CN102453757B - Application of micro RNA and antisense nucleic acid thereof in diagnosis, prevention, treatment and/or prognosis evaluation of myocardial ischemic injury - Google Patents
Application of micro RNA and antisense nucleic acid thereof in diagnosis, prevention, treatment and/or prognosis evaluation of myocardial ischemic injury Download PDFInfo
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Abstract
The invention provides an application of micro RNA and antisense nucleic acid thereof in the diagnosis, prevention, treatment and/or prognosis evaluation of myocardial ischemic injury. The micro RNA provided by the invention is nucleic acid or a bioactive functional fragment or variant thereof containing the following sequence: 5'-ACAGCAGGCACAGACAGGCAG-3'. The micro RNA can be used as a new biological marker to be applied to the diagnosis and/or prognosis evaluation of myocardial ischemic injury. In addition, the antisense nucleic acid of the micro RNA can be used as a new drug to be applied to the prevention and/or treatment of myocardial ischemic injury.
Description
Technical field
The invention belongs to diagnosis, prevention and the treatment field of biological medicine engineering field and heart disease, the new purposes that relates to the little RNAs of a kind of endogenic non-coding and antisense nucleic acid thereof, be specifically related to a kind of Microrna (microRNA, miRNA) and antisense nucleic acid thereof and diagnosing, preventing and/or treating the purposes in myocardial ischemic injury.
Background technology
At present, cardiovascular disorder is the first killer who causes mankind's death, and ischemia injury is the important pathological factor of many cardiovascular disordeies, and the major cause of myocardial ischemic injury causes because of anoxic.How effectively prevention and treatment myocardial ischemic injury are current medical science and biological study problem demanding prompt solution.
Microrna (microRNA, miRNA) be that a class is extensively present in nonprotein that eukaryote, length the are 21-25nt little RNA that encodes, they can be expressed with sequence-specific mode regulatory gene, can not be ignored all playing a part aspect growth, apoptosis, metabolism and human diseases.The physiological and pathological regulatory mechanism of miRNA is a new discipline of being paid much attention in recent years.
Summary of the invention
Therefore, the object of the invention is, provide a kind of Microrna and antisense nucleic acid thereof in the medicine for the preparation of diagnosis, prevention, treatment and/or prognosis evaluation myocardial ischemic injury or the purposes in test kit.
Another object of the present invention is that the test kit or the medicine that comprise above-mentioned Microrna and antisense nucleic acid thereof are provided.
The object of the invention is to be achieved through the following technical solutions.On the one hand, the invention provides a kind of Microrna and antisense nucleic acid thereof in the medicine for the preparation of diagnosis, prevention, treatment and/or prognosis evaluation myocardial ischemic injury or the purposes in test kit, wherein said Microrna is nucleic acid or its bioactive functions fragment or variant: the 5 '-ACAGCAGGCACAGACAGGCAG-3 ' (SEQ ID NO.1) that comprises following sequence.
Preferably, described miRNA antisense nucleic acid is nucleic acid or its bioactive functions fragment or variant: the 5 '-CUGCCUGUCUGUGCCUGCUGU-3 ' (SEQ IDNO.2) that comprises following sequence.
Preferably, described myocardial ischemic injury comprises stenocardia, Acute Myocardial Infarction, ventricular arrhythmia, coronary atherosclerotic heart disease.
On the other hand, the invention provides a kind of for diagnosing and/or the test kit of prognosis evaluation myocardial ischemic injury, described test kit comprises for the probe of specific detection Microrna or primer, and wherein said Microrna is nucleic acid or its bioactive functions fragment or variant: the 5 '-ACAGCAGGCACAGACAGGCAG-3 ' (SEQ ID NO.1) that comprises following sequence.
Preferably, the probe or the primer that in described test kit, comprise have sequence shown below:
5’-ATTGACAGCAGGCACAGAC-3’(SEQ?ID?NO.3)；
5’-GTGCAGGGTCCGAGGT-3’(SEQ?ID?NO.4)。
It is a kind of for preventing and/or treating the pharmaceutical composition of heart disease that the present invention also provides, it comprises miRNA antisense nucleic acid and pharmaceutically acceptable virus, carrier or the auxiliary material for the treatment of significant quantity, and wherein said miRNA antisense nucleic acid is nucleic acid or its bioactive functions fragment or variant: the 5 '-CUGCCUGUCUGUGCCUGCUGU-3 ' (SEQ ID NO.2) that comprises following sequence.
The content of the miRNA antisense nucleic acid preferably, comprising in described pharmaceutical composition is 0.5-2 μ g.
Preferably, described pharmaceutically acceptable carrier or auxiliary material are selected from chitosan, cholesterol, liposome, nano particle etc.
Preferably, described pharmaceutical composition is with mode administration oral or injection; Preferably, described drug administration by injection mode is selected from intravenous injection, intramuscular injection, intracoronary injection and myocardial injection.
In sum, the inventor finds by lot of experiments, and the damaging action of one of miRNAs member's miR-214 to anoxic myocardial, for novel targets is found in the treatment of myocardial ischemia.Particularly, the inventor carries out anoxic, hypoxia/reoxygenation and H to H9C2 myocardial cell
2o
2process, Real-time PCR method is determined the expression of miR-214 in anoxic H9C2 cell, finds that miR-214 is at anoxic, hypoxia/reoxygenation and H
2o
2the H9C2 cells of processing increases, and in westenblotting technical measurement cell, inhibitor of apoptosis protein Bcl-W expresses minimizing.And adopt cell transfecting technology that miR-214 inhibitor (being miR-214 antisense nucleic acid) is proceeded to anoxic and H
2o
2in the H9C2 cell of processing, mtt assay is measured H9C2 cell mortality, finds that miR-214 inhibitor reduces cell mortality, finds that after testing in cell, inhibitor of apoptosis protein Bcl-W expression increases.In addition, adopt the adenovirus of the miR-214 plasmid transfection amplification building to increase anoxic, hypoxia/reoxygenation and H
2o
2the mortality ratio of the H9C2 cell of processing, and the expression of Bcl-W albumen in H9C2 cell is reduced.In further testing, find that the adenovirus perfusion of miR-214 plasmid transfection amplification increases isolated rat Reperfusion Heart myocardial cell's apoptosis, and reduce its cardiac function and recover, also increase its heart myocardium cell necrosis and myofiber simultaneously and dissolve.
Therefore, miR-214 can be used as a kind of new biomarker and is applied to diagnosis and/or the prognosis evaluation in myocardial ischemic injury.In addition, the antisense nucleic acid of miR-214 can be used as a kind of novel medicine preventing and/or treating for myocardial ischemic injury.
Accompanying drawing explanation
Below, describe by reference to the accompanying drawings embodiment of the present invention in detail, wherein:
Fig. 1 is the expression of miR-214 in the H9C2 of anaerobic treatment cell; The anaerobic treatment time is respectively 1h, 2h, and 4h, 8h, along with anoxic time lengthening, miR-214 genetic expression increases gradually.
Fig. 2 is the expression of miR-214 in the H9C2 cell of hypoxia/reoxygenation processing; The anaerobic treatment time is respectively 1h, 2h, 4h, 8h, then the oxygen supply time is 1h; The anoxic 2h again miR-214 genetic expression of oxygen supply 1h treatment group reaches maximum.
Fig. 3 is that miR-214 is at H
2o
2expression in the H9C2 cell of processing; H2O2 processes 0.5h, 1h, along with H
2o
2concentration increases, and miR-214 genetic expression increases gradually.
Fig. 4 is the impact of miR-214 inhibitor on the H9C2 cell mortality of anaerobic treatment; MiR-214 inhibitor has reduced the H9C2 cell mortality of anaerobic treatment 2h.
Fig. 5 is that miR-214 inhibitor is to H
2o
2the impact of the H9C2 cell mortality of processing; MiR-214 inhibitor has reduced H
2o
2process the H9C2 cell mortality of 1h.
Fig. 6 is that BcL-W albumen is at H
2o
2expression in the H9C2 cell of processing; H
2o
2concentration is respectively 20 μ M, 40 μ M, and the treatment time is 1h; H
2o
2processing has reduced the expression of BcL-W albumen in H9C2 cell.
Fig. 7 is the impact of miR-214 inhibitor on BcL-W protein expression; MiR-214 inhibitor has increased the protein expression of BcL-W.
Embodiment
Referring to specific embodiment, the present invention is described.It will be appreciated by those skilled in the art that these embodiment are only for the present invention is described, the scope that it does not limit the present invention in any way.
The experiment material and the reagent that in following embodiment, use are as follows:
H9C2 cell is purchased from ATCC (the biological product of USS collecting center)
TRIquick reagent is purchased from Beijing Suo Laibao Science and Technology Ltd.;
DNTPs is purchased from Shanghai Bo Ya Bioisystech Co., Ltd;
M-MLV reversed transcriptive enzyme is purchased from Amersham company;
Ribonuclease inhibitor (Ribonuclease Inhibitor) and Real-time RT PCR test kit are all purchased from TaKaRa company;
Primer sequence entrusts Invitrogen company synthetic.
embodiment 1: miR-214 increases at the H9C2 of anaerobic treatment cells
The present embodiment is chosen the H9C2 cell of logarithmic phase, and by put into hypoxemia incubator in the cell of logarithmic phase, (Changsha Hua Xi Electronic Science and Technology Co., Ltd. produces.Model: YCP-30SQ), carry out anoxic (≤1%) 1h, 2h, 4h, 8h processing.Afterwards, TRIquick reagent extracts total RNA, preparation c-DNA, and Real-time RT PCR method detects the expression amount of miR-214, and with the cell without anaerobic treatment in contrast.
1, total RNA extracts:
Adopt TRIquick reagent to extract respectively cell total rna.
(1) anoxic 1h, 2h, 4h, 8h remove nutrient solution after processing, and directly add TRIquick lysing cell, 10cm in culture dish
2area adds 1ml TRIquick.With sampler piping and druming, mix.
(2) 4 ℃ of centrifugal 10min of 12000rpm, get supernatant.Every use 1ml TRIquick adds 0.2ml chloroform in sample, builds pipe lid, thermal agitation 15s, and room temperature is placed 5min.
(3) 4 ℃ of centrifugal 15min of 12000rpm, sample is divided into three layers.RNA is mainly at colourless water.Water is transferred in new pipe.
(4) in the water in transferring to new pipe, add isopyknic ice-cold Virahol, mix, room temperature is placed 20min.
(5) 4 ℃ of centrifugal 10min of 12000rpm, remove supernatant.Add 1ml 75% ethanol (with the preparation of RNase-free water) washing precipitation.4 ℃ of centrifugal 5min of 7000rpm, abandon supernatant.
(6) room temperature is placed airing, adds appropriate RNase-free water, fully dissolves RNA.
(7) preserve RNA sample for-80 ℃.
2, the c-DNA of miR-214 preparation
1) design primer sequence:
MiR-214 reverse transcription (RT) primer sequence following (SEQ ID NO.5):
5’-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCTGCCT-3’；
U6 reverse transcription (RT) primer sequence following (SEQ ID NO.6):
5’-AACGCTTCACGAATTTGCGT-3’。
2) c-DNA of miR-214 preparation:
5*RT damping fluid: 4 μ l
DNTPs：8μl
MiR-214RT primer: 1 μ l
Ribonuclease inhibitor (Ribonuclease Inhibitor): 1 μ l
RNA：1μl
DEPC water: 4 μ l
M-MLV reversed transcriptive enzyme: 1 μ l
Before and after adding M-MLV, fully mix, 42 ℃ of 1h, 95 ℃ of 5min, preserve sample for-20 ℃.
The cDNA sequence following (SEQ ID NO.7) of prepared miR-214:
CACGTGTATGAGTGTACTTCCTGAATAAAGTCAAACGTCTAACTTCTTTTTAGTTCCATAATGTTTAATGTTTAATTCTATTGTGTGTTTTTCTCCTTTCCCTTTATCCCCTCTCCTTCAATCACCAAATCTGGAAAACAGGCTGATTGTATCTGTCCAAGAAAAAAGGAACCGCGAAGGAGCCATGGTCCTGGATGGACAGAGTTGTCATGTGTCTGCCTGTCTACACTTGCTGTGCAGAACATCCGCTCACCTGTACAGCAGGCACAGACAGGCAGTCACATGACAACCCAGCCTGAATGACAACCAGCCATTGAAAGAAAGCTGCCCTCACACCATAGCATCTACACCAAGAGCTACAACCACAGTGAGGGGTTTGGGGGCCTGGGGCTTTTCTGTCGGCTTATTAAAATAAACTCGTATGTAATCCTTCTATTTTGATTCGTATATTAAAACCTACCCCCCAAGTAAAGGGAGTGGTGCC
3) c-DNA of U6 preparation:
5*RT damping fluid: 4 μ l
DNTPs：8μl
U6RT primer: 1 μ l
Ribonuclease inhibitor (Ribonuclease Inhibitor): 1 μ l
RNA：1μl
DEPC water: 4 μ l
M-MLV reversed transcriptive enzyme: 1 μ l
Before and after adding M-MLV, fully mix, 42 ℃ of 1h, 95 ℃ of 5min, preserve sample for-20 ℃.
3, Real-time PCR method detects the expression amount of miR-214
Adopt Real-time RT PCR test kit (purchased from TaKaRa company) to detect the expression amount of miR-214.
1) design primer sequence is as follows:
MiR-214 upstream primer (SEQ ID NO.3):
5’-ATTGACAGCAGGCACAGAC-3’
MiR-214 downstream primer (SEQ ID NO.4):
5’-GTGCAGGGTCCGAGGT-3’
U6 upstream primer (SEQ ID NO.8):
5’-CTCGCTTCGGCAGCACA-3’
U6 downstream primer (SEQ ID NO.9):
5’-AACGCT?TCA?CGA?ATT?TGCGT-3’
2) miR-214Real-time RT PCR reaction system is as follows:
Mix：12.5μl
MiR-214 upstream primer: 0.5 μ l
MiR-214 downstream primer: 0.5 μ l
ROX dyestuff: 0.5 μ l
c-DNA：2μl
Distilled water: 9 μ l
U6Real-time RT PCR reaction system is as follows:
Mix：12.5μl
U6 upstream primer: 0.5 μ l
U6 downstream primer: 0.5 μ l
ROX dyestuff: 0.5 μ l
c-DNA：2μl
Distilled water: 9 μ l
Real-time RT PCR reaction conditions is as follows:
①95℃：30s
②95℃：5s
③58℃：30s
④72℃：30s
Circulation step is 2. to 4. 45 times.
3) data processing:
MiR-214 expression amount calculates with following formula:
Δ Ct
sample=Ct
sample-Ct
sample U6
Δ Ct
contrast=Ct
contrast-Ct
contrast U6
Δ Δ Ct=Δ Ct
sample-Δ Ct
contrast
MiR-214 relative expression quantity changes=2
-Δ Δ Ct
Concrete outcome as shown in Figure 1.Visible, along with the prolongation of anaerobic treatment time, miR-214 genetic expression increases gradually.
embodiment 2: the H9C2 cells that miR-214 processes at hypoxia/reoxygenation increases
The present embodiment is chosen the H9C2 cell of logarithmic phase, puts into hypoxemia incubator and carries out anoxic (≤1%) 1h, 2h, 4h, 8h processing, afterwards, then cell is moved on to CO
2incubator (SANYO GS company.Model: continue to cultivate 1h MCO15AC).
TRIquick reagent extracts total RNA, preparation c-DNA, and Real-time RT PCR method detects the expression amount of miR-214, and concrete method is referring to embodiment 1.
Experimental result as shown in Figure 2.Visible, the anoxic 2h again miR-214 genetic expression of oxygen supply 1h treatment group reaches maximum.
embodiment 3: miR-214 is at H
2o
2the H9C2 cells of processing increases
The present embodiment is chosen the H9C2 cell of logarithmic phase, adopts the H of different concns
2o
2(20 μ M, 40 μ M, 80 μ M) process different time (0.5h, 1h, 2h).
TRIquick reagent extracts total RNA, preparation c-DNA, and Real-time RT PCR method detects the expression amount of miR-214, and concrete method is referring to embodiment 1.
Experimental result as shown in Figure 3.Visible, H
2o
2process 0.5h, 1h, along with H
2o
2concentration increases, and miR-214 genetic expression increases gradually.
In addition (Methods in molecularbiology (C1ifion, N.J.) 1995 is shown in concrete operations, also to adopt westen blotting technology; 49:423-437) detect H
2o
2the expression of BcL-W albumen in the H9C2 cell of processing, and take actin albumen as contrast.
Electrophoresis detection the results are shown in Figure 4, visible, works as H
2o
2concentration is respectively 20 μ M, 40 μ M, and the treatment time is while being 1h, H
2o
2processing has reduced the expression of BcL-W albumen in H9C2 cell.
embodiment 4: miR-214 inhibitor reduces the mortality ratio of the H9C2 cell of anaerobic treatment
The present embodiment is chosen the H9C2 cell of logarithmic phase, by cell transfecting technology, will respectively the miR-214 inhibitor (being synthetic miR-214 anti sense nucleotide sequence) of difference amount (2 μ g, 4 μ g, 8 μ g) be proceeded in H9C2 cell, carry out anoxic 2h processing, (J Immunol Methods is shown in concrete operations to adopt mtt assay, 1983,65 (1～2): the mortality ratio of 55) measuring H9C2 cell.
Experimental result as shown in Figure 5.Visible, miR-214 inhibitor has reduced the mortality ratio of the H9C2 cell of anaerobic treatment 2h.
embodiment 5: miR-214 inhibitor reduces H
2o
2the mortality ratio of the H9C2 cell of processing
The present embodiment is chosen the H9C2 cell of logarithmic phase, will respectively 4 μ gmiR-214 inhibitor (being synthetic miR-214 anti sense nucleotide sequence) be proceeded in H9C2 cell, with 20 μ M H by cell transfecting technology
2o
2process 1h, not add the negative contrast of cell of miR-214 inhibitor, adopt mtt assay to measure the mortality ratio of H9C2 cell.
Experimental result as shown in Figure 6.Visible, miR-214 inhibitor has reduced H
2o
2process the mortality ratio of the H9C2 cell of 1h.
In addition, also adopt westen blotting technology for detection miR-214 inhibitor to H
2o
2the impact of BcL-W protein expression in the H9C2 cell of processing.
Electrophoresis result is shown in Fig. 7, shows that miR-214 inhibitor has increased H
2o
2the expression of BcL-W albumen in the H9C2 cell of processing.
embodiment 6: the structure of cDNA plasmid
The present embodiment adopts RT-PCR, DNA purifying and connection, transformed competence colibacillus cell construction plasmid, and wherein building the cDNA sequence that plasmid adopts is the cDNA sequence of the prepared miR-214 of embodiment 1.
1) RT-PCR reaction
RT-PCR the primer sequence (primer that comprises the section of DNA sequence (500bp) of miR-214 sequence):
Ad-rno-214-F (upstream primer, SEQ ID NO.10):
5’-AGACTCGAGCACGTGTATGAGTGTACTTCC-3’
Ad-rno-214-R (downstream primer, SEQ ID NO.11):
5’-AATACTAGTGGCACCACTCCCTTTACTTGG-3’
RT-PCR reaction system:
2*PCR?TaqMix：20μl
Ad-rno-214-F：2μl
Ad-rno-214-R：2μl
DNA：2μl
Distilled water: 14 μ l
Mix the centrifugal 5min of 12000rpm.
2*PCR TaqMix (purchased from Beijing Mei Laibo medical science and technology company limited) wherein.
RT-PCR reaction conditions:
①95℃：5min
②95℃：30s
③56℃：30s
④72℃：1min
⑤72℃10min
Circulation step is 2. to 4. 35 times.
2) RT-PCR reaction product purifying:
RT-PCR reaction product purifying adopts DNA purification kit (purchased from TIANGEN Biotech (Beijing) Co., Ltd.), and concrete operations are as follows:
(1) RT-PCR reaction product is carried out agarose gel electrophoresis: deposition condition: 8% sepharose.Constant voltage (100 volts) 45min.
(2) single target DNA band is cut and puts into clean centrifuge tube from sepharose, take weight.
(3) in blob of viscose, add 3 times of volume sol solutions PN.10min are placed in 50 ℃ of water-baths, dissolve completely to glue.
(4) previous step gained solution is added in an adsorption column CA2, room temperature is placed 2min, and the centrifugal 60s of 12000rpm, outwells the waste liquid in collection tube, and adsorption column CA2 is put into collection tube.
(5) in adsorption column CA2, add 600 μ l rinsing liquid PW, the centrifugal 60s of 12000rpm, outwells the waste liquid in collection tube, and adsorption column CA2 is put into collection tube.
(6) in adsorption column CA2, add 600 μ l rinsing liquid PW, the centrifugal 60s of 12000rpm, outwells waste liquid.
(7) adsorption column CA2 is put into collection tube, the centrifugal 2min of 12000rpm, is placed in room temperature by adsorption column CA2 and places 10min.
(8) adsorption column CA2 is put in a clean centrifuge tube, to the appropriate elution buffer EB of the unsettled dropping in adsorption film mid-way, room temperature is placed 2min, and the centrifugal 2min of 12000rpm collects DNA solution.
3) connect
The RT-PCR reaction product of purifying is connected with Shuttle Vector 1.0-CMV carrier:
Shuttle Vector 1.0-CMV carrier double digestion:
(1) enzyme is cut system and condition:
speI：2μl
xhoI：2μl
NEB?buffer2：4μl
DNA solution: 32 μ l
Mix, centrifugal, 37 ℃ of water-bath 4h, 65 ℃ of water-bath 20min termination reactions.
(2) enzyme is cut product purification:
Enzyme is cut product purification with RT-PCR reaction product purifying.
The RT-PCR reaction product of purifying is cut with enzyme, the Shuttle Vector 1.0-CMV carrier of purifying is connected:
The DNA ligation kit (purchased from precious biotechnology (Dalian) company limited) that adopts TaKaRa company to produce.
Linked system and condition:
Solution?I：5μl
DNA solution: 2.5 μ l
Shuttle Vector 1.0-CMV carrier soln: 2.5 μ l
Mix gently, centrifugal, 16 ℃ are spent the night.
4) transform
Adopt and connect product transformed competence colibacillus cell, concrete operations are as follows:
Get competent cell suspension 50 μ l, add above-mentioned connection product, mix, standing 30min in ice bath.
Centrifuge tube is placed in to 42oC water-bath and places 60s, then fast pipe is transferred in ice bath, the cooling 2min of cell.
In centrifuge tube, add the aseptic LB substratum of 300 μ l (not containing microbiotic), mix and be placed on 37 ℃ of shaking table shaking culture 1h.
Get the above-mentioned liquid medium of 200 μ l, be applied to (containing acillin) on LB plate 37 ℃ of overnight incubation.
5) extract plasmid:
Bacterium colony on the above-mentioned culture plate of picking, joins in 3ml LB substratum (containing acillin), mixes to be placed on 37 ℃ of shaking table shaking culture and to spend the night.
The bacterium liquid of above-mentioned overnight incubation is joined in 250ml LB substratum to (containing acillin), mix and be placed on 37 ℃ of shaking table shaking culture and spend the night.
Extract plasmid and adopt the large extraction reagent kit of high purity plasmid (purchased from TIANGEN Biotech (Beijing) Co., Ltd.), experimental procedure is as follows:
Get the bacterium liquid of the above-mentioned incubated overnight of 100ml, the centrifugal 3min of room temperature 10000rpm collects bacterium.Remove supernatant as far as possible.
To leaving in the centrifuge tube of bacterial sediment, add 10ml solution P1, the bacterial cell that thoroughly suspends precipitation.
In centrifuge tube, add 10ml solution P2, leniently spin upside down 6-8 time immediately, fully mix, room temperature is placed 5min.
In centrifuge tube, add 10ml solution P4, leniently spin upside down 6-8 time immediately, fully mix, room temperature is placed 10min.The centrifugal 5-10min of 10000rpm, all pours solution in strainer CS, slowly pushes away handle and filters, and filtrate collection is in clean 50ml pipe.
Virahol to adding 0.35 times of filtrate volume in filtrate, turns upside down and fully mixes.
4 ℃ of centrifugal 30min of 10000rpm, outwell supernatant liquor gently, are upside down on thieving paper.
Xiang Guanzhong adds the abundant rinsing precipitation of 6ml 70% ethanol, and 4 ℃ of centrifugal 10min of 10000rpm, outwell supernatant liquor gently.Repeat this step once.
The uncovered room temperature of centrifuge tube is placed to 20min.After fully being volatilized, ethanol adds 1-1.5ml elution buffer TB, fully dissolution precipitation.
embodiment 7: the adenovirus of miR-214 plasmid transfection amplification increases anoxic, hypoxia/reoxygenation and H
2o
2the expression of the mortality ratio of the H9C2 cell of processing and reduction Bcl-W albumen
1) miR-214 plasmid transfection and adenovirus amplification:
The FuGENE HD transfection Reagent transfection reagent transfected HEK 293 that adopts Roche company to produce, transfection step is as follows:
(1) HEK293 cell 24h before transfection is inoculated in the cell cultures dish of 2 10cm, and making it cell convergence rate when transfection is 50%-70%.
(2) cell before transfection for 12h the DMEM without dual anti-(10%FBS) change liquid.
(3) inhale 15 μ l transfection reagents and 135 μ l DMEM (serum-free, without dual anti-) and mix the standing 5min of room temperature.Then mix the standing 25min～30min of room temperature with the 20 μ l DNA that mix in advance and 130 μ l DMEM (serum-free, without dual anti-).
(4) siphon away the substratum in 10cm ware, the mixed solution in (3) is added in 10cm ware, add again 8ml DMEM (10%FBS, without dual anti-), 37 ℃, 5%CO simultaneously
2cultivate 10～12h.
(5) substratum is siphoned away, change 37 ℃ of 8ml DMEM (10%FBS has dual anti-), 5%CO
2cultivate 7-12d.
2) collection of primary virus
(1) when cell occurs after cytopathic effect (CPE) phenomenon, 7-12d, cell can become circle and come off, and now can receive cell.
(2) by nutrient solution together with cell inspiration 15ml centrifuge tube, 2000rpm/min, centrifugal 5min.
(3) remove supernatant, in precipitation, add 400 μ l serum-free DMEM substratum suspension cells, in-70 ℃ of preservations.
(4) cell of collecting is in-70 ℃ and 37 ℃ of multigelations 3 times.
(5) 4 ℃, 5000rpm/min, centrifugal 10min.
(6) draw supernatant (primary virus) ℃ frozen in another sterile eppendorf tubes-70.
3) viral enrichment
(1) HEK293 cell is laid in 10cm ware with 50%-70% convergence rate, with the volume ratio of 30-50%, adds containing viral supernatant liquor.Within 2-3 days after infection, there is obvious lysis and CPE phenomenon.Within after infecting 3-5 days, by primary collection virus method, collect virus.
(2), for further amplification, the viral supernatant of above-mentioned collection is further infected to HEK293 cell, to obtain the virus of q.s.
4) adenovirus infection H9C2 cell and processing
By the good H9C2 cell of growth conditions with 2 * 10
5individual cells/well is inoculated in 6 orifice plates, when complete adherent growth to 60～80% of cell merges, gives recombinant adenovirus with 2 * 10
5after PFU/ml cells infected, continue after culturing cell 24h, carry out respectively following processing:
(1) anaerobic treatment: by the H9C2 cell of adenovirus infection with do not have the H9C2 cell infecting to put into hypoxemia incubator, carry out anoxic (≤1%) 2h and process.
(2) hypoxia/reoxygenation is processed: by the H9C2 cell of adenovirus infection with not have the H9C2 cell infecting to put into hypoxemia incubator, carry out after anoxic (≤1%) 2h processing, then cell is moved on to CO
2in incubator, continue to cultivate 1h.
(3) H
2o
2process: at the H9C2 of adenovirus infection cell with in the H9C2 cell that does not have to infect, add respectively H
2o
2(concentration is 20 μ M), continues to cultivate 1h.
5) measure the mortality ratio of H9C2 cell
Adopt mtt assay to measure the mortality ratio of H9C2 cell, concrete operations are as follows:
(1) cell suspension is with 1000rpm centrifugal 10 minutes, abandons supernatant liquor;
(2) precipitation adds 0.5-1ml MTT, blows and beats into suspension;
At (3) 37 ℃, be incubated 2 hours;
(4) add 4-5ml acidifying Virahol (constant volume), beat;
(5) 1000rpm is centrifugal, gets supernatant liquor microplate reader or spectrophotometer 570nm colorimetric, acidifying Virahol zeroising.
Result shows, adenovirus increase anoxic, hypoxia/reoxygenation and the H of the amplification of miR-214 plasmid transfection
2o
2the mortality ratio of the H9C2 cell of processing.
6) measure the expression of Bcl-W albumen in H9C2 cell
(1) extraction of collecting cell and albumen, concrete operations are as follows:
A) culture dish that covers with cell is placed on ice, with pipette sucking-off nutrient solution.Fully washed cell is surperficial in culture dish to add enough cold PBS, to wash away residual substratum, outwells PBS, repeats above operation 2-3 time.In washing, blot as far as possible residual PBS the last time, as far as possible in operation on ice.
B) in culturing bottle, add cold RIPA lysis buffer 1ml.Then with cell sleaker, along bottle wall, start cell scraping.
C) cell pyrolysis liquid that sucking-off is scraped is placed in 1.5ml centrifuge tube (being placed on ice).
D) sample inserts ice chest and carries out ultrasonicly, and ultrasound intensity is as the criterion not produce foam, ultrasonic each 2-3 second, repeats 3-4 time.Centrifugal 10000rpm, 10 minutes, leaves and takes supernatant.
E) take out cell pyrolysis liquid in a small amount and survey its protein concentration, packing is kept at-70 ℃.
(2) westen blotting determines the expression of Bcl-W albumen in H9C2 cell, and experimental procedure is as follows:
A) get the cell pyrolysis liquid (volume * protein concn) of equal in quality, and add isopyknic 2 * electrophoresis sample loading buffer, in boiling water bath 8 minutes, centrifugal 5 minutes of 12000rpm.
B) loading
C) electrophoresis (concentrated glue 20mA, separation gel 35mA)
D) electric transferring film instrument transferring film (100mA 40 minutes)
E) seal 1 hour, primary antibodie (1: 1000) overnight incubation (4 degree).(room temperature) hatched 2 hours in two anti-(1: 2000).
F) luminous.
Result shows, adenovirus reduction anoxic, hypoxia/reoxygenation and the H of the amplification of miR-214 plasmid transfection
2o
2the expression of Bcl-W albumen in the H9C2 cell of processing.
embodiment 8: the adenovirus perfusion of miR-214 plasmid transfection amplification increases isolated rat Reperfusion Heart myocardial cell's apoptosis, reduction heart function recovery, increases myocardium cell necrosis and myofiber and dissolves
1) isolated rat heart Ischemia Reperfusion injecting method:
Rats by intraperitoneal injection 20% urethane (5mL/kg), after anesthesia, vena femoralis injection 0.1% heparin (0.6mL/kg), after 2～3min, take out rapidly heart, be placed on Langendorff perfusion device aorta retroperfusion Krebs-Henseleit (K-H) liquid (mmol/L): NaCl, 118.0; KCl, 4.7; KH
2pO
4, 0.93; MgSO
47H
2o, 1.2; CaCl
2, 1.5; NaHCO
3, 25; C
6h
12o
6, 11.0; PH 7.4.37 ℃ of constant temperature, constant currents are poured into, and with 95%O
2-5%CO
2gas mixture is saturated.Employing stops filling with 120min, fills with again 60min and copies I/R damage model.Stopping filling with first 5 minutes, with adding 10
7the adenovirus perfusate perfused hearts of PFU/L.
2) measure myocardial cell's apoptosis:
Adopt Roche TUNEL cell apoptosis detection kit to measure the apoptosis of cardiac muscular tissue, experimental procedure is as follows:
Cardiac muscular tissue is conventional fixing, specimens paraffin embedding slices.
PBS washes cell once, adds 0.1%TritonX-1000.1% Trisodium Citrate, hatches 2min on ice.PBS washes 2 times, dry sample 5min.
In each section, add 50 μ l Tunel mix reagents.2 negative controls add 50 μ l Labelsolution (vial 2).[preparation of Tunel mix reagent: Label solution (vial 2): enzyme liquid (vial1)=9: 1].
Shake slide glass, guarantee that mix reagent covers whole tissue.
Covered, uses tin tissue packaging, places 1 hour in incubator.
PBS washes 3 times.Mounting.Sample is direct-detection under fluorescent microscope, and excitation wavelength is 450-500nm.Emission wavelength is 515-565nm.
Result shows, the adenovirus perfusion increase isolated rat Reperfusion Heart myocardial cell's of miR-214 plasmid transfection amplification apoptosis.
3) detect the heart function of isolated heart, concrete operations are as follows:
In the preparation process of isolated rat Reperfusion Heart model, the heart catheter inserting with sacculus through Atrioventricular valve supports left ventricle, and Filled Balloon is pressed left ventricular end diastolic and is less than 10mmHg (1mmHg=0.133kPa).Connect pressure transducer and input computer, monitoring heart function.Parameters of left ventricular function comprises: left ventricle maximum collapse is pressed (LVESP), the maximum diastolic pressure (LVEDP) of left ventricle, the maximum velocity of variation (+dp/dtmax) of left ventricular systolic pressure, the maximum velocity of variation (dp/dtmax) of left ventricular diastolic pressure, heart rate (HR).
Result shows, the adenovirus perfusion reduction isolated rat Reperfusion Heart heart function recovery of miR-214 plasmid transfection amplification.
4) detect myocardium cell necrosis and myofiber and dissolve situation:
Heart is left and taken myocardium of apex of heart tissue after pouring into and finishing, and 4% formaldehyde is fixed, specimens paraffin embedding slices, and HE dyeing, micro-Microscopic observation myocardium cell necrosis and myofiber dissolve situation.
Result shows, the adenovirus perfusion increase isolated rat Reperfusion Heart myocardium cell necrosis of miR-214 plasmid transfection amplification and myofiber dissolving.
Claims (6)
- The probe of specific detection Microrna or primer for the preparation of diagnosis and/or the medicine of prognosis evaluation myocardial ischemic injury or the purposes in test kit, the nucleic acid that wherein said Microrna is following sequence:5’-ACAGCAGGCACAGACAGGCAG-3’（SEQ?ID?NO.1）；Wherein said myocardial ischemic injury comprises stenocardia and ventricular arrhythmia.
- 2. the antisense nucleic acid of Microrna as claimed in claim 1 is for the preparation of preventing and/or treating the medicine of myocardial ischemic injury or the purposes in test kit, the nucleic acid that the antisense nucleic acid of wherein said Microrna is following sequence:5-CUGCCUGUCUGUGCCUGCUGU-3’（SEQ?ID?NO.2）；Wherein said myocardial ischemic injury comprises stenocardia and ventricular arrhythmia.
- 3. a miRNA antisense nucleic acid is in the purposes for the preparation of preventing and/or treating in the pharmaceutical composition of heart disease, described pharmaceutical composition comprises miRNA antisense nucleic acid and pharmaceutically acceptable virus, carrier or the auxiliary material for the treatment of significant quantity, the nucleic acid that wherein said miRNA antisense nucleic acid is following sequence:5’-CUGCCUGUCUGUGCCUGCUGU-3’（SEQ?ID?NO.2）；Wherein said heart disease comprises stenocardia and ventricular arrhythmia.
- 4. purposes according to claim 3, is characterized in that, the content of the miRNA antisense nucleic acid comprising in described pharmaceutical composition is 0.5-2 μ g.
- 5. purposes according to claim 3, is characterized in that, described pharmaceutically acceptable carrier or auxiliary material are selected from chitosan, cholesterol, liposome, nano particle etc.
- 6. according to the purposes described in any one in claim 3 to 5, it is characterized in that, described pharmaceutical composition is with mode administration oral or injection; Preferably, described drug administration by injection mode is selected from intravenous injection, intramuscular injection, intracoronary injection and myocardial injection.
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| Altered microRNA expression in human heart disease;Sadakatsu Ikeda et al.;《Physiol Genomics》;20070821 * |
| MicroRNA Expression Signature and the Role of MicroRNA-21 in the Early Phase of Acute Myocardial Infarction;Shimin Dong et al.;《THE JOURNAL OF BIOLOGICAL CHEMISTRY》;20090825 * |
| MicroRNAs: novel regulators in cardiac development and disease;Thomas Thum et al.;《Cardiovascular Research》;20081231 * |
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