CN105106942B - Dual specificity phosphatase enzyme 14(DUSP14) treating function and application in myocardial hypertrophy - Google Patents
Dual specificity phosphatase enzyme 14(DUSP14) treating function and application in myocardial hypertrophy Download PDFInfo
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Abstract
The invention discloses a kind of dual specificity phosphatase enzyme 14(DUSP14) function and application in myocardial hypertrophy are being treated, belong to the function and application field of gene.Present invention determine that the correlation between the expression and myocardial hypertrophy of DUSP14, result of study shows in the model that myocardial hypertrophy occurs, and the expression of DUSP14 and normally organizes and compares significant decrease;Inhibit DUSP14 expression to significantly promote myocardial hypertrophy, fibrosis, deteriorate heart function, promotes DUSP14 overexpression then to significantly suppress myocardial hypertrophy, fibrosis, protect heart function.Therefore; DUSP14 can be used as target gene; for screening cardioprotection function, anti-cardiac fibrosis and/or prevention, alleviation and/or the drug for treating myocardial hypertrophy; it is used to prepare cardioprotection function, resisting cardiac hypertrophy and/or prevention, alleviation and/or the drug for treating myocardial hypertrophy, provides an effective new way for the treatment of myocardial hypertrophy.
Description
Technical field
The invention belongs to the function of gene and application field, in particular to a kind of dual specificity phosphatase enzyme 14(DUSP14)
Treat the function and application in myocardial hypertrophy.
Background technique
Myocardial hypertrophy is cardiac muscle to chronobiological mechanical pressure or the increased compensatory response of volumetric loading, is common in high blood
The cardiovascular diseases such as pressure, aortic stenosis are mainly shown as that cardiac muscle cell's volume increases, albumen synthesis increases, extracellularly
Matrix such as increases at the features [1-3].Hypertension, Senile degenerative aortic valve disease are in rise year by year trend in China.By high blood
Myocardial hypertrophy caused by the diseases such as pressure, hypertensive heart disease disease incidence are consequently increased.Although myocardial hypertrophy can initially make the heart
Myocyte increases, and myocardial contractive power is reinforced, and is a kind of compensatory mechanism for maintaining normal cardiac output, but the pressure of long duration
Or volumetric loading is overweight, can cause myocardial remodelling, simultaneously because myocardium requirementing keto quantity increases, and coronary artery blood supply relative deficiency,
Cause myocardial ischemia, cardiac muscle cell apoptosis, and then lead to decompensation, so as to cause heart failure, malignant arrhythmia, even sudden
It waits indefinitely [4,5].Research shows that with the occurrence and development of heart left chamber plumpness, myocardial ischemia, ventricular arrhythmia, heart failure,
The incidence of the cardiovascular events such as sudden death increases 6~10 times [6].
It is now recognized that myocardial hypertrophy is the complicated dynamic process that one kind of multiple factors participate in adjusting.Research finds long-term
Biomethanics pressure and/or volumetric loading are excessive, increase ventricle wall stress, lead to myocardial hypertrophy.In addition, Angiotensin II
The various extracellular stimulus signals such as (Ang II), Endothelin (ET), catecholamine, transforming growth factor-β (TGF-β) can induce core
The change of interior gene expression, so as to cause cardiac myocyte hypertrophy [7-11].The pathological process of myocardial hypertrophy from molecular level
Point three links: it is gene transcription activating in the appearance of extracellular plumpness stimulus signal, intracellular signal transduction and core, finally induce cell
Loose character mutation occurs.Research at present has shown that many A signal pathways participate in the process of myocardial hypertrophy.Wherein, calcium tune nerve
Phosphatase calcineurin/NFAT, mitogen-activated protein kinase (MAPK) and PI3K/Akt/GSK3 signal beta Signal Transduction Pathways with
And by this three accesses downstream transcription factor MCIP1.4 adjusted, NF- κ B, AP-1, MEF2, mTOR etc. in myocardial hypertrophy
Play an important role [1,2,12-17] in occurrence and development.Calcineurin (calcineurin) is one kind by calcium ion
And the multifunctional signal enzyme that calmodulin is adjusted, it can be by making nuclear factor of activated T cells (nuclear factor of
Activated T cells, NFAT) indexing enters core, adjust the expression [18,19] of loose gene (ANP, BNP) in core.MAPK packet
Include three subfamilies [20]: ERKs, JNKs and p38-MAPK.Under the MAPKs activation promotion of phosphorylation is related with myocardial hypertrophy
The transcriptional activity of transcription factor NF-KB, AP-1, MEF2, NFAT etc. is swum, and adjusts cytogene transcription and albumen synthesis, is caused
Cardiac myocyte hypertrophy leads to myocardial hypertrophy [20].
Dual specificity phosphatase enzyme (Dual Specificity Phosphatase, DUSP) is Protein-tyrosine-phosphatase I
An important member in type subfamily can also remove phosphorylated tyrosine dephosphorylation to phosphorylation serine/threonine
Phosphorylation.The domains characteristic and catalyst mechanism of dual specificity phosphatase enzyme are similar to typical tyrosine phosphatase.But double spies
The catalytic structure of specific phosphatase is more shallow more wider than typical tyrosine phosphatase, this feature is considered being their ability to quickly
The reason of identifying two or more amino acid residues.Because the tyrosine phosphatase activity of dual specificity phosphatase enzyme compares serine/threonine
Phosphatase activity is 40-500 times high, so being classified as a kind of tyrosine phosphatase without individually classifying.Have now been found that 61
The kind form dual specificity phosphatase enzyme different with structure, is generally classified as seven classes: slingshots, PRLs
(phosphatases of regenerating liver), Cdc14 phosphatases (Cdc is celldivision
cycle), PTENs (phosphatase and tensin homologues deleted on chromosome 10),
myotubularins, MKPs (mitogen activated protein kinase phosphatases) and
atypical DUSPs。
DUSP14 is a kind of atypical DUSP(atypia dual specificity phosphatase enzyme), molecular weight 22.26kDa, almost
There is expression in all cell lines and tissue, cellular localization experiment display DUSP14 is present in cytoplasm.DUSP14 exists for the first time
It being found in saccharomycete, experiment confirms that DUSP14 can be interacted with T cell Co stituation factor CD28 to adjust the secretion of IL-2,
The exogenous DUSP14 of GST label can result in ERK, JNK, p38MAPK dephosphorylation, in T cell, inactive DUSP14
The phosphorylation level of ERK and JNK can be made to increase, and p38 phosphorylation level does not change.DUSP14 can be by adjusting ERK
Activity influence beta Cell of islet proliferation.
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Summary of the invention
For the defect and deficiency for solving the above-mentioned prior art, it is an object of the invention to determine the expression of DUSP14 and cardiac muscle
Plump correlation, provide a kind of DUSP14 prepare cardioprotection function, anti-cardiac fibrosis and/or prevention, alleviate and/
Treat the new opplication in the drug of myocardial hypertrophy.
The purpose of the invention is achieved by the following technical solution:
The present invention has determined the relationship between DUSP14 expression and myocardial hypertrophy by test:
1, the expression of cardiac myocyte hypertrophy marker ANP, Myh7 are obviously raised when myocardial hypertrophy occurs, the expression of DUSP14
It is obvious to lower
The present invention selects normal person and dilated cardiomyopathy heart respectively, normal sham-operation (Sham) mouse and passes through
Aorta arch constriction operation (AB) causes the mouse heart of myocardial hypertrophy, has detected the albumen table of ANP, Myh7, DUSP14 respectively
Up to situation.The result shows that the cardiac myocyte hypertrophy mark in the mouse heart of dilated cardiomyopathy and generation myocardial hypertrophy
The expression of object ANP, Myh7 are obviously raised, and (Fig. 1, Fig. 2) is obviously lowered in the expression of DUSP14.
2, DUSP14 interferes (Adsh DUSP14) and is overexpressed (Ad DUSP14) adenovirus to the heart induced through Ang II
The influence of myocyte hypertrophy model
Present invention discover that in menses angiotensin II(Ang II) induction external myocardial hypertrophy model in, DUSP14 crosses table
Hypertrophy up to cardiac muscle cell is obvious suppressed, and DUSP14 does not express cardiac muscle cell and obvious loose (Fig. 3) occurs.
3, DUSP14 gene knockout significantly promotes myocardial hypertrophy, fibrosis, deteriorates heart function
The present invention selects wild-type mice, DUSP14 knock out mice to test, and every kind of mouse is divided into artificial hand
Art group and operation group, every group of 10 mouse.Operation group gives aorta arch constriction operation, and sham-operation group refuses arch of aorta contracting
It is narrow, the measurement of plump cardiac myocytes, fibrosis and heart function is then carried out by each group mouse to sham-operation group and operation group,
The influence for the myocardial hypertrophy that research DUSP14 gene knockout induces aorta arch constriction.The result shows that knocking out DUSP14 gene institute
The DUSP14 defect of cause significantly deteriorates myocardial hypertrophy, fibrosis and heart function (Fig. 4, Fig. 5, Fig. 6, Figure 10).
4, DUSP14 gene overexpression significantly suppresses myocardial hypertrophy and its fibrosis, improves heart function
The present invention selects heartspecific DUSP14 transgenic mice and nontransgenic mice to test, and small by every kind
Mouse is divided into sham-operation group and operation group, every group of 10 mouse.Operation group gives aorta arch constriction operation, and sham-operation group is not led
Then arch of aorta constriction carries out plump cardiac myocytes, fibrosis and heart function by each group mouse to sham-operation group and operation group
The measurement of energy, the influence for the myocardial hypertrophy that research DUSP14 gene overexpression induces aorta arch constriction.The result shows that crossing table
Myocardial hypertrophy and fibrosis are significantly inhibited up to DUSP14 gene, is protected heart function (Fig. 7, Fig. 8, Fig. 9, Figure 11).
It can be seen from the above result that the expression of DUSP14 compares significant drop with normal group in the model that myocardial hypertrophy occurs
It is low;Inhibit DUSP14 expression to significantly promote myocardial hypertrophy, fibrosis, deteriorate heart function, promotes DUSP14 to be overexpressed then significant
Myocardial hypertrophy, fibrosis are inhibited, heart function is protected.Therefore DUSP14 has protection heart function and inhibits myocardial hypertrophy and fiber
The effect of change, especially DUSP14 have the work for being able to suppress the generation of myocardial hypertrophy related disease caused by aorta arch constriction
With.
A kind of function of DUSP14 in myocardial hypertrophy, being mainly reflected in DUSP14 has protection heart function and inhibits cardiac muscle
Plump effect, especially DUSP14 have the function of the generation of myocardial hypertrophy caused by being able to suppress aorta arch constriction.
For the above-mentioned function of DUSP14, provide the application of DUSP14 a kind of, be mainly reflected in DUSP14 cardioprotection,
Application in anti-cardiac fibrosis and treatment myocardial hypertrophy, especially DUSP14 are screening or are preparing cardioprotection function, the anti-heart
It is applied in dirty fibrosis and/or the drug of prevention, alleviation and/or treatment myocardial hypertrophy.The application includes DUSP14 direct
As cardioprotection function, anti-cardiac fibrosis and/or prevention, alleviation and/or the effective component for treating myocardial hypertrophy drug, energy
Enough promote DUSP14 expression chemical substance be used as indirectly cardioprotection function, anti-cardiac fibrosis and/or prevention, alleviate and/
Or the effective component for the treatment of myocardial hypertrophy drug.
A kind of drug of cardioprotection function includes DUSP14.
A kind of drug of anti-cardiac fibrosis includes DUSP14.
It is a kind of prevention, alleviate and/treatment myocardial hypertrophy drug, include DUSP14.
The invention has the following beneficial effects:
Present invention finds the new function of DUSP14 gene, i.e., have the function of being capable of cardioprotection, the anti-heart for DUSP14 gene
Dirty fibrosis and the effect for inhibiting myocardial hypertrophy.Therefore, DUSP14 can be used as target gene, for screening cardioprotection function, resisting
Cardiac fibrosis and/or prevention, alleviation and/or the drug for treating myocardial hypertrophy are used to prepare cardioprotection function, anti-cardiac muscle fertilizer
Thick and/or prevention, the drug for alleviating and/or treating myocardial hypertrophy provide an effective new way for the treatment of myocardial hypertrophy
Diameter.
Detailed description of the invention
Fig. 1 is the protein expression of ANP, Myh7, DUSP14 in normal person and dilated cardiomyopathy patients (expanding heart trouble) heart
Situation and statistics histogram;(0.05 vs normal person's group of *: p <) is lowered in the expression of dilated cardiomyopathy patients heart DUSP14.
Fig. 2 be mouse in sham-operation (Sham) and aorta arch constriction operation (AB) afterwards heart ANP, Myh7, DUSP14
Expression and statistics histogram, GAPDH be used as internal reference, wherein 4W indicate 4 weeks, 8W expression 8 weeks;The heart of myocardial hypertrophy occurs
(0.05 vs wild-type mice Sham group of *: p <) is lowered in the expression of dirty DUSP14.
Fig. 3 is SD suckling mouse primary cardiomyocytes adenovirus AdshRNA, AdshDUSP14, AdGFP and AdDUSP14 sense
Dye counts histogram through the post-stimulatory immunofluorescence of Ang II and cell surface area, and the interference adenovirus of DUSP14 promotes the heart
The overexpression adenovirus of myocyte hypertrophy, DUSP14 inhibits cardiac myocyte hypertrophy (0.05 vs PBS group of *: p <, #:p < 0.05
Vs Ang II group).
Fig. 4 be HW/BW after wild-type mice (WT) and DUSP14 knock out mice (DUSP14-KO) AB perform the operation 4 weeks,
The statistics histogram of LW/BW and HW/TL;Wherein, HW is cardiac mass, and BW is weight, and LW is lungs total amount, and TL is tibia length
(0.05 vs wild type Sham group of *: p <, 0.05 vs wild type AB group of #:p <).
Fig. 5 is wild-type mice (WT) and ventricle is transversal after DUSP14 knock out mice (DUSP14-KO) AB operation 4 weeks
Face and heart tissue HE dyeing, WGA dyeing and cardiomyocytes cross-sectional area statistics histogram (0.05 vs wild type of *: p <
Sham group, 0.05 vs wild type AB group of # p <).
Fig. 6 is wild-type mice (WT) and heart tissue after DUSP14 knock out mice (DUSP14-KO) AB operation 4 weeks
(0.05 vs wild type Sham group of *: p <, 0.05 vs of #:p < are wild for sirius red stains and left room area of collagen statistics histogram
Raw type AB group).
Fig. 7 is statistics histogram (*: the p < 0.05 of HW/BW, LW/BW and HW/TL after NTG and TG mouse AB performs the operation 4 weeks
Vs NTG Sham group, 0.05 vs NTG AB group of #:p <).
Fig. 8 is ventricle cross section and heart tissue HE dyeing, WGA dyeing and cardiac muscle after NTG and TG mouse AB performs the operation 4 weeks
Cell cross section product statistics histogram (0.05 vs NTG Sham group of *: p <, 0.05 vs NTG AB group of #:p <).
Fig. 9 is heart tissue sirius red stains and left room area of collagen statistics column after NTG and TG mouse AB performs the operation 4 weeks
Scheme (0.05 vs NTG Sham group of *: p <, 0.05 vs NTG AB group of #:p <).
Figure 10 is wild-type mice (WT) and heart function after DUSP14 knock out mice (DUSP14-KO) AB operation 4 weeks
The statistics histogram of testing result;EF is ejection fraction, and FS is shortening fraction, and LVEDd is left ventricular end diastolic diameter,
LVSDd is left room end systolic diameter (0.05 vs wild type Sham group of *: p <, 0.05 vs wild type AB group of #:p <).
Figure 11 is the statistics histogram of heart function testing result after NTG and TG mouse AB performs the operation 4 weeks;EF is ejection fraction,
FS is shortening fraction, and LVEDd is left ventricular end diastolic diameter, and LVSDd is left room end systolic diameter (0.05 vs of *: p <
NTG Sham group, 0.05 vs NTG AB group of #:p <).
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
Animal for research and raising
Experimental animal: select 8-10 week old, weight in 23.5-27.5g, background is the wild-type mice of male C57BL/6
(name WT), whole body DUSP14 knock-out mice (DUSP14-KO), heartspecific DUSP14 transgenic mice (DUSP14-TG)
And nontransgenic mice (NTG, littermate control nontransgenic mice) is experimental subjects.
(1) building of systemic DUSP14 knock out mice:
Systemic DUSP14 knock out mice is constructed using CRISPR-Cas9 technology.Firstly, being set by online CRISPR
Meter tool (http://crispr.mit.edu) predicts the boot sequence of mouse DUSP14, designs 2 single-stranded oligo:
Oligo1:TAGGATGACGCAGGTGATGCCGC,
Oligo2:AAACGCGGCATCACCTGCGTCAT.
Oligo1 and oligo2 anneal to form double-stranded DNA, and double-stranded DNA is connected into the pUC57-sgRNA through BsaI digestion
(Addgene 51132) constructs sgRNA expression vector.
Using the sgRNA expression vector of above-mentioned building as template, using following primer by PCR amplification contain T7 promoter and
The DNA fragmentation of boot sequence:
Forward primer:GATCCCTAATACGACTCACTATAG,
Reverse primer:AAAAAAAGCACCGACTCGGT.
It is carried out using the PCR product expanded as template using MEGAshortscript Kit(Ambion, AM1354) external
Transcription;Cas9 plasmid (Addgene 44758) passes through T7 Ultra kit(Ambion, Am1345) it is transcribed.It will transcribe
The mRNA of the Cas9 and boot sequence RNA that arrive use miRNeasy Micro Kit(Qiaen, 217084) after purification, pass through
5247 micro-injection system of FemtoJet is injected into the unicellular fertilized eggs of wild type C57BL/6 mouse.It chooses through micro- note
The fertilized eggs survived after penetrating are transplanted in healthy female mice fallopian tubal, obtain F0 for mouse after gestation by 21 days.
F1 generation and F2 identify that wild-type mice includes the DNA sequence dna of one section of long 347bp, and is mutated small for mouse by PCR
The DNA sequence dna of mouse (DUSP14 knock out mice) containing 291bp.Final protein product is detected by western blot
Identification.Wherein, PCR identifies that primer is as follows:
Primers-F:5 '-GGAGAGCTGCAAAACATAATGC-3 ',
Primers-R:5 '-CAGCTGTCAAAGCCCACATA-3 '.
Testing mouse used is mutant homozygote.
(2) building of heartspecific DUSP14 transgenic mice:
It is complete with following primer PCR amplification mouse DUSP14 using wild type C57BL/6 mouse DUSP14 gene cDNA as template
Long gene (NCBI, DUSP14 mus NM_011909.2):
Upstream primer: TGCTCTAGAGCCACCATGAGCTCCAGAGGTCACAG,
Downstream primer: TGCTCTAGACTAAATCCCCCAATAAGGCA.
Amplification DUSP14 full-length gene and pCAG-loxP-CAT-loxP plasmid (pCAG-loxP-CAT-loxP plasmid by
Beijing Union Medical College basis institute teacher Yang Qinglin provides, and preparation process is referring to document: Kim T, Zhelyabovska O,
Liu J, et al. Generation of an Inducible, Cardiomyocyte-Specific Transgenic
Mouse Model with PPAR b/d Overexpression[J]. Peroxisome Proliferator-
Activated Receptors (PPARs), 57.) through XbaI(NEB, # R0145L) it connects after digestion, form pCAG-
LoxP-CAT-loxP-DUSP14-hGHpA carrier (CAG (chicken beta-actin)-CAT (chloramphenicol
Acetyltransferase)-DUSP14-hGHpA), the plasmid of building is injected into wild type by micro-injection system
In the unicellular fertilized eggs of C57BL/6 mouse, which enters in healthy female mice, and development obtains F0 for mouse, will obtain
F0 for mouse and α-MHC-Cre mouse hybrid, and by tamoxifen induction (tamoxifen 80mg/kg/D, intraperitoneal injection,
Continued stimulus 5 days) obtain heartspecific DUSP14 transgenic mouse (above-mentioned transgenic mice is prepared referring to following documents:
Jiang DS, Bian ZY, Zhang Y, Zhang SM, Liu Y, Zhang R et al. Role of
interferon regulatory factor 4 in the regulation of pathological cardiac
hypertrophy. Hypertension 2013;61:1193-1202.).
Feeding environment: all experiment mices are raised in SPF grades of Experimental Animal Centers, and SPF grades of size mouse feeds are purchased from north
Capital Fukang Biotechnology Co., Ltd.Rearing conditions: room temperature is between 22-24 DEG C, and humidity is between 40-70%, light and shade alternating
Lighting hours is 12h, and free water is ingested.
Expression of 1 DUSP14 of embodiment in normal person and Mutation of Patients with Cardiomyopathy heart
Normal Human Heart's (dead individual contributed of non-cardiac reason), dilated cardiomyopathy heart is selected (to do the heart
The receptor of dirty transfer operation patient displacement, DCM), protein is extracted to heart and carries out SDS-PAGE- western blot test
(Western blot), binding specificity know DUSP14 albumen and cardiac myocyte hypertrophy marker ANP(Millipore,
AB2232), Myh7(santa cruz, sc53090) antibody detected, measure its DUSP14(Abnova, PAB4143)
Expression, GAPDH(Cell Signaling Technology, 2128) it is used as internal reference.Testing result is as shown in Figure 1, the expanding heart
The expression of cardiac myocyte hypertrophy marker ANP, Myh7 in patients with myopathy heart are obviously raised, and the expression of DUSP14 is obviously lowered
(Fig. 1).
Expression of 2 DUSP14 of embodiment in wild-type mice sham-operation group and myocardial hypertrophy model group heart
1. establishing mouse cardiac muscle hypertrophy model using aorta arch constriction operation (AB), model manipulation process:
1.1 Preoperative Method
(1) it anaesthetizes: first weighing to mouse, the amount of anaesthetic (3% yellow Jackets) needed for calculating according to 90mg/kg weight passes through
Intraperitoneal injection, and record injection time point.Press from both sides tail, folder toe without significant reaction and mouse it is in good condition for anesthesia Success criteria (one
As after injection about 10min have reaction with the small mousetrap toe of about 50min after anesthesia, 30min or so is best after anesthesia without significant reaction
Operating time).
(2) art area prepares: by the left chest of mouse, left side chest and the skin unhairing of left fore oxter.With wet yarn after shaving
Cloth wiping art area removes deratization hair, is advisable with not influencing surgical field of view.
(3) trachea cannula: the upper front tooth of mouse is fixed on V shaped slab inclined-plane with rubber band, and rapidly passes through trachea cannula
Glottis is properly inserted intratracheally, and subsequent right lateral position is placed on heating cushion (heating cushion need to preheat in advance), then by trachea cannula
It is connect with ventilator, fixed mouse.If the thorax fluctuating of mouse is consistent with breathing unit frequency, illustrate trachea cannula success.
1.2 aorta arch constriction arts (AB)
AB art myocardial hypertrophy model group: taking right lateral position, and mouse left fore is placed in above right fore, and will with medical adhesive tape
Two forelimbs are fixed.It is encased inside cotton swab below right chest, raises thorax, is successively 75% alcohol to field of operation with the tincture of iodine and volume fraction
Domain skin degerming.Left hand holds ophthalmic tweezers and has pinched left skin of chest, and the right hand holds eye scissors and cuts off skin about 1cm, successively separates flesh
Meat and soft tissue slightly push left lung aside with cotton swab in 2-3 rib horizontal opening thoracic cavity, and dissociate arch of aorta descending branch, and 7-0 is performed the operation
Suture pass through blood vessel, and one section of 26G(25.0-27.5g mouse is placed in parallel above blood vessel) or 27G(23.5-25.0g) infuse
Emitter syringe needle ligatures blood vessel and syringe needle together, then extracts syringe needle out i.e. and can reach the Vasoconstriction of respective degrees.Ligation finishes
It successively sutures afterwards, closes thoracic cavity, be inserted into thoracic cavity from sealing with syringe and extract 1cc gas out to restore negative pressure in thoracic cavity, pull out
Rapid skin suture notch after syringe out.
Only threading does not ligature sham-operation group (Sham) after free descending aorta out, remaining step is the same as AB art myocardial hypertrophy
Model group.
1.3 postoperative care
In operation after the ligation of arch of aorta descending branch, there is autonomous respiration to mouse, kickback occurs in folder toe, extraction tracheae
Intubation, and mouse is put into the rearging cage of padding, feed and the drinking water crossed equipped with high pressure sterilization, continue to raise in receptacle
Observation.
2. materials
Assay balance is opened, is returned to zero spare.It is re-weighed and puts to death mouse.The curved tweezer of ophthalmology lives the vessel pedicle below auricle, cuts
Lower heart, is immediately placed on sterile gauze, gently squeezes heart intracavity liquid, after dipping in dry surface liquid, weighs and record, by heart
It is put into corresponding cryopreservation tube, is immediately placed in liquid nitrogen container, be used for molecular Biological Detection after -80 DEG C of refrigerators save.
3. expression of the DUSP14 in Sham group mouse and myocardial hypertrophy model group mouse heart
4 weeks and 8 weeks hearts, mention heart after selecting wild-type mice Sham group and myocardial hypertrophy model group AB to perform the operation respectively
Protein is taken to carry out SDS-PAGE- western blot test (Western blot), binding specificity identifies DUSP14 albumen and the heart
The antibody of myocyte hypertrophy marker ANP, Myh7 are detected, and measure the expression of its DUSP14, GAPDH is as internal reference, detection
As a result as shown in Figure 2.Expression of the cardiac myocyte hypertrophy marker in AB postoperative ANP, Myh7 is obviously raised, and the expression of DUSP14 exists
The postoperative obvious downward of AB.Show that the expression that the heart DUSP14 of myocardial hypertrophy occurs is lowered.
3 DUSP14 of embodiment interferes (Adsh DUSP14) and is overexpressed (Ad DUSP14) adenovirus and stimulates Ang II
Primary cardiomyocytes hypertrophy influence
1. primary newborn SD rat myocardial cells culture
(1) newborn 1 day Sprague-Dawley suckling mouse 8,75% alcohol disinfecting below neck, with eye scissors and microforceps
Heart is removed, is put into the glass dish for filling 10mL DMEM/F12 liquid.Another is taken again, repeats above procedure.
(2) heart is cleaned with DMEM/F12 culture medium, and heart is cut into 1-2mm3Fragment.It is transferred to and is placed with rotor
In serum bottle, DMEM/F12 is sucked, pancreatin digestive juice is added.Revolving speed is 120r/min, digests 15min, the static several seconds, discards
Supernatant.
(3) pancreatin digestive juice is added, revolving speed 120r/min digests 15min.Static several seconds, Aspirate supernatant are used
The DMEM/F12 culture medium of 20% calf serum terminates digestion, is placed in 4 DEG C of refrigerators and saves.The step is repeated, circulation is several times.
Taking should exhaust when supernatant as far as possible, when tissue block bleaches and obviously becomes smaller, terminate digestion.
(4) myocardial cell suspensions that will be gathered are centrifuged 8min with 1500rpm revolving speed, discard supernatant liquid.In centrifuge tube
Appropriate culture medium is added, cell is resuspended in soft piping and druming, is concentrated in 1 50mL centrifuge tube, and cell suspension is filtered with 40 μm of cell
Net filtration.
(5) by cell inoculation in the culture dish of 100mm, adherent 90min when poor draws not adherent cell suspension mistake
Filter.Brdu(final concentration 0.1mM is added according to the total amount of cell suspension), after mixing, it is added to the coated device of 0.1% gelatin
In ware.
(6) jog cell dispersion, whirlpool does not rock.37℃,5% CO2It is incubated for 48 hours and is cleaned 1 time with PBS, replacement culture
Base.
2. DUSP14 interferes (Adsh DUSP14) and is overexpressed (Ad DUSP14) adenovirus to the heart induced through Ang II
The influence of myocyte hypertrophy model
AdshRNA(silencing RNA containing shRNA() adenovirus, be used as control), Adsh DUSP14(contain shRNA-DUSP14
The adenovirus of (silencing RNA-DUSP14 fusion protein)), AdGFP(green fluorescent protein containing GFP() adenovirus, be used as control)
And the Ad DUSP14(- DUSP14 of green fluorescent protein containing GFP-DUSP14(fusion protein) adenovirus) (the structure of these adenovirus
Process is built referring to document: Chen K, Gao L, Liu Y, et al. Vinexin- β protects against
cardiac hypertrophy by blocking the Akt-dependent signalling pathway[J].Basic Res Cardiol, 2013,108 (2): 1-14.) adenovirus 10 MOIs infect 3 days primary cardiac muscles of culture respectively
Cell is small with 1 μM of Angiotensin II (Ang II) (being purchased from Sigma company, A9525) or control PBS stimulation 48 after 12 hours
When, then carry out immunofluorescent test.The result shows that myocardial cell surface area after Adsh DUSP14 adenovirus infection compared with
AdshRNA control group increases, and the myocardial cell surface area through AdDUSP14 adenovirus infection is then more obvious than control group AdGFP
It reduces (Fig. 3), i.e. the interference adenovirus of DUSP14 promotes cardiac myocyte hypertrophy, and the overexpression adenovirus of DUSP14 inhibits cardiac muscle thin
Born of the same parents are loose.
4 myocardial hypertrophy model mice myocardial hypertrophy of embodiment and fibrosis detection and heart function detection
1. establishing mouse cardiac muscle hypertrophy model using aorta arch constriction operation (AB)
Select 8-10 week old, weight 23.5-27.5g wild-type mice (WT), DUSP14 knock out mice
(DUSP14-KO), heartspecific DUSP14 transgenic mice (DUSP14-TG) and nontransgenic mice (NTG) are respectively divided into vacation
Operation group (Sham) and AB art myocardial hypertrophy model group, i.e. WT Sham group, WT AB group, DUSP14-KO Sham group, DUSP14-
KO AB group, DUSP14-TG Sham group, DUSP14-TG AB group, NTG Sham group, NTG AB group, every group of 10 mouse.Modeling
Method is the same as embodiment 2.The detection of the AB detection for carrying out myocardial hypertrophy and fibrosis respectively and heart function in postoperative 4 weeks.
2. materials
(1) previous work: preparing the urine cup of 10% formaldehyde of volume fraction equipped with 20mL in advance, and posts label (mouse volume
Number, group, type of surgery and materials the date).The culture dish for filling 10% KCl solution of mass fraction is placed at materials.It opens
Assay balance returns to zero spare.It is re-weighed and puts to death mouse.
(2) draw materials: the curved tweezer of ophthalmology lives the vessel pedicle below auricle, cuts heart, is immediately placed in 10% KCl of mass fraction
In solution.It after cardiac arrest after diastole, is placed on sterile gauze, gently squeezes heart intracavity liquid, after dipping in dry surface liquid,
It weighs and records, heart is put into corresponding urine cup, detected after fixed 48h for pathology.
(3) measurement of correlation and calculating: mouse lung is taken out, filter paper blots after trimming, weighs and records.Cut off mouse hind leg
Skin at shin bone, measures and records tibia length.Calculate the ratio (HW/BW) of heart weight and weight, the ratio of lung weight and weight
(LW/BW) and the heart weighs the ratio (HW/TL) with tibia length.
3. pathology detect
3.1 prepare paraffin specimen slice
Primary operational program includes trimming heart → embedding frame processing → flowing water flushing → dehydration → transparent → waxdip → packet
Bury → slice → spread out it is spare after piece → dry or toast.
3.2 hematoxylin-eosins (HE) dyeing
Key step are as follows: paraffin specimen is taken to be sliced 55 DEG C of baking 30min → dimethylbenzene 5min, 3 times → 100% alcohol 1min
→ 95% alcohol of alcohol 1min → 70% 1min → distilled water 1min → haematoxylin solution (Zhuhai shellfish rope, BA-4021) 5min → water
Wash the hydrochloride alcohol of 1min → 1% (take 3mL concentrated hydrochloric acid and 70% alcohol of 297mL be sufficiently mixed uniformly) 1-3s → washing 1min →
Scott liquid (sodium bicarbonate 0.35g, epsom salt 2g, the two are dissolved in 100mL distilled water) 1min → washing 1min → Yihong is molten
Liquid (Zhuhai shellfish rope, BA-4024) 3-5min → distilled water washes away loose colour → 70% alcohol of alcohol 1s → 95% alcohol of 1s → 100%
30s, 3 times → dimethylbenzene 2min, 3 times → take advantage of the interior drying of the not dry mounting → draught cupboard immediately of dimethylbenzene, microscope is taken pictures.
HE dyes picture statistics: every picture selects 3 or more clear borders, and the approximately centrally located cell of core is used
6.0 software circle cell area of Image-Pro Plus.
3.3 wheat germ aggregate (WGA) dyeing
Key step: the paraffin specimen slice that 30min is toasted through 60 DEG C is placed in dimethylbenzene 5min × 3 time → 100% second
Alcohol 5min × 2 time → 95% ethyl alcohol of ethyl alcohol 5min → 70% 5min → distilled water rinsing 5min × 2 time → PBS rinses 5min → PBS
Rinsing 10min → discard PBS is added dropwise 37 DEG C of pancreatin working solution (DIG-3008, Foochow step new) and is protected from light incubation 20min → PBS drift
5min × 3 time → taking-up slice is washed, dries the liquid (tissue is sure not drying) around tissue with filter paper, changes stroke circle with group and lays flat
→ 37 DEG C of dropwise addition 488 working solution of WGA-Alexa Flour (10 μ g/mL), which is protected from light, is incubated for 2h → discards dye liquor, PBS in wet box
5min × 3 time → SlowFade Gold antifade reagent with DAPI mounting → viewed under fluoroscopy is rinsed, is shown
Micro mirror is taken pictures.
3.4 Picro-Sirius reds (PSR) dyeing
Key step are as follows: paraffin specimen is taken to be sliced 55 DEG C of baking 30min → dimethylbenzene 2min, 3 times → 100% alcohol 1min
→ 95% alcohol of alcohol 1min → 70% 1min → flowing water rinses 10min → 0.2% phosphomolybdic acid 2min of distilled water 1min → mass fraction
→ 0.1% sirius red picric acid solution drop dyed in tissue, wet box 90min → go residul liquid-removing → 0.01N hydrochloric acid 4s →
70% alcohol, 1 time → 90% alcohol, 1 time → 100% alcohol 30s, 3 times → dimethylbenzene 2min, 3 times → take advantage of the not dry lid glass immediately of dimethylbenzene
Piece mounting, microscope are taken pictures.
PSR dyes picture statistics (6.0 software of Image-Pro Plus): the collagen ratio=area of collagen/(gross area-sky
Fine flour product) × 100%.
Cardiac muscular tissue is made of cardiac muscle cell and interstitial tissue, and heart is a terminal differentiation organ, and cardiac muscle cell loses increasing
Ability is grown, the reaction of cardiac muscle cell caused by various physiology or pathological stimuli can only be the volume increase of individual cells and cannot
Quantitatively it is proliferated.Therefore, in the pathophysiological process of myocardial hypertrophy, it is mainly shown as that cardiac muscle cell's volume increases, muscle segment
Quantity increases, cell arrangement disorder, and cardiac mesenchymal changes proliferation and conversion including Cardiac Fibroblasts, collagenous fibres density
Increase, collagen secretion increases, collagen proportional balancing method imbalance etc..
Phenotypic results after WT and DUSP14-KO mouse uses AB art to establish myocardial hypertrophy model are shown in Fig. 4-6.Sham(is false
Operation) difference in group between HW/BW, LW/BW and HW/TL of WT and DUSP14-KO mouse is not statistically significant;WT mouse
AB postoperative 4 weeks HW/BW, LW/BW, HW/TL are higher than its Sham group;AB postoperative 4 weeks, HW/BW, LW/BW of DUSP14-KO mouse
And HW/TL rises (Fig. 4) compared with WT mouse.HE dyeing and WGA stained slice can be observed: Sham group heart no significant difference,
AB group increases compared with the heart of Sham group, and the heart of DUSP14-KO mouse is significantly greater than WT group mouse;Sham group myocardium myo fibril
It is neat, fine and close to tie up cell arrangement, form is complete, and karyon and nucleolar structure are clear;AB group myofilament is disorganized, loose, and cardiac muscle is thin
Cell space product significantly increases, and form is irregular, born of the same parents' nuclear hyperchromatism, increase, deformity, and kernel is fuzzy, and DUSP14-KO group is then than WT group cell
Loose obvious, difference is statistically significant (Fig. 5).After PSR dyeing, find AB group myocardium of ventricle interstitial collagen content compared with Sham group
Increase, collagen increase becomes apparent around arteries, and collagen thickening is disorganized at network-like;DUSP14-KO mouse AB art
Myocardial collagen network content and perivascular collagen content then increase more (Fig. 6) more postoperative than WT mouse AB afterwards.Result above is said
It is bright after the modeling of AB art, apparent myocardial hypertrophy and fibrosis, the myocardial hypertrophy and fibrosis of DUSP14-KO mouse occur for mouse
Degree is greater than WT mouse.
Fig. 7-9 is that NTG and DUSP14-TG mouse uses AB art to establish the phenotypic results after myocardial hypertrophy model.AB postoperative 4
The degree that HW/BW, LW/BW and HW/TL of all TG mouse increase is significantly less than NTG mouse (Fig. 7).HE dyeing and WGA dyeing are cut
Piece can be observed: AB group increases compared with the heart of Sham group, and the degree that the postoperative TG mouse heart of AB increases is small much smaller than NTG
Mouse;TG mouse AB postoperative myocardial cell cross section product is significantly less than NTG mouse AB group (Fig. 8).PSR is dyed as it can be seen that TG mouse AB
Postoperative myocardial interstitial collagen content and perivascular collagen content are less than NTG mouse AB group (Fig. 9).These results suggest that through AB art
After modeling, the myocardial hypertrophy and degree of fibrosis of DUSP14-TG mouse are less than NTG mouse.
4. ultrasound detection heart function
4.1 early-stage preparations
(1) Anesthesia machine prepares: first connecting the intake interface on oxygen cylinder and Anesthesia machine, then to unscrew dosing mouth on Anesthesia machine close
Capping, tightens sealing cover after being rapidly added isoflurane to safe scale.Total valve on oxygen cylinder is unscrewed, flow control valve is adjusted
Knob, outlet pressure maintain 0.2-0.3mPa.
(2) mouse to be measured prepares: after mouse to be detected is anaesthetized rapidly with isoflurane, left anterior pectorial region shaving, by what is handled well
Mouse head protrudes into anesthetic conduit pullover, the narcosis for maintaining mouse stable with 1.5-2.0% isoflurane.
The detection of 4.2 heart functions
Mouse takes left lateral position or dorsal position, and uniformly smears ultrasonic coupling agent (Tianjin Cheng Xin company) in shaving area.It adopts
With high-frequency ultrasound in diagnosis instrument, frequency 15MHz, selection standard papillary muscles of left ventricle short axis view is measured in left room diastasis
Diameter (LVEDd), left room end systolic diameter (LVESd), ejection fraction (EF) and shortening fraction (FS).
Figure 10 is that WT and DUSP14-KO mouse uses AB art to establish the heart function testing result after myocardial hypertrophy model.With
WT Sham group is compared, and is shown decreased cardiac function and myocardial hypertrophy within WT mouse AB postoperative 4 weeks, is mainly shown as myocardial hypertrophy
Index LVEDd, LVESd increases, and reflects index EF, FS of heart function and then decline.AB postoperative 4 weeks, DUSP14-KO mouse cardiac muscle
The degree and reflect that the degree of the index decline of heart function is greater than WT mouse that plump index increases.
Figure 11 is that NTG and DUSP14-TG mouse uses AB art to establish the ultrasonic testing results after myocardial hypertrophy model.As a result
Show AB postoperative 4 weeks, compared with NTG mouse, the degree that the index of TG mouse cardiac muscle plumpness increases and the index for reflecting heart function
The degree of decline is then less than NTG group.
It can be seen from the above result that in the myocardial hypertrophy disease model caused by aorta arch constriction, DUSP14 gene defect
Myocardial hypertrophy, fibrosis are significantly promoted, heart function is deteriorated, DUSP14 gene overexpression significantly suppresses myocardial hypertrophy, fiber
Change, protects heart function.Therefore DUSP14 gene has the function of protecting heart function and inhibits myocardial hypertrophy and fibrosis, especially
DUSP14 gene has the function of the generation of myocardial hypertrophy related disease caused by being able to suppress aorta arch constriction.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (6)
1. application of the dual specificity phosphatase enzyme 14 in the drug for screening anti-cardiac fibrosis.
2. application of the dual specificity phosphatase enzyme 14 in the drug for preparing anti-cardiac fibrosis.
3. application according to claim 2, it is characterised in that: be used as anti-cardiac fibrosis including dual specificity phosphatase enzyme 14
Drug effective component.
4. dual specificity phosphatase enzyme 14 is applied in screening prevention, the drug alleviated and/or treat myocardial hypertrophy.
5. dual specificity phosphatase enzyme 14 is applied in preparation prevention, the drug alleviated and/or treat myocardial hypertrophy.
6. application according to claim 5, it is characterised in that: including dual specificity phosphatase enzyme 14 as prevention, alleviate and/
Or the effective component of the drug for the treatment of myocardial hypertrophy.
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Dual-Specificity Phosphatases as Molecular Targets for Inhibition in Human Disease;Pablo Rı´os et al.;《ANTIOXIDANTS & REDOX SIGNALING》;20140430;2251-2273 |
Negative-Feedback Regulation of CD28 Costimulation by a Novel Mitogen-Activated Protein Kinase Phosphatase, MKP6;Francesc Marti et al.;《J Immunol》;20010101;197-200 |
p38MAPK 信号通路与心脏重构关系的研究进展;赵金红等;《医学综述》;20130731;2312-2314 |
The Krüppel-Like Factor Gene Target Dusp14 Regulates Axon Growth and Regeneration;Keiichiro Iwao et al.;《ARVO Annual Meeting Abstract》;20140430;1447 |
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