CN114480618A - Marker for predicting male reproductive function decline and application thereof - Google Patents
Marker for predicting male reproductive function decline and application thereof Download PDFInfo
- Publication number
- CN114480618A CN114480618A CN202210031782.XA CN202210031782A CN114480618A CN 114480618 A CN114480618 A CN 114480618A CN 202210031782 A CN202210031782 A CN 202210031782A CN 114480618 A CN114480618 A CN 114480618A
- Authority
- CN
- China
- Prior art keywords
- pla2g2d
- reproductive function
- detecting
- male reproductive
- predicting
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 230000001850 reproductive effect Effects 0.000 title claims abstract description 38
- 230000008717 functional decline Effects 0.000 title claims abstract description 24
- 239000003550 marker Substances 0.000 title claims abstract description 12
- 102100026828 Group IID secretory phospholipase A2 Human genes 0.000 claims abstract description 35
- 101000983153 Homo sapiens Group IID secretory phospholipase A2 Proteins 0.000 claims abstract description 35
- 210000000582 semen Anatomy 0.000 claims abstract description 35
- 238000000034 method Methods 0.000 claims abstract description 19
- 101150039930 PLA2G2D gene Proteins 0.000 claims abstract description 5
- 230000014509 gene expression Effects 0.000 claims description 15
- 239000003153 chemical reaction reagent Substances 0.000 claims description 14
- 230000007423 decrease Effects 0.000 claims description 13
- 108020004999 messenger RNA Proteins 0.000 claims description 10
- 102000007469 Actins Human genes 0.000 claims description 9
- 108010085238 Actins Proteins 0.000 claims description 9
- 238000011529 RT qPCR Methods 0.000 claims description 6
- 210000001124 body fluid Anatomy 0.000 claims description 6
- 238000002965 ELISA Methods 0.000 claims description 5
- 239000010839 body fluid Substances 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims 5
- 238000004519 manufacturing process Methods 0.000 claims 1
- 201000010063 epididymitis Diseases 0.000 abstract description 32
- 230000032683 aging Effects 0.000 abstract description 6
- 230000002381 testicular Effects 0.000 abstract description 6
- 230000021595 spermatogenesis Effects 0.000 abstract description 5
- 238000001574 biopsy Methods 0.000 abstract description 2
- 230000035800 maturation Effects 0.000 abstract description 2
- 210000000918 epididymis Anatomy 0.000 description 15
- 210000001550 testis Anatomy 0.000 description 11
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 9
- 241000699670 Mus sp. Species 0.000 description 8
- 238000001514 detection method Methods 0.000 description 8
- 230000035558 fertility Effects 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 238000012163 sequencing technique Methods 0.000 description 6
- 239000000243 solution Substances 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 4
- 210000002149 gonad Anatomy 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 230000000920 spermatogeneic effect Effects 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 101100028420 Mus musculus Pla2g2d gene Proteins 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 230000002710 gonadal effect Effects 0.000 description 3
- 229940088597 hormone Drugs 0.000 description 3
- 239000005556 hormone Substances 0.000 description 3
- 239000006166 lysate Substances 0.000 description 3
- 238000002205 phenol-chloroform extraction Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000010839 reverse transcription Methods 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 102000012673 Follicle Stimulating Hormone Human genes 0.000 description 2
- 108010079345 Follicle Stimulating Hormone Proteins 0.000 description 2
- 102000009151 Luteinizing Hormone Human genes 0.000 description 2
- 108010073521 Luteinizing Hormone Proteins 0.000 description 2
- 230000006044 T cell activation Effects 0.000 description 2
- MUMGGOZAMZWBJJ-DYKIIFRCSA-N Testostosterone Chemical compound O=C1CC[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 MUMGGOZAMZWBJJ-DYKIIFRCSA-N 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 230000007850 degeneration Effects 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 229940028334 follicle stimulating hormone Drugs 0.000 description 2
- 238000013467 fragmentation Methods 0.000 description 2
- 238000006062 fragmentation reaction Methods 0.000 description 2
- 230000000977 initiatory effect Effects 0.000 description 2
- 229940040129 luteinizing hormone Drugs 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 102000042759 phospholipase A2 family Human genes 0.000 description 2
- 108091082051 phospholipase A2 family Proteins 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 238000012257 pre-denaturation Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000011534 wash buffer Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 1
- 208000006545 Chronic Obstructive Pulmonary Disease Diseases 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000021588 free fatty acids Nutrition 0.000 description 1
- 238000010230 functional analysis Methods 0.000 description 1
- 150000002327 glycerophospholipids Chemical class 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 108010067479 inhibin B Proteins 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000013188 needle biopsy Methods 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 229960003604 testosterone Drugs 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/34—Genitourinary disorders
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Urology & Nephrology (AREA)
- Microbiology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Pathology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Cell Biology (AREA)
- Biophysics (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a marker for predicting male reproductive function decline and application thereof, and relates to the technical field of biology. The invention relates to a marker for predicting male reproductive function decline, which comprises a PLA2G2D gene. The male reproductive function decline is predicted by detecting PLA2G2D in seminal plasma, the seminal plasma is easier to obtain relative to other tissues, and compared with testicular biopsy and puncture, the method belongs to non-invasive examination, and relieves the pain of a testee; the PLA2G2D in seminal plasma expresses and rises in early stage of reproductive function aging and gives an early warning in time, so that the reproductive function can be prevented and controlled from declining as soon as possible, and the hysteresis of testicular spermatogenesis and epididymal sperm maturation is avoided.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a marker for predicting male reproductive function decline and application thereof.
Background
With the gradual delay of the reproductive age of male in China, the problem of the decline of the fertility of old male needs to be paid high attention. The aging of gonads (testis, epididymis and the like) caused by factors such as aging, diseases and the like is a core pathogenesis of the decline of the fertility of the old men, the aging of the gonads is recognized at an early stage, the degeneration of the spermatogenic function of the gonads is prevented, and the method is important for preventing and treating the decline of the fertility of the old men. However, at present, a reliable early warning evaluation index for fertility decline of old men, namely a method for predicting male reproductive function decline is lacked. At present, the markers clinically used for evaluating male spermatogenic function mainly come from blood and semen, including semen convention, sperm acrosome enzyme, sperm DNA fragmentation rate, serum hormone and the like.
Serum hormone indexes include Follicle Stimulating Hormone (FSH), Luteinizing Hormone (LH), testosterone, inhibin B, etc. In practical clinical application, the indexes have limited effect on predicting male reproductive function decline particularly at an early stage. The reason is that the hormone in blood can only indirectly reflect the function of testis and can not reflect the function of epididymis; the generation of testicular sperms and the maturity of epididymal sperms have hysteresis, and the conventional semen inspection results only can reflect the quality of the spermatogenic quality in a certain past semen period, so that early warning cannot be realized; testis and epididymis tissue samples can be obtained through needle biopsy, but the extensive clinical application is difficult to realize as an invasive examination means. Therefore, the research on a new method for predicting the male reproductive function decline is a clinical problem which needs to be solved at present and is also a problem which needs to be solved for preventing and treating the fertility decline of old men.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a marker for predicting male reproductive function decline and application thereof. Several studies have shown that as men age, their reproductive function gradually declines, especially as the sperm concentration begins to decline slowly after age 40 and sharply after age 55. In the early reproductive function decline stage of old men, various clinical indexes such as semen routine examination and the like are usually in a normal range. Early identification of male gonadal deterioration and prevention of spermatogenic function degeneration are important for preventing and treating fertility decline of old men. According to the invention, a large number of experiments show that seminal plasma PLA2G2D can be expressed and early warned in time in the early stage of male reproductive function aging, has sufficient scientificity and practicability, and can fill the gap that the current lack of reliable early warning and evaluation indexes for the decline of fertility of old men is provided.
In order to achieve the purpose, the invention adopts the technical scheme that: a marker for predicting male reproductive function decline comprising the PLA2G2D gene. It was shown that PLA2G2D encodes a secreted member of the phospholipase a2 family, and that members of the phospholipase a2 family hydrolyse the sn-2 fatty acid ester bond of glycerophospholipids, producing lysophospholipids and free fatty acids. The gene may be involved in inflammation and immune responses, as well as weight loss associated with chronic obstructive pulmonary disease. Through a great deal of research, the inventor of the application finds that the expression of seminal plasma PLA2G2D is gradually increased along with the increase of the male age and the decline of the reproductive function, and the PLA2G2D can be used as a marker for predicting the decline of the male reproductive function.
The invention also provides application of the PLA2G2D in preparing a detection reagent for predicting male reproductive function decline.
As a preferred embodiment of the use of the invention, the reagent comprises a reagent for detecting the mRNA expression of PLA2G2D in a body fluid.
As a preferred embodiment of the application of the invention, the reagent comprises a reagent for detecting the expression of PLA2G2D protein in body fluid.
As a preferred embodiment of the use according to the invention, the body fluid is seminal plasma. Research shows that the testicular spermatogenesis and epididymis spermatogenesis have hysteresis, and the semen routine and other series of examination results can only reflect the quality of spermatogenesis in a certain past sperm cycle, and early warning cannot be realized. The invention detects the expression condition of PLA2G2D in seminal plasma, and the detection result is more accurate.
The invention also provides application of the reagent for detecting the seminal plasma PLA2G2D protein by an ELISA method in preparing products for predicting male reproductive function decline.
The invention also provides a kit for predicting male reproductive function decline, which comprises a reagent for detecting the seminal plasma PLA2G2D protein by an ELISA method.
The invention also provides application of a reagent for detecting the primers of the PLA2G2D by using a qRT-PCR method in preparation of products for predicting male reproductive function decline.
The invention also provides a kit for predicting male reproductive function decline, which comprises a primer for detecting PLA2G2D and a primer for detecting beta-Actin by adopting a qRT-PCR method.
As a preferred embodiment of the kit, the sequence of the primer for detecting PLA2G2D is shown as SEQ ID NO.1 and SEQ ID NO. 2; the sequences of the primers for detecting the beta-Actin are shown as SEQ ID NO.3 and SEQ ID NO. 4.
The invention has the beneficial effects that: the marker for predicting male reproductive function decline and the application thereof provided by the invention are used for predicting male reproductive function decline by detecting PLA2G2D in seminal plasma, wherein the seminal plasma is easier to obtain relative to other tissues, and compared with testicular biopsy and puncture, the marker belongs to noninvasive examination and relieves the pain of a testee; the PLA2G2D in seminal plasma expresses and rises in early stage of reproductive function aging and gives an early warning in time, so that the reproductive function can be prevented and controlled from declining as soon as possible, and the hysteresis of testicular spermatogenesis and epididymal sperm maturation is avoided.
Drawings
FIG. 1 Is a thermal map of the differential mRNA of common transcriptomics sequencing of 3-and 21-month-old male mouse testis (Te), epididymal head (Ca), epididymis body (Co), epididymal tail (Cd).
FIG. 2 Is a diagram of the overall functional analysis of the mRNA of the epididymal initiation part (Is), epididymal head (Ca), epididymis body (Co) and epididymal tail (Cd) of male mice testis (Te) of 3 and 21 months old by sequencing.
FIG. 3 is a graph showing the expression of PLA2G2D in seminal plasma free mRNA of healthy men of different ages.
FIG. 4 is a graph showing the expression of PLA2G2D protein levels in seminal plasma of healthy men of different ages.
Detailed Description
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Example 1
In this example, the differential mRNA of gonads of 3-and 21-month-old male mice, including 4 regions of testis (Te) and epididymis, i.e., initiation (Is), epididymis head (Ca), epididymis (Co), and epididymis tail (Cd), was analyzed for overall function by ordinary transcriptomic sequencing and PCR.
(1) The experimental method comprises the following steps: a. general transcriptomics sequencing methods: respectively taking tissues of each part of testis and epididymis of male mice with the age of 3 months and 21 months, adding RNA lysate, extracting total RNA of the tissues by a phenol chloroform extraction method, and constructing an mRNA sequencing library, wherein the library construction step comprises the processes of mRNA enrichment and fragmentation, mRNA reverse transcription, joint addition, PCR enrichment and the like. And detecting by a sequencer to obtain original data, performing genome comparison after sample quality control and filtration, calculating the gene expression quantity of each sample, and performing annotation to obtain differentially expressed gene data. b, PCR method: respectively taking 3-month-old and 21-month-old male mice, taking tissues of each part of testis and epididymis, adding RNA lysate, extracting total RNA of the tissues by a phenol chloroform extraction method, and detecting the concentration and purity of the RNA by using Nanodrop 2000. A reverse transcription reaction was carried out in a system of 500ng RNA/10. mu.L to obtain a cDNA solution and the concentration thereof was examined. The cDNA solution concentration was adjusted to 2.5 ng/. mu.L for qRT-PCR detection. Taking beta-Actin as an internal reference gene, detecting 3 auxiliary holes for each gene, and setting the following reaction programs: after pre-denaturation at 95 ℃ for 30s, establishing 50 cycles of template amplification steps, and treating at 95 ℃ for 5 s; treating at 60 deg.C for 20 s. Finally, establishing a dissolution curve program to detect the product specificity, and treating for 5s at 95 ℃; treating at 60 deg.C for 1 min; treating at 95 deg.C for 5 s. After the reaction is finished, the cycle number of each secondary pore is derived, and the fluctuation of the cycle number among the secondary pores is kept within +/-0.3 cycle. And (4) making a difference value between the mean value of the number of target gene cycles and the mean value of the number of beta-Actin cycles to obtain a cycle threshold value of each target gene and calculate the level of the relative expression quantity.
(2) The results of the experiment are shown in FIGS. 1 to 2. As can be seen in fig. 1. As can be seen from fig. 2(a), the T cell activation pathways are most significantly different among them; further analysis of the T cell activation pathway revealed that Pla2g2d was expressed differently in gonadal testis (Te), epididymal origin (Is), epididymal head (Ca), epididymis (Co), and epididymal tail (Cd), as shown in fig. 2 (b); as can be seen from the sequencing result of fig. 2(c), Pla2g2d of 21-month-old male mice showed significantly higher expression in gonad testis (Te), epididymal origin (Is), epididymal head (Ca), epididymis (Co), and epididymal tail (Cd) than that of 3-month-old male mice; from the PCR results shown in FIG. 2(d), it was found that the expression of Pla2g2d in 21-month-old male mice was significantly increased in gonadal testis (Te), epididymal origin (Is), epididymal head (Ca), epididymis body (Co) and epididymal tail (Cd) as compared with 3-month-old male mice.
Example 2
In the embodiment, the expression of PLA2G2D mRNA in seminal plasma of healthy men of different ages is detected by using a qRT-PCR method.
(1) The experimental method comprises the following steps: the study subjects are males aged 30-45 years, which are divided into 30-31 years, 32-33 years, 34-35 years, 36-37 years, 38-39 years, 40-41 years, 42-43 years and more than 44 years according to the ages, and semen is collected after 3-5 days of abstinence of all subjects. Centrifuging semen sample at 4 deg.C and 3000 Xg for 10min to remove sperm and round cells, centrifuging at 4 deg.C and 12000 Xg for 30min to remove cell debris, collecting supernatant, and storing at-80 deg.C. 0.2mL of seminal plasma is taken, RNA lysate is added, free total RNA is extracted by a phenol chloroform extraction method, and the concentration and purity of the RNA are detected by using Nanodrop 2000. Reverse transcription was performed in a 500ng RNA/10. mu.L system. As shown in Table 1, PLA2G2D is used as a target gene, and the primer sequence of PLA2G2D is shown as SEQ ID NO.1 and SEQ ID NO. 2; beta-Actin is taken as an internal reference gene, and the primer sequence of the beta-Actin is shown as SEQ ID NO.3 and SEQ ID NO. 4. Each gene was tested in 3 subpores and the following reaction program was set up: after pre-denaturation at 95 ℃ for 30s, establishing 99 cycles of template amplification steps, and treating at 95 ℃ for 5 s; treating at 60 deg.C for 20 s. Finally, establishing a dissolution curve program to detect the product specificity, and treating for 5s at 95 ℃; treating at 60 deg.C for 1 min; treating at 95 deg.C for 5 s. After the reaction is finished, the number of cycles of each secondary hole is led out, and the fluctuation of the number of cycles among the secondary holes is kept within +/-0.3 cycle. The mean value of PLA2G2D cycles is different from the mean value of beta-Actin cycles to obtain the delta Ct value (cycle threshold) of the PLA2G2D gene.
TABLE 1
(2) The results of the experiment are shown in FIG. 3. As can be seen from FIG. 3, the delta Ct value of the PLA2G2D gene gradually decreased with the age of men and the decline of reproductive function, indicating that the expression of PLA2G2D in seminal plasma gradually increased with the age of men and the decline of reproductive function.
Example 3
In the embodiment, the expression of the PLA2G2D protein in seminal plasma of healthy men of different ages is detected by an ELISA method.
(1) The experimental method comprises the following steps: the study subjects are males aged 30-45 years, which are divided into groups aged 30-35 years and groups aged more than 35 years, and semen is collected after all subjects refrain from taking semen for 3-5 days. Centrifuging the semen sample at 4 deg.C and 3000 Xg for 15min to remove sperm and round cells to obtain seminal plasma. The detection of PLA2G2D protein in seminal plasma by ELISA kit includes the following steps: a. adding a standard substance and a sample into an enzyme label plate, and incubating for 1 hour at 37 ℃; b. after the incubation is finished, removing the sample in the step a, adding Detection Reagent A, and incubating for 1 hour at 37 ℃; c. after the incubation is finished, washing the mixture for 3 times by using 1 multiplied by Wash Buffer, adding Detection Reagent B, and incubating the mixture for 30 minutes at 37 ℃; d. discarding the Detection Reagent B after finishing the incubation, washing 5 times by using 1 × Wash Buffer, adding TMB Substrate, and incubating for 20 minutes at 37 ℃; e. after the incubation is completed, Stop Solution is added to terminate the reaction, and the OD value is detected under 450nm spectrophotometry and the OD value is used for the conversion of the sample concentration.
(2) The results of the detection are shown in FIG. 4. As can be seen from fig. 4, PLA2G2D in seminal plasma was significantly expressed after entering the age group (>35 years) prone to male reproductive failure.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
SEQUENCE LISTING
<110> secondary first hospital of Zhongshan university
<120> marker for predicting male reproductive function decline and application thereof
<130> 2022.01.11
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 19
<212> DNA
<213> Artificial Synthesis
<400> 1
gcagaggcca acccaaaga 19
<210> 2
<211> 21
<212> DNA
<213> Artificial Synthesis
<400> 2
gcagtcgctt ctggtaggtg t 21
<210> 3
<211> 18
<212> DNA
<213> Artificial Synthesis
<400> 3
cgggaaatcg tgcgtgac 18
<210> 4
<211> 21
<212> DNA
<213> Artificial Synthesis
<400> 4
ggaaggaagg ctggaagagt g 21
Claims (10)
1. A marker for predicting male reproductive failure, comprising a PLA2G2D gene.
Use of PLA2G2D in the manufacture of a test agent for predicting decline in male reproductive function.
3. The use of claim 2, wherein the agent comprises an agent for detecting mRNA expression of PLA2G2D in a bodily fluid.
4. The use of claim 2, wherein the agent comprises an agent for detecting expression of PLA2G2D protein in a bodily fluid.
5. The use according to any one of claims 3 to 4, wherein the body fluid is seminal plasma.
Application of reagent for detecting seminal plasma PLA2G2D protein by ELISA method in preparation of products for predicting male reproductive function decline.
7. A kit for predicting male reproductive function decline, which is characterized by comprising a reagent for detecting seminal plasma PLA2G2D protein by an ELISA method.
Application of a reagent for detecting a primer of PLA2G2D by a qRT-PCR method in preparing a product for predicting male reproductive function decline.
9. A kit for predicting male reproductive function decline is characterized by comprising a primer for detecting PLA2G2D by adopting a qRT-PCR method and a primer for detecting beta-Actin.
10. The kit according to claim 9, wherein the primer for detecting PLA2G2D has a sequence shown in SEQ ID No.1 and SEQ ID No. 2; the sequences of the primers for detecting the beta-Actin are shown as SEQ ID NO.3 and SEQ ID NO. 4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210031782.XA CN114480618B (en) | 2022-01-12 | 2022-01-12 | Marker for predicting male reproductive function decline and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210031782.XA CN114480618B (en) | 2022-01-12 | 2022-01-12 | Marker for predicting male reproductive function decline and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114480618A true CN114480618A (en) | 2022-05-13 |
| CN114480618B CN114480618B (en) | 2023-05-26 |
Family
ID=81511357
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210031782.XA Active CN114480618B (en) | 2022-01-12 | 2022-01-12 | Marker for predicting male reproductive function decline and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114480618B (en) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003060132A1 (en) * | 2002-01-17 | 2003-07-24 | Tanabe Seiyaku Co., Ltd. | Novel phospholipase a2 and gene thereof |
| CN102348810A (en) * | 2009-03-11 | 2012-02-08 | 雀巢产品技术援助有限公司 | Tissue-specific aging biomarkers |
| WO2018076061A1 (en) * | 2016-10-26 | 2018-05-03 | The University Of Melbourne | An assay and method of treatment |
| CN110732017A (en) * | 2019-11-08 | 2020-01-31 | 中山大学 | Use of SP in preparing medicine for treating male sterility |
| CN111944895A (en) * | 2020-08-28 | 2020-11-17 | 中山大学附属第一医院 | Kit for predicting semen collection fate of non-obstructive azoospermia patient |
| CN112695106A (en) * | 2021-02-18 | 2021-04-23 | 河南省农业科学院畜牧兽医研究所 | Method for rapidly and auxiliarily detecting growth traits of cattle by using PLA2G2A gene CNV marker and special kit |
| CN113667742A (en) * | 2021-09-07 | 2021-11-19 | 中山大学附属第一医院 | Kit for preoperative evaluation of azoospermia based on seminal plasma exosome mRNA expression profile |
-
2022
- 2022-01-12 CN CN202210031782.XA patent/CN114480618B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003060132A1 (en) * | 2002-01-17 | 2003-07-24 | Tanabe Seiyaku Co., Ltd. | Novel phospholipase a2 and gene thereof |
| CN102348810A (en) * | 2009-03-11 | 2012-02-08 | 雀巢产品技术援助有限公司 | Tissue-specific aging biomarkers |
| WO2018076061A1 (en) * | 2016-10-26 | 2018-05-03 | The University Of Melbourne | An assay and method of treatment |
| CN110732017A (en) * | 2019-11-08 | 2020-01-31 | 中山大学 | Use of SP in preparing medicine for treating male sterility |
| CN111944895A (en) * | 2020-08-28 | 2020-11-17 | 中山大学附属第一医院 | Kit for predicting semen collection fate of non-obstructive azoospermia patient |
| CN112695106A (en) * | 2021-02-18 | 2021-04-23 | 河南省农业科学院畜牧兽医研究所 | Method for rapidly and auxiliarily detecting growth traits of cattle by using PLA2G2A gene CNV marker and special kit |
| CN113667742A (en) * | 2021-09-07 | 2021-11-19 | 中山大学附属第一医院 | Kit for preoperative evaluation of azoospermia based on seminal plasma exosome mRNA expression profile |
Non-Patent Citations (3)
| Title |
|---|
| KUN LI ET AL.: "Secretory Phospholipase A2 Group IID Is Involved in Progesterone-Induced Acrosomal Exocytosis of Human Spermatozoa", JOURNAL OF ANDROLOGY * |
| 冯瑞祥 等: "精液中性粒细胞弹性蛋白酶的研究进展", 中华男科学杂志 * |
| 谢海锋 等: "磷脂酶A_2亚型及其与男性生殖相关性研究", 中国细胞生物学学报 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114480618B (en) | 2023-05-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US6664056B2 (en) | Non-invasive prenatal monitoring | |
| US20080108071A1 (en) | Methods and Systems to Determine Fetal Sex and Detect Fetal Abnormalities | |
| CN105039530B (en) | Mark and its application that mitochondria correlation refining miRNA occurs as mankind's severe asthenospermia | |
| EP1422297A1 (en) | Method of testing for bronchial asthma | |
| CN112251508B (en) | Seminal plasma exosome tsRNA marker related to non-obstructive azoospermia diagnosis and application thereof | |
| EP3074538A2 (en) | Method for predicting congenital heart defect | |
| CN102933723A (en) | Methods and kits for diagnosing conditions related to hypoxia | |
| CN109136358B (en) | Reagent for identifying and diagnosing residual sperms in testis of NOA patient and application of miRNA in reagent | |
| WO2017023194A1 (en) | Immunodiagnostic method and set for the analysis of dna molecules of trec and krec and of the number of dna genome equivalents | |
| CN113355407B (en) | tsRNA marker for diagnosing systemic lupus erythematosus and clinical application | |
| CN111944895B (en) | Kit for predicting semen collection fate of non-obstructive azoospermia patient | |
| CN114231612A (en) | MiRNA marker related to active tuberculosis and application thereof | |
| CN109423514A (en) | Recurrent spontaneous abortion related microRNA and its application | |
| CN114480618A (en) | Marker for predicting male reproductive function decline and application thereof | |
| CN108300788A (en) | A kind of micro RNA combination and its application for detecting light-duty brain trauma | |
| CN111944894A (en) | Molecular marker for prenatal noninvasive diagnosis of cleft lip and palate, neural tube malformation and congenital heart disease and application thereof | |
| KR102497167B1 (en) | Method for diagnosing pre-eclampsia using ratio of expression levels of miR-155-5p and miR-1290-3p | |
| CN109136359B (en) | Reagent for identifying and diagnosing residual sperms in testis of NOA patient and application of piRNA in reagent | |
| CN114107461A (en) | Circular RNA marker and application thereof in diagnosis and prediction of recurrent abortion pathological pregnancy | |
| CN112176052A (en) | Plasma exosome tsRNA marker related to non-obstructive azoospermia diagnosis and application thereof | |
| CN114032297B (en) | Serum/plasma exosome miRNA marker related to ICP (inductively coupled plasma) auxiliary diagnosis and application thereof | |
| CN115976200B (en) | Kit for evaluating recurrent abortion risk related to endometrial receptivity and application of kit | |
| CN112195232B (en) | Plasma exosome miRNA marker related to primary non-obstructive azoospermia diagnosis and application thereof | |
| CN116179681B (en) | Cyclic RNAZBTB10 and detection primer and application thereof | |
| KR102604625B1 (en) | Method for evaluating muscle aging by using autophagy-related gene of pbmc |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |