CN101940780A - Zinc finger protein ZBTB20 in chondrocyte-specific knockout mouse model and application thereof - Google Patents
Zinc finger protein ZBTB20 in chondrocyte-specific knockout mouse model and application thereof Download PDFInfo
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Abstract
The invention relates to a ZBTB20 in a chondrocyte-specific knockout mouse model and application thereof, belonging to the technical field of genes, in particular to a function and an application of a zinc finger protein. The invention discloses the application of the zinc finger protein ZBTB20 in the preparation of a combination for promoting or inhibiting the proliferation of chondrocytes of mammals. The invention also discloses a method for screening the combination for promoting or inhibiting the proliferation of the chondrocytes of the mammals based on the zinc finger protein ZBTB20 used as a target point. The invention first points out the importance of the zinc finger protein ZBTB20 in the aspect of adjusting the proliferation of the chondrocytes, thereby providing an effective target point for the research on diseases related to cartilage growth.
Description
Technical field
The invention belongs to the gene technology field, more specifically, the present invention relates to a kind of function and purposes of zinc finger protein.
Background technology
Skeletal system is the important component part of vertebrates body, mainly finishes its growth course with intramembranous ossification and endochondral ossification mode.Endochondral ossification is one of main mode of skeleton development, and it has constituted most of skeleton of skeletal system, comprises vertebrae, bones of limbs, basion and a part of clavicle.Cartilage is the first step of endochondral ossification, finally is formed on the skeletal tissue of playing structure, motion and protective effect in the vertebrates growth course.The cartilage development process is a succession of multi-level and strict regulation and control incident, and it comprises the condensing of mesenchyme cartilage CFU-GM (mesenchymal progenitor cell), differentiating cartilage-forming cell, and the pattern of chondrify tissue formation skeletal tissue is built up.
Endochondral ossification can be divided into cartilage period and growth plate development period take place on order.At the initial period of this process, mesenchymal cell is not to be divided into osteoblast, but is divided into chondrocyte, forms the cartilage original hase.The cartilage original hase is the blank of following skeleton, and chondrocyte differentiation and the arrangement in order that original hase is interior forms the growth plate structure, and the cartilaginous tissue by entering in the constantly alternative growth of endochondral osseous tissue forms skeleton at last.The cartilage-derived growth plate structure generally is divided into four districts, i.e. tranquillization district (R), breeding blanket (P), preceding hypertrophic zone (PH) and hypertrophic zone (H).The tranquillization district is that some are in intermitotic cell, and cell is little and rounded, arranges closely, is about to enter the breeding blanket.The breeding blanket is made up of the vigorous flat cylindrical cell of a group splitting ability, arranges more neatly, and cell volume is bigger.Preceding hypertrophic zone is equivalent to a transition region, is prepared to the hypertrophy differentiation by vegetative state for chondrocyte, and this regional cell becomes big than P district cell, and cell becomes circle by flat column, and cell is arranged loose.The hypertrophic zone is the zone of high mature, it is the sophisticated sign of chondrocyte, the cell volume in this period is big, arrangement is loose random, cell finally entered eventually and became the terminal hypertrophic chondrocyte after the differentiation of end in period this, be accompanied by the apoptosis of terminal hypertrophic chondrocyte, osteoblast replaces chondrocyte gradually, and its excretory bone matrix has replaced cartilage matrix, has finally realized ossified.
Endochondral ossification is actually cell proliferation, the atomization of a complexity.In this course, the extracellular matrix that the chondrocyte secretion is different is expressed different marker molecules.Endochondral ossification is a process that is subjected to tight regulation and control, the local cytokine that produces such as BMP, VEGF, FGF, Ihh, PTHrP, vitamin D etc., by endocrine and paracrine approach such as growth hormone (GH), thyroxin (thyroxin), parathyroid hormone (PTH), insulin growth like factor (IGF), and act on the chemical factors of cartilage such as oxygen concentration, ionic strength, mechanical pressure etc. and can act on the chondrocyte and the cell of other type on every side.These signals activate various transcription factor by all means, as chondrocyte being grown vital Sox9, Runx2, regulate hypoxic inducing factor-1-α (HIF-1 α) of chondrocyte hypoxia reaction or the like, thereby start the expression of series of genes, endochondral ossification harmonious orderly ground is carried out.
Dyschondroplasia and dysplasia are the results who is subjected to the comprehensive adjustment effect of hormone, multiple heredity, environmental factors etc.The research of knock out mice has confirmed that fully many transcription factor, somatomedin and receptor thereof comprise FGF and receptor, IGF and receptor thereof etc., causes cartilage development various imbalances in various degree to occur, and takes place closely related with cartilage disease.
However, this area is still unclear to pathogenetic understanding of chondrocyte proliferation, growth and cartilage development relevant disease at present, has many blank.
Summary of the invention
The object of the present invention is to provide function of a kind of zinc finger protein ZBTB20 (hereinafter to be referred as ZBTB20) and uses thereof.
Another object of the present invention is to provide based on above-mentioned described ZBTB20 is target spot, and screening is for promoting cartilage in mammals cell proliferation and the method that promotes the active substance that skeleton forms.
In a first aspect of the present invention, the purposes of a kind of ZBTB20 or its inhibitor is provided, can be used for preparing the combination material that promotes chondrocyte proliferation.
In another preference, described compositions or its inhibitor are to generation and the secretion capacity of raising mammal to IGF-1, thus the propagation of promotion chondrocyte.
In another preference, described compositions also is used to prevent and treat the cartilage in mammals impaired development.
In another preference, described ZBTB20 is selected from:
(i) aminoacid sequence shown in the SEQ ID:1; Or
(ii) the aminoacid sequence shown in the SEQ ID NO:1 is formed through a series of replacement, disappearance or interpolation, and can promote the cartilage in mammals cell proliferation by (i) deutero-albumen.
Provide the purposes of a kind of ZBTB20 of screening in a second aspect of the present invention, be used to screen the material that promotes the cartilage in mammals cell proliferation.
In a third aspect of the present invention, a kind of a kind of method that promotes the potential material of cartilage in mammals cell proliferation of screening is provided, described method comprises:
(a) candidate substances is contacted with the system that contains ZBTB20;
(b) observe the influence of candidate substances for the expression activity of ZBTB20;
Wherein, if described material can suppress expression or the activity of ZBTB20, show that then this candidate substances is the potential material that promotes chondrocyte proliferation.
In another preference, step (a) comprising: in the test group, candidate substances is joined in the system that contains zinc finger protein; And/or
Step (b) comprising: detect expression or the activity of ZBTB20 in the system of test group, and compare with matched group, wherein said matched group is not for adding the system that contains ZBTB20 of described candidate substances;
(preferably significantly be lower than, if the expression of ZBTB20 or activity are lower than statistically in the test group as low 20%; Preferred low 40%; Preferred low 60% or lower) matched group just shows that this material is the potential material that promotes chondrocyte proliferation.
In another preference, also comprise step:
The potential material that obtains is carried out further cell experiment and/or zoopery, to select to promoting the material of chondrocyte proliferation.
In a fourth aspect of the present invention, provide a kind of with the rodentine application of ZBTB20 gene knockout, be used for model as research cartilage and/or skeleton development.
In another preference, described rodent is selected from rat or mice.
In another preference, described ZBTB20 knock out mice is the mice of homozygous deletion ZBTB20 gene.
In another preference, described ZBTB20 knock out mice is used for the screening of the medicine of cartilage development relevant disease as the animal model of cartilage development and bone development research.
In a fifth aspect of the present invention, the purposes of a kind of ZBTB20 or its encoding gene is provided, be used to prepare the reagent or the test kit of diagnosis cartilage development relevant disease.
In a sixth aspect of the present invention, the method that (non-therapeutic ground) regulates chondrocyte proliferation in a kind of external or body is provided, described method comprises: expression or the activity of ZBTB20 in the reduction chondrocyte.
Others of the present invention are because the disclosure of this paper is conspicuous for a person skilled in the art.
Description of drawings
The immunohistochemical staining analysis of Fig. 1 .ZBTB20 specifically expressing in the normal mouse chondrocyte.This figure is the fluorescence staining result who adopts the monoclonal antibody of anti-ZBTB20.
Fig. 2 .ZBTB20 conditional gene targeting route sketch map.
The evaluation of Fig. 3 .ZBTB20 chondrocyte specificity knock-out mice model.
Fig. 3 A extracts the mousetail genomic DNA, carries out electrophoresis result behind the pcr amplification with the primer of the allelic P1 of ZBTB20 and P2 primer and Cre gene.Wherein ,+/ +/Cre represents the Cre transgenic, and F/F represents the flox allele homozygote of ZBTB20, and F/F/Cre represents flox allele homozygote and the Cre transgenic of ZBTB20, and F/+ represents the flox allele heterozygote of ZBTB20.
Fig. 3 B utilizes the immunofluorescence histochemistry to knocking out the dyeing of mice and littermate control mouse hind leg joint part.
The body weight of Fig. 4 ZBTB20 cartilage specificity knock-out mice CZB20KO and tail are long to be analyzed
Fig. 4 A is the body weight change situation of male CZB20KO mice (right side) at institute's monitoring time (1～5 monthly age) point, with brood male wild mouse (left side) in contrast.Each time point P＞0.1.
Fig. 4 B is the tail length situation of change of male CZB20KO mice (right side) at institute's monitoring time (1～5 monthly age) point, with brood male wild mouse (left side) (Con) in contrast.Each time point P＜0.001).
Fig. 5. IGF-1 expresses and changes in the cartilage specificity knock-out mice CZB20KO body
Fig. 5 is the horizontal situation of change of former generation chondrocyte culture IGF-1mRNA of CZB20KO mice (right side) and littermate control (left side) thereof, P＜0.05.
Fig. 6. cartilage specificity knock-out mice CZB20KO skeletal form changes and chondrocyte proliferation changes
Fig. 6 A is the knee joint A Er XINLAN dyeing of embryo 17.5 days (E17.5) CZB20KO mice (right side), and contrast (left side) is a wild-type mice, and blue region is the chondrocyte dyeing part.
Fig. 6 B is the knee joint A Er XINLAN dyeing of birth back the 5th day (P5) CZB20KO mice (right side), and contrast (left side) is a wild-type mice, and blue region is the chondrocyte dyeing part.
Fig. 6 C is that (knee joint A Er XINLAN F) dyes 3 monthly age CZB20KO mices for D, E, and (A, B C) are wild-type mice, and blue region is the chondrocyte dyeing part in contrast.
Fig. 6 D is the X-ray living imaging figure of 12 monthly age CZB20KO mices (left side) and littermate control mice (right side).
Fig. 6 E is the cell counting analysis (P＜0.005) of the 4th day (P4) CZB20KO mice (right side) in birth back and the single costicartilage separator of littermate control mice (left side).
The specific embodiment
The inventor points out ZBTB20 regulating action to chondrocyte secretion IGF-1 first through long term studies, thereby described zinc finger protein ZBTB20 can regulate chondrocyte proliferation by the expression-secretion that influences IGF-1.Thereby ZBTB20 can be used as the target spot of screening of medicaments, is used to screen by regulating ZBTB20 expression or the active material of regulating chondrocyte proliferation.Finished the present invention on this basis.
ZBTB20 and uses thereof
ZBTB20, be called DPZF (Dendritic Cell-Derived POZ Zinc Finger) again, ZNF288, it is a novel transcription factor of forming by 741 aminoacid, its N end contains the POZ domain, the C end contains 5 C2H2 Zinc finger domains, belongs to BTB zinc finger protein subfamily, and its biological function is still indeterminate.The ZBTB20 wide expression is in mammal, and it is a high conservative in various mammals, has very high albumen homology.
The inventor analyzes discovery, ZBTB20 is specificity overexpression in chondrocyte, utilization has successfully been set up the ZBTB20 mouse model that specificity is rejected in chondrocyte based on the conditional gene targeting technology of Cre-loxP, find that there is the growth plate structure in this mouse model, the vertebra development exists unusual, mainly shows as the knee joint growth plate and increases slightly the broadening of growth plate hypertrophic zone, and find epiphyseal growth plate structure disturbance, irregular arrangement or discontinuous during 3 monthly ages.Analyze and find its growth plate chondrocyte hyper-proliferative, and apoptosis does not have significant difference.The cervical vertebra of the 3 monthly age mice of analysis discovery simultaneously increases thick and is curved little with cervical vertebra place angle of bend, and cervical vertebra is elongated.The thoracic vertebra projection is on the profile and present projection.These change to some pathological states of the proliferation of chondrocytes has some similar.The inventor has found the inside and outside regulating action of ZBTB20 to chondrocyte secretion IGF-1 first, proposes the generating process that the afunction of ZBTB20 in chondrocyte may cause cartilage proliferation and cartilage relevant disease.
In the present invention, used ZBTB20 can be naturally occurring, such as its can separated purification from mammal.In addition, described ZBTB20 can also can be an artificial preparation, such as producing the ZBTB20 of reorganization according to the technique for gene engineering of routine.
Any suitable ZBTB20 is all applicable to the present invention.Described ZBTB20 comprises ZBTB20 or its bioactive fragment of total length.Preferably, the aminoacid sequence of described ZBTB20 can be basic identical with the sequence shown in the SEQ ID NO:1.
The aminoacid sequence of the ZBTB20 that passes through replacement, disappearance or the interpolation of one or more amino acid residues and form is also included among the present invention.ZBTB20 or its bioactive fragment comprise the alternative sequence of a part of conserved amino acid, and the sequence of described amino acid replacement does not influence its activity or kept its part activity.Suitably replace aminoacid and be the known technology in this area, described technology can, be implemented and guarantee not change the biological activity of known molecular at an easy rate.These technology are recognized those skilled in the art, in general, can't change biological activity at the non essential amino acid area change single amino acids of a peptide species.
The bioactive fragment of any ZBTB20 all can be applied among the present invention.Here, the implication of the bioactive fragment of ZBTB20 is meant a peptide species, and it still can keep all or part of function of the ZBTB20 of total length.Generally, described bioactive fragment keeps the activity of 50%, 60% to 99% or 100% total length ZBTB20 at least.
The present invention also can adopt all or part of amino acid whose ZBTB20 modified or improvement, such as, the ZBTB20 that can be modified or improve in order to promote half-life, effectiveness, metabolism and/or proteic effectiveness.Described can be the conjugate of a kind of ZBTB20 through the zinc finger protein of modifying or improve, or it can substituted or artificial aminoacid.Described process is modified or the ZBTB20 of improvement can be gene or can certain difference be arranged with the ZBTB20 or the gene of natural product coexistence, but also can regulate chondrocyte proliferation, and can not bring other untoward reaction or toxicity.That is to say that the version of the biological function of any biological activity that does not influence ZBTB20 or perhaps gene all can be used among the present invention.
(such as the homology with ZBTB20 is 60% or higher to any and described ZBTB20 homology height, preferably, homology is 80% or higher, and is preferred, and homology is 90% or higher) and protein with ZBTB20 identical function be also included within the present invention.
Based on the inventor's new discovery, the invention provides the purposes of ZBTB20, be used to promote the cartilage in mammals cell to improve multiplication capacity.Described ZBTB20 can be directly as the propagation of medicament adjusting cartilage in mammals cell.Perhaps, can screen the material that promotes the cartilage in mammals cell proliferation based on described ZBTB20.
Inhibitor of zinc finger protein and uses thereof
The inhibitor of described ZBTB20 is meant any material of transcribing and translating that can suppress the activity of ZBTB20, the stability of destroying ZBTB20, reduction ZBTB20 effective acting time or play obstruction ZBTB20, these materials can be used for the present invention, as expression or the active material that promotes chondrocyte proliferation by suppressing ZBTB20.Described inhibitor comprises (but being not limited to): antagonist, blocker etc.
The material of the various active mechanisms of any adjusting ZBTB20 also can be used for the present invention, as the active substance to the cartilage in mammals cell proliferation.
Screening promotes the material of chondrocyte proliferation
After getting the influence of the described ZBTB20 of cicada, can adopt several different methods well known in the art to screen the potential material that promotes the cartilage in mammals cell proliferation for the cartilage in mammals cell.And then can from described potential material, find for the material that promotes chondrocyte proliferation.
Therefore, the invention provides a kind of method of screening the potential material that promotes the cartilage in mammals cell proliferation, may further comprise the steps:
Candidate substances is contacted with the system that contains ZBTB20; Observe the influence of candidate substances for the expression activity of ZBTB20; Wherein, if described material can suppress expression or the activity of ZBTB20, show that then this candidate substances is the potential material that promotes chondrocyte proliferation.
In the present invention, described system is selected from (but being not limited to): solution system, cell system, subcellular fraction system, organizational framework, organ systems or animal system.
In optimal way of the present invention, when screening,, matched group can also be set in order to be easier to screen the change of observing ZBTB20, described matched group is can be the system that contains ZBTB20 of not adding described candidate substances.
The material that these Preliminary screening are come out can become the screening storehouse of a ZBTB20, can further carry out cell experiment and or zoopery to these materials.Can be so that finally can therefrom filter out for promoting the real useful medicine of cartilage in mammals cell proliferation.
Therefore, the present invention also comprises the material of a class by the promotion cartilage in mammals cell proliferation of screening technique acquisition of the present invention.
The present invention also provides in a kind of body or the method (the particularly method of non-therapeutic) of external promotion cartilage in mammals cell proliferation, comprises the expression or the activity that reduce ZBTB20 in the chondrocyte.
Should understand, when being used to regulate chondrocyte proliferation, the used ZBTB20 or the effective dose of its inhibitor can change with the order of severity of object to be treated, concrete condition decides according to the individual instances tried, and these factors all can be in the scope that skilled doctor or nutritionist can judge.
After the purposes that gets the described ZBTB20 of cicada or its inhibitor, can adopt several different methods well known in the art that described even ZBTB20 or its inhibitor are carried out, perhaps their encoding gene or their pharmaceutical composition deliver medicine to mammal.Preferably, can adopt the means of gene therapy to carry out.Such as, can be directly with ZBTB20 or its inhibitor by delivering medicine to the experimenter such as methods such as injections; Perhaps, can be delivered on the target spot by the ceneme (such as expression vector or virus etc.) that certain approach will carry ZBTB20 or its inhibitor.And the ZBTB20 that makes it expression activity its inhibitor of storing up a utensil for the right time, concrete condition also need be decided on the type of described inhibitor, and these all are well-known to those skilled in the art.
Preferably, the gene of coding ZBTB20 its inhibitor or the carrier that the carries described gene method by routine can be incorporated into the expression of realization ZBTB20 in the target cell tissue or its inhibitor.Described target tissue is including, but not limited to cartilaginous tissue or chondrocyte.
Animal model
The present invention also provides a kind of ZBTB20 gene knockout rodent that can be used as cartilage development or bone development.Described animal model utilization is set up based on Cre-loxP conditional gene targeting technology, specificity has been rejected the ZBTB20 gene in the chondrocyte (tissue), this animal model cartilage development exists obviously unusual, mainly showing as the joint increases slightly, growing article plate hypertrophic zone broadens, the chondrocyte proliferation hyperenergia, the growth plate structural arrangement is irregular; Either side of spinous processes makes that to present the back of the body protruding, and cervical vertebra is elongated and present flexibility and diminish.This changes similar to the pathological characters of some bone development relevant diseases.This animal model can be used for the screening of bone development treating correlative diseases medicine.
Preparing the technology that the animal gene technology that knocks out of the animal model of this cartilage development and osteodysplasty also can adopt those skilled in the art to be familiar with carries out.
The invention has the advantages that:
Disclose the regulating action of ZBTB20 to the chondrocyte proliferation ability first, described ZBTB20 can suppress the multiplication capacity of chondrocyte and suppress the release of IGF-1.Therefore, ZBTB20 can be directly as medicine with the hyper-proliferative of regulating chondrocyte and the releasability of IGF-1; Perhaps can be used as the target spot of screening of medicaments, be used to screen expression or the active material of regulating the multiplication capacity of chondrocyte or regulating chondrocyte generation IGF-1 by regulating zinc finger protein.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment to be only applicable to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: lab guide (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise percentage ratio and mark calculate by weight.
Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, similar or impartial method and the material of any and contained content all can be applicable among the present invention.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
I. material and method
1. the preparation of gene knockout mice
Select the target spot of the 6th exon of ZBTB20 as gene targeting, make up the targeting vector that the 6th exon two flank introns contain the loxP site, transfection mouse embryo stem cell (ES), screening homologous recombination clone, with the reorganization positive colony ES injection cell to the mice blastaea, migrate to then in the pregnant Mouse Uterus of wait upon, grow and be chimeric Mus, obtain carrying the ZBTB20 allele mice in loxP site through hypergamasis, with this mice and the copulation of protamine (Protamine) promoter Cre transgenic mice, screening is removed neomycin resistance gene Neo by homologous recombination but still is kept complete ZBTB20 the 6th exon and the loxP mice in flank loxP site; With this mice with carry the copulation of mice II Collagen Type VI promoter Cre transgenic mice, in the chondrocyte of expressing the II Collagen Type VI, reject 6 exons that draw of ZBTB20 gene by the homologous recombination specificity of Cre mediation.
2. genotype identification
Extract the mousetail genomic DNA, (shown in Figure 2 with allelic P1 of ZBTB20 and P2 primer, be respectively 5 '-GGCACCCTTT AAATCCAATC AT-3 ' (SEQ ID NO:2) and 5 '-CTCTCCCCTCCTCCCTCTG-3 ' (SEQ ID:3), and the primer of Cre gene (is respectively 5 '-GGCGGATCCGAAAAGAAAAA-3 ' (SEQ ID:4), carry out pcr amplification with 5 '-CAGATGGCGCGGCAACAACAACC-3 ' (SEQ ID:5), obtain the 700bp band and represent ZBTB20 wild-type allele band, the 800bp band represents to contain the ZBTB20 allele in loxP site, the 400bp band is represented the Cre gene, thereby the genotype of mice is comprehensively judged.Extract the genomic DNA of F/F/Cre different tissues of mice, carry out pcr amplification, analyze the specificity and the efficient thereof of ZBTB20 gene knockout in chondrocyte.
3. immunohistochemical analysis
Get the section of mouse hind leg knee joint routine paraffin wax, after the boiling method antigen retrieval, hatch with mouse-anti ZBTB20 labeling of monoclonal antibody, use the mice monoclonal antibody of corresponding fluorescein-labeled two anti-(Alexa 594 or Alexa 488) labellings then, available from Vector company) hatch, mounting after the rinsing is observed coloration result under fluorescence microscope.
4. Ah's XINLAN-fast red colouring
Get the mouse hind leg skeleton specimen, through fixing back routine paraffin wax embedding, slicing treatment, earlier with A Xinlan dyeing, the washing back with routinely method behind the fast red colouring dewater, transparent and mounting.
5.HE dyeing
Get the mouse hind leg skeleton, after fixing, decalcification, carry out conventional stand sheet after paraffin embedding, the section routinely, drag for enter conventional dewaxing rehydration behind sheet, the roasting sheet after, then, use haematoxylin dyeing 5min, wash from the beginning.Use ammonia treatment 1min then, be put in the 2min that dyes in the eosin stain after the washing from the beginning.Conventional method dehydration, transparent, mounting.Microscopy.
6. the separation of chondrocyte and experiment in vitro
Utilize the collagenase digesting method, the chondrocyte in separation and Culture former generation from the articular cartilage of newborn mice (P2-P5) and costicartilage.
II. specific embodiment
Embodiment 1ZBTB20 is specific expressed in chondrocyte
Normal mouse cartilaginous tissue paraffin section has shown that through the coloration result of the monoclonal antibody of anti-ZBTB20 ZBTB20 expresses, and sees Fig. 1 in chondrocyte.
The flow process of specificity rejecting ZBTB20 gene is seen Fig. 2 in chondrocyte.Select the target spot of the 6th exon of ZBTB20 as gene targeting, make up the targeting vector that the 6th exon two flank introns contain the loxP site, transfection mouse embryo stem cell (ES), screening homologous recombination clone, with the reorganization positive colony ES injection cell to the mice blastaea, migrate to then in the pregnant Mouse Uterus of wait upon, grow and be chimeric Mus, obtain carrying the ZBTB20 allele mice in loxP site through hypergamasis, with this mice and the copulation of protamine (Protamine) promoter Cre transgenic mice, screening is removed neomycin resistance gene Neo by homologous recombination but still is kept complete ZBTB20 the 6th exon and the loxP mice in flank loxP site; With this mice with carry the copulation of mice II Collagen Type VI promoter Cre transgenic mice, in the chondrocyte of expressing the II Collagen Type VI, reject 6 exons that draw of ZBTB20 gene by the homologous recombination specificity of Cre mediation.
The genotype identification of ZBTB20 chondrocyte specificity knock-out mice the results are shown in Figure 3.
Fig. 3 A extracts the mousetail genomic DNA, primer with the allelic P1 of ZBTB20 and P2 primer and Cre gene carries out pcr amplification, amplified production is carried out electrophoresis, the 700bp band that electrophoresis result obtains is represented ZBTB20 wild-type allele band, the 800bp band represents to contain the ZBTB20 allele in loxP site, the 400bp band is represented the Cre gene, thereby the genotype of mice is comprehensively judged.
Fig. 3 B, is hatched with mouse-anti ZBTB20 labeling of monoclonal antibody after the boiling method antigen retrieval for getting the section of mouse hind leg knee joint routine paraffin wax, and with corresponding fluorescein-labeled two anti-hatching, mounting after the rinsing is observed coloration result under fluorescence microscope then.The result shows that obvious positive signal is not seen in the cartilage zone of CZB0 knock-out mice, and the diagram redness is a ZBTB20 strong positive signal.
The foundation of this animal model of presentation of results is successful.
The variation of the biochemical indicator of embodiment 3ZBTB20 knock out mice
Measuring the ZBTB20 cartilage specificity, to reject body weight and the tail of mice long, physical signs analysis results such as height, and with contrast (wild-type mice) and compare.The mice of measuring branch sex according to circumstances carries out statistical analysis.
The result compares with matched group as shown in Figure 4, and the body weight of knock out mice does not have obvious change, tail and lacks (P＜0.01).
The variation of the IGF-1 secretion level of embodiment 4ZBTB20 knock-out mice
QPCR analyzes the interior IGF-1mRNA of former generation chondrocyte and changes, and Fig. 5 is seen in P＜0.05.
Embodiment 5ZBTB20 knock-out mice cartilage morphosis changing features and cell number mutation analysis
Knee cartilage tissue to ZBTB20 knock-out mice (CZB20KO) and wild-type mice (contrast) carries out HE dyeing and the chromoscopy of A Er XINLAN.
See Fig. 6 A, Fig. 6 B, 6C and Fig. 6 D.The result shows that ZBTB20 chondrocyte specificity strikes chondrocyte form and the structure of mice CZB20KO does not have the significance change, but chondrosin long slab significance increases slightly on the whole, hypertrophic zone broadening, and 3 monthly age mice growth plate structural arrangement are irregular.This shows that ZBTB20 chondrocyte knock-out mice exists unusually aspect cartilage development.
BrdU mixes method monitoring mice chondrocyte proliferation situation difference.The results are shown in Figure 6E, the result shows that the chondrocyte proliferation ability that ZBTB20 chondrocyte specificity strikes mice CZB20KO strengthens than littermate control mice significance.This shows CZB20KO mice chondrocyte proliferation ability grow.
Utilize collagenase digestion, the chondrocyte of the costicartilage of separation of C ZB20KO and control mice carries out cell counting and makes pairing t-test statistical procedures, the results are shown in Figure 6F.
The result shows that compare with control mice, the chondrocyte quantity significance of CZB20KO mice increases (P＜0.001).This multiplication capacity that shows chondrocyte indirectly is strong.
With isolating former generation chondrocyte be study subject, the expression of endogenous ZBTB20 in the cell before and after the detection candidate substances stimulates, utilize the immunoblotting of quantitative PCR and anti-ZBTB20 antibody to detect, cell ZBTB20 after the analyzing and processing utilizes the ELISA method to detect the variation of somatomedin secretion levels such as IGF-1 in the cells and supernatant.
Test group: the chondrocyte culture of adding candidate substances;
Matched group: the chondrocyte culture of not adding candidate substances.
If compare with matched group, the expression of ZBTB20 weakens in the chondrocyte in the test group, illustrates that then this candidate substances is can reduce ZBTB20 to express, thereby promotes the material of cartilage in mammals cell proliferation.
All quote in this application as a reference at all documents that the present invention mentions, just quoted separately as a reference as each piece document, after having read above-mentioned teachings of the present invention, those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (10)
1. the purposes of a zinc finger protein ZBTB20 or its inhibitor is characterized in that, is used to prepare the compositions that promotes the cartilage in mammals cell proliferation.
2. purposes as claimed in claim 1 is characterized in that described compositions also can be used for preventing and treating the hyper-proliferative of cartilage in mammals cell.
3. purposes as claimed in claim 1 is characterized in that, described zinc finger protein ZBTB20 is selected from:
(i) aminoacid sequence shown in the SEQ ID:1; Or
(ii) the aminoacid sequence shown in the SEQ ID NO:1 is formed through a series of replacement, disappearance or interpolation, and can promote the cartilage in mammals cell propagation by (i) deutero-albumen.
4. the purposes of a zinc finger protein ZBTB20 is characterized in that, is used to screen the material that promotes the cartilage in mammals cell proliferation.
5. method of screening the potential material that promotes the cartilage in mammals cell proliferation is characterized in that described method comprises:
(a) candidate substances is contacted with the system that contains zinc finger protein ZBTB20;
(b) observe the influence of candidate substances for the ZBTB20 expression activity;
Wherein, if described material can suppress expression or the activity of zinc finger protein ZBTB20, show that then this candidate substances is the potential material that promotes chondrocyte proliferation.
6. method as claimed in claim 5 is characterized in that, step (a) comprising: in the test group, candidate substances is joined in the system that contains zinc finger protein; And/or
Step (b) comprising: detect expression or the activity of ZBTB20 in the system of test group, and compare with matched group, wherein said matched group is not for adding the system that contains zinc finger protein ZBTB20 of described candidate substances;
If the expression of zinc finger protein ZBTB20 or activity are lower than matched group statistically in the test group, just show that this material is the potential material that promotes chondrocyte proliferation.
7. method as claimed in claim 5 is characterized in that, also comprises step: the potential material that obtains is carried out further cell experiment and/or zoopery, to select promoting the material of chondrocyte proliferation.
8. the purposes of a zinc finger protein ZBTB20 gene knockout rodent is characterized in that, is used for the model as the research skeleton development.
9. the purposes of a zinc finger protein ZBTB20 or its encoding gene is characterized in that, is used to prepare bad reagent or the test kit of diagnosis skeleton development.
10. the external or interior method of regulating chondrocyte proliferation of body is characterized in that described method comprises: the expression or the activity that suppress zinc finger protein ZBTB20 in the chondrocyte.
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