CN110779946A - Application of biopsy tissue metabolite detection reagent in preparation of prostate cancer diagnosis reagent, kit and qualitative and quantitative analysis method - Google Patents
Application of biopsy tissue metabolite detection reagent in preparation of prostate cancer diagnosis reagent, kit and qualitative and quantitative analysis method Download PDFInfo
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- CN110779946A CN110779946A CN201910898295.1A CN201910898295A CN110779946A CN 110779946 A CN110779946 A CN 110779946A CN 201910898295 A CN201910898295 A CN 201910898295A CN 110779946 A CN110779946 A CN 110779946A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N24/00—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects
- G01N24/08—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects by using nuclear magnetic resonance
Abstract
The provided biopsy tissue metabolite group can be used for diagnosing and distinguishing prostatic hyperplasia and prostatic cancer, has the characteristics of high detection speed, high repeatability and high diagnosis sensitivity, can be used as an auxiliary method of traditional histopathological section analysis, and the detection kit can be used for diagnosing the prostatic cancer, quickens the diagnosis speed and improves the diagnosis convenience and standardization.
Description
Technical Field
The invention specifically relates to the technical field of analytical chemistry and clinical medicine, and particularly relates to an application of a detection reagent for biopsy tissue metabolites in preparation of a prostate cancer diagnostic reagent, a kit and a qualitative and quantitative analysis method.
Background
Prostate cancer is one of the most common cancers in men, the incidence and the death rate of the prostate cancer are respectively positioned at the second and the fifth, 130 new prostate cancer patients are added all over the world in 2018, and the number of new deaths reaches 35.9 ten thousand (1). How to effectively prevent and control and treat the prostate cancer is urgent, but the clinical diagnosis and treatment of the prostate cancer often has the situations of over-diagnosis or misdiagnosis and the like, and is extremely unfavorable for the treatment of the prostate cancer, so the accurate typing diagnosis is very critical for the accurate treatment of the prostate cancer.
The current methods for diagnosing and typing the prostate cancer mainly comprise anus digital diagnosis, prostate specific antigen determination, imaging examination and puncture biopsy under the guidance of rectal ultrasound, but the former three diagnostic methods have poor specificity and great misdiagnosis rate. The tissue sample can be obtained by puncture biopsy under the guidance of rectal ultrasound for pathological section examination, and the method can be used as a gold standard for prostate cancer diagnosis and typing, but a prostate cancer diagnosis marker based on biopsy tissue is still lacking at present. The tissue metabolism marker can assist pathological section diagnosis on one hand, and has the advantages of high analysis speed, strong operability and the like.
Metabonomic analysis technology refers to the application of advanced analysis methods to determine metabolites in biological samples as much as possible, and analyze the specific changes of the metabolites due to diseases or drug actions so as to identify metabolic markers in the processes of disease development or drug treatment. Metabonomics has been primarily tried and applied as a tissue diagnosis technology in the field of prostate cancer research, for example, as early as 2009, researchers such as Sreekumar reported in Nature journal utilize the metabonomics technology to identify sarcosine in urine as a diagnostic marker of prostate cancer. Berez-rambala et al found that amino acids such as branched-chain amino acids, glutamic acid and glycine in urine and metabolites such as arginine and acylcarnitine in blood could well distinguish prostate cancer from prostate hyperplasia patients. Schmidt et al have reported that acylcarnitines, glycerophospholipids, and sphingolipid metabolites in plasma can be used for risk assessment of prostate cancer. Researchers such as Kumar found that alanine, pyruvate and glycine in serum could well distinguish low and high grade prostate cancer. In addition, Lima et al summarize tissue metabolites with significant differences between prostate cancer patients and normal persons, mainly including citric acid, spermine, choline, lactic acid, alanine, and glutamic acid.
Therefore, the content change of the micromolecular metabolites in the biological sample is measured, so that the metabolic change of the prostate cells can be known, and the diagnosis and the differentiation of the prostate cancer patients can be assisted. The method has the advantages of simplicity, convenience and rapidness, and has important significance for clinical diagnosis of the prostate cancer.
Disclosure of Invention
In order to solve the technical defects in the prior art, the invention provides an application of a detection reagent for biopsy tissue metabolites in preparing a prostate cancer diagnosis reagent, a kit and a qualitative and quantitative analysis method, provides an auxiliary diagnosis method based on metabolic analysis for prostate cancer biopsy tissue pathological section analysis, and provides a novel prostate biopsy tissue metabolite combination which can be used as a marker for prostate cancer diagnosis, and can be synchronously analyzed and determined when the metabolic markers exist in biopsy tissues. In addition, the invention provides a metabolic marker group detection kit based on biopsy tissues, which is used for diagnosing and distinguishing prostate cancer patients, accelerating the diagnosis speed and improving the diagnosis convenience.
The technical solution adopted by the invention is as follows: the application of a detection reagent for biopsy tissue metabolites in preparing a prostate cancer diagnostic reagent is disclosed, wherein the biopsy tissue metabolites are one or more of lactic acid, alanine, glycine and choline. The diagnosis includes patients with prostate hyperplasia and prostate cancer.
A kit for diagnosing prostate cancer based on a biopsy tissue metabolite, which comprises a chemical monomer or a mixture of the biopsy tissue metabolite as a standard substance of a diagnostic kit.
The kit also comprises a solvent for extracting the metabolites of the biopsy tissue and reagents required for nuclear magnetic resonance spectroscopy.
The solvent for extracting the biopsy tissue metabolite comprises methanol and chloroform.
The standard substance is above analytical grade. The metabolic markers can be accurately qualitatively and quantitatively analyzed by using the standard substance, so that the detection standardization is facilitated, and the reproducibility is improved.
The reagents required for NMR spectroscopy include deuterium oxide and 2,3, 3-trimethylsilylpropionic acid (TSP).
A qualitative and quantitative analysis method for the metabolites of biopsy tissues, which is characterized in that the metabolites of biopsy tissues are qualitatively and quantitatively analyzed by a nuclear magnetic resonance spectrometer. The method has the characteristics of high analysis speed, high reproducibility, accurate quantification and the like.
The invention has the beneficial effects that: the invention provides an application of a detection reagent for biopsy tissue metabolites in preparing a prostate cancer diagnosis reagent, a kit and a qualitative and quantitative analysis method, the provided biopsy tissue metabolite group can be used for diagnosing and distinguishing prostatic hyperplasia and prostate cancer, has the characteristics of high detection speed, high repeatability and high diagnosis sensitivity, can be used as an auxiliary method of traditional histopathological section analysis, and the detection kit can be used for prostate cancer diagnosis, quickens the diagnosis speed, and improves the diagnosis convenience and standardization.
Drawings
FIG. 1 is a hydrogen spectrum of metabolites from prostate biopsy based on NMR spectroscopy.
FIG. 2 is metabolic marker screening: wherein (A) the difference in metabolic patterns of biopsy tissues of patients with prostate hyperplasia and prostate cancer is examined based on partial least squares discriminant analysis; (B) screening important metabolic markers based on Variable image for the project (VIP).
FIG. 3 is a graph showing the relative content change of metabolic markers in biopsies from patients with prostate hyperplasia and prostate cancer: wherein, (A) lactic acid; (B) alanine; (C) glycine; (D) choline. BPH, prostate hyperplasia patients; HSPC, prostate cancer patients.
FIG. 4 is a Receiver Operating Characteristic (ROC) curve analysis of individual metabolic markers: wherein, (A) lactic acid; (B) alanine; (C) glycine; (D) choline.
FIG. 5 is a Receiver Operating Characteristic (ROC) curve analysis of the metabolic marker combinations.
Detailed Description
According to the evaluation standard of the ROC curve, the area value AUC under the ROC curve is closer to 1, and the diagnosis effect is better. When the single prostate tissue metabolism marker group provided by the invention is used for diagnosing and distinguishing patients with prostatic hyperplasia and prostate cancer, the AUC value is larger than 0.87. The combined application of the four metabolites can realize better diagnosis and differentiation effects, the AUC value is 0.95 (0.902-0.985), and the clinical diagnosis significance is stronger.
The first embodiment is as follows: differential metabolic profiling of biopsy tissue from patients with prostate hyperplasia and prostate cancer
Prostate tissue collection
After the volunteers signed informed consent, biopsy tissue specimens were collected from 39 prostate hyperplasia patients and 64 prostate cancer patients from the affiliated renji hospital of shanghai university of transportation. Prostate hyperplasia and prostate cancer patients are included in the standard reference "prostate cancer diagnostic treatment guidelines" by the chinese urology surgery association 2014 edition. The prostate tissue specimen is collected and then is rapidly stored in a refrigerator at the temperature of minus 80 ℃ for standby.
Analytical procedure
Prostate tissue metabolite extraction
Frozen tissue in 4
oAfter slight thawing under C, a certain amount is weighed and placed in a centrifuge tube, and 4 ml/g precooled methanol and 0.85 ml/g precooled deionized water are added. After mixing, the tissue was disrupted with a hand-held homogenizer and then 2 ml/g precooled chloroform and 2 ml/g precooled deionized water were added. The mixture was shaken vigorously on a shaker for 10 s, placed on ice for 15 min, and then at 4
oC and 10,000
gWas centrifuged for 15 min under the conditions of (1). Carefully sucking the supernatant into a new centrifuge tube, freeze-drying for 24 hours, and dissolving the lyophilized powder in 0.6 ml of heavy water (D)
2O) contains 0.05% of 2,3, 3-trimethylsilylpropionic acid (TSP). Transferring the solution to be detected into a 5 mm nuclear magnetic tube for metabolite detection.
Nuclear magnetic resonance spectrometer analysis
Hydrogen spectra of metabolites from tissue samples were obtained using a 600 MHz NMR spectrometer (AVANCE III, Bruker BioSpin, Rheinstetten, Germany) at 25
oAnd C, collecting under the environment. Metabolite signals were acquired using a standard pre-saturated water signal monopulse sequence (ZGPR). The main acquisition parameter settings include: data points were collected, 156K; relaxation decay time, 4 s; spectral width, 10,822.5 Hz; acquisition time, 3.03 s/scan.
Qualitative and quantitative analysis of metabolites
TSP peaks in all nmr spectra were localized at 0 ppm, followed by manual phase and baseline corrections (Topspin 3.0, Bruker BioSpin, Rheinstetten, Germany). Metabolite signals in the hydrogen spectra were identified in combination with the Chenomx NMR suite software (v 7.0, Chenomx inc., Edmonton, AB, Canada) and the human metabolome Database (HumanMetabolome Database). Figure 1 shows a hydrogen profile and metabolite identification of prostate tissue samples. The metabolite relative content was calculated using the metabolite peak area and the TSP peak area of known concentration.
Metabolic marker identification and screening
Log relative metabolite content values (log)
10) Transformation, and then the difference of metabolic patterns of the prostate hyperplasia patients and the prostate cancer patients is examined by using partial least squares discriminant analysis, as shown in fig. 2A, the obvious metabolic pattern difference exists between two groups of patients. Meanwhile, metabolites which contribute to the classification of two groups of patients are screened by calculating Variable Projection Importance (VIP), and as shown in FIG. 2B, 4 important metabolites, namely lactic acid, alanine, glycine and choline, are screened when the threshold value of the VIP value is set to 1.5. And drawing a standard curve by using the standard substance of the metabolite to quantitatively analyze the corresponding metabolite in the tissue sample. As shown in fig. 3, the relative content of the above 4 important metabolites significantly increased in the biopsy tissue of prostate cancer patients (HSPC) compared to the prostate hyperplasia patients (BPH) by standard quantitative determination, with the average increase greater than 1.5 times. Therefore, the content of the 4 important metabolites is obviously different in biopsy tissues of prostate hyperplasia patients and prostate cancer patients, and the metabolic markers can be used for diagnosing and distinguishing the prostate hyperplasia patients and the prostate cancer patients.
Example two: evaluation of the Performance of 4 potential metabolic markers for diagnosis and differentiation of prostatic hyperplasia and prostate cancer based on the ROC curve
The ROC curve was used to further verify the ability of the 4 potential metabolic markers to be used for diagnosis and differentiation of prostate hyperplasia and prostate cancer. As shown in FIG. 4, when lactic acid, alanine, glycine and choline are used alone to diagnose and differentiate patients with prostatic hyperplasia and prostate cancer, the area under the ROC curve (AUC) values are all greater than 0.872, which has strong clinical diagnosis significance. As shown in FIG. 5, all AUC values were greater than 0.923 when the above 4 metabolic markers were used in combination. It can be seen that the combination of one or more of the above 4 metabolic markers has good performance and clinical diagnosis significance for diagnosing and distinguishing patients with prostatic hyperplasia and prostate cancer.
Example three: preparation of diagnostic kit
The diagnosis kit prepared based on the biopsy tissue metabolism marker provided by the invention is used for assisting the histopathological section examination to carry out rapid diagnosis on prostate cancer, and comprises the following parts:
the standard substance of the metabolic marker comprises lactic acid, alanine, glycine and choline which are respectively packaged.
Biopsy tissue metabolite extraction reagents, including methanol and chloroform, were separately packaged.
Reagents required for NMR spectroscopy analysis, including heavy water and 2,3, 3-trimethylsilylpropionic acid (TSP), were separately packaged.
The detailed flow introduction of sample pretreatment, data pretreatment and analysis.
The skilled person should understand that: although the invention has been described in terms of the above specific embodiments, the inventive concept is not limited thereto and any modification applying the inventive concept is intended to be included within the scope of the patent claims.
The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and embellishments within the scope of the invention may occur to those skilled in the art without departing from the principle of the invention, and are considered to be within the scope of the invention.
Claims (6)
1. The application of the detection reagent for the biopsy tissue metabolites in the preparation of the prostate cancer diagnostic reagent is characterized in that the biopsy tissue metabolites are one or more of lactic acid, alanine, glycine and choline.
2. A kit for diagnosing prostate cancer based on the metabolite of the biopsy tissue of claim 1, comprising a chemical monomer or mixture of the metabolite of the biopsy tissue of claim 1 as a standard for a diagnostic kit.
3. The kit of claim 2, further comprising a solvent for extraction of metabolites from biopsy tissue and reagents required for nmr spectroscopy.
4. The kit for diagnosing prostate cancer according to claim 3, wherein said solvents for extracting metabolites from biopsy tissue include methanol and chloroform.
5. The kit of claim 3, wherein the reagents required for NMR spectroscopy comprise deuterium oxide and 2,3, 3-trimethylsilylpropionic acid (TSP).
6. A method for qualitative and quantitative analysis of a metabolite of a biopsy according to claim 1, wherein the qualitative and quantitative analysis of the metabolite of a biopsy according to claim 1 is performed by nmr spectroscopy.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111796099A (en) * | 2020-06-03 | 2020-10-20 | 温州医科大学 | Use and kit for preparing diagnostic reagent for differentiating hormone sensitivity and castration resistance of prostate cancer, and method and device thereof |
| CN114487215A (en) * | 2022-01-25 | 2022-05-13 | 广州市番禺区中心医院 | Biomarker and kit for detecting benign prostatic hyperplasia |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108344830A (en) * | 2017-01-22 | 2018-07-31 | 中国科学院大连化学物理研究所 | Urine sample composite marker object and detection kit and method for diagnosis of prostate cancer |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108344830A (en) * | 2017-01-22 | 2018-07-31 | 中国科学院大连化学物理研究所 | Urine sample composite marker object and detection kit and method for diagnosis of prostate cancer |
Non-Patent Citations (2)
| Title |
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| ORLA TEAHAN等: "《Metabolic signatures of malignant progression in prostate epithelial cells》", 《THE INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111796099A (en) * | 2020-06-03 | 2020-10-20 | 温州医科大学 | Use and kit for preparing diagnostic reagent for differentiating hormone sensitivity and castration resistance of prostate cancer, and method and device thereof |
| CN114487215A (en) * | 2022-01-25 | 2022-05-13 | 广州市番禺区中心医院 | Biomarker and kit for detecting benign prostatic hyperplasia |
| CN114487215B (en) * | 2022-01-25 | 2024-02-06 | 广州市番禺区中心医院 | Biomarker and kit for detecting benign prostatic hyperplasia |
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