CN104922657A - Medicine for treating myocardial infarction - Google Patents
Medicine for treating myocardial infarction Download PDFInfo
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- CN104922657A CN104922657A CN201510197193.9A CN201510197193A CN104922657A CN 104922657 A CN104922657 A CN 104922657A CN 201510197193 A CN201510197193 A CN 201510197193A CN 104922657 A CN104922657 A CN 104922657A
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- medicine
- myod
- myocardial infarction
- transcription factor
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Abstract
The invention discloses a medicine for treating myocardial infarction. The medicine contains transcription factor MyoD as an effective component and the MyoD accounts for 70-85 percent of effective components. According to experimental research results, the medicine for treating myocardial infarction can effectively realize intervention treatment of myocardial infarction.
Description
Technical field
The present invention relates to a kind of medicine for the treatment of myocardial infarction, be specifically related to the medicine of the treatment myocardial infarction containing transcription factor MyoD being effective ingredient.
Background technology
MiRNA is that a class is about 21-25nt, the strand non-coding RNA of high conservative in evolution, and major part is produced by the intron region of gene, mainly through suppressing mRNA translation or promoting its degraded thus regulate gene expression.Single miRNA directly can regulate the expression of up to a hundred genes, and then indirectly plays regulating and controlling effect to thousands of gene expressions, plays an important role in a series of physiological process such as cell proliferation, apoptosis, differentiation, metabolism.
Transcription factor (transcription factor, TF) be the combined protein matter that a class comprises multiple isolated area, it take part in activation or the suppression of transcription, and transcription factor and miRNA play important regulating and controlling effect at the gene transcription level of higher eucaryotic cells biology and post-transcriptional level.Present the harmony of height between transcription factor and miRNA, both are closely connected in the regulation and control of transcriptional level and post-transcriptional level.At transcriptional level, transcription factor participates in the expression of regulation and control several genes as a kind of important regulatory factor.Recent researches shows that transcription factor can the expression of direct regulation and control miRNA, and take part in various biological processes such as comprising cardiovascular disease generation.The regulated and control network that transcription factor and miRNA are formed is extensively studied, and previously research shows, transcription factor directly can be combined in the promoter region of miRNA and regulate and control the expression of miRNA.The network that this discovery makes TF and miRNA 2 kinds be in different regulation and control level organically contacts together.The analysis of full-length genome microRNA microRNA target prediction also shows, the target spot of microRNA is all transcription factor mostly.
The miR-206 of the mankind lays respectively on No. 6 chromosome, and quite conservative.MiR-206 is one of miR-1 family member of muscle specific expression.Research prove, miR-206 is specifically expressing in vertebrate skeletal muscle only, and in slow muscle high expressed.Mankind MyoD gene is positioned at chromosome 11p 15.4, total linear DNA 6490bp, mRNA 1974bp, protein 318 aminoacid.MyoD is sarcoplast and myoblast differentiation in other types cell transformation is play an important role in the process of myotube; In special flesh gene transcription regulation, MyoD plays a part main switch.This research is intended on the basis of early-stage Study, prediction and the checking of the combination of MyoD and miR-206 promoter is carried out by two luciferase report gene experiment, preliminary study transcription factor MyoD on the impact of miR-206 promoter activity, thus provides foundation for the regulated and control network of miR-206 and action principle.
Summary of the invention
Technical problem to be solved by this invention is as myocardial infarction provides a kind of medicine compositions.For solving the problem, the present inventor has found a kind of medicine for the treatment of myocardial infarction through concentrating on studies, it is characterized in that, is effective ingredient containing MyoD.
A kind of medicine for the treatment of myocardial infarction of the present invention, preferred effective ingredient accounts for described medicine gross weight and is: described MyoD accounts for 70 ~ 85%.
A kind of medicine for the treatment of myocardial infarction of the present invention, more preferably described MyoD accounts for 85%.
A kind of medicine for the treatment of myocardial infarction of the present invention, also comprises pharmaceutically acceptable carrier and/or excipient.
The present invention also provides the application of a kind of medicine in treatment myocardial infarction, and it is characterized in that, it is effective ingredient that described medicine contains MyoD.
The present invention also provides the application of a kind of medicine in treatment myocardial infarction, and preferred described effective ingredient accounts for described medicine gross weight and is: described MyoD accounts for 70% ~ 85%.
The present invention also provides the application of a kind of medicine in treatment myocardial infarction, and more preferably described MyoD accounts for 85%.
Detailed description of the invention
To embodiments of the present invention be illustrated below, however embodiments of the present invention not limit by following detailed description of the invention.
1. from gene library, obtain MyoD
1. gene library: will containing certain biological heterogeneic many DNA fragmentation, the colony importing recipient bacterium stores, and the different genes of each recipient bacterium respectively containing this biology, is called gene library.
2. the kind of gene library: 1, genomic library; 2, portion gene library.
Utilize round pcr amplifying target genes
1. PCR implication: be and copy the ribose synthetic technology of specific DNA fragments in vitro.Full name is polymerase chain reaction.
2. PCR principle: DNA double chain copies.
3. precondition: the nucleotide sequence having one section of known genes of interest, so that according to this sequent synthesis primer.
4. amplification procedure: unwind after genes of interest DNA heated denaturalization as strand, primer is combined with strand respective complementary sequence, extends under the effect of archaeal dna polymerase, and repetitive cycling like this repeatedly.
5. result: the amount of the genes of interest after each circulation doubles, exponential form amplification.
2. vector construction
1) form
(1) genes of interest; (2) promoter; (3) terminator; (4) marker gene.
3. function
1) make genes of interest stable existence in recipient cell, and can the next generation be entailed.
2) genes of interest expressed and play a role.
4. step
1) with certain restriction endonuclease cutting plasmid, appearance cohesive end otch is made it.
2) with restriction endonuclease cutting genes of interest of the same race, identical cohesive end is produced.
3) genes of interest fragment is inserted plasmid otch, add appropriate DNA ligase, make genes of interest and plasmid form recombiant plasmid.
4) formation of expression vector: genes of interest+promoter+terminator+marker gene
5. target imports
1) transform
Genes of interest enters in recipient cell, and in recipient cell, maintain process that is stable and that express.
2) acceptor specy
(1), microorganism: bacterial cell
(2), plant cell: ovum, germ cell, somatic cell
(3), zooblast: germ cell, embryonic cell
6. Testing and appraisal
(1), detect:
1., detection is imported: whether detect on recipient cell chromosomal DNA containing genes of interest.
DNA molecule hybridize method (DNA probe method).
2., detection of expression: whether testing goal gene completes is transcribed out mRNA.
Molecular hybridization detects RNA.
Whether testing goal gene completes expression and translates into protein.
Extract protein, carry out antigen with corresponding antibody---antibody hybridization.
(2), qualification: qautobiology level is identified, qualification resistance etc.
6. blood plasma miRNA extraction step
1) isopyknic blood plasma and 2 × Denaturing reagent fully mix, and room temperature places 5 minutes.
2) phenol-chloroform doubling Plasma volumes is added, put upside down mixing 60 seconds, centrifugal 15 minutes of 16000 × g (as centrifugal effect is bad, namely biphase liquid level separation is bad needs recentrifuge, recentrifuge 5 minutes after maybe all aqueous phase sucking-offs to newly being managed) under room temperature.
3) reclaim in aqueous phase to clean tube of upper strata, record recycle-water phase volume.
4) add the dehydrated alcohol of 1.25 times of aqueous phase volumes and fully mix.
5) add 700ul (being at every turn no more than) dehydrated alcohol mixed liquor on Filter column, centrifugal 30 seconds of 10000 × g, abandon filter liquor (repeatedly cross post and use same Filter column and same collecting pipe).
6) add 700ul Wash I on Filter column, centrifugal 20 seconds of 10000 × g, abandons filter liquor.
7) add 500ul Wash 2/3 on Filter column, centrifugal 20 seconds of 10000 × g, abandons filter liquor, repeats 1 time.
8) recentrifuge 1 minute, takes out liquid residue in filter post.
9) add the RNase-free water of preheating 95 DEG C to filter membrane center, centrifugal 50 seconds of 10000 × g, eluent stream fluid is stored in-80 DEG C.
The detection of 7.miRNA quality
Get the RNA sample 1ul of mixing, make the light absorption value of blank mensuration 260,280nm with RNase-free water.Nucleic acid purity and concentration is calculated according to A260/A280.
8.miRNA gene microarray analysis
MiRNeasy mini test kit is used to carry out the purification of total serum IgE.Then detect concentration and the purity of RNA with spectrophotometer, 1% agarose gel electrophoresis detects the integrity of RNA.Be separated the RNA obtained and adopt miRCURY
tMhy3
tM/ Hy5
tMpower labelling kit carries out labelling to miRNA.The sample that labelling is good and miRCURY
tMlNA Array (v.18.0) is hybridized.Gather image after sample hybridization, last image adopts scanning analysis software to carry out data analysis.CHD group standard value compared with Normal group standard value higher than 2.0 times or namely think that there is significance raises or down regulation trend lower than 2.0 times.MiR-96 gene chip analysis becomes Bioisystech Co., Ltd to complete by upper Haikang.
9. transcription factor binding site puts (TFBS) prediction
First on UCSC bioinformatics website, (http://genome.ucsc.edu) searches for the sequence within hsa-miR-206 upstream region of gene promoter region 2000bp; Apply TRANSFAC (http://www.gene-regulation.com/pub/databases.html) and TFSEARCH (http://www.cbrc.jp/research/db/TFSEARCH.html) online tool more further, whether prediction hsa-miR-206 exists transcription factor binding site within genomic upstream 2000bp is put (TFBS).
The beyond thought effect acquired by medicine of the present invention is introduced successively below according to result order.
The first, the display of this result of study originally, includes altogether 240 routine object of study in, wherein CHD group 120 example, male 77 example, women 43 example; Normal group 120 example, male 57 example, women 63 example.The CHD group mean age 64.70 ± 6.79; 63.69 ± 5.96. the smoking of Normal group age, hypertension, diabetes, triglyceride, the equal not statistically significant of LDL-C and HDLIC difference.
Second, use the scanning of GenePix 4000B chip scanner, the raw florescent intensity data of chip image complete analysis by GenePix Pro 6.0 software, revised by original value subtracting background value, and do standardization by intermediate value, and calculate the standard value of miRNA in 3 patients with coronary heart disease and 3 normal control samples and the ratio of standard value between any two respectively. with the miRNA picking out differential expression in the figure of volcano.
3rd, with MEV software, cluster analysis is carried out to differential expression miRNA.Result shows: result display by miR-96 gene chip detection and after statistical analysis, the miRNA molecule totally 33 that differential expression multiple is greater than 5 times.
4th, the essential information of selected 4 kinds of miRNA and has-miR-574-5p, has-miR-206, has-miR-303a-3p and has-miR-383.
5th, Bioinformatics Prediction MyoD is miR-206 gene promoter area transcription factor.Applying biological informatics software TRANSFAC and TFSEARCH predicts transcription factor respectively, selects the common factor alternatively transcription factor of 2 software prediction results.Found that there are 2 transcription factor MyoD binding sites in miR-206 upstream region of gene promoter region.Therefore, Bioinformatics Prediction is thought, MyoD is the transcription factor of miR-206 gene.
According to above each experimental result, after can learning the medicine using invention, the technique effect obtained has: compared with matched group, uses MyoD to have therapeutic intervention effect to myocardial infarction.
Claims (6)
1. treat a medicine for myocardial infarction, be effective ingredient, it is characterized in that containing transcription factor MyoD, described MyoD accounts for effective ingredient 70% ~ 85%.
2. medicine as claimed in claim 1, it is characterized in that, described MyoD accounts for effective ingredient 85%.
3. medicine as claimed in claim 1 or 2, is characterized in that, also comprise pharmaceutically acceptable carrier and/or excipient.
4. the application of medicine in therapeutic intervention myocardial infarction, is characterized in that, it is effective ingredient that described medicine contains transcription factor MyoD.
5. apply as claimed in claim 4, it is characterized in that, the percentage ratio that described effective ingredient accounts for described medicine gross weight is: described MyoD accounts for 70% ~ 85%.
6. apply as claimed in claim 5, it is characterized in that, described MyoD accounts for 85%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510197193.9A CN104922657A (en) | 2015-04-22 | 2015-04-22 | Medicine for treating myocardial infarction |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201510197193.9A CN104922657A (en) | 2015-04-22 | 2015-04-22 | Medicine for treating myocardial infarction |
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| CN201510197193.9A Pending CN104922657A (en) | 2015-04-22 | 2015-04-22 | Medicine for treating myocardial infarction |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003064637A1 (en) * | 2001-11-06 | 2003-08-07 | Medtronic, Inc. | Method and system for myocardial infarction repair |
| US20080206863A1 (en) * | 1993-11-03 | 2008-08-28 | Diacrin, Inc. | Embryonic stem cells capable of differentiating into desired cells lines |
-
2015
- 2015-04-22 CN CN201510197193.9A patent/CN104922657A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080206863A1 (en) * | 1993-11-03 | 2008-08-28 | Diacrin, Inc. | Embryonic stem cells capable of differentiating into desired cells lines |
| WO2003064637A1 (en) * | 2001-11-06 | 2003-08-07 | Medtronic, Inc. | Method and system for myocardial infarction repair |
Non-Patent Citations (3)
| Title |
|---|
| ANDREW H WILLIAMS等: "MicroRNA control of muscle development and disease", 《CURRENT OPINION IN CELL BIOLOGY》 * |
| 范慧敏等: "MyoD和Cx43基因转染成纤维细胞后心肌移植治疗大鼠心肌梗死", 《中华实验外科杂志》 * |
| 薛胜将 等: "microRNAs与心血管疾病", 《生命科学》 * |
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