CN102719436A - Oligonucleotides and usage thereof in preparation of medicine for preventing and curing myocardial hypertrophy and heart failure - Google Patents
Oligonucleotides and usage thereof in preparation of medicine for preventing and curing myocardial hypertrophy and heart failure Download PDFInfo
- Publication number
- CN102719436A CN102719436A CN2012102150127A CN201210215012A CN102719436A CN 102719436 A CN102719436 A CN 102719436A CN 2012102150127 A CN2012102150127 A CN 2012102150127A CN 201210215012 A CN201210215012 A CN 201210215012A CN 102719436 A CN102719436 A CN 102719436A
- Authority
- CN
- China
- Prior art keywords
- oligonucleotide
- myocardial hypertrophy
- aacgtt
- oligonucleotides
- heart failure
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Abstract
The invention belongs to the technical field of medicines, and particularly relates to 5'-AACGTT-3'-containing oligonucleotides, a functional analogue of the oligonucleotides, and usage of the oligonucleotides or the functional analogue of the oligonucleotides for preventing and curing myocardial hypertrophy and chronic heart failure. The oligonucleotides and the usage realize the function of the 5'-AACGTT-3'-containing oligonucleotides by using myocardial cells and animal hypertrophy models acknowledged in the academia at home and abroad. The experimental result shows that the 5'-AACGTT-3'-containing oligonucleotides can obviously restrain the expression of a landmark protein ANF (atrial natriuretic factor) and a beta-MHC (major histocompatibility complex) gene of the myocardial hypertrophy, and can restrain the increase of a superficial area of the myocardial cell, and the restrain function of the oligonucleotides depends on the dosage of the oligonucleotides.. In the whole experiment, the 5'-AACGTT-3'-containing oligonucleotides can obviously restrain the myocardial hypertrophy and the increase of the heart-body ratio, and the happening of myocardial necrosis can be reduced. The experiment result disclosed by the invention prompts that the 5'-AACGTT-3'-containing oligonucleotides is the medicine for effectively curing the myocardial hypertrophy and the heart failure.
Description
Technical field
The invention belongs to biotechnology and biological medicine technology field, related to a kind of oligonucleotide and be used to prepare prevention and the purposes of treatment pathologic myocardial hypertrophy and heart failure drugs.
Background technology
Chronic heart failure claims that again congestive heart failure is meant because myocardial contraction reduces; Having under the prerequisite of abundant venous return; Heart is discharged the absolute or minimizing relatively of blood volume; Can not satisfy a kind of pathological state of body tissue's organ metabolism needs, be the end stage eventually of the heart disease that causes of Different types of etiopathogenises.Along with the change of living standards of the people and mode of life, add the aging of intensification of competition and society, sickness rate in heart failure is the trend that increases year by year.Therefore, how to prevent and treat heart failure and more seem important and urgent.
Clinical data is in recent years summed up and is shown, pathologic myocardial hypertrophy, remodeling ventricle are characteristics in heart failure, also is simultaneously decision sickness rate in heart failure, the key factor of course of disease development and mortality ratio.Cause the typical disease of pathologic myocardial hypertrophy to comprise hypertension, myocardial infarction, myocardosis and valvular heart disease etc.Myocardial hypertrophy is accompanied by the change of several genes usually, and research shows that ANF and belta-MHC are the significant albumen of most typical myocardial hypertrophy; Simultaneously, be accompanied by the development of myocardial hypertrophy, the mesenchymal cell such as the fibroblast proliferation of cardiac muscle; Cardiac fibrosis; And then the reduction of the conformability of heart, penetrate the blood function and also receive tangible influence, and the incidence of irregular pulse, heart failure and sudden death is increased.Therefore, myocardial hypertrophy is the core procedure of the pathological change of chronic heart failure, uses drug intervention and reverses the effective means that myocardial hypertrophy is the control chronic heart failure.At present; Aspect myocardial hypertrophy and in heart failure the control, beta-blocker, angiotensin converting enzyme inhibitor (ACE I), angiotensin receptor antagonist (ARB), diuretic(s), aldosterone receptor antagonist and the purple foxglove class preparation of mainly containing commonly used.Yet; The prolonged application diuretic(s) can cause the reperfusion injury of drug resistance and kidney; Use deterioration and hyperpotassemia that ACE I, ARB and beta-blocker then can cause hypertension, renal function, therefore, press for and seek the treatment that new treat-ment is used for myocardial hypertrophy and heart failure.
Oligonucleotide is to contain one type of single stranded DNA sequence that contains the non-cytosine(Cyt) that methylates-guanine dinucletide motif, and it is mainly through (Toll-like receptor9, hormesis is brought into play in combination TLR9) with Toll appearance receptor 9.Research shows, the natural immune system of oligonucleotide ability direct activation body, and inducing generation is master's immunne response with the Th1 type, the indirect activation acquired immunity.Therefore, oligonucleotide is widely used in the treatment of tumour, virus infection and anaphylactic disease, and before clinical and in the clinical and experimental study, has obtained gratifying achievement.What is interesting is that cause PI3K signal path to activate behind many TLR receptor activations, the latter can effectively prevent the pathologic myocardial hypertrophy, but so far, does not see that oligonucleotide has the report that improves remodeling ventricle, myocardial hypertrophy effect.The present invention provides effective anti-myocardial hypertrophy of a kind of novelty and method in heart failure, will new selection be provided for the clinical treatment heart failure.
Summary of the invention
Technical problem to be solved by this invention is that the oligonucleotide and the functional analogue thereof that contain 5 '-AACGTT-3 ' are prevented and treated the application in myocardial hypertrophy, the heart failure drugs in preparation, reaches the purpose of heart diseases such as control heart failure.
The present invention at first provides a kind of oligonucleotide that contains sequence fragment 5 '-AACGTT-3 ' that can effectively suppress the incidence and development of myocardial hypertrophy and chronic heart failure.
The present invention has provided the oligonucleotide sequence and the functional analogue thereof of the generation and the development that contain above-described sequence fragment and can effectively suppress pathologic myocardial hypertrophy and chronic heart failure simultaneously.
Oligonucleotide sequence and functional analogue thereof that the present invention provides above-described oligonucleotide sequence fragment 5 '-AACGTT-3 ' simultaneously and contained this sequence fragment 5 '-AACGTT-3 (comprise myocardial hypertrophy, remodeling ventricle in the preparation treatment heart, cerebrovascular disease; Acute and chronic heart failure; And myocardial hypertrophy irregular pulse or the sudden death relevant) purposes in the pharmaceutical prepn with heart failure.Above-described oligonucleotide sequence and functional analogue thereof the purposes in preparation treatment hypertension, coronary heart disease, valvular heart disease, myocardiac pharmaceutical prepn also is provided simultaneously.
Described oligonucleotide sequence and functional analogue thereof can be used separately, or self combined utilization, or with the medication combined application of one or more other treatment cardiovascular disorder.The medicine of described treatment cardiovascular disorder can be beta-blocker, angiotensin converting enzyme inhibitor (ACE I), angiotensin receptor antagonist (ARB), diuretic(s), aldosterone receptor antagonist and purple foxglove class preparation.
Oligonucleotide sequence of the present invention and functional analogue thereof can be used separately or used with the pharmacology acceptable carrier as the effective constituent in the drug regimen when giving individuality.
Described oligonucleotide sequence and functional analogue thereof can be implemented through route of administration such as enteron aisle, non-enteron aisle (like subcutaneous injection, intravenous injection and intramuscular injection etc.) and external application and suctions when using individuality.
The application dose of described oligonucleotide sequence and functional analogue thereof is the effective dose of treatment.
Term among the present invention
Can be only if lay special stress on, the term among the present invention have by the ordinary meaning that the technician understood in the field under the present invention.If the conflict on the implication occurs, should defer to explanation among the present invention, define or explain.
" pathologic myocardial hypertrophy and chronic heart failure "
" pathologic myocardial hypertrophy " among the present invention and " chronic heart failure " are the pathologic myocardial hypertrophy and the chronic heart failure of modern medicine definition; Wherein the pathologic myocardial hypertrophy is that a kind of that the myocardial cell is produced under various pathological stimuli is the compensatory change of principal character with myocardial cell's increase; Chronic heart failure is meant because myocardial contraction reduces; Having under the prerequisite of abundant venous return; Heart is discharged the absolute or minimizing relatively of blood volume, can not satisfy a kind of pathological state of body tissue's organ metabolism needs.Research shows, comprise the appearance that all is accompanied by the pathologic myocardial hypertrophy in generation and the evolution of multiple cardiovascular disordeies such as coronary heart disease, valvular heart disease, hypertension and congenital heart disease, and the end stage eventually of multiple cardiovascular disorder all shows as heart failure.Therefore, the pathologic myocardial hypertrophy that relates to of this institute and treatment in heart failure include but not limited to the treatment of multiple cardiovascular disordeies such as coronary heart disease, valvular heart disease, hypertension and congenital heart disease.
" individuality "
Individuality is meant that the human individual and the non-human vertebrates of using oligonucleotide of the present invention are individual.Non-human vertebrates individuality comprises the non-human primate, farming animals and pet animals.
" disease-resistant rational myocardial hypertrophy and heart failure drugs "
Disease-resistant rational myocardial hypertrophy and heart failure drugs are meant that individuality is used to treat pathologic myocardial hypertrophy and medicine in heart failure; Include but not limited to beta-blocker; Angiotensin converting enzyme inhibitor (ACE I); Angiotensin receptor antagonist (ARB), diuretic(s), aldosterone receptor antagonist and purple foxglove class preparation.The oligonucleotide that contains 5 '-AACGTT-3 ' or its functional analogue among the present invention can be used separately, or several kinds of combined utilization, or the drug application acceptable carrier, or with one or more other the medication combined application of disease-resistant rational myocardial hypertrophy.The oligonucleotide of 5 '-AACGTT-3 ' provided by the invention or its functional analogue can be used with other disease-resistant rational myocardial hypertrophy medicine simultaneously, or use sequentially.
" oligonucleotide "
Oligonucleotide is by sugar (like ribodesose or ribose), and the mononucleotide of phosphate group and based composition is connected and the molecule that forms, and wherein glycan molecule and base connect into nucleosides (nucleoside); Nucleosides is connected to form Nucleotide (nucleotide) by phosphate group; The base that forms nucleosides has pyrimidine and purine, and pyrimidine has thymus pyrimidine (thymine is abbreviated as T or t) and cytosine(Cyt) (cytosine; Be abbreviated as C or c); Purine has VITAMIN B4, and (adenine is abbreviated as A or a) and guanine (guanine is abbreviated as G or g).Oligonucleotide can be strand also can be double-stranded.In the present invention, " oligonucleotide " (Oligodeoxynucleotide ODN) can use its english abbreviation ODN to replace.
" chemically modified "
Compare with the DNA of nature, the oligonucleotide among the present invention can be through various chemically modifieds, and the position of modification can occur in the phosphodiester bond between the nucleosides, and ribose units is or/and organic base (uridine is abbreviated as U or u for A, T, C, G, uridylic).Can modify between the synthesis phase of oligonucleotide or after synthetic.Chemically modified between synthesis phase can be modified at the inside or the 5` end of oligonucleotide.Oligonucleotide after synthetic can but be not limited to carry out chemically modified at reactive group (like the phosphoric acid or the hydroxyl of 5` or 3` end).The professional can understand the concrete mode of these chemically modifieds.Chemically modified among the present invention comprises the backbone modification of oligonucleotide.Wherein, the non-bridge property phosphoric acid Sauerstoffatom at least one internucleotide linkage is replaced by sulphur atom.Nonionic DNA analogue also can take place in the skeleton of oligonucleotide; Modify like alkyl, fragrance-phosphonate (charged phosphonate Sauerstoffatom is replaced by alkyl, aromatic base), the part of the Sauerstoffatom in phosphodiester and the alkyl phosphotriester is modified by alkylation for another example.Oligonucleotide can also be the mosaic of phosphorothioate and phosphodiester.Chemically modified comprises that also base substitutes, and substitutes like C-5 propine pyrimidine and the alternative purine of 7-deaza-7.Chemically modified also comprises base modification.The base of base in chemically being different from typical nature of modifying, but have their basic chemical structures.Oligonucleotide among the present invention can also be with cytosine derivative or thymidine derivative modified.Here cytosine derivative is meant cytosine(Cyt) appearance nucleosides (except cytosine(Cyt)).The thymidine verivate is meant the phonetic appearance of thymus gland nucleosides (not comprising thymus pyrimidine).In addition, the modification of the oligonucleotide among the present invention can be two or divalent alcohol of a terminal connection at oligonucleotide, like TEG or six terepthaloyl moietie.
" treatment effective dose "
In order to treat the pathologic myocardial hypertrophy, the dosage of giving individual applications is the treatment effective dose.The effective dose of this treatment is meant in the process of treatment pathologic myocardial hypertrophy, gives can produce ideal prevention or the oligonucleotide that contains 5 '-AACGTT-3 ' of result of treatment or the dosage of its functional analogue behind the individuality.The oligonucleotide that contains 5 '-AACGTT-3 ' among the present invention or its functional analogue can directly or be put on the pharmacology and use in the acceptable carrier.This " dosage " how much be decided by the standard technique that those skilled in the art should be known, also to comprise the severity that is not limited to individual body weight, age, healthy state and disease with reference to other factors.Oligonucleotide among the present invention can be as treatment or repeatedly treatment separately.The oligonucleotide that contains 5 '-AACGTT-3 ' among the present invention or its functional analogue be applied at every turn individual dosage range from 1 μ g to 100mg.In order to reach the ideal result of treatment, those skilled in the art can adjust the dosage of using, like the attending doctor on the basis of medical judgment reliably, the dose titration of making, its dosage range can be 10 times to 1000 times of aforementioned range.
" pharmaceutical composition "
Pharmaceutical composition refers to the mixture of acceptable carrier composition on the oligonucleotide that contains 5 '-AACGTT-3 ' and functional analogue and the pharmacology of treatment effective dose.Pharmaceutical composition can comprise oligonucleotide that contains 5 '-AACGTT-3 ' and the functional analogue thereof among one or more the present invention.Pharmaceutical composition can but be not limited to the aqueous solution, salts solution, particle, aerosol, tablet, pulvis, capsule, suppository, emulsion, drops and other suitable materials.Under various situation, pharmaceutical composition must be aseptic and stable.
" pharmacology acceptable carrier "
The pharmacology acceptable carrier is meant weighting agent, thinner or the encapsulating substance of one or more solids or liquid, and this carrier is fit to the oligonucleotide among the present invention is applied to individuality.This carrier can be organic, inorganic, natural or synthetic.This carrier comprises the carrier of the oligonucleotide among various solution, thinner, solvent, dispersion agent, liposome, emulsion, sugar-coat, antiseptic-germicide, anti-mycotic agent and other suitable the present invention.The selection of pharmacology acceptable carrier is decided by to contain among the present invention the oligonucleotide of 5 '-AACGTT-3 ' or the application mode of its functional analogue.The carrier that injectable is used comprises water, saline water, balanced salt solution, glucose solution, glycerine etc.
Advantage of the present invention and beneficial effect:
The invention provides the oligonucleotide or its functional analogue that contain 5 '-AACGTT-3 ', and use the purposes that this oligonucleotide or its functional analogue are used to prepare treatment myocardial hypertrophy medicine.Oligonucleotide or its functional analogue that contains 5 '-AACGTT-3 ' provided by the invention contains 5 '-AACGTT-3 ' sequence.Provided by the inventionly contain the oligonucleotide of 5 '-AACGTT-3 ' or its functional analogue all can suppress myocardial hypertrophy in cell and experimentation on animals generation and development.Simultaneously, the oligonucleotide or its functional analogue that contain 5 '-AACGTT-3 ' are immunomodulator, and human body is not seen obvious toxic and side effects.
Description of drawings:
Fig. 1 is the restraining effect of oligonucleotide (ODN) to the mRNA expression of the ANF in the loose process of neonatal cardiac myocytes and β-MHC; A is the restraining effect that each oligonucleotide is expressed ANF, and B is the restraining effect that each oligonucleotide is expressed β-MHC; Control group: solvent control group; Model group: the post-stimulatory myocardial hypertrophy model group of Racemic isoproterenol.
Fig. 2 is the restraining effect of oligonucleotide (ODN) to ANF protein expression in the myocardial hypertrophy process; A is that IIF detects the restraining effect of oligonucleotide (ODN) to ANF protein expression in the myocardial hypertrophy process, control group: solvent control group; Model group: the post-stimulatory myocardial hypertrophy model group of Racemic isoproterenol; The RJL01 group: oligonucleotide RJL01 adds the Racemic isoproterenol stimulating group; B is the statistical procedures to A figure;
Fig. 3 is the dose curve that oligonucleotide (ODN) suppresses the expression of ANF and β-MHC in the myocardial hypertrophy process; A is the restraining effect that different concns oligonucleotide RJL01 expresses ANF, and B is the restraining effect that different concns oligonucleotide RJL01 expresses β-MHC;
Fig. 4 is the time curve that oligonucleotide (ODN) suppresses the expression of ANF and β-MHC in the myocardial hypertrophy process; A is that different time points adds the restraining effect that oligonucleotide RJL01 expresses ANF, and B is that different time points adds the restraining effect that oligonucleotide RJL01 expresses β-MHC;
Fig. 5 is the restraining effect that oligonucleotide (ODN) increases the myocardial cell's surface-area in the myocardial hypertrophy process; A is that IIF detects the restraining effect that oligonucleotide (ODN) increases the myocardial cell's surface-area in the myocardial hypertrophy process, control group: solvent control group; Model group: the myocardial hypertrophy model group that Racemic isoproterenol stimulates; The RJL01 group: oligonucleotide RJL01 adds the Racemic isoproterenol stimulating group; B is the statistical procedures to A figure;
Fig. 6 is the restraining effect of oligonucleotide (ODN) to the loose model of mouse cardiac muscle; The A oligonucleotide is to the restraining effect of ventricular hypertrophy, and B is the restraining effect of oligonucleotide to cardiac weight; C is the restraining effect of oligonucleotide to heart body ratio; D is the provide protection of oligonucleotide to myocardium cell necrosis, control group: solvent control group; Model group: the loose model group of the mouse cardiac muscle that Racemic isoproterenol stimulates; RJL01 group: behind the injection oligonucleotide RJL01 in the model group of injection Racemic isoproterenol.
Embodiment
Oligonucleotide among the embodiment is synthetic by TAKARA company, and handles through no thermal source.Below be the sequence and the title of the oligonucleotide (ODN) used in an embodiment:
RJL-1(SEQ?ID?NO:1):5’-tcgtcgttaa?cgttaacgct?tag-3’(23bp)
RJL-2(SEQ?ID?NO:2):5’-tcttcgttaa?cgttaacgat?gacgta-3’(26bp)
RJL-3(SEQ?ID?NO:3):5’-taaacgaacg?ttttaacgtt?cgttta-3’(26bp)
RJL-4(SEQ?ID?NO:4):5’-tcgtcgttaa?cgttaagacg?taggg-3’(25bp)
RJL-5(SEQ?ID?NO:5):5’-tcgcgaacgt?tcgccgcgtt?cgaacgcggg-3’(30bp)
Oligonucleotide (ODN) is to the restraining effect of the mRNA expression of the ANF in the loose process of neonatal cardiac myocytes and β-MHC
The separation of 1 neonatal cardiac myocytes, myocardial hypertrophy model induce the processing with oligonucleotide
(1) plant and instrument, equipment, reagent and material
Cryogenic refrigerator, carbonic acid gas incubator, Bechtop, inverted microscope, Tissue Culture Flask, horizontal centrifuge, sample injector, the dropper of all size etc.ODN: synthetic by Takara company, the OPC rank, its phosphoric acid skeleton is full thio-modification except that special indicating.With PBS (prescription is with reference to " molecular cloning experiment guide (third edition) ") dissolving, ultraviolet spectrophotometer is quantitative.Racemic isoproterenol (Racemic isoproterenol), pancreatin, II Collagen Type VI enzyme, ITS and Laminin are all purchased the company in Sigma.HBSS, DMEM, two anti-(PS) and FBS purchase the company in Gibco.
(2) method
The sacrificed by decapitation neonate rat is opened its thoracic cavity, takes out heart, and the aorta that prunes away is put ventricle to the capsule that contains HBSS, and it is cut into four fritters; Be transferred to the ventricle that cuts in the culturing bottle that contains 1% pancreatin solution, put into refrigerator and preserve 10-12h for 4 ℃; The separation I liquid (DMEM+7%FBS+1%PS) that in culturing bottle, adds the 10ml neonatal cardiac myocytes, 37 ℃ of water-bath 3min; Liquid in the sucking-off bottle adds 10mlII Collagen Type VI enzyme solution (1mg/ml), 37 ℃ of water-bath jog 2min; Sucking-off liquid adds 10mlII Collagen Type VI enzyme solution again, and 6-10min is shaken in 37 ℃ of water-baths; Collect collagenase/cell suspension in the 50ml centrifuge tube, and place on ice; In remaining ventricular organization, continue to add II Collagen Type VI enzyme solution, repeating step is until can't see the ventricular muscles fritter; With the cell suspension that 100 order nylon net filters are collected, the centrifugal 5min of 500rpm; Separation I liquid suspension cell deposition with neonatal cardiac myocytes; The good cell suspension that suspends is transferred in the culturing bottle, places incubator to cultivate 75min; With the cell suspension sucking-off, be transferred in another culturing bottle, place CO2gas incubator to cultivate 24h; To separate the sucking-off of I liquid and be replaced by after neonatal cardiac myocytes separates II liquid (DMEM+1%ITS+1%PS) and continue to cultivate 12h, add ODNRJL01, RJL02; RJL03, RJL04, RJL05 is to final concentration 10mg/ml; Behind the effect 12h; Add Racemic isoproterenol to final concentration 10 μ mol/ml, after continuing to hatch 48h, carry out subsequent experimental; Control group wherein is the solvent control group, only adds the solvent contrast of PBS in the experimentation; Model group is the post-stimulatory myocardial hypertrophy model group of Racemic isoproterenol, does not carry out the stimulation of ODN, only adds Racemic isoproterenol effect 48h.
2, extraction, rt and the real-time quantitative PCR of total RNA
(1) plant and instrument, equipment, reagent and material
Cryogenic refrigerator, real-time quantitative PCR appearance (Eppendorf), ultraviolet spectrophotometer, refrigerated centrifuge, various droppers, centrifuge tube etc.
Trizol purchases in Takara, reversed transcriptive enzyme, and Oligo dT, dNTPs, SYBR-green etc. all purchase in Invitrogen.
(2) method:
The extraction of total RNA: the treated cell of PBS washing 3 times, add Trizol1ml, blow and beat 10 times after, collect in the centrifuge tube, leave standstill 5min; Add chloroform 200 μ l, vibration mixing 2min leaves standstill 15min, and 12000 leave heart 15min; Get in the new centrifuge tube of honest and upright and thrifty 500 μ l to, add Virahol 500 μ l, leave standstill 15min, 12000 leave heart 10min; Abandon supernatant, add 75% ethanol 1ml, 12000 leave heart 5min, abandon supernatant; Dry 10min under the room temperature does not have enzyme water dissolution deposition with 20 μ l, carries out subsequent experimental as template.
RT-PCR：
1) following ingredients is added the PCR pipe that places on ice:
Oligo?dT 1μl
Template ribonucleic acid 1 μ g
No enzyme water complements to 10 μ l
2) mixing is gently hatched 6min for 70 ℃, places 5min on ice;
3) following composition adding is somebody's turn to do in the pipe:
4) 42 ℃ of 60min, 70 ℃ of 10min termination reactions are cDNA, place 4 ℃ of preservations to carry out subsequent experimental.
Real-time quantitative PCR
Reaction system:
Response procedures:
1. 50 ℃ 2 minutes
2. 95 ℃ 2 minutes
3. 95 ℃ 15 seconds
4. 60 ℃ 1 minute
5. return step 3., repeat 3. and 4. 29 times
6. solubility curve
Primer sequence:
ANF:5' holds primer: gggggtagga ttgacaggat
3' holds primer: ctccaggagg gtattcacca
β-MHC:5' holds primer: cctcgcaata tcaagggaaa
3' holds primer: tacaggtgca tcagctccag
18s:5' holds primer: accgcagcta ggaataatgga
3' holds primer: gcctcagttc cgaaaacca
3, experimental result:
The result shows, in the loose model of neonatal cardiac myocytes, after Racemic isoproterenol is handled 48h, compares with control group, and the mRNA of ANF and β-MHC expresses significantly and raises, and wherein the up-regulated of ANF is about 7 times, about 3 times of the up-regulated of β-MHC.After each oligonucleotide was handled 12h, the expression of ANF and β-MHC significantly descended, compare with model group, both P 0.001, have significant difference (Fig. 1).
Conclusion: oligonucleotide can significantly suppress the mRNA up-regulated of ANF and β-MHC in the loose model of myocardial cell.
Oligonucleotide (ODN) is to the restraining effect of ANF protein expression in the myocardial hypertrophy process
1, inducing of the separation of neonatal cardiac myocytes and myocardial hypertrophy: method is noted cell inoculation in the petridish that is covered with the 20mm deckglass with embodiment 1.
2, indirect immunofluorescence detects the proteic expression of ANF
(1) plant and instrument, equipment, reagent and material
PI (sigma), ANF monoclonal antibody (mouse) is purchased the Cruz in Santa, FITC-fluorescence two anti-purchasing in Invitrogen, Laser Scanning Confocal Microscope (Leica, Germany).
(2) method:
Nutrient solution is removed in suction, adds an amount of PBS and cleans 3 times; Add 4% formaldehyde solution, room temperature is 20min fixedly; Add an amount of PBS and fully clean, add 0.5%Triton100 ,-20 ℃ of penetrating 10min; 2%BSA seals 1h; Add ANF one anti-(1:200 dilution) 400 μ l, incubated at room 1h, PBS cleans 3 times; Add fluorescence two anti-(1:200 dilution) 400 μ l, incubated at room 1h, PBS cleans 3 times; PI redyes 2min, and PBS cleans 2 times.80% glycerine mounting uses the confocal fluorescent microscope to take pictures, analyze.
3, experimental result:
The result shows, in the loose model of neonatal cardiac myocytes, compares with control group, and myocardial cell's ANF expresses significantly and raises, and the cell of ANF positive expression can reach more than 40% of whole cells.After oligonucleotide RJL01 handled 12h, the expression of ANF significantly descended, and the cell of ANF positive expression only reaches 10% of whole cells, compare with model group, both P 0.001, have significant difference (Fig. 2).
Conclusion: oligonucleotide RJL01 is the up-regulated of the ANF in the loose model of myocardial cell significantly.
The dose curve that ANF in oligonucleotide (ODN) the inhibition myocardial hypertrophy model and β-MHC express
1, inducing of the separation of neonatal cardiac myocytes and myocardial hypertrophy: method is with embodiment 1.
In the experiment of dose curve; RJL01 with final concentration 20,10,5,2,1,0.5 μ g/ml stimulates neonatal cardiac myocytes respectively; Control group and model group add PBS, behind the effect 12h, add Racemic isoproterenol to final concentration 10 μ mol/ml; After continuing to hatch 48h, carry out subsequent experimental.
2, extraction, rt and the real-time quantitative PCR of total RNA: method is with embodiment 1.
3, experimental result
The result shows; Concentration is that the ODN RJL01 of 1 μ g/ml promptly can effectively suppress ANF and the up-regulated of β-MHC in the myocardial hypertrophy process, and along with the increase of ODN concentration, and the restraining effect of these two kinds of genes is strengthened (* gradually; Compare with model group, P < 0.05; * compares with model group, P 0.001, Fig. 3).
Conclusion: RJL01 is dose-dependence for the restraining effect of the up-regulated of ANF and β-MHC in the myocardial hypertrophy process.
The time curve that ANF in oligonucleotide (ODN) the inhibition myocardial hypertrophy model and β-MHC express
4, inducing of the separation of neonatal cardiac myocytes and myocardial hypertrophy: method is with embodiment 1.
In the experiment of time curve; Respectively before adding Racemic isoproterenol 12,3 with 1h and add 1,3 after the Racemic isoproterenol and 12h to add final concentration be that the RJL01 of 5 μ g/ml stimulates; Behind the Racemic isoproterenol effect 48h, carry out subsequent experimental again.
5, extraction, rt and the real-time quantitative PCR of total RNA: method is with embodiment 1.
6, experimental result
The result shows, add Racemic isoproterenol before 12,31 with 1h and after adding Racemic isoproterenol, 3h adds ODN RJL01 and all can effectively suppress the up-regulated of ANF and β-MHC in the myocardial hypertrophy process (* compares with model group, and P < 0.05; * compares with model group, P 0.001, Fig. 4).
This up-regulated to ANF and β-MHC in the myocardial hypertrophy mechanism of conclusion: RJL01 has prevention and therapeutic effect.
The restraining effect that oligonucleotide (ODN) increases the myocardial cell's surface-area in the myocardial hypertrophy process
1, inducing of the separation of neonatal cardiac myocytes and myocardial hypertrophy: method is with embodiment 1.
2, IIF detects the expression of α-actin
PI (sigma), ANF monoclonal antibody (mouse) is purchased the Cruz in Santa, FITC-fluorescence two anti-purchasing in Invitrogen.
(2) method:
Nutrient solution is removed in suction, adds an amount of PBS and cleans 3 times; Add 4% formaldehyde solution, room temperature is 20min fixedly; Add an amount of PBS and fully clean, add 0.5%Triton100 ,-20 ℃ of penetrating 10min; 2%BSA seals 1h; Add ANF one anti-(1:200 dilution) 400 μ l, incubated at room 1h, PBS cleans 3 times; Add fluorescence two anti-(1:200 dilution) 400 μ l, incubated at room 1h, PBS cleans 3 times; PI redyes 2min, and PBS cleans 2 times.80% glycerine mounting uses the confocal fluorescent microscope to take pictures, and Application of I mage J software pair cell surface-area is measured.
3, experimental result
The loose model group of myocardial cell is compared with control group, and myocardial cell's surface-area has increased 3 times.After ODN RJL01 handled 12h, tangible increase did not appear in myocardial cell's surface-area again, compare with model group, both P 0.001, have significant difference (Fig. 5).
Conclusion: RJL01 can significantly suppress the increase of myocardial cell's surface-area in the myocardial hypertrophy process.
Oligonucleotide (ODN) is to the restraining effect of the loose model of mouse cardiac muscle
1, the foundation of the loose model of mouse cardiac muscle and the injection of ODN
With 6～8 ages in week, 18～22g C57BL/6 mouse is divided into 4 groups, and 8 every group, 3 back subcutaneous injections every afternoon concentration is the Racemic isoproterenol 200 μ l of 5mg/ml, injects continuously 6 days, can form the loose model of mouse cardiac muscle.Begin abdominal injection previous day in the injection Racemic isoproterenol of ODN, and the time is points in mornings 8, injects continuously 6 days, and concentration is 250 μ g/ml.Control group injection PBS.Concrete grouping is following:
Experiment is divided into groups:
Control group, back subcutaneous injection 200 μ l PB, continuous 6 days;
Model group, subcutaneous injection 200 μ l concentration in back are the Racemic isoproterenol of 5mg/ml, continuous 6 days;
The ODN group, abdominal injection 200 μ l concentration are the RJL01 of 250 μ g/ml, continuous 6 days; Be the Racemic isoproterenol 6 days of 5mg/ml again since back subcutaneous injection in second day 200 μ l concentration.
2, experimental result
Behind the 24h of injection, phenylethyl barbituric acid injecting anesthetic mouse is weighed the last time; And then open the thoracic cavity, take pictures, weigh after obtaining heart.Heart carries out Paraformaldehyde 96 to be fixed, section, HE dyeing.
The result shows, compares with control group, and the cardiac weight of model group mouse obviously increases, and the ratio of cardiac weight and mouse body weight (heart body ratio) increases, and the big area tissue necrosis occurs in the HE dyeing demonstration heart.After injection ODN RJL01, to compare with model group, the cardiac weight of ODN group descends, and heart body is than descending, and the necrosis area of tissue obviously reduces, both P 0.05, have significant difference (Fig. 6).
Conclusion: injection RJL01 has the loose model of the myocardial cell of mouse and significantly alleviates effect.
Claims (10)
1. oligonucleotide that contains sequence fragment 5 '-AACGTT-3 ' that can effectively suppress myocardial hypertrophy and chronic heart failure incidence and development.
2. functional analogue that contains the described sequence of claim 1 and can effectively suppress the generation and the development of myocardial hypertrophy and chronic heart failure.
3. oligonucleotide sequence according to claim 2 and functional analogue thereof is characterized in that two ends of sequence 5 '-AACGTT-3 ' can add the nucleotide sequence that other are made up of 4 kinds of Nucleotide A, T, C and G arbitrarily.
4. oligonucleotide sequence according to claim 2 and functional analogue thereof is characterized in that sequence 5 '-AACGTT-3 ' can be through chemically modified, and the position of chemically modified can be phosphodiester bond and/or ribose and/or the base between nucleosides; Arbitrary oligonucleotide is defined as the functional analogue of corresponding corresponding oligonucleotide in the oligonucleotide sequence of chemically modified.
5. the described oligonucleotide that contains sequence fragment 5 '-AACGTT-3 ' of claim 1 prevents and treats the purposes in myocardial hypertrophy, the remodeling ventricle pharmaceutical prepn in preparation.
6. the described oligonucleotide that contains sequence fragment 5 '-AACGTT-3 ' of claim 1 is in the preparation prevention and treat the purposes in the pharmaceutical prepn of acute and chronic heart failure.
7. the purposes of the described oligonucleotide that contains sequence fragment 5 '-AACGTT-3 ' of claim 1 in the pharmaceutical prepn of preparation prevention and treatment myocardial hypertrophy irregular pulse relevant or sudden death with heart failure.
8. the purposes in myocardial hypertrophy, the remodeling ventricle pharmaceutical prepn is prevented and treated to each described oligonucleotide sequence and functional analogue thereof in preparation in the claim 2 to 4.
In the claim 2 to 4 each described oligonucleotide sequence and functional analogue thereof in preparation prevention and treat the purposes in the pharmaceutical prepn of acute and chronic heart failure.
10. each described oligonucleotide sequence and functional analogue thereof the purposes in the pharmaceutical prepn of preparation prevention and treatment myocardial hypertrophy irregular pulse relevant or sudden death in the claim 2 to 4 with heart failure.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012102150127A CN102719436A (en) | 2012-06-27 | 2012-06-27 | Oligonucleotides and usage thereof in preparation of medicine for preventing and curing myocardial hypertrophy and heart failure |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012102150127A CN102719436A (en) | 2012-06-27 | 2012-06-27 | Oligonucleotides and usage thereof in preparation of medicine for preventing and curing myocardial hypertrophy and heart failure |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102719436A true CN102719436A (en) | 2012-10-10 |
Family
ID=46945305
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012102150127A Pending CN102719436A (en) | 2012-06-27 | 2012-06-27 | Oligonucleotides and usage thereof in preparation of medicine for preventing and curing myocardial hypertrophy and heart failure |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102719436A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018192505A1 (en) * | 2017-04-18 | 2018-10-25 | Changchun Huapu Biotechnology Co., Ltd. | Immunomodulatory polynucleotides and uses thereof |
| CN108913655A (en) * | 2018-07-16 | 2018-11-30 | 浙江大学 | The method for establishing " humanized " myocardial hypertrophy model based on multipotential stem cell technology |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1745089A (en) * | 2002-12-23 | 2006-03-08 | 戴纳伐克斯技术股份有限公司 | Immunostimulatory sequence oligonucleotides and methods of using the same |
| CN101380477A (en) * | 2008-09-28 | 2009-03-11 | 中国医学科学院医药生物技术研究所 | Use of CR-1 siRNA in treating myocardial hypertrophy induced by AngII |
| CN101522222A (en) * | 2006-08-01 | 2009-09-02 | 得克萨斯系统大学董事会 | Identification of a micro-RNA that activates expression of beta-myosin heavy chain |
-
2012
- 2012-06-27 CN CN2012102150127A patent/CN102719436A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1745089A (en) * | 2002-12-23 | 2006-03-08 | 戴纳伐克斯技术股份有限公司 | Immunostimulatory sequence oligonucleotides and methods of using the same |
| CN101522222A (en) * | 2006-08-01 | 2009-09-02 | 得克萨斯系统大学董事会 | Identification of a micro-RNA that activates expression of beta-myosin heavy chain |
| CN101380477A (en) * | 2008-09-28 | 2009-03-11 | 中国医学科学院医药生物技术研究所 | Use of CR-1 siRNA in treating myocardial hypertrophy induced by AngII |
Non-Patent Citations (3)
| Title |
|---|
| 杨光等: "CT-1反义脱氧寡核苷酸、AG490、川芎嗪抑制AngII诱导的心肌细胞肥大", 《哈尔滨医科大学学报》 * |
| 陆莹等: "HDAC2-反义脱氧寡核苷酸抑制AngII诱导的心肌细胞肥大", 《心血管康复医学杂志》 * |
| 陆莹等: "反义寡核苷酸和丙戊酸对心肌细胞肥大组蛋白脱乙酰基酶2的影响", 《中国老年学杂志》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018192505A1 (en) * | 2017-04-18 | 2018-10-25 | Changchun Huapu Biotechnology Co., Ltd. | Immunomodulatory polynucleotides and uses thereof |
| CN110753756A (en) * | 2017-04-18 | 2020-02-04 | 长春华普生物技术股份有限公司 | Immunomodulatory polynucleotides and uses thereof |
| US11326170B2 (en) | 2017-04-18 | 2022-05-10 | Changchun Huapu Biotechnology Co. Ltd. | Immunomodulatory polynucleotides and uses thereof |
| CN110753756B (en) * | 2017-04-18 | 2023-09-01 | 华普生物技术(河北)股份有限公司 | Immunomodulatory polynucleotides and uses thereof |
| CN108913655A (en) * | 2018-07-16 | 2018-11-30 | 浙江大学 | The method for establishing " humanized " myocardial hypertrophy model based on multipotential stem cell technology |
| CN108913655B (en) * | 2018-07-16 | 2022-07-15 | 浙江大学 | Method for establishing human-derived myocardial hypertrophy model based on pluripotent stem cell technology |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103080314B (en) | Dominant mutant genes expression inhibitor | |
| CN102921003B (en) | Stabilized immune modulatory RNA (SIMRA) compounds | |
| CN109414448A (en) | For reducing the nucleic acid molecules of PAPD5 or PAPD7 mRNA treatment hepatitis B infection | |
| CN101072866A (en) | Method of altering cell properties by administering RNA | |
| CN105907756A (en) | Extended Dicer Substrate Agents And Methods For The Specific Inhibition Of Gene Expression | |
| KR20100057022A (en) | Micro-rnas that control myosin expression and myofiber identity | |
| CN104540527A (en) | Inhibitors of the MIR-15 family of micro-RNAs | |
| CN102335189B (en) | Medicament composition containing antisense polynucleotide targeting miR-214 | |
| CN108064175A (en) | Inhibitor of MYH7B and application thereof | |
| Fan et al. | Cardiomyocytes from CCND2-overexpressing human induced-pluripotent stem cells repopulate the myocardial scar in mice: A 6-month study | |
| CN102719436A (en) | Oligonucleotides and usage thereof in preparation of medicine for preventing and curing myocardial hypertrophy and heart failure | |
| CN107459546B (en) | A kind of tRF-Gly antisenses chain inhibitor and its application | |
| CN101392251A (en) | Micro RNA capable of inducing stem cell to differentiate toward osteoblasts and use thereof | |
| CN106390123B (en) | MiR-29 and its inhibitor are preparing the application in anti-organ-graft refection's drug | |
| CN107385033A (en) | The application of piRNA 5938 and its antisensenucleic acids in diagnosis, prevention, treatment, prognosis evaluation ischemic heart disease | |
| CN104995300B (en) | The adjusting of RNA activity and vasopermeability | |
| CN104561000B (en) | Inhibit nucleic acid oligomer and its application of CD44 genes | |
| CN101390872B (en) | Use of ginsenoside Rb1 in preparing medicine for treating dilated cardiomyopathy | |
| CN104622874B (en) | Application of the CCR4 antagonists in cancer growth and transfer is suppressed | |
| CN104334195A (en) | Composition comprising material for inhibiting scf or receptor thereof for treating or preventing diseases associated with vascular permeability | |
| CN103361428B (en) | The application in preparing chronic pain diagnosis marker and medicine of the miRNA-132 compound | |
| CN104436195B (en) | Purposes of the miR-155 in preparation prevention and treatment acute lung injury drug | |
| CN107375261A (en) | Application of the pinostrobin in osteoporosis is prevented and treated | |
| Rannou et al. | Human MuStem cell grafting into infarcted rat heart attenuates adverse tissue remodeling and preserves cardiac function | |
| CN102973953B (en) | MiRNA (micro ribose nucleic acid)-874 and application of miRNA-874antisense nucleotide |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20121010 |