CN104491879A - MiRNA-2861 antisense nucleotide pharmaceutical composition and application thereof - Google Patents
MiRNA-2861 antisense nucleotide pharmaceutical composition and application thereof Download PDFInfo
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- CN104491879A CN104491879A CN201410803184.5A CN201410803184A CN104491879A CN 104491879 A CN104491879 A CN 104491879A CN 201410803184 A CN201410803184 A CN 201410803184A CN 104491879 A CN104491879 A CN 104491879A
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Abstract
The invention relates to a miRNA-2861 antisense nucleotide pharmaceutical composition and application thereof. The sequence of miRNA-2861 antisense nucleotide is 5'-CCGCCCGCCGCCAGGCCCC-3'. The miRNA-2861 antisense nucleotide pharmaceutical composition comprises over-expressed miRNA-2861 antisense nucleotide vectors, viruses or accessories, wherein the infection titers of the over-expressed miRNA-2861 antisense nucleotide vectors are 1016PFU; the vectors are cholesterol, nano-particles or lipidosome; virus vectors are one or more of adenovirus vectors, lentiviral vectors and retroviral vectors; the accessories are one or more of mannitol, a phosphate buffer solution and normal saline. The miRNA-2861 antisense nucleotide pharmaceutical composition is used for preventing and treating myocardial hypertrophy, myocardial fibrosis, coronary heart diseases and heart failure by virtue of an oral taking or injection manner. The miRNA-2861 antisense nucleotide pharmaceutical composition has the beneficial effects that the formula is reasonable, the pharmacological effect is reliable, a preparation process is simple, the treatment effect is obvious, the application range is wide, and the use is environmentally friendly.
Description
Technical field:
The present invention relates to new drug of a kind of endogenous non-coding tiny RNA s and uses thereof, especially a kind of medical composition and its use of miRNA-2861 antisense nucleotide treatment heart disease, belongs to biological pharmacy technical field.
Background technology:
Cardiovascular disease mainly comprises hypertension, coronary heart disease and congestive heart failure etc., cardiovascular disease is the number one killer of human health, have 1,007 million peoples to die from cardiovascular disease in worldwide every year, its mortality rate is close to the summation of all cancer mortalities.Along with living standards of the people improve the change with dietary structure day by day, cardiovascular disease mortality rate is obvious ascendant trend, and having exceeded cancer becomes the first cause of death, and prevention and therapy cardiovascular disease remains the significant task of Med Biol.Myocardial hypertrophy refers to that the general name thickening the increase of setting one's heart myocyte's volume with cellular level organizing level to set one's heart muscular tissue causes heart size to increase a kind of disease occurred because myocardial cell volume increases.This disease is occurred by the Co stituation of some physiology and pathological factor, is the comprehensive performance of many cardiovascular disease; Myocardial hypertrophy is myocardiac one, and be a kind of responsing reaction that myocardial cell is directed to hematodinamics increase, multiple situation can cause the increase of blood in human body in hydraulic power, as hypertension, and valvular heart disease.Long-term over loading hemodynamics stimulation can cause the myocardial cell remodeling process being increased to feature with myocardial cell volume, is exactly myocardial hypertrophy.It is generally acknowledged now the myocardial hypertrophy of existence two kinds of situations, one is normal physiological hypertrophy, and such as after birth, supporter grows the myocardial hypertrophy of generation, also has the myocardial hypertrophy that physical training causes; Another is pathologic myocardial hypertrophy, its early sign is that locular wall and interventricular septum thicken, myocardium shrinkage function strengthens, therefore compensatory hypertrophy is regarded as, if the state of an illness is not eased always, can be there is ventricle wall interstitial fibrosis, mycardial contractility functional disorder and energy metabolism in the myocardial hypertrophy later stage, the exception of gene expression and Electrophysiological characteristics, finally causes cardiac failure.In view of myocardial hypertrophy is a Progressive symmetric erythrokeratodermia irreversible process, and finally can cause heart failure, having there is it a landmark cardiac morphology change of clinical pathological changes in modern medicine, thinks that it is the risk factor causing heart disease and cardiac sudden death as heart.
Along with the upsurge of recent miRNA research application, endogenous non-coding tiny RNA s appears as the center factor of a Gene expression and regulation, participate in many important physiological process, for miRNA, the feature of control methods determines that its target gene can not only have one, be the mode of one-to-many, the function of miRNA studied carefully abundant in content; MiRNA has important function for maintenance heart normal physiological function, and the miRNA unconventionality expression in heart is relevant to the generation of many heart diseases.Therefore, using the target spot of miRNA as cardiac disease treatment, exploitation related drugs has potential clinical value.; Although increasing miRNA is found as the biomarker of human diseases and therapy target, the crucial miRNA in cardiac hypertrophy and myocardial fibrosis heart disease does not still determine, its function played is the challenge to this field scientific research personnel.
Summary of the invention:
The object of the invention is to the deficiency overcoming prior art existence, seek to provide a kind of miRNA-2861 antisense nucleotide for the preparation of diagnosis, prevention or the medical composition and its use for the treatment of heart disease, determine or find the miRNA expressed in heart of cardiac myocyte hypertrophy, determine its effect in heart disease further, and be applied to the Diagnosis and treatment of these heart diseases.
In order to realize foregoing invention object, the antisense base sequences of the miRNA-2861 of the present invention's design is 5 '-CCGCCCGCCGCCAGGCCCC-3 '; Adopt common process to be made into pharmaceutical composition, comprising infection titer is 10
16pFU process LAN miRNA-2861 antisense nucleoside acid vectors, virus or adjuvant, described carrier is cholesterol, nano-particle or liposome; Viral vector is one or more in adenovirus vector, slow virus carrier, retroviral vector; Described adjuvant is one or more in mannitol, phosphate buffer, normal saline;
Pharmaceutical dosage form is one or more in oral formulations, ejection preparation, tablet, therapy in dry powder form, the preferably little aqueous injection of injection or injection freeze-dried powder.
MiRNA-2861 antisense nucleotide pharmaceutical composition of the present invention, is used for prevention and therapy myocardial hypertrophy, myocardial fibrosis, coronary heart disease and heart failure by oral or injection.
The pharmaceutical composition that the present invention relates to is expressed and is significantly raised in the myocardial cell and cardiac hypertrophy of hypertrophy, the expression of miRNA-2861 is strengthened by building miRNA-2861 process LAN adenovirus, find the generation of miRNA-2861 process LAN at cellular level energy inducing cardiomyocytes hypertrophy, the expression of loose gene has obvious increase compared with matched group; Be significantly improved compared with matched group in the myocardial cell of hypertrophy, the detection that it carries out loose index is found, index such as the cell surface of myocardial hypertrophy amasss, the ratio of albumen/DNA and the restructuring of sarcomere also have obvious increase compared with matched group, the generation of myocardial hypertrophy can be induced, as the biomarker of the heart diseases such as early diagnosis and early prevention myocardial hypertrophy, myocardial fibrosis; To myocardial hypertrophy and myocardial fibrosis, there is protective effect, find in the antisense nucleotide of cellular level by transfection miRNA-2861, the cardiac myocyte hypertrophy that loose stimulating factor is induced can be suppressed, comprise loose index such as cell surface to amass, the ratio of albumen/DNA and the restructuring of sarcomere also with compared with stimulating group have obvious reduction; Also the loose effect of loose model can be suppressed animal to comprise cardiac phenotype, heart weight ratio, the cardiomyocytes cross-sectional area (WGA dyeing) of marginal zone and myocardial fibrosis (Masson dyeing) horizontally through the antisense nucleotide of injection external source miRNA-2861 and have obvious reduction, while cardiac function also improve significantly.
Compared with prior art, its drug regimen composition formula is reasonable in the present invention, and pharmacology is reliable, and processing technology is simple, and therapeutic effect is obvious, applied range, and environment for use is friendly.
Accompanying drawing illustrates:
Fig. 1 is the change of miRNA-2861 expression in the cardiac myocyte hypertrophy process of inducing through cardiac hypertrophy stimulating factor PE (phyenlephrinium) and Ang II (angiotensinⅡ);
Fig. 2 carries out PE process to the primary cell through the transfection of miRNA-2861 antisense nucleotide, on a cellular level to the suppression situation of the hypertrophy that PE induces;
Fig. 3 is the result of the test schematic diagram of the sarcomere restructuring to myocardial cell after carrying out PE process through the primary cell of miRNA-2861 antisense nucleotide transfection;
The testing result schematic diagram of the loose model that Fig. 4 can suppress PE to induce for mouse mainline miRNA-2861 antisense nucleotide (antagomir);
The testing result schematic diagram of the loose model of mouse heart that Fig. 5 can suppress coarctation of aorta (TAC) to be induced for mouse mainline miRNA-2861 antisense nucleotide (antagomir).
Detailed description of the invention:
Also by reference to the accompanying drawings further elaboration is made to the present invention below by embodiment.
Embodiment 1,
MiRNA-2861 antisense nucleotide process LAN adenovirus vector and negative control adenovirus build, the present embodiment adopts synthetic miRNA-2861 antisense base sequences, sub-clone is connected into the pSilencer Adeno 1.0-CMV system of Ambion company, builds miRNA-2861 antisense nucleotide process LAN adenovirus.Its miRNA-2861 antisense nucleotide nucleotide sequence is as shown in following SEQ ID NO:1: 5 '-CCGCCCGCCGCCAGGCCCC-3 '
According to company's description step, by this carrier linearizing for Pac I and linearizing adenoviral backbone (AdenovirTs LacZ Backbone) cotransfection HEK-293 cell, encapsidated adenovirus virus, virus is collected in amplification, measures virus titer; By empty carrier and adenoviral backbone according to above-mentioned steps cotransfection HEK-293 cell, packaging negative control adenovirus.
The recombinant adenovirus phosphate buffer of the process LAN miRNA-2861 antisense nucleotide of above-mentioned gained is diluted for infection titer 1x10
16pFU/ml, aseptic subpackaged in ampoule bottle by 1ml amount, to obtain final product.
Embodiment 2,
The change of miRNA-2861 expression under PE and Ang II stimulates, the present embodiment is for cardiac myocyte hypertrophy model, (rat neonatal rat is purchased from Beijing Medical University to adopt the method set up to cultivate rat neonatal rat primary cardiomyocytes, rat strains is Wistar, the preparation of rat neonatal rat primary cardiomyocytes can see with Publication about Document: W.-Q.Tan, etal, Foxo3a Inhibits Cardiomyocyte Hypertrophy through Transactivating Catalase J Biol Chem.2008October 31; 283 (44): 29730-29739), myocardial cell PE and Ang II is processed, the different time cultivated, extract the total serum IgE of cell, Real-Time Fluorescent Quantitative PCR Technique detects the expression of miRNA-2861, as shown in Figure 1A, 1B, wherein, Figure 1A is rat neonatal rat primary cardiomyocytes miRNA-2861 expression in time after PE process; Figure 1B is rat neonatal rat primary cardiomyocytes miRNA-2861 expression in time after Ang II processes, vertical coordinate represents with the expression of miRNA-2861 in untreated Neonatal Rat Primary Cardiomyocytes for benchmark, the expression of miRNA-2861 in Neonatal Rat Primary Cardiomyocytes in PE or Ang II processing procedure.MiRNA-2861 expression processes within 6 hours at PE and Ang II significant rising.
Embodiment 3,
The hypertrophy that miRNA-2861 antisense nucleotide can suppress PE to induce on a cellular level, the present embodiment is to after primary cardiomyocytes transfection miRNA-2861 antisense nucleotide (anta-2861), effectively can suppress the expression of miRNA-2861, cell PE processes myocardial cell simultaneously, and use the negative control (anta-NC) of miRNA-2861 antisense nucleotide to process simultaneously, after 24 hours, myocardial cell is carried out to the detection of myocardial hypertrophy index, as myocardial cell surface area (Fig. 2 A), albumen/DNA (Fig. 2 B) detects, the cardiac myocyte hypertrophy that the results show miRNA-2861 antisense nucleotide can effectively suppress PE to induce.
Embodiment 4,
The restructuring of the myocardial cell sarcomere that miRNA-2861 antisense nucleotide can suppress PE to induce on a cellular level, the present embodiment is to after primary cardiomyocytes transfection miRNA-2861 antisense nucleotide (anta-2861) and negative control (anta-NC), with PE, myocardial cell is processed, after process 24 hours, restructuring myocardial cell being carried out to sarcomere detects, as shown in Figure 3, the restructuring of the sarcomere of the results show miRNA-2861 antisense nucleotide myocardial cell that can effectively suppress PE to induce.
Embodiment 5,
The loose model that mouse mainline miRNA-2861 antisense nucleotide (antagomir) can suppress PE to induce, the present embodiment buries pump process after two weeks to mice PE, the heart of mice has obvious loose phenotype, and adds miRNA-2861 antisense nucleotide (anta-2861) simultaneously and obviously can weaken cross-sectional area size (WGA dyeing) (as Fig. 4 A) and the heart weight ratio (as Fig. 4 B) that loose effect that PE induces comprises myocardial cell; Simultaneously to negative control (anta-NC) and the PE of injected in mice miRNA-2861 antisense nucleotide, can not weaken the loose effect of PE, experimental result confirms the generation of the cardiac hypertrophy that miRNA-2861 antisense nucleotide can suppress PE to induce in integral level.
Embodiment 6,
The loose model of the mouse heart that mouse mainline miRNA-2861 antisense nucleotide (antagomir) can suppress coarctation of aorta (TAC) to be induced, the present embodiment can induce the generation of loose phenotype after three weeks with the method process of coarctation of aorta (Thoracic aorta constriction, TAC) to mice; Add miRNA-2861 antisense nucleotide (anta-2861) simultaneously and obviously can weaken the loose effect that TAC induces; The group adding the negative contrast (anta-NC) of miRNA-2861 antisense nucleotide and TAC group than significantly change namely can not weaken the loose effect (Fig. 5) that TAC induces, comprise the cross-sectional area size (WGA (Wheat germ agglutinin) dyeing) (Fig. 5 B) of heart weight ratio (Fig. 5 A) and myocardial cell; Experimental result confirms the generation of the cardiac hypertrophy that miRNA-2861 antisense nucleotide can suppress TAC to induce in integral level.
。
Sequence table
SEQ ID NO:1
5’- CCGCCCGCCGCCAGGCCCC-3’
Claims (3)
1. a miRNA-2861 antisense nucleotide pharmaceutical composition, is characterized in that the antisense base sequences of miRNA-2861 is 5 '-CCGCCCGCCGCCAGGCCCC-3 '; It is 10 that pharmaceutical composition comprises infection titer
16pFU process LAN miRNA-2861 antisense nucleoside acid vectors, virus or adjuvant, described carrier is cholesterol, nano-particle or liposome; Viral vector is one or more in adenovirus vector, slow virus carrier, retroviral vector; Described adjuvant is one or more in mannitol, phosphate buffer, normal saline.
2. a kind of miRNA-2861 antisense nucleotide pharmaceutical composition according to claim 1, is characterized in that pharmaceutical dosage form is one or more in oral formulations, ejection preparation, tablet, therapy in dry powder form.
3. a purposes for miRNA-2861 antisense nucleotide pharmaceutical composition, it is characterized in that by oral or injection for prevention and therapy myocardial hypertrophy, myocardial fibrosis, coronary heart disease and heart failure.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103476947A (en) * | 2011-03-02 | 2013-12-25 | 格路福生物制药公司 | Enhanced biodistribution of oligomers |
| US20140087964A1 (en) * | 2012-09-24 | 2014-03-27 | University Of Virginia Patent Foundation | Compositions and methods for detecting aberrant regulation, expression, and levels of hgh |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103476947A (en) * | 2011-03-02 | 2013-12-25 | 格路福生物制药公司 | Enhanced biodistribution of oligomers |
| US20140087964A1 (en) * | 2012-09-24 | 2014-03-27 | University Of Virginia Patent Foundation | Compositions and methods for detecting aberrant regulation, expression, and levels of hgh |
Non-Patent Citations (2)
| Title |
|---|
| HUI LI等: "A novel microRNA targeting HDAC5 regulates osteoblast differentiation in mice and contributes to primary osteoporosis in humans", 《THE JOURNAL OF CLINICAL INVESTIGATION》 * |
| 刘唯: "miR-X/miR-2861表达簇与转录因子Runx2调控环路在成骨细胞分化中的功能研究", 《中国博士学位论文全文数据库 基础科学辑》 * |
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