Summary of the invention
Object of the present invention is just to provide a kind ofly grows the gene relevant with function and regulatory pathway thereof and application to cardiac system.
First aspect present invention, provides the purposes of a kind of Isl1 gene, albumen or its promoter, for the preparation of the pharmaceutical composition promoting HCN4 gene or protein expression, or for the preparation of the transcriptional activation agent of HCN4.
In another preference, described pharmaceutical composition or transcriptional activation agent are also for prevention or treatment sinuatrial node (SAN) abnormal relevant disease.
In another preference, described Isl1 gene or albumen come from mammal, more preferably derive from mice, rat or people.
In another preference, the Isl1 gene expression product that described Isl1 gene, albumen or its promoter comprise, promoted type microRNA, promoted type transcription regulatory factor or promoted type targeting micromolecular compound.
In another preference, described pharmaceutical composition comprises described Isl1 gene, albumen or its promoter and pharmaceutically acceptable carrier.
In another preference, described pharmaceutical composition is also containing HCN4 albumen or the promoter (accelerative activator) activating HCN4 protein active.
In another preference, the abnormal relevant disease of described sinus node cells comprises: sick sinus syndrome or conducting system of heart are correlated with arrhythmia.
In another preference, described conducting system of heart is correlated with that arrhythmia comprises atrial fibrillation, room is flutterred, Fang Zao, room morning, preexcitation syndrome, arrhythmia.
Second aspect present invention, provide a kind of for promoting the pharmaceutical composition of HCN4 gene or protein expression, described pharmaceutical composition contains Isl1 gene, albumen or its promoter and pharmaceutically acceptable carrier.
Third aspect present invention, a kind of screening, for promoting the method for the compound of HCN4 gene or protein expression and/or activity, comprises step:
I (), at experimental group, adds test compounds in cell culture system, and measure Isl1 expression and/or activity in cell culture system; At matched group, in identical cell culture system, do not add test compounds, and measure Isl1 expression and/or activity in cell culture system;
Wherein, if the Isl1 expression in experimental group and/or activity are greater than matched group, then illustrate that this test compounds is the compound for promoting HCN4 gene or protein expression and/or activity.
In another preference, also comprise step:
(ii) to the compound obtained in (i), its facilitation to HCN4 expression and/or activity is measured further; Wherein, if the expression of HCN4 and/or activity are obviously greater than matched group in experimental group, then illustrate that this compound is the compound for promoting HCN4 gene or protein expression and/or activity.
Fourth aspect present invention, provide a kind of zooblast of gene recombinaton, described zooblast contains an inducible expression box, described inducible expression box comprises inducible promoter, and the Isl1 coded sequence to be connected with described inducible promoters and termination codon, thus can express described Isl1 albumen under inductive condition, and Isl1 expressing quantity is relevant to inductive condition;
Further, described zooblast is not the cell of constitutive expression Isl1 coded sequence.
In another preference, described zooblast comprises myocardial cell, pacemaker cell and endocardial cells, pulmonary artery and aortal smooth muscle cell.
In another preference, described zooblast is somatic cell.
In another preference, described inducible expression box is the expression cassette of external source.
In another preference, described Isl1 expressing quantity and inductive condition are proportionate.
In another preference, described Isl1 expressing quantity and the level of derivant or concentration are proportionate.
Fifth aspect present invention, a kind of processing method of external non-therapeutic, comprises step:
A (), under certain density derivant exists, cultivates the zooblast of the gene recombinaton described in fourth aspect present invention, thus induce in described zooblast and express Isl1 albumen; With
B () detects expression and/or the activity level of albumen in described zooblast.
In another preference, in step (b), described albumen comprises: Isl1 albumen, HCN4 albumen or other albumen.
In another preference, in step (b), also comprise and detect parameter relevant to heart cell function (especially pacemaker cell function) in described zooblast.
In another preference, in step (a), under the inducer concentrations corresponding to predetermined expression, cultivate described zooblast, wherein said predetermined expression is, with constitutive expression Isl1 albumen and in the wild-type animal cell of identical type compared with Isl1 protein expression level, Isl1 protein expression level is about 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110% or 120% of wild type.
In another preference, described derivant is tamoxifen.
In another preference, described certain density derivant is 1-50mg/ml, more preferably, is 5-20mg/ml.
Sixth aspect present invention, a kind of method building heart disease animal pattern, comprises step:
A () provides a zooblast, described zooblast is not the cell of constitutive expression Isl1 coded sequence, and described zooblast contains an inducible expression box, described inducible expression box comprises inducible promoter, and the Isl1 coded sequence to be connected with described inducible promoters and termination codon, thus can express described Isl1 albumen under inductive condition, and Isl1 expressing quantity is relevant to inductive condition; With
B () is by described zooblast regeneration animal, thus obtained heart disease animal pattern.
Should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and can combining mutually between specifically described each technical characteristic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, tiredly no longer one by one to state at this.
Detailed description of the invention
The present inventor is through extensive and deep research, overcome the technical barrier that the early stage Isl1 gene delection of fetal development causes animal model mortality first, establish the mouse model that the low expression of Islet1 and condition knock out, through experimental studies have found that this expression model, at fetal development middle and advanced stage, the low expression of Islet1 can result in serious decreased heart rate or arrhythmia, and this kind of embryo can be survived mostly to birth.
Experiment proves, even if Isl1 gene wide expression is during fetal development, but its early expression and middle and advanced stage express the regulating and controlling effect that rises different, the congenital cardiac arrhythmia that embryo's middle and advanced stage low expression Isl1 can cause birth filial generation serious, the embryonic death that the low expression of the more early stage Isl1 of this consequence or disappearance cause, can bring larger misery and burden for family and society.
In addition, the present inventor also uses HCN4-CreERT2 mice and ties up to specific knockdown Islet1 in pacemaker, experiment shows, knocks out Islet1 result in and lowly to Islet1 express the similar phenotype of mice, with the loss of pacemaker cell in growth early stage (E9.5).Knock out Islet1 in growth middle and advanced stage (E12.5), mutation shows serious heart rate slowly and arrhythmia and without the loss of pacemaker pacemaker cell, this illustrates that Islet1 has important regulating and controlling effect to a conducting system of heart function.Confirm thus, Isl1 gene, as the upstream gene of HCN4 path, in the middle and advanced stage of pacemaker cell is grown, serves important regulating and controlling effect.On this basis, the present invention is completed.
Term
As used herein, in the formation that term " the second cardiac field " refers to heart and growth course, there is part cardiac precursors to derive from front dorsal part mesoderm, and this region is defined as the second cardiac field.
Term " the second cardiac field precursor " refers to that deriving from the second cardiac field can become the cell mass of ripe heart cell by differentiation and development further.
Term " pacemaker precursor " refers to can become the cell mass of ripe pacemaker cell by differentiation and development further.
As used herein, term " sinuatrial node abnormal " refers under pathology or physiologic factor, and the pace-making autonomy of sinus node cells and rhythmicity are affected the sinus node dysfunction caused, and cause the situation of related pathologies symptom.Sinuatrial node is extremely usual to be caused by congenital factor and autonomic nerve influence factor etc.
As used herein, term " sinuatrial node abnormal relevant disease " refers to sinuatrial node pace-making and conduct relevant arrhythmia.The abnormal relevant disease of usual sinuatrial node comprises sick sinus syndrome or conducting system of heart and to be correlated with arrhythmia.
As used herein; term " conducting system of heart be correlated with arrhythmia " refers to directly or indirectly to affect due to sinuatrial node exception or functional defect cardiac rhythm and conduction and causes ARR pathologic conditions, comprise atrial fibrillation, room flutterred, Fang Zao, room morning, preexcitation syndrome, arrhythmia.
As used herein, term " Cre ", " Cre enzyme " are used interchangeably, and all refer to Cre recombinase, are the I type topoisomerase of bacteriophage P1, carry out Site-specific recombinase for the DNA between catalysis loxP site.In the present invention, be mainly used in knocking out the specific gene between LoxP site.
As used herein, term " fetal development is early stage " refers to that early embryonic development comprises some stages such as fertilization, the spilting of an egg, blastaea and implantation, gastrula and germinal layer formation, differentiation of germinal layers and the formation of idiosome embryo.Corresponding rodent fetal development is E7.5-E11.5 in early days, and it is the 1-3 month that corresponding human embryos are grown in early days.
As used herein, term " fetal development middle and advanced stage " refers to that almost all histoorgan was formed with ripe until the period of birth.Corresponding rodent fetal development middle and advanced stage and E12.5-E21, it is the 4-9 month that corresponding human embryos grow middle and advanced stage.
Term " Tbx18 pedigree ": Tbx18 transcription factor is expressed in proepicardium cell and epicardial cell, important regulating action has been formed to the growth of heart, therefore can specificity marker heart, and the proepicardium cell of heart and epicardial cell pedigree be defined as Tbx18 pedigree.
As used herein, term " the compound mutant mice of Isl1 " refers to the compound sudden change Mus of Isl1 (Isl1nLacZ/f
neo), namely pass through Isl1f
neothe mouse model that /+Mus system and the hybridization of Isl1 nLacZ/+ Mus system obtain.
" tamoxifen " (tamoxifen) is a kind of exogenous estrogen analog, can combine and make Cre enter nucleus and LoxP site is combined to knock out specific gene by the estrogen receptor in chimeric recombinase.
Term " Tbx18 pedigree ": Tbx18 transcription factor is expressed in proepicardium cell and epicardial cell, important regulating action has been formed to the growth of heart, therefore can specificity marker heart, and the proepicardium cell of heart and epicardial cell pedigree be defined as Tbx18 pedigree.
Islet1
Isl1 gene source is in No. 5 chromosome long arm, and the about 2.4kb of total length, 349 aminoacid of encoding, derives from coded sequence (Genbank accession number BC132609.1), Protein Information (Genbank accession number AAI32610.1)
Islet1 is a homologous structure domain transcription factor, whole body knocks out disappearance completely and the embryo's Deaths (E9.5-10.5) that Islet1 causes the cardiac structure deriving from Islet1 precursor, which prevent the further research acted in pacemaker Islet1.
Isl1 of the present invention has the homology of height in several species, and Isl1 used in the present invention can come from any mammal, comprises (but being not limited to): people, rodent (as mice, rat), chimpanzee, cattle, dog, cat etc.In addition, Isl1 comprises wild type Isl1 and saltant type Isl1 and active fragment thereof.
HCN4
HCN4(hyperpolarization-activated cyclic nucleotide-gated channel 4) gene source is in Chromosome 9, mRNA total length 3.7kb, to encode 1201 aminoacid, its nucleotide sequence is (gene ID NM_001081192.) as shown in SEQ ID NO.:3, and the protein sequence of its coding is as shown in SEQ ID NO.:4 (NP_001074661.1).
Just express at heart crescent phase (Cardiac Crescent) HCN4.In period of development and one-tenth human heart, HCN4 indicates sinuatrial node specifically.
The mankind, the variation of HCN4 causes bradycardia.In the animal patterns such as mice, knock out HCN4 completely and cause serious frequent sinus heart beating to stop fighting, and embryo's Deaths (E9.5-10.5).But knock out HCN4 at adult mouse heart and do not cause dead mouse, the basal heart rate/rule of mutation is normal, but has intermittent cardiac arrest.These researchs show that HCN4 is required to early stage heart pace-making function.HCN4 and calcium current clock (Calcium clock) determine the high autorhythm of pacemaker cell, in the adjustment of the autorhythmic generation of the sinus of heart and heart rate, play pivotal role.In the manhood, other mechanism comprises calcium current clock and relevant Function protein may play a role jointly.
Detection method and test kit
The invention still further relates to quantitative and qualitative detection people Isl1 protein level or mRNA level in-site diagnostic testing process.These tests are known in the art.The people Isl1 protein level detected in test, may be used for detecting or the abnormal relevant disease of prediction sinuatrial node.
A kind of method that whether there is Isl1 albumen in sample that detects utilizes the specific antibody of Isl1 albumen to detect, and it comprises: contacted with Isl1 protein specific antibody by sample; Observe and whether form antibody complex, define antibody complex and just represent in sample to there is Isl1 albumen.
Isl1 albumen or its polynucleotide can be used for the Diagnosis and Treat of Isl1 protein related diseases.Part or all of polynucleotide of the present invention can be used as probe and is fixed in microarray or DNA chip, for analyzing Differential expression analysis and the gene diagnosis of gene in tissue.The antibody of anti-Isl1 can be fixed on protein chip, for detecting the Isl1 albumen in sample.
Present invention also offers a kind of test kit detecting the abnormal relevant disease of sinuatrial node, it contains the primer pair of specific amplification Isl1 and/or Isl1 specific antibody and label or description.
Wherein, described label or description indicate following content: the expression of Isl1 gene or albumen and/or activity significantly lower than normal population, then illustrate, this detected object suffers from the probability of the abnormal relevant disease of sinuatrial node higher than normal population.
The inventive method and test kit can be used for detecting people's amniotic fluid samples, tissue sample, blood sample, blood serum sample or humoral sample, for the abnormal relevant congenital cardiac arrhythmia of early screening sinuatrial node, are conducive to prenatal and postnatal care.
Promoter and pharmaceutical composition
Utilize albumen of the present invention, by various conventional screening assays, can filter out, with Isl1 albumen, interactional material occur, especially promoter etc.
The promoter of Isl1 albumen of the present invention, when carrying out using (administration) on treating, can promote expression and/or the activity of Isl1 albumen, and then promotes HCN4 gene or protein expression, or activates transcribing of HCN4.In another preference, the Isl1 gene expression product that described Isl1 promoter comprises, promoted type microRNA, promoted type transcription regulatory factor or promoted type targeting micromolecular compound.
Usually, these promoter can be formulated in nontoxic, inertia with in pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although pH value can with being formulated the character of material and disease to be treated and changing to some extent.The pharmaceutical composition prepared can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intraperitoneal, intravenous, subcutaneous, Intradermal or topical.
Present invention also offers a kind of pharmaceutical composition, it contains the Isl1 albumen of the present invention of safe and effective amount or its promoter and pharmaceutically acceptable carrier or excipient.This kind of carrier comprises (but being not limited to): saline, buffer, glucose, water, glycerol, ethanol and combination thereof.Pharmaceutical preparation should match with administering mode.Pharmaceutical composition of the present invention can be made into injection form, such as, be prepared by conventional method with normal saline or the aqueous solution containing glucose and other adjuvant.The pharmaceutical composition of such as Tablet and Capsula and so on, is prepared by conventional method.Pharmaceutical composition such as injection, solution, Tablet and Capsula should aseptically manufacture.The dosage of active component is treatment effective dose, such as every day about 1 microgram-10 mg/kg body weight.
Universal method
Transgenic mice
The structure of Isl1-nLacZ gene knock-in mice (being designated as Isl1nLacZ/+) is by conventional method, mice 1 to chromosomal wherein one in Isl1 initiation site insert nLacZ, a kind of preferred visible Song of construction method, M.R., Sun, Y., Bryson, A., Gill, G.N., Evans, S.M., and Pfaff, S.L.2009.Islet-to-LMO stoichiometries control the function of transcription complexes that specify motor neuron and V2a interneuron identity.Development136:2923-2932.In this mice, the Isl1 of item chromosome can not normal expression, and another item chromosome can normal expression Isl1.
TnT-Cre mice system (being designated as cTnT-Cre), cTnT-Cre by fix a point the method knocked in by mice 1 to chromosomal wherein one after the initiation site of cTnT, insert recombinase gene cre, Cre by the regulation and control of cTnT promoter only at the Expression in Myocardium of differentiation.In this mice, item chromosome can express recombinase gene cre.This mice is purchased from south, Shanghai model organism Science and Technology Ltd..
Jiao is shown in by the structure of Isl1floxed mice system, K., Kulessa, H., Tompkins, K., Zhou, Y., Batts, L., Baldwin, H.S., this Mus of and Hogan, B.L.2003.An essential roleof Bmp4in the atrioventricular septation of the mouse heart.Genes & development 17:2362-2367. system replaces with two ends four exon with LoxP site mice 1 to chromosomal Isl1 the 4th exon by the method for homologous recombination.
CTnT-Cre; Isl1 mice is: be obtained by cTnT-Cre mice system and the hybridization of Isl1floxed mice system.Hybridize mice can be expressed recombinase gene Cre and can be identified the loxp site of inserting Isl1 the 4th exon two ends thus be deleted by Isl1 the 4th exon, makes Isl1 gene cannot normal expression.This mice is that cTnT-Cre mice system and the hybridization of Isl1 floxed mice system obtained.
Isl1 is low, and expression (is designated as Isl1f
neoisl1 the 4th exon to be replaced with the 4th exon with loxp site and screening-gene neo by homologous recombination by/the+) structure of mice system.A kind of preferred visible Sun of construction method, Y., Dykes, I.M., Liang, X., Eng, S.R., Evans, S.M., and Turner, E.E.2008.A central role for Islet1in sensory neuron development linking sensory and spinal gene regulatory programs.Nature neuroscience 11:1283-1293. neo in this mice can disturb the allelic normal expression of the Isl1 at neo place, because the compensation Isl1 of other item chromosome expresses normal in chimeric mice; But in pure and mild mice, make the expression of Isl1 significantly reduce, death in p1 days after birth.
Tamoxifen induction type HCN4-Cre ERT2(is designated as HCN4-CreERT2) mice builds available homologous recombination technique and carries out.A kind of preferred method is that the cDNA segment one being comprised CreERT2 sequence is inserted at the position near HCN4 gene start codon (ATG), builds transgenic animal model.A kind of preferred construction method is visible
liang X,
wang G,
lin L,
lowe J,
zhang Q,
bu L,
chen yH,
chen J,
sun Y,
evans SM.HCN4 Dynamically Marks the First HeartField and Conduction System Precursors.
circ Res.2013 these mices of Jun 6.Epublication wherein item chromosome can express CreERT2, and another expresses HCN4.
The compound sudden change Mus of Isl1 (is designated as Isl1nLacZ/f
neo): by by Isl1f
neo/+Mus system and the hybridization of Isl1nLacZ/+ Mus system obtain.Mice 1 pair of chromosome that hybridization obtains, wherein one is inserted nLacZ at Isl1 initiation site, makes Isl1 can not normal expression.In another item chromosome Isl1 gene intron in insert neo gene, interference Isl1 expression.
HCN4-CreERT2/Rosa-LacZ, YFP mice: HCN4-CreERT2 mice system is hybridize with the Isl1 flox/flox mice with Rosa-LacZ or YFP genetic background, to obtain HCN4-CreERT2/Rosa-YFP or LacZ mice.This mice can be expressed the loxP site in the middle of CreERT2 identifiable design Rosa-YFP or Rosa-LacZ and its middle gene order be deleted, and is the expression that Rosa can start YFP or LacZ.
The Cre enzymatic activity be to induce HCN4-CreERT2 mice, gives 150-300ul tamoxifen (10mg/ml) to the female Mus of different brephic pregnancies required time point is oral.Early stage to embryo, tamoxifen is in mice embryonic E9.5 administration, and embryo samples is 32-36 hour upon administration, about period of embryo E11 days results.To late embryo stage, tamoxifen is in mice embryonic E11.5 administration, and embryo samples is about gathered in the crops upon administration for period of embryo E14.5 days.The enable expression observing HCN4-CreERT2 after tamoxifen induction Cre expresses of the Rosa26-YFP genetic background that transgenic mice has.
Immunofluorescence dyeing and RNA in situ hybridization
X-gal dyes, and the method using conventional procedures of immunofluorescence and RNA in situ hybridization carries out, and representational method is visible:
Liang,X.,Zhou,Q.,Li,X.,Sun,Y.,Lu,M.,Dalton,N.,Ross,J.,Jr.,and Chen,J.2005.PINCH1plays an essential role in early murine embryonic development but is dispensable in ventricular cardiomyocytes.Molecular and cellular biology25:3056-3062;
Liang,X.,Sun,Y.,Schneider,J.,Ding,J.H.,Cheng,H.,Ye,M.,Bhattacharya,S.,Rearden,A.,Evans,S.,and Chen,J.2007.Pinch1 is required for normal development of cranial and cardiac neural crest-derived structures.Circ Res 100:527-535。
Use primary antibodie is as follows: mice anti-Islet1/2 monoclonal antibody (DSHB), rabbit anti-Islet1(abcam), rat anti-HCN4(abcam), rabbit anti-Cx40(Santa Cruz), sheep anti-Tbx3(Santa Cruz), mice anti-Nkx2.5(Santa Cruz), rat anti-Brdu(abcam).Two anti-employ Alexa 488 or 594 labeled(Invitrogen).TUNEL staining employs kits manuals (purchased from Roche).
BrdU staining: pregnancy female Mus lumbar injection 500 μ l BrdU (premix liquid of particular point in time, Ambion) 3 times, 3 hours, interval, BrdU dyeing is undertaken by conventional method, representational method is shown in Sun, Y., Dykes, I.M., Liang, X., Eng, S.R., Evans, S.M., and Turner, E.E.2008.A central role for Islet1 in sensory neuron development linking sensory and spinal gene regulatory programs.Nature neuroscience 11:1283-1293.
Cell counting: to specific budding heart SAN region samples section, slice thickness 10um, each sample is chosen 4 serial section and carried out dyeing and counting positive cell.In cell proliferation experiment and cell apoptosis assay, to the BrdU in venous sinus and SAN region on the right side of mutated embryonic and contrast embryo, positive and TUNEL positive cell counts.In order to study the expression of Isl1 in growth course, the Isl1 positive and HCN4 positive cell are counted, in order to calculate the ratio of Isl1 and HCN4 positive cell.To the different samples of each time point, the section that the minimum analysis of each sample 4 to 6 is corresponding, the minimum analysis of each experiment 3 pairs of samples.
Nano-ChIP and real-time quantitative PCR (qPCR):
Nano-ChIP of the present invention and real-time quantitative PCR (qPCR) are undertaken by conventional method, and representational method is as follows:
Adopt FACS-sorted method, be separated HCN4-CreERT2; Isl1f/f sudden change Mus embryo and HCN4-CreERT2; Isl1 matched group embryo (Rosa-YFP background) SAN pacemaker cell is used for qPCR and analyzes.Gather in the crops and perform the operation the Isl1 sudden change and matched group embryo SANs that are separated E11 days YFP positives, use collagenase/dispase(1mg/ml, Roche)/trypsin(0.1%) mixture slaking liquid digests 10 minutes.Collect the pacemaker cell (YFP is positive) be suspended in pace-making Digestive system, FACS sorted(BD FACSAria) isolated cell be stored in-80C.RNA uses RNeasy Mini kit(Qiagen) preparation.QPCR uses SYBR green method.
Nano-ChIP experimental technique: HCN4-nEGFP embryo SAN cell collects (E13.5 to E18.5) at late embryo stage, and cell harvesting and FACS-sorted method are as previously mentioned.About 40,000SAN cells use 1%formaldehyde at normal temperature crosslinked 10 minutes.Cell uses glycine cold quenching 5 minutes and rinses 2 times with frozen water pre-cooling PBS.Chromatin uses ultrasound wave to be fragmented into 200-800bp size segment, and uses 1 μ g Isl1 antibody (39.4D5, DSHB) co-precipitation.Oppositely be cross-linked subsequently and purification, the DNA of ChIP DNA and input is amplified (60).After amplification, DNA uses qPCR qualification, and associated regulatory region is by confirming after analytical data.
Ultrasound cardiograph detects:
Pregnant mouse uses isoflurane anesthesia.Use ultrasound cardiograph model for Visualsonics Vevo770 high-res ultrasonic system, use RMV704 (40MHz) probe.Each embryo detects separately, and obtains independently image and data.Heart rate and every heart function parameter is obtained by acquisition B-mode and pulse Doppler image.After ultrasound cardiograph detects, solution cuts embryo, corresponding with ultrasound cardiograph testing result respectively, and carries out genotype and Phenotype detection.
Statistical analysis method
All data use meansigma methods (mean) ± SEMb to represent, t-test uses 2 groups of independent test results mutually to verify.Think that experimental result has significant difference as P<0.05.
Beneficial effect of the present invention
1. present invention demonstrates in embryo development procedure, Isl1 gene or albumen play an important role to the formation in sinus node cells whole growth phase and function point analysis, especially for the formation of sinus node cells middle and advanced stage and function, the disappearance of Isl1 or variation can not cause embryonic death, but cause postnatal fetus to suffer from arrhythmia to be the various congenital heart diseases of cardinal symptom.
2. the present invention have also demonstrated the upstream gene as HCN4, and the regulation and control of Isl1 to HCN4 play an important role, thus the middle and advanced stage of regulation and control embryo sinus node cells is grown.
3. the present invention can be used for congenital heart disease, is especially Prenatal Screening and the diagnosis of the various congenital heart diseases of cardinal symptom with arrhythmia, is conducive to prenatal and postnatal care.
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition, the people such as such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise percentage ratio and number are percentage by weight and parts by weight.
Embodiment 1Isl1 and HCN4 is at heart SAN cells
In order to study the effect of Isl1 in SAN formation and function, first analyzing the expression of Isl1 in SAN growth course, and using several SAN cell and myocardium of atrium cell antibody (HCN4 Tbx3 Cx40 is purchased from abcam company) as labelling.
HCN4 is pacemaker cell channel protein, starts in early days to express, in SAN differentiation and development process, play pivotal role at heart development.The venous sinus (sinus venosus (SV)) of myocardium is expressed in, pacemaker cell hair growth promoting region high expressed HCN4(Figure 1A of body early embryo) at E9.5, Isl1.
At E10.5, Isl1 and HCN4 coexpression in embryo SAN head (Figure 1B) and afterbody (Fig. 1 C), venous valve (vv), and around the atrial myocardium (DM) (Figure 1A, B, arrow) of dorsal mesocardium.
Early stage after fetal development late period and birth, Isl1 and HCN4 coexpression is in sinus node cells (Fig. 1 D, E, I).
The expression of Isl1 in SAN reduced gradually along with the age.The pacemaker cell quantitative proportion of Isl1 and HNC4 coexpression significantly reduces after birth for 2 weeks, and reduces (Fig. 1 L) gradually along with the age increases.
Tbx3 and CX40 dyes the boundary that can mark between SAN and atrial myocardium altogether.Find at embryo E10.5 days, Tbx3 in SAN head and afterbody and venous valve and Isl1 coexpression (Fig. 1 F, G).At atrial myocardium, Isl1 is expressed in SAN(Fig. 1 H, J in Cx40 expression).
Result indicates Isl1 and grows and playing an important role in function at SAN.
Embodiment 2 reduction that Isl1 expresses in Isl1 compound sudden change Mus embryo causes the minimizing of arrhythmia and SAN cell
The compound mutant mice model of the Isl1 (Isl1nLacZ/f that a 2.1 structures Isl1 not exclusively expresses
neo+), its Phenotype comparatively whole body knock-out mice is light, can survive slightly for a long time (death in E12.5 days), but comparatively Isl1 not exclusively to express mice system Phenotype heavier.
The compound mutant mice model of Isl1 uses Isl1 core LacZ to knock in mice system (Isl-nLacZ), and Isl1 low expression mice system (Isl1f
neocontaining neomycin site in this mice Isl1 intron of /+, Isl1 normal expression can be disturbed) hybridization forms.
The compound mutant mice of result: Isl1 (Isl1 nLacZ/f
neo) not exclusively express compared with Isl1 the Isl1 that less amount is expressed by mice system, embryo can be caused in E11.5 death (54).In tissue slice X-gal dyeing display Isl1-nLaz mice, the expressive site of Isl1-nLaz colour developing and endogenous Isl1 is completely the same.
Consistent with shown in Fig. 1, at E9.5Isl1-nLacZ at dorsal mesocardium, around SAN and SAN, atrial endocardium expresses (Fig. 2 A, A' arrow).
SAN(Fig. 2 D, D' is expressed in) at E11.5, Isl1-nLacZ.
At the compound mutant mice of E9.5 and E11.5Isl1 at SV myocardium, SAN, the Isl1-nLacZ positive cell quantity at dorsal mesocardium position significantly reduces (Fig. 2 B, B', E, E', arrow).
Isl1 and HCN4 antibody carries out immunofluorescence dyeing and shows that Isl1 and HCN4 positive cell significantly reduces (Fig. 2 C, C') in the E9.5 days compound mutant SV of Isl1.
Conclusion: Isl1 is to SAN cell, and SV myocardial cell, propagation and the survival of the cardiac progenitor cell at heart rear portion are all necessary.For this reason, use BrdU as cell proliferation marker, use TUNEL dyeing observation of cell apoptosis.Find E9.5 embryo, SV myocardium BrdU labeled cell quantity significantly reduces, and SV right corner apoptosis quantity significantly increases (Fig. 2 G, G').
2.2 in order to study the function of the compound mutant pacemaker cell of Isl1 further, and using ultrasound cardiograph detects the changes in heart rate of mutant mice E9.5 to E11.5.Marked the position of each embryo in uterus and detect each embryo heart rate.
The compound mutant of result: Isl1 is compared obviously slack-off at the heart rate of E9.5 with the heart rate of matched group E9.5, at Isl1 compound mutant E11.5 decreased heart rate more significantly (Fig. 2 H).Find that the compound mutated embryonic of Isl1 there will be intermittent cardiac extra systole, its occurrence frequency increases along with the growth of embryo simultaneously.Partial embryonic generation Prolonged Cardioplegia is had in experiment testing process.
The sudden change Mus of the early stage low expression of conclusion: Isl1 dies from arrhythmia.
In the compound mutated embryonic of embodiment 3Isl1, Isl1 expresses to reduce and causes the key gene that SNA function is relevant to express reduction
RNA in situ hybridization result shows, and HCN4, Shox2 and Tbx3 express (Fig. 3 A-C, A'-C', arrow) in the SV of matched group E9.5, and in the SV of the compound mutant E9.5 of Isl1, express significantly reduction (Fig. 3 F-H, F'-H', arrow).
Cx40 and Nkx2.5 expresses at matched group embryonic myocardium layer, but is not expressed in SAN (Fig. 3 D, D', E, E', arrow).In the compound mutated embryonic of Isl1, find that ectopic expression appears in Shox2 and Tbx3, and do not find that the phenomenon (Fig. 3 I, I', J, J', arrow) of ectopic expression appears in CX40 and NKX2.5.
In the compound mutated embryonic of conclusion: Isl1, Isl1 expresses reduction and can cause HCN4, and the gene expression that the SNA functions such as Shox2 with Tbx3 are relevant reduces, and Isl1 has regulated and controled the expression of these genes, is its upstream gene.
Embodiment 4Isl1 to break up myocardial cell be still required
In the compound mutated embryonic of Isl1, broken up SAN pacemaker cell (E9.5-E11.5), in the second heart district cardiac progenitor cell and SV, SAN myocardium, the expression of Isl1 all significantly reduces.Therefore, Troponin-Cre(cTnT-Cre is used) mice breaking up specific knockdown Isl1 in myocardial cell, breaking up the effect in sinus node cells in order to observe Isl1.
CTnT-Cre knocks out Isl1 mice (cTnT-Cre; Isl1 mice is) and the Phenotype of Isl1 compound sudden change Mus similar, this sudden change Mus is in period of embryo's i.e. E9.5-E11.5 death in early days, and mutated embryonic shows the slack-off and premature beat of obvious heart rate, sees Fig. 7.
Conclusion: experiment shows, even if myocardial cell is ripe, it is required that the pacing function of expression to myocardial cell of Isl1 is still.
Embodiment 5 uses HCN4-CreERT2 knock-out mice SAN cell ISl1 can cause embryo E11.5 arrhythmia at E9.5, and sinus node cells reduces and death during embryonic period
Because Isl1 continuous expression and expression time in SAN will be longer than cTnT-Cre; Therefore need to use additive method research to grow the effect of Isl1 in late period in SAN.
5.1 in E9.5 days, the SV main pacemaker areas as heart, and SAN formed distinguishable organizational structure as far back as E11.5 days, the ripe also functionating at E13.5 days.Therefore main research is the above-described SAN stage of development.Induced by tamoxifen, HCN4-Cre can express in conducting system of heart.Use HCN4-CreERT2 and the Isl1f/f mouse hybrid with Rosa26-LacZ or YFP background, to follow the trail of the expressive site of Cre.Derivable HCN4Cre mice is adopted to be that (HCN4-CreERT2) builds the gene knock-out mice model of specific Isl1 in SAN pacemaker cell.
Result: detect embryo heart rate with ultrasound cardiograph by every day after giving tamoxifen at E9.5 days.Find, compare with matched group, HCN4-CreERT2; Isl1 sudden change Mus heart rate is significantly slack-off at E10.5 days.HCN4-CreERT2; Isl1 mutant heart rate reduces gradually along with growth course, stops jumping for a long time, most HCN4-CreERT2 at E11.5 days generation hearts; Isl1 mutant embryos is at death in E11.5 days (Fig. 4 A).
5.2 in order to detect SAN cell at HCN4-CreERT2; Distribution situation in Isl1, uses Rosa-LacZ dyeing to follow the tracks of SAN cell development and distribution situation.After embryo E9.5 gives tamoxifen, get embryo in E10.5-E11 and E11.5 and carry out X-gal dyeing (Fig. 4 B-F).
Result: matched group E10.5 and 11.5 embryos, the X-gal positive cell of HCN4-CreERT2 institute labelling is present in SV and SAN (Fig. 4 B, E, red arrow, b1, b2), this consistent with the expressive site of endogenous HCN4mRNA (Fig. 3 A).But at E10.5-E11 mutated embryonic, part X-gal positive cell disappearance (Fig. 4 E, F) in SV region.Similar with matched group in E11.5, X-gal+ positive cell distributed areas, but in SV, HCN4-CreERT2 positive cell quantity significantly reduces (Fig. 4 B, C, red arrow, b1, b2, c1, c2, D).
5.3 application immunofluorescence dyeings detect the expression of E11 days Isl1 before SAN cell loss.
Result: significantly reduce the expression of Isl1 in SV and SAN (Fig. 4 G, H) in the induction of E9.5 days tamoxifens, and Isl1 has no change in pharyngeal and expression that is dorsal mesocardium region simultaneously.
HCN4-CreERT2; In SV and the SAN region of Isl1 mutated embryonic, the expression of HCN4 and Tbx3 then significantly reduces (Fig. 4 I-L), and this shows that Isl1 has regulated and controled the expression of HCN4 and Tbx3 simultaneously.
In addition, experiment proves, in the SAN region that HCN4 expresses, the cell quantity of TUNEL labelling significantly increases (Fig. 8), and the cell quantity of BrdU labelling reduces.
Conclusion: Isl1 grows in the survival of SAN cell and breeding and has early interimly played important function.
Embodiment 6 uses HCN4-CreERT2 to knock out Isl1 in E11.5 embryo SAN cell can cause arrhythmia, reduces the propagation of SAN cell.
Growing late period and the effect in its pacing function in order to study Isl1 at SAN, growing at SAN and having knocked out Isl1 late period.
6.1 gave tamoxifen at E11.5 days, significantly slowed down (Fig. 5 A) in the super detection of the heart of Isl1 mutated embryonic heart rate after 24 and 72 hours.But with embryo, to knock out Isl1 in early days different, and after knocking out Isl1 late period, most mutated embryonic still can be survived.
Gave tamoxifen at E13.5 days, find that mutated embryonic heart rate significantly reduces (Fig. 5 B) after 48 hrs.Research also finds that matched group embryo heart rate increases gradually in Development Mouse Embryo, but HCN4-CreERT2; Isl1 mutated embryonic heart rate has no increase, and it is irregular to show changes in heart rate.(Fig. 5 B).
E14.5 days heart (induction of E11.5 tamoxifen) X-gal and galactosidase immunofluorescences that are overall and section are dyeed display altogether, X-gal positive cell quantity (Fig. 5 D, D', H) comparatively matched group (Fig. 5 C in Isl1 mutated embryonic SAN, C', G) slightly reduce.Quantitative analysis display (each SAN counts 10 sections), in the SAN of Isl mutated embryonic quantity (175.5 ± 11.7/ section) (Fig. 5 D') of X-gal positive cell comparatively matched group (205.6 ± 17.6/ cut into slices) (Fig. 5 C') decrease 15%.
After conclusion: SAN grows and knocks out Isl1 late period, most of embryo can be survived, but still can cause serious arrhythmia.
HCN4-CreERT2 in 6.2Isl1 mutated embryonic SAN; The proliferative cell (BrdU+) of Rosa-YFP positive cell obviously reduces.Contrary, early stage in growth, HCN4-CreERT2; Isl1 mutated embryonic SAN apoptosis has no and increases.
HCN4-CreERT2; The expression of the display of SAN section immunofluorescence dyeing result Isl1, HCN4 and the Tbx3 of Isl1 sudden change Mus significantly reduces (Fig. 5 E-H).Although but the expression of Tbx3 reduces, and in no evidence display Isl1 mutant SAN, there is the ectopic expression of NKX2.5.
The minimizing of conclusion: Isl1 causes the minimizing of multiple gene (as HCN4), and therefore, Isl1 may serve important upstream regulating and controlling effect in conducting system of heart.
Embodiment 7Isl1 has regulated and controled the key transcription factor that SAN grows and function is necessary, ion channel, and cell cycle, the expression of cell survival gene.
In order to study the downstream target gene that the phenotypic Isl1 of aforementioned Isl1 sudden change Mus may be caused potential, use qRT-PCR to E11.5 days HCN4-CreERT2; Isl1 sudden change Mus and matched group SAN cell have carried out analyzing (tamoxifen is induced at E9.5) (Fig. 6 A-C).Relevant to Isl1 or relevant with Isl1 Phenotype gene is selected to analyze.
7.1 experiments find, a series of signal pathway gene down-regulated expression, comprises cell proliferation (Ccnd1, Ccnd2, Ccne1, Ccnb1, Plk1, Aurka and Cdkn1), cell survival (Bcl2) (Fig. 6 A) and heart and SAN grow necessary transcription factor (Shox2, Isl1, Mef2c, Tbx3) (Fig. 6 B).In these genes, Ccnd1 (encoding cyclin D1) and Mef2c has been proved to be the direct downstream target gene of Isl1.Meanwhile, the down-regulated expression (Atp1b1, Atp1b2, Atp2a1, Kcne1, Kcna5 and Cacna1h) of a series of ion channel gene relevant with conducting system function is also had.Gja5 and Gjd3(encodes Cx40 and Cx30.2 respectively) expression also obviously reduce.Gja1 and Gja4(encodes Cx43 and Cx37 respectively) expression then have and increase (Fig. 6 C).In addition, also find that the expression of the gene M yh6 relevant with mankind's sick sinus syndrome in Isl1 mutated embryonic SAN reduces (Fig. 6 C).
Key factor HCN4 and Tbx3 in 7.2SAN growth course is at HCN4-CreERT2; Expression in Isl1 mutated embryonic SAN significantly reduces.In order to study the direct downstream target gene whether these 2 genes are Isl1, the Mus SAN cell that suddenlys change of E14.5 to E17.5HCN4-nEGFP after FACS purification is carried out nano-ChIP analysis (60) (51).
QPCR analyzes the gene expression abundance of different DNA in ChIP result.In order to identify that Isl1 is at evolution conservative region (ECRs) possible binding site (YTAATR), checked the region of mice HCN4 and Tbx3 genomic upstream and each 5kb in downstream.
Result: 2 ECRs may include Isl1 binding site (Fig. 6 D, F).Tbx3-1 (chr5:119671762-119672016) is positioned at the 5 ' non-coding region of Tbx3, includes 2 adjacent TAAT sites at transcriptional start site (TSS) downstream 1271bp and 1270bp.Tbx3-2 (chr5:119684628-119685046) holds in the ECR of adjacent 465bp at a Tbx33 ', containing multiple potential Isl1 binding site.
ChIP-PCR have also discovered 2 high abundance site locus (Fig. 6 E, F) at HCN4.HCN4-2(chr9:58842538-58842979) be positioned in a 442bp intron ECR, this site has been proved to be as being an effective enhancer, and there is multiple Isl1 binding site in this region.HCN4-1(chr9:58820136-58820197) be positioned at a non-conservative region, be positioned at 3376bp place, HCN4 transcriptional start site upstream, include a potential Isl1 binding site.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.