CN104846086A

CN104846086A - Application of plasma miR-15b as molecular marker in sperm generation and functional evaluation

Info

Publication number: CN104846086A
Application number: CN201510219996.XA
Authority: CN
Inventors: 许文明; 陈艳; 廖惠娟; 岳焕勋; 杨明; 孙华钦
Original assignee: West China Second University Hospital of Sichuan University
Current assignee: West China Second University Hospital of Sichuan University
Priority date: 2015-05-04
Filing date: 2015-05-04
Publication date: 2015-08-19

Abstract

The invention provides application of a plasma miR-15b as a molecular marker in sperm generation and functional evaluation. MiRNA in the plasma of a testee is extracted and subjected to reverse transcription, a template cDNA obtained is subjected to real-time fluorescent quantitative PCR (polymerase chain reaction), and an expression of the miR-15b in the plasma is compared to that of a normal plasma sample. The miRNA miR-15b playing an important role in the process of sperm generation is discovered, the expressional differences of the miR-15b in male aspermatism and severe oligozoospermia is discovered, and evident changes in the expression of the miR-15 in the plasma can be detected; the miR-15 is suitable for laboratory detection methods of evaluating sperm quality and function, and detection and clinical consultation are provided for male patients with infertility caused by unknown clinical reasons.

Description

Blood plasma miR-15b produces and the application in functional assessment at sperm as molecular marked compound

Technical field

The invention belongs to medical detection technology, be specifically related to blood plasma miR-15b and produce and the application in functional assessment at sperm as molecular marked compound.

Background technology

For many years, traditional semen routine analysis is the most basic clinical indices for judging male fertility always.Still unclear to the real sterile reason of the male sterility patient of the overwhelming majority clinically.Especially nearly 1/3rd infertile patients clinically, the Sperm routine analysis result of the male sex is all normal and close normal.Clinically this kind of patient is divided into Unexplained infertility.Therefore, for a long time, there is very large difficulty in the clinical diagnosis to male sterility.

The diagnostic method of traditional male sterility is the activity analysis utilizing sperm, the methods such as sperm count, and these class methods have following feature:

1, sperm count is subject to external influence many factors, as smoking, drink etc. all can cause the fluctuation of result larger;

Whether 2, the activity analysis of sperm needs expensive instrumental analysis, and have in the recent period with experimenter and to have sexual intercourse etc. because have significant correlation;

3, current method is all the detection method based on seminal fluid, and the process of collecting sample is comparatively loaded down with trivial details.

And it is simple, direct to adopt the detection method of the molecular marked compound of blood plasma to have sampling, the feature of the birth level of patient directly can be reflected.As 201310112391.1, name is called the application for a patent for invention of " biomarker of male sterility and application thereof ", detect the level of at least one instruction male sterility biomarker in experimenter's body fluid, wherein at least one instruction male sterility biomarker is selected from acetylcarnitine, aspartic acid, carnitine C3:1, ketone carnitine, leucyl proline(Pro), carnitine C8, methyl xanthine, carnitine C10:2, carnitine C10:1, VITAMIN B4; Described body fluid is selected from experimenter's urine, serum, blood plasma, blood, seminal fluid, sperm, refining, saliva.On the one hand, this detection method needs the instrument of the costlinesses such as high performance liquid chromatography analyser, on the other hand, needs to detect multiple biomarker simultaneously.

201310722584.9, name is called the application for a patent for invention of " a kind of gene tester of spermatid apoptosis-related genes ", by the genomic dna of extracting mouth epithelial cells sample, individual NOS3 gene, FASLG gene SNP s locus gene portable is analyzed again through quantitative fluorescent PCR reaction detection, for all persons under inspection provide spermatid apoptosis-related genes to detect, and in conjunction with in Chinese population distinctive to azoospermia, the relevant environmental exposure of aspermia or oligospermia and the sterile onset risk of mode of life influence factor to the male sex carry out forecast assessment.This detection method extracts the DNA of mouth epithelial cells, gene pleiomorphism and the correlation circumstance without sperm is observed after carrying out gene amplification, the correlation circumstance of itself and azoospermia is observed from the level of DNA, its DNA makes a variation and azoospermia is only correlation research, not to the information of the concrete dependency of the parameter associations such as detection method and sperm generation, not there is practicality.

Summary of the invention

In order to solve the problems of the technologies described above, the invention provides a kind of blood plasma miR-15b and producing and the application in functional assessment at sperm as molecular marked compound.Present invention finds a kind of Microrna (miRNA) gene miR-15b played an important role in spermatogenesis, miR-15b without the differential expression in essence and serious few essence (being less than 5,000,000/ml), and can detect its noticeable change expressed the male sex in blood plasma.

In order to realize foregoing invention object, the present invention adopts following technical scheme:

Blood plasma miR-15b produces and the application in functional assessment at sperm as molecular marked compound, it is characterized in that: extract the miRNA in experimenter's blood plasma, carry out reverse transcription reaction, the template cDNA obtained is carried out real-time fluorescence quantitative PCR, and the expression and the normal plasma sample that detect miR-15b in blood plasma compare.

In the process of real-time fluorescence quantitative PCR, the amplification of target sequence and the detection of fluorescent signal are carried out simultaneously, quantitative PCR apparatus whole process gathers fluorescent signal, and experiment terminates post analysis software and automatically sets threshold value by mathematical algorithm deduction autofluorescent background signal thus obtain the Ct value of each sample.

The gene order of miR-15b of the present invention is (SEQID NO:1): 5 '-TAGCAGCACA TCATGGTTTA C.

Real-time fluorescence quantitative PCR reaction of the present invention adopts Taqman probe method.

Preferably, adopt Life technologies company to provide, article No. is the Taqman probe of Assay ID000390.

Reverse transcription of the present invention adopts the Reverse Transcription box P/N 4366596 of Life Technologies company.

Present method, has the following advantages relative to traditional motility of sperm compared with the method for counting:

1, the sample size needed for detection is few, and speed is fast, and the time is short, can be used for the test in laboratory method assessing sperm quality and function, can be clinical agnogenic male sterility patient and provides detection and on.

2, this test item adopts and detects from blood plasma, but not from seminal fluid separated sperm sample, be a kind of fast with the new method of screening spermatogenesis obstacle accurately.

3, present method is based on the principle of quantitative PCR.Result is reproducible, objective and accurate, is easy at different feeler mechanisies and hospital laboratory, the popularizations such as andrology laboratory.

Accompanying drawing explanation

Fig. 1 is the interpretation of result of animal model.

A () is constructed by the transgenic mice of PGK2 promotor at the special process LAN miR-15b of androgone.

B () PCR identifies positive transgene mouse model.

C the testis H & E dyeing of () miR-15b transgenic mice shows spermatogenesis defect, prematurity spermatid comes off.The left side is the section of normal control mice testis.

The sperm count result of collecting in (d) epididymis.

The diameter of the convoluted seminiferous tubule of (e) miR-15b transgenic mice.

F the miR-15b of () miR-15b transgenic mice blood plasma expresses.

The expression of the downstream gene Cdc25a of (g) Western blot result display miR-15b regulation and control.

Fig. 2 is the distribution plan of the miR-15b relative expression quantity obtained after testing without smart person and normal subjects.

Embodiment

Below in conjunction with embodiment, essentiality content of the present invention is described in further detail.

Embodiment 1

Blood plasma miR-15b produces and the application in functional assessment at sperm as molecular marked compound, extract the miRNA in experimenter's blood plasma, carry out reverse transcription reaction, the template cDNA obtained is carried out real-time fluorescence quantitative PCR, the expression and the normal plasma sample that detect miR-15b in blood plasma compare.

Embodiment 2

Described real-time fluorescence quantitative PCR reaction adopts Taqman probe method.

Embodiment 3

Described real-time fluorescence quantitative PCR reaction adopts Taqman probe method, and provided by Life technologies company, article No. is Assay ID000390.

Embodiment 4

Described reverse transcription adopts the Reverse Transcription box P/N 4366596 of Life Technologies company.

Specific experiment operation of the present invention is as follows:

1, RNA extracts: collect 3ml whole blood with the heparin tube containing EDTA antithrombotics, 3000g, 4 DEG C of centrifugal 10min, collecting the yellow translucent liquid in upper strata is exactly blood plasma, nearly 500ul blood plasma, extract test kit with mirVana PARIS Kit (Life technologies, AM1556) blood plasma and extract RNA.Concrete operations are:

A. by 2X denaturation buffer 37 DEG C placement before using.

B. 500ul plasma sample adds isopyknic 2X denaturation buffer, is adding 1ml chloroform vortex 60s, 10000g, 5min, and room temperature is placed.

C. transferred to by supernatant in new EP pipe, add 100% ethanol of 1.25 times of volumes, it is centrifugal to add filtration core, abandons solution.

D. washing lotion 1, washs once, 10000g, 30s.

E. washing lotion 2/3, washes twice, 10000g, 30s.

F. air separation.

G. the elutriant adding 100ul 95 DEG C of preheatings dissolves collects RNA.

2, reverse transcription: the primer that reverse transcription system adopts comes from Reverse Transcription box P/N 4366596 (Life company, the U.S.).

1) by table 1 order preparation reverse transcription reaction system

Table 1

85 DEG C of reaction 5min, 60 DEG C of reaction 5min, 4 DEG C of preservations, place 2min on ice.

2) the 7ul mixed solution of table 2 is added

Table 2

16 DEG C of reaction 30min, 42 DEG C of reaction 5min, 85 DEG C of reaction 5min, 4 DEG C of preservations.

3, quantitative fluorescent PCR: the expression detecting miR-15b in tested sample and normal plasma sample by Taqman probe method, the primer of pcr amplification reaction system provides for Life technologies company, and article No. is Assay ID000390.

PCR reaction system is prepared by table 3

Table 3

50 DEG C of reaction 2min, (95 DEG C of reaction 10min, 95 DEG C of reaction 15s, 60 DEG C of reaction 60s) 40cycles.Detect the Ct value of tested sample and quality control product, and adopt 2-Ct method to be converted into relative expression quantity.

Experimentation on animals

The present invention by animal model, builds the transgenic mice of process LAN miR-15b, finds at the special process LAN miR-15b of androgone by the key protein Cdc25a in Inhibit sperm Hemapoiesis process, thus the generation of Inhibit sperm cell.Experimentation on animals demonstrates the biomarker that miR-15b can be used as the generation of reflection sperm.

Fig. 1 (a) is constructed by the transgenic mice of PGK2 promotor at the special process LAN miR-15b of androgone.Fig. 1 (b) PCR identifies positive transgene mouse model.The testis H & E dyeing of Fig. 1 (c) miR-15b transgenic mice shows spermatogenesis defect, and prematurity spermatid comes off.The left side is the section of normal control mice testis.The sperm count result display sperm count collected in Fig. 1 (d) epididymis significantly reduces (n=3-6; P<0.05); Significantly reducing appears in the diameter of the convoluted seminiferous tubule of Fig. 1 (e) miR-15b transgenic mice; The miR-15b of the blood plasma of Fig. 1 (f) miR-15b transgenic mice expresses and significantly raises (n=3; P=0.05); The expression of downstream gene Cdc25a of Fig. 1 (g) Western blot result display miR-15b regulation and control significantly declines (n=9; P<0.05).

Clinical experiment

The present invention to the male sex of 12 routine normal reproduction and the 14 routine male sex without smart person, extract the peripheral blood of experimenter respectively, extract the RNA molecule in blood plasma, adopt the fluorescent quantitative PCR technique of Taqman method to the microRNA molecules of circulation, the gene expression abundance of miR-15b detects, and adopts 2-Ct method to be converted into relative expression quantity.Ct=Ct goal gene (miR-15b)-Ct reference gene (β-actin).

Interpretation of result: the relative expression quantity mean value of normal subjects is 1.42+/-0.28(average +/-standard deviation), the relative expression quantity without smart person is 23.16+/-7.51(average +/-standard deviation).Fig. 2 is the distribution plan detecting the miR-15b relative expression quantity obtained, and the expression amount without the blood plasma miR-15b of smart person obviously rises relative to the expression amount of normal subjects.This result display blood plasma miR-15b is a molecular marked compound well evaluating mankind spermatozoon situation occurred.

Sequence table

<110> West China No.2 Hospital, Sichuan University

<120> blood plasma miR-15b produces and the application in functional assessment at sperm as molecular marked compound

<130> 2015

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 21

<212> DNA

<213> miR-15b

<400> 1

tagcagcaca tcatggttta c 21

Claims

1. blood plasma miR-15b produces and the application in functional assessment at sperm as molecular marked compound, it is characterized in that: extract the miRNA in experimenter's blood plasma, carry out reverse transcription reaction, the template cDNA obtained is carried out real-time fluorescence quantitative PCR, and the expression and the normal plasma sample that detect miR-15b in blood plasma compare.

2. blood plasma miR-15b according to claim 1 produces and the application in functional assessment at sperm as molecular marked compound, it is characterized in that: described real-time fluorescence quantitative PCR reaction adopts Taqman probe method.

3. blood plasma miR-15b according to claim 2 produces and the application in functional assessment at sperm as molecular marked compound, it is characterized in that: adopt Life technologies company to provide, article No. is the Taqman probe of Assay ID000390.

4. blood plasma miR-15b according to claim 1 produces and the application in functional assessment at sperm as molecular marked compound, it is characterized in that: described reverse transcription adopts the Reverse Transcription box P/N 4366596 of Life Technologies company.