Application of two dipeptides in preparation of kit for diagnosing and distinguishing prostatic cancer and benign prostatic hyperplasia

Abstract

The invention discloses application of two dipeptides in preparation of a kit for diagnosing and distinguishing prostatic cancer and benign prostatic hyperplasia. It is found that the combination of the dipeptide H-Asp-Phe-OH (DF) and the H-Leu-Ala-OH (LA) and the PSA has high diagnostic value for distinguishing the prostatic cancer and the benign prostatic hyperplasia, the accuracy rate of diagnostic distinguishing is high, and the combined diagnostic effect of the dipeptide H-Asp-Phe-OH (DF), the H-Leu-Ala-OH (LA) and the PSA is remarkably superior to the diagnostic effect of singly distinguishing the prostatic cancer and the benign prostatic hyperplasia. The probe has a prospect of being developed into a kit for diagnosing and distinguishing prostatic cancer and benign prostatic hyperplasia.

Description

Technical Field

[0001] The present invention belongs to the field of disease detection and diagnosis, relates to the application of biomarkers in disease diagnosis, and specifically relates to the application of two dipeptides in the preparation of a kit for diagnosing and distinguishing prostate cancer from benign prostatic hyperplasia. Background Art

[0002] Prostate cancer (PCa) is the most common malignant tumor in men. In recent years, with the aging of the population and changes in lifestyle, the incidence of prostate cancer in my country has continued to grow. Since the early symptoms of prostate cancer are not obvious, most prostate cancer patients in my country have already undergone local progression or distant metastasis when diagnosed, resulting in poor prognosis. Although prostate-specific antigen (PSA) has been widely used in the early diagnosis of prostate cancer, patients with non-tumor conditions such as benign prostatic hyperplasia (BPH) will also have elevated plasma PSA levels. Due to its poor specificity, PSA often leads to overdiagnosis and overtreatment, which is difficult to meet clinical needs. Therefore, finding other ideal biomarkers combined with PSA for joint diagnosis and improving the ability of PSA to distinguish between prostate cancer and benign prostatic hyperplasia is the key to promoting the accurate screening of prostate cancer.

[0003] Small peptide metabolites are a class of small molecule metabolites composed of ten or fewer amino acids connected by peptide bonds, with an average molecular weight of less than 1000. With the rapid development of modern biology and nutrition, in the 1960s, scientists discovered that in addition to amino acids, some peptides in the digestion products of proteins in the small intestine can enter the intestinal mucosal cells intact; by the 1990s, Professor Zou Yuandong and many other scientists confirmed that peptides are the main form of protein absorption by the human body. Peptides have a variety of biological activities and are widely involved in physiological processes such as cell proliferation, anti-inflammation, immune regulation and anti-oxidation. Many diseases can lead to changes in the expression quantity and structure of small peptides in urine, which is of great significance for the diagnosis of diseases.

[0004] In recent years, with the rapid development of metabolomics and proteomics, it has been revealed that urine metabolites can be used as potential markers for tumor diagnosis, becoming an important way to find disease diagnostic markers. Summary of the invention

[0005] The present invention aims to overcome the shortcomings of the prior art and provide the use of dipeptides H-Asp-Phe-OH (DF) and H-Leu-Ala-OH (LA) in the preparation of a kit for diagnosing and distinguishing prostate cancer from benign prostatic hyperplasia.

[0006] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0007] The application of dipeptide H-Asp-Phe-OH and H-Leu-Ala-OH combined with prostate specific antigen in the preparation of a kit for diagnosing and distinguishing prostate cancer from prostate hyperplasia.

[0008] Preferably, the kit is a urine detection kit.

[0009] More preferably, the kit contains reagents for detecting H-Asp-Phe-OH, H-Leu-Ala-OH and prostate specific antigen.

[0010] Beneficial effects:

[0011] The present invention finds that the dipeptide H-Asp-Phe-OH (DF) and H-Leu-Ala-OH (LA) combined with prostate-specific antigen (PSA) have a high diagnostic value for distinguishing prostate cancer from benign prostatic hyperplasia, and the accuracy of the diagnostic distinction is high. The combined diagnostic effect of the three is significantly better than the diagnostic effect of PSA alone for distinguishing prostate cancer from benign prostatic hyperplasia, and has the prospect of being developed into a kit for diagnosing and distinguishing prostate cancer from benign prostatic hyperplasia. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The difference in the content of dipeptide DF and LA in urine between patients with prostate cancer and benign prostatic hyperplasia;

[0013] Figure 2 The ROC curve and diagnostic cutoff of dipeptide DF and LA combined with PSA in the validation set 1 for distinguishing between prostate cancer and benign prostatic hyperplasia patients; for comparison, the ROC curve of PSA alone for distinguishing between prostate cancer and benign prostatic hyperplasia patients is also provided;

[0014] Figure 3 To verify the accuracy of dipeptide DF and LA combined with PSA in distinguishing prostate cancer from benign prostatic hyperplasia patients in set 2. DETAILED DESCRIPTION

[0015] The essential contents of the present invention are described in detail below with reference to the accompanying drawings and embodiments, but the protection scope of the present invention is not limited thereto.

[0016] Example 1: Diagnostic performance for prostate cancer and benign prostatic hyperplasia

[0017] 1. Experimental Sample

[0018] A total of 63 prostate cancer patients (PCA) and 58 prostate hyperplasia patients (BPH) from Zhongda Hospital Affiliated to Southeast University were randomly divided into test set samples, validation set 1 samples, and validation set 2 samples. Among them: the test set samples included 14 prostate cancer patients and 22 BPH patients; validation set 1 samples included 24 prostate cancer patients and 21 BPH patients; validation set 2 samples included 25 prostate cancer patients and 15 BPH patients.

[0019] Inclusion criteria: Prostate cancer patients were diagnosed by pathological tissue and had not received radiotherapy or chemotherapy; prostate hyperplasia patients were confirmed to be benign lesions by puncture biopsy.

[0020] Exclusion criteria: ① patients who do not meet the relevant diagnostic criteria for prostate cancer and benign prostatic hyperplasia; ② patients with other prostate diseases, such as prostatitis; ③ patients with other serious liver, kidney, heart, lung and hematopoietic system diseases.

[0021] 2. Experimental Methods

[0022] 1. Collection and storage of urine samples

[0023] A urine catheter was used to collect the subjects' first fresh midstream urine of about 10 to 12 minutes in the morning, which was packaged and stored at -80°C for long term storage.

[0024] 2. Determination of the content of dipeptide DF and LA

[0025] The content of dipeptide DF and LA was determined by conventional methods in the field, as follows: after the dipeptide DF and LA were derivatized with the EOTMBA reagent, the content of dipeptide DF and LA in the urine of the research subjects was detected by Shimadzu LCMS-ultra-high performance liquid phase triple quadrupole mass spectrometer. The specific experimental steps are as follows: accurately aspirate an appropriate amount of urine sample and mix it with the derivatization reagent EOTMBA methanol solution, react at 60°C for 1.5h; detect it by Shimadzu LCMS-8045 ultra-high performance liquid phase triple quadrupole mass spectrometer, and the chromatographic column is ACQUITYUPLC HSS T3 column (2.1×100nm, 1.8μM); the column temperature is controlled at 40°C. The mobile phase system A phase is 10mM, pH=3 ammonium formate aqueous solution, and B phase is pure acetonitrile, and the flow rate is set to 0.35mL / min. The autosampler is controlled at 10°C, and the injection volume is 1μL. The elution gradient was set as follows: 0-1min, 10% B; 1-2min, 10-15% B; 2-4min, 15-20% B; 4-5min, 20%; 5-12min, 20-25% B; 12-16min, 25% B; 16-18min, 25%-30% B; 18-18.1min, 100% B; 18.1-19min, 100% B; and finally returned to the initial gradient balance for 3min. After the test, the ratio of the concentration of the small peptide derivative to be tested to the concentration of the isotope-labeled small peptide derivative was used as the ordinate, and the small peptide concentration was used as the abscissa to draw a standard curve, and finally the dipeptide content in the sample was detected.

[0026] 3. Determination of prostate-specific antigen (PSA) content

[0027] The PSA content in urine was detected using a commercially available PSA detection kit according to the kit instructions.

[0028] 4. Data processing methods

[0029] The content of the target dipeptide was expressed as mean ± standard deviation, and the Student's t-test was used to compare the differences in metabolite content between different groups. The diagnostic efficacy of dipeptides DF and LA for prostate cancer and prostate hyperplasia was evaluated by drawing the receiver operating characteristic (ROC) curve and calculating the area under the curve (AUC) and its 95% confidence interval (CI). DeLong's test was used to compare whether there was a difference in the AUC of different models, so as to comprehensively evaluate the application value of DF and LA in urine combined with PSA as a diagnostic biomarker for prostate cancer.

[0030] The calculation of diagnostic evaluation index is as follows:

[0031]

[0032] Sensitivity is also called the true positive rate, that is, the probability that a person is actually sick and is correctly diagnosed as sick by the diagnostic test. Sensitivity = [a / (a+c)]×100%; specificity is also called the true negative rate, that is, the probability that a person is actually not sick and is correctly diagnosed as not sick by the diagnostic test. Specificity = [d / (b+d)]×100%.

[0033] 3. Experimental Results

[0034] 1. Differences in the levels of dipeptides DF and LA in urine of patients with prostate cancer and benign prostatic hyperplasia

[0035] In the test set, the levels of DF and LA dipeptides in urine of patients with prostate cancer were significantly higher than those of patients with prostate hyperplasia. The results are shown in the following table and Figure 1 shown.

[0036]

[0037] 2. Efficacy of dipeptide DF and LA combined with PSA in distinguishing prostate cancer from benign prostatic hyperplasia

[0038] In validation set 1, the levels of dipeptide DF and LA and PSA in urine of each patient were measured, ROC curve was drawn and the area under the curve (AUC) and its 95% CI were calculated to evaluate the diagnostic efficacy of dipeptide DF and LA combined with PSA for prostate cancer and prostate hyperplasia. Figure 2 As shown. In validation set 1, the AUC value of urine DF and LA dipeptide combined with PSA for distinguishing prostate cancer from benign prostatic hyperplasia patients was as high as 0.932 (sensitivity was 0.929, specificity was 0.818), and the optimal cutoff value was 0.263. The AUC value of PSA alone for distinguishing prostate cancer from benign prostatic hyperplasia patients was only 0.86. Those skilled in the art know that when the AUC is 0.5, it means that the diagnosis is meaningless; when the AUC is 0.5-0.7, it means that the diagnostic accuracy is low; when the AUC is 0.7-0.9, it means that the diagnostic accuracy is moderate; when the AUC is greater than 0.9, it means that the diagnostic accuracy is high. The experimental results show that the diagnostic differentiation efficiency of dipeptide DF and LA combined with PSA for prostate cancer and prostate hyperplasia is significantly better than that of PSA alone for prostate cancer and prostate hyperplasia.

[0039] 5. The accuracy of urine dipeptide DF and LA combined with PSA in distinguishing prostate cancer from benign prostatic hyperplasia

[0040] In validation set 2, the content of dipeptide DF, LA and PSA in the urine of each patient was measured, and the cutoff value of 0.263 obtained in validation set 1 was used as the diagnostic threshold. The prediction of benign prostatic hyperplasia was below this threshold, and the prediction of prostate cancer was above this threshold. The number of samples correctly predicted was divided by the total number of samples, which is the accuracy of the target dipeptide DF and LA combined with PSA in distinguishing prostate cancer from benign prostatic hyperplasia. The results are shown in Figure 3 As shown, in validation set 2, the accuracy of the combined use of dipeptide DF and LA in urine and PSA in distinguishing prostate cancer from benign prostatic hyperplasia patients was 77.5% (31 / 40), close to 80%, with a high accuracy rate. It has considerable development and application prospects in the field of prostate cancer diagnosis.

[0041] In summary, dipeptide DF and LA combined with PSA have high diagnostic value for distinguishing prostate cancer from benign prostatic hyperplasia, and the accuracy of diagnostic distinction is high. The combined diagnostic effect of the three is significantly better than the diagnostic effect of PSA alone in distinguishing prostate cancer from benign prostatic hyperplasia, and has the prospect of being developed into a diagnostic kit for distinguishing prostate cancer from benign prostatic hyperplasia.

[0042] Example 2: Diagnostic kit for distinguishing prostate cancer from prostate hyperplasia

[0043] A urine test kit for diagnosing and differentiating prostate cancer from benign prostatic hyperplasia, comprising reagents for detecting DF and LA dipeptides and prostate-specific antigen.

[0044] The purpose of the above-mentioned embodiments is to specifically introduce the essential content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to the specific embodiments.

Claims

1. Application of dipeptides H-Asp-Phe-OH and H-Leu-Ala-OH in combination with prostate-specific antigen in the preparation of a diagnostic kit for distinguishing prostate cancer from prostate hyperplasia.

2. The use according to claim 1, characterized in that: The kit is a urine detection kit.

3. The use according to claim 1 or 2, characterized in that: The kit contains reagents for detecting H-Asp-Phe-OH, H-Leu-Ala-OH and prostate-specific antigen.

Images