CN115232141A - Fluorescent dye for detecting male sperm quality and application thereof - Google Patents
Fluorescent dye for detecting male sperm quality and application thereof Download PDFInfo
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- CN115232141A CN115232141A CN202211025958.7A CN202211025958A CN115232141A CN 115232141 A CN115232141 A CN 115232141A CN 202211025958 A CN202211025958 A CN 202211025958A CN 115232141 A CN115232141 A CN 115232141A
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- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 47
- 150000001875 compounds Chemical class 0.000 claims abstract description 15
- 238000001514 detection method Methods 0.000 claims abstract description 8
- 150000003839 salts Chemical class 0.000 claims abstract description 8
- 210000000582 semen Anatomy 0.000 claims description 21
- 238000005406 washing Methods 0.000 claims description 21
- 238000010186 staining Methods 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 239000012153 distilled water Substances 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 9
- 238000002156 mixing Methods 0.000 claims description 8
- -1 nitro, hydroxy Chemical group 0.000 claims description 6
- 229910052739 hydrogen Inorganic materials 0.000 claims description 5
- 239000001257 hydrogen Substances 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- 125000000217 alkyl group Chemical group 0.000 claims description 4
- 229910052736 halogen Inorganic materials 0.000 claims description 4
- 150000002367 halogens Chemical class 0.000 claims description 4
- 150000002431 hydrogen Chemical class 0.000 claims description 4
- 238000004043 dyeing Methods 0.000 claims description 3
- 230000000694 effects Effects 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims 1
- 239000000243 solution Substances 0.000 description 22
- 210000000170 cell membrane Anatomy 0.000 description 18
- 239000000975 dye Substances 0.000 description 14
- 230000002438 mitochondrial effect Effects 0.000 description 11
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 7
- 210000003470 mitochondria Anatomy 0.000 description 7
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 6
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 6
- 239000007853 buffer solution Substances 0.000 description 6
- 238000007865 diluting Methods 0.000 description 6
- 239000011550 stock solution Substances 0.000 description 6
- 239000000725 suspension Substances 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 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 description 4
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 4
- 230000004899 motility Effects 0.000 description 3
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000005138 cryopreservation Methods 0.000 description 2
- 239000012074 organic phase Substances 0.000 description 2
- 238000001303 quality assessment method Methods 0.000 description 2
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical class O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- NJYFRQQXXXRJHK-UHFFFAOYSA-N (4-aminophenyl) thiocyanate Chemical compound NC1=CC=C(SC#N)C=C1 NJYFRQQXXXRJHK-UHFFFAOYSA-N 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- DZBUGLKDJFMEHC-UHFFFAOYSA-N benzoquinolinylidene Natural products C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 1
- 238000004820 blood count Methods 0.000 description 1
- 125000001309 chloro group Chemical group Cl* 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000035558 fertility Effects 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 125000001153 fluoro group Chemical group F* 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000013441 quality evaluation Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
- C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
- C07D493/10—Spiro-condensed systems
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B57/00—Other synthetic dyes of known constitution
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- 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—Electro-optical investigation, e.g. flow cytometers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1003—Carbocyclic compounds
- C09K2211/1007—Non-condensed systems
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/302—Stain compositions
-
- 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
- G01N2015/1006—Investigating individual particles for cytology
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
Abstract
The invention relates to a fluorescent dye for detecting male sperm quality and application thereof, in particular to a compound shown as a formula (I) or a salt thereof. The detection method provided by the invention has the advantages of stronger objectivity, higher accuracy and higher precision in microscopic examination of sperm quality.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a fluorescent dye for detecting male sperm quality and application thereof.
Background
Sperm quality assessment is an important indicator of male fertility. The traditional sperm quality evaluation technology adopts a phase contrast microscope and a special blood cell counting plate, and is observed by naked eyes, and mainly evaluates and analyzes the number of sperms, the motility of the sperms and the morphology of the sperms. In the last two decades, with the continuous progress of computer technology, a special analysis software combined with a high resolution image acquisition device, namely a computer aided sperm analysis system (CASA), has been developed clinically, and the quality of the sperm is identified by analyzing the movement track, speed and dynamic parameters of the sperm, so that the research on the quality of the sperm is greatly improved. Although the CASA can measure parameters such as sperm density and motility, the CASA has certain limitations, and cannot detect the change of the internal structure of the sperm, particularly, the sperm subjected to cryopreservation or stress is easy to damage due to plasma membrane integrity and mitochondrial activity, so that the quality of the sperm is obviously influenced. In the last decade, a fluorescent CASA technology combining a fluorescent microscope and a computer technology appears, and the fluorescent CASA technology has the advantages that acridine orange nucleic acid fluorescent dye is adopted to dye sperm DNA, sperm cells can be well distinguished from impurities, and the accuracy of sperm cell counting is improved. Although the CASA can measure parameters such as sperm density and motility, the CASA has certain limitations, and cannot detect the change of the internal structure of the sperm, particularly, the sperm subjected to cryopreservation or stress is easy to damage due to plasma membrane integrity and mitochondrial activity, so that the quality of the sperm is obviously influenced.
Whether conventional or computer-assisted sperm quality assessment methods (including fluorescent CASA) are used, it is not possible to assess the quality of all sperm cells. Therefore, a new fluorescent dye and a more precise sperm quality detection method need to be found, so that the accuracy and precision of the experiment can be improved, and the sperm quality can be conveniently, rapidly and objectively detected.
Disclosure of Invention
The invention aims to solve the defects in the prior art, and provides a fluorescent dye for detecting the quality of male sperms and application thereof.
In order to achieve the above object, the present invention provides the following technical solutions.
In a first aspect, the present invention provides a compound of formula (I), or a salt thereof:
wherein:
R 1 、R 2 each independently selected from hydrogen, C 1-6 Alkyl, nitro, hydroxy and halogen.
In some preferred embodiments, the compounds of the invention are of formula (I), or a salt thereof, wherein:
R 1 、R 2 each independently selected from hydrogen and C 1-3 Alkyl, nitro, hydroxy and halogen;
further preferably, R 1 、R 2 Each independently selected from hydrogen, methyl, ethyl, propyl, nitro, hydroxy, fluoro and chloro.
In some embodiments, the compound of formula (I) is the following compound:
the invention also provides application of the compound shown in the formula (I) or the salt thereof in preparing a fluorescent dye for detecting the quality of male sperms.
In a third aspect, the present invention provides a method for detecting male sperm quality by using a fluorescent dye represented by formula (I), which comprises the following steps:
(1) Preparing a fluorescent dye: respectively dissolving the fluorescent dye shown in the formula (I) and the Syto16 in distilled water, and storing;
(2) Staining semen: mixing the fluorescent dye shown in the formula (I) and Syto16 with semen to be detected for dyeing;
(3) And (3) detection: detecting the sperms in the step (2) by adopting a flow cytometer;
wherein the fluorescent dye shown in the formula (I) has a structure shown in the formula (I):
further preferably, the step (1) is specifically: respectively dissolving the fluorescent dye shown in the formula (I) and the Syto16 in distilled water to prepare storage liquid with the concentration of 1mg/mL, and storing at-20 ℃ according to the storage requirement of the reagent.
Further preferably, the semen to be detected in the step (2) is a fresh liquefied human semen sample, and is prepared by washing with BSA-PBS buffer solution.
Further preferably, when the fluorescent dye shown in the formula (I) and the Syto16 are mixed with the semen to be detected in the step (2), the sequence of adding the fluorescent dye has no influence.
Further preferably, the dyeing time in the step (2) is 3-10min.
Further preferably, the step (2) further comprises the steps of placing, centrifuging, washing and resuspending after the staining; more preferably, the step (2) specifically includes diluting the two storage solutions of the fluorescent dye and Syto16 shown in formula (I) to 100 μ g/mL, taking a certain amount of semen, adding the two dye solutions, mixing (the order of adding the dye solutions has no influence), so that the final concentration of the dye solution of the sample to be detected is 10 μ g/mL, placing in the dark at room temperature for 3-10min, centrifuging (300 × g,5 min), washing with 0.01M PBS (ph 7.4), repeatedly washing and centrifuging for 2~3 times, resuspending the PBS, and detecting by a flow cytometer.
Further preferably, the flow cytometer parameters of step (3) are: forward scattering FSC and side scattering SSC are linearly amplified, fluorescence channels FL1 and FL2 are logarithmically amplified, the fluorescence intensity of each sperm Syto16 and the fluorescent dye shown in the formula (I) is measured, and about 10000 to 20000 sperms are detected in each suspension.
Compared with the prior art, the invention has the remarkable advantages that:
firstly, the invention designs and synthesizes the fluorescent dye shown in the formula (I) with a novel structure, combines the fluorescent dye shown in the formula (I) with the Syto16 staining and flow cytometry to detect the sperm quality, compared with the FITC and the Syto16 staining, the detection method of the invention detects more sperm quantity, can accurately detect the survival ratio of the sperm, conveniently and quickly distinguishes the sperm in each state under the condition of not damaging the sperm, and further objectively and accurately evaluates the sperm quality.
Secondly, the invention can obtain a plurality of parameters through one-time detection, not only can clearly distinguish sperms in different states and display the proportions of the sperms to obtain the death rate, the survival rate, the plasma membrane damage rate and the mitochondria activity proportion of the sperms, but also can avoid the phenomenon of lower precision rate caused by subjective factors, simultaneously reduce the influence of human factors on the detection result and ensure the accuracy and precision of the detection result.
Detailed Description
The following representative examples are intended to better illustrate the invention and are not intended to limit the scope of the invention. The materials used in the following examples are all commercially available unless otherwise specified.
EXAMPLE 1 preparation of the fluorescent dye FITC-TRI
In a reaction flask, fluorescein isothiocyanate (FITC, 3210mg, 8.24mmol) was dissolved in N, N-dimethylformamide (100 mL), and triethylamine (1.14mL, 8.24mmol), p-thiocyanoaniline (CAS number: 15191-25-0, 1125mg, 7.49mmol) were added to the solution. The reaction was left overnight in the dark and at room temperature with constant stirring. Subsequently, water (100 mL) was added and extracted with ethyl acetate (100 mL. Times.2), the organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated to give the fluorescent dye FITC-TRI. ESI-MS m/z =540.1[ 2 ] M +1] + 。
EXAMPLE 2 preparation of fluorescent dye FITC-NO
In a reaction flask, fluorescein isothiocyanate (FITC, 3210mg, 8.24mmol) was dissolved in N, N-dimethylformamide (100 mL), and triethylamine (1.14mL, 8.24mmol), 2-nitro-4-thiocyananilide (CAS No.: 54029-45-7, 1462mg, 7.49mmol) were added to the solution. The reaction was left overnight in the dark and at room temperature with constant stirring. Subsequently, water (100 mL) was added and extracted with ethyl acetate (100 mL. Times.2), the organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated to give the fluorescent dye FITC-NO. ESI-MS m/z =585.1[ 2 ] M +1] + 。
EXAMPLE 3 preparation of the fluorescent dye FITC-M1
The title compound was obtained by the same procedure as in example 1 except for substituting paracyanoanilide for 2-methyl-4-thiocyanoanilide (CAS No.: 99055-48-8). ESI-MS m/z =554.1[ 2 ] M +1] + 。
EXAMPLE 4 preparation of fluorescent dye FITC-M2
The title compound was obtained by the same procedure as in example 1 except for substituting paracyanoanilide for 3-methyl-4-thiocyanoanilide (CAS No.: 1087413-12-4). ESI-MS m/z =554.1[ 2 ] M +1] + 。
Experimental example 1
Collecting fresh liquefied human semen sample, washing with BSA-PBS buffer solution, and adjusting sperm cell number to 10 in reaction tube 7 Per mL;
preparing a fluorescent dye: the fluorescent dye prepared in example 1 and Syto16 were dissolved in distilled water, respectively, to prepare 1mg/mL stock solutions, which were stored at-20 ℃.
Staining semen: diluting the two storage solutions to 100 mu g/mL, taking 160 mu L of sperms with salinity stress of 15min, 30min, 1h, 2h and 4h respectively, placing the 160 mu L of sperms in a 1.5 mL centrifuge tube, adding 20 mu L of two dye solutions respectively, mixing to ensure that the final concentration of the dye solution of the sample to be detected is 10 mu g/mL, placing the sample to be detected in the dark at room temperature for 10min, centrifuging (300 Xg, 5 min), washing with 0.01M PBS (pH7.4), repeatedly washing and centrifuging for 2 times, resuspending the PBS, and detecting by a flow cytometer.
Flow cytometer parameter setting: forward scatter FSC and side scatter SSC were amplified linearly, fluorescence channels FL1 and FL3 were amplified logarithmically, and the fluorescence intensity of the fluorescent dye prepared in example 1 and Syto16 was measured for each sperm, with approximately 20000 sperm detected per suspension.
FCM test results are shown in table 1: the sperm stained with the fluorochrome or Syto16 prepared in example 1 and detected by FCM were distributed in different quadrants, R1 representing dead sperm with plasma membrane disruption and mitochondrial inactivity; r2 represents sperm with disrupted plasma membrane but still active mitochondria, i.e. about to die; r3 represents sperm with normal plasma membrane but no mitochondrial transmembrane potential; r4 represents normal viable sperm.
TABLE 1
Experimental example 2
Collecting fresh liquefied human semen sample, washing with BSA-PBS buffer solution, and adjusting sperm cell number to 10 in reaction tube 7 Per mL;
preparing a fluorescent dye: the fluorescent dye prepared in example 2 and Syto16 were dissolved in distilled water to prepare 1mg/mL stock solutions, which were stored at-20 ℃.
Staining semen: diluting the two storage solutions to 100 mu g/mL, taking 160 mu L of sperms with salinity stress of 15min, 30min, 1h, 2h and 4h respectively, placing the 160 mu L of sperms in a 1.5 mL centrifuge tube, adding 20 mu L of two dye solutions respectively, mixing to ensure that the final concentration of the dye solution of the sample to be detected is 10 mu g/mL, placing the sample to be detected in the dark at room temperature for 10min, centrifuging (300 Xg, 5 min), washing with 0.01M PBS (pH7.4), repeatedly washing and centrifuging for 2 times, resuspending the PBS, and detecting by a flow cytometer.
Flow cytometer parameter setting: forward scatter FSC and side scatter SSC were amplified linearly, fluorescence channels FL1 and FL3 were amplified logarithmically, and the fluorescence intensity of the fluorescent dye prepared in example 2 and Syto16 was measured for each sperm, with approximately 20000 sperm per suspension.
The FCM test results are shown in table 2: the sperm stained with the fluorochrome or Syto16 prepared in example 2 and detected by FCM were distributed in different quadrants, R1 represents dead sperm with plasma membrane disruption and mitochondrial inactivity; r2 represents sperm with disrupted plasma membrane but still active mitochondria, i.e. about to die; r3 represents sperm with normal plasma membrane but no mitochondrial transmembrane potential; r4 represents normal viable sperm.
TABLE 2
Experimental example 3
Collecting fresh liquefied human semen sample, washing with BSA-PBS buffer solution, and adjusting sperm cell number to 10 in reaction tube 7 Per mL;
preparing a fluorescent dye: the fluorescent dye prepared in example 3 and Syto16 were dissolved in distilled water, respectively, to prepare 1mg/mL stock solutions, which were stored at-20 ℃.
Staining semen: diluting the two storage solutions to 100 mu g/mL, taking 160 mu L of sperms stressed by salinity for 15min, 30min, 1h, 2h and 4h respectively, placing the 160 mu L sperms in a 1.5 mL centrifuge tube, adding 20 mu L sperms respectively into the two dye solutions, mixing to ensure that the final dye solution concentration of a sample to be detected is 10 mu g/mL, placing the sample to be detected in a dark place for 10min at room temperature, centrifuging (300 Xg, 5 min), washing with 0.01M PBS (pH7.4), repeatedly washing and centrifuging for 2 times, resuspending the PBS, and detecting by a flow cytometer.
Flow cytometer parameter setting: forward scatter FSC and side scatter SSC were amplified linearly, fluorescent channels FL1 and FL3 were amplified logarithmically, and the fluorescent intensity of the fluorescent dye prepared in example 3 and Syto16 was measured for each sperm, with approximately 20000 sperm per suspension.
The FCM test results are shown in table 3: the sperm stained with the fluorochrome or Syto16 prepared in example 3 and detected by FCM were distributed in different quadrants, R1 represents dead sperm with plasma membrane disruption and mitochondrial inactivity; r2 represents sperm with disrupted plasma membrane but still active mitochondria, i.e. about to die; r3 represents sperm with normal plasma membrane but no mitochondrial transmembrane potential; r4 represents normal viable sperm.
TABLE 3
Experimental example 4
Taking a fresh liquefied human semen sample, washing the sample by BSA-PBS buffer solution, and adjusting the number of spermatids to be 10 in a reaction test tube 7 Per mL;
preparing a fluorescent dye: the fluorescent dye prepared in example 4 and Syto16 were dissolved in distilled water, respectively, to prepare 1mg/mL stock solutions, which were stored at-20 ℃.
Staining semen: diluting the two storage solutions to 100 mu g/mL, taking 160 mu L of sperms with salinity stress of 15min, 30min, 1h, 2h and 4h respectively, placing the 160 mu L of sperms in a 1.5 mL centrifuge tube, adding 20 mu L of two dye solutions respectively, mixing to ensure that the final concentration of the dye solution of the sample to be detected is 10 mu g/mL, placing the sample to be detected in the dark at room temperature for 10min, centrifuging (300 Xg, 5 min), washing with 0.01M PBS (pH7.4), repeatedly washing and centrifuging for 2 times, resuspending the PBS, and detecting by a flow cytometer.
Flow cytometer parameter setting: forward scatter FSC and side scatter SSC were amplified linearly, fluorescence channels FL1 and FL3 were amplified logarithmically, and the fluorescence intensity of the fluorescent dye prepared in example 4 and Syto16 was measured for each sperm, and approximately 20000 sperm were detected for each suspension.
The FCM test results are shown in table 4: the sperm stained with the fluorochrome or Syto16 prepared in example 4 and detected by FCM were distributed in different quadrants, R1 representing dead sperm with plasma membrane disruption and mitochondrial inactivity; r2 represents sperm with disrupted plasma membrane but still active mitochondria, i.e. about to die; r3 represents sperm with normal plasma membrane but no mitochondrial transmembrane potential; r4 represents normal viable sperm.
TABLE 4
Comparative example
Collecting fresh liquefied human semen sample, washing with BSA-PBS buffer solution, and adjusting sperm cell number to 10 in reaction tube 7 Per mL;
preparing a fluorescent dye: FITC and Syto16 were dissolved in distilled water to prepare a stock solution with a concentration of 1mg/mL, and the stock solution was stored at-20 ℃.
Staining semen: diluting the two storage solutions to 100 mu g/mL, taking 160 mu L of sperms with salinity stress of 15min, 30min, 1h, 2h and 4h respectively, placing the 160 mu L of sperms in a 1.5 mL centrifuge tube, adding 20 mu L of two dye solutions respectively, mixing to ensure that the final concentration of the dye solution of the sample to be detected is 10 mu g/mL, placing the sample to be detected in the dark at room temperature for 10min, centrifuging (300 Xg, 5 min), washing with 0.01M PBS (pH7.4), repeatedly washing and centrifuging for 2 times, resuspending the PBS, and detecting by a flow cytometer.
Flow cytometer parameter setting: forward scatter FSC and side scatter SSC were amplified linearly, fluorescence channels FL1 and FL3 were logarithmically amplified, and fluorescence intensity was measured for FITC and Syto16 for each sperm, and for each suspension, approximately 20000 sperm were detected.
The FCM test results are shown in table 5: the sperm stained with FITC or Syto16 and detected by FCM are distributed in different quadrants, R1 represents dead sperm with plasma membrane destroying mitochondria and without activity; r2 represents sperm with disrupted plasma membrane but still active mitochondria, i.e. about to die; r3 represents sperm with normal plasma membrane but no mitochondrial transmembrane potential; r4 represents normal viable sperm.
TABLE 5
Although the present invention has been described in detail above, those skilled in the art will appreciate that various modifications and changes can be made to the present invention without departing from the spirit and scope of the invention. The scope of the invention is not to be limited by the above detailed description but is only limited by the claims.
Claims (10)
1. A compound represented by the formula (I):
wherein:
R 1 、R 2 each independently selected from hydrogen, C 1-6 Alkyl, nitro, hydroxy and halogen.
2. The compound of formula (I), or a salt thereof, as claimed in claim 1, wherein R is 1 、R 2 Each independently selected from hydrogen, C 1-3 Alkyl, nitro, hydroxy and halogen.
3. The compound of formula (I), or a salt thereof, according to claim 1, wherein the compound of formula (I) is the following compound:
4. Use of a compound of formula (I), or a salt thereof, according to any one of claims 1-3, in the preparation of a fluorescent dye for detecting male sperm quality.
5. A method for detecting the quality of male sperm using a compound represented by the formula (I) or a salt thereof as described in any one of claims 1 to 3, comprising the steps of:
(1) Preparing a fluorescent dye: respectively dissolving the fluorescent dye shown in the formula (I) and the Syto16 in distilled water, and storing;
(2) Staining semen: mixing the fluorescent dye shown in the formula (I) and Syto16 with semen to be detected for dyeing;
(3) And (3) detection: detecting the sperms in the step (2) by adopting a flow cytometer;
wherein the fluorescent dye represented by the formula (I) has the structure shown in claim 1.
6. A method of detecting sperm quality as described in claim 5, wherein said step (1) is specifically a step of: respectively dissolving the fluorescent dye shown in the formula (I) and the Syto16 in distilled water to prepare storage liquid with the concentration of 1mg/mL, and storing at-20 ℃ according to the storage requirement of the reagent.
7. A method of detecting sperm quality as described in claim 5, wherein the semen to be detected in step (2) is a freshly liquefied human semen sample prepared by washing with BSA-PBS buffer.
8. A method of measuring sperm quality as described in claim 5, wherein the fluorescent dye of formula (I) and Syto16 are mixed with the semen being examined in step (2) without any effect on the order of addition of the fluorescent dye.
9. A method of detecting sperm cell quality as described in claim 5, wherein said staining time of step (2) is between 3 and 10min.
10. A method of detecting sperm cell quality as described in claim 5, wherein said staining of step (2) is followed by the further steps of placement, centrifugation, washing, and resuspension.
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Citations (5)
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|---|---|---|---|---|
| US7070917B1 (en) * | 1999-03-05 | 2006-07-04 | Preben Christensen | Determination of sperm concentration and viability for artificial insemination |
| US20080199430A1 (en) * | 2007-02-16 | 2008-08-21 | Dyn-Bioshaf (2006) Ltd. | Methods and kits for qualifying sperm cells |
| CN101762684A (en) * | 2008-12-24 | 2010-06-30 | 上海市计划生育科学研究所 | Method for extracorporeally detecting survival rate of sperms |
| CN108398407A (en) * | 2018-01-25 | 2018-08-14 | 迈克博（天津）生物科技有限公司 | A kind of human sperm's motility rate and its kit of the two-parameter detection of DNA damage and analysis |
| CN111879686A (en) * | 2020-08-03 | 2020-11-03 | 贵州大学 | Method for detecting semen quality of mammal by flow cytometry |
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- 2022-08-25 CN CN202211025958.7A patent/CN115232141A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7070917B1 (en) * | 1999-03-05 | 2006-07-04 | Preben Christensen | Determination of sperm concentration and viability for artificial insemination |
| US20080199430A1 (en) * | 2007-02-16 | 2008-08-21 | Dyn-Bioshaf (2006) Ltd. | Methods and kits for qualifying sperm cells |
| CN101762684A (en) * | 2008-12-24 | 2010-06-30 | 上海市计划生育科学研究所 | Method for extracorporeally detecting survival rate of sperms |
| CN108398407A (en) * | 2018-01-25 | 2018-08-14 | 迈克博（天津）生物科技有限公司 | A kind of human sperm's motility rate and its kit of the two-parameter detection of DNA damage and analysis |
| CN111879686A (en) * | 2020-08-03 | 2020-11-03 | 贵州大学 | Method for detecting semen quality of mammal by flow cytometry |
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