CN106399364A - Zebrafish model of thrombocytopenia - Google Patents
Zebrafish model of thrombocytopenia Download PDFInfo
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- CN106399364A CN106399364A CN201610301527.7A CN201610301527A CN106399364A CN 106399364 A CN106399364 A CN 106399364A CN 201610301527 A CN201610301527 A CN 201610301527A CN 106399364 A CN106399364 A CN 106399364A
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- thrombocytopenia
- brachydanio rerio
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Abstract
The invention provides a zebrafish model of thrombocytopenia. Specially, the invention provides an application of an mplsmu40 mutant zebrafish in preparing an animal model of the thrombocytopenia. With the application of the zebrafish Mpl mutant provided by the invention, studies on human thrombocytopenia as well as large-scale screening of alternative medicines for treating the thrombocytopenia can be implemented.
Description
Technical field
The application is related to biological technical field, in particular it relates to be used for platelet development research, blood little
Plate reduces the zebra fish model of disease research and its extensive drug candidate screening.
Background technology
Platelet is an indispensable part in blood system, and its major function is blood coagulation and only
Blood, repairs damaged blood vessel.When platelet goes wrong, body can assume morbid state, for example
Blood platelet disorder, is that (blood is little due to platelet counts minimizing (thrombocytopenia) or hypofunction
Plate functional defect) cause tampon to form bad and bleeding and cause.Thrombocytopenia is periphery
Thrombocytopenia in blood and cause the disease of mucocutaneous and visceral hemorrhage, be clinical common with blood coagulation
One group of disease with the characteristics of dysfunction, bleeding.
In order to preferably study hematoblastic development and function, needing to search out one being capable of specific mark
Hematoblastic gene, so that build the transgenic lines of blood-platelet specific labelling, real-time tracking platelet
Source and differentiation.
MPL (MPL proto-oncogene, thrombopoietin receptor) is to promote thrombocytopoiesis
The receptor of plain TPO, is a kind of hemopoietic growth factor, regulates and controls multiple hemopoietic ancestrals thin in the mankind with mice
Born of the same parents and hematoblastic generation.
Embryo is particularly in order to preferably study the function of MPL and understand human platelet's minimizing disease
The related mechanism of budding thrombocytopenia, needs the mutant for building thrombocytopenia, from
And set up corresponding disease model.Especially, the blood lacked in prior art as embryonic development period is little
Plate reduces the model of disease and is suitable for the dynamic of the drug candidate of extensive stable screening thrombocytopenia
Thing model.The hematoblastic related mechanism of embryonic development period is particularly to preferably study platelet,
Need to build the hematoblastic transgenic zebrafish of specific marker, it is possible to use which carries out Real Time Observation embryo
The hematoblastic generation of tire phase and development.Very necessary to hematoblastic research.
Brachydanio rerio spawning is more, is fertilized in vitro, and ectogenesises and body early embryo is transparent are suitable in embryo
Period of development is observed and for high-throughout drug candidate screening on a large scale.Brachydanio rerio and the blood of people
Liquid composition and mechanisms of gene regulation similar cause Brachydanio rerio have been used for research hematopoietic development1.
Brachydanio rerio has been used for the new molecular mechanism for illustrating some hematopoietic diseases as disease pattern animal2With
The new medicine of screening3.
Existing document report CD41 can be used as a hematoblastic label4, have been built up in document
One kind can be with the hematoblastic transgenic zebrafish of labelling, and cell sorting experiment is proved
CD41:GFPhighFluorescently-labeled cell be platelet.So the transgenic zebra can be utilized
Fish system carries out thrombocytopenic screening mutant, and the experiment of Drug therapy is proved.
Interleukin 11 (IL-11) be a kind of hemopoietic growth factor, can promote hematopoietic stem cell with
And the propagation of macrophage CFU-GM, and inducing macrophage is ripe, so as to increased platelets counts quantity.This
It is existing in mice it is demonstrated experimentally that and also Successful utilization that class medicine is used for being lifted number of platelets
Thrombocytopenia in the clinical treatment mankind5 , 6.
For convenience to hematoblastic research, monitoring platelet treating correlative diseases effect etc., Neng Goute
The hematoblastic Brachydanio rerio transgenic lines of heterolabeling are particularly important.
Content of the invention
The following is the general introduction to the theme for describing in detail herein.This general introduction is not will in order to limit right
The protection domain that asks.
The first aspect of the application is preparing thrombocytopenia there is provided a kind of mutant Brachydanio rerio
Purposes in animal model, wherein described mutant Brachydanio rerio are mplsmu40Mutant.In some realities
Apply in scheme, the thrombocytopenia is caused by the knockout of mpl genes.Further real
Apply in scheme, the thrombocytopenia can remission after being treated by hemopoietic growth factor,
The hemopoietic growth factor is preferably interleukin 11 (IL-11).
The first aspect of the application additionally provides a kind of mutant Brachydanio rerio in screening to thrombocytopenia
Purposes in the effective medicine of disease, wherein described mutant Brachydanio rerio are mplsmu40Mutant.One
In a little embodiments, the thrombocytopenia is caused by the knockout of mpl genes.Further
Embodiment in, the thrombocytopenia can after being treated by hemopoietic growth factor symptom delay
Solution, the hemopoietic growth factor are preferably interleukin 11 (IL-11).Further implementing
In scheme, the screening may comprise steps of：A () is processed with the drug candidate of same concentrations and is received
1 day to 2.5 days after essence, the mutant Brachydanio rerio of preferably 2 days and wild type siblings fish control
Embryo；(b) one to hematoblastic number is observed two days later, to determine the drug candidate to the blood
Platelet reduces the therapeutic effect of disease.
The first aspect of the application is additionally provided one kind and is screened to platelet using mutant Brachydanio rerio
The method for reducing the effective medicine of disease, wherein described mutant Brachydanio rerio are mplsmu40Mutant.?
In some embodiments, the thrombocytopenia is caused by the knockout of mpl genes.Entering one
In the embodiment of step, the thrombocytopenia can after being treated by hemopoietic growth factor symptom
Alleviate, the hemopoietic growth factor is preferably interleukin 11 (IL-11).Further real
Apply in scheme, the screening may comprise steps of：A () is processed with the drug candidate of same concentrations
After fertilization 1 day to 2.5 days, the mutant Brachydanio rerio of preferably 2 days and the control of wild type siblings fish
Embryo；(b) one to hematoblastic number is observed two days later, to determine the drug candidate to described
The therapeutic effect of thrombocytopenia.
The first aspect of the application additionally provides a kind of mutant Brachydanio rerio and is preparing coagulation disorderses
Animal model in purposes, wherein described mutant Brachydanio rerio be mplsmu40Mutant.At some
In embodiment, the coagulation disorderses are caused by the knockout of mpl genes.
The first aspect of the application is related generally to but is not limited to：
A. the mutant of mpl gene orders disappearance, by TALEN targeted gene disruption technology, is obtained
Brachydanio rerio.
B. embryonic stage, using the probe of labelling different phase myeloid cell carry out WISH experiments come
The detailed platelet count purpose of detection changes, and with CD41 labellings maturation platelet cell, and is coagulated
The coagulation function of blood experiment detection mutant juvenile fish.
C. the animal model as thrombocytopenia carries out the drug screening of thrombocytopenia.
By the Mpl mutant mpl for obtainingsmu40, find that there is following phenotype：
1. Mpl mutants mplsmu40Zebrafish embryo number of platelets is substantially reduced.
2. simultaneously in young stage, Mpl mutant mplsmu40Significantly affects the clotting mechanism in body.
The clotting time of mutant significantly extends, and amount of bleeding significantly increases.
3. interleukin-11 has been used to mutant mplsmu40Treated, as a result proved,
Interleukin-11 can effectively improve mutant mplsmu40Brachydanio rerio number of platelets.
Progress of the first aspect of the application relative to prior art：
The research of period of embryo's blood platelet disorder can be carried out using Brachydanio rerio；And stablize as which has
The characteristic of heredity can carry out prolonged sickness tracking and high-flux medicaments sifting.Brachydanio rerio using the present invention
Mpl mutant mplsmu40, can carry out thrombocytopenia be particularly embryonic development period platelet subtract
The research of few disease, and for thrombocytopenia treatment drug candidate Large-scale Screening.
In the first aspect of the application, Mpl mutant mplsmu40There is the platelet of stable heredity
The phenotype of minimizing, can carry out the thrombocytopenia that thrombocytopenia is particularly embryonic development period
Research.Additionally, the Mpl mutant mpl of the applicationsmu40Can also be used to patulous research thrombosis, coagulate
The various disease mechanisms related to platelet such as blood dysfunction.
The second aspect of the application provides a kind of transgenic zebrafish and is preparing platelet and its precursor
Purposes in the animal model of cell specific marker, wherein described transgenic zebrafish is
Tg(mpl:eGFP).In some embodiments, in the transgenic zebrafish, myeloid cell with into
Ripe erythrocyte is not by specific marker.In some embodiments, the high expression of the transgenic zebrafish
Platelet marker gene, low expression medullary system gene and hemoglobin gene, wherein described platelet labelling
Gene includes cd41, lrrc32, gp9, kif1b, nfe2, the medullary system gene include mpo, lyz,
Mfap4, the hemoglobin gene include hbbe1, hbae1.In some embodiments, described
In transgenic zebrafish, GFP+Cell number significantly increase in response to thrombopoietin.
The second aspect of the application additionally provides a kind of transgenic zebrafish and is preparing the dynamic of clotting assay
Purposes in thing model, wherein described transgenic zebrafish are Tg (mpl:eGFP).In some embodiment party
In case, in the transgenic zebrafish, myeloid cell is with mature erythrocyte not by specific marker.?
In some embodiments, the high expression platelet marker gene of the transgenic zebrafish, low expression medullary system
Gene and hemoglobin gene, wherein described platelet marker gene include cd41, lrrc32, gp9,
Kif1b, nfe2, the medullary system gene include mpo, lyz, mfap4, and the hemoglobin gene includes
hbbe1、hbae1.In some embodiments, in the transgenic zebrafish, GFP+Cell number
Mesh significantly increases in response to thrombopoietin.
The second aspect of the application additionally provides a kind of transgenic zebrafish and is preparing for screening increase
Purposes in the animal model of platelet count purpose medicine, wherein described transgenic zebrafish is
Tg(mpl:eGFP).In some embodiments, in the transgenic zebrafish, myeloid cell with into
Ripe erythrocyte is not by specific marker.In some embodiments, the high expression of the transgenic zebrafish
Platelet marker gene, low expression medullary system gene and hemoglobin gene, wherein described platelet labelling
Gene includes cd41, lrrc32, gp9, kif1b, nfe2, the medullary system gene include mpo, lyz,
Mfap4, the hemoglobin gene include hbbe1, hbae1.In some embodiments, described
In transgenic zebrafish, GFP+Cell number significantly increase in response to thrombopoietin.
The second aspect of the application further provides for a kind of transgenic zebrafish of use mpl genetic marker,
Can with specific marker platelet, can with this hematoblastic generation of transgenic zebrafish Real Time Observation,
Ripe, differentiation and thrombosiss etc..The transgenic zebrafish of the mpl genetic markers can be with
Effect of drugs for the various treatments for platelet relevant disease is monitored, it is possible to using Brachydanio rerio
Unique advantage carry out Real Time Observation curative effect of medication.The transgenic zebrafish of the mpl genetic markers is made
For extremely convenient efficiently animal model, with more excellent for hematoblastic development and function is studied
Gesture.The transgenic zebrafish is Tg (mpl:eGFP).
The second aspect of the application is related generally to：
A. by the transposon-mediated technology of Tol2, pTol2-mpl promoter eGFP are obtained and turns base
Because of Brachydanio rerio.
B. embryonic stage, using different transgenic lines Brachydanio rerio cell markings carry out antibody staining contaminate altogether come
Detection mpl:Whether the cell of eGFP labellings is platelet.Simultaneously also using fluorecyte method for separating
The difference of the cell-specific markers gene of the different hemocyte transgenic lines of comparison.And with known
Tg(CD41:EGFP) platelet cell and hematopoietic stem cell of transgenic lines labelling is compared.
C. mpl is checked:Whether eGFP is labeled as platelet cell, carries out blood vessel injury experiment, and
Tpo (thrombopoietin) proliferative induction is tested.
By the Tg (mpl for obtaining:EGFP) transgenic zebrafish, finds that there is following phenotype：
①mpl:EGFP transgenic lines obtain heritable F0, and for transgenic zebrafish, (in F0 generations, refer in particular to sieve
The heritable transgenic of the first generation chosen is maternal or male parent), the cell position of labelling and mpl
Probe In Situ Hybridization expression position is identical.
2. by comparing with medullary system transgenic lines and red system's transgenic lines Brachydanio rerio, cell sorting result
Represent, mpl:eGFP+The high expression platelet surface proteins gene of cell, the base of low expression other pedigrees
Cause.Antibody staining result shows, mpl:eGFP+Not with medullary system or hemoglobin co expression.With
Known CD41:eGFP+Cell compares, mpl:eGFP+Cell higher expression platelet labelling base
Cause.
3. injury experiment shows, mpl under degree of impairment:eGFP+Cell can arrive wound, as
One of hematoblastic function performance.Under the stimulation of Tpo, mpl:eGFP+Cellular response Tpo letter
Number, presents the state that significantly increase.
4. drug treating experiment show, give thrombocytosiss medicine interleukin 11 process with
Afterwards, mpl is shown:eGFP+Cytosis, it was demonstrated that the effectiveness of thrombocytosiss medicines.
The application can be very good the advantage for showing Brachydanio rerio Real Time Observation, by blood-platelet specific labelling
Out, later platelet development is studied and relevant disease research and is clinically used for increasing blood little
The drug screening of plate number provides good animal model.
In this application, Tg (mpl:EGFP before) transgenic zebrafish system can be by Brachydanio rerio platelet
Somatic cell and blood-platelet specific are labeled as green.The transgenic lines have the blood of stable heredity little really
Plate fluorescent labeling, can be used to the hematoblastic generation of patulous research, differentiation, ripe, function (example
Formation such as thrombosis), and the various disease mechanisms related to platelet and carry out drug screening.
By the Tg (mpl for obtaining:EGFP) transgenic zebrafish, finds that there is following purposes：1. green
Color fluorescent specific marked Brachydanio rerio platelet.2. fluorescence signal is from zebrafish embryo after fertilization 2
It starts to occur, and hereafter gradually increases.3. the transgenic lines become a most specificity so far
The hematoblastic transgenic zebrafish system of labelling.For hematoblastic development and research and the drug sieve of function
Choosing provides an extremely easy instrument.
Term as used herein " dpf " refers to the natural law of after fertilization.For example, " 3dpf " refers to after fertilization
3rd day, " 8dpf " referred to after fertilization the 8th day.Term as used herein " hpf " refers to the hour of after fertilization
Number.For example, " 72hpf " refers to after fertilization the 72nd hour.
Term " GFP used in this application+" refer to cell with green fluorescence.
Term as used herein " wild type " or " WT " are all referring to wild-type zebrafish.
Description of the drawings
Fig. 1：TALEN targeted gene disruptions technology obtains Brachydanio rerio mutant mplsmu40, it is a kind of
Thrombocytopenic mutant
(A) TALEN targeted gene disruptions design, selects Brachydanio rerio mpl gene transcripts
Mpl-001 (ENSDART00000124917), 1 exon sequence carry out gene knockout as target spot.
(B) mutant mpl is obtained through sequencingsmu40In the mutation of target position sequence -8bp+48bp, and dash forward
After change, Mpl albumen terminates in advance, disappearance critical function region.(C) immunochemistry coloration result.Tail
Portion's signaling point is CD41:eGFPhighCell, labelling maturation platelet.Mutant mplsmu40In,
Signaling point is significantly reduced, and illustrates that ripe number of platelets is significantly reduced.(D) WISH detections wild type is same
Born of the same parents fish and mutant mplsmu40Mpl gene expressions, mutant mplsmu40The mRNA of middle mpl genes
Expression is substantially reduced.Afterbody partial enlargement frame is 100 × amplification.(E) platelet related gene
Mrna expression amount result, black bar represents wild type siblings fish, and gray bars represent mutant
mplsmu40.Shown in abscissa, the mrna expression amount of these genes is in mutant mplsmu40In all obvious
Lower (every group of n=30).By t- inspections, significant difference show that ns is not significantly different from, * P<0.05,
**P<0.01, * * * P<0.001.
Fig. 2：Brachydanio rerio mutant mplsmu40Coagulation disorderses are shown as in clotting assay
(A) the zebrafish embryo tail veins of acupuncture 3dpf, it is illustrated that stop afterbody when flowing out for blood
Visual field wild type siblings fish (WT, left column) and mutant (mplsmu40, right column), mutant mplsmu40
Amount of bleeding is more, and wound sludged blood is larger.(B) clotting assay processes wild type siblings fish and mutant
mplsmu40Each group at least 10, basis of microscopic observation its from damage start to stopping of bleeding time.
Process in 3dpf and 6dpf respectively, statistical analysiss blood coagulation duration, mutant mplsmu40Afterbody blood
Pipe blood coagulation duration is significantly more than wild type siblings fish (every group of n >=10).Significant difference is checked by t-
Go out, ns is not significantly different from, * P<0.05, * * P<0.01, * * * P<0.001.
Fig. 3：Interleukin 11 (IL-11) can effectively improve Brachydanio rerio mutant mplsmu40Blood
Platelet number
(A) upper figure is the mutant mpl for not injecting IL-11smu40Matched group, figure below are injection IL-11
Mutant mpl afterwardssmu40Treatment group, it is illustrated that signaling point is CD41:eGFPhighCell, represent blood
The number of platelet.Mutant mpl after injection IL-11smu40Signaling point showed increased.(B, C) is right
Wild type and mutant mplsmu40IL-11 injections are carried out simultaneously, black bar represents matched group and (only notes
Penetrate distilled water), gray bars represent IL-11 injection groups (injection IL-11).(B) in, result shows note
Penetrating IL-11 causes the mrna expression amount of cd41 genes in wild type siblings fish to increase relative to matched group
Many about 40% (P=0.016)；(C) in, result shows, injection IL-11 causes mutant mplsmu40In
The mrna expression amount of cd41 genes increases about 97% (P=0.005) relative to matched group.As a result illustrate,
Mutant mplsmu40In response to the treatment of IL-11, and compared with the effect in wild type siblings fish,
IL-11 can significantly more lift mutant mplsmu40Number of platelets.
Fig. 4：Using the transposon-mediated technology of Tol2, pTol2-mpl promoter-eGFP transgenic
The acquisition of Brachydanio rerio
(A) the transposon-mediated Analyzing on Building A Planning Scheme of Tol2, selects Brachydanio rerio mpl gene transcripts
Till mpl-201 (ENSDART00000057297), two exon ATG sequences, 4641bp forward
As mpl promoter regions.(B) above, small icon has gone out zebrafish embryo phase hemopoietic position schematic diagram.
AGM:Aorta-gonad-mesonephros；CHT：Afterbody blood island；It is wild type embryos 2.5dpf on the left of figure below
AGM regions and the diagram of 2.5dpf and 5dpf CHT regions mpl gene hybridization in situ；Right side is
Tg(mpl:EGFP) embryo carries out GFP antibody staining results.Both expressive site results are identical.(C)
In Tg (mpl:EGFP) carry out GFP and Lcp1 (myeloid cell expressing gene) antibody to contaminate in embryo altogether
As a result, on：Red is Lcp1+Cell, in：Green is GFP+Cell, under：It is superimposed upon for both
Diagram together, is as a result without any overlap cell.Illustrate that Lcp1 and GFP does not have co expression
Cell, i.e. GFP not labelling myeloid cells.(D) in Tg (mpl:EGFP) GFP is carried out in embryo
Contaminated with Hbae1 (hemoglobin alpha, all mature erythrocytes of labelling) antibody altogether, on：Red is Hbae1+
Cell, in：Green is GFP+Cell, under：For the diagram that both are superimposed, it is as a result do not have
Any overlap cell.Illustrate that Hbae1 and GFP do not have the cell of co expression, i.e. GFP not labelling
Mature erythrocyte.(E) Tg (mpl of the embodiment of the present application 5:EGFP) with Tg (coronin1a:eGFP)
Embryo carry out GFP fluidic cell sortings, the wherein all myeloid cells of coronin1a labellings respectively.
Both GFP+Cell carries out the testing result of qPCR.As a result show mpl-GFP+Cell with
coronin1a-GFP+Cell is compared, high expression platelet gene cd41, and low expression or does not express marrow
It is cell gene of expression.(F)Tg(mpl:EGFP) with Tg (gata1:DsRed embryo) enters respectively
Row green fluorescence GFP and the sorting of red fluorescence DsRed fluidic cells, wherein gata1 labellings are all red
It is cell.Both GFP+And DsRed+Cell carries out the testing result of qPCR.As a result show
mpl-GFP+Cell and gata1-DsRed+Cell is compared, high expression platelet gene cd41 and lrrc32,
And low expression hemoglobin gene (hbbe1, hbae1) (every group of n=30).Significant difference is checked by t-
Draw, ns is not significantly different from, * P<0.05, * * P<0.01, * * * P<0.001.(G)Tg(mpl:eGFP)
With Tg (CD41:EGFP embryo) carries out GFP fluidic cell sortings, wherein cd41 labellings speckle respectively
Horse fish hematopoietic stem cell and platelet cell.Both GFP+Cell carries out the testing result of qPCR.Knot
Fruit shows mpl-GFP+Cell and CD41-GFP+Cell, although high expression platelet marker gene
(from left to right successively：Gp9, kif1b, lrrc32, nfe2), but these platelet marker gene exist
mpl-GFP+Than in CD41-GFP in cell+Expression in cell is significantly higher.
Fig. 5：Tg(mpl:EGFP) transgenic zebrafish labelling cell is platelet cell, can respond
In injury response and Tpo and interleukin 11.
(A) in 4dpf Tg (mpl:EGFP blood vessel injury experiment is carried out in embryo), and damage location is neck
Portion's blood vessel, GFP+Cellular response damage, and damage location can be arrived at, this is one of platelet function.
(B) in Tg (mpl:EGFP the mRNA of microinjection tpo in embryo), in the embryo of 3dpf,
The result of GFP antibody stainings shows that the embryo for having injected tpo mRNA significantly increases GFP+Thin
Born of the same parents' number.(C) with proceed to Tg (mpl:EGFP) the fish wild type born of the same parents of transgene background
Tg(mpl:EGFP) compare, in thrombocytopenic mplsmu40；Tg(mpl:EGFP) in mutant
mpl-GFP+Cell is substantially reduced.(D) interleukin 11 can significantly lift Tg (mpl:eGFP)
Middle mpl-GFP+Cell number.By t- inspections, every group of n >=10, significant difference show that ns does not have
Significant difference, * P<0.05, * * P<0.01, * * * P<0.001.
After reading and understanding accompanying drawing and describing in detail, it can be appreciated that in terms of other.
Specific embodiment
By way of example the application will be described in further detail below, so that this area skill
Art personnel can put into practice the application.It should be appreciated that other embodiment can be adopted, and can do
Go out appropriate change without departing from spirit herein or scope.In order to avoid for making art technology
Personnel can put into practice unnecessary details for the application, and description may be eliminated for this area skill
Some information known for art personnel.Therefore, described in detail below not should with restricted meaning come
Understand, and the scope of the present invention is limited solely by the scope of the following claims.
Below example facilitates a better understanding of the application, but is not intended to limit the model of the application
Enclose.
Each raw material used in following embodiments, in addition to particularly pointing out, can be obtained from commercially available.
Materials and methods
TALEN targeted gene disruption technology
TALEN target spots identification module is built by Beijing only Shang Lide bio tech ltd, will
TALEN plasmid pairs by micro-injection method be expelled to zebrafish embryo unicellular in can achieve target base
Because knocking out7.In experiment, Brachydanio rerio mpl gene transcripts mpl-001 are devised, on an exon
One section of sequence TTTGGGATGCACCT (SEQ ID NO:1) as TALEN interval regions,
The target spot identification TALEN module plasmids of synthesis the right and left, are oriented knockout to which.
The transposon-mediated technology of Tol2
Promoter sequence is obtained from NCBI, and bis- extras of Brachydanio rerio mpl gene transcripts mpl-201 show
Till sub- ATG sequences, 4641bp is used as mpl promoter regions forward.By promoter sequence and eGFP
Sequence is connected with transposon sequence, clones, is converted, and is prepared into pTol2-mpl promoter-eGFP eventually
Plasmid.
Brachydanio rerio screening mutant
The first generation mutant that TALEN microinjections are obtained is F0 generations, by individual for each F0 generation with
Wild type crosses obtain F1 generation, F1 generation adult fish is cut tail sequencing and obtains different sequence insertions or disappearance
Mutant, whether reduced using mpl in situ hybridizations signal, CD41:Whether GFP fluorescence signals subtract
Carry out phenotypic screen less, through sequencing, obtain target spot interval region series jump (- 8bp+48bp)
(TTTGAAAAGACTTTGAAGACTTGAAAAGACTTTTGCTTTTGAAAAG
ACTTTGCT)(SEQ ID NO:2) mplsmu40Mutant Brachydanio rerio.
Brachydanio rerio transgenic lines are screened
The first generation mutant that pTol2-mpl promoter-eGFP microinjections are obtained is F0 generations,
In F0 generations, are chosen with fluorescent labeling and the correct embryo in luciferase expression position in 3dpf to 4dpf,
By each GFP+F0 is cultivated to individual sexual maturity for individuality, obtains F1 generation with wild type crosses, with
When the whether stable heredity fluorescent labeling of detection F1 generation 3-4dpf embryo.Through fluorescent labeling phenotypic screen,
Obtain the hematoblastic transgenic zebrafish strain Tg (mpl of stable hereditary labelling Brachydanio rerio:eGFP).
The raising and breeding of Brachydanio rerio
Wild type, mutant mplsmu40Brachydanio rerio and transgenic zebrafish system Tg (mpl:EGFP training)
Foster method is as described in the prior art8.For all experiments mentioned in this article, zebrafish embryo quilt
It is placed in the embryo containing 0.003% phenylthiourea (phenylthiourea, PTU) and 0.002% methylene blue
In culture fluid, to remove embryo's pigment.
Whole mount in situ hybridization (whole-mount in situ hybridization, WISH)
The synthesis of the antisense RNA probes of digoxigenin labeled and WISH are operated according to standard test
Operating process is completed9.The mpl probe sequences for using are：
AACAGCAGGTGACATAGAGTTCAGGTGCCACACTTCTGATCTGATTC
AAATCATTTGCAAATGGAGGGGAGACTTATATAAGGACAATACATACA
GCTTCTACTATAAACAATTAAACAGAAGTTCATGGAGCAGTTGGAAG
TTGTGTCCCAATTGCAATAACTCCATCCATCAGTGTGTCCTGTATGGC
CAGAAGTCCAATGTCTTTAAGTTTTACCTCAATACAGGGTTGCAACCA
TTCAGCCGAACATTTTATGCAGAGACTTTCTATATGAACAGTAAAGTT
CAGACAAGGCCTCCAGAAGGTCTGAAGGTACAGATTGGGGAAGAAA
GGCTTTGTTTGACATGGGATTCACCATTTCTGATCATTTCTAAGCATCT
AATGTACCAGATCCGTTATCAGCATCATGAAGAGAATCAGTGGAAGG
GTTTTAAAGCTTCTGGATCCAAGACCAGCACTTGTCTAGATGTGCAC
AGAGGGGGTCGATACACCATCCAGGTTCGAGCACAACCCAATGGATC
TGTGTACAGCGGAAACTGGAGTGACTGGTCAAAACCTGTTACAACC
AACCTACCTTTAAGCAAAGAGTGGATTATTTTTGTTTGCATACCAGTG
GCTCTGATCATCATTGCAACTGCAGTCATCTCTTTCTTCTCCAGATACT
TCCGAAAGGTCAAGAGGTCCCTGTGGCCACCAGTACCAAACCTTAA
CAAGGTCCTGGAAAACATCCTGACTGATATCAGTGGATCACACTGGG
AGCCAACCTTCAACATTAAGCAATGTGATGATGACACTGCTACATCA
GTGGTGGAGGTTCTCACTGAGGGGGAATCTGCGGTCAAAACCTGTA
AGAACACCTGTCCTCTGCTCTCTGAGCACAGTGAAAAACATGGAGA
ACATTTCAGAGAGGACTTGGAAATGGCGCAAGATTATGTGATTTTGA
ACAACAATATAATCCCCTGCCTTACGGGAAACGACTACGTGTATAAGG
ATGTTGCTTCAACACATCTGGCCAATGAAAAACAACACTCTTGCTCC
AGCACTTCTTACACCTCCCTACCAGATCACACCACAGATATTCTCAAC
CAATCCTATCTCCTTTTGGCAGAACAATCCGATCTTGGGGCGTATCAA
ACAACCTGTGGCCAGTACACCAATCTGGAGATCACAGCAGTATCATG
TGTAGCAATTGGAGAGTGAGGTTTACTGTCAAAATGAAGCTCATAAT
CATGTATATATTAAGGGTTCTCTCACAAATGTAAAAAAGACATTCAGT
TTCTGTGGAATGAATGAGAAACAAAGGAAAAGCAAAGCTTACGTTA
ATGGAGCACTTATGAAGGTAATC(SEQ ID NO:17).
Immunochemistry dyes (Immunochemistry staining)
With Anti-GFP antibody (Biotin) (ab6658) one anti-(purchase is from abcam companies),
Donkey anti-Goat IgG(H+L)Secondary Antibody,Alexa488
Conjugate bis- anti-(purchase is from invitrogen companies), donkey anti-rabbit Alexa-555 (purchases
From Invitrogen), and Lcp1 and Hbae1 mono- is anti-(being presented by Hong Kong University of Science and Thchnology)10, will mutation
Body mplsmu40With transgenic strain Brachydanio rerio Tg (CD41:EGFP) copulation4, so as to be carried
Tg(CD41:EGFP) the mutant mpl of transgene backgroundsmu40.Immunochemistry dyeing is carried out to which, is grasped
Make method as known in the art11；Using same method to carrying Tg (mpl:EGFP) the transgenic back of the body
The Brachydanio rerio system of scape carries out immunochemistry dyeing, the highlighted green fluorescence of fluorescence microscopy Microscopic observation thin
Born of the same parents, its quantity will represent the quantity of platelet cell.
qRT-PCR
Extracted using Roche Tripure RNA Isolation Reagent (purchase is from Roche companies)
The total serum IgE of zebrafish embryo, its operating procedure are executed according to related description.Inverted using M-MLV
Record enzyme (Promega), prepares cDNA, and its operating procedure is executed according to related description.Use zebra
Fish gene β-actin carry out qRT-PCR as internal reference, and operational approach is as known in the art12.
QRT-PCR primer sequences
Brachydanio rerio afterbody clotting assay
Microinjection pin (borosilicate glass capillaries using 0.03mm diameters
1B100F-4, buys from World Precision Instruments Inc companies), as the instrument of damage,
Choosing the after fertilization zebrafish embryo of 72 to 84 hours carries out afterbody clotting assay.By zebrafish embryo
It is placed in the embryo medium containing 0.02% tricaine (tricaine) and anaesthetizes, is subsequently placed in agarose
On flat board.Needle stick injuries are carried out using microinjection from tail inner side angiosomeses.Quantity is wild type
Fish born of the same parents and mutant mplsmu40Each more than 110.Basis of microscopic observation timing, from the beginning of damage
Formed to sludged blood, stopping of bleeding.
Drug study
From the huge and grain injection recombination human interleukins-11 of Qilu Pharmaceutical Co., Ltd.'s production, choosing
The zebrafish embryo for taking after fertilization 48-52hpf carries out drug injection.Choose cervical region or yolk sac surface
Intravascular injection.Concentration is 6 μ g/ μ L, each embryo's medication about 5ng.After observation one day, to Brachydanio rerio
Embryo carries out platelet count.
Brachydanio rerio cervical region clotting assay
Microinjection pin (borosilicate glass capillaries using 0.03mm diameters
1B100F-4, buys from World Precision Instruments Inc companies), as the instrument of damage,
The zebrafish embryo for choosing after fertilization 4dpf carries out cervical region clotting assay.Zebrafish embryo is placed on and is contained
Have in the embryo medium of 0.02% tricaine (tricaine) and anaesthetize, be subsequently placed on agarose plate.
Needle stick injuries are carried out using microinjection from angiosomeses on the upside of cervical region yolk sac.Quantity be 10 with
On.Start to platelet arrival injury region thrombosiss to examine under a microscope timing from damage.
Tpo is tested
From mMSSAGE mMACHINE SP6Kit (AM1340) purchases certainlyPublic
Department, external synthesis tpo mRNA, chooses the Tg (mpl of 1 cell stage of after fertilization:EGFP) zebra
Fish embryo, carries out unicellular microinjection.Concentration is 800g/ μ L, each embryo's consumption about 400pg.
After observation 3 days, GFP antibody stainings are carried out to zebrafish embryo.
Fluidic cell is sorted
Choose each transgenic lines Brachydanio rerio i.e. Tg (mpl:eGFP)、Tg(coronin1a:eGFP)10All
Myeloid cell specific marker transgenic lines, Tg (gata1:DsRed)13Whole erythroid cellses specificity marks
Note transgenic lines, Tg (CD41:eGFP)4Platelet and hemopoietic stem cell marker transgenic lines zebra
Fish, in 4dpf periods, each transgenic lines take the zebrafish embryo of more than 200 and carry out green fluorescence
Or red fluorescent cell sorting experiment, test described in concrete grammar reference literature14.
The acquisition of the mutant Brachydanio rerio of embodiment 1.mpl gene order disappearance
In this experiment, Brachydanio rerio mpl gene transcripts mpl-001 are devised
ENSDART00000124917, on an exon section sequence TTTGGGATGCACCT
As TALEN interval regions, synthesize the target spot identification TALEN module plasmids of the right and left, to which
Knockout is oriented, a disappearance 8bp has been obtained, that the Mpl albumen of insertion 48bp terminates in advance is prominent
Variant mplsmu40(referring to Figure 1A, Figure 1B).
Mpl gene expressions
In order to detect the effectiveness of mutation, using mpl as labelling platelet and platelet precursor cells
Marker gene.Using hybridization in situ technique, wild type siblings fish and the mpl of 3dpf is detectedsmu40Mutation
The mRNA expressions of mpl genes in body.
As a result as shown in Figure 1 D, signaling point represents the mRNA expressions of mpl genes.As a result
Show, mutant mplsmu40In mpl genes mrna expression amount compared with wild type siblings fish
Have and obviously reduce.
The mutant Brachydanio rerio of 2. embodiment of the present invention 1 of embodiment can be used as the dynamic of thrombocytopenia
Thing model
Number of platelets
The zebrafish embryo of 5dpf is taken, by mutant mplsmu40With transgenic strain Brachydanio rerio
Tg(CD41:EGFP) copulation4, obtain and carry Tg (CD41:EGFP) the mutant of transgene background.
Wild type siblings fish is equally processed.To carrying Tg (CD41:EGFP) transgene background
mplsmu40Mutant and wild type siblings fish carry out immunochemistry dyeing, in fluorescence microscopy Microscopic observation blood
The cell of highlighted fluorescence in the platelet flowed in liquid, signaling point is CD41:eGFPhighSignaling point,
Its quantity will represent the quantity of platelet cell.
As a result as shown in Figure 1 C, mplsmu40In mutant, number of platelets is compared with wild type siblings fish
It is greatly reduced.
The mRNA expression of multiple genes related to platelet
Have chosen these existing document reports of mpl, cd41, lrrc32, gp1bb, fog1, nfe2 and blood
The related gene of platelet15-18, to 5-dpf wild type siblings fish and mutant mplsmu40Zebrafish embryo
Carry out fluorescent quantitative PCR experiment.In these genes, Lrrc32, Gp1bb are platelet membrane proteins；
Fog1 and Nfe2, is to produce to mammal megalokaryocyte and ripe related transcription regulatory factor.
As a result as referring to figure 1e, show, these genes are in mutant mplsmu40In mRNA water
Flat substantially lower than wild type siblings fish, illustrates mutant mplsmu40Strictly thrombocytopenic prominent
Variant.
In a word, result above shows, in mutant mplsmu40In, due to the knockout of mpl genes,
Hematoblastic number is substantially reduced.Thus, the mpl of the embodiment of the present invention 1smu40Mutant can conduct
The animal model of thrombocytopenia.
Embodiment 3. is carried out by thrombocytopenia using the mutant Brachydanio rerio of the embodiment of the present invention 1
The coagulation disorderses model construction for causing.
Due to mutant mplsmu40Show as thrombocytopenia, it is possible to studied with the mutant
Effect of the platelet to Brachydanio rerio hemostasis-coagulation, and then build Brachydanio rerio coagulation disorderses experimental model19.
First with the zebrafish embryo of 3dpf, per group at least 10, respectively to wild type siblings fish group and
Mutant mplsmu40Group Brachydanio rerio carry out tail veins needle stick injuries, by optical microscope mirror under
Wound clot size after 20 times of observation blood vessel injury, and record each zebrafish embryo from the beginning of damage
Time of the time of bleeding to stopped bleeding.
Fig. 2A show wound clot size after blood vessel injury, and left figure is wild type siblings fish, right figure
For mutant mplsmu40, can be clearly distinguished from out compared to wild type siblings fish, mplsmu40Mutant
After damage, bleeding amount of wound is more, and the clot that wound is formed about is larger.The specific experiment of blood coagulation duration
Data see the table below.Respectively the Brachydanio rerio of 3-dpf and 6-dpf is tested, difference has been carried out to data
This t is checked, to determine wild type siblings fish group and mutant mplsmu40With the presence or absence of significantly between two groups of group
Sex differernce.As a result see Fig. 2 B, show the cartogram of every group of blood coagulation duration.mplsmu40Mutant
Blood coagulation duration is significantly longer than wild type siblings fish.
Result above shows, mplsmu40Mutant can be used as coagulation disorderses caused by thrombocytopenia
Model, be beneficial to follow-up study.
Embodiment 4. carries out drug candidate screening using the mutant Brachydanio rerio of the embodiment of the present invention 1
Due to mplsmu40Mutant shows as thrombocytopenia, so have chosen clinical for platelet
The common drug for reducing disease carries out Drug therapy to which.IL-11 is have selected, which passes through to increase blood system ancestral
The effect that breeds to obtain platelet increase of cell.As shown in Figure 3A, after IL-11 is injected one day,
Hematoblastic number in observation mutant.Upper figure is matched group (only injecting distilled water), and figure below is note
IL-11 groups are penetrated, it can clearly be seen that CD41:eGFPhighSignaling point increases.Simultaneously also using qPCR
Experiment carries out quantitative analyses, as a result as shown in Fig. 3 B-C, in wild type siblings fish and mutant mplsmu40
The expression for occurring the mRNA of cd41 genes after middle injection IL-11 is raised, but in mutant
mplsmu40In rise multiple significantly bigger 97% (P=0.005), compared to wild type siblings fish in
40% (P=0.016).The result explanation of Fig. 3 B-C, mutant mplsmu40In response to the treatment of IL-11,
And compared with the effect in wild type siblings fish, IL-11 can significantly more lift mutant
mplsmu40Number of platelets.
Result above shows, it is possible to use the mpl of the present inventionsmu40Mutant is as animal model, right
The alternative treatment medicine of those thrombocytopenia caused for mpl genetic flaws carries out drug sieve
Choosing.
Embodiment 5.Tg (mpl:EGFP) the acquisition of transgenic zebrafish
In this experiment, bis- exon ATG sequences of Brachydanio rerio mpl gene transcripts mpl-201 are devised
Only it is classified as, 4641bp is used as mpl promoter regions forward, by this section of sequence (SEQ ID NO:30)
It is inserted in pTol2 Transposon plasmids, synthesizes pTol2-mpl promoter-eGFP, to wild type
Zebrafish embryo DNA carries out gene insertion, and having obtained one being capable of hematoblastic turn of specific marker
Gene Brachydanio rerio system Tg (mpl:eGFP).
Fig. 4 A show the transposon-mediated Analyzing on Building A Planning Scheme of Tol2.In Fig. 4 B, top small icon
Zebrafish embryo phase hemopoietic position schematic diagram, wherein AGM are gone out:Aorta-gonad-mesonephros；CHT：
Afterbody blood island.In the figure of lower section, left side is wild type embryos 2.5-dpf AGM regions and 2.5-dpf
With the result of 5-dpf CHT regions mpl gene hybridization in situ, right side is to transgenic zebrafish system
Tg(mpl:EGFP) embryo carries out the result of GFP antibody stainings.Both expressive site results are identical,
The position of the luciferase expression position strictly mpl gene expressions of GFP is described.
Result above shows, obtains transgenic zebrafish system Tg (mpl:eGFP).
The research of the feature of the transgenic lines Brachydanio rerio of 6. embodiment of the present invention 5 of embodiment
Mpl not labelling mature erythrocyte and myeloid cell
Take the zebrafish embryo Tg (mpl of 4dpf:EGFP), immunochemistry dyeing is carried out to which, glimmering
Viewed under light microscopy Lcp1 (myeloid cell)20With Hbae1 (mature erythrocyte)21Coloration result, letter
Number point is respectively Lcp1, Hbae1 and mpl-GFP+Signaling point, its quantity will represent various types of cells
Quantity.
As a result as shown in Fig. 4 C-D, Tg (mpl:EGFP) there is no mpl in transgenic zebrafish embryo
With lcp1 or mpl and the cell of hbae1 co expression.Illustrate, the Tg (mpl of the present invention:eGFP)
Transgenic zebrafish system Tg (mpl:EGFP, in), myeloid cell and mature erythrocyte be not by specificity
Labelling.
Compare with already present Brachydanio rerio transgenic lines, mpl-GFP
+
The high expression platelet labelling of cell
Gene, low expression other pedigree genes.
Using mpl as labelling platelet and the marker gene of platelet precursor cells, coronin1a conducts
The marker gene of labelling myeloid cell, marker gene of the gata1 as the whole erythroid cellses of labelling, profit
Fluidic cell sorting technology is used, the Tg (coronin1a of 4dpf are detected:eGFP)10,
Tg(gata1:DsRed)13, Tg (CD41：eGFP)4Tg (mpl with the embodiment of the present invention 5:eGFP)
Middle DsRed+Cell and GFP+In mRNA expressions.
As a result as shown in Fig. 4 E-F, Tg (mpl:EGFP GFP in)+The high expression platelet base of cell
Because of (cd41, lrrc32), low expression does not express medullary system gene (mpo, lyz, mfap4)22-24With blood red
Protein gene (hbbe1, hbae1)21.As a result show, mpl not labelling mature erythrocyte and myeloid cell.
Further, gp9, lrrc32, kif1b, nfe2 have chosen15,17,18,25These existing document reports
The gene related to platelet, to 4-dpf Tg (CD41:eGFP)4With the embodiment of the present invention 5
Tg(mpl:EGFP) zebrafish embryo carries out green fluorescent protein GFP airflow classifications respectively, will sort
To cell carry out fluorescent quantitative PCR experiment.
As a result as shown in Figure 4 G, show, these genes related to platelet are in mpl-GFP+Thin
Than in CD41-GFP in born of the same parents+Cell in have significantly higher expression, the embodiment of the present invention 5 is described
Tg (mpl:EGFP) transgenic lines Brachydanio rerio is strictly the hematoblastic transgenic lines speckle of more specific mark
Horse fish.
Embodiment 7. using the embodiment of the present invention 5 transgenic lines Brachydanio rerio carry out blood coagulation model and
Tpo and interleukin 11 propagation platelet response mechanism build.
Due to this transgenic lines Brachydanio rerio can fluorescent visual labelling well platelet, so can
To study effect of the platelet to Brachydanio rerio hemostasis-coagulation with this transgenic strain, so as to build Brachydanio rerio
Clotting assay model19.First with the zebrafish embryo of 4dpf, per group at least 10, to this
Tg (the mpl of bright embodiment 5:EGFP) transgenic lines zebrafish embryo carries out Cervical Vessels needle stick injuries,
GFP after the lower 64 times of observations blood vessel injury of optical microscope+Whether cell reaches injury region.
As shown in Figure 5A, mpl-GFP after blood vessel injury+Cell can reach injury region, little as blood
One of function of plate, left figure are 1min after damaging, and right figure is 3min, and 3min thrombus areas compare 1min
Thrombus area is big, illustrates that thrombus area is increased over.
As platelet mainly generates approach for Tpo/Mpl, when Tpo is given, platelet can be responded
Breed in Tpo in a large number26.By immunofluorescence dyeing technology, in Tg (mpl:EGFP injection in)
After tpo mRNA, mpl-GFP is observed+Cell number whether can showed increased.
As shown in Figure 5 B, to unicellular period Tg (mpl:EGFP) microinjection tpo mRNA are carried out,
Detection 3dpf Tg (mpl:EGFP) zebrafish embryo GFP+Cell proliferative conditions.As a result show, Tpo
So that GFP+Cell showed increased.
Mutant Brachydanio rerio mpl using the embodiment of the present invention 1smu40, we observe and subtract in platelet
Mpl-GFP in the middle of few disease model+Whether cell can be reduced.
As shown in Figure 5 C, by immunofluorescence dyeing technology for detection mplsmu40；Tg(mpl:EGFP) (will
The mpl of embodiment 1smu40Mutants homozygous and the Tg (mpl of embodiment 5:EGFP) transgenic lines zebra
Fish hybridizes, and obtains existing mpl to its offspring's screeningsmu40Gene mutation has Tg (mpl again:EGFP) turn
The Brachydanio rerio system of genetic background, is named as mplsmu40；Tg(mpl:EGFP in)), 4-dpf Brachydanio rerio
Embryo GFP+Cell with proceed to Tg (mpl:EGFP) the wild type siblings fish of transgene background is compared,
Statistical result showed GFP+Cell number is substantially reduced.
Interleukin 11 be we have also investigated as one of hematoblastic medicine of increase, if also may be used
To act on Tg (mpl:EGFP) so that GFP+Cytosis.
As shown in Figure 5 D, the Brachydanio rerio embryo of IL-11 is being injected by immunofluorescence dyeing technology for detection
Tire Tg (mpl:EGFP in), compared to matched group, GFP+Cell significantly increases, and as a result this is described
Bright Tg (mpl:EGFP) transgenic zebrafish can be used as antiplatelet drug screening model.
Result above shows, Tg (mpl:EGFP) transgenic lines really can be used as blood-platelet specific mark
The Visualization Model of note, is beneficial to follow-up study.
In sum, the preferred embodiment of the application is these are only, the application is not intended to limit
Protection domain, therefore, all any modifications that is made within spirit herein and principle, equivalent
Replace, improve etc., should be included within the protection domain of the application.
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Claims (13)
1. purposes of a kind of mutant Brachydanio rerio in the animal model for preparing thrombocytopenia, its
Described in mutant Brachydanio rerio be mplsmu40Mutant.
2. purposes as claimed in claim 1, wherein described thrombocytopenia is by mpl genes
Knockout cause.
3. purposes as claimed in claim 1, wherein described thrombocytopenia are being given birth to by hemopoietic
Remission after long factor in treatment, the hemopoietic growth factor are preferably interleukin
-11(IL-11).
4. a kind of mutant Brachydanio rerio screening to the purposes in the effective medicine of thrombocytopenia,
Wherein described mutant Brachydanio rerio is mplsmu40Mutant.
5. purposes as claimed in claim 4, wherein described thrombocytopenia is by mpl genes
Knockout cause.
6. purposes as claimed in claim 4, wherein described thrombocytopenia are being given birth to by hemopoietic
Remission after long factor in treatment, the hemopoietic growth factor are preferably interleukin
-11(IL-11).
7. purposes as claimed in claim 4, wherein described screening are comprised the following steps：
A () processes after fertilization 1 day to 2.5 days with the drug candidate of same concentrations, preferably 2 days described
Mutant Brachydanio rerio and the embryo of wild type siblings fish control；
B () is to hematoblastic number is observed two days later, little to the blood to determine the drug candidate
Plate reduces the therapeutic effect of disease.
8. a kind of utilization mutant Brachydanio rerio to be screening the method to the effective medicine of thrombocytopenia,
Wherein described mutant Brachydanio rerio is mplsmu40Mutant.
9. method as claimed in claim 8, wherein described thrombocytopenia is by mpl genes
Knockout cause.
10. method as claimed in claim 8, wherein described thrombocytopenia are being given birth to by hemopoietic
Remission after long factor in treatment, the hemopoietic growth factor are preferably interleukin
-11(IL-11).
11. methods as claimed in claim 8, wherein described screening are comprised the following steps：
A () processes after fertilization 1 day to 2.5 days with the drug candidate of same concentrations, preferably 2 days described
Mutant Brachydanio rerio and the embryo of wild type siblings fish control；
B () is to hematoblastic number is observed two days later, little to the blood to determine the drug candidate
Plate reduces the therapeutic effect of disease.
A kind of purposes of the 12. mutant Brachydanio rerio in the animal model for preparing coagulation disorderses, its
Described in mutant Brachydanio rerio be mplsmu40Mutant.
13. purposes as claimed in claim 12, wherein described coagulation disorderses are by mpl bases
The knockout of cause causes.
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| CN117417882A (en) * | 2023-10-18 | 2024-01-19 | 中国科学院水生生物研究所 | Preparation method and equipment of single cell suspension of zebra fish embryo |
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