Auxiliary diagnostic kit for prostatitis caused by sex hormone imbalance

Abstract

The present invention relates to the technical field of inflammation detection, and in particular to an auxiliary diagnosis kit for sex hormone imbalance type prostatitis. Clusterin in prostatic fluid can be used as an auxiliary diagnosis marker for sex hormone imbalance type prostatitis, so sex hormone imbalance type prostatitis can be diagnosed by detecting the level of Clusterin in prostatic fluid. In the present invention, anti-human Clusterin antibodies are prepared into a detection kit, which can be used for auxiliary diagnosis of sex hormone imbalance type prostatitis.

Description

Technical Field

[0001] The invention relates to the technical field of inflammation detection, and in particular to an auxiliary diagnosis kit for sex hormone imbalance type prostatitis. Background Art

[0002] Prostatitis can be divided into acute bacterial prostatitis, chronic bacterial prostatitis, chronic prostatitis / chronic pelvic pain syndrome (CP / CPPS) and asymptomatic prostatitis. CP / CPPS is also called chronic non-bacterial prostatitis (CNP). Sex hormone imbalance is the main cause of CNP, and chronic non-bacterial prostatitis caused by sex hormone imbalance is called sex hormone imbalance prostatitis.

[0003] Since the serum estrogen or androgen levels of most CP / CPPS patients are still within the normal range (the normal range of sex hormones is currently wide), relying solely on serum sex hormone levels may not be enough to assess prostate function, increasing the difficulty of diagnosing sex hormone imbalance prostatitis. Currently, the diagnosis of sex hormone imbalance prostatitis relies on the clinical experience of doctors, which may result in diagnostic errors, lack of standardization, or low efficiency.

[0004] Clusterin (CLU) has been used to diagnose prostate cancer, but whether it can be used to diagnose sex hormone imbalance-type prostatitis remains unknown. Summary of the invention

[0005] Based on this, the present invention provides an auxiliary diagnosis kit for sex hormone imbalance type prostatitis, which at least solves one problem in the prior art.

[0006] In a first aspect, the present invention provides an auxiliary diagnostic kit for sex hormone imbalance type prostatitis, which comprises an anti-human Clusterin antibody.

[0007] Clusterin in prostatic fluid can be used as an auxiliary diagnostic marker for sex hormone imbalance prostatitis, so sex hormone imbalance prostatitis can be diagnosed by detecting the level of Clusterin in prostatic fluid. In the present invention, anti-human Clusterin antibodies are prepared into a detection kit, which can be used for auxiliary diagnosis of sex hormone imbalance prostatitis.

[0008] In some optional embodiments, the anti-human Clusterin antibody is coated on an ELISA plate, and the anti-human Clusterin antibody is the antibody with the product number 12289-1-AP of Wuhan Tri-Eagle Biotechnology Co., Ltd.

[0009] In some optional embodiments, the auxiliary diagnostic kit for sex hormone imbalance type prostatitis also includes an enzyme-labeled secondary antibody, a substrate solution, a standard, a diluent, a washing solution, and a termination reaction solution.

[0010] In some optional embodiments, the enzyme-labeled secondary antibody is a horseradish peroxidase-labeled secondary antibody.

[0011] In some optional embodiments, the substrate solution is a 3,3',5,5'-tetramethylbenzidine (TMB) solution containing 1% H2O2.

[0012] In some optional embodiments, the standard product is lyophilized powder of human Clusterin protein.

[0013] In some optional embodiments, the diluent is deionized water containing 0.1% BSA and 0.05% NaN3.

[0014] In some optional embodiments, the washing solution is PBS buffer (phosphate buffered saline) containing 0.05% Tween-20.

[0015] In some optional embodiments, the termination reaction solution is 1 mol / L H2SO4 solution.

[0016] In a second aspect, the present invention provides the use of an antibody against Clusterin in the preparation of an auxiliary diagnostic kit for sex hormone imbalance type prostatitis, wherein the antibody against Clusterin is an anti-human Clusterin antibody.

[0017] Due to the adoption of the above technical solution, the embodiments of the present invention have at least the following beneficial effects:

[0018] (1) Clusterin was found to be a marker for prostatitis caused by sex hormone imbalance. Clusterin antibodies were made into a detection kit, which can effectively assist in the diagnosis of prostatitis caused by sex hormone imbalance.

[0019] (2) Using the auxiliary diagnostic kit for sex hormone imbalance type prostatitis, the concentration of Clusterin in the prostatic fluid (≥2.65 μg / ml) can be used to determine whether patients with sex hormone imbalance type prostatitis have hormone imbalance, thereby reducing the misdiagnosis rate and formulating personalized treatment plans for patients, thereby improving clinical treatment effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a photograph of the prostate appearance of a rat after orchiectomy in an example of the present invention.

[0021] Figure 2 This is the H&E staining result of rat prostate tissue in the example of the present invention.

[0022] Figure 3 The RT-PCR results of the mRNA expression of the main inflammatory factors IL-6, IL-1β and TNF-α in the prostate tissue of rats in the examples of the present invention; in the figure, ** indicates p < 0.01, *** indicates p < 0.001.

[0023] Figure 4 The serum hormone levels of male rats under different treatments in the examples of the present invention; in the figure, *** means p < 0.001.

[0024] Figure 5 The expression of differentially expressed genes (DEGs) in rat prostate tissue in the examples of the present invention.

[0025] Figure 6 This is the GO-P enrichment analysis result of DEGs in the examples of the present invention.

[0026] Figure 7 This is a heat map of differential gene expression related to neural development in the examples of the present invention.

[0027] Figure 8 This is the expression heat map of ion transport-related DEGs in the examples of the present invention.

[0028] Fig. 9 The expression verification results of the main differentially expressed genes Sema4a and Clu in tissues in the examples of the present invention are shown in the figure; in the figure, * indicates p < 0.05, and *** indicates p < 0.001.

[0029] Fig.10 This is the verification result of the expression of Sema4a protein and Clusterin protein in tissues in the examples of the present invention.

[0030] Fig.11 This is a statistical graph of the protein level of Clusterin in the prostatic fluid of 39 patients with prostatitis collected in the examples of the present invention; in the figure, *** indicates p < 0.001.

[0031] Fig.12 This is a statistical graph of the protein level of Sema4a in the prostatic fluid of 39 patients with prostatitis collected in the examples of the present invention.

[0032] Fig.13 This is the ROC curve for Clusterin diagnosis in the embodiment of the present invention. DETAILED DESCRIPTION

[0033] The following is a clear and complete description of the concept of the present invention and the technical effects produced, so as to fully explain the purpose, scheme and effect of the present invention.

[0034] The present invention aims to explore biomarkers for diagnosing sex hormone imbalance type prostatitis. For this purpose, a CNP rat model with sex hormone imbalance was established to verify the expression and diagnostic effect of biomarkers in sex hormone imbalance CNP.

[0035] In one embodiment of the present invention, 20 male Sprague-Dawley rats (purchased from Hunan Slake Jingda Experimental Animal Co., Ltd.) aged between 3 and 4 months and weighing between 250 and 350 grams were used. Under pathogen-free conditions, the rats were placed in a constant temperature room with an ambient temperature of 22±2°C, a humidity of 55±5%, good ventilation, and a light cycle of 08:00 to 20:00. The rats were randomly divided into two groups: a sham operation group (SN group, n=10) and a model group (SP group, n=10). After the SN group rats underwent surgical incision, the incision was sutured; after the SP group rats had bilateral testicular ligation, estradiol was subcutaneously injected daily at a dose of 0.25 mg / kg. One month later, the rats were euthanized. Serum samples of the rats were collected, and a small portion of the prostate tissue (ventral lobe) was fixed in 4% paraformaldehyde for pathological examination and IHC detection, and the remaining prostate tissue samples were frozen at -80°C for subsequent analysis.

[0036] The prostate tissues of rats were fixed with paraformaldehyde for 24 hours and then made into paraffin-embedded sections (about 5 μm). The sections were stained with hematoxylin and eosin, and the infiltration of inflammatory cells in the prostate of each group was observed under an optical microscope.

[0037] After dewaxing and hydration, paraffin-embedded sections were heated and boiled in a microwave oven for antigen retrieval in citrate buffer (pH 6.0, 10 mM). Prostate tissue sections were incubated with Clusterin antibody (12289-1-AP, Wuhan Sanying Biotechnology Co., Ltd.) and sema4A antibody (27359-1-AP, Wuhan Sanying Biotechnology Co., Ltd.) at 4°C overnight, and then incubated with biotinylated goat anti-rabbit IgG (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) at 37°C. The immune complex was detected with DAB (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.), and then restained with hematoxylin to observe the sections. Testosterone and estradiol levels in rat plasma were measured using ELISA kits (R&D Systems, USA).

[0038] like Figure 1 As shown in the figure, the seminal vesicle and prostate tissues of the rats in the SP group showed obvious atrophy, and the volume was reduced by about 3-4 times compared with the SN group. The tissues of the rats in the SP group were dark red with blurred stripes.

[0039] like Figure 2As shown in the figure, H&E staining results showed that there was inflammatory cell infiltration in the prostate tissue of rats in the SP group. A large number of inflammatory cell infiltrations were found in the glandular cavity and between the glands, with a wide range and high degree. Abnormal lymph nodes can also be found in the glandular cavity (indicated by the arrow in the figure).

[0040] like Figure 3 As shown in the figure, RT-PCR results showed that the inflammatory factors in the prostate tissue of rats in the SP group were upregulated, and the expression of IL-6 was about 1.7 times that of the SN group (p=0.0007); the expression level of IL-1β increased by about 1 times compared with the SN group (p=0.0063); the expression level of TNF-α also increased significantly, which was 5 times that of the SN group (p=0.0041). The above results indicate that the prostatitis model was successfully established.

[0041] In order to better observe the changes in sex hormones in rats, a ligated group (SN (ligated) group) was prepared. The rats in the SN (ligated) group only had their testicles ligated without injecting estradiol. The androgen level in normal rats is 331ng / dl, and the serum estrogen level is 1.668pg / ml. Figure 4 As shown in the figure, compared with the SN group, the serum testosterone levels of rats in the SP group and the SN (ligated) group decreased by about 65 times (p<0.001). In addition, the estradiol levels in the SP group and the SN ligated group increased significantly, especially in the SP group (p<0.001). In terms of the testosterone / estradiol ratio (T / E2), both the SP group and the SN ligated group decreased by about 200 times (p<0.001), indicating that the sex hormone imbalance prostatitis model was successfully established.

[0042] To better understand the changes in prostate-related gene expression in rats after ligation, RNA sequencing analysis was performed on rat prostate samples (ventral lobe). Prostate mRNA was extracted from rat prostate tissue and converted into double-stranded complementary DNA. The resulting products were then sequenced on a machine, and the resulting data were uploaded to a database for analysis, and analyzed and plotted using the R language pheatmap package. A heat map was then generated to show the expression of gene clusters in the samples, with the Q value set to 0.05 or the FDR set to 0.001. In addition, the R language DESeq2 package (version 1.4) was used for further analysis. A hypergeometric distribution-based test was used to determine differentially expressed genes associated with phenotypic variation, and the Phyper function in the R language was used to perform enrichment analysis on GO data (GeneOntology, http: / / www.geneontology.org) and KEGG genetic variation data (https: / / www.kegg.jp) to study their functions; only candidate genes with a Q value of 0.05 were considered significantly enriched.

[0043] like Figure 5 As shown in the figure, a total of 18,556 DEGs were found between the SP and SN groups (10,159 upregulated DEGs and 8,397 downregulated DEGs). Some significantly differentially expressed genes, such as Andpro, were only expressed in rats. Therefore, |log2(SP / SN)|>2, p<0.05, q<0.001 were used to screen DEGs (a total of 2,003 DEGs were screened) to explore their functions. Among them, Sema4A and Clusterin became key differentially expressed proteins, and their roles in neuroinflammation and immune regulation may promote the occurrence and development of prostatitis associated with sex hormone imbalance ( Figure 6 ).

[0044] like Figure 7 and Figure 8 As shown, Sema4a and Clusterin were significantly upregulated in the SP group, highlighting their possible involvement in the pathological process.

[0045] Total RNA was extracted from rat prostate tissues, and RT-PCR was performed to verify the expression and distribution of Sema4a and Clusterin. Total RNA was obtained from prostate samples using TransZol Up (Beijing Tiangen Biochemical Technology Co., Ltd.) according to the manufacturer's protocol and then converted into cDNA. Real-time quantitative PCR (qPCR) was performed using the SYBR kit (Beijing Quanshijin Biotechnology Co., Ltd.) on the QuantStudio™ 6 Flex Real-Time PCR System (Thermo Fisher Scientific, USA) to detect the mRNA expression levels of major inflammatory factors and significantly differentially expressed genes. GAPDH was used as a normalization control, and 2 -ΔΔCt The relative mRNA expression was calculated by qPCR. The primer sequences used for qPCR are as follows:

[0046] GAPDH: forward primer was 5′-GCATCTTCTTGTGCAGTGCC-3′, and reverse primer was 5′-TACGGCCAAATCCGTTCACA-3′;

[0047] IL-1β: forward primer was 5′-TTGAGTCTGCACAGTTCCCC-3′, and reverse primer was 5′-TCCTGGGGAAGGCATTAGGA-3′;

[0048] TNF-α: forward primer was 5′-GGCTTTCGGAACTCACTGGA-3′, and reverse primer was 5′-GGGAACAGTCTGGGAAGCTC-3′;

[0049] IL-6: forward primer was 5′-GACCAAGACCATCCAACT-3′, reverse primer was 5′-TAGGTTTGCCGAGTAGAC-3′;

[0050] Sema4A: forward primer was 5′-GGACCCCCTTCCTTGCCTTG-3′, and reverse primer was 5′-CTCCCGCATGGTACTTGACTC-3′;

[0051] Cluterin: the forward primer was 5′-CTCTCAGCAATCGGGTGAAGTA-3′ and the reverse primer was 5′-TGGCCTCTTTGGAGTCTGGA-3′.

[0052] like Fig. 9 As shown, the results of RT-PCR analysis showed that the expression of Sema4a and Clusterin in the prostate tissue of the SP group rats was upregulated compared with that of the SN group rats. The Sema4a level in the SP group was twice that of the SN group, while the Clusterin level in the SP group was about 25 times that of the SN group.

[0053] Immunohistochemical analysis showed that the expression of Sema4A and Clusterin was increased in the prostate tissue of the SP group compared with the SN group. These two genes were diffusely expressed throughout the prostate tissue of SP, and strong staining was observed in more than 50% of the cells, especially in epithelial cells and inflammatory cells ( Fig.10 ).

[0054] In addition, 39 patients diagnosed with CP / CPPS were selected from the First Affiliated Hospital of Gannan Medical University, and prostate fluid samples were collected through prostate massage. All patients signed informed consent and agreed to participate in the relevant research. Patients who met the following criteria were included in the study: (1) men clinically diagnosed with chronic prostatitis / chronic pelvic pain syndrome (CP / CPPS) according to the National Institutes of Health (NIH) classification system; (2) symptoms lasting at least 3 months, manifested as dysuria, perineal discomfort or pain; (3) agreed to undergo serum sex hormone testing, including testosterone and estradiol levels, and calculated the testosterone:estradiol ratio; (4) provided written informed consent. Exclusion criteria were as follows: (1) acute urinary tract infection or acute prostatitis at enrollment; (2) known prostate disease such as prostate hyperplasia / prostate cancer, or other systemic diseases that affect hormone levels; (3) history of endocrine disease, including thyroid dysfunction or Cushing's syndrome; (4) use of hormone therapy or drugs that affect the hypothalamus-pituitary-gonadal axis in the past 3 months; (5) previous surgery that affects prostate or gonadal function, such as prostatectomy or orchiectomy; (6) severe mental illness that may hinder the accuracy of symptom reporting; (7) participating in other clinical trials that may lead to overlapping results. The patients were divided into two groups: hormone balance group and hormone imbalance group. Specifically, patients with a ratio of testosterone (T) to estradiol (E2) in prostatic fluid (T / E2) greater than 20 or less than 10 were classified as hormone imbalance group, and others were classified as hormone balance group.

[0055] Clusterin and Sema4a concentrations in the prostatic fluid of patients were also quantitatively detected using ELISA kits produced by R&D Systems (Cat. No. DCLU00) and Ray Biotec (Cat. No. ELH-SEMA4A-1), respectively. Given the high viscosity of prostatic fluid, the samples were diluted 100-fold and 10-fold, respectively, to meet the detection range specified in the test instructions.

[0056] Statistical analysis was performed using SPSS 20.0 (IBM, USA), and the results were expressed as mean ± standard error of the mean (SEM). The normality of the data was tested using the Kolmogorov-Smirnov test. For data sets that met the normal distribution, the Student's t-test was used for statistical comparison. If the normality assumption was violated, the Mann-Whitney test was used. Statistical significance was set at p < 0.05. Receiver operating characteristic curves (ROC) were generated to evaluate the diagnostic performance of Clusterin. The area under the curve (AUC) was calculated, with an AUC value of 0.5 indicating no discrimination and an AUC value of 1.0 indicating complete discrimination. Sensitivity and specificity were derived from the ROC curve, and the Youden index was used to determine the optimal cutoff point. All statistical tests were two-sided.

[0057] As shown in Table 1 and Fig.11 As shown, the Clusterin level of patients in the hormone imbalance group was significantly increased, about five times that of patients in the hormone balanced group (p<0.001).

[0058] Table 1 Patient information table

[0059]

[0060] The level of Sema4a was higher in the hormone imbalance group, but there was no significant difference ( Fig.12 ).

[0061] In addition, if Fig.13 As shown in the figure, the area under the ROC curve of Clusterin was 0.922±0.048 (p<0.001), and the critical value of Clusterin was set at 2.65 μg / ml. The sensitivity of this critical value was 100%, and the specificity was 82.4%, indicating that the level of Clusterin in prostatic fluid of 2.65μg / ml can be used as an effective diagnostic criterion for hormone imbalance CNP (p<0.001).

[0062] In another embodiment of the present invention, a diagnostic kit for prostatitis of sex hormone imbalance type is provided. The kit is used for the diagnostic of prostatitis of sex hormone imbalance type, and mainly detects the biomarker Clusterin in prostatic fluid. The kit includes the following components:

[0063] ELISA plate coated with anti-human Clusterin antibody: Anti-human Clusterin antibody is used to specifically bind Clusterin, 0.2 mg of anti-human Clusterin antibody is frozen and stored after antibody coating, incubation, blocking, washing, lyophilization;

[0064] Enzyme-labeled secondary antibody: The secondary antibody that binds to the anti-human Clusterin antibody is responsible for signal amplification; the enzyme-labeled secondary antibody is horseradish peroxidase-labeled secondary antibody, with a concentration of 1 mg / mL and a content of 0.5 mL;

[0065] Substrate solution: used to react with enzyme-labeled secondary antibody to produce a measurable signal (such as color change); the substrate solution is 3,3',5,5'-tetramethylbenzidine (TMB) solution containing 1% H2O2, with a concentration of 3 mg / mL and a content of 3 mL;

[0066] Standards: including human Clusterin protein lyophilized powder, used to establish a standard curve to ensure the accuracy of the measurement results, with a content of 100 μg;

[0067] Diluent: used to dilute samples and standards; the diluent is deionized water containing 0.1% BSA and 0.05% NaN3, a total of 50 mL;

[0068] Washing solution: used to maintain the stability of the reaction system and the activity of the antibody; the washing solution is PBS buffer containing 0.05% Tween-20;

[0069] Termination reaction solution: used to terminate the TMB reaction. The termination reaction solution is H2SO4 solution with a concentration of 1 mol / L and a content of 20 ml.

[0070] The process of using the auxiliary diagnostic kit for sex hormone imbalance prostatitis for testing can include the following steps:

[0071] Take a sample of prostatic fluid and dilute it in a certain ratio;

[0072] The diluted samples were added to the coated well plate to react with anti-human Clusterin antibodies to bind to the target biomarker;

[0073] Use wash buffer to remove nonspecific binders;

[0074] Add enzyme-labeled secondary antibodies to bind to the markers and generate signals;

[0075] Adding the substrate solution produces a measurable color change, and the absorbance at 450 nm is detected using an enzyme reader;

[0076] The concentration of Clusterin is quantified by detecting the color change of the reaction (the absorbance can be measured by a microplate reader) to obtain the diagnostic result.

[0077] In other embodiments, the auxiliary diagnostic kit for sex hormone imbalance type prostatitis includes anti-human Sema4a antibodies and / or anti-human PSA antibodies, which can improve the accuracy of detecting sex hormone imbalance type prostatitis.

[0078] The above is only a preferred embodiment of the present invention. The present invention is not limited to the above implementation. As long as the technical effect of the present invention is achieved by the same or equivalent means, it should belong to the protection scope of the present invention. Within the protection scope of the present invention, its technical scheme and / or implementation method can have various modifications and changes.

Claims

1. Application of Clusterin antibody in the preparation of an auxiliary diagnostic kit for sex hormone imbalance type prostatitis, characterized in that: The antibody against Clusterin is an anti-human Clusterin antibody.

2. The use according to claim 1, characterized in that: The anti-human Clusterin antibody was coated on an ELISA plate. The anti-human Clusterin antibody was the antibody with the catalog number 12289-1-AP from Wuhan Sanying Biotechnology Co., Ltd.

3. The use according to claim 1, characterized in that: The auxiliary diagnostic kit for sex hormone imbalance type prostatitis includes enzyme-labeled secondary antibody, substrate solution, standard substance, diluent, washing solution and termination reaction solution.

4. The use according to claim 3, characterized in that: The enzyme-labeled secondary antibody was horseradish peroxidase-labeled secondary antibody.

5. The use according to claim 3, characterized in that: The substrate solution was a 3,3',5,5'-tetramethylbenzidine solution containing 1% H2O2.

6. The use according to claim 3, characterized in that: The standard product is lyophilized powder of human Clusterin protein.

7. The use according to claim 3, characterized in that: The diluent was deionized water containing 0.1% BSA and 0.05% NaN3.

8. The use according to claim 3, characterized in that: The washing solution was PBS buffer containing 0.05% Tween-20.

9. The use according to claim 3, characterized in that: The reaction was terminated with 1 mol / L H2SO4 solution.

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