Erythroblast simulation particle and the preparation method and application thereofTechnical field
The invention belongs to technical field of medical detection, and in particular to a kind of erythroblast simulation particle and preparation method thereofWith application.
Background technique
The mature erythrocyte of normal people does not contain nucleus, and main component is protein and iron, has the energy of transport oxygenPower, erythroblast belong to the initial puerilism during red blood cell development, are generally present in marrow, but some pathology feelingsIt also will appear under condition, in peripheral blood erythroblast (NRBC), the detection to erythroblast in blood of human body, can beThe diagnosis of disease provides foundation.
Its clinical meaning is as follows:
1, hyperplastic anemia: being most commonly in various anaemias, acute posthemorrhagic anemia, megaloblastic anemia, seriousHypochrosis microcytic anemia；It is common to there is metarubricyte or rubricyte；Occurring erythroblast in peripheral blood indicates boneErythron obvious proliferation in marrow；
2, erythremic myelosis, erythroleukemia: erythroneocytosis paraplasm and being released into blood in marrow, with pronormoblast, early childrenRed blood cell is common；
3, extramedullary hematopoiesis: when myelofibrosis, the hematopoiesis function of the organized renewings embryonic stage such as spleen, liver, lymph node, theseFor tissue due to a lack of the ability of regulation and control discharged to haemocyte, inmature haemocyte largely enters peripheral blood；The children of each stage of development is red thinBorn of the same parents are visible, and visible immature granulocyte and megacaryocyte；
4, other: such as metastatic carcinoma of bone marrow, severe depletion of oxygen.
Currently, more and more cellanalyzers have the function of detecting erythroblast, in order to guarantee the standard of measured valueTrue property need to carry out quality control to it, that is, the quality-control product containing erythroblast simulation particle is used to be detected.
United States Patent (USP) US5559037 (Kim et al.) discloses a kind of fluidic cell for erythroblast and leucocyteThe method of analysis of accounts.The method use fluorescence, low angle light scattering and axis light loss mensurations, so as to from white blood sampleTell NRBC.United States Patent (USP) US5879900 (Kim et al.) further discloses a kind of discrimination NRBC, leucocyte and impairedThe method of leucocyte, and the leucocyte difference method for distinguishing in blood sample is provided by flow cytometer, this method lacksPoint is that accuracy is not high.
United States Patent (USP) US7176031 and US6962817 are disclosed using artificial compound particle simulation people's erythroblastMethod.The defect of this method is compound particle, and production process is complex and costly.United States Patent (USP) US7354767 is disclosedThe method for preparing erythroblast simulation particle using general mammalian red blood cell (being free of nucleus).This method preparationSimulation particle and people's erythroblast nucleus size it is close, suitable for the blood using impedance method detection erythroblastCytoanalyze.But this simulation particle is free of nucleus, is unsuitable for simulating people's erythroblast by specific fluorescent dyeingFluorescent characteristic afterwards.Technical solution disclosed in United States Patent (USP) US7195919 is by connecting large biological molecule in erythrocyte surface(such as: nucleic acid, skin chain), can simulate the fluorescent characteristic of people's erythroblast.This method is disadvantageous in that workSkill is complicated, and the large biological molecule higher cost as raw material.
United States Patent (USP) US6723563 and US6653137 propose with birds (such as turkey or chicken), reptiles (such as alligator) orThe red blood cell of fish (such as toadfish) is that raw material prepares erythroblast simulation particle.United States Patent (USP) US6406915, US6403377,US6399388, US6221668 and US6200500 disclose the side that erythroblast simulation particle is prepared using turkey erythrocytesMethod.United States Patent (USP) US6448085 discloses the method for preparing erythroblast simulation particle using chicken red blood cell.United States Patent (USP)US6187590 and US5858790, which is disclosed, prepares erythroblast simulation particle using the red blood cell of turkey, chicken and frog crouching class fishMethod.United States Patent (USP) US7285417, US7135341 and US7198953 disclose that be prepared with core using alligator red blood cell redThe method of cell simulation particle.The common ground of these technical solutions is: use is with nucleolate animal erythrocyte, including birdClass, reptiles and the red blood cell of fish simulate people's erythroblast.This method is disadvantageous in that, most birds,Reptiles and the red blood cell of fish are oval, larger with mankind's erythroblast morphological differences of subcircular.Therefore certainIn the case of, these animal erythroblasts can not simulate the feature of people's erythroblast well.For example, with fluidic cellWhen art detects cell, elliptical erythrocyte is inconsistent by flow chamber direction, thus generates the very big forward direction of difference in sizeScattered light signal, and round or subcircular cell is then not in this situation.
It can be seen that the method for preparing erythroblast simulation particle in the prior art all has certain disadvantage, needA kind of preparation method for the erythroblast simulation particle that simple process, yield are high, product stability is good.
Summary of the invention
In view of this, the purpose of the present invention is how to provide, a kind of simple process, yield are high, product stability is good coreThe preparation method of red blood cell simulation particle, and the erythroblast simulation particle for providing this method preparation is used as with fluorescence-scatteringLight method is the application of the Quality Control object of the cellular blood analyzer of testing principle.
The technical solution that the present invention realizes that above-mentioned purpose is taken is as follows.
The preparation method of erythroblast simulation particle, steps are as follows:
Step 1: separation and Washed Red Blood Cells, obtain purification of erythropoietin；
Step 2: load agent is added, agent will be loaded under the action of pulse electromagnetic field with diluted purification of erythropoietinIt is introduced into purification of erythropoietin；
The load agent is artificial synthesized DNA or RNA segment, and the additive amount for loading agent is greater than 1.5 μ g/L and in 3.0 μWithin g/L；
Step 3: cell fixer is added to being introduced into the cell after load agent, 18-28 DEG C of fixation washs cell, with thinBorn of the same parents save liquid and save, and obtain erythroblast simulation particle.
Preferably, in the step 1, red blood cell is human red blood cells or the MCV mammal similar with human red blood cellsRed blood cell.
Preferably, in the step 2, with diluted purification of erythropoietin to 1012-1014A/L.
Preferably, in the step 2, the pH value of dilution is 7.1-7.5, by ATP, MgCl2、KH2PO4、NaHCO3、Glucose and water composition, the concentration of ATP are 0.1%-1.0%, MgCl2Concentration be 0.1%-0.5%, KH2PO4Concentration is0.5%-3.0%, NaHCO3Concentration is 0.05%-0.3%, the concentration 0.01%-0.1% of glucose.
Preferably, in the step 2, load agent will be introduced into purification of erythropoietin under the action of pulse electromagnetic fieldProcess are as follows: electroporation apparatus 90-120V voltage stimulate 4-8 seconds.
Preferably, in the step 1 and step 3, the detergent for washing use includes buffer, preservative, inorganicSalt and water.
Preferably, in the step 3, cell fixer includes fixative, buffer, disodium ethylene diamine tetraacetate(EDTA-2Na), preservative and water；Fixative is one of formaldehyde, acetaldehyde, glutaraldehyde, paraformaldehyde, methanol, acetone or moreKind；Set time is 3-5h.
Preferably, in the step 3, cell-preservation liquid includes buffer, nutritional ingredient, preservative and water.
The present invention also provides the erythroblast simulation particles of the preparation method of above-mentioned erythroblast simulation particle preparation.
The present invention also provides above-mentioned erythroblast simulation particles as using nucleic acid fluorescent dyeing as the blood of testing principleThe application of the Quality Control object of cytoanalyze.
Compared with prior art, the invention has the benefit that
The preparation method of erythroblast simulation particle of the invention, by nucleic acid object by way of electric pulse stimulation cellMatter introduce mature erythrocyte in prepare erythroblast simulation particle, the simulation particle preparation step is simple, agents useful for same atPoint more single, products collection efficiency obtained is higher, is suitble to a large amount of production.
Detailed description of the invention
The erythroblast simulation particle that nucleic acid substances are introduced without electric pulse stimulation cell is shown in Fig. 1, with maturationThe whole blood quality control materials of red blood cell analogies 1:4 mixed preparing blood analyser in mass ratio is being inspection with nucleic acid fluorescent dyeingIt surveys on the blood cell analyzer of principle and tests picture (erythroblast channel is only provided).
Erythroblast simulation particle prepared by embodiment 1 is shown in Fig. 2, presses quality with mature erythrocyte analogiesThan the whole blood quality control materials of 1:4 mixed preparing blood analyser, using nucleic acid fluorescent dyeing as the blood cell of testing principle pointPicture is tested in analyzer (erythroblast channel is only provided).
Erythroblast simulation particle prepared by embodiment 2 is shown in Fig. 3, presses quality with mature erythrocyte analogiesThan the whole blood quality control materials of 1:4 mixed preparing blood analyser, using nucleic acid fluorescent dyeing as the blood cell of testing principle pointPicture is tested in analyzer (erythroblast channel is only provided).
Erythroblast simulation particle prepared by embodiment 3 is shown in Fig. 4, presses quality with mature erythrocyte analogiesThan the whole blood quality control materials of 1:4 mixed preparing blood analyser, using nucleic acid fluorescent dyeing as the blood cell of testing principle pointPicture is tested in analyzer (erythroblast channel is only provided).
Specific embodiment
For a further understanding of the present invention, the preferred embodiment of the invention is described below, but it is to be understood that thisA little descriptions are only further explanation the features and advantages of the present invention, rather than limiting to the claimed invention.
The preparation method of erythroblast simulation particle of the invention, first separation and Washed Red Blood Cells, obtain purifying red thinBorn of the same parents；Then purification of erythropoietin is added in dilution, load agent is added, the effect through impulse electric field imported into purifying for agent is loadedIn red blood cell, cell fixer is finally added into the cell being introduced into after loading agent, 18-28 DEG C of fixation washs cell, use cellIt saves liquid to save, obtains erythroblast simulation particle.
In above-mentioned technical proposal, red blood cell can be the red blood cell of people, be also possible to MCV value and the close mammal of peopleRed blood cell, with new blood preparation the present invention in analogies better effect.Separation method is by centrifugation, washing, filteringMode remove leucocyte and blood platelet in blood sample, wherein filtering can use leukocyte depletion filter remove leucocyte.
In above-mentioned technical proposal, the pH value of dilution is 7.1-7.5；Dilution is by ATP, MgCl2、KH2PO4、NaHCO3、Glucose and water composition；The concentration of ATP is 0.1%-1.0%；MgCl2Concentration be 0.1%-0.5%；KH2PO4Concentration is0.5%-3.0%；NaHCO3Concentration is 0.05%-0.3%；The concentration 0.01%-0.1% of glucose；General dilutedPurification of erythropoietin is to 1012-1014A/L.
In above-mentioned technical proposal, load agent be artificial synthesized RNA segment, load agent additive amount be greater than 1.5 μ g/L andWithin 3.0 μ g/L.It can be the 200- of arbitrary sequence to the sequence of artificial synthesized DNA or RNA segment without particular/special requirementThe segment of 1000bp；The expression vector selection being attached thereto uses pET-15b.Concretely: will be shown in sequence 1 or sequence 2DNA through expression vector is extracted and isolated and purified in artificial synthesized rear clone to pET-15b carrier, after amplification, most afterwards through digestionA large amount of DNA fragmentations are obtained after purification, i.e. load agent.
In above-mentioned technical proposal, load agent will be introduced into the process in purification of erythropoietin under the action of pulse electromagnetic fieldAre as follows: electroporation apparatus 90-120V voltage stimulates 4-8 seconds.
In above-mentioned technical proposal, the set time of fixer is 3-5h.
In above-mentioned technical proposal, cell fixer, cell-preservation liquid are used when carrying out carrying out washing treatment to red blood cell and productDetergent, be substance commonly used in the art, be not particularly limited.Wherein, detergent is conventional isotonic solution, including bufferingLiquid, preservative, inorganic salts and water, cell fixer include fixative, buffer, EDTA-2Na, preservative and water, can also be wrappedInclude nutriment；Wherein, fixative be one of formaldehyde, acetaldehyde, glutaraldehyde, paraformaldehyde, methanol, acetone or a variety of, oftenThe concentration of kind fixative is 0.1-2.0mL/L.Cell-preservation liquid includes buffer, nutritional ingredient, preservative and water.Above-mentioned technologyIn scheme, one of preservative Proclin-150, Proclin-200, Proclin-300 and Proclin-500 or twoKind；Buffer is PBS buffer solution, D-HANKS buffer, HEPES buffer solution, citrate buffer solution, sodium chloride buffer, boric acidOne of buffer is a variety of；Nutriment is glucose, mannitol, adenine etc..Commonly, detergent is by 20.0g/LDisodium hydrogen phosphate, 3.0g/L sodium dihydrogen phosphate, 5.5g/L sodium chloride, 0.2g/L Proclin-200 and 1L purified water composition；CarefullyBorn of the same parents' fixer is by 0.3-2.0mL/L acetaldehyde, 0.1-0.2mL/L glutaraldehyde, 2.0g/L sodium dihydrogen phosphate, 0.15g/L EDTA-2Na, 8.0g/L glucose, 2.5g/L adenine, 0.2g/LProclin-200 and 1L purified water composition；Cell-preservation liquid by0.2-0.5/L citric acid, 1.5-2.0g/L sodium citrate, 1.0g/L sodium dihydrogen phosphate, 8.0-15.0g/L glucose, 12.0g/LMannitol, 2.5g/L adenine, 0.3g/LProclin-200 and 1L purified water composition.
The present invention is further illustrated with reference to embodiments, and chemical reagent employed in embodiment is that analysis is pure, can be led toIt crosses commercially available.
Embodiment 1
The preparation method of erythroblast simulation particle: anticoagulant people's whole blood is placed in centrifuge, 3000rpm centrifugation10min removes supernatant, cleans sedimentation cell with detergent, and 3000rpm is centrifuged 10min, removes supernatant, washes repeatedly 2-3It is secondary, leucocyte-removing is removed with leukocyte depletion filter, obtains purification of erythropoietin.Purification of erythropoietin is diluted to 10 with dilution 112A/L,Load agent 1 is added according to 1.51 μ g/L, electroporation apparatus 100V voltage stimulates 5 seconds, into the purification of erythropoietin after electro photoluminescenceCell fixer 1 is added and fixes 3h at room temperature.It is washed repeatedly reaction product 3-4 times with detergent, with cell-preservation liquid 1 to productIt suspends, obtains Quality Control object.It is the blood cell analyzer of testing principle to above-mentioned Quality Control quality testing to nucleic acid fluorescent dyeingIt surveys, as a result (only provides erythroblast channel image) referring to fig. 2.
Load agent 1: by DNA shown in sequence 1 through being extracted in artificial synthesized rear clone to pET-15b carrier after amplificationAnd expression vector is isolated and purified, most obtain a large amount of DNA fragmentations after purification through digestion afterwards, i.e. load agent 1.
Detergent: 20.0g/L disodium hydrogen phosphate, 3.0g/L sodium dihydrogen phosphate, 5.5g/L sodium chloride, 0.2g/LProclin-200,1L purified water.
Dilution 1:1.0g/L magnesium chloride, 12.0g/L potassium dihydrogen phosphate, 1.2g/L sodium bicarbonate, 5.0g/LATP, 0.4g/L glucose, 1L purified water；PH value 7.2.
Cell fixer 1:2.0mL/L acetaldehyde, 0.1mL/L glutaraldehyde, 2.0g/L sodium dihydrogen phosphate, 0.15g/LEDTA-2Na, 8.0g/L glucose, 2.5g/L adenine, 0.2g/LProclin-200,1L purified water.
Cell-preservation liquid 1:0.2g/L citric acid, 1.5g/L sodium citrate, 1.0g/L sodium dihydrogen phosphate, 8.0g/L grapeSugar, 12.0g/L mannitol, 0.3g/LProclin-200,1L purified water.
Embodiment 2
The preparation method of erythroblast simulation particle: anticoagulant people's whole blood is placed in centrifuge, 3000rpm centrifugation10min removes supernatant, cleans sedimentation cell with detergent, and 3000rpm is centrifuged 10min, removes supernatant, washes repeatedly 2-3It is secondary, leucocyte-removing is removed with leukocyte depletion filter, obtains purification of erythropoietin.Purification of erythropoietin is diluted to 10 with dilution 212A/L,Load agent 2 is added according to 2.0 μ g/L, electroporation apparatus 100V voltage stimulates 8 seconds, adds into the purification of erythropoietin after electro photoluminescenceEnter cell fixer 2 and fixes 4h at room temperature.With detergent wash repeatedly reaction product 3-4 times, with cell-preservation liquid 2 to product intoRow suspends, and obtains Quality Control object.To blood cell analyzer that nucleic acid fluorescent dyeing is testing principle to above-mentioned Quality Control analyte detection,As a result referring to Fig. 3 (only providing erythroblast channel image).
Load agent 2: by DNA shown in sequence 2 through being extracted in artificial synthesized rear clone to pET-15b carrier after amplificationAnd expression vector is isolated and purified, most obtain a large amount of DNA fragmentations after purification through digestion afterwards, i.e. load agent 2.
Detergent: 20.0g/L disodium hydrogen phosphate, 3.0g/L sodium dihydrogen phosphate, 5.5g/L sodium chloride, 0.2g/LProclin-300,1L purified water.
Dilution 2:5.0g/L magnesium chloride, 12.0g/L potassium dihydrogen phosphate, 1.2g/L sodium bicarbonate, 8.0g/LATP, 0.6g/L glucose, 1L purified water；PH value 7.4.
Cell fixer 2:0.5mL/L formaldehyde, 0.1mL/L glutaraldehyde, 2.0g/L sodium dihydrogen phosphate, 0.15g/LEDTA-2Na, 0.2g/LProclin-500,1L purified water.
Cell-preservation liquid 2:0.5g/L citric acid, 2.0g/L sodium citrate, 1.0g/L sodium dihydrogen phosphate, 15.0g/L grapeSugar, 2.5g/L adenine, 0.3g/LProclin-300,1L purified water.
Embodiment 3
The preparation method of erythroblast simulation particle: selected people's anticoagulated whole blood is placed in centrifuge, 3000rpm fromHeart 10min removes supernatant, cleans sedimentation cell with detergent, and 3000rpm is centrifuged 10min, removes supernatant, repeated washing2-3 times, leucocyte-removing is removed with leukocyte depletion filter, obtains purification of erythropoietin.Purification of erythropoietin is diluted to 10 with dilution 312A/Load agent 1 is added according to 3.0 μ g/L in L, and electroporation apparatus 100V voltage stimulates 6 seconds, into the purification of erythropoietin after electro photoluminescenceCell fixer 3 is added and fixes 5h at room temperature.It is washed repeatedly reaction product 3-4 times with detergent, with cell-preservation liquid 3 to productIt suspends, obtains Quality Control object.It is the blood cell analyzer of testing principle to above-mentioned Quality Control quality testing to nucleic acid fluorescent dyeingIt surveys, as a result (only provides erythroblast channel image) referring to fig. 4.
Load agent 1: by DNA shown in sequence 1 through being extracted in artificial synthesized rear clone to pET-15b carrier after amplificationAnd expression vector is isolated and purified, most obtain a large amount of DNA fragmentations after purification through digestion afterwards, i.e. load agent 1.
Detergent: 20.0g/L disodium hydrogen phosphate, 3.0g/L sodium dihydrogen phosphate, 5.5g/L sodium chloride, 0.2g/LProclin-300,1L purified water.
Dilution 3:5.0g/L magnesium chloride, 11.5.0g/L potassium dihydrogen phosphate, 2.0g/L sodium bicarbonate, 10.0g/LATP,1.0g/L glucose, 1L purified water；PH value 7.5.
Cell fixer 3:0.3mL/L formaldehyde, 0.2mL/L glutaraldehyde, 2.0g/L sodium dihydrogen phosphate, 0.15g/LEDTA-2Na, 0.2g/LProclin-300,1L purified water.
Cell-preservation liquid 3:0.5g/L citric acid, 2.0g/L sodium citrate, 1.0g/L sodium dihydrogen phosphate, 15.0g/L grapeSugar, 0.3g/LProclin-300,1L purified water.
As attached drawing 1-4 as it can be seen that by erythroblast Quality Control object made from various embodiments of the present invention in blood analyserIt tests and analyzes, the simulation particle for not loading nucleic acid substances can not see the part of erythroblast on scatter plot, such as Fig. 1 instituteShow；And by electromagnetic pulse, treated, it can be seen that having obvious boundary with mature erythrocyte area, leucocyte area on scatter plotThe erythroblast area of limit, as shown in Figures 2 to 4.In summary, preparation method simple process of the invention, yield are high, suitableProduce in enormous quantities, erythroblast simulation particle obtained is as using fluorescence-scattered light method as the cellular blood of testing principle pointThe application of the Quality Control object of analyzer has good prospect.
The foregoing description of the disclosed embodiments enables those skilled in the art to implement or use the present invention.Various modifications to these embodiments will be readily apparent to those skilled in the art, as defined hereinGeneral Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, of the inventionIt is not intended to be limited to the embodiments shown herein, and is to fit to and the principles and novel features disclosed herein phase oneThe widest scope of cause.
Sequence table
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