CN110286080A - Ejaculated sperm cells rapid typing detection reagent box and detection method - Google Patents
Ejaculated sperm cells rapid typing detection reagent box and detection method Download PDFInfo
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- CN110286080A CN110286080A CN201811250704.9A CN201811250704A CN110286080A CN 110286080 A CN110286080 A CN 110286080A CN 201811250704 A CN201811250704 A CN 201811250704A CN 110286080 A CN110286080 A CN 110286080A
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- 239000012981 Hank's balanced salt solution Substances 0.000 claims abstract description 28
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- DHNRXBZYEKSXIM-UHFFFAOYSA-N chloromethylisothiazolinone Chemical compound CN1SC(Cl)=CC1=O DHNRXBZYEKSXIM-UHFFFAOYSA-N 0.000 claims description 3
- BEGLCMHJXHIJLR-UHFFFAOYSA-N methylisothiazolinone Chemical compound CN1SC=CC1=O BEGLCMHJXHIJLR-UHFFFAOYSA-N 0.000 claims description 3
- BFMYDTVEBKDAKJ-UHFFFAOYSA-L disodium;(2',7'-dibromo-3',6'-dioxido-3-oxospiro[2-benzofuran-1,9'-xanthene]-4'-yl)mercury;hydrate Chemical compound O.[Na+].[Na+].O1C(=O)C2=CC=CC=C2C21C1=CC(Br)=C([O-])C([Hg])=C1OC1=C2C=C(Br)C([O-])=C1 BFMYDTVEBKDAKJ-UHFFFAOYSA-L 0.000 claims description 2
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1434—Optical arrangements
-
- 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
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Dispersion Chemistry (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a kind of Ejaculated sperm cells rapid typing detection reagent box, including PBS solution, without calcium HBSS buffer, fixer, staining reagent and quality-control product；The PBS solution and without including one of bovine serum albumin(BSA), fetal calf serum, glycerin blood serum, albumen, glycerol or a variety of in calcium HBSS buffer；The staining reagent includes cell-permeant agent and fluorescent labeled antibody.The present invention also provides a kind of methods for carrying out the detection of Ejaculated sperm cells fast typing using mentioned reagent box.The present invention can be used for androgone type and quantity in Ejaculated sperm cells and quickly judge, and then it is used for obstructive or Non-obstructive Azoospermia differentiation, change China at present clinically to aiming at the problem that clinical assessment of current Seminal cytology lacks quick and precisely detection reagent, guidance is provided when carrying out therapeutic scheme selection for clinician, solves the problems, such as patient conscientiously.
Description
Technical field
The present invention relates to medical detection field, in particular to a kind of Ejaculated sperm cells rapid typing detection reagent box and detection side
Method.
Background technique
Big data shows that China's reproduction age population is about 2.3 hundred million, wherein be about 40,000,000 with infertile population,
Male patient is about 12,000,000, the detection of these patients depends on parting and monitoring to Ejaculated sperm cells.
Sperm is also former thin containing seminaferous epithelium cast-off cells, urogenital tract cast-off cells etc., such as essence in addition to sperm
Born of the same parents, sperm mother cell, spermatoblast, sertoli cell, interstitial glands, epithelial cell, leucocyte etc..Azoospermia is suffered from
Person can judge whether to belong to obstructive or non-obstructivity without essence by androgone type, quantity in assessment Ejaculated sperm cells
Sub- disease.Currently, to the assessment of Seminal cytology by conventional film-making, dyeing, and colouring method mostly uses improvement Pasteur or fast
Speed dyeing.It is simple that cell type cannot quickly be identified by dyeing.Therefore, if can by cell surface Specific marker into
Row fluorescent marker, it will help quickly identify cell type and each cell by stages.
Flow cytometry (Flow Cytometry, FCM) is using flow cytometer as detection means, by target molecules
Specificity fluorescent label fast, accurately physicochemical property to individual cells (or particle) can carry out multi-parameter and quantitatively divide
The technology of analysis and sorting, thus be the most powerful to solve the above problems, but so far, there has been no related kits to go out
It is existing.
Summary of the invention
In view of the above-mentioned deficiencies in the prior art, the technical problem to be solved by the present invention is that providing a kind of Ejaculated sperm cells
Rapid typing detection reagent box and detection method.
In order to solve the above technical problems, the technical solution adopted by the present invention is that: a kind of Ejaculated sperm cells fast typing detection examination
Agent box, including PBS solution, without calcium HBSS buffer, fixer, staining reagent and quality-control product；
The PBS solution and without in calcium HBSS buffer include bovine serum albumin(BSA), fetal calf serum, glycerin blood serum, egg
One of white, glycerol is a variety of；
The staining reagent includes cell-permeant agent and fluorescent labeled antibody.
Preferably, the PBS solution further includes potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, potassium chloride and deionization
Water.
Preferably, the no calcium HBSS buffer further includes potassium chloride, potassium dihydrogen phosphate, sodium bicarbonate, sodium chloride, phosphorus
Sour disodium hydrogen, glucose, ethylenediamine tetra-acetic acid and deionized water.
It preferably, further include sodium hydroxide or hydrochloric acid in the no calcium HBSS buffer.
Preferably, the fluorescent labeled antibody is the antibody after fluorochrome label, for identifying cell type；
Wherein, the antibody include: for identification the CD45 of leucocyte, for identification EpCAM, CK18 of epithelial cell or
SCA, DAZL or TRA98 of CK19 and for identification spermatogonium；
Wherein, the fluorescent dye includes one of APC, FITC, PerCP, PI or a variety of, the fluorescent dye and anti-
Body can be matched arbitrarily.
Preferably, the quality-control product is chicken red blood cell.
Preferably, the fixer is -20 DEG C of warp treated ice ethyl alcohol overnight.
Preferably, further include in the fixer bovine serum albumin(BSA), fetal calf serum, glycerin blood serum, albumen, in glycerol
It is one or more.
It preferably, further include 5-Chloro-2-methyl-4-isothiazolin-3-one, the different thiophene of 2- methyl -4- in the PBS solution
Oxazoline -3- ketone, quaternary ammonium salt, TritonX-100 and dimethyl sulfoxide.
It is including following the present invention also provides a kind of method for carrying out the detection of Ejaculated sperm cells fast typing using mentioned reagent box
Step:
1) fresh spermatozoa sample is obtained, supernatant is removed in centrifugation, it is added without calcium HBSS buffer or containing the PBS solution of EDTA, then
It is centrifuged spare, what cannot be used immediately is fixed in fixer；
2) step 1) treated sample of sperm is adjusted in no calcium HBSS buffer after the washing of no calcium HBSS buffer
Whole end cell concentration, adds staining reagent, carries out cell dyeing；
3) it is detected using flow cytometer, to be identified to the cell type in sample of sperm, wherein using chicken
Red blood cell is as quality-control product.
The beneficial effects of the present invention are:
Ejaculated sperm cells rapid typing detection reagent box of the invention can be used for androgone type and quantity in Ejaculated sperm cells
Quickly judgement, and then it is used for obstructive or Non-obstructive Azoospermia differentiation, change China at present clinically to for mesh
The clinical assessment of preceding Seminal cytology lacks the problem of quick and precisely detection reagent, when carrying out therapeutic scheme selection for clinician
Guidance is provided, solves the problems, such as patient conscientiously.Domestic flow cytometer reagent can be altered in steps to import reagent in the present invention
It relies on, substantially reduces the application cost of flow cytometer.Kit of the invention can be stablized preservation 1 year or more at 4 DEG C, have
Good product development potential quality.The method provided by the invention for carrying out the detection of Ejaculated sperm cells fast typing using the kit has
The advantages of easily operated, repeated strong, cost is relatively low and high-throughput detection, full and accurate clinic can be provided for patient and patient
Data.
Detailed description of the invention
Fig. 1 is the spermatoblast of the fresh separated in a kind of embodiment of the invention；
Fig. 2 is chicken red blood cell (RBC) and testing result of times body cell in streaming in a kind of embodiment of the invention；
Fig. 3 is the Ejaculated sperm cells flow cytometer detection result in a kind of embodiment of the invention.
Specific embodiment
The present invention will be further described in detail below with reference to the embodiments, to enable those skilled in the art referring to specification
Text can be implemented accordingly.
It should be appreciated that such as " having ", "comprising" and " comprising " term used herein are not precluded one or more
The presence or addition of a other elements or combinations thereof.
A kind of Ejaculated sperm cells rapid typing detection reagent box of the present embodiment, including PBS solution, without calcium HBSS buffer,
Fixer, staining reagent and quality-control product.
The PBS solution and without in calcium HBSS buffer include bovine serum albumin(BSA), fetal calf serum, glycerin blood serum, egg
One of white, glycerol is a variety of, to prevent from being crushed in nuclear membrane earthquake.
Wherein, the PBS solution further includes potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, potassium chloride and deionized water.
Wherein, the no calcium HBSS buffer further includes potassium chloride, potassium dihydrogen phosphate, sodium bicarbonate, sodium chloride, phosphoric acid hydrogen
Disodium, glucose, ethylenediamine tetra-acetic acid and deionized water.It further include sodium hydroxide or hydrochloric acid in the no calcium HBSS buffer,
To adjust pH.
The staining reagent includes cell-permeant agent and fluorescent labeled antibody.The fluorescent labeled antibody is through fluorescent dye
Antibody after label, for identifying cell type；
Wherein, the antibody include: for identification the CD45 of leucocyte, for identification EpCAM, CK18 of epithelial cell or
SCA, DAZL or TRA98 of CK19 and for identification spermatogonium；The fluorescent dye includes APC (allophycocyanin), FITC
One of (fluorescein isothiocynate), PerCP (perdinin-Chlorophyll-protein complex), PI (propidium iodide) or
A variety of, the fluorescent dye can be matched arbitrarily with antibody.The fluorescence of other quantum dots with identical emission spectrum also may be selected
Dyestuff.Wherein, CD45: leukocyte common antigen, EpCAM: epithelial cell adhesion molecule, CK18/19: cytokeratin, SCA:
Spermatozoa-coating antigen, DAZL: spermatogenesis albumen, TRA98: sperm-specific antigen.The resolving method of each cell in sperm are as follows:
Spermatoblast or sperm correspond to 1 times of body, and first spermatocyte corresponds to 4 times of bodies, and leucocyte corresponds to CD45, and epithelial cell is corresponding
EpCAM, CK18 or CK19, spermatogonium correspond to SCA, DAZL or TRA98.
The quality-control product is chicken red blood cell.Chicken red blood cell detects peak on flow cytometer and kisses substantially with sperm monoploid peak
It closes, can be used for determining spermatoblast position.The specific production method of chicken red blood cell can be found in patent 201610242531.0.
Wherein, fixer can not contain aldehyde, because will lead to cell agglomerate.In an advantageous embodiment, fixer is warp -20
The ice ethyl alcohol that DEG C overnight treated ice ethyl alcohol, preferably volume fraction are 70%.It further include that ox blood is pure in the fixer
One of albumen, fetal calf serum, glycerin blood serum, albumen, glycerol are a variety of, can prevent from being crushed in nuclear membrane earthquake.
In one embodiment, PBS solution formula are as follows: potassium dihydrogen phosphate 0.27g, disodium hydrogen phosphate 1.42g, sodium chloride 8g
With potassium chloride 0.2g, add 1L deionized water dissolving, solution end PH is 7.4 ± 0.05；
Without calcium HBSS buffer formulation are as follows: potassium chloride 0.4g, potassium dihydrogen phosphate 0.06g, sodium bicarbonate 0.35g, sodium chloride
8g, disodium hydrogen phosphate 0.048g, glucose 1g and ethylenediamine tetra-acetic acid (EDTA) 0.29g, are dissolved in 1L deionized water, hydrogen-oxygen
Change sodium or hydrochloric acid solution adjusts PH to 7.4 ± 0.05；
Fixer is 70% ethyl alcohol, formula are as follows: 75ml ethyl alcohol is mixed with 25ml ultrapure water, is placed in -20 and is spent night.
In another embodiment, PBS solution and without further include in calcium HBSS buffer mass fraction be 1-10% ox
Seralbumin or fetal calf serum or glycerin blood serum, albumen or glycerol.
In another embodiment, further include in fixer mass fraction be 1-10% bovine serum albumin(BSA) or tire ox blood
Clear or glycerin blood serum, albumen or glycerol.
It in another embodiment, further include 5-Chloro-2-methyl-4-isothiazolin-3-one, 2- first in the PBS solution
Base -4- isothiazoline -3- ketone, quaternary ammonium salt, TritonX-100 and dimethyl sulfoxide.Wherein, 5- chloro-2-methyl -4- isothiazoline -
3- ketone has very strong inhibition and killing effect, and germicidal efficiency is high, and degradability is good, has and does not generate residual, safe operation, compatibility
The advantage that property is good, stability is strong, use cost is low；2-methyl-4-isothiazolin-3-one plays sterilization, sterilizing；5- is chloro-
2-methyl-4-isothiazolin-3-one, 2-methyl-4-isothiazolin-3-one and quaternary ammonium salt are used in compounding, and can reach enhancing sterilizing
The effect of ability and stability, to extend the reagent holding time.Dimethyl sulfoxide can improve cell permeability, and pH is kept to stablize.
TritonX-100 plays solubilising, promotes discrete, promotes cell dispersion, detects convenient for instrument, while reducing the table of dilution
Face tension to reduce the generation of bubble, give to measurement bring interference by elimination bubble.
It is including following the present invention also provides a kind of method for carrying out the detection of Ejaculated sperm cells fast typing using mentioned reagent box
Step:
1) fresh spermatozoa sample is obtained, supernatant is removed in centrifugation, it is added without calcium HBSS buffer or containing the PBS solution of EDTA, then
It is centrifuged spare, what cannot be used immediately is fixed in fixer；
2) step 1) treated sample of sperm is adjusted in no calcium HBSS buffer after the washing of no calcium HBSS buffer
Whole end cell concentration, adds staining reagent, carries out cell dyeing；
3) it is detected using flow cytometer, to be identified to the cell type in sample of sperm, wherein using chicken
Red blood cell is as quality-control product.
In one embodiment, the method for carrying out the detection of Ejaculated sperm cells fast typing using the kit, including following step
It is rapid:
1) fresh spermatozoa sample is obtained, every pipe 0.5-1ml is placed on ice chest and saves.3000rpm, 4 degrees Celsius of centrifugations
After 10min, supernatant being removed, the PBS of the EDTA containing 0.29g/L is added or being washed without calcium HBSS, it is spare to be repeated 1 times centrifugation, cannot be immediately
What is used is fixed on 70% ice ethyl alcohol being pre-chilled at -20 DEG C.
2) step 1) treated it is fresh or fixed after spermatoblast, after the washing of no calcium HBSS solution, in no calcium
It is 1 × 10 that whole cell concentration is adjusted in HBSS6/ ml, is added seriation staining reagent, and the staining reagent includes cell-permeant agent
(triton x-100 of 0.1-0.5%) and fluorescent labeled antibody.The antibody include: for identification the CD45 of leucocyte, be used for
EpCAM, CK18 or CK19 of identification epithelial cell and for identification SCA, DAZL or TRA98 of spermatogonium；The fluorescence dye
Material includes APC, FITC, PerCP, PI or the quantum dot with identical emission spectrum.Fluorescent dye can be matched arbitrarily with antibody.Ginseng
It is fluorescence excitation and the emission spectrum of each fluorescent marker according to table 1.
Table 1
3) it is detected using flow cytometer, to be identified to the cell type in sample of sperm, wherein using chicken
For red blood cell as quality-control product, quality-control product detects peak and sperm monoploid peak after ibid staining procedure on flow cytometer
Substantially it coincide, can be used for determining spermatoblast position.It is chicken red blood cell (RBC) and inspection of times body cell in streaming referring to Fig. 2
Survey result.The resolving method of each cell in sperm are as follows: spermatoblast or sperm correspond to 1 times of body, and first spermatocyte corresponds to 4
Times body, leucocyte correspond to CD45, and epithelial cell corresponds to EpCAM, CK18 or CK19, and spermatogonium corresponds to SCA, DAZL or TRA98.
It is Ejaculated sperm cells flow cytometer detection result referring to Fig. 3.
Although the embodiments of the present invention have been disclosed as above, but its is not only in the description and the implementation listed
With it can be fully applied to various fields suitable for the present invention, for those skilled in the art, can be easily
Realize other modification, therefore without departing from the general concept defined in the claims and the equivalent scope, the present invention is simultaneously unlimited
In specific details.
Claims (10)
1. a kind of Ejaculated sperm cells rapid typing detection reagent box, which is characterized in that including PBS solution, without calcium HBSS buffer, solid
Determine liquid, staining reagent and quality-control product；
The PBS solution and without include bovine serum albumin(BSA) in calcium HBSS buffer, it is fetal calf serum, glycerin blood serum, albumen, sweet
One of oil is a variety of；
The staining reagent includes cell-permeant agent and fluorescent labeled antibody.
2. Ejaculated sperm cells rapid typing detection reagent box according to claim 1, which is characterized in that the PBS solution is also
Including potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, potassium chloride and deionized water.
3. Ejaculated sperm cells rapid typing detection reagent box according to claim 2, which is characterized in that the no calcium HBSS is slow
Fliud flushing further include potassium chloride, potassium dihydrogen phosphate, sodium bicarbonate, sodium chloride, disodium hydrogen phosphate, glucose, ethylenediamine tetra-acetic acid and
Deionized water.
4. Ejaculated sperm cells rapid typing detection reagent box according to claim 3, which is characterized in that the no calcium HBSS is slow
It further include sodium hydroxide or hydrochloric acid in fliud flushing.
5. Ejaculated sperm cells rapid typing detection reagent box according to claim 4, which is characterized in that the fluorescent marker is anti-
Body is the antibody after fluorochrome label, for identifying cell type；
Wherein, the antibody includes: EpCAM, CK18 or CK19 of the CD45 of leucocyte, epithelial cell for identification for identification
And SCA, DAZL or TRA98 of spermatogonium for identification；
Wherein, the fluorescent dye includes one of APC, FITC, PerCP, PI or a variety of, and the fluorescent dye and antibody can
Any matching.
6. Ejaculated sperm cells rapid typing detection reagent box according to claim 5, which is characterized in that the quality-control product is chicken
Red blood cell.
7. Ejaculated sperm cells rapid typing detection reagent box according to claim 1, which is characterized in that the fixer is
Through -20 DEG C of treated ice ethyl alcohol overnight.
8. Ejaculated sperm cells rapid typing detection reagent box according to claim 7, which is characterized in that in the fixer also
Including one of bovine serum albumin(BSA), fetal calf serum, glycerin blood serum, albumen, glycerol or a variety of.
9. Ejaculated sperm cells rapid typing detection reagent box according to claim 2, which is characterized in that in the PBS solution
Further include 5-Chloro-2-methyl-4-isothiazolin-3-one, 2-methyl-4-isothiazolin-3-one, quaternary ammonium salt, TritonX-100 and
Dimethyl sulfoxide.
10. a kind of kit using as described in any one of claim 1-9 carries out the detection of Ejaculated sperm cells fast typing
Method, which comprises the following steps:
1) fresh spermatozoa sample is obtained, supernatant is removed in centrifugation, is added without calcium HBSS buffer or containing the PBS solution of EDTA, then be centrifuged
Spare, what cannot be used immediately is fixed in fixer；
2) step 1) treated sample of sperm adjusts eventually in no calcium HBSS buffer after the washing of no calcium HBSS buffer
Cell concentration adds staining reagent, carries out cell dyeing；
3) it is detected using flow cytometer, to be identified to the cell type in sample of sperm, wherein red thin using chicken
Born of the same parents are as quality-control product.
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