CN108007754A - A kind of one step decoration method of cast-off cells, dye combinations used and kit - Google Patents
A kind of one step decoration method of cast-off cells, dye combinations used and kit Download PDFInfo
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- CN108007754A CN108007754A CN201610929313.4A CN201610929313A CN108007754A CN 108007754 A CN108007754 A CN 108007754A CN 201610929313 A CN201610929313 A CN 201610929313A CN 108007754 A CN108007754 A CN 108007754A
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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
- 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
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- 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
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
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- General Health & Medical Sciences (AREA)
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Abstract
The invention belongs to cell pathology field, and in particular to a kind of one step decoration method of cast-off cells, dye combinations used and kit.The one step dyeing, which refers to, synchronizes the nucleus and cytoplasm of cast-off cells dyeing, including carries out a step dyeing to cast-off cells using fluorochrome combinations, and the fluorochrome combinations contain nucleus fluorescent dye and cytoplasm fluorescent dye.The present invention uses fluorochrome combinations to synchronize dyeing to the nucleus and cytoplasm of cast-off cells, and need not clean background fluorescence, so as to realize rapid dyeing and detection.The present invention is excited using single ultraviolet source and is obtained good effect, can be simplified light source and be avoided irradiating the complexity to form image twice.
Description
Technical field
The invention belongs to cell pathology field, and in particular to a kind of one step decoration method of cast-off cells, dye combinations used
And kit.
Background technology
Exfoliative cytology inspection refers to the epithelial cell that collection body lumen organ surface comes off, and microscope is used after dyed
Observe its form and make diagnosis.It is widely used in precancerous lesion examination.The main source of cast-off cells has：Detachment of cervix
Cell, dropsy of serous cavity cast-off cells and urinary system cast-off cells.
It is to utilize the biochemical composition of various structures in cell different that conventional Off, which falls cell dyeing method, with acidophilia or thermophilic alkali
Dyestuff reacts and shows different colors, and the form and structure for making cell are easily recognized.It is common to have Pasteur and Rui-Ji Shi dyes
Color.
Papanicolaou's vaginal smear technique：This decoration method is most common method in cast-off cells dyeing.Its feature is nuclear structures
Clearly, cytoplasm color separation is obvious, and transparency is good, and endochylema is bright-colored.And sample is not easy to decolourize, easy to preserve for a long time.Pasteur
Dyeing is not notable to DNA content change in infantile tumour nucleus.So the tumour patient confirmed at present with microscope is mostly evening
Phase.It is insensitive that its main cause is that pap staining increases the intracellular minim DNA of infantile tumour.Papanicolaou staining process is complicated.Together
When, the experience and visual fatigue of doctor also influence quality and the judgement of diagosis.
Auspicious-Ji's Albert'stain Albert method：This decoration method colours preferably nucleus using Ji Shi methods, but cytoplasm coloring is poor.It is auspicious
Family name's method endochylema color tinted clear.Mutual deficiency can be made up after both are compound.The shortcomings that auspicious Ji's Albert'stain Albert is display cell color
Difference is weak, easily dyes fuzzy color group, makes Cell microstructure be not easy to recognize.Dyeing course needs 30 minutes.
Chromosome quantitative and DNA content reflection cell growth and differentiation state in cell, measure the change pair of DNA content
Judge the differentiation of cell, the property of tumour and prognosis are of great significance.Carrying out cast-off cells DNA content (times body) detection
When, the quality of nucleus DNA dyeing quality directly affects the accuracy of diagnosis.
Feulgen decoration methods：This decoration method is a kind of more method to nucleus DNA dyeing of application at present.The party
Method has the characteristics that the main method (CN 02181534A) as the dyeing of cast-off cells DNA ploidy body to DNA preferably selectivity.But
There are several shortcomings for Feulgen dyeing：
(1) dying operation process is complicated, and stringent with liquid and reaction condition requirement, time-consuming (a few hours).
(2) specificity of dyeing is not strong, redyes HE or EA36 backgrounds and easily contaminates altogether, directly affects the accuracy of interpretation.
(3) Feulgen reagents do not show the form of cytoplasm, consequently, it is desirable to do second of dyeing only to cell nuclear reaction
(HE,EA36)。
(4) stained cells section is easily faded and can not be preserved for a long time.
(5) effect of dyeing is very different with Pasteur.Doctor can not adopt (pap staining) experience diagosis of accumulation.By
In the above the shortcomings that, Feulgen dyeing is never clinically widely used.
The content of the invention
In order to overcome the problems of in the prior art, it is an object of the invention to provide a kind of one step of cast-off cells dye
Color method, dye combinations used and kit.
To achieve these goals and other related purposes, the present invention adopt the following technical scheme that：
The first aspect of the present invention, there is provided a kind of one step decoration method of cast-off cells, the step dyeing refer to thin to coming off
The nucleus and cytoplasm of born of the same parents synchronizes dyeing, including carries out a step dyeing, institute to cast-off cells using fluorochrome combinations
State fluorochrome combinations and contain nucleus fluorescent dye and cytoplasm fluorescent dye.
The nucleus fluorescent dye refers to the fluorescent dye dyed to the nucleus of cast-off cells.The cytoplasm
Fluorescent dye refers to the fluorescent dye dyed to the cytoplasm of cast-off cells.
The nucleus fluorescent dye is to the wavelength of fluorescence sent after nuclear targeting with cytoplasm fluorescent dye to cell
The wavelength of fluorescence sent after pulp color is different, can distinguish.
The second aspect of the present invention, there is provided a kind of one step decoration method fluorochrome combinations of cast-off cells, the fluorescence
Dye combinations contain nucleus fluorescent dye and cytoplasm fluorescent dye.
The nucleus fluorescent dye refers to the fluorescent dye dyed to the nucleus of cast-off cells.The cytoplasm
Fluorescent dye refers to the fluorescent dye dyed to the cytoplasm of cast-off cells.
The nucleus fluorescent dye is to the wavelength of fluorescence sent after nuclear targeting with cytoplasm fluorescent dye to cell
The wavelength of fluorescence sent after pulp color is different, can distinguish.
The third aspect of the present invention, there is provided a kind of one step decoration method dye liquor of cast-off cells, including foregoing cast-off cells
One step decoration method fluorochrome combinations and fluorescence preserve liquid.
The fourth aspect of the present invention, there is provided foregoing one step decoration method fluorochrome combinations of cast-off cells foregoing come off
The purposes of one step decoration method dye liquor of cell, the purposes are selected from any one of following or multinomial：
(1) step for cast-off cells dyes；
(2) it is used to prepare cast-off cells detection reagent.
The fifth aspect of the present invention, there is provided a kind of cast-off cells detection method, including：
(1) dyeing is synchronized to the nucleus and cytoplasm of cast-off cells using one step decoration method of cast-off cells, including
Cast-off cells are carried out with a step dyeing using fluorochrome combinations, the fluorochrome combinations contain nucleus fluorescent dye and thin
Endochylema fluorescent dye；
(2) in details in a play not acted out on stage, but told through dialogues environment, the cast-off cells after dyeing in step (1) are observed using fluorescence microscope.
The sixth aspect of the present invention, there is provided a kind of cast-off cells detection kit, including：Foregoing one step of cast-off cells dyeing
Method is with one step decoration method dye liquor of fluorochrome combinations and/or foregoing cast-off cells.
Compared with prior art, the present invention has the advantages that：
The present invention uses fluorochrome combinations to synchronize dyeing to the nucleus and cytoplasm of cast-off cells, and need not
Background fluorescence is cleaned, so as to realize rapid dyeing and detection.The present invention is excited using single ultraviolet source and obtained good
Effect, can simplify and light source and avoid irradiating the complexity to form image twice.
Brief description of the drawings
Fig. 1：Fluorescence is carried out to the cast-off cells from cervix using the fluorescent dye working solution that embodiment 1 has configured
Coloration result figure.
Fig. 2：Fluorescent staining cellular morphology in Fig. 1 is converted into gray level image, the part outlined is complete squamous
Epithelial cell.
Fig. 3：Fluorescent staining cellular morphology in Fig. 1 is converted into gray level image, the part outlined is the cell of cell
Core, the size and clean mark of core are visible.
Fig. 4：The fluorescent dye that embodiment 1 has configured preserves the stability of liquid fluorescent staining.
Fig. 5：The fluorescent dye that embodiment 1 has configured preserves the time of liquid fluorescent staining.
Fig. 6：Fluorescence is carried out to the cast-off cells from cervix using the fluorescent dye working solution that embodiment 2 has configured
Coloration result figure.
Fig. 7：Fluorescent staining cellular morphology in Fig. 6 is converted into gray level image, the part outlined is complete squamous
Epithelial cell.
Fig. 8：Fluorescent staining cellular morphology in Fig. 6 is converted into gray level image, the part outlined is the cell of cell
Core, the size and clean mark of core are visible.
Fig. 9：The fluorescent dye that embodiment 2 has configured preserves the stability of liquid fluorescent staining.
Figure 10：The fluorescent dye that embodiment 2 has configured preserves the time of liquid fluorescent staining.
Figure 11：It is glimmering to being carried out from the cast-off cells of cervix using the fluorescent dye working solution that embodiment 3 has configured
Light coloration result figure.
Figure 12：Fluorescent staining cellular morphology in Figure 11 is converted into gray level image, the part outlined is complete squama
Columnar epithelium cell.
Figure 13：Fluorescent staining cellular morphology in Figure 11 is converted into gray level image, the part outlined is the thin of cell
Karyon, the size and clean mark of core are visible.
Figure 14：The stability for the fluorescent dye working solution fluorescent staining that embodiment 3 has configured.
Figure 15：The time for the fluorescent dye working solution fluorescent staining that embodiment 3 has configured.
Embodiment
First, one step decoration method of cast-off cells
The one step decoration method of cast-off cells of the present invention, refers to synchronize dye to the nucleus and cytoplasm of cast-off cells
Color, including one step dyeing is carried out to cast-off cells using fluorochrome combinations, the fluorochrome combinations contain cell nuclear fluorescence
Dyestuff and cytoplasm fluorescent dye.
The nucleus fluorescent dye refers to the fluorescent dye dyed to the nucleus of cast-off cells.The cytoplasm
Fluorescent dye refers to the fluorescent dye dyed to the cytoplasm of cast-off cells.
Further, nucleus fluorescent dye is to the wavelength of fluorescence that is sent after nuclear targeting and cytoplasm fluorescent dye pair
The wavelength of fluorescence sent after cytoplasm dyeing is different, can distinguish.For example, can by human eye or optical image sensor (CCD,
CMOS etc.) distinguish.
Further, the nucleus fluorescent dye is selected from：DAPI, Syto DNA, Hoechst33342, PI (iodate third
Pyridine), EB (ethidium bromide)；The cytoplasm fluorescent dye is selected from：AO, FITC (fluorescein isothiocynate), CPO, phalloidine.
In preference, the fluorochrome combinations are made of DAPI and AO.
DAPI refers to 4,6 diamidines -2-phenylindone.The affinity that DAPI is combined with endonuclear DNA is high.Therefore,
The fluorescence of DAPI transmittings mainly reflects endonuclear DNA distributions.
AO refers to fluorescent dye acridine orange.AO is mainly combined with the RNA in cytoplasm.Therefore, the fluorescence reflection of AO transmittings
The form and RNA metabolic informations of cytoplasm.
The present invention is by extensive and deep experiment discovery, the fluorochrome combinations being made of AO and DAPI, therebetween
Without influencing each other without bad.Will not produce and mutually be quenched, and A0 combined with cell nuclear dna after the faint fluorescent orange launched simultaneously
The intense blue fluorescence for not disturbing DAPI to be launched after being combined with cell nuclear dna.In being total to for DAPI and two kinds of fluorescent dyes of AO
Under same-action, the fluorescence color of nucleus and cytoplasm is distinguished substantially, is obtained respectively in the different color channels of image sensor
Take the information (blueness) of nucleus and the information (green) of cytoplasm.This knowledge for taking figure mode to contribute to artificial intelligence system
, do not classify and judge, greatly improve the recall rate of cast-off cells.
The fluorochrome combinations being made of AO and DAPI, another feature are when they are separately existed in solution, are only sent
Faint fluorescence.After fluorescent dye is combined with nucleic acid molecules, under exciting light (320nm--400nm) irradiation, fluorescence production
The rate of coming into force greatly improves.This feature makes that nucleic acid domain of the existence has strong fluorescence and free nucleic acid regional background fluorescence is extremely weak.This
After feature avoids fluorescent staining, the step of flushing repeatedly to obtain clean background.This makes whole staining reaction very simple
It is single and effective.Thus greatly reduce the complexity of manpower and automation process.
When further, using by the fluorochrome combinations that DAPI and AO are formed cast-off cells are carried out with step dyeing,
DAPI is with working concentration scope during mixing with cells：0.025~0.50ug/ml.AO and working concentration model during mixing with cells
It is 1.0~10ug/mL to enclose.
Further, DAPI is with working concentration scope during mixing with cells：0.025~0.05ug/ml.
Further, DAPI is with working concentration scope during mixing with cells：0.05~0.50ug/ml
In preference, DAPI is 0.025ug/ml, 0.05ug/ml or 0.5ug/ml with working concentration during mixing with cells
.
Further, AO and working concentration scope during mixing with cells are 1.0~5.0ug/mL.
Further, AO and working concentration scope during mixing with cells are 5.0~10.0ug/mL.
In preference, 1.0ug/mL, 5.0ug/mL or 10.0ug/mL.
When further, using by the fluorochrome combinations that DAPI and AO are formed cast-off cells are carried out with step dyeing,
DAPI and AO is dissolved in fluorescence and preserves in liquid.
Further, the pH value range that the fluorescence preserves liquid is 2~6.More preferably 3.5.
Further, the fluorescence preserves liquid and is selected from：Phosphate buffer, HEPES buffer solution, acetate buffer solution or citric acid
Buffer solution.
In preference, the fluorescence preserves liquid and selects citrate-phosphate salt buffer.
Further, after carrying out a step dyeing to cast-off cells using fluorochrome combinations, without rinsing, it is glimmering to remove background
Light.
Further, the step decoration method only needs 5min to complete to dye.
Further, the cast-off cells derive from hydrothorax, ascites, urine, sputum or uterine neck.
Further, the catching method of the cast-off cells is selected from：Liquid basal cell method, sedimentation, centrifugal process or micro-fluidic
Method.
Further, the cast-off cells are dyed after being prepared into cast-off cells smear.
The cast-off cells smear is to be transferred to cast-off cells on transparent carrier to be prepared.The transparent carrier
Selected from glass slide or chip.
2nd, one step decoration method fluorochrome combinations of cast-off cells
The one step decoration method fluorochrome combinations of cast-off cells of the present invention, it is glimmering containing nucleus fluorescent dye and cytoplasm
Photoinitiator dye.
Further, nucleus fluorescent dye to the fluorescence that is sent after nuclear targeting with cytoplasm fluorescent dye to cell
The fluorescence sent after pulp color is different, can distinguish.
Further, the nucleus fluorescent dye is selected from：DAPI, Syto DNA, Hoechst33342, PI (iodate third
Pyridine), EB (ethidium bromide)；The cytoplasm fluorescent dye is selected from：AO, FITC (fluorescein isothiocynate), CPO, phalloidine.
Further, the fluorochrome combinations are made of DAPI and AO.
Further, the mass ratio range of DAPI and AO is (1~100)：(5~5000).
3rd, one step decoration method dye liquor of cast-off cells
The one step decoration method dye liquor of cast-off cells of the present invention, including one step decoration method fluorescent dye of foregoing cast-off cells
Combination and fluorescence preserve liquid.
Preferably, the pH value range that the fluorescence preserves liquid is 2~6.More preferably 3.5.
Further, the fluorescence preserves liquid and is selected from：Phosphate buffer, HEPES buffer solution, acetate buffer solution or citric acid
Buffer solution.
In preference, the buffer solution selects citrate-phosphate salt buffer.
The present invention also provides foregoing one step decoration method fluorochrome combinations of cast-off cells or one step of foregoing cast-off cells to contaminate
The purposes of color method dye liquor, the purposes are selected from any one of following or multinomial：
(1) step for cast-off cells dyes；(2) it is used to prepare cast-off cells detection reagent.
4th, cast-off cells detection method
The cast-off cells detection method of the present invention, including：
(1) dyeing is synchronized to the nucleus and cytoplasm of cast-off cells using foregoing one step decoration method of cast-off cells,
Including carrying out a step dyeing to cast-off cells using fluorochrome combinations, the fluorochrome combinations contain nucleus fluorescent dye
With cytoplasm fluorescent dye；
(2) in details in a play not acted out on stage, but told through dialogues environment, the cast-off cells after dyeing in step (1) are observed using fluorescence microscope.
Further, in step (1), the cast-off cells derive from hydrothorax, ascites, urine, sputum or uterine neck.
Further, in step (1), the catching method of the cast-off cells is selected from：Liquid basal cell method, sedimentation, centrifugation
Method or microfluidic method.
Further, in step (1), the cast-off cells are dyed after being prepared into cast-off cells smear.
The cast-off cells smear is to be transferred to cast-off cells on transparent carrier to be prepared.The transparent carrier
Selected from glass slide or chip.
Further, nucleus fluorescent dye to the fluorescence that is sent after nuclear targeting with cytoplasm fluorescent dye to cell
The fluorescence sent after pulp color is different, can distinguish.
Further, the nucleus fluorescent dye is selected from：DAPI, Syto DNA, Hoechst33342, PI (iodate third
Pyridine), EB (ethidium bromide)；The cytoplasm fluorescent dye is selected from：AO, FITC (fluorescein isothiocynate), CPO, phalloidine.
In preference, the fluorochrome combinations are made of DAPI and AO.
DAPI refers to 4,6 diamidines -2-phenylindone.The affinity that DAPI is combined with endonuclear DNA is high.Therefore,
The fluorescence of DAPI transmittings mainly reflects endonuclear DNA distributions.
AO refers to fluorescent dye acridine orange.AO is mainly combined with the RNA in cytoplasm.Therefore, the fluorescence reflection of AO transmittings
The form and RNA metabolic informations of cytoplasm.
The present invention is by extensive and deep experiment discovery, the fluorochrome combinations being made of AO and DAPI, therebetween
Without influencing each other without bad.Will not produce and mutually be quenched, and A0 combined with cell nuclear dna after the faint fluorescent orange launched simultaneously
The intense blue fluorescence for not disturbing DAPI to be launched after being combined with cell nuclear dna.In being total to for DAPI and two kinds of fluorescent dyes of AO
Under same-action, the fluorescence color of nucleus and cytoplasm is distinguished substantially, is obtained respectively in the different color channels of image sensor
Take the information (blueness) of nucleus and the information (green) of cytoplasm.This knowledge for taking figure mode to contribute to artificial intelligence system
, do not classify and judge, greatly improve the recall rate of cast-off cells.
It is to be preserved DAPI and AO respectively in conventional method.DAPI generally use purified water Fresh simultaneously adds
Anti- quencher, it is necessary to -20 DEG C of preservations, in room temperature generally can only 24 it is small when it is effective.This occupation mode is difficult clinically to promote.
AO is prepared using phosphate buffer, and when the outfit rear chamber middle benefit gas term of validity is 24 small, long-term preservation is protected under the conditions of being also required to -20 DEG C
Deposit.When they are separately existed in solution, faint fluorescence is only sent.And in the present invention, DAPI and AO are put together guarantor
Fluorescence is stored in preserve in liquid, it is glimmering under exciting light (320nm--400nm) irradiation after fluorescent dye is combined with nucleic acid molecules
Light generation efficiency greatly improves.This feature make nucleic acid domain of the existence have strong fluorescence and free nucleic acid regional background fluorescence pole
It is weak.After fluorescent staining, the step of flushing repeatedly to obtain clean background.This makes whole staining reaction non-
It is often simple and effective.Thus greatly reduce the complexity of manpower and automation process.
When further, using by the fluorochrome combinations that DAPI and AO are formed cast-off cells are carried out with step dyeing,
DAPI is with working concentration scope during mixing with cells：0.025~0.50ug/ml.AO and working concentration model during mixing with cells
It is 1.0~10ug/mL to enclose.
Further, DAPI is with working concentration scope during mixing with cells：0.025~0.05ug/ml.
Further, DAPI is with working concentration scope during mixing with cells：0.05~0.50ug/ml
In preference, DAPI is 0.025ug/ml, 0.05ug/ml or 0.5ug/ml with working concentration during mixing with cells
.
Further, AO and working concentration scope during mixing with cells are 1.0~5.0ug/mL.
Further, AO and working concentration scope during mixing with cells are 5.0~10.0ug/mL.
In preference, 1.0ug/mL, 5.0ug/mL or 10.0ug/mL.
When further, using by the fluorochrome combinations that DAPI and AO are formed cast-off cells are carried out with step dyeing,
DAPI and AO is dissolved in fluorescence and preserves in liquid.
Further, the pH value range that the fluorescence preserves liquid is 2~6.More preferably 3.5.
Further, the fluorescence preserves liquid and is selected from：Phosphate buffer, HEPES buffer solution, acetate buffer solution or citric acid
Buffer solution.
In preference, the fluorescence preserves liquid and selects citrate-phosphate salt buffer.
Further, after carrying out a step dyeing to cast-off cells using fluorochrome combinations, without rinsing, it is glimmering to remove background
Light.
Further, the step decoration method only needs to complete within 5 minutes to dye.
The fluorescent dye different to two kinds, traditional exciting method are the light sources using two kinds of different wave lengths.Such as：For
DAPI passes through the ultraviolet light frequently with 350nm, and the exciting light of AO is at 460nm (detection RNA).In instrument manufacturing, due to two
Complexity and cost are increased in the use of kind light source.Meanwhile two light source irradiations need to be imaged twice, pass through image procossing in the later stage
It is for composite to incite somebody to action image twice.Such process can extend image processing time, the time of delay report diagnosis.
And in the present invention, it is preferable that in step (2), laser is carried out using single ultraviolet light during observation, the ultraviolet light
Wave-length coverage is：320~400nm.Using single ultraviolet source, light source can be simplified and avoid irradiating to form answering for image twice
Polygamy, achieves good effect.
The detection method is external detection method.
Pass through fluorescence counterstain, the recall rate of lifting infantile tumour (in cast-off cells).There is provided for pathologist a kind of fast
Speed, automatically, the method for accurately screening heteroploid cell.
5th, cast-off cells detection kit
The cast-off cells detection kit of the present invention, including：Foregoing one step decoration method fluorochrome combinations of cast-off cells
And/or foregoing one step decoration method dye liquor of cast-off cells.
The present invention uses fluorochrome combinations to synchronize dyeing to the nucleus and cytoplasm of cast-off cells, and need not
Background fluorescence is cleaned, so as to realize rapid dyeing and detection.The present invention is excited using single ultraviolet source and obtained good
Effect, can simplify and light source and avoid irradiating the complexity to form image twice.
Before the specific embodiment of the invention is further described, it should be appreciated that protection scope of the present invention is not limited to down
State specific specific embodiment；It is also understood that the term used in the embodiment of the present invention is specific specific in order to describe
Embodiment, the protection domain being not intended to be limiting of the invention.The test method of actual conditions is not specified in the following example,
Usually according to normal condition, or the condition proposed by according to each manufacturer.
When embodiment provides number range, it should be appreciated that except non-invention is otherwise noted, two ends of each number range
Any one numerical value can be selected between point and two endpoints.Unless otherwise defined, in the present invention all technologies for using and
Scientific terminology is identical with the normally understood meaning of those skilled in the art of the present technique.Except used in embodiment specific method, equipment,
Outside material, according to grasp of the those skilled in the art to the prior art and the record of the present invention, it can also use and this
Any method, equipment and the material of the similar or equivalent prior art of method, equipment described in inventive embodiments, material come real
The existing present invention.
Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method using this technology lead
Molecular biology, biochemistry, chromatin Structure and the analysis of domain routine, analytical chemistry, cell culture, recombinant DNA technology and
The routine techniques of association area.These technologies existing perfect explanation in the prior art, for details, reference can be made to Sambrook etc.
MOLECULAR CLONING：A LABORATORY MANUAL, Second edition, Cold Spring Harbor
Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley＆Sons, New York, 1987and periodic updates；the
Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN
STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN
ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic
Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin
Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
Embodiment 1
First, fluorescent dye buffer solution configures
1st, 0.1M citric acid mother liquors：2.1g monohydrate potassiums are weighed, are dissolved in 100ml dH2In O；
2nd, 0.2M disodium hydrogen phosphates：7.16 grams of disodium hydrogen phosphate dodecahydrates are weighed, are dissolved in 100ml dH2In O；
3rd, 10mM citrate-phosphates salt buffer, 0.1M NaCl：Take 9.92ml 0.1M citric acids, 5.46ml 0.2M phosphorus
Sour disodium hydrogen, 1.7g NaCl, add water to 200ml and stirring and dissolving, adjustment PH to 3.5.
Above 10mM citrate-phosphate salt buffers, PH3.5,0.1M NaCl, that is, fluorescent dye preserve liquid.
2nd, fluorescent dye working solution configures
1st, accurate weighing acridine orange (AO) 5mg, is dissolved in 10ml 10mM citrate-phosphate salt buffers, as acridine
Orange (AO) mother liquor (0.5mg/ml), 2-8 DEG C of preservation；
2nd, accurate weighing DAPI 0.25mg, are dissolved in 10mldH2In O, as DAPI mother liquors (0.025mg/ml), -20 DEG C
Preserve；
3rd, 500ul acridine oranges (AO) mother liquor and 100ulDAPI mother liquors are taken, adds 10mM citrate-phosphates salt buffer extremely
50ml is made into fluorescent dye working solution (5ug/ml AO, 0.05ug/ml DAPI).
3rd, fluorescent staining is carried out to cast-off cells using the fluorescent dye working solution configured
1st, fluorescent staining is carried out to the cast-off cells from cervix using the fluorescent dye working solution configured：
Specifically, including step：(1) cast-off cells from cervix are obtained：A variety of catching methods can be used to obtain
Cast-off cells, such as：The cast-off cells layer or colony that liquid basal cell method, sedimentation, centrifugal process and microfluidic method obtain.
(2) cast-off cells smear is obtained：By step (1) obtain cast-off cells be transferred to transparent carrier (glass slide or
Chip) on, obtain cast-off cells smear；
(3) fluorescent staining：The cast-off cells smear that step (2) is obtained, the fluorescence being configured using the present embodiment
Dyestuff working solution carries out several minutes of dyeing；Without rinsing；
(4) scanning：In details in a play not acted out on stage, but told through dialogues environment, using fluorescence microscope to coming off carefully after fluorescent staining in step (3)
Born of the same parents are scanned and graphical analysis；Using a kind of ultraviolet excitation (320~400nm of wavelength), figure is taken by colour TV camera and is led to
Cross Computer digital image analysis to classify to cell, registration is carried out to cell DNA content and detects that abnormal DNA ploidy body is thin
Born of the same parents.
As a result, as shown in Figure 1, the image shot with colour imagery shot under 20 times of fluorescence microscopes, cast-off cells source
In cervix；It can be seen that squamous cell and lymphocyte；Nucleus and cytoplasm can substantially be distinguished；Cell type also clearly may be used
Identification.
Fluorescent staining cellular morphology in Fig. 1 is converted into gray level image, as shown in Fig. 2, the part outlined is complete
Squamous cell.As shown in figure 3, the part outlined is the nucleus of cell, the size and clean mark of core are visible.
4th, the fluorescent dye working solution stability configured is grown 7 days
Experimental design is that the one bottle of fluorescent dye configured working solution is used continuously seven days.Every morning is dyed once.
For cell derived in same cell pool, which mixed five normal person's cervical cell samples and in cell-preservation liquid (BD
Product) in suspend and refrigeration.Cell pool refrigerates about seven days in 4 DEG C in the entire experiment process.First and third, seven shown in lower Fig. 4
It scanning figure.Compare the nuclear fluorescence intensity and endochylema fluorescence intensity of first day and the 7th day and find that they do not fail.
Substantially remained in through computer graphical analysis fluorescence intensity in same level.
5th, the fluorescent dye working solution configured carries out fluorescent staining and tends towards stability in 5 minutes, you can completes dyeing
The cast-off cells smear that step (2) is obtained, the fluorescent dye working solution point being configured using the present embodiment
Do not carry out dyeing 2 minutes, 5 minutes, dyeing 10 minutes；Without rinsing.
The results are shown in Figure 5, and from left from the right side, the display fluorescent staining in 2 minutes of first library professor is weaker, and endochylema colour developing is unknown
It is aobvious；Second library professor, 5 minutes display core is obvious with endochylema dyeing and stablizes；3rd library professor, 10 minutes Fig. 8 are shown carefully
Born of the same parents' fluorescent staining intensity and 5 minutes difference are small, and endochylema slightly has partially red.It can be seen from the above that it has been configured using the present embodiment glimmering
Photoinitiator dye working solution carries out fluorescent staining and tends towards stability for 5 minutes, you can completes dyeing.
Embodiment 2
First, fluorescent dye buffer solution configures
1st, 0.1M citric acid mother liquors：2.1g monohydrate potassiums are weighed, are dissolved in 100ml dH2In O；
2nd, 0.2M disodium hydrogen phosphates：7.16 grams of disodium hydrogen phosphate dodecahydrates are weighed, are dissolved in 100ml dH2In O；
3rd, 10mM citrate-phosphates salt buffer, 0.1M NaCl：Take 9.92ml 0.1M citric acids, 5.46ml 0.2M phosphorus
Sour disodium hydrogen, 1.7g NaCl, add water to 200ml and stirring and dissolving, adjust PH to 2.
Above 10mM citrate-phosphate salt buffers, PH 2,0.1M NaCl, that is, fluorescent dye preserve liquid.
2nd, fluorescent dye working solution configures
1st, accurate weighing acridine orange (AO) 100mg, is dissolved in 10ml 10mM citrate-phosphate salt buffers, as a word used for translation
Pyridine orange (AO) mother liquor (10mg/ml), 2-8 DEG C of preservation；
2nd, accurate weighing DAPI 0.25mg, are dissolved in 10mldH2In O, as DAPI mother liquors (0.025mg/ml), -20 DEG C
Preserve；
3rd, 5ul acridine oranges (AO) mother liquor and 1000ulDAPI mother liquors are taken, adds 10mM citrate-phosphates salt buffer to 50ml
It is made into fluorescent dye working solution (1.0ug/ml AO, 0.50ug/ml DAPI).
3rd, fluorescent staining is carried out to cast-off cells using the fluorescent dye working solution configured
1st, fluorescent staining is carried out to the cast-off cells from cervix using the fluorescent dye working solution configured：
Specifically, including step：(1) cast-off cells from cervix are obtained：A variety of catching methods can be used to obtain
Cast-off cells, such as：The cast-off cells layer or colony that liquid basal cell method, sedimentation, centrifugal process and microfluidic method obtain.
(2) cast-off cells smear is obtained：By step (1) obtain cast-off cells be transferred to transparent carrier (glass slide or
Chip) on, obtain cast-off cells smear；
(3) fluorescent staining：The cast-off cells smear that step (2) is obtained, the fluorescence being configured using the present embodiment
Dyestuff working solution carries out several minutes of dyeing；Without rinsing；
(4) scanning：In details in a play not acted out on stage, but told through dialogues environment, using fluorescence microscope to coming off carefully after fluorescent staining in step (3)
Born of the same parents are scanned and graphical analysis；Using a kind of ultraviolet excitation (320~400nm of wavelength), figure is taken by colour TV camera and is led to
Cross Computer digital image analysis to classify to cell, registration is carried out to cell DNA content and detects that abnormal DNA ploidy body is thin
Born of the same parents.
As a result, as shown in fig. 6, the image shot with colour imagery shot under 20 times of fluorescence microscopes, cast-off cells source
In cervix；It can be seen that squamous cell and lymphocyte；Nucleus and cytoplasm can substantially be distinguished；Cell type also clearly may be used
Identification.
Fluorescent staining cellular morphology in Fig. 6 is converted into gray level image, as shown in fig. 7, the part outlined is complete
Squamous cell.As shown in figure 8, the part outlined is the nucleus of cell, the size and clean mark of core are visible.
4th, the fluorescent dye working solution stability configured is grown 7 days
Experimental design is that the one bottle of fluorescent dye configured working solution is used continuously seven days.Every morning is dyed once.
For cell derived in same cell pool, which mixed five normal person's cervical cell samples and in cell-preservation liquid (BD
Product) in suspend and refrigeration.Cell pool refrigerates about seven days in 4 DEG C in the entire experiment process.First and third, seven shown in lower Fig. 9
It scanning figure.Compare the nuclear fluorescence intensity and endochylema fluorescence intensity of first day and the 7th day and find that they do not fail.
Substantially remained in through computer graphical analysis fluorescence intensity in same level.
5th, the fluorescent dye working solution configured carries out fluorescent staining and tends towards stability in 5 minutes, you can completes dyeing
The cast-off cells smear that step (2) is obtained, the fluorescent dye working solution point being configured using the present embodiment
Do not carry out dyeing 2 minutes, 5 minutes, dyeing 10 minutes；Without rinsing.
The results are shown in Figure 10, and from left from the right side, the display fluorescent staining in 2 minutes of first library professor is weaker, and endochylema develops the color not
Substantially；Second library professor, 5 minutes display core is obvious with endochylema dyeing and stablizes；3rd library professor, 10 minutes Fig. 8 are shown
Cell fluorescence staining power and 5 minutes difference are small, and endochylema slightly has partially red.It can be seen from the above that be configured using the present embodiment
Fluorescent dye working solution carries out fluorescent staining and tends towards stability for 5 minutes, you can completes dyeing.
Embodiment 3
First, fluorescent dye buffer solution configures
1st, 0.1M citric acid mother liquors：2.1g monohydrate potassiums are weighed, are dissolved in 100ml dH2In O；
2nd, 0.2M disodium hydrogen phosphates：7.16 grams of disodium hydrogen phosphate dodecahydrates are weighed, are dissolved in 100ml dH2In O；
3rd, 10mM citrate-phosphates salt buffer, 0.1M NaCl：Take 9.92ml 0.1M citric acids, 5.46ml 0.2M phosphorus
Sour disodium hydrogen, 1.7g NaCl, add water to 200ml and stirring and dissolving, adjust pH to 6.
Above 10mM citrate-phosphate salt buffers, PH 6,0.1M NaCl, that is, fluorescent dye preserve liquid.
2nd, fluorescent dye working solution configures
1st, accurate weighing acridine orange (AO) 1mg, is dissolved in 10ml 10mM citrate-phosphate salt buffers, as acridine
Orange (AO) mother liquor (0.1mg/ml), 2-8 DEG C of preservation；
2nd, accurate weighing DAPI 1.25mg, are dissolved in 10mldH2In O, as DAPI mother liquors (0.125mg/ml), -20 DEG C
Preserve；3rd, 5000ul acridine oranges (AO) mother liquor and 10ulDAPI mother liquors are taken, add 10mM citrate-phosphates salt buffer to 50ml i.e.
It is made into fluorescent dye working solution (10ug/ml AO, 0.025ug/ml DAPI).
3rd, fluorescent staining is carried out to cast-off cells using the fluorescent dye working solution configured
1st, fluorescent staining is carried out to the cast-off cells from cervix using the fluorescent dye working solution configured：
Specifically, including step：(1) cast-off cells from cervix are obtained：A variety of catching methods can be used to obtain
Cast-off cells, such as：The cast-off cells layer or colony that liquid basal cell method, sedimentation, centrifugal process and microfluidic method obtain.
(2) cast-off cells smear is obtained：By step (1) obtain cast-off cells be transferred to transparent carrier (glass slide or
Chip) on, obtain cast-off cells smear；
(3) fluorescent staining：The cast-off cells smear that step (2) is obtained, the fluorescence being configured using the present embodiment
Dyestuff working solution carries out several minutes of dyeing；Without rinsing；
(4) scanning：In details in a play not acted out on stage, but told through dialogues environment, using fluorescence microscope to coming off carefully after fluorescent staining in step (3)
Born of the same parents are scanned and graphical analysis；Using a kind of ultraviolet excitation (320~400nm of wavelength), figure is taken by colour TV camera and is led to
Cross Computer digital image analysis to classify to cell, registration is carried out to cell DNA content and detects that abnormal DNA ploidy body is thin
Born of the same parents.
As a result, as shown in figure 11, the image shot with colour imagery shot under 20 times of fluorescence microscopes, cast-off cells source
In cervix；It can be seen that squamous cell and lymphocyte；Nucleus and cytoplasm can substantially be distinguished；Cell type also clearly may be used
Identification.
Fluorescent staining cellular morphology in Figure 11 is converted into gray level image, as shown in figure 12, the part outlined has been
Whole squamous cell.As shown in figure 13, the part outlined is the nucleus of cell, and the size and clean mark of core can
See.
4th, the fluorescent dye working solution stability configured is grown 7 days
Experimental design is that the one bottle of fluorescent dye configured working solution is used continuously seven days.Every morning is dyed once.
For cell derived in same cell pool, which mixed five normal person's cervical cell samples and in cell-preservation liquid (BD
Product) in suspend and refrigeration.Cell pool refrigerates about seven days in 4 DEG C in the entire experiment process.First and third shown in lower Figure 14,
The scanning figure of seven days.Compare the nuclear fluorescence intensity and endochylema fluorescence intensity of first day and the 7th day and find that they do not decline
Move back.Substantially remained in through computer graphical analysis fluorescence intensity in same level.
5th, the fluorescent dye working solution configured carries out fluorescent staining and tends towards stability in 5 minutes, you can completes dyeing
The cast-off cells smear that step (2) is obtained, the fluorescent dye working solution point being configured using the present embodiment
Do not carry out dyeing 2 minutes, 5 minutes, dyeing 10 minutes；Without rinsing.
As a result as shown in figure 15, from left from the right side, the display fluorescent staining in 2 minutes of first library professor is weaker, and endochylema develops the color not
Substantially；Second library professor, 5 minutes display core is obvious with endochylema dyeing and stablizes；3rd library professor, 10 minutes Fig. 8 are shown
Cell fluorescence staining power and 5 minutes difference are small, and endochylema slightly has partially red.It can be seen from the above that be configured using the present embodiment
Fluorescent dye working solution carries out fluorescent staining and tends towards stability for 5 minutes, you can completes dyeing.
The above, be only presently preferred embodiments of the present invention, not to the present invention in any form with substantial limitation,
It should be pointed out that for those skilled in the art, on the premise of the method for the present invention is not departed from, can also make
Some improvement and supplement, these are improved and supplement also should be regarded as protection scope of the present invention.All those skilled in the art,
Without departing from the spirit and scope of the present invention, when made using disclosed above technology contents it is a little more
Dynamic, modification and the equivalent variations developed, are the equivalent embodiment of the present invention；Meanwhile all substantial technologicals pair according to the present invention
The variation, modification and evolution for any equivalent variations that above-described embodiment is made, still fall within the scope of technical scheme
It is interior.
Claims (21)
1. a kind of one step decoration method of cast-off cells, the step dyeing, which refers to, synchronizes the nucleus and cytoplasm of cast-off cells
Dyeing, including one step dyeing is carried out to cast-off cells using fluorochrome combinations, it is glimmering that the fluorochrome combinations contain nucleus
Photoinitiator dye and cytoplasm fluorescent dye.
2. one step decoration method of cast-off cells according to claim 1, nucleus fluorescent dye after nuclear targeting to sending
Wavelength of fluorescence and cytoplasm fluorescent dye cytoplasm is dyed after the wavelength of fluorescence that sends it is different, can distinguish.
3. one step decoration method of cast-off cells according to claim 1, it is characterised in that the nucleus fluorescent dye choosing
From：DAPI、Syto DNA、Hoechst 33342、Hoechest33258、PI、EB；The cytoplasm fluorescent dye is selected from：AO、
FITC, CPO, phalloidine.
4. one step decoration method of cast-off cells according to claim 1, it is characterised in that the fluorochrome combinations are by DAPI
And AO compositions.
5. one step decoration method of cast-off cells according to claim 4, it is characterised in that glimmering using being made of DAPI and AO
When photoinitiator dye combination carries out cast-off cells the dyeing of one step, DAPI is with working concentration scope during mixing with cells：0.025~
0.50ug/ml；AO is 1.0~10ug/mL with working concentration scope during mixing with cells.
6. one step decoration method of cast-off cells according to claim 4, it is characterised in that glimmering using being made of DAPI and AO
When photoinitiator dye combination carries out cast-off cells the dyeing of one step, DAPI and AO are dissolved in same fluorescence and preserve in liquid.
7. one step decoration method of cast-off cells according to claim 5, it is characterised in that the fluorescence preserves the pH value model of liquid
Enclose is 2.0~6.0.
8. one step decoration method of cast-off cells according to claim 5, it is characterised in that the fluorescence preserves liquid and is selected from：Phosphorus
Acid buffer, HEPES buffer solution, acetate buffer solution or citrate buffer solution.
9. one step decoration method of cast-off cells according to claim 1, it is characterised in that using fluorochrome combinations to coming off
After cell carries out a step dyeing, without rinsing.
10. one step decoration method of cast-off cells according to claim 1, it is characterised in that the step decoration method only needs
5min can complete to dye.
11. a kind of one step decoration method fluorochrome combinations of cast-off cells, it is characterised in that the fluorochrome combinations contain carefully
Karyon fluorescent dye and cytoplasm fluorescent dye.
12. fluorochrome combinations according to claim 11, it is characterised in that nucleus fluorescent dye is to nuclear targeting
The wavelength of fluorescence that the wavelength of fluorescence sent afterwards is sent after being dyed from cytoplasm fluorescent dye to cytoplasm is different, can distinguish.
13. fluorochrome combinations according to claim 11, it is characterised in that the nucleus fluorescent dye is selected from：
DAPI、Syto DNA、Hoechst 33342、Hoechest33258、PI、EB；The cytoplasm fluorescent dye is selected from：AO、
FITC, CPO, phalloidine.
14. fluorochrome combinations according to claim 11, it is characterised in that the fluorochrome combinations are by DAPI and AO
Composition.
15. a kind of one step decoration method dye liquor of cast-off cells, it is characterised in that the dye liquor includes such as claim 11~14 times
One one step decoration method fluorochrome combinations of cast-off cells and fluorescence preserve liquid.
16. one step decoration method dye liquor of cast-off cells according to claim 15, it is characterised in that the fluorescence preserves liquid
PH value range be 2~8.
17. one step decoration method dye liquor of cast-off cells according to claim 15, it is characterised in that the fluorescence preserves liquid
It is selected from：Phosphate buffer, HEPES buffer solution, acetate buffer solution or citrate buffer solution.
18. one step decoration method fluorochrome combinations of cast-off cells or such as claim as described in any one of claim 11~14
The purposes of any one of 15~17 one step decoration method dye liquors of cast-off cells, the purposes are selected from following：
(1) it is used for the one-step method fluorescent staining of cast-off cells；(2) it is used to prepare cast-off cells detection reagent.
19. a kind of cast-off cells detection method, including：(1) contaminated using one step of cast-off cells as described in any one of claim 1~10
Color method synchronizes dyeing to the nucleus and cytoplasm of cast-off cells；(2) in details in a play not acted out on stage, but told through dialogues environment, using fluorescence microscope to step
Suddenly the cast-off cells in (1) after dyeing are observed.
20. cast-off cells decoration method according to claim 19, it is characterised in that in step (2), using single during observation
Ultraviolet light is excited, and the wave-length coverage of the ultraviolet light is：320~400nm.
21. a kind of cast-off cells detection kit, including as claim comes off as described in any one of claim 11~14
One step decoration method fluorochrome combinations of cell and/or the one step decoration method of cast-off cells as described in any one of claim 15~17
Use dye liquor.
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| Publication number | Priority date | Publication date | Assignee | Title |
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