CN101122601A - Method for separating and authenticating erythroblast of blood - Google Patents
Method for separating and authenticating erythroblast of blood Download PDFInfo
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- CN101122601A CN101122601A CNA2007101519665A CN200710151966A CN101122601A CN 101122601 A CN101122601 A CN 101122601A CN A2007101519665 A CNA2007101519665 A CN A2007101519665A CN 200710151966 A CN200710151966 A CN 200710151966A CN 101122601 A CN101122601 A CN 101122601A
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Abstract
The invention discloses a method for separating and appraising nucleated red blood cells in blood, including the following steps: (1) confecting reagent; (2) preparing anti-degumming slide; (3) selecting the staining method for nucleated red blood cells; (4) getting nucleated red blood cells from blood through microoperation. The method for separating and appraising nucleated red blood cells in blood can effectively identify nucleated red blood cells. Nucleated blood cell can be detected in 40 samples and the detection rate is 100 percent. Each sample is detected to have 2*106 to 6*106 /ml nucleated blood cell. In the invention, after the aniline being stained, the nucleated red blood cell is easy to be identified and skilled microoperation can quickly obtain the single nucleated red blood cell, so that the invention lays a good primary foundation to apply the single red blood cell in gene analysis.
Description
Technical field
The present invention relates to blood separation identification and detection technical field, the method for erythroblast in particularly a kind of isolation identification blood.
Background technology
Blood sample is separated into various cellular components, just need correspondingly regulate the processing/process technology of blood sample.The conventional method that whole blood sample is separated into its various cellular components is to use centrifugation method.Though this kind method is effectively, need colored very big work, efficient quite low and need be with the cell part of manually controlling in the blood sample.
The separation of erythroblast is the beginning of genetic analysis, also is one of step of comparatively difficulty.Nowadays the various countries scholars have attempted various method and have separated erythroblast, but do not find direct, reliable, high efficiency separating pathway yet.
When attempting to carry out this kind separation of the cell part in the blood sample with chemical agent, for various reasons, it is satisfied fully that the result can not make us all the time.More precisely, cell population or external life intensity and survivability all depends on the consistent accurate physiological environment of chemical equilibrium that keeps in a kind of and actual eucaryotic cell structure of preservation and the cell in vivo.Cell permeability of the membrane and the chemical equilibrium of transporting in the characteristic may command cell.The change of physiological environment can be brought out replying of cell membrane again or change.Cell membrane is replied and is being " defensive " in nature; That is to say that the physiologic response of cell membrane is the chemical equilibrium that is suitable for keeping in the cell, therefore is that the survivability of continuity and uninterrupted cell is fully recognized that now cell can only tolerate the change of the desirable physiological environment of this kind in certain limits; And, recognize that also cell will suffer nonvolatil damage if surpass this kind limit.Recognizing further also that in the art the change of this kind physiological environment (that is, even the toxicity of diluent media-use distilled water) can influence the haemocylolysis of cell.
The approach that obtains erythroblast at present mainly contains two: 1. directly obtain.Utilize erythroblast surface antigen such as CD71, GPA, CD36 etc. serve as a mark, by magnetic active cell separating method (magneticactivated sorting, MACS) or the fluorescence-activated cell sorting method (fluorescenceactivated cellsorting, FACS) technology is obtained erythroblast, or utilizes that the Y globin chain serves as a mark in the blood, after method Direct Recognition such as immunohistochemistry, micromanipulation is obtained.2. obtain indirectly.The most representative with micrurgy, dyeing is discerned from form earlier, and single erythroblast is caught in micromanipulation by high power, is applied to genetic analysis again.Directly obtain method cost height, or specificity is not strong, vulnerable to pollution, or complex operation, the technical requirement height, its reliability, practicality are doubted, and are difficult to apply.The method of obtaining indirectly can effectively be avoided polluting, and along with the development of unicellular round pcr, this method is regarded as obtaining the comparatively ideal approach of erythroblast.
Summary of the invention
Technical matters to be solved by this invention is: set up the method for erythroblast in a kind of blood of isolation identification fast and effectively, be convenient to micromanipulation, can obtain erythroblast efficiently and accurately and carry out follow-up cell evaluation and genetic analysis.
For solving the problems of the technologies described above, the invention provides the method for erythroblast in a kind of isolation identification blood, may further comprise the steps:
(1) reagent preparation
(1) poly-D-lysine working fluid: the poly-D-lysine storing solution fully mixes with distilled water at 1: 10, and is standby behind the bubble collapse;
(2) PH7.4 contains the 0.01mol/L PBS of 0.5%BSA:
Sodium chloride 6.5g
Na
2HPO
4·12H
2O 4g
NaH
2PO
4·2H
2O 0.35g
Add distilled water 800ml, regulate pH value to 7.4, distilled water is mended to 1000ml, and 0.01mol/L PBS behind the autoclaving, adds 5g BSA, 40 ℃ of preservations;
(3) contain KOH 200mmol/L, the alkaline cell pyrolysis liquid of DTT 50mmol/L:
400mmol/L?KOH 1ml
1mol/L?DTT 100ul
Sterilized water for injection 850ul fully mixes, and 0.22 μ m millipore filter filters the back packing, and-20 ℃ of preservations are standby;
(2) the anti-slide that comes unstuck of preparation
(1) pre-service of microslide
Common microslide is scrubbed with hot suds, and the tap water cleaning is clean, and oven dry places the sulfuric acid cleaning solution to soak more than 24 hours, the tap water flushing is spent the night, distilled water flushing 3 times, oven dry, 95% alcohol immersion 24 hours, 15 pounds of autoclaving 20min are dried in distilled water flushing 3 times;
(2) the poly-D-lysine bag is by microslide
Pretreated slide was dipped up and down in the poly-D-lysine working fluid 10～20 seconds, divided and fall apart on the slide frame, oven dry below 60 ℃, 4 ℃ of preservations are standby;
(3) select the erythroblast colouring method;
(4) micrurgy obtains erythroblast in the blood.
Described step (three) may further include following steps:
The single density gradient centrifugation is collected nucleated blood cell:
1. blood and equivalent contain the 0.01mmol/L PBS mixing of 0.5%BSA, be superimposed on the lymphocyte separation medium gently, make both form an interface clearly, the centrifugal 20min of 1800r/min level, draw the mononuclearcell layer of tunica albuginea shape, move to and use in advance in the moistening clean glass centrifuge tube of PBS;
2. add the 0.01mmol/L PBS washed cell of 0.5%BSA more than 2 times, the centrifugal 10min of 1500r/min level abandons supernatant, triplicate;
3. with smear after the extremely suitable concentration of 0.01mmol/L PBS diluting cells, air dry.
Described step (three) may further include following steps:
Adopt Rui Shi-Giemsa staining method that nucleated blood cell is dyeed:
1. slide places and dyes on the horse, drips 3～5 Rui Shi-Jim Sa complex staining liquid, makes it cover sample 1min rapidly;
2. drip 5～10 of damping fluids, rubber pipette bulb is aimed at slide and is blown, and damping fluid is fully mixed with dye liquor, dyeing 5～10min;
3. distilled water flushing is dried.
Described step (three) may further include following steps:
Adopt the benzidine staining method that nucleated blood cell is dyeed:
1. slide places 1% biphenylamine methanol solution to soak 2min;
2. in solution, drip 10～20 30%H
2O
2, making the volumetric ratio of itself and 1% biphenylamine methanol solution is 1: 50, mixing, slide continue to soak 1～2min;
3. distilled water fully washes, and dries;
4. haematoxylin is redyed.
Described step (four) may further include following steps:
1. the slide for preparing is positioned under the inverted phase contrast microscope, and 400 times of following observation identification erythroblasts of light microscopics are also counted;
2. around erythroblast, drip 0.25% Proteinase K of 10～20ul, make it to relax, with the single absorption of micromanipulator from slide;
3. the cell that is drawn to is moved in the special-purpose thin-walled reaction tube of PCR that fills 2.5ul alkalescence cell pyrolysis liquid, centrifugal back is preserved in-20 ℃ of low temperature refrigerators and is equipped with inspection.
Described method may further include the following step of isolating mononuclearcell from peripheral blood:
(1) the centrifugal 10min of blood sample 2000r/min, 80% blood plasma in the every pipe of usefulness suction pipe sucking-off;
(2) then add PBS and dilute, this liquid contains a whole leucocyte group, comprises granulocyte, lymphocyte, monocyte and part red blood cell;
(3) get 2ml lymphocyte layering liquid in centrifuge tube, the cell suspension with the about 2ml of capillary syring absorption adds on the layering liquid gently simultaneously; With horizontal rotor hydro-extractor 2000r/min, behind the centrifugal 20min, as seen be divided into multilayer, orlop is a red blood cell, and the middle layer is a layering liquid, and the superiors are blood plasma; Be the fine and close white layer of skim between plasma layer and layering liquid, be the mononuclearcell layer;
(4) be inserted into the mononuclearcell layer with capillary syring, draw this layer, put into another test tube; With Hanks liquid washing, centrifugal.
Described method may further include the purification procedures of following erythroblast:
(1) preparation of nylon hair post:
1. getting the nylon hair spends the night with 0.2mol/L HCl immersion treatment;
2. clean HCl with distilled water, put 37 ℃ of dry for standby;
3. take by weighing 90～120mg nylon hair, evenly disperse, put in the Hanks liquid and soak;
4. get the 1ml syringe, outlet connects plastic tube, clamps;
5. the nylon hair evenly is packed in the syringe emptying air, the high 7～8cm of post;
6. each is used will to make each good post autoclaving;
(2) the nylon hair separates erythroblast:
1. get the mononuclearcell of separation, cell is made into 400/ml with cell culture fluid;
2. melt nylon hair post, emit Hanks liquid with the speed of 18～20 of per minutes, pre-temperature to 37 ℃ cell culture fluid washs nylon hair post;
3. cell suspension is added nylon Mao Zhuzhong, treat that cell suspension all enters nylon Mao Zhuhou, add the 0.2ml cell culture fluid immediately, clamp plastic tube; In 37 ℃ saturation vapour CO2gas incubator, left standstill 1 hour;
4. with the cell culture fluid wash-out cell of the pre-temperature of 5ml, with the cell culture fluid washing nylon hair post of the pre-temperature of 5ml, clean the cell that adheres to again to 37 ℃ to 37 ℃;
5. add the 2ml cell culture fluid, clamp plastic tube, nylon hair post is put refrigerator and cooled but; Cell culture fluid with 4 ℃ of precoolings divides wash-out nylon hair post 3～4 times, each 2ml; Clamp plastic tube earlier, push the nylon hair repeatedly with glass bar or tinsel and be beneficial to adherent cell and come off, and then wash-out;
6. centrifugal, 1000rpm, 10 minutes, remove supernatant, use cell culture fluid washed cell 1 time; Counting cells, with cell culture fluid that cell is resuspended.
Described method may further include the purification procedures of following erythroblast:
1) anticoagulant dilutes with isopyknic physiological saline;
2) slow being added on the Ficoll liquid level casually, it is clear that the interface of blood and Ficoll keeps;
3) 2000rpm, centrifugal 20min;
4) draw nebulous mononuclearcell layer;
5) 1000rpm, centrifugal 15min;
6) supernatant discarded, washing precipitation;
7) 1000rpm, centrifugal 15min;
8) triplicate the 6th), 7) step;
9) abandon supernatant, precipitation.
The instrument and equipment that uses in the described method can be following equipment:
RPM worker's 1640 nutrient culture media: U.S. Gibc company;
Cell separation liquid: Shanghai China smart biological high well-behaved Ri Zhi company limited;
Hemagglutinin PHA: Guangzhou Medicine Industry Inst;
P-III type inverted microscope: Japanese Nikon company;
24 porocyte culture plates: U.S. Corning company;
CO
2Incubator: U.S. Forma scientific company;
DL-CJ-2N high-performance aseptic experiment platform: Harbin Donglian Electronic ﹠ Technology Development Co., Ltd.'s Beijing Company;
0412-1 type trace low speed centrifuge: Shanghai Surgical Operation Equipment Factory.
In the described method collocation method of PBS solution can for:
NaCL 18.8g, KCl 0.2g, Na
2HPO
43.488g, add distilled water to 1000ml, transferring pH value is 7.4,15 pounds of autoclavings, 4 ℃ of preservations.
The method of erythroblast can effectively be discerned erythroblast in the isolation identification blood of the present invention, and 40 routine sample standard deviations can detect erythroblast, recall rate 100%, and every example is found erythroblast 2 * 10
6～6 * 10
6Individual/ml.Erythroblast is easy to identification behind the benzidine staining of the present invention, and skilled micromanipulation can be obtained single erythroblast rapidly, carries out genetic analysis and lays good basis in early stage for using single erythroblast.Erythroblast can be applicable to some genetic analysis and examination.Realize that micrurgy obtains single erythroblast and carries out genetic analysis, at first must from blood, discern and separate erythroblast.The present invention adopts benzidine staining method identification erythroblast, by micromanipulation it is separated from blood, for genetic analysis and examination lay the first stone.
Description of drawings
Fig. 1 is the described erythroblast Rui Shi of the embodiment of the invention-Giemsa staining figure;
Fig. 2 is the described erythroblast benzidine staining of embodiment of the invention figure.
Embodiment
(1) main agents preparation
1. poly-D-lysine working fluid: the poly-D-lysine storing solution fully mixes with distilled water at 1: 10, treats behind the bubble collapse standby;
2. the 0.01mol/L PBS (PH7.4) that contains 0.5%BSA
Sodium chloride 6.5g
Na
2HPO
4·12H
2O 4g
NaH
2PO
4·2H
2O 0.35g
Add distilled water 800ml, regulate pH value to 7.4, distilled water is mended to 1000ml and promptly is made into.0.01mol/L PBS behind the autoclaving, adds 5g BSA, 40 ℃ of preservations.
3. alkaline cell pyrolysis liquid (containing KOH 200mmol/L, DTT 50mmol/L)
400mmol/L?KOH?1ml
1mol/L?DTT 100ul
Sterilized water for injection 850～900ul fully mixes, and 0.22 μ m millipore filter filters the back packing, and-20 ℃ of preservations are standby.
(2) the anti-slide preparation of coming unstuck
1. the pre-service of microslide
Common microslide is scrubbed with hot suds, and the tap water cleaning is clean, and oven dry places the sulfuric acid cleaning solution to soak more than 24 hours, the tap water flushing is spent the night, distilled water flushing 3 times, oven dry, 95% alcohol immersion 24 hours, 15 pounds of autoclaving 20min. are dried in distilled water flushing 3 times
2. the poly-D-lysine bag is by microslide
Pretreated slide was dipped up and down in the poly-D-lysine working fluid 10～20 seconds, divided and fall apart on the slide frame, oven dry below 60 ℃, 4 ℃ of preservations are standby.
(3) selection of erythroblast colouring method
From peripheral blood, isolate mononuclearcell:
(1) the centrifugal 10min of blood sample 2000r/min is with most blood plasma in the every pipe of suction pipe sucking-off;
(2) then add PBS and dilute, this liquid contains a whole leucocyte group, comprises granulocyte, lymphocyte, monocyte and part red blood cell;
(3) get 2ml lymphocyte layering liquid in centrifuge tube, the cell suspension with the about 2ml of capillary syring absorption adds on the layering liquid gently simultaneously.With horizontal rotor hydro-extractor 2000r/min, behind the centrifugal 20min, as seen be divided into multilayer, orlop is a red blood cell, and the middle layer is a layering liquid, and the superiors are blood plasma.Be the fine and close white layer of skim between plasma layer and layering liquid, be the mononuclearcell layer;
(4) be inserted into the mononuclearcell layer with capillary syring, draw this layer, put into another test tube; With Hanks liquid washing, centrifugal, be mixed with suitable cell concentration at last.
The erythroblast separation and purification:
1) system of nylon hair post is respectively:
1. getting the nylon hair spends the night with 0.2mol/L HCl immersion treatment;
2. clean HCl with distilled water, each is used to put 37 ℃ of oven dry;
3. take by weighing 90～120mg nylon hair, evenly disperse, put in the Hanks liquid and soak;
4. get the 1ml syringe, outlet connects plastic tube, clamps;
5. the nylon hair evenly is packed in the syringe emptying air, the high about 7～8cm of post;
6. each is used will to make each good post autoclaving;
(2) the nylon hair separates erythroblast:
1. get the mononuclearcell of separation, cell is made into 400/ml with cell culture fluid;
2. melt nylon hair post, emit Hanks liquid with the speed of 18～20 of per minutes, pre-temperature to 37 ℃ cell culture fluid washs nylon hair post.
3. cell suspension is added nylon Mao Zhuzhong, treat that cell suspension all enters nylon Mao Zhuhou, add the 0.2ml cell culture fluid immediately, clamp plastic tube; In 37 ℃ saturation vapour CO2gas incubator, left standstill 1 hour;
4. with the cell culture fluid wash-out cell of the pre-temperature of 5ml, with the cell culture fluid washing nylon hair post of the pre-temperature of 5ml, clean the cell that adheres to again to 37 ℃ to 37 ℃.
5. add the 2ml cell culture fluid, clamp plastic tube, nylon hair post is put refrigerator and cooled but; Cell culture fluid with 4 ℃ of precoolings divides wash-out nylon hair post 3～4 times, each 2ml.Clamp plastic tube earlier, push the nylon hair repeatedly with glass bar or tinsel and be beneficial to adherent cell and come off, and then wash-out;
6. centrifugal, 1000rpm, 10 minutes, remove supernatant, use cell culture fluid washed cell 1 time; Counting cells, with cell culture fluid that cell is resuspended;
Instrument:
1.RPM worker's 1640 nutrient culture media (dry powder): U.S. Gibc company
2. cell separation liquid ((Ficoll): Shanghai China smart biological high well-behaved Ri Zhi company limited
3. hemagglutinin PHA (10mg/ props up): Guangzhou Medicine Industry Inst
4.P-III type inverted microscope: Japanese Nikon company
5.24 porocyte culture plate: U.S. Corning company
6.CO
2Incubator: U.S. Forma scientific company
7.DL-CJ-2N high-performance aseptic experiment platform: Harbin Donglian Electronic ﹠ Technology Development Co., Ltd.'s Beijing Company
8.0412-1 type trace low speed centrifuge: Shanghai Surgical Operation Equipment Factory
PBS solution: NaCL 18.8g, KCl 0.2g, Na
2HPO
43.488g, add distilled water to 1000ml, transferring pH value is 7.4,15 pounds of autoclavings, 4 ℃ of preservations.
With the erythroblast in the Ficoll density separation blood sample, concrete steps are as follows:
1) anticoagulant dilutes with isopyknic physiological saline;
2) slow being added on the Ficoll liquid level casually, it is clear that the interface of blood and Ficoll keeps;
3) 2000rpm, centrifugal 20min;
4) draw nebulous mononuclearcell layer;
5) 1000rpm, centrifugal 15min;
6) supernatant discarded, washing precipitation;
7) 1000rpm, centrifugal 15min;
8) repetition one order 6), the 7) step;
9) abandon supernatant, precipitation.
The single density gradient centrifugation is collected nucleated blood cell:
1. blood and equivalent contain the 0.01mmol/L PBS mixing of 0.5%BSA, be superimposed on the lymphocyte separation medium gently, make both form an interface clearly, the centrifugal 20min of 1800r/min level, draw the mononuclearcell layer of tunica albuginea shape, move to and use in advance in the moistening clean glass centrifuge tube of PBS.
2. add the 0.01mmol/L PBS washed cell of 0.5%BSA more than 2 times, the centrifugal 10min of 1500r/min level abandons supernatant.Repeat once.
3. with smear after the extremely suitable concentration of 0.01mmol/L PBS diluting cells, air dry.The nucleated blood cell colouring method:
(1) Rui Shi-Giemsa staining method
1. slide places and dyes on the horse, drips 3～5 Rui Shi-Jim Sa complex staining liquid, makes it cover sample, about 1min rapidly.
2. drip 5～10 of damping fluids, rubber pipette bulb is aimed at slide and is blown, and damping fluid is fully mixed with dye liquor, dyeing 5～10min.
3. distilled water flushing is dried.
(2) benzidine staining method
1. slide places 1% biphenylamine methanol solution to soak 2min.
2. in solution, drip 10～20 30%H
2O
2, making the volumetric ratio of itself and 1% biphenylamine methanol solution is 1: 50, mixing, slide continue to soak 1～2min.
3. distilled water fully washes, and dries.
4. haematoxylin is redyed.
(4) micrurgy obtains erythroblast in the blood
1. the slide for preparing is positioned under the inverted phase contrast microscope, and 400 times of following observation identification erythroblasts of light microscopics are also counted.
2. around erythroblast, drip 0.25% Proteinase K of 10～20ul, make it to relax, with the single absorption of micromanipulator from slide.
3. the cell that is drawn to is moved in the special-purpose thin-walled reaction tube of PCR that fills 2.5ul alkalescence cell pyrolysis liquid, centrifugal back is preserved in-20 ℃ of low temperature refrigerators and is equipped with inspection.
The result:
One, the enrichment of erythroblast
The isolated cell of single density gradient centrifugation contains the lymphocyte more than 90%, a small amount of monocyte and erythroblast or the like.
Two, erythroblast dyeing
As shown in Figure 1, be the described erythroblast Rui Shi of the embodiment of the invention-Giemsa staining figure.Wherein, Rui Shi-Giemsa staining is that the erythroblast kytoplasm is hyacinthine under 1000 times of light microscopics, and karyon is mazarine, is difficult to sometimes under 400 times of light microscopics distinguish with other cell especially lymphocyte.
As shown in Figure 2, be the described erythroblast benzidine staining of embodiment of the invention figure.Wherein, the erythroblast benzidine staining: visible erythroblast tenuigenin is pale brown look under the optical microscope, and haematoxylin is redyed the back karyon and is blue, obviously distinguishes over other cell, and recognition effect is better than Rui Shi-Giemsa staining.
The erythroblast recall rate:
40 routine sample standard deviations can detect erythroblast, recall rate 100%, and every example is found 2～6/ml of erythroblast, obtains 157 altogether.
Mononuclearcell mainly comprises lymphocyte, erythroblast and monocyte in the peripheral blood, and its volume, form and density are different with other cells.Red blood cell and multinuclear granulocyte density are bigger, be about 1.090, and mononuclearcell density are 1.075～1.090, and blood platelet is 1.030～1.035.If utilize a kind of density between 1.075～1.092 and the solution (layering liquid) that oozes such as be bordering on and do density gradient centrifugation, just the haemocyte of various different densities can be separated.Separating medium of the present invention is that density is 1.077 lymphocyte separation medium, after horizontal is centrifugal, can occur the liquid and the cell band of several different levels in the centrifuge tube, the mononuclearcell suitable with layering liquid density is intensive in the interface of plasma layer and layering liquid, is the tunica albuginea shape.Use the inventive method isolated cells, mononuclearcell purity can reach 95%, and the cell pick-up rate reaches more than 80%.
When separating erythroblast with micrurgy, identification is a committed step.Rui Shi commonly used in the prior art-Giemsa staining identification erythroblast, yet, use the method for prior art, erythroblast is in enrichment process, passed through repeatedly washing, centrifugal, cell all has in various degree distortion, shrinkage after the film-making, is difficult for the identification erythroblast with above-mentioned dyeing under 400 times of light microscopics, and separating difficulty is big.Haemoglobin in the red blood cell or hematin have the activity of peroxidase sample, can make hydrogen peroxide discharge nascent oxygen, colourless biphenylamine is oxidized to benzidine blue, and then becomes brown compound.Therefore, the present invention adopts benzidine staining, and the erythroblast kytoplasm is dyed pale brown look, carry out haematoxylin again and redye, make karyon be coloured to blueness, 400 times of light microscopics can be known identification down, in conjunction with skilled micromanipulative technique, can catch single erythroblast rapidly, exactly.
Method of inspection recall rate 100%, 40 routine sample standard deviation of the present invention can detect erythroblast, recall rate 100%, and every example is found 2 * 106～6 * 106/ml of erythroblast, frequency of occurrences height.
Use the erythroblast in the inventive method isolation identification blood, testing cost is low, simple to operate, benzidine staining one haematoxylin is redyed back erythroblast recognition effect under 400 times of light microscopics and is better than Rui Shi-Giemsa staining, can track down and arrest the purpose cell faster under the mirror, the later stage micrurgy of being more convenient for is caught erythroblast.
Use authentication method of the present invention, single erythroblast can be discerned and obtain to micrurgy efficiently and accurately behind the benzidine staining, in conjunction with the whole genome amplification technology, can be applicable to gene analysis technique.
Claims (10)
1. the method for erythroblast in the isolation identification blood is characterized in that, may further comprise the steps:
(1) reagent preparation
(1) poly-D-lysine working fluid: the poly-D-lysine storing solution fully mixes with distilled water at 1: 10, and is standby behind the bubble collapse;
(2) PH 7.4 contains the 0.01mol/L PBS of 0.5%BSA:
Sodium chloride 6.5g
Na
2HPO
4·12H
2O 4g
NaH
2PO
4·2H
2O 0.35g
Add distilled water 800ml, regulate pH value to 7.4, distilled water is mended to 1000ml, and 0.01mol/L PBS behind the autoclaving, adds 5g BSA, 40 ℃ of preservations;
(3) contain KOH 200mmol/L, the alkaline cell pyrolysis liquid of DTT 50mmol/L:
400mmol/L?KOH 1ml
1mol/L?DTT 100ul
Sterilized water for injection 850ul fully mixes, and 0.22 μ m millipore filter filters the back packing, and-20 ℃ of preservations are standby;
(2) the anti-slide that comes unstuck of preparation
(1) pre-service of microslide
Common microslide is scrubbed with hot suds, and the tap water cleaning is clean, and oven dry places the sulfuric acid cleaning solution to soak more than 24 hours, the tap water flushing is spent the night, distilled water flushing 3 times, oven dry, 95% alcohol immersion 24 hours, 15 pounds of autoclaving 20min are dried in distilled water flushing 3 times;
(2) the poly-D-lysine bag is by microslide
Pretreated slide was dipped up and down in the poly-D-lysine working fluid 10～20 seconds, divided and fall apart on the slide frame, oven dry below 60 ℃, 4 ℃ of preservations are standby;
(3) select the erythroblast colouring method;
(4) micrurgy obtains erythroblast in the blood.
2. the method for erythroblast is characterized in that in the isolation identification blood according to claim 1, and described step (three) further may further comprise the steps:
The single density gradient centrifugation is collected nucleated blood cell:
1. blood and equivalent contain the 0.01mmol/L PBS mixing of 0.5%BSA, be superimposed on the lymphocyte separation medium gently, make both form an interface clearly, the centrifugal 20min of 1800r/min level, draw the mononuclearcell layer of tunica albuginea shape, move to and use in advance in the moistening clean glass centrifuge tube of PBS;
2. add the 0.01mmol/L PBS washed cell of 0.5%BSA more than 2 times, the centrifugal 10min of 1500r/min level abandons supernatant, triplicate;
3. with smear after the extremely suitable concentration of 0.01mmol/L PB S diluting cells, air dry.
3. the method for erythroblast is characterized in that in the isolation identification blood according to claim 2, and described step (three) further may further comprise the steps:
Adopt Rui Shi-Giemsa staining method that nucleated blood cell is dyeed:
1. slide places and dyes on the horse, drips 3～5 Rui Shi-Jim Sa complex staining liquid, makes it cover sample 1min rapidly;
2. drip 5～10 of damping fluids, rubber pipette bulb is aimed at slide and is blown, and damping fluid is fully mixed with dye liquor, dyeing 5～10min;
3. distilled water flushing is dried.
4. the method for erythroblast is characterized in that in the isolation identification blood according to claim 2, and described step (three) further may further comprise the steps:
Adopt the benzidine staining method that nucleated blood cell is dyeed:
1. slide places 1% biphenylamine methanol solution to soak 2min;
2. in solution, drip 10～20 30%H
2O
2, making the volumetric ratio of itself and 1% biphenylamine methanol solution is 1: 50, mixing, slide continue to soak 1～2min;
3. distilled water fully washes, and dries;
4. haematoxylin is redyed.
5. according to the method for erythroblast in any described isolation identification blood among the claim 1-4, it is characterized in that described step (four) further may further comprise the steps:
1. the slide for preparing is positioned under the inverted phase contrast microscope, and 400 times of following observation identification erythroblasts of light microscopics are also counted;
2. around erythroblast, drip 0.25% Proteinase K of 10～20ul, make it to relax, with the single absorption of micromanipulator from slide;
3. the cell that is drawn to is moved in the special-purpose thin-walled reaction tube of PCR that fills 2.5ul alkalescence cell pyrolysis liquid, centrifugal back is preserved in-20 ℃ of low temperature refrigerators and is equipped with inspection.
6. the method for erythroblast is characterized in that in the isolation identification blood according to claim 5, further comprises the following step of isolating mononuclearcell from peripheral blood in the described method:
(1) the centrifugal 10min of blood sample 2000r/min, 80% blood plasma in the every pipe of usefulness suction pipe sucking-off;
(2) then add PBS and dilute, this liquid contains a whole leucocyte group, comprises granulocyte, lymphocyte, monocyte and part red blood cell;
(3) get 2ml lymphocyte layering liquid in centrifuge tube, the cell suspension with the about 2ml of capillary syring absorption adds on the layering liquid gently simultaneously; With horizontal rotor hydro-extractor 2000r/min, behind the centrifugal 20min, as seen be divided into multilayer, orlop is a red blood cell, and the middle layer is a layering liquid, and the superiors are blood plasma; Be the fine and close white layer of skim between plasma layer and layering liquid, be the mononuclearcell layer;
(4) be inserted into the mononuclearcell layer with capillary syring, draw this layer, put into another test tube; With Hanks liquid washing, centrifugal.
7. the method for erythroblast is characterized in that in the isolation identification blood according to claim 6, further comprises the purification procedures of following erythroblast in the described method:
(1) preparation of nylon hair post:
1. getting the nylon hair spends the night with 0.2mol/L HCl immersion treatment;
2. clean HCl with distilled water, put 37 ℃ of dry for standby;
3. take by weighing 90～120mg nylon hair, evenly disperse, put in the Hanks liquid and soak;
4. get the 1ml syringe, outlet connects plastic tube, clamps;
5. the nylon hair evenly is packed in the syringe emptying air, the high 7～8cm of post;
6. each is used will to make each good post autoclaving;
(2) the nylon hair separates erythroblast:
1. get the mononuclearcell of separation, cell is made into 400/ml with cell culture fluid;
2. melt nylon hair post, emit Hanks liquid with the speed of 18～20 of per minutes, pre-temperature to 37 ℃ cell culture fluid washs nylon hair post;
3. cell suspension is added nylon Mao Zhuzhong, treat that cell suspension all enters nylon Mao Zhuhou, add the 0.2ml cell culture fluid immediately, clamp plastic tube; In 37 ℃ saturation vapour CO2gas incubator, left standstill 1 hour;
4. with the cell culture fluid wash-out cell of the pre-temperature of 5ml, with the cell culture fluid washing nylon hair post of the pre-temperature of 5ml, clean the cell that adheres to again to 37 ℃ to 37 ℃;
5. add the 2ml cell culture fluid, clamp plastic tube, nylon hair post is put refrigerator and cooled but; Cell culture fluid with 4 ℃ of precoolings divides wash-out nylon hair post 3～4 times, each 2ml; Clamp plastic tube earlier, push the nylon hair repeatedly with glass bar or tinsel and be beneficial to adherent cell and come off, and then wash-out;
6. centrifugal, 1000rpm, 10 minutes, remove supernatant, use cell culture fluid washed cell 1 time; Counting cells, with cell culture fluid that cell is resuspended.
8. the method for erythroblast is characterized in that in the isolation identification blood according to claim 6, further comprises the purification procedures of following erythroblast in the described method:
1) anticoagulant dilutes with isopyknic physiological saline;
2) slow being added on the Ficoll liquid level casually, it is clear that the interface of blood and Ficoll keeps;
3) 2000rpm, centrifugal 20min;
4) draw nebulous mononuclearcell layer;
5) 1000rpm, centrifugal 15min;
6) supernatant discarded, washing precipitation;
7) 1000rpm, centrifugal 15min;
8) triplicate the 6th), 7) step;
9) abandon supernatant, precipitation.
9. the method for erythroblast is characterized in that in the isolation identification blood according to claim 8, and the instrument and equipment that uses in the described method is following equipment:
RPM worker's 1640 nutrient culture media: U.S. Gibc company;
Cell separation liquid: Shanghai China smart biological high well-behaved Ri Zhi company limited;
Hemagglutinin PHA: Guangzhou Medicine Industry Inst;
P-III type inverted microscope: Japanese Nikon company;
24 porocyte culture plates: U.S. Corning company;
CO
2Incubator: U.S. Forma scientific company;
DL-CJ-2N high-performance aseptic experiment platform: Harbin Donglian Electronic ﹠ Technology Development Co., Ltd.'s Beijing Company;
0412-1 type trace low speed centrifuge: Shanghai Surgical Operation Equipment Factory.
10. the method for erythroblast is characterized in that in the isolation identification blood according to claim 9, and the collocation method of PBS solution is in the described method:
NaCl18.8g, KCl 0.2g, Na
2HPO
43.488g, add distilled water to 1000ml, transferring pH value is 7.4,15 pounds of autoclavings, 4 ℃ of preservations.
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