CN111518868A

CN111518868A - Application of PGAM5 as diagnosis marker and treatment target of oligoasthenospermia

Info

Publication number: CN111518868A
Application number: CN202010581380.8A
Authority: CN
Inventors: 黄宁; 董锡阳; 张亚卓; 张曼玉; 孙胜楠
Original assignee: Shandong Lifei Biological Industry Co ltd
Current assignee: Shandong Lifei Biological Industry Co ltd
Priority date: 2020-06-23
Filing date: 2020-06-23
Publication date: 2020-08-11

Abstract

The invention belongs to the technical field of oligozoospermia diagnosis, and particularly relates to application of PGAM5 as an oligozoospermia diagnosis marker and a treatment target. The invention is based on a proteomics method, analyzes protein data in normal and oligozoospermia patient samples, detects protein abundance in the samples by taking BSA protein signal intensity as a standard, and obtains differential protein in normal and diseased samples. The method provided by the invention is used for detecting and confirming that PGAM5 is obviously reduced in severe asthenospermia samples, has good reliability as a diagnosis biomarker, and is applied to development of related detection reagents and kits. Meanwhile, the research result is helpful for determining the pathogenesis of oligospermia and developing a target drug for the oligospermia, and has important clinical significance.

Description

Application of PGAM5 as diagnosis marker and treatment target of oligoasthenospermia

Technical Field

The invention belongs to the field of oligozoospermia diagnostic markers, and particularly relates to application of PGAM5 as a diagnostic marker or a treatment target for oligozoospermia, asthenospermia and even severe oligozoospermia in preparation of a diagnostic reagent or a medicament for treating oligozoospermia, asthenospermia and severe oligozoospermia.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

According to the world health organization investigation, 15% of fertile couple have sterility problems, and infertility has become a global medical and social problem affecting human health and social development (Turner, T.T. and J.J.Lysiak, oxidative stress: a common factor in physiological stress.J. Androl,2008.29(5): p.488-98). By definition by the world health organization, asthenospermia is meant less than 32% of the sperm in the semen that move forward. Asthenospermia is one of the major causes of male infertility. Asthenospermia accounts for about 19% of male infertility, while oligospermia or teratospermia accounts for 63% of male infertility. Although many factors such as lifestyle, environmental pollution, varicocele, and infection may cause asthenospermia, which seriously affects the quality of life of people.

Asthenospermia refers to a condition in which less than 50% of the sperm in forward movement (grade a and b) or less than 25% of the sperm in grade a movement in the semen parameters, which is also called asthenospermia. The oligospermia refers to abnormal expression that the number of sperms in one milliliter of semen is less than 2000 ten thousand, and when the density of the sperms is less than or equal to 1 × 10/mL, the oligospermia is diagnosed as severe. The concept of oligospermia and asthenospermia is different, the diagnosis standard is different, but the oligospermia and asthenospermia belong to the quality problem of sperms, and the causes of the diseases are similar. At present, there is no unified understanding on the induction factors and development mechanism of oligospermia, and the factors are generally considered to be related to infection, gene mutation, vitamin deficiency and the like. Aiming at the treatment of oligospermia, no effective medicine exists, the traditional Chinese medicine for nourishing yin and tonifying kidney is mostly adopted for clinical treatment, or the traditional Chinese medicine and western medicine are combined for treatment, and the treatment mode mainly supplements vitamins and tonifies essence, qi and blood deficiency of kidney. In order to overcome the medical problem, the development of related mechanism research is beneficial to determining the pathogenesis of oligospermia and correspondingly researching and developing corresponding target drugs.

PGAM5(The protein phosphate mutase 5) is a serine/threonine protein phosphatase, activation of PGAM5 can lead to linear fragmentation of mitochondria arranged in a string, and is related to apoptosis, necrosis and autophagy of mitochondria, and PGAM5 plays a pivotal role in a pathway of cellular necrosis caused by various causes, and is involved in cellular necrosis caused by, for example, excessive increase of oxygen radicals and excessive leakage of calcium ions, but The mechanism of cellular necrosis caused by PGAM5 is currently poorly understood.

Disclosure of Invention

Based on the research background, the invention aims to screen and research related targets and pathways of oligoasthenospermia based on sperm proteomics, and provide a basis of related mechanisms for diagnosis and treatment of severe oligoasthenospermia. According to the screening result of the invention, the protein content of PGAM5 in the oligozoospermia sample is obviously reduced, namely the relative abundance of PGAM5 in healthy and oligozoospermia samples is different, indicating that PGAM5 is expected to be a diagnosis and treatment target of oligozoospermia.

Based on the research results, the invention provides the following technical scheme:

in a first aspect of the invention, the invention provides the use of PGAM5 as a biomarker in the preparation of a product for diagnosing oligospermia, asthenospermia or severe oligospermia.

The invention detects the relative abundance of protein in sperm samples of patients with normal and severe oligozoospermia by a proteomics method, wherein the expression difference of PGAM5 in the normal sample and the patient sample is obvious, the expression content of PGAM5 in the patient sample is reduced four times, and the obvious correlation between PGAM5 and the diseased condition is proved.

The diagnostic product comprises a diagnostic reagent or a diagnostic kit; the diagnostic reagent includes but is not limited to a reagent for measuring the content of PGAM5 in the sample by an immunoblotting method, an enzyme-linked immunosorbent assay method, a chromatographic analysis method or a genetic engineering method; the diagnostic kit comprises the reagent for detecting the content of PGAM5 in the sample.

In a second aspect of the invention, the invention provides the use of PGAM5 as a biomarker in the preparation of a medicament for the treatment of oligospermia, asthenospermia and severe oligospermia.

In some specific embodiments, the use comprises the use of an active substance that modulates the level of PGAM5 for the preparation of a medicament for the treatment of oligospermia, asthenospermia and severe oligospermia. Specifically, the active substance for regulating the content of the PGAM5 is an agonist of PGAM 5.

In a third aspect of the invention, a kit for diagnosing oligoasthenospermia is provided, wherein the kit comprises a reagent for detecting the content of PGAM5 in a sperm sample.

In a fourth aspect of the present invention, there is provided a medicament for treating oligoasthenospermia, which comprises an active ingredient that modulates the expression level of PGAM 5.

Preferably, the active ingredient is a PGAM5 agonist.

In a fifth aspect of the present invention, there is provided a method for diagnosing asthenospermia, comprising the steps of: extracting a sperm sample of an object to be detected, detecting the detection abundance of PGAM5 protein in the sperm, and judging the patient with oligozoospermia when the detection abundance is less than 0.00591.

The beneficial effects of one or more technical schemes are as follows:

the invention takes the sperm sample of oligoasthenospermia as a research object, detects the relative abundance of the protein in the normal sample and the patient sample, and obtains the differential protein with higher detection value by screening. Through mass spectrometry data analysis, the expression content of PGAM5 in a patient sample is remarkably reduced by 4.1 times, the ROC analysis result shows that the PGAM5 used as a diagnostic marker has a good diagnostic effect, and the false positive probability applied to individual detection is 12.7%, so that the protein is proved to be a marker with higher accuracy and diagnostic value.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a ROC plot of the relative abundance of the PGAM5 protein assay in example 1.

FIG. 2 is a graph comparing the intensity of PGAM5 protein detection in healthy and oligozoospermic samples in example 1.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

As described in the background, the present invention addresses the deficiencies of the prior art by providing the use of PGAM5 as a diagnostic marker and therapeutic target for severe oligoasthenospermia.

In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.

Example 1

Detection method

In this embodiment, a method for detecting oligoasthenospermia markers based on sperm proteomics is provided, wherein the samples used in the following embodiments are all from subsidiary reproductive hospitals of Shandong university, the forward movement of sperm of samples of oligoasthenospermia is between 7.8% and 17.3, and the concentration of sperm is between 3.8 and 14.8 million/mL, and the detection method comprises the following steps:

(1) extracting sperm cell holoprotein: washing a sperm sample by adopting DPBS, adding RIPA lysate into the sperm sample, performing ultrasonic treatment for 1-2 min, putting the sperm sample on ice, incubating the sperm sample for 30min for lysis, centrifuging the sperm sample for 20min at 14,000g, taking supernatant protein clear liquid, and determining the protein concentration in the clear liquid by utilizing a Bradford method.

(2) Enzymolysis of proteins using an ultrafiltration-assisted sample preparation (FASP) -based method: adding 300 μ g protein solution into 10KD ultrafiltration tube, shaking at 600RPM for 1min,14000g, centrifuging for 20min, adding 100 μ L8M Urea, shaking at 600RPM for 1min, and centrifuging at 14000g, centrifuging for 20min, and repeating once; add 100. mu.L of 50mM NH₄HCO₃Shaking at 600RPM for 1min, centrifuging at 14000g for 20min, and repeating twice; the cartridge of the ultrafiltration tube was replaced and 1 fmol/. mu.g BSA (19.929 ng BSA should be added for 300. mu.g protein) was added, and 50mM NH was added₄HCO₃The total solution volume was kept at 50-60uL, trypsin 3-6. mu.g was added and digestion was carried out overnight at 37 ℃.

(3) Desalting the peptide segment after enzymolysis by a ziptip method to prepare a sample mixed peptide segment;

(4) high Performance Liquid Chromatography (HPLC) separation of mixed peptide fragments:

phase A: 2% CH₃CN，98％H₂O，NH₃H₂Adjusting the pH value to 10.0;

phase B: 98% CH₃CN，2％H₂O。

Mu.g of the mixed peptide fragment was dissolved in 80. mu. L A phase, and the sample amount was 80. mu.L. The mixed peptide fragment solution was separated on a C18 XBidge BEH-130 column according to its hydrophilicity, and the mobile phase gradient was set as: 0-3min, 100% phase A, 3-25min, and the flow rate is always kept at 0.7 mL/min. The UV detector is set to be 214nm, liquid phase separation fractions are collected by a 1.5mL centrifuge tube at about 10min according to a chromatogram of sample separation, 1min, namely 0.7mL, is collected in each tube, and finally 90 components are collected, and the 90 components are numbered from 1 to 90 according to the collection time sequence. 90 fractions were dried in a SpeedVac under vacuum. Dissolving 90 components with acetonitrile water mixed liquor (50% acetonitrile and 50% water), and combining the 90 components into 20 components in a crossed manner, namely combining five components 1,21,41,61 and 81 into a component No. 1, combining five components 2,22,42,62 and 82 into a component No. 2, and so on to obtain a component No. 1-20 finally. The 20 fractions finally obtained were dried in a SpeedVac under vacuum and stored at-80 ℃.

(5) Nano flow liquid chromatography-tandem mass spectrometry

Nano-flow liquid chromatography separation: phase A: 10% CH₃CN,90％H₂O, 1% formic acid; phase B: 1% formic acid in 90% CH₃CN,10％H₂And (4) in O.

Each sample was separately dissolved with 13.5. mu. L A phases, the sample introduction volume was 4. mu.L, the nanoflow liquid mass spectrometry system was Orbitrap Elite (Thermo Scientific), the self-made pre-column and analytical column were separately equilibrated with 4. mu. L A phases before sample separation, the specifications of the pre-column and analytical column were, respectively, pre-column (4cm × 150. mu.m I.D., C18 filler particle size 5. mu.m,

) Analytical column (30cm × 75 μm I.D., packed with C18 packing, particle size 3 μm,

dr. maisch GmbH, Germany). After the equilibrium, the sample is loaded on a pre-column under the drive of the phase A, and then the liquid phase separation is carried out under different gradients. The 150min chromatographic gradient varied as follows: 5-32% of mobile phase B for 100min, 32-80% of mobile phase B for 20min, and 80% of mobile phase B for 30min, wherein the flow rate is always kept at 300 nL/min. The sample subjected to nanoflow liquid phase separation directly enters an ESI ion spray source and enters an Orbitrap Elite mass spectrometer for mass spectrum detection.

Collecting mass spectrum data: 350-1800m/z full scan with a resolution of 60,000(m/z 200). In secondary atlas scanning, the activation time was 10ms and the isolation width was 2 m/z. The fragmentation mode is induced-induced dissociation (CID), the normalized collision energy is set to 35%, and the dynamic discharge time is 90 s.

(6) Mass spectrometric data analysis

The obtained mass spectrum RAW data is converted into MGF files by utilizing the Proteome scanner software, and a Uniprot Human database is searched by utilizing Mascot and Maxquant.

The Maxquant search library parameters are, protease: trypsin (Trypsin); at most, the number of missed cleavage sites is allowed: 2, the number of the cells is 2; fixing and modifying: alkylation of cysteine; first order spectral search error: 20 ppm; minimum peptide length: 7 amino acids; peptide, protein and protein modified FDR were set at 1%.

Filtration of protein data from Maxquant contaminating and anti-pool protein information was analyzed as follows: protein data obtained by Maxquant searching are screened by detecting the number of specific peptide segments (unique peptides >3), and then the relative detection abundance of protein in each sample is calculated by taking the BSA protein signal intensity as a standard. The proteins from the normal and diseased groups were screened for their relative abundance by T-test (p <0.05) and ratio (ratio >2) to obtain differential proteins. Then, SPSS software is used for making a difference protein ROC curve, and the area under the curve (AUC) is calculated, so that the diagnostic value of the difference protein ROC curve is judged.

Second, analysis of experimental results

Through mass spectrometry data analysis, 11 pieces of specific peptide segments are detected by the PGAM5 protein, the protein reliability is high, the protein is found to be obviously reduced by 4.1 times in a sample with little asthenospermia through comparison, and the p value is 7.0E-11< <0.05.

The ROC analysis in fig. 1 showed that AUC of PGAM5 protein was 0.940>0.7, indicating a better diagnostic effect, with a sensitivity of 85.7% and specificity of 87.3% at 0.00591 relative assay abundance. When the relative test abundance is less than 0.00591 when the individual test is performed, the patient is judged to be a patient with weak sperm (the false positive rate is 12.7%).

Comparing the relative abundance of the PGAM5 protein detected in the healthy and oligozoospermic samples (fig. 2), it can be seen that this protein is 0.013 in the healthy human samples and 0.003 in the pathological samples (solid line in the figure), and the difference in the number of bits (dotted line) is far, indicating that this protein is increased to a greater extent in the oligozoospermic samples.

In view of the above results, the PGAM5 protein can be used as a potential specific protein marker for asthenospermia, thereby predicting this disorder.

Example 2 validation of clinical assays

The collected 10 groups of oligospermia patient sperm were first subjected to protein extraction and nanofluidic liquid chromatography-tandem mass spectrometry according to the method of example 1, with triplicates per group of data, and then the mass spectrometry data were analyzed. Table 1 shows the results of the analysis, and 10 persons were detected to have oligoasthenospermia by the above method. The accuracy of the group data is up to 100%.

TABLE 1

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The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

The application of PGAM5 as a biomarker in preparing a product for diagnosing oligospermia, asthenospermia or severe oligospermia.
2. Use of PGAM5 as a biomarker for the manufacture of a diagnostic product for oligozoospermia, asthenospermia or severe oligozoospermia according to claim 1, wherein the diagnostic product comprises a diagnostic reagent or a diagnostic kit.
3. The use of PGAM5 as a biomarker for the preparation of diagnostic products for oligospermia, asthenospermia or severe oligospermia according to claim 2, wherein the diagnostic reagents include but are not limited to reagents for measuring the content of PGAM5 in a sample by immunoblotting, enzyme linked immunosorbent assay, chromatography or genetic engineering; the diagnostic kit comprises the reagent for detecting the content of PGAM5 in the sample.
The application of PGAM5 as a biomarker in preparing medicines for treating oligospermia, asthenospermia and severe oligospermia.
5. Use of PGAM5 as a biomarker for the manufacture of a medicament for the treatment of oligospermia, asthenospermia and severe oligospermia according to claim 4, wherein the use comprises the use of an active substance which modulates the content of PGAM5 for the manufacture of a medicament for the treatment of oligospermia, asthenospermia and severe asthenospermia.
6. The use of PGAM5 as a biomarker for the manufacture of a medicament for the treatment of oligospermia, asthenospermia and severe oligospermia according to claim 5, wherein the active substance that modulates the level of PGAM5 is an agonist of PGAM 5.
7. A kit for diagnosing oligoasthenospermia is characterized by comprising a reagent for detecting the content of PGAM5 in a sperm sample.
8. A medicament for the treatment of oligoasthenospermia, which comprises an active ingredient that modulates the level of expression of PGAM 5.
9. The medicament for treating oligoasthenospermia according to claim 8, wherein the active ingredient is a PGAM5 agonist.
10. A method for diagnosing asthenospermia, which comprises the steps of: extracting a sperm sample of an object to be detected, detecting the detection abundance of PGAM5 protein in the sperm, and judging the patient with oligozoospermia when the detection abundance is less than 0.00591.