CN104007103A - Sperm detection method and kit thereof - Google Patents
Sperm detection method and kit thereof Download PDFInfo
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- CN104007103A CN104007103A CN201310055555.1A CN201310055555A CN104007103A CN 104007103 A CN104007103 A CN 104007103A CN 201310055555 A CN201310055555 A CN 201310055555A CN 104007103 A CN104007103 A CN 104007103A
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Abstract
The invention discloses a sperm detection method, a kit and application thereof. By means of the method or the kit, the use quantity of sperm samples can be reduced, the oxidative stress level of seminal plasma is conveniently detected, the leukocyte pollution degree in the seminal plasma and the oxidative stress capacity of sperms can be detected, and the detection rate is improved.
Description
Technical field
Relate to a kind of detection method and detection kit of biological sample, relate in particular to the method for inspection and the test kit of human seminal fluid's sample.
Background technology
Normal semen is a kind of potpourri, is mixed by the juice of testis and epididymis and suspend sperm wherein and the secretion of prostate, seminal vesicle and bulbourethral gland in when ejaculation.The final potpourri penetrating is a kind of liquid of thickness, be that ejaculum penetrates continuously for several times with lumps, by the relatively demonstration before and after vasectoray, with regard to volume, have 90% to be secretion from attached body of gland, be mainly wherein prostate and seminal vesicle, small part is from bulbourethral gland and epididymis.
Two main quantizating index of seminal fluid: 1. sperm sum, the degree of mobility of piping system after the production of sperm situation of reaction testis and testis; 2. volume, the secretion capacity of reaction body of gland.
Sperm function is mainly subject to the impact of vigor, motion, form and the refining composition of sperm self.Under rigid condition, semen sample quality is mainly subject to the secretion of testicular spermatogenic function, attached body of gland, the especially high febrile illness of recent ill situation, and other is as the impact of the length of ascetic time.
In seminal fluid, often contain certain Nonsperm cell composition, part is close with Relationship with Clinical, generally includes urogenital tract epithelial cell, prostatic cell, androgone and leucocyte, and latter two is referred to as " circle cell ".In the smear of these cell components after dyeing, (amplify 1000 times) and be difficult to difference, but can differentiate by superoxide superoxide zymotechnic and full leucocyte monoclonal antigen technology.
There is at present suitable evidence to show, a small amount of leucocyte plays an important role in adjusting autoimmune response, but under pathologic condition, is also the extremely metabolic product of leucocyte of excessive existence in sex genital tract, can affect the function of mankind spermatozoon, reduce sperm motility and insemination ability.It is generally acknowledged, the leucocyte in seminal fluid mainly causes spermatoblast membrane lipid peroxidating approach to damage sperm function by discharging active oxygen (Reactive Oxygen Species, ROS).Owing to having complicated oxidation and antioxidant system in seminal fluid, there is certain limitation by morphologic detection semen white blood cell quantity merely, therefore the method for evaluating leukocytic oxidative stress level in seminal fluid reactive oxygen species, particularly seminal fluid will be that the fabulous of traditional detection method supplemented.
Whether in detection refining, activated oxidized water is gentle comes to exist leucocyte to pollute in fast detecting seminal fluid by free radical generative capacity, and can assess the oxidative stress ability of sperm, the total quality of reflection sperm generative process, have great significance researching and analysing in the cause of disease of male sterility and treatment, and can apply and the assessment of male sterility therapeutic intervention effect.
The collection of sample and inspection are all carried out according to normalization procedure, and its result is just valuable.Described in this chapter, operating process is identified as the basic composition step of carrying out semen evaluation.
Summary of the invention
The application relates generally to a kind of method or kit of checking seminal fluid oxidative stress.
In some embodiments, provide a kind of method of checking seminal fluid oxidative stress, described method comprises,
To the superoxide enzyme solutions that adds 10 μ l-100 μ l luminol solutions and 10 μ l-100 μ l in 20 μ l-200 μ l seminal fluid samples, after reaction, obtain potpourri A, record the spectrophotometric value Dr of potpourri A;
In potpourri A, add 10 μ l-100 μ l fMLP solution, after reaction, obtain potpourri B, record the spectrophotometric value Df of potpourri B; And
In potpourri B, add 10 μ l-100 μ l PMA solution, after reaction, obtain mixture C, record the spectrophotometric value Dp of mixture C;
Determine according to the logarithm value of Df/Db ratio whether this seminal fluid is leukocyte sperm disease, and/or determine that according to the logarithm value of Dp/Dr ratio whether the oxidative stress value of sperm is normal.
In some embodiments, provide a kind of kit of checking seminal fluid oxidative stress, described kit comprises,
Luminol solution;
Superoxide enzyme solutions;
FMLP solution;
PMA solution; And
Optionally, described kit also comprises positive reference substance (for example, 0.02% H
2o
2, dobell's solution) and/or negative control product (for example, PBS).
The working concentration of the luminol solution that uses in described method or kit in some embodiments,, superoxide enzyme solutions, fMLP solution, PMA solution is:
Luminol solution (2.5mM);
Superoxide enzyme solutions (12.5IU/ml);
FMLP solution (0.2mM); And
PMA solution (0.4 μ M).
In some embodiments, described kit can provide the 10 μ l-100 μ l luminol solutions of single part, superoxide enzyme solutions, 10 μ l-100 μ l FMLP solution and the 10 μ l-100 μ l PMA solution of 10 μ l-100 μ l etc. that single uses to detect reagent.Therefore, described kit comprises at least a such detection reagent.
In some embodiments, the disclosed kit of the application also comprises standard items, and it passes through comprised detection reagent, and especially single part is detected after reagent detection, can draw out the typical curve of ROS.In other embodiments, described kit comprises negative standard items and/or the positive criteria product stated.In some embodiments, described positive criteria product are that leucocyte pollutes reference substance.Logarithm value >=1.25 of Df/Db ratio, illustrate in described seminal fluid sample to be leukocyte sperm disease.
In some embodiments, described kit is the kit for implementing the disclosed method of the application.
Whether the oxidative stress value that in some embodiments, the logarithm value of described Dr/Db ratio judges refining in seminal fluid sample is in normal range.Be generally, logarithm value≤0.75 of Dr/Db ratio, illustrates that the oxidative stress ability of refining in described seminal fluid sample is normal.
In some embodiments, the logarithm value of described Df/Dbr ratio judges in seminal fluid sample whether exist leucocyte to pollute.Be generally, in the described seminal fluid sample of logarithm value >=1.25 explanation of Df/Db ratio, exist leucocyte to pollute.
In some embodiments, the logarithm value of described Dp/Dr ratio is more than or equal to 0.9, illustrates that the sperm oxidative stress ability in described seminal fluid sample is normal.Be generally, it is normal that the logarithm value of Dp/Dr ratio is more than or equal in the described seminal fluid sample of 0.9 explanation sperm oxidative stress ability.
In some embodiments, the detection consumption of described semen sample compared with the prior art, obviously reduces, for example, and 200 μ l, 150 μ l, 100 μ l, 75 μ l, 50 μ l or 30 μ l.Because the seminal fluid amount of samples of the disclosed method of the application or the use of its kit reduces, so can utilize limited seminal fluid sample to carry out revision test or other goal analysis.
In some embodiments, the disclosed method of the application and kit can analyze that leucocyte in seminal fluid pollutes and the oxidative stress ability of sperm simultaneously, it is the vigor of sperm, thereby can in once checking, identify sterility because leucocyte pollutes or sperm motility reduces and causes, or the two all exists.
Therefore, whether the detection method of the disclosed seminal fluid oxidative stress of the application and kit thereof be for existing the oxidative stress ability of leucocyte pollution, refining and sperm that a kind of new appraisal procedure and means are provided in fast detecting seminal fluid.
Brief Description Of Drawings
Figure 1A and Figure 1B show respectively the log-linear relation of leucocyte content and values of chemiluminescence.Figure 1A is under fMLP stimulates, the log-linear relation of leucocyte content and values of chemiluminescence; Figure 1B is under PMA stimulates, the log-linear relation of leucocyte content and values of chemiluminescence.
Fig. 2 A and Fig. 2 B show respectively the chemiluminescence figure of the seminal fluid that use luminol-peroxidase system observes.Fig. 2 A is presented in the situation that has leucocyte pollution, adds after leucocyte specific probe fMLP, produces a ROS peak.Do not have leucocyte to pollute if Fig. 2 B shows, the ROS peak of fMLP reaction disappears, and causes a chemiluminescence signal being produced by sperm significantly by PMA.
Embodiment
On the one hand, the application relates to a kind of method of inspection sheet part seminal fluid oxidative stress.
On the other hand, the application relates to a kind of kit of inspection sheet part seminal fluid oxidative stress.
In some embodiments, provide a kind of method of inspection sheet part seminal fluid oxidative stress, described method comprises,
Get 20 μ l-200 μ l seminal fluid samples after counting in sample hose, the luminous value data 1 that record sample are Db;
To the superoxide enzyme solutions (12.5IU/ml) that adds 10 μ l-100 μ l luminol solutions (2.5mM) and 10 μ l-100 μ l in 20 μ l-200 μ l seminal fluid samples, after reaction, obtain potpourri A, record the luminous value Dr of potpourri A;
In potpourri A, add 10 μ l-100 μ l fMLP solution (0.2mM), after reaction, obtain potpourri B, record the luminous value Df of potpourri B; And
In potpourri B, add 10 μ l-100 μ l PMA solution (0.4 μ M), after reaction, obtain mixture C, record the luminous value Dp of mixture C;
The logarithm value of Dr/Db ratio determines that whether the refining oxidative stress ability in this seminal fluid is normal, and/or the logarithm value of Df/Db ratio determines whether this seminal fluid is leukocyte sperm disease, and/or determines the oxidative stress value of sperm according to the logarithm value of Dp/Dr ratio.
In some embodiments, provide a kind of kit of checking seminal fluid oxidative stress, described kit comprises,
Luminol solution
Superoxide enzyme solutions;
FMLP solution;
PMA solution; And
Optionally, described kit also comprises positive reference substance (for example, 0.02% H
2o
2, dobell's solution, pH8.7) and/or negative control product (for example, PBS, pH8.7).
In some embodiments, described kit can provide the 10 μ l-100 μ l luminol solutions of single part, superoxide enzyme solutions, 10 μ l-100 μ l FMLP solution and the 10 μ l-100 μ l PMA solution of 10 μ l-100 μ l etc. that single uses to detect reagent.
In some embodiments, the consumption of the luminol solution in described method or kit, superoxide enzyme solutions, fMLP solution and PMA solution or single part of consumption are 1/2nd of wish detection seminal fluid volumes.For example, in the time that the seminal fluid of wish detection is 100 μ l, the consumption of luminol solution, superoxide enzyme solutions, fMLP solution and PMA solution is 50 μ l; In the time that the seminal fluid of wish detection is 200 μ l, the consumption of luminol solution, superoxide enzyme solutions, fMLP solution and PMA solution is 100 μ l; In the time that the seminal fluid of wish detection is 150 μ l, the consumption of luminol solution, superoxide enzyme solutions, fMLP solution and PMA solution is 75 μ l; In the time that the seminal fluid of wish detection is 75 μ l, the consumption of luminol solution, superoxide enzyme solutions, FMLP solution and PMA solution is about 35-40 μ l; In the time that the seminal fluid of wish detection is 50 μ l, the consumption of luminol solution, superoxide enzyme solutions, fMLP solution and PMA solution is 25 μ l.
In some embodiments, luminol solution, superoxide enzyme solutions, fMLP solution and PMA solution in described method or kit can be concentrates, in use, utilize analysis buffer, such as PBS damping fluid, physiological saline, ringer's solution etc. are diluted to the concentration using in described method.Therefore, in some embodiments, in the disclosed kit of the application, also comprise analysis buffer or dilution such as PBS damping fluid, physiological saline, ringer's solution, cell culture medium, balanced salt solution or its combination etc., it can be for dilute sample or reagent, or use as negative control product.Correspondingly, in some embodiments, the disclosed method of the application also comprises the step of carrying out dilute sample or diluting concentrated luminol solution, superoxide enzyme solutions, fMLP solution and PMA solution with the above-mentioned analysis buffer such as PBS damping fluid, physiological saline, ringer's solution, cell culture medium, balanced salt solution or its combination etc. or dilution, so that it reaches working concentration, the concentration that described method or kit use.
The working concentration of the luminol solution that uses in described method or kit in some embodiments,, superoxide enzyme solutions, fMLP solution, PMA solution is:
Luminol solution (2.5mM);
Superoxide enzyme solutions (12.5IU/ml);
FMLP solution (0.2mM); And
PMA solution (0.4 μ M).
In some embodiments, detect for convenient, the reaction described in the disclosed method of the application is to leave standstill 3-15 minute in room temperature, or bathes standing completing for 3-10 minute or approximately 5 minutes 37 ° of C temperature.
In some embodiments, the disclosed kit of the application also comprises standard items, and it passes through comprised detection reagent, and especially single part is detected after reagent detection, can draw out the typical curve of ROS.
In other embodiments, described kit also comprises negative control product and/or positive reference substance.Described positive control for example, 0.02% H
2o
2, dobell's solution, pH8.7; Negative control product for example, PBS, pH8.7.
In some embodiments, described positive criteria product are that leucocyte pollutes reference substance, and the logarithm value of the disclosed Df/Db ratio of its application determines whether this seminal fluid is leukocyte sperm disease.
Whether the oxidative stress value that in some embodiments, the logarithm value of described Dr/Db ratio judges refining in seminal fluid sample is in normal range.Be generally, logarithm value≤0.75 of Dr/Db ratio, illustrates that the oxidative stress ability of refining in described seminal fluid sample is normal.
In some embodiments, the logarithm value of described Df/Db ratio judges in explanation seminal fluid sample and exists leucocyte to pollute.Be generally, in the described seminal fluid sample of logarithm value >=1.25 explanation of Df/Db ratio, exist leucocyte to pollute.
In some embodiments, the logarithm value of described Dp/Dr ratio is more than or equal to 0.9, illustrates that the sperm oxidative stress ability in described seminal fluid sample is normal.Be generally, it is normal that the logarithm value of described Dp/Dr ratio is more than or equal in the described seminal fluid sample of 0.9 explanation sperm oxidative stress ability.
In some embodiments, described kit also comprises the instructions that instructs this kit of use.Therefore, in some embodiments, described kit is the kit of implementing the method for the disclosed inspection seminal fluid of the application oxidative stress.
In some embodiments, the detection limit of described semen sample compared with the prior art, obviously reduce, for example, 200 μ l, 150 μ l, 100 μ l, 75 μ l, 50 μ l or 30 μ l, be generally the untreated seminal fluid of 100 μ l, for example, without separation, centrifugal seminal fluid, can complete the detection of the disclosed seminal fluid response to oxidative stress of the application, thereby avoid spermatoblast to produce infringement, can therefrom analyze again the oxidative stress ability of leukocytic pollution and sperm.Because the seminal fluid sample size of the disclosed method of the application or the use of its kit reduces, so can utilize limited seminal fluid sample to carry out revision test or other goal analysis.
In some embodiments, a described point shading value utilizes Chemiluminescence Apparatus to detect, and conventionally detects 3,5,10,12,15 or 20 seconds, more generally detects 10 seconds.
The disclosed analysis buffer of the application or dilution include but not limited to cell culture medium, damping fluid, ringer's solution, physiological saline and balanced salt solution.The example of cell culture medium comprises BME, MEM, DMEM, IMEM, HAM F12, PRMI1640, M199 etc.The example of damping fluid comprises phosphate, acetate, citrate, borate and carbonate.
In some embodiments, described analysis buffer or dilution are isotonic buffer solution, are preferably isotonic phosphate buffer liquid (PBS).
The disclosed term of the application " individuality ", refers to mammal or people, includes but not limited to people, primate, ox, horse, pig, sheep, goat, dog, cat and the rodent such as rat and mouse.
Above-mentioned disclosure has been described the application's disclosed method kit and application thereof generally, and those skilled in the art adjust agents useful for same concentration and consumption as required, and can combine arbitrarily described embodiment.Following embodiment further carries out exemplary explanation to the application.Describing these embodiment is only the disclosed technical scheme of explanation the application, instead of limits its scope.Although used special term and value herein, these terms and value are understood to exemplary equally, do not limit the scope of the application's claims protection.
embodiment
Embodiment 1 leucocyte oxidative stress gradient experiment
Material: leucocyte (blood separates and obtains); Detection kit comprises luminol (luminol), peroxidase (HRP), each reagent component of fMLP and PMA.
Instrument: the Berthold Detection Systems tube-type chemical luminous tester SmartLine of company or 96 orifice-plate type OrionII.
Experimental procedure:
Cell gradient dilution: utilize PBS damping fluid to dilute cell by following concentration (cells/ml).
4.5×10
5/ml；2.25×10
5/ml；4.5×10
4/ml；2.25×10
4/ml；4.5×10
3/ml；2.25×10
3/ml；4.5×10
2/ml；2.25×10
2/ml。
Get the luminol that adds 100 μ l after 100 μ l, the HRP of 100 μ l, then adds the fMLP of 100 μ l, detects luminous value, after luminous value falls after rise, adds the PMA of 100 μ l, detects luminous.
Blank Baseline detection: 100 μ l cell+100 μ l luminol+100 μ l HRP
Pattern detection: 100 μ l cell+100 μ l luminol+100 μ l HRP+100 μ l FMLP
100μl?cell+100μl?luminol+100μl?HRP+100μl?PMA
Detection time: every 0.5min mono-surveys, and surveys altogether 3min.
Experimental result and analysis:
1. blank baseline value is in 70 ~ 90 left and right
2. from measurement result, under fMLP stimulates, there is peak value in 2min left and right in cell, declines subsequently, drops to 200 ~ 300 in 10min left and right.
After under PMA stimulates, after stimulating along with the time progressively raises, there is a long-term plateau when rising in luminous value after certain value.
3. from the result of gradient dilution, detect luminous value and reduce along with the minimizing of cell number.
1.5min fMLP luminous value is as shown in table 1 below, substantially to 10
2the individual order of magnitude.
The leukocytic fMLP luminous value of table 1 1.5min
| Cell/ml | 4.5×10 5 | 2.25×10 5 | 4.5×10 4 | 2.25×10 4 | 4.5×10 3 | 2.25×10 3 | 4.5×10 2 | 2.25×10 2 | Blank |
| Shading value | 106723 | 85120 | 16232 | 8565 | 1890 | 1286 | 226 | 141 | 103 |
The leukocytic PMA luminous value of 3.0min is as shown in table 2 below, substantially to 10
2the individual order of magnitude.
| Cell/ml | 4.5×10 5 | 2.25×10 5 | 4.5×10 4 | 2.25×10 4 | 4.5×10 3 | 2.25×10 3 | 4.5×10 2 | 2.25×10 2 | Blank |
| Shading value | 227344 | 146061 | 18902 | 12310 | 2487 | 1677 | 334 | 166 | 103 |
Get the measurement data of FMLP, PMA peak value, luminous value and cell concentration are all taken the logarithm, mapping, calculates R2, comprises 4.5 × 10
5~ 2.25 × 10
2the result of lower 8 luminous values of variable concentrations as shown in FIG. 1A and 1B.
In embodiment 2 seminal fluid, leucocyte pollutes with the detection of sperm oxidative stress ability and tests 1
One, research object
Given birth in the male sex by investigation health in 6000 examples, randomly draw, gather the semen sample of ascetic 2-7 days, as educating normal healthy controls.Inclusive criteria: live together 1 year with interior fertility, age <40 year when semen collection.
And adopt coding schedule to randomly draw the heredity of Gynaecology &. Obstetrics Hospital Attached to Fudan Univ collection love and diagnosis and treatment center out-patient with 1:10 ratio, inclusive criteria: the not contraception above in 2 years of living together, sexual life normally and is not educated.Exclusion standard: clinical diagnosis is sterile, azoospermia, the aspermia or oligospermia patient that female factors causes.
With reference to WHO standard, by seminal parameters, infertile patients is divided into 2 groups:
Seminal parameters normal group: sperm concentration>=15 × 10
6/ ml, forward direction motile sperm rate>=32% or forward direction motile sperm rate+non-forward direction motile sperm rate>=40%, normal morphology rate>=4%
Few asthénospermie group: sperm concentration>=or <15 × 10
6/ ml, forward direction motile sperm rate <32% is forward direction motile sperm rate+non-forward direction motile sperm rate <40% simultaneously, normal morphology rate>=4%.
Finally, according to the check result of seminal fluid generalized case, research object is divided into 3 groups, seminal parameters normally can be educated group (230 example), seminal parameters normal sterile group (166 example) and few sterile group of asthénospermie (234 example).
Two, method
(1) seminal fluid collecting and seminal fluid conventional sense
Press WHO " human seminal fluid's inspection and processing laboratory manual the 5th edition "
[2]seminal fluid collecting requirement, collects fresh semen.After liquefying completely, seminal fluid use CASA system to detect sperm concentration, the indexs such as kinetic parameter; According to WHO standard, adopt Diff-Quik decoration method to evaluate sperm morphological index.Record rate of teratosperm, head defect, stage casing defect, afterbody defect.
(2) chemoluminescence method detects seminal fluid ROS level
Oxygen radical can react with luminol, under the catalysis of enzyme, produces bioluminescence, detects the light intensity producing by Chemiluminescence Apparatus, calculates the amount of oxygen radical.Formyl tripeptides (fMLP) and phorbol (PMA) are the stimulants that can excite oxidative stress.FMLP, as leukocytic probe, can stimulate nadph oxidase system wherein to produce oxygen radical; And PMA can enter spermatoblast, activate spermatoblast, produce oxidative stress.
Be suitable for instrument:
The Berthold Detection Systems tube-type chemical luminous tester SmartLine of company or micro-pore plate type Chemiluminescence Apparatus Orionll.
Detecting step:
The fresh semen of above-mentioned collection, is no less than 1ml, avoids centrifugal or concuss.Seminal fluid sample detects in 1 hour as best, but should not exceed 2 hours.
Get 100 μ l seminal fluid in 2.0ml centrifuge tube or in microwell plate micropore, detect 10 seconds, record data 1 are Db;
Add 50 μ l luminols and 50 μ l superoxide enzyme solutions, after room temperature fully mixes, 37 ° of C temperature are bathed and are left standstill 5 minutes, detect 10 seconds, and record data 2 are Dr;
Add 50 μ l fMLP in above-mentioned centrifuge tube, piping and druming gently, 37 ° of C temperature are bathed lucifuge and are left standstill 15 minutes, detect 10 seconds, and record data 3 are Df;
Add 50 μ l PMA in above-mentioned centrifuge tube, piping and druming gently, 37 ° of C temperature are bathed lucifuge and are left standstill 10 minutes, detect 10 seconds, and record data 4 are Dp.
According to data 1,2,3,4 draw the ROS curve map of this sample.
Negative, the positive detects with reference to product:
Negative and positive with reference to product, detection is used for Quality Control and positive control, uses the quality that reaction detection is tested each detection.Its detecting step is with the detection of seminal fluid.
[reference value]
1, refining normal oxidation stress be worth: logarithm value≤0.75 of Dr/Db ratio.
2, leucocyte pollutes: logarithm value >=1.25 of Df/Db ratio are judged as leukocyte sperm disease;
3, sperm normal oxidation stress be worth: the logarithm value >0.9 of Dp/Dr ratio.
(4) statistical analysis
Use SAS9.1.3 software package to carry out statistical study
Three, result
From Fig. 2 A, in the situation that existing leucocyte to pollute, add after leucocyte specific probe fMLP, produce a ROS peak.Then add PMA, a lasting extensive chemical luminous signal of the common generation of sperm and leucocyte group.From Fig. 2 B, if do not have leucocyte to pollute, the ROS peak of fMLP reaction disappears, and only has PMA to cause a chemiluminescence signal being produced by sperm significantly.
1, generalized case and semen efamination result
The normal male sex's 166 examples of sterile, seminal parameters: their sperm concentration>=15 × 10
6/ ml, forward direction motile sperm rate>=32% or forward direction motile sperm rate+non-forward direction motile sperm rate>=40%, normal morphology rate>=4%.
The male sex's 234 examples of sterile, few asthénospermie: their sperm concentration>=or <15 × 10
6/ ml, forward direction motile sperm rate <32% is forward direction motile sperm rate+non-forward direction motile sperm rate <40% simultaneously, normal morphology rate>=4%.
Educate, seminal parameters normal male 230 examples: their sperm concentration>=15 × 10
6/ ml, forward direction motile sperm rate >32% is forward direction motile sperm rate+non-forward direction motile sperm rate >40% simultaneously, normal morphology rate>=4%.
Each group check result meets the grouping feature requirement of each group.
2, refining oxidative stress level
Calculate the logarithm value of Dr/Db ratio, carry out t inspection and can find, sterile group of crowd's refining oxidative stress value, apparently higher than normally educating group, has statistical significance (table 1).
Table 1* refining oxidative stress level
*: because of between two relatively time all ratio be normal distribution, otherness between any two relatively adopts " parametric test ".The normal sterile and few weak smart sterile group of comparison of seminal parameters, SIG > 0.05, does not have significant difference.Two sterile group respectively compared with can educating group, the equal < 0.05 of SIG, has significant difference.
3, leucocyte pollutes inspection situation
Calculate the logarithm value of Df/Dr ratio, and carry out can finding after t inspection, the value of leukocyte sperm disease, apparently higher than non-leukocyte sperm disease, has statistical significance; And the semen white blood cell of two sterile group pollution ratio is apparently higher than educating group, has statistical significance (table 2), this makes cell contamination clear and can cause the mobility of sperm to decline, and then causes sterile.
Table 2 semen white blood cell pollutes inspection situation
4, the oxidative stress ability of sperm
Calculate the logarithm value of Dp/Df ratio, add the logarithm of the ROS/ refining ROS of Sample producing after PMA, and carry out can finding after t inspection, can educate the PMA oxidative stress level of group apparently higher than sterile group, there is statistical significance (table 3).
The oxidative stress ability of table 3 sperm
*: because of between two relatively time all ratio be normal distribution, otherness between any two relatively adopts " parametric test ".The normal sterile and few weak smart sterile comparison of seminal parameters, the equal > 0.05 of SIG, does not have significant difference.Seminal parameters is normal sterile and few weak smart sterile respectively compared with can educating group, and the equal < 0.05 of SIG, has significant difference.Sperm motility in this normal sterile group of seminal parameters of explanation obviously declines, and is to cause sterile another one main cause.And in the normal infertile patients of free of contamination seminal parameters, the oxidative stress ability that almost all shows as sperm declines.
In embodiment 3 seminal fluid, leucocyte pollutes with the detection of sperm oxidative stress ability and tests 2
Get 50 μ l seminal fluid in 2.0ml centrifuge tube or in microwell plate micropore, detect 10 seconds, record data 1 are Db;
Add 25 μ l luminols and 50 μ l superoxide enzyme solutions, after room temperature fully mixes, 37 ° of C temperature are bathed and are left standstill 5 minutes, detect 10 seconds, and record data 2 are Dr;
Add 25 μ l fMLP in above-mentioned centrifuge tube, piping and druming gently, 37 ° of C temperature are bathed lucifuge and are left standstill 15 minutes, detect 10 seconds, and record data 3 are Df;
Add 25 μ l PMA in above-mentioned centrifuge tube, piping and druming gently, 37 ° of C temperature are bathed lucifuge and are left standstill 10 minutes, detect 10 seconds, and record data 4 are Dp.
Testing result is identical with the trend of 50 μ l.
Four, discuss
In normal fertility semen, leucocyte density is generally less than 1 × 10
6/ ml, wherein granulocyte accounts for 50～60%, and macrophage accounts for 20～30%.Leucocyte has panimmunity function, comprise and engulf lethal effect, antigen presentation utmost point secretion etc., in body, can play engulf and kill multiple pathogenic microorganisms, process the cell of senescense and damnification, by effective antigenic component submission to lymphocyte and effect such as secretion various bioactivators etc.Contained phagocyte in seminal fluid, what they were main act as engulfs foreign matter and bacterium, under pathologic condition, also can engulf sperm under the effect of AsAb, and sperm concentration and vigor are declined.In addition, can also have influence on sperm function by discharging ROS.
In seminal fluid, the leucocyte of excessive generation is the main contributions person of seminal fluid ROS, document shows, the active oxygen that leucocyte produces in stress situation, can reach a sperm identical produce in stress situation more than 100 times, so, even quantity of leucocyte is far below spermatoblast in seminal fluid, its ROS producing has a huge impact the chemiluminescence signal of sperm suspension.In addition, the rising of the active oxygen in seminal fluid is relevant with many factors, as smoking, and genital tract infection, varicocele, environmental pollution, the use of hormone etc.Active oxygen occurs to regulate eupyrene sperm function at low concentration, and viability and the function of high concentration ROS harm sperm.But in seminal fluid, also exist the purge mechanism of ROS, only have when the generation of ROS is during much larger than the removing ability of polyphenoils, just can cause the generation of oxidative stress.Therefore, measure merely seminal fluid reactive oxygen species and calculate leukocytic quantity and can not really reflect leukocytic oxidative stress ability in seminal fluid, with method described in the application or the disclosed leucocyte specific probe of kit formyl tripeptides (fMLP) induction leucocyte generation active oxygen, consider refining reactive oxygen species and sperm concentration simultaneously, also evaluate the impact of leucocyte on semen quality with the active oxygen index of leucocyte, can overcome now methodical limitation.
In this application, measure the level of refining ROS in seminal fluid by chemiluminescence method, pass through formyl tripeptides (fMLP) as the special probe of leucocyte simultaneously, stimulate leukocytic NADPH oxidation superoxide enzyme system to produce oxygen radical.With the active oxygen index of leucocyte, the active oxygen/refining active oxygen/sperm concentration also producing with fMLP, finds, this index value is higher, and the activity ratio of sperm is poorer, and each group difference is remarkable.In our research, also find leucocyte ROS index in azoospermia group, few asthénospermie group apparently higher than Sperm Parameters normal group, that is to say, in balance after the impact of refining ROS level and sperm concentration, the ROS that in azoospermia group, few asthénospermie group seminal fluid, leucocyte produces is apparently higher than Sperm Parameters normal group, this is also showing to lack asthénospermie and asthénospermie group, in seminal fluid, there is excessive leucocyte, and the ability that leucocyte produces ROS is stronger, may be the major reason that causes motility of sperm low.Visible, carry out the detection of leucocyte pollution, refining and sperm oxidative stress ability simultaneously, can identify exactly patients with infertility, improve recall rate, and give a clue for successive treatment.
In addition, the disclosed method of the application or kit can utilize the seminal fluid sample of smaller size smaller can complete leucocyte in seminal fluid and produce the detection of ROS and the analysis of sperm oxidative stress ability, make it possible to utilize limited seminal fluid sample to carry out revision test or other goal analysis, can reflect exactly again leucocyte pollution condition and the sperm oxidative stress ability of seminal fluid simultaneously.
Although be appreciated that the present invention is illustrated with certain form, the present invention is not limited to content shown in this instructions and that describe.It should be apparent to those skilled in the art that under the prerequisite that does not depart from scope of the present invention and also can make a variety of changes.These change all in the scope of protection of present invention.
Claims (10)
1. check a kit for individual seminal fluid oxidative stress, described kit comprises,
Luminol solution;
Superoxide enzyme solutions;
FMLP solution;
PMA solution; And
Optionally, positive reference substance and/or negative control product.
2. kit as claimed in claim 1, it comprises 10 μ l-100 μ l luminol solutions, superoxide enzyme solutions, 10 μ l-100 μ lFMLP solution and 10 μ l-100 μ l PMA solution of 10 μ l-100 μ l etc. that at least a single uses and detects reagent.
3. kit as described in claim 1-2 any one, the amount of luminol solution, superoxide enzyme solutions, FMLP solution and PMA solution that wherein said a single uses is 1/2 to 1 times that wish detects seminal fluid volume.
4. kit as described in claim 1-3 any one, when the seminal fluid wherein detecting when wish is 100 μ l, the consumption of luminol solution, superoxide enzyme solutions, FMLP solution and PMA solution that described a single uses is 50 μ l;
In the time that the seminal fluid of wish detection is 200 μ l, the consumption of luminol solution, superoxide enzyme solutions, FMLP solution and PMA solution that described a single uses is 100 μ l;
In the time that the seminal fluid of wish detection is 150 μ l, the consumption of luminol solution, superoxide enzyme solutions, FMLP solution and PMA solution that described a single uses is 75 μ l;
In the time that the seminal fluid of wish detection is 75 μ l, the consumption of luminol solution, superoxide enzyme solutions, FMLP solution and PMA solution that described a single uses is about 35-40 μ l; Or
In the time that the seminal fluid of wish detection is 50 μ l, the consumption of luminol solution, superoxide enzyme solutions, FMLP solution and PMA solution that described a single uses is 25 μ l.
5. kit as described in claim 1-4 any one, also comprises feminine gender and/or positive criteria product.
6. kit as described in claim 1-5 any one, also comprises and instructs the instructions that uses this kit.
7. kit as described in claim 1-6 any one, wherein said positive criteria product are that leucocyte pollutes reference substance, logarithm value >=1.25 of its Df/Db ratio claimed in claim 1 illustrate in described seminal fluid sample to be leukocyte sperm disease.
8. kit as described in claim 1-7 any one, wherein said positive criteria product are the standard items of reflection normal oxidation stress ability, the logarithm value of its Dr/Db ratio claimed in claim 1 is less than or equal to 0.75, and the logarithm value of Dp/Dr ratio is more than or equal to 0.9.
9. as claim 1-8 any one kit, wherein said individuality is selected from mammal or people, includes but not limited to people, primate, ox, horse, pig, sheep, goat, dog, cat and the rodent such as rat and mouse.
10. check a method for patient's seminal fluid oxidative stress, described method comprises,
Get 20 μ l-200 μ l seminal fluid samples after counting in sample hose, the luminous value data 1 that record sample are Db;
To the superoxide enzyme solutions (12.5IU/ml) that adds 10 μ l-100 μ l luminol solutions (2.5mM) and 10 μ l-100 μ l in 20 μ l-200 μ l seminal fluid samples, after reaction, obtain potpourri A, record the luminous value Dr of potpourri A;
In potpourri A, add 10 μ l-100 μ l FMLP solution (0.2mM), after reaction, obtain potpourri B, record the luminous value Df of potpourri B; And
In potpourri B, add 10 μ l-100 μ l PMA solution (0.4 μ M), after reaction, obtain mixture C, record the luminous value Dp of mixture C;
Can judge that according to the logarithm value of Dr/Db ratio whether this refining oxidative stress is normal, and/or determine according to the logarithm value of Df/Db ratio whether this seminal fluid is leukocyte sperm disease, and/or determine that according to the logarithm value of Dp/Dr ratio whether the oxidative stress value of sperm is normal; And
Optionally, described method is the method that implements the claims kit described in 1-9 any one.
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| CN106932597A (en) * | 2017-03-29 | 2017-07-07 | 山东大学 | Purposes of 1 lysine of generation mass shift of ATP5A1 protein 53s in the few weak smart diagnostic reagent of severe is prepared |
| CN106996981A (en) * | 2017-03-29 | 2017-08-01 | 山东大学 | Purposes of 6 N 114.04278 of AKAP4 protein 18s in the few weak smart diagnostic reagent of severe is prepared |
| CN106996980A (en) * | 2017-03-29 | 2017-08-01 | 山东大学 | Purposes of the lysine of 733 generation mass shifts of AKAP4 albumen in the few weak smart diagnostic reagent of severe is prepared |
| CN106996979A (en) * | 2017-03-29 | 2017-08-01 | 山东大学 | Purposes of 6 N 113.05347 of AKAP4 protein 18s in the few weak smart diagnostic reagent of severe is prepared |
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| CN106932597A (en) * | 2017-03-29 | 2017-07-07 | 山东大学 | Purposes of 1 lysine of generation mass shift of ATP5A1 protein 53s in the few weak smart diagnostic reagent of severe is prepared |
| CN106996981A (en) * | 2017-03-29 | 2017-08-01 | 山东大学 | Purposes of 6 N 114.04278 of AKAP4 protein 18s in the few weak smart diagnostic reagent of severe is prepared |
| CN106996980A (en) * | 2017-03-29 | 2017-08-01 | 山东大学 | Purposes of the lysine of 733 generation mass shifts of AKAP4 albumen in the few weak smart diagnostic reagent of severe is prepared |
| CN106996979A (en) * | 2017-03-29 | 2017-08-01 | 山东大学 | Purposes of 6 N 113.05347 of AKAP4 protein 18s in the few weak smart diagnostic reagent of severe is prepared |
| CN107015005A (en) * | 2017-03-29 | 2017-08-04 | 山东大学 | Purposes of the threonine of 64 generation mass shifts of GAPDHS albumen in the few weak smart diagnostic reagent of severe is prepared |
| CN106932597B (en) * | 2017-03-29 | 2018-07-20 | 山东大学 | Purposes of the lysine of 1 generation mass shift of ATP5A1 protein 53s in preparing the few weak smart diagnostic reagent of severe |
| CN106996981B (en) * | 2017-03-29 | 2018-11-02 | 山东大学 | Purposes of 6 N-114.04278 of AKAP4 protein 18s in preparing the few weak smart diagnostic reagent of severe |
| CN106996979B (en) * | 2017-03-29 | 2018-11-02 | 山东大学 | Purposes of 6 N-113.05347 of AKAP4 protein 18s in preparing the few weak smart diagnostic reagent of severe |
| CN110286080A (en) * | 2018-10-25 | 2019-09-27 | 中国科学院苏州生物医学工程技术研究所 | Ejaculated sperm cells rapid typing detection reagent box and detection method |
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