CN102166346B

CN102166346B - Lrrc10 micromolecules and application thereof to preparation of medicaments for treating myocardial hypertrophy

Info

Publication number: CN102166346B
Application number: CN 201110103175
Authority: CN
Inventors: 吴秀山
Original assignee: Hunan Normal University
Current assignee: Hunan Normal University
Priority date: 2011-04-25
Filing date: 2011-04-25
Publication date: 2013-05-01
Anticipated expiration: 2031-04-25
Also published as: CN102166346A

Abstract

The invention relates to leucine rich repeat connectin 10 (Lrrc10) micromolecules and application thereof to the preparation of medicaments for treating myocardial hypertrophy. The Lrrc10 micromolecules contain 72 amino acids. A construction method comprises the following steps of: (1) fishing protein molecules which are interactive with a serum response factor (SRF) by a yeast two-hybrid method; (2) screening candidate genes for reducing expression at a heart by using a myocardial hypertrophy model, and analyzing to obtain an Lrrc10 gene which is expressed in the heart specially; and (3) taking the Lrrc10 gene as candidate gene molecules to identify functions. Lrrc10 micromolecule-containing medicaments can inhibit the myocardial hypertrophy effectively and can be used for treating the myocardial hypertrophy caused by pathological stimulation.

Description

Lrrc10 micromolecule and the application in preparation treatment myocardial hypertrophy medicine thereof

Technical field

The present invention relates to a kind of leucine Abundant protein 10(Lrrc10 of specific heart expression) micromolecule and construction method and the application in preparation treatment myocardial hypertrophy medicine.

Background technology

Heart disease is one of Major Risk Factors that affects in recent decades human life's health, and its final result causes human heart depleted.The patient is before heart failure, its heart can experience the process of a hyperplasia, it is myocardial hypertrophy, this process stimulates in certain extraneous pathology by the myocardial cell effect, and then some signal paths of active cell inside and finishing, these signal paths comprise MAPK signal path, g protein coupled receptor signal path, calcineurin-NFAT path and PI3K-AKT path etc.Signal path is made of specific receptor, signal transduction pathway and effect terminal.So, in theory, when heart suffers that some pathological signals stimulate, if can suppress the activation of these paths, just can effectively treat and prevention of cardiac.

Because it is exclusive that signal path is not heart organ institute in the organism; various signal paths are prevalent in each tissue and cell of organism; these signal paths all are vital to the function of body, so the medicine of these signal paths of targeting some side effect can occur usually.Just show the immunosuppressant reaction and to the toxic action of kidney such as the suppressive drug that is directed to calcineurin-NFAT path exploitation.Screen a kind of new higher tissue-specific small-molecule drug target spot that has, become a major challenge of developing novel low side effect treatment heart disease medicine.

Summary of the invention

The Lrrc10 small molecular protein and the construction method thereof that the purpose of this invention is to provide a kind of specific heart expression, and the application of Lrrc10 small molecular protein in the low side effect small-molecule drug of preparation treatment myocardial hypertrophy.

Technical scheme of the present invention is:

Studies show that SRF gene (gene library registration number GI:61743976) is the important protein factor of keeping the heart normal function.Utilize the mice transgenic technology, cross to express in myocardial cell so that SRF is moderate, just can be stronger induce myocardial hypertrophy, make mice very fast death owing to heart failure after birth.In myocardial cell, even slight the crossing of SRF expressed, also can make mouse heart cross presenility.Utilize gene Knockout, SRF gene specificity in mouse heart is knocked out, also can cause myocardial hypertrophy and heart failure.But studies show that the suitable expression of reduction SRF in mouse heart can make heart obtain a better function, shows the phenomenon that is not easy to occur heart failure under pathology stimulates.In addition, research is also found, the content of SRF in the mouse heart is to raise gradually along with the increase of Mouse Age.In mouse heart, reduce the expression of SRF, can make heart be tending towards rejuvenation.SRF is a very famous transcription factor, and stronger expression is all arranged in heart and muscle.As a transcription factor, it often and other the cofactor of transcribing interact, regulate and control together the expression of downstream gene.Although SRF is not the tissue-specific factor, some cofactor interactional with it has tissue specificity; Specific physiological process is regulated and control in the two combined effect, thereby can realize popularity and the specificity of SRF on its function.

Lrrc10 gene (gene library registration number GI:63003902) is the functional gene of an intronless, and coding contains the albumen of 7 LRR conserved domains, and the LRR domain has the function that the effect framework is provided for protein interaction.Studies show that, Lrrc 10 genes have a large amount of and specific expression in the mouse heart tissue, heart in the mice embryonic phase has extremely similar expressive site to NKX2-5, and the Subcellular Localization experiment shows that it has expression at Cytoplasm and nucleus, expresses raising after the birth.The neonatal cardiac myocytes of further separating experimental results show that Lrrc 10 genes on the Z-line and transverse tubule is expressed.But Brachydanio rerio and mice LRRC10 are opposite in the developmental effect of embryo heart, and without impact, and mice Lrrc 10 expresses at specific heart mice Lrrc 10 on its heart development, point out this gene may be relevant with the function that is maintained in systemic heart.The similarity of Lrrc 10 genes in human and mice is up to the 84%(amino acid levels), point out this gene in keeping the human adult cardiac function, to play an important role.

The present invention has set up following Lrrc10 micromolecule construction method: (1) is angled by the method for yeast two-hybrid and is got the interactional protein molecular with SRF; (2) also pass through correlation analysis by the myocardial hypertrophy model discrimination at the candidate gene of heart down-regulated expression, obtain a leucine Abundant protein 10(Lrrc10 specific expressed in heart) micromolecule; (3) with it as candidate molecules, carry out Function Identification.

The present invention has made up the adenovirus vector that contains Lrrc10 gene (gene library registration number GI:63003902); further packed the adenovirus that contains Lrrc10 by transfection 293A cell; myocardial cell with Adenovirus Transfection new life rat of former generation; the stimulation (FBS) of phyenlephrinium (PE) and 10% hyclone 48 hours; by Cardiac-specific antibody α-actin immunofluorescence dyeing; under Laser Scanning Confocal Microscope, observe; choose 100 cells; with SPOT software its cell surface is amassed dimension analysis; find that Lrrc10 crosses the myocardial cell size that expression can suppress to be caused by PE and 10%FBS significantly and becomes large, show that Lrrc10 is a new Cardioprotective factor.

In order further to detect Lrrc10 gene transcripting suppressioning action to the SRF gene in myocardial cell, the present inventor is in the myocardial cell of the myocardial cell that infects the Lrrc10 adenovirus and infection GFP (gene library registration number GI:211909965) empty plasmid adenovirus, carried out the experiment of chromatin co-immunoprecipitation, select the classical target gene Egr-1 of SRF to detect, discovery under the stimulation of serum, cross expression Lrrc10 obviously make SRF in conjunction with the DNA of Egr-1 gene (gene library registration number GI:31317226) promoter region in conjunction with minimizing.The Egr-1 gene is the booster of a myocardial hypertrophy, and this result has further verified the defencive function of Lrrc10 gene pairs heart.Meanwhile, the present inventor also in 293T cell line, utilizes the method for gel blocking, and same the discovery expressed in the situation of Lrrc10 excessively, and the DNA joint efficiency of SRF and Egr-1 gene promoter region reduces.In order further to understand, the mechanism of action that Lrrc10 suppresses the SRF transcriptional activity, we have detected SRF and have crossed the expression of expressing in the Lrrc10 myocardial cell, find that SRF is not affected, but the result of immunofluorescence shows that SRF can enter rapidly nuclear under the stimulation of serum, and in crossing expression Lrrc10 myocardial cell, SRF this enters the nuclear effect and is subject to obvious inhibition.SRF is the transcription factor of classics, and its function realizes by transcriptional control downstream target gene.These results prove that Lrrc10 regulates and control SRF downstream target gene by the nuclear mechanism that enters that suppresses SRF.

In order to verify that whether Lrrc10 suppresses the protection factor of myocardial hypertrophy in the Mice Body; the present invention has further made up specific heart and has crossed the Lrrc10 transgenic mice of expression; Founder goes down to posterity successfully for verifying through PCR; Northern blot shows; compare with brood negative mice, transgenic mice Lrrc10 expresses in heart obviously and raises.After mice was subject to the ISO stimulation, obvious loose phenotype appearred in wild-type mice, and being embodied in the heavy tibia Length Ratio of the heart increases, and ventricle thickness increases, fibrosis, and loose significant gene, ANF, SKA, β MHC express obvious the rise.And the heavy tibia Length Ratio of the transgenic mice heart is so unobvious, faint fibrosis occurs, and loose significant gene raises and significantly suppressed.And in the situation that is not subject to the pathology stimulation, wild-type mice and transgenic mice do not have notable difference.By detecting in the body, a series of target genes of SRF comprise that obvious downward modulation all appears in SMA, CTGF, the isogenic expression of Egr-1, ET-1, this explanation, and Lrrc10 suppresses the transcriptional activity of SRF really in Mice Body.

Lrrc10 is a small molecular protein, molecular weight is 32kd, it contains 7 LRR domains, in order to develop the demand of small-molecule drug, the present invention is directed to the different structure territory of Lrrc10, its protein molecular is carried out the truncate of different levels, with different truncate expression plasmids and SRF and ANF fluorescence report plasmid, corotation 293T cell is found, only comprises the truncate of the 6th and the 7th LRR domain, just can suppress fully the transcriptional activity of SRF.Its domain that blocks body is one and contains 72 amino acid whose peptide molecules that aminoacid sequence is:

MGNTIRALVAFIPADRCQNYVVRDLREMPLDKMVDLSGSQLRRFPLHVCSFRELVKLYLSDNHLNSLPPELG。

This section aminoacid sequence is connected into carrier for expression of eukaryon, changes eukaryotic cell over to, can obtain the peptide molecule albumen of this section inhibition SRF activity, this just provides good application foundation for the small-molecule drug of preparation Lrrc10.

The micromolecular medicine of Lrrc10 that contains of the present invention, energy establishment myocardial hypertrophy can be used for treating the myocardial hypertrophy that is caused by the pathology stimulation.

The invention has the beneficial effects as follows: the present invention with the myocardial cell of former culture as target cell, adenovirus vector-mediated, and transgenic animal, prove that the Lrrc10 gene is inhibited to myocardial hypertrophy in vitro and in vivo.Pass through the cytology, histology and molecular biology method, prove that all inhibitory action is fairly obvious, and because the Lrrc10 transgenic mice does not have obvious phenotype at normal physiological level, the Lrrc10 gene is the specific heart expressing gene simultaneously, and this molecule can be prepared into the medicine of the little treatment myocardial hypertrophy of toxic and side effects low pathogenicity.Add the Lrrc10 truncate SRF is had stronger inhibitory action, for the small-molecule drug of exploitation preparation take the Lrrc10 gene as target spot provides foundation.Because SRF is relevant with the senescence of heart, not only can be used as treatment myocardial hypertrophy disease but also can be used as the health medicine that prevents the heart premature aging so cross the medicine of expressing the Lrrc10 gene again.

Description of drawings

Fig. 1 is Lrrc10 and SRF interaction collection of illustrative plates;

Fig. 2 is Lrrc10 and SRF direct interaction collection of illustrative plates;

Fig. 3 is Lrrc10 down-regulated expression collection of illustrative plates in the loose model of mice;

Fig. 4 is Lrrc10 down-regulated expression collection of illustrative plates in the mice hypertrophic cardiomyocytes;

Fig. 5 is Lrrc10 middle down-regulated expression collection of illustrative plates in the loose specimen of the mankind;

Fig. 6 is the transcriptional activity collection of illustrative plates that Lrrc10 suppresses SRF;

Fig. 7 is that adenovirus mediated Lrrc10 crosses expression map in myocardial cell;

Fig. 8 is that Lrrc10 inhibition myocardial cell size becomes large collection of illustrative plates;

Fig. 9 is the ANF rise collection of illustrative plates that Lrrc10 suppresses PE and 10%FBS stimulation;

Figure 10 be Lrrc10 cause SRF to Egr-1 promoter part DNA in conjunction with reducing collection of illustrative plates;

Figure 11 be Lrrc10 cause SRF to Egr-1 promoter part DNA in conjunction with reducing collection of illustrative plates;

Figure 12 is the evaluation collection of illustrative plates of transgenic positive mice;

Figure 13 is transgenic positive mice Lrrc10 protein expression level rising collection of illustrative plates;

Figure 14 is transgenic positive mice Lrrc10 transcriptional expression level rising collection of illustrative plates;

Figure 15 is transgenic positive mice Lrrc10 transcriptional expression level rising collection of illustrative plates;

Figure 16 is that transgenic mice suppresses the ANF expression rising collection of illustrative plates that ISO induces;

Figure 17 is the increase sketch map that transgenic mice suppresses the heavy tibia lenth ratio of the heart of ISO stimulation

Figure 18 is that the total ventricle thickness of transgenic mice increases suppressed collection of illustrative plates;

Figure 19 is transgenic mice after ISO stimulates, and fibrosis (blueness) is than the faint collection of illustrative plates of wild-type mice;

Figure 20 be wild type and transgenic mice under the normal physiological state SRF with signal without obvious change, under ISO stimulated, SRF's reduced collection of illustrative plates with signal in the transgenic mice;

Figure 21 expresses obviously in ANF, the β MHC wild type heart under ISO stimulates to raise, and the rise of transgenic mice ANF, β MHC is suppressed collection of illustrative plates;

Figure 22 expresses obviously in ANF, the β MHC wild type heart under ISO stimulates to raise, and the rise of transgenic mice ANF, β MHC is suppressed collection of illustrative plates.

Figure 23 is SMA, CTGF, Egr-1, the ET-1 downward modulation collection of illustrative plates than wild-type mice in transgenic mice

Figure 24 was that expression Lrrc10 increases SRF in endochylema, reduced collection of illustrative plates in karyon.

Figure 25 is that each fragment of Lrrc10 splits sketch map;

Figure 26 is that Lrrc10 is to the transcriptional activity inhibitory action collection of illustrative plates of SRF.

The specific embodiment

The present invention will be further described below in conjunction with the drawings and specific embodiments.

Lrrc10 micromolecule construction method: (1) is angled by the method for yeast two-hybrid and is got the interactional protein molecular with SRF (gene library registration number GI:61743976); (2) by the myocardial hypertrophy model discrimination at the candidate gene of heart down-regulated expression and pass through correlation analysis, obtain a leucine Abundant protein 10(Lrrc10 specific expressed in heart), it as candidate molecules, is carried out Function Identification (specifically referring to hereinafter described each embodiment).

By co-immunoprecipitation and GST-Pull down experiment, the result shows that Lrrc10 can interact with SRF really.Simultaneously, we detect the protein content of Lrrc10 in the specimen of human myocardium's hypertrophy, find to compare with normal heart in 6 myocardial hypertrophy Pathologic specimens, and the protein content of Lrrc10 is obviously on the low side.This result shows that Lrrc10 participates in the pathogenetic process of human myocardial hypertrophy disease.We enter the 293T cell to Lrrc10 gene, SRF and ANF report carrier cotransfection, find that the Lrrc10 gene can suppress SRF significantly to the activation of ANF report carrier uciferase activity, and in not having the 293T cell of SRF gene overexpression, Lrrc10 does not have any impact to ANF luciferase reporting activity.This presentation of results, Lrrc10 can suppress the transcriptional activity of SRF.Because the ANF gene is an important symbol gene of myocardial hypertrophy, so showing Lrrc10 simultaneously, this result can and then suppress the generation of myocardial hypertrophy again.

Embodiment 1 Lrrc10 and the interactional checking of SRF

With the HEK293T cell of pCMV-tag-2B-LRRC10 and the cultivation of pCMV-myc-FHL2 carrier cotransfection 100 mm plates, behind the transfection 48hr, remove cell culture fluid, wash 3 times with PBS (room temperature) is adherent.400 μ l cell pyrolysis liquids (50 mM Tris, pH 7.5,25 mM HEPES, 100 mM NaCl, 1% Triton X-100, protease inhibitor cocktail) cell lysis, centrifugal 10 min of 12000 rpm collect supernatant.Measure protein concentration, add 2 μ g and resist-monoclonal antibody (Sigma company) of Flag or the monoclonal antibody (Santa Cruz Biotechnology) of anti--myc in above-mentioned cell lysate 1000 μ g total cell proteins, 4 ℃ of shaking tables are hatched 4h.Add afterwards 20 μ l protein-A/G plus-agarose, 4 ℃ of shaking tables are hatched 2hr again.4000 rpm(1000g) centrifugal.Carefully remove supernatant.Wash three times with lysis buffer or PBS, all use 2500 rpm rotating speeds centrifugal at every turn.After for the last time centrifugal, go supernatant to add 30 μ l, 2 * SDS sample-loading buffer.Sample boils 8～9 min, and the wide glue hole of per 1 mm adds 5-10 μ l sample.The SDS-PAGE protein isolate, the wet method transferring film, with anti--Flag(Sigma) or anti--myc monoclonal antibody (Santa Cruz Biotechnology) carry out Western-blot and detect.Testing result finds that Lrrc10 can coprecipitation phenomena occur with SRF, illustrates that the two has the interaction (see figure 1)

Change pGEX-4T-1-LRRC10, pGEX-4T-1-LRR12 and pGEX-4T-1-LRR123LINK carrier over to e. coli bl21, through IPTG induced protein prokaryotic expression, albumen after the expression is with paddy cystamine peptide sepharose 4B 4B purification under suitable time-temperature.PCMV-myc-FHL2 and pcDNA3.1A-myc-SRF change respectively the HEK293T cell transient expression that the 100mm culture dish is cultivated over to, behind the transfection 48hr, remove cell culture fluid, clean adherent washing 3 times with PBS (room temperature), then use 400ul Triton-glycerol cell pyrolysis liquid (1% Triton X-100,10% glycerol, 25 mM HEPES, 150 mM NaCl, 2 mM EDTA, protease inhibitor cocktail) cell lysis, 12, the centrifugal 10min of 000rmp collects supernatant.Paddy cystamine peptide sepharose 4B 4B 10ul in connection with GST-LRRC10, GST-LRR12 and GST-LRR123LINK adds respectively in the upper albumin, hatches 4hr for 4 ℃, and the centrifugal collection agarose of 4000rpm pearl is with cell pyrolysis liquid washing 3 times.Remove supernatant, add 30 μ l SDS-sample-loading buffers.Sample boils 3min, the glue hole loading 10 μ l that each 1mm is wide.SDS-PAGE protein isolate (preparing simultaneously 2 SDS-PAGE glue), use coomassie brilliant blue staining for one, another wet transferring film, carry out Western-blot with anti--myc monoclonal antibody (Santa Cruz Biotechnology) and detect, the result shows that direct interaction (see figure 2) occurs for SRF energy and Lrrc10.

The correlation analysis of example 2 Lrrc10 and myocardial hypertrophy

6-8 week FVB Strains of Mouse adopts the subcutaneous injection isoproterenol, and 30mg/kg/d injected 7 days continuously, made its myocardial hypertrophy.After core dirty extraction mRNA and protein, take FVB mice of the same age, injecting normal saline compares, and utilizes the method for the method of Real-time PCR and Western-blot to detect the expression (see figure 3) of Lrrc10.Separate former generation neonatal cardiac myocytes, utilize the phyenlephrinium of 100uM concentration and 10% hyclone to stimulate, make its cardiac myocyte hypertrophy.Extract respectively loose myocardial cell and untreated myocardial cell mRNA, utilize the expression water (see figure 4) of the method Lrrc10 of Real-time PCR; Extract respectively human normal cardiac tissue and heart failure specimen histone, utilize comprehensive all results of expression (see figure 5) of the method detection Lrrc10 of Western-blot to show that expression pattern and the myocardial hypertrophy of Lrrc10 present negative correlation.

Example 3 is estimated Lrrc10 to the impact of SRF transcriptional activity

With plasmid pCMV-Tag2B-LRRC10 and ANF-Luc transfection to the 293T cell.Behind the cell culture 48h, thin tniema, the detection of its step and fluorescence activity is summarized as follows: after cell is washed 2 times with PBS, add lysate 80 μ L/holes,-80 ℃ of refrigerators are placed 15min, place 15 min for 37 ℃, make the more abundant cracking of cell, then scrape with cell and scrape rapidly cell, be transferred to the microcentrifugal tube of 1.5 mL, and at 12000 rpm, 4 ℃ of centrifugal 15s get supernatant 20 μ L in-80 ℃ of temporary transient preservations.Thaw on ice before the measurement, fluorescent color-developing agent liquid is placed thawed on ice simultaneously, open the fluorescence measurement instrument, suitable parameter is set, the ratio of fluorescent color-developing agent according to 40 μ L/ pipes is added in the 20 μ L cell pyrolysis liquids, and places rapidly in the fluorescence measurement instrument record data.Get supernatant 20 μ L and 10 μ L ONPG assay mixings and put into 37 ℃ of calorstats cultivation reaction 30 min, add again 50 μ L trisbase cessation reactions, add at last the 300 μ L aquesterilisa light absorption value of spectrophotometric instrumentation 420 nm, record data are observed its transfection efficiency.Experiment is triplicate altogether, repeats two at every turn.With biometrics Epidemiological Analysis experimental data.The result shows that Lrrc10 can effectively suppress SRF transcriptional activity (see figure 6)

The outer foundation of crossing expression system of example 4 Lrrc10 genosomes

PCR obtains connecting the T-carrier behind the genes of interest ORF, a large amount of enzyme action after order-checking is correct, enzyme action product agarose gel electrophoresis, genes of interest DNA fragment and T-carrier segments are separated as far as possible, reclaim purification genes of interest DNA fragment, be connected into the TRACK carrier, utilize the PmeI linearization for enzyme restriction, electricity changes the BJ5183 competence over to, extracts recombiant plasmid, obtains adenoviral plasmid.With adenoviral plasmid, the PacI linearization for enzyme restriction changes the 293A cell over to, and green fluorescence all appears in the band cell, and nearly 50% becomes circle when hiking up, and collecting cell after the freeze thawing three times, obtains virion.With viral infection, former generation myocardial cell, after 2 days, collect myocardial cell, extract albumen, utilize the method for Western-blot to detect the expression of Lrrc10 label H A. the outer mistake of display body expressed successfully as a result.(see figure 7)

Example 5 experiment in vitro proof Lrrc10 suppresses myocardial hypertrophy

Separate former generation myocardial cell, hungry 24 as a child, carry respectively the adenovirus of Lrrc10 gene and GFP gene, recession virus removal in 5 hours, adding the phyenlephrinium and 10% hyclone that utilize 100uM concentration stimulated after 48 hours, myocardial cell is carried out immunofluorescence experiment, take a picture, with SPOT software statistics cell size size.Extract simultaneously the albumen of myocardial cell, utilize the method for Western-blot to detect the expression of loose significant gene.Found that the cell of Lrrc10 viral infection, can suppress the myocardial hypertrophy that phyenlephrinium and 10% hyclone are induced.(see figure 8).

Example 6: the protein expression level of ANF during protein blot hybridization check Lrrc10 crosses and expresses in the myocardial cell

Behind the Adenovirus Transfection myocardial cell of former generation, PE and 10%FBS stimulated 48 hours, collected albumen, about 20 micrograms are at 10% polyacrylamide albumin glue electrophoresis, as confidential reference items, be transferred to pvdf membrane with GAPDH behind the protein electrophoresis, use respectively ANF and GAPDH antibody (SANTA CRUZ company) hybridization.Be presented at not to cross by Fig. 9 and express in the myocardial cell of Lrrc10, after PE and 10%FBS stimulated, ANF expressed obviously and rises, and ANF almost can not detect in crossing the myocardial cell of expressing Lrrc10.

Example 7: the chromatin co-immunoprecipitation detects SRF to the binding ability of its target molecule Egr-1

Former generation infection of cardiac myocytes GFP and Lrrc10 cross the adenovirus of expression, it is a reaction that 106 myocardial cell are collected in trypsinization, with cell suspension in 20 milliliters DMEM, the formaldehyde (ultimate density is 1%) that adds 560 microlitres 37%, in 37 degree shaking tables, processed 10 minutes, add 3 milliliters 1 mole every liter glycine (final concentration is 0.125 mole every liter), processed in the shaking table 3 minutes, 400rmp 10 minutes is with 10 milliliters, be added with the PBS of the pre-cooling of protease inhibitor, washed cell changes in 15 milliliters the pipe, has added proteinase inhibitor C HIP SDS Lysis buffer with 100 microlitres, suspension cell goes in 1.5 milliliters of EP pipes.30% energy, 1.5 minutes once, it is directly ultrasonic to pop one's head in, point sample detects, ultrasonic fragment in 1KB, centrifugal 10 minutes of 14000rmp, supernatant changes in the new EP pipe.Surveying the OD value, probably is the every microlitre of 1 microgram.Add 9 times of volumes interpolation the CHIP diluent of protease inhibitor, get 20 microlitres and contrast as INPUT.Every pipe adds the ProteinA Agarose of 50 microlitres, 4 degree upsets, 30 minutes (go out non-specific binding).300rmp3 minute, get supernatant and change in the new EP pipe, add the antibody of 3 micrograms, 4 degree overnight incubation.The ProteinA Agarose that adds 60 microlitre muddinesses, 4 degree 1 hour, removed supernatant in 300rmp3 minute, with 1 milliliter of less salt, high salt, LiCl solution, 3-5 minute each, with the TE washing liquid wash twice, 3-5 minute each, with lancet head sucking-off supernatant (must blot only not so have non-specific), add 250 microlitre eluents, greenhouse upset 15 minutes, 2000rmp got supernatant in 2 minutes, repeated once, supernatant with 500 microlitres, the NaCl that adds 250 microlitres, the method for phenol chloroform is extracted DNA. and is used following primer to do the PCR reaction: c-Egr-1-s:CCCACCACTCTTGGATGGGAGGGCTTCAC, c-Egr-1-a:TCGGCCTCTATTTCAAGGGTCTGGAACAGC; C-β-globin-s:CAGCGTTTTCTTCAGAGGGAGTACCCAGAG, c-β-globin-a:TCAGAAGCAAATGTGAGGAGCGACTGATCC. Figure 10 be presented at SRF in the myocardial cell that Lrrc10 crosses expression to the promoter of Egr-1 in conjunction with minimizing.

Example 9: gel retardation assasy detects in the cell that Lrrc10 crosses table SRF to the binding ability of its target gene Egr-1

Express in the plasmid transfection 293T cell of Lrrc10 crossing, it is for subsequent use to extract nucleoprotein.With the nucleotide sequence justice of Egr-1SRF calmodulin binding domain CaM be: the ACAGACCTTATTTGGGCAGCGCCTTATATGGAGTGGCCCAA antisense is 5 ' TTGGGCCACTCCATATAAGGCGCTGCCCAAATAAGGTCTGT3 ' annealing, annealing way is, at first nucleotide sequence is diluted to the every microlitre of 3 micrograms, respectively getting 1 microlitre mixes with annealing liquid, the prescription of annealing liquid is 100mM KAc, 30mM HEPES 2mM MgAc, in the PCR instrument, 94C 4min 80C 15min 75C 4min 70C 15min 25C 10S 4C 10min. after the annealing, get the oligonucleotide that 3.42ul is equivalent to 100pm, the reaction buffer of 4ul 5x, 4ul CoCl2 solution 1ul DIG-ddUTP solution, the 1ul terminal transferase adds water to 20ul .37C 15min. and place on ice, add 2ul 0.2M EDTA (PH=8.0) cessation reaction.Get the nucleotide of 2ulDIG labelling, 4ul binding buffer (Tris pH7.5 0.1M KCl 0.5M DTT 10Mm), 1ul playDI-DC 4ug nucleoprotein, room temperature 25 minutes, UV-crosslinked twice, add bromophenol blue, 95C water-bath 5min is 5min on ice, western uses the antibody of DIG as primary antibodie, colour developing.Figure 11 showed express Lrrc10 SRF to the promoter of Egr-1 in conjunction with minimizing.

Example 10: the foundation of specific heart Lrrc10 transgenic mice

The Lrrc10 fragment is connected into the SK carrier, then be connected into again the promoter of Myh6 gene at Lrrc10 fragment money, utilize KpnI and SacII linearization for enzyme restriction, by the microinjection mode, fragment is injected in the mouse fertilized egg, and the method by embryo transfer is transplanted to it in the fallopian tube of pseudo-fetus mice (seeing Table 1).In the offspring of pregnant mouse, by the method for PCR, detect the head person of building mice (seeing Figure 12). going down to posterity by the head person of building mice obtains the transgenic mice of energy genetic stability.

Sheet name section injection ovum number is transplanted the ovum number and is transplanted the conceived Mus of Mus number and count filial mice and count head and build Mus and count positive rate
	PMHC-Lrrc10 382 377 16 5 27 5 18.5%

Table 1: microinjection situation

Example 11: detect Lrrc10 and in the transgenic mice heart, cross expression

Extract the transgenic mice myocardium protein, about 50 micrograms are transferred to pvdf membrane at 10% polyacrylamide albumin glue electrophoresis behind the protein electrophoresis, use respectively Lrrc10 and β-actin antibody (SANTA CRUZ company) hybridization.Figure 13 shows that protein expression raises in the Lrrc10 transgenic mice heart.Extract transgenic mice heart RNA, be inverted to cDNA by counter-rotating test kit (TAKARA company), detect the variation of Lrrc10 transcriptional level by the mode of Real-time PCR, with GAPDH as confidential reference items.The forward primer of Lrrc10 is: 5'GGATGTGGTCTGTGAACT 3' downstream primer is 5'GGAAGTAGCGGATACTGT 3'; The forward primer of GAPDH is: 5 ' AGCCCAGAACATCATCCCT3 ' downstream primer is: GGTCCTCAGTGTAGCCCAAG. is shown in Figure 14, and the transcriptional level of Lrrc10 significantly raises.Get 15 microgram RNA, in processing the device of RNA, gel electrophoresis 2-3 hour, 100 volts, it is changed in the nitrocellulose filter, once UV-crosslinked, energy 1200,80 degree bakings 1 hour, prehybridization 6 as a child adds the probe with isotope 32P labelling, hybridized 18 hours, wash film three times, tabletting develops after one week to-80 degree.Shown in Figure 15, the Lrrc10 transcriptional level obviously raises.The Lrrc10 probe is from cDNA, middle amplification out, forward primer: GTCGTCGACTCCCTGACAGAGTTGGTG downstream primer: TCAAGCGTAATCTGGAACATCGTATGGGTA AGAGCTGGAAGGAAG 3'

Example 12: transgenic mice perfusion isoproterenol ISO

Get 5 pairs of brood wild type and Lrrc10 transgenic mices with the sex contrast of the same age, ISO is dissolved in 0.9% the normal saline, ISO amount with per kilogram of body weight subcutaneous injection 30mg every day, injection is 7 days continuously, get in addition 5 pairs 5 pairs brood wild type and Lrrc10 transgenic mices with the sex contrast of the same age, injecting normal saline is done contrast.

Example 13: the protein expression level of ANF in the protein blot hybridization check transgenic mice

Extract the transgenic mice myocardium protein, about 50 micrograms are transferred to pvdf membrane at 10% polyacrylamide albumin glue electrophoresis behind the protein electrophoresis, use respectively ANF and GAPDH antibody (SANTA CRUZ company) hybridization.Figure 16 shows that ANF protein expression in the heart that mice ISO stimulates raises.And obviously suppress in the rise of the total ANF of transgenic mice.

Example 13: histologic analysis transgenic mice phenotype

Dissect mice, get its heart, weigh is measured hind leg tibia length, statistical analysis cardiac weight and tibia lenth ratio, and Figure 17 shows, transgenic mice has obviously suppressed the increase of the heavy tibia lenth ratio of the heart of ISO stimulation.Get its heart tissue, 4% paraformaldehyde fixedly spends the night, tissue is carried out serial ethanol dehydration: 70% ethanol (30 minutes), 80% ethanol (30 minutes), 90% ethanol (30 minutes), 95% ethanol (30 minutes), dehydrated alcohol (30 minutes), dehydrated alcohol (30 minutes), dimethylbenzene (15 minutes), dimethylbenzene (15 minutes).The tissue that will take off water goes in the paraffin (melted paraffin wax spends the night in 60 ℃ of placements) of handling well, and wax is 2 hours thoroughly, and wax is changed twice in the centre.Tissue is forwarded in the embedding frame of preheating, carefully remove the bubble in the base plate, room temperature is placed, and dewaxing is solidified.The finishing wax stone is to required size.In rotary microtome (MICROM HM340E) section, 37 ℃ of dried overnight.

The dyeing of haematoxylin Yihong:With paraffin organization serial ethanol (100%, 95%, 90%, 80%, the 70%) rehydration of cutting into slices.Place in the Harris haematoxylin (SIGMA) and dyeed 1 minute and 30 seconds, then the flowing water punching is 5 minutes, acidic alcohol differentiation 30 seconds, and oil blackeite was carried out in the flowing water punching in 5 minutes again.95% ethanol 1 minute, then eosin stain dyeing is 1 minute.Section after the dyeing is through the ethanol dehydration of 95%, 100%, 100% concentration, dimethylbenzene transparent (2 minutes * 2 times), neutral gum mounting.Figure 18 shows the increase that ventricle thickness stimulates at mice ISO, and increases suppressed at the total ventricle thickness of transgenic mice.

Dyeing:Tissue slice takes off cured to water.Ambient temperature overnight in Bouin ' s fixative.The color of the upper fixative of the abundant rinsing of distilled water place to go section, haematoxylin dyeing 5 minutes, distilled water flushing 5 minutes.Deionized water infiltrates 5-10 minute, Biebrich Scalet-Acid Fucshin dyeing 5 minutes, deionized water infiltrates 5-10 minute, and section places Salkowski's solution to place 5 minutes, with Annilline Blue solution-dyed 5 minutes, section placed 1% acetic acid 2 minutes.Differentiation, dehydration and the mounting of section.Figure 19 shows transgenic mice after ISO stimulates, and fibrosis is faint than wild-type mice.

Immunohistochemistry:Paraffin section 58oC baking 2 hours, dewaxing, serial ethanol rehydration, 3% hydrogen peroxide at room temperature was hatched 10 minutes, to eliminate the activity of endogenous peroxydase.Distillation washing 2 minutes * 3 times, PBS soaked 5 minutes.Tissue slice is placed in 400ml 0.1 * sodium citrate buffer solution, and microwave-oven-heating 4 minutes repeats twice, then makes its natural cooling, adds the confining liquid of lowlenthal serum, incubated at room 30 minutes.The serum deprivation that inclines drips SRF (SANTA CRUZ) dilution of 1:50 dilution, the 4oC refrigerator overnight.PBS washes, 5 minutes * 3 times, the biotinylation that drips the 1:200 dilution two anti-(middle mountain biotech company), 37oC was hatched 30 minutes, and PBS washes, 5 minutes * 3 times, drip the Radix Cochleariae officinalis enzyme labelling strepto-avidin (middle mountain biotech company) of 1:200 dilution, 37oC was hatched 30 minutes, and PBS washes, 5 minutes * 3 times, microscopically is observed DAB colour developing situation.The flowing water flushing, serial ethanol dehydration, neutral gum mounting.Figure 20 be presented at wild type and transgenic mice under the normal physiological state SRF with signal without obvious change, under ISO stimulated, SRF's reduced with signal in the transgenic mice.

The expression of example 14 Northern hybridization check ANF, β MHC

15 microgram wild types and genetically modified heart tissue mRNA, slow electrophoresis 100v is 2-4 hour on agarose gel (1.0%).With distillation washing twice, 9. with the capillary transfer method DNA is transferred on the nitrocellulose filter from agarose gel.After shift finishing, with the position of pencil labelling gel well, filter membrane is placed in the middle of two groups of 3M filter paper, did roasting 1 hour for 80 ℃.Filter membrane is placed prehybridization solution, 65 ℃ of incubations 1 hour, use new prehybridization solution instead, add dna probe and the salmon sperm DNA of degeneration, 65 ℃ of hybridization are spent the night, with filter membrane in 1xSSC, 0.1%SDS medium temperature chamber washed 20 minutes, subsequently film is placed 0.2xSSC, gentleness is shaken rinsing three times in 65 ℃ of shaking baths of 0.1%SDS, each 1 hour.Place two 3M filter paper to pause drying filter membrane, then wrap filter membrane with preservative film, stick several fluorescence labels, in order to calibrate the autoradiography band in the position of filter membrane in the future.Filter membrane is placed X-ray sheet folder, added screen exposure 48 hours, development, photographic fixing in-70 ℃.Figure 21 shows, express obviously in ANF, the β MHC wild type heart under ISO stimulates and raise, and the rise of transgenic mice ANF, β MHC is suppressed.Primer sequence is as follows

Northern-β-MHC -S	5’-TGCAAAGGCTCCAGGTCTGAGGGC-3’
		NorthernβMHC -S	5’-GCCAACACCACCCT GTCCAAGTTC-3’;
Northern-ANF-S	5’-CTGCTAGACCACCTGGAGGAG-3’
		Northern-ANF-A	5’-CCAGGAG GGTATTCACCACCAC-3’

Example 15 Realtime PCR detect the expression of ANF, β MHC, Egr-1 etc.

The total RNA of the cell or tissue that extracts measures concentration, and drawing 1-2 μ g is cDNA with the reverse transcription of reverse transcription test kit, and 2.SYBR Green mixes the expression (Roche LightCycler 2.0 system) of method testing goal gene, and primer sequence is as follows:

RT-Egr1-Sense1:5' TTTGCCTCCGTTCCACCTGC 3'

RT-Egr1-ANTI1: 5' TGCCAACTTGATGGTCTAGCGC 3'

Tm-SMA fw: 5-CAGCAAACAGGAATACGACGAA-3

Tm-SMA-bw: 5-TGTGTGCTAGAGGCAGAGCAG-3

Tm-ET-1 fw: 5-TGCCACCTGGACATCATCTG-3

Tm-ET-1 bw: 5-AGAACCTCCCAGTCCATACGG-3

ANF-RT-S:GCCGGTAGAAGATGAGGTCA

ANF-RT-A:GGGCTCCAATCCTGTCAATC

βMHC –RT-S:GTGAAGGGCATGAGGAAGAGT

βMHC –RT-A:AGGCCTTCACCTTCAGCTGC

Figure 22 shows, express obviously in ANF, the β MHC wild type heart under ISO stimulates and raise, and the rise of transgenic mice ANF, β MHC is suppressed.Figure 23 shows that SMA, CTGF, Egr-1, ET-1 obvious downward modulation occurs than wild-type mice in transgenic mice

Example 16 extracts nucleus and cytoplasm protein detects the content of SRF in caryoplasm

In crossing expression Lrrc10 cell, the trypsinization collecting cell, add the CERI that 100 microlitres are added with protease inhibitor and inhibitors of phosphatases in each 10cm ware cell, shook ice bath 15 minutes 5 seconds at a high speed, add the CERII5.5 microlitre, shook 5 seconds at a high speed, ice bath 1 minute shook 5 seconds at a high speed, centrifugal 5 minutes of 4 degree 16000g get immediately supernatant and are plasmosin.Blot fully for precipitation, avoid the pollution of plasmosin, add added protease inhibitor and inhibitors of phosphatases NER, frozen-thawed twice, ultrasonic 2 minutes, centrifugal 20 minutes of 16000g got supernatant, is nucleoprotein.Get 10 microgram albumen, be WESTERN. Figure 24 and show, cross expression Lrrc10 SRF is increased in endochylema, in karyon, reduce.

Example 17 is estimated each domain of Lrrc10 to SRF transcriptional activity inhibitory action

As shown in figure 25, with each domain of Lrrc10, split and connect into expression vector, carry out according to the method described above co-immunoprecipitation experiment and fluorescing system report activity analysis.Find that any one fragment can interact with SRF, can suppress its transcriptional activity, and one of them to contain 72 amino acid whose peptide molecule inhibitions more obvious, its aminoacid sequence is:

MGNTIRALVAFIPADRCQNYVVRDLREMPLDKMVDLSGSQLRRFPLHVCSFRELVK LYLSDNHLNSLPPELG(sees Figure 26).

＜110〉Hunan Normal University

＜120〉Lrrc10 micromolecule and the application in preparation treatment myocardial hypertrophy medicine thereof

<130>

<140>

<141>

<160>

<210> 1

<211> 29

<212> DNA

＜213〉artificial sequence

<220>

＜223〉according to the specific nucleotide sequence design, with the primer as pcr amplification.

<400> 1

CCCACCACTC TTGGATGGGA GGGCTTCAC 29

<210> 2

<211> 30

<212> DNA

＜213〉artificial sequence

<220>

<400> 2

TCGGCCTCTA TTTCAAGGGT CTGGAACAGC 30

<210> 3

<211> 30

<212> DNA

＜213〉artificial sequence

<220>

<400> 3

CAGCGTTTTC TTCAGAGGGA GTACCCAGAG 30

<210> 4

<211> 30

<212> DNA

＜213〉artificial sequence

<220>

<400> 4

TCAGAAGCAA ATGTGAGGAG CGACTGATCC 30

<210> 5

<211> 41

<212> DNA

＜213〉artificial sequence

<220>

<400> 5

ACAGACCTTA TTTGGGCAGC GCCTTATATG GAGTGGCCCA A 41

<210> 6

<211> 41

<212> DNA

＜213〉artificial sequence

<220>

<400> 6

TTGGGCCACT CCATATAAGG CGCTGCCCAA ATAAGGTCTG T 41

<210> 7

<211> 18

<212> DNA

＜213〉artificial sequence

<220>

<400> 7

GGATGTGGTC TGTGAACT 18

<210> 8

<211> 18

<212> DNA

＜213〉artificial sequence

<220>

<400> 8

GGAAGTAGCG GATACTGT 18

<210> 9

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

<400> 9

AGCCCAGAAC ATCATCCCT 19

<210> 10

<211> 20

<212> DNA

＜213〉artificial sequence

<220>

<400> 10

GGTCCTCAGT GTAGCCCAAG 20

<210> 11

<211> 27

<212> DNA

＜213〉artificial sequence

<220>

<400> 11

GTCGTCGACT CCCTGACAGA GTTGGTG 27

<210> 12

<211> 45

<212> DNA

＜213〉artificial sequence

<220>

<400> 12

TCAAGCGTAA TCTGGAACAT CGTATGGGTA AGAGCTGGAA GGAAG 45

<210> 13

<211> 24

<212> DNA

＜213〉artificial sequence

<220>

<400> 13

TGCAAAGGCT CCAGGTCTGA GGGC 24

<210> 14

<211> 24

<212> DNA

＜213〉artificial sequence

<220>

<400> 14

GCCAACACCA CCCTGTCCAA GTTC 24

<210> 15

<211> 20

<212> DNA

＜213〉artificial sequence

<220>

<400> 15

CTGCTAGACCA CCTGGAGGAG 21

<210> 16

<211> 22

<212> DNA

＜213〉artificial sequence

<220>

<400> 16

CCAGGAGGGT ATTCACCACC AC 22

<210> 17

<211> 20

<212> DNA

＜213〉artificial sequence

<220>

<400> 17

TTTGCCTCCG TTCCACCTGC 20

<210> 18

<211> 22

<212> DNA

＜213〉artificial sequence

<220>

<400> 18

TGCCAACTTG ATGGTCTAGC GC 22

<210> 19

<211> 22

<212> DNA

＜213〉artificial sequence

<220>

<400> 19

CAGCAAACAG GAATACGACG AA 22

<210> 20

<211> 21

<212> DNA

＜213〉artificial sequence

<220>

<400> 20

TGTGTGCTAG AGGCAGAGCA G 21

<210> 21

<211> 20

<212> DNA

＜213〉artificial sequence

<220>

<400> 21

TGCCACCTGG ACATCATCTG 20

<210> 22

<211> 21

<212> DNA

＜213〉artificial sequence

<220>

<400> 22

AGAACCTCCC AGTCCATACG G 21

<210> 23

<211> 20

<212> DNA

＜213〉artificial sequence

<220>

<400> 23

GCCGGTAGAA GATGAGGTCA 20

<210> 24

<211> 20

<212> DNA

＜213〉artificial sequence

<220>

<400> 24

GGGCTCCAAT CCTGTCAATC 20

<210> 25

<211> 21

<212> DNA

＜213〉artificial sequence

<220>

<400> 25

GTGAAGGGCA TGAGGAAGAG T 21

<210> 26

<211> 20

<212> DNA

＜213〉artificial sequence

<220>

<400> 26

AGGCCTTCAC CTTCAGCTGC 20

Claims

1. the Lrrc10 micromolecule of a specific heart expression is characterized in that, Lrrc10 is a small molecular protein, and aminoacid sequence is:

MGNTIRALVAFIPADRCQNYVVRDLREMPLDKMVDLSGSQLRRFPLHVCSFRELVKLYLSDNHLNSLPPELG。

2. the as claimed in claim 1 application of the Lrrc10 micromolecule of specific heart expression in preparation treatment myocardial hypertrophy disease medicament.