CN106754691A - A kind of method from poultry whole blood sample quick separating high-purity red blood cell - Google Patents
A kind of method from poultry whole blood sample quick separating high-purity red blood cell Download PDFInfo
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- CN106754691A CN106754691A CN201611139082.3A CN201611139082A CN106754691A CN 106754691 A CN106754691 A CN 106754691A CN 201611139082 A CN201611139082 A CN 201611139082A CN 106754691 A CN106754691 A CN 106754691A
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Abstract
A kind of method from poultry whole blood sample quick separating high-purity red blood cell, is related to poultry blood to separate identification detection technique field.Lymphocyte separation medium surface is then added to after the poultry blood sample of sodium citrate or anticoagulant heparin is first mixed with PBS or physiological saline, through horizontal centrifuge centrifugal treating, discard the red blood cell of the blood plasma after centrifugation, lymphocyte, lymphocyte separation medium and the superiors, obtain high-purity poultry red blood cell, purity reaches more than 99.9%, there is no the pollution of other cells, especially lymphocyte in blood, for the biological function for studying poultry red blood cell provides extraordinary biological sample.The present invention establishes a kind of method that fast and easy efficiently separates high-purity poultry red blood cell, is easy to micromanipulation, and can efficiently and accurately obtain high-purity poultry red blood cell carries out follow-up cellular identification and biological function research.
Description
Technical field
Identification detection technique field is separated the present invention relates to poultry blood.
Background technology
I. Siegel are found that the panimmunity function of red blood cell within 1981, and to contemplate and there is red blood cell in serum
Immunological regulation system and red blood cell kill the effect of cause of disease.The 1950's, it is immune that Nelson has found that red blood cell has first
Adhesion function, the compound of adhesion is more easy to by leukocytes phagocytic.Now it has proven convenient that red blood cell energy self regulating and control and participation immunity of organism
Regulation and control, with adhesion, kill antigen, transmit antigenic information and remove the effect of immune complex in circulation, are whole organism rabbits
Important component in epidemic disease system.The new ideas of " erythrocyte immune system ", have updated understanding of the people to erythrocytic function,
Erythrocyte immune is studied to be developed rapidly.
Red blood cell derives from marrow hemopoietic stem cells with other cells, and the major function that red blood cell is thought in the past is transport
Gas and regulation acid-base balance.Current research shows, red blood cell goes to recognize, adheres to, concentrates, kills by itself C3b receptor
Antigen and removing harmful substance, are the overall indispensable constituents of rabbit epidemic disease system.
Red blood cell can remove circulation rabbit epidemic disease compound, promote the function red blood cell of phagocytosis, similar rabbit epidemic disease cell also to have
There are identification, storage, the function of present antigen and effector cell's sample to act on.Additionally, red blood cell also has regulation and control to immunologic function
Effect.Red blood cell has regulating and controlling effect to phagocyte, lymphocyte, and promotes T cell to break up.Red blood cell can also directly strengthen
The antitumor activity of NK cells, significantly improves the activity that Lymphokine kills (LAK) cell.Influence hematid immunity function
Influence factor mainly have, the red blood cell Immunoregulation factor in serum, the regulation and control of neuroendocrine system and temperature adjusting.
Animal erythrocyte immune Research is started late, the red-cell immune adherence measure side that the nineteen eighty-two such as Guo Feng sets up
Method, domestic Zhang Decheng (1992) etc. take the lead in determining red blood cell C3b and the IC rosette rate of various healthy animals, including right
The measure of the poultry such as chicken, duck, goose, has thus started the research to poultry hematid immunity function.Then, domestic scholars are to health
The side such as hematid immunity function of poultry, ill poultry and vaccine immunity poultry and carried out that following several respects are more deep to grind
Study carefully.
1st, the hematid immunity function of normal poultry：
Research shows, not only the red blood cell of the mammal such as horse, ox, sheep, pig, donkey has an immune function, chicken, duck, goose,
The red blood cell of the birds such as dove, crane quail equally has Immune Adhesive Activity, it was demonstrated that erythrocyte immune system hypothesis is not only dynamic in lactation
Set up in thing, be applied equally to birds.
2nd, influence of the disease to poultry hematid immunity function：
The red corpuscle immunity activity of ill poultry has substantially change.Suffer from fowl cholera, bird flu, On Infectious Bursal Disease of The Chicken, chicken biography
The erythrocyte immune of the poultry of the disease such as metachromia laryngotracheitis, infectious bronchitis of chicken, duck virus hepatitis, chicken inclusion body hepatitis
Adhesion function is damaged, and C3b rosette rates are substantially reduced.Global-worm illness chicken red blood cell C3b rosette rates are significantly reduced, and IC rosette rates are raised.
The erythrocyte C3b rosette and IC rosette rates of fluorine poisoning diseased chicken are reduced, and have the time effect of dosage one.To Marek's disease
Research is relatively more, but result is not the same.Height (1999) research of learning military affairs shows, the strong poison people of 1 age in days AA chick Marek's disease
After work infection, red blood cell C3b and IC rosette rate is first raised and reduced afterwards.The research of Bao Endong shows that Marek's disease is sent out naturally
Diseased chicken C3b rosette rates reduction, IC rosette rates slightly have reduction.The author thinks, the reason for above result of study is inconsistent, may with receive
Examination chicken age and kind difference are relevant, it is also possible to which, with the inadequate specification of detection method, standard is not enough unified relevant.
3rd, influence of the vaccine immunity to hematid immunity function：
Li Shufang etc. (2002) researchs show that after 3 days, C3b and IC rosette rates are significantly reduced infectious laryngotracheitis vaccine immunity,
9 days after immune, rosette rate is recovered to normal level, and erythrocyte immune can be eliminated using Chinese medicine reinforcing agent while vaccine immunity
The adverse reaction of function transience reduction afterwards.Bao Endong etc. (2000) comparative studies Marek's disease 18 mouthfuls of instar embryos it is immune and
The immune influence to chick Exploration of Red Cell Immuno-adhesive Function of 1 Japanese instar chickling, it is found that fetal immune and Immune Profile In Chicks cause chicken red
It is bigger that the rising of cell C3b rosette rates, the reduction of IC rosette rates, but embryo exempt from group C3b rosette rate elevation amplitudes；IC rosette rates foretell drop amplitude
It is bigger.Author thinks that erythrocyte immune adherence inhibiting factor activity foretells drop during embryo exempts from chicken serum so that red blood cell Immunologic function is more
Play well.
In general, the primary stage is also rested on for the research of Immune Function of Animal Erythrocytes both at home and abroad, is only stopped
In red blood cell rosettes test experience.The factor of major limitation erythrocytic function research is the red blood cell for being difficult to obtain high-purity
Individuality, excludes the pollution of other cells in blood.
The content of the invention
For above present Research, the present invention seeks to propose that a kind of quick, efficient, high-purity separates poultry red blood cell
Method, is beneficial to the research for poultry erythrocytic function.
The inventive method is：After the poultry blood sample of sodium citrate or anticoagulant heparin is first mixed with PBS or physiological saline again
Lymphocyte separation medium surface is added to, through horizontal centrifuge centrifugal treating, the blood plasma after centrifugation, lymphocyte, lymph is discarded thin
Born of the same parents' separating liquid and the red blood cell of the superiors, obtain high-purity poultry red blood cell.
By the method for the invention efficiently, the poultry red blood cell purity for obtaining reaches more than 99.9%, does not have in blood other
The pollution of cell, especially lymphocyte, for the biological function for studying poultry red blood cell provides extraordinary biological sample.
The present invention establishes a kind of method that fast and easy efficiently separates high-purity poultry red blood cell, is easy to micromanipulation, can efficiently,
Obtaining high-purity poultry red blood cell exactly carries out follow-up cellular identification and biological function research.
Further, the density of lymphocyte separation medium of the present invention is 1.119.In order that separate red blood cell contamination
The probability for drenching blood mini-bus cell is preferably minimized, and the lymphocyte separation medium that the present invention selects SIGMA companies density to be 1.119 enters
The separation of row red blood cell.
The poultry blood sample is 1 with the mixed volume ratio of PBS or physiological saline:1.In order to reach lymphocyte and red blood cell
The optimum efficiency of separation, poultry blood sample is mixed by this volume ratio in advance with PBS or physiological saline.
The eccentricity of the horizontal centrifuge is 200g, the side that centrifugal treating is stopped naturally after 20 minutes using centrifuge
Formula.Centrifugal condition operation can at utmost avoid separate red blood cell from being polluted by lymphocyte.
Brief description of the drawings
Fig. 1 is the form under high-purity red blood cell light microscope.
Fig. 2 is high-purity red blood cell Wright's staining result.
Specific embodiment
Further describe the present invention with reference to specific embodiment, advantages of the present invention and feature will be with description and
It is apparent.But these embodiments are only exemplary, do not constitute any limitation to the scope of the present invention.People in the art
Member to the details of technical solution of the present invention and form it should be understood that can enter without departing from the spirit and scope of the invention
Row modification is replaced, but these modifications and replacement are each fallen within protection scope of the present invention.
First, the separation of high-purity poultry red blood cell：
Using sodium citrate or anticoagulant heparin venae vasorum collection poultry blood.
With density-gradient centrifugation method, the tool of separating red corpuscle from the sodium citrate or the poultry blood sample of anticoagulant heparin that obtain
Body step is as follows：
1st, collection 2mL poultry blood sample mixes with the PBS or physiological saline of 2mL.
2nd, lentamente by 4mL separation of lymphocytes that the density that 4ml mixed blood samples are added to SIGMA Companies is 1.119
On liquid, blood keeps clear with the interface of lymphocyte separation medium.
3rd, being placed in the trial jar with blood and lymphocyte separation medium carries out centrifugal treating on horizontal centrifuge, adjust water
The eccentricity of flat centrifuge be 200g, room temperature centrifugation 20min, the brake system that can not be carried using centrifuge at the end of centrifugation,
Centrifuge is allowed to be tapered off naturally after terminating.
4th, centrifugation discards the red blood cell of blood plasma, lymphocyte, lymphocyte separation medium and the superiors after terminating, obtain surplus
Excess matter.
5th, the surplus materials for obtaining step 4 adds PBS or physiological saline to 10mL, is 200g in eccentricity at room temperature
Horizontal centrifuge in centrifugal treating 20min, supernatant discarded, take precipitation with PBS or brine.
6th, again by the sediment after washing by mixing with PBS or physiological saline twice, through centrifugation, the place of supernatant discarded
Reason, obtains the sediment for washing three times.
7th, the sediment LCF for washing three times is suspended again.
2nd, the morphological observation of separating red corpuscle：
By isolated poultry red blood cell, optical microphotograph Microscopic observation, form ovalisation, as shown in figure 1, simultaneously naked eyes are placed in
It can be seen that obvious nucleus is present.Red blood cell is counted using cell counting count board, every 1000 red blood cells just see 1-2
The lymphocyte of pollution, therefore reach more than 99.9% using the poultry red blood cell purity that the method for the present invention is separate.
3rd, Wright Stain is dyeed to poultry nucleated blood cell：
（1）Slide is placed on dye horse, is added dropwise 3~5 and is dripped Rui Shi complex staining liquid, makes its rapid covering sample 1min.
（2）Buffer solution 5~10 is added dropwise to drip, rubber pipette bulb alignment slide is blown, and makes buffer solution be sufficiently mixed with dye liquor, dyes 5-
10 min。
（3）Distilled water is rinsed, and is dried.
（4）Observation by light microscope.
The poultry red blood cell of dyeing is placed in optical microphotograph Microscopic observation, as shown in Figure 2：Form ovalize, nucleus
Into bluish violet.Same that red blood cell is counted using cell counting in the different visual field of microscope, every 1000 red thin
Born of the same parents see 1-2 pollution lymphocyte, therefore using the method for the present invention separate poultry red blood cell purity reach 99.9% with
On.
Claims (4)
1. a kind of method from poultry whole blood sample quick separating high-purity red blood cell, it is characterised in that by sodium citrate or heparin
The poultry blood sample of anti-freezing is then added to lymphocyte separation medium surface after first mixing with PBS or physiological saline, through horizontal centrifuge from
Heart treatment, discards the red blood cell of the blood plasma after centrifugation, lymphocyte, lymphocyte separation medium and the superiors, obtains high-purity poultry
Red blood cell.
2. according to claim 1 from the method for poultry whole blood sample quick separating high-purity red blood cell, it is characterised in that：Institute
The density for stating lymphocyte separation medium is 1.119.
3. according to claim 1 from the method for poultry whole blood sample quick separating high-purity red blood cell, it is characterised in that：Institute
It is 1 that poultry blood sample is stated with the mixed volume ratio of PBS or physiological saline:1.
4. from the method for poultry whole blood sample quick separating high-purity red blood cell, its feature according to claim 1 or 2 or 3
It is：The eccentricity of the horizontal centrifuge is 200g, and centrifugal treating is after 20 minutes by the way of centrifuge stops naturally.
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| CN101122601A (en) * | 2007-09-24 | 2008-02-13 | 孙艳萍 | Method for separating and authenticating erythroblast of blood |
| CN101463071A (en) * | 2009-01-13 | 2009-06-24 | 张振冬 | Preparation of flounder erythrocyte outer membrane protein |
| CN103760333A (en) * | 2009-10-13 | 2014-04-30 | 艾博生物医药(杭州)有限公司 | Method for separating red blood cells in blood sample, and applications |
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Application publication date: 20170531 |