052] Figure 3 This is a statistical graph showing the diagnostic efficiency of FGF9 levels in prostatic fluid from 311 patients with benign prostatic hyperplasia in predicting symptom progression. Detailed Implementation

[0053] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0054] Example 1

[0055] Prostatic fluid was collected from patients who developed urinary retention symptoms (progressive BPH) after more than 3 months of drug treatment, and from patients with benign prostatic hyperplasia (non-progressive BPH, IPSS < 20) who maintained mild to moderate symptoms after more than 3 months of drug treatment. The prostatic fluid was analyzed using ELISA. Figure 1 This figure illustrates the difference in FGF9 concentration in prostatic fluid between patients with progressive benign prostatic hyperplasia (BPH) and those with non-progressive BPH. The graph shows a clear difference between the FGF9 values ​​in progressive BPH samples (boxes) and those in non-progressive BPH samples (dots). Patients with progressive BPH have significantly higher FGF9 concentrations in their prostatic fluid than those with non-progressive BPH. While the FGF9 value in non-progressive BPH samples is well below 100, the value in progressive BPH samples is significantly higher, often more than twice that of non-progressive BPH samples. This difference can be used as a basic method to determine positive progression of BPH.

[0056] Prostatic fluid samples were collected from patients who presented for the first time with a diagnosis of benign prostatic hyperplasia (BPH) and severe voiding symptoms (IPSS ≥ 20). The concentration of FGF9 in the prostatic fluid was measured using ELISA. Patients were simultaneously treated with a dual therapy regimen of finasteride and tamsulosin (a classic treatment for BPH). Patients with FGF9 levels < 100 pg / ml showed significant symptom improvement after 3 months of treatment. However, patients with FGF9 levels ≥ 100 pg / ml did not show significant symptom improvement after treatment. Figure 2 The study showed the symptom improvement in patients with benign prostatic hyperplasia (BPH) whose prostatic fluid concentrations were <100 pg / ml and ≥100 g / ml after treatment with a combination of finasteride and tamsulosin.

[0057] Figure 2This study is based on a long-term (3-month) medication follow-up record of patients with mild to moderate benign prostatic hyperplasia (BPH). Patients were divided into three groups based on their relative FGF9 levels: the faster the FGF9 level decreased, the more significant the improvement in BPH symptoms. Higher relative FGF9 levels resulted in less effective medication (more severe BPH symptoms). Conversely, lower relative FGF9 levels showed significant long-term improvement with medication (the line showing the most pronounced decrease, the bottom line). Figure 2 This also confirms that the relative value of FGF9 can serve as a measure of the improvement or progression of BPH symptoms.

[0058] Based on the statistical analysis of diagnostic efficiency from a sample of 311 patients, prostatic fluid samples were collected from outpatients diagnosed with BPH, and FGF9 levels were measured using ELISA. Patients were then treated with a dual-drug regimen of finasteride and tamsulosin. Follow-up was conducted for 3 years. Using urinary retention as a positive indicator, ROC curves were plotted, and the area under the curve (AUC) and its 95% CI were calculated to evaluate the predictive value of FGF9 in prostatic fluid for BPH progression. ROC analysis results showed an AUC value as high as 0.911 (sensitivity 0.909, specificity 0.914). Figure 3 This is a statistical graph showing the diagnostic efficiency of FGF9 levels in prostatic fluid from 311 patients with benign prostatic hyperplasia in predicting symptom progression. Figure 3 The statistical analysis of diagnostic efficiency is based on a sample of 311 patients. The AUC diagnostic sensitivity (accuracy) value is 0.911, which is higher than the target of 0.8. This indicates that the concentration of FGF9 in prostatic fluid can be used as an indicator to predict whether patients diagnosed with benign prostatic hyperplasia will experience symptom progression in the future. This method has high diagnostic efficiency and can be used as a non-invasive detection method.

[0059] In this embodiment, a non-invasive method for detecting benign prostatic hyperplasia is also provided, which diagnoses the condition by detecting the content of a specific protein, FGF9, in prostatic fluid.

[0060] The specific process includes:

[0061] The experimental group and control group were designed. The experimental group consisted of patients with mild to moderate symptomatic benign prostatic hyperplasia (IPSS<20), whose prostatic fluid was collected. The standard used to prepare the standard curve was a 500 pg / ml FGF9 standard.

[0062] Then, the concentration of FGF9 in the sample was tested using ELISA. The concentration was calculated based on the OD value measured by a spectrophotometer. Samples with a concentration greater than 100 pg / ml were considered positive.

[0063] The specific method includes the following steps:

[0064] 1. Selecting test subjects: Select patients with mild to moderate symptoms of benign prostatic hyperplasia (IPSS questionnaire <20) and inform them of the testing method and precautions.

[0065] 2. Collection of prostatic fluid from the subject: Prostatic fluid is collected using prostate massage. The specific method is as follows: The subject needs to lie on their side with an empty bladder after urination, while medical staff perform prostate massage to collect the prostatic fluid into a container.

[0066] 3. Prepare the experimental control group: Prepare FGF9 standard at a concentration of 500 pg / ml in the laboratory. FGF9 is a fibroblast growth factor that has been shown to play an important role in the occurrence and development of BPH.

[0067] The specific steps for preparing an FGF9 standard with a concentration of 500 pg / ml are as follows:

[0068] 1) Centrifuge FGF9 before opening the container.

[0069] 2) Weigh 50ug of reagent FGF9 and dissolve it in 0.5ml of physiological saline to prepare a 100ug / ml concentration solution.

[0070] 3) Take 5 μL of the above mixture, dissolve it in 1 mL of physiological saline, shake to mix thoroughly, and prepare a 500 pg / mL FGF9 standard solution.

[0071] 4. Perform ELISA testing: Place the prostate fluid of the subject and the experimental control group into the ELISA kit and perform the test according to the kit's instructions (the steps will be described in detail below). The ELISA kit can be used to measure the concentration of FGF9 in prostate fluid.

[0072] After collecting the prostate fluid from the subjects, the FGF9 antibody solution was prepared according to laboratory methods: the FGF9 antibody was dissolved in 50 mM carbonate coating buffer (pH 9.6) to obtain an FGF9 antibody solution with an antibody concentration of 10 μg / ml. 100 μl / well was added to a 96-well microplate and incubated overnight at 4°C. The next day, the coating solution was discarded, and the plate was washed three times with PBST. 150 μl of 1% (w / v) BSA was added to each well, and the plate was blocked at 37°C for 1 hour. After washing three times with PBST, the plate was ready for use.

[0073] Among them, the FGF9 antibody selected was the product with catalog number LAA036Hu71 from Wuhan Yunclone Technology Co., Ltd.

[0074] The specific ELISA testing procedure after preparation is as follows:

[0075] 1) Sample addition: Set up standard wells, sample wells, and blank wells (i.e., FGF9 concentration of 0). Set up 7 standard wells, and add 100 μL of serially diluted FGF9 standard at concentrations of 500 pg / ml, 250 pg / ml, 125 pg / ml, 62.5 pg / ml, 31.25 pg / ml, 15.625 pg / ml, and 7.8125 pg / ml. Add 100 μL of physiological saline to the blank wells, and add 100 μL of prostatic fluid from the patients to be tested to the remaining wells. Cover the ELISA plate with a membrane and incubate at 37°C for 1 hour.

[0076] 2) Discard the liquid, shake dry, no washing required.

[0077] 3) Add 100 μL of FGF9 antibody solution to each well, cover the ELISA plate with a membrane, and incubate at 37°C for 1 hour.

[0078] 4) Discard the liquid in the wells. Wash each well with 350 μL of PBS for 1-2 minutes, then gently tap the plate on absorbent paper to remove all liquid from the wells. Repeat the washing process 3 times. After the final wash, aspirate or pour out the remaining PBS, invert the plate onto absorbent paper, and blot away any remaining liquid from the wells.

[0079] 5) Add 100 μL of horseradish peroxidase (HRP)-labeled avidin solution to each well, cover the microplate with a membrane, and incubate at 37°C for 30 minutes.

[0080] 6) Discard the liquid in the wells. Wash each well with 350 μL of PBS for 1-2 minutes. Gently tap the plate on absorbent paper to remove all liquid from the wells. Shake dry. Repeat the washing process 5 times. After the last wash, aspirate or pour out the remaining PBS. Invert the plate onto absorbent paper and blot away any remaining liquid in the wells.

[0081] 7) Add 90 μL of TMB chromogenic solution to each well, cover the microplate with a membrane, and incubate at 37°C in the dark (control the reaction time to 10-20 minutes, not exceeding 30 minutes. The reaction can be stopped when the first 3 standard wells show a clear gradient of blue color, and the gradient of the last 4 wells is not obvious).

[0082] 8) Add 50 μL of stop solution (2 mol / L H2SO4) to each well to terminate the reaction. The blue color will immediately turn yellow. The order in which the stop solution is added to each well should be the same as the order in which the substrate solution is added. If uneven coloring occurs, gently shake the ELISA plate to mix the solutions evenly.

[0083] 9) After ensuring that there are no water droplets at the bottom of the ELISA plate and no air bubbles in the wells, immediately measure the optical density (OD value) of each well at a wavelength of 450nm using an ELISA reader. Based on the OD values ​​of different standards in the standard wells and the concentration of FGF9 in the different standards, calculate the relationship between the OD value and the FGF9 concentration to obtain a standard curve and a linear formula. Then, based on the linear formula and the OD value of the prostatic fluid of the patient to be tested in the sample wells, calculate the concentration of FGF9 in the prostatic fluid of the patient to be tested.

[0084] When the calculated concentration of FGF9 in the prostatic fluid of the patient being tested is greater than twice the concentration of the standard, it indicates that the patient is BPH positive.

[0085] By following the steps above, BPH can be accurately detected without invasively affecting the patient's body.

[0086] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. Application of reagents for detecting FGF9 protein content in prostatic fluid in the preparation of kits for predicting clinical progression in patients with benign prostatic hyperplasia after treatment with a combination of finasteride and tamsulosin.

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