CN102205124A - Composition for inhibition of expression of miR-27b, medicines containing the same, and use for the same - Google Patents
Composition for inhibition of expression of miR-27b, medicines containing the same, and use for the same Download PDFInfo
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Abstract
The invention relates to a composition for specifically inhibiting the expression of miR-27b and medicines containing the composition. The composition has the nucleotide sequence 5' -GCAGAACUUAGCCACUGUGAA-3' of functional molecules produced in vivo. The inhibition of the expression of miR-27b in vivo can alleviate cardiac hypertrophy and heart damage caused by stress load. The invention determines the functions of miR-27b in cardiac hypertrophy through myocardial cell experiments. A mouse model of cardiac hypertrophy caused by TAC surgically created stress load is treated by packaging adenovirus for the specific inhibition of the expression of miR-27b and synthesizing an inhibitor against miR-27b, which brings out that cardiac hypertrophy and fibrosis caused by TAC operation can be alleviated effectively by respectively utilizing two inhibitors to treat the TAC postoperative mouse, with obvious thinning of ventricular wall thickness and obvious remission of systolic function of heart.
Description
Technical field
The present invention relates to the application of miR-27b as the target molecules of cardiac disease treatment, relating in particular to a kind of is the medicine of target treatment heart disease with miR-27b.
Background technology
In recent years, the sickness rate of heart disease was trend of rising gradually, and most of heart disease patients finally can death because form heart failure, added up according to World Health Organization (WHO), and heart disease has become and causes one of main causes of death at present.The generation of heart failure and development are the complicated change procedures that multiple factor participates in, and myocardial hypertrophy and myocardial fibrosis are two wherein important factors.Myocardial hypertrophy normally under numerous pathology stimulate, as myocardial ischemia, hypertension, cardiac valve defective etc., the hypertrophic response that cardiac muscle takes place mainly shows as ventricle wall to thicken, the myocardial cell volume increases and some period of embryo's gene expression rising etc.Although the early stage compensatory phase of myocardial hypertrophy is considered to a kind of adaptation response of heart, favourable and the function that strengthens heart, cardiac muscle is in plump state for a long time can cause arrhythmia, heart failure and cardiac sudden death.Therefore the research molecular mechanism that forms myocardial hypertrophy will help us better to understand heart failure to form early stage mechanism, help the early diagnosis and the control of heart failure, and can be clinically that the gene therapy of heart disease provides important target molecules.
The recent miRNAs that studies show that is also playing the part of extremely important role in the heart disease generating process.When horizontal large artery trunks constriction or mistake expression activity calcineurin (calcineurin), the expression of miRNA changes in the mouse heart tissue, part miRNA expression variation is similar to the miRNAs variation in the human failure heart tissue, and experiment in vivo and vitro shows that the expression of crossing of some of them miRNA is enough to bring out myocardial cell generation hypertrophy.Cause the plump early stage miR-1 expression of mouse cardiac muscle at TAC and obviously reduce, in-vitro transfection miR-1 can suppress the myocardial hypertrophy that ET-1 causes.In addition, find that the miR-133 expression reduces in plump specimen of human myocardium and mouse cardiac muscle hypertrophy model, external mistake is expressed the generation that miR-133 suppresses myocardial hypertrophy, and striking of miR-133 subtracts the generation that can promote myocardial hypertrophy.Other has two research groups to utilize knock out mice and the function of transgenic mice technical research miR-208 in heart, finds that miR-208 is playing important regulatory role aspect myocardial cell growth that stress rely on and the Gene regulation.Except performance function in myocardial hypertrophy, also have some miRNA to be proved and bringing into play important function aspect adjusting myocardial fibrosis and the apoptosis of cardiac muscle.In the pathologic myocardial hypertrophy, the expression of miR-133 and miR-30 reduces, and the CTGF expression raises, and fibrosis takes place easily change, and illustrates that miR-133 and miR-30 have brought into play important function in regulating the myocardial fibrosis process.In acute myocardial infarction, the downward modulation of miR-29 family molecule expression.By the analysis of its target molecule being found the target molecule of its effect mostly is the fibrosis associated protein, the downward modulation of miR-29 raises these developed by molecule, causes Fibrotic generation.These studies show that miRNAs has brought into play important function really in heart disease.
In view of the critical function that miRNAs brings into play in heart, in recent years, researcher had been carried out utilization successively and had been carried out the treatment and the prevention of heart disease at the adjusting of miRNA both at home and abroad, had obtained some good results.Application at present form more widely is Antagomir, and Antagomir designs according to the ripe body sequence of microRNA, through the single stranded RNA of special marking and modification, is specifically designed to the efficient blocker that suppresses endogenous microRNA.There was the scholar to be reported in 2008 and utilizes the expression of Antagomir-21 inhibition miR-21 can effectively prevent and treat myocardial hypertrophy and the heart failure that raises and cause by the pressure load in the heart tissue.In mice, strike and subtract the myocardial hypertrophy that miR-23a can prevent isoproterenol to cause.Use Antagomir-320 in the body and can alleviate the cardiac fibrosis that causes by ischemia-reperfusion.There is the result of study of two external research groups to show that miR-210 and miR-494 can be used as the new treatment target molecules of treatment ischemic heart disease recently again.
The miR-27b expression in the human specimen of mouse model that myocardial hypertrophy takes place and aortosclerosis that studies show that in the past obviously raises, point out it in the heart disease generating process, may bring into play important effect, but still be not reported about its concrete function in heart.
Summary of the invention
The purpose of this invention is to provide a kind of chemical compound of the miR-27b of inhibition expression and the medicine that contains this chemical compound.
The miR-27b that discovers to the function of miR-27b in heart brings into play critical function in myocardial hypertrophy, and that the expression that suppresses miR-27b in vivo can be alleviated the myocardial hypertrophy and the cardiac function that are caused by pressure load is impaired, illustrates that miR-27b can become the new target molecules of cardiac disease treatment.
For this reason, the invention provides specificity and suppress the application of chemical compound in the medicine of preparation treatment heart disease that miR-27b expresses.Wherein, described heart disease is myocardial hypertrophy, myocardial fibrosis and the core function abnormality that causes thereof.
The invention provides a kind of medicine for the treatment of heart disease, it comprises that specificity suppresses the chemical compound that miR-27b expresses.Wherein, described chemical compound is small molecule disturbance ribonucleic acid, small molecule disturbance ribonucleic acid chemical modification object or the carrier that produces small molecule disturbance ribonucleic acid in vivo etc.
Preferably, described small molecule disturbance ribonucleic acid is siRNA or shRNA.
Preferably, the nucleotides sequence of described siRNA is classified 5 '-GCAGAACUUAGCCACUGUGAA-3 ' as.
Preferably, described carrier is the viral vector of expression of exogenous gene box for containing with 5 '-GCAGAACTTAGCCACTGTGAA-3 '.Wherein, described viral vector can be used adenovirus vector.
Viral vector linearisation with described inhibition miR-27b expresses can obtain the linear dsdna molecule.Linear dsdna molecule transfectional cell can obtain to suppress the virus that miR-27b expresses.The genomic DNA of the virus that this inhibition miR-27b expresses is described linear dsdna molecule.
The present invention provides also that two kinds of treatment heart diseases are plump as cardiac muscle, the medicine of myocardial fibrosis and the core function abnormality that causes thereof, and its effective ingredient is not the virus of described inhibition miR-27b expression and RNA inhibitor (5 '-GCAGAACUUAGCCACUGUGAA-3 ').
The medicine of above-mentioned treatment heart disease can be by the administration of intravenous injection mode, and adenovirus dosage is 2.5 * 10
10Pfu, only injection is once; The dosage of RNA inhibitor is 80mg/kg/d and injected in continuous three days.
The present invention determines the function of miR-27b in myocardial hypertrophy by the myocardial cell experiment, by the packing specificity suppress that miR-27b expresses adenovirus and synthetic inhibitor at miR-27b, the pressure load that TAC operation is set up causes that the mouse model of myocardial hypertrophy treats, the result shows that these two kinds of inhibitor all can alleviate myocardial hypertrophy and fibrosis that TAC operation causes effectively, the obvious attenuation of mouse core chamber wall thickness, cardiac systolic function has also obtained tangible alleviation, so miR-27b can be used as the new target molecules of cardiac disease treatment.
Description of drawings
The external myocardial cell dimension measurement of Fig. 1 result.
Fig. 2 utilizes and suppresses the tactful sketch map that miR-27b carries out cardiac disease treatment.
Fig. 3 Ad-anti-miR-27b suppresses the detection of effect.
The detection of miR-27b expression in the mouse heart tissue of Fig. 4 TAC operation back.
Fig. 5 utilizes Ad-anti-miR-27b and Antagomir-27b TAC operation mice to be treated the detection of back miR-27b expression.
Fig. 6 utilizes Ad-anti-miR-27b that TAC operation mice is treated back cardiac function testing result.
Fig. 7 utilizes Antagomir-27b that TAC operation mice is treated back cardiac function testing result.
Fig. 8 utilizes Ad-anti-miR-27b that TAC operation mice is treated the back cardiomorphology and observes and the molecular marker testing result.
Fig. 9 utilizes Antagomir-27b that TAC operation mice is treated the back cardiomorphology and observes and the molecular marker testing result.
Figure 10 utilizes Ad-anti-miR-27b that TAC operation mice is treated back heart tissue observed result.
Figure 11 utilizes Antagomir-27b that TAC operation mice is treated back heart tissue observed result.
Figure 12 utilizes Ad-anti-miR-27b and Antagomir-27b that TAC operation mice is treated the result that the back is detected miR-27b target molecule PPAR-γ expression.
The specific embodiment
A first aspect of the present invention is that miR-27b crosses packing and the cell in vitro experiment of expressing adenovirus, may further comprise the steps:
The gene order that will comprise the miR-27b precursor is connected into the pAd-Track carrier, recombinates in the BJ5183 bacterium with the pAd-easy carrier after Pme I linearisation, obtains pAd-miR-27b.The carrier transfection 293A cell after Pac I linearisation is reclaimed that obtains was obtained to express the adenovirus (pAd-miR-27b) of miR-27b, infect myocardial cell 48h with pressing 100moi after the virus amplification, (simultaneously with Ad-GFP as negative control, with Ad-GFP infect add simultaneously that PE handles as positive control), measure by immunofluorescence dyeing and measuring software, analyze miR-27b and cross the influence of expression the myocardial cell size, extract cell RNA simultaneously, detect the variation of loose marker gene after the reverse transcription.
A second aspect of the present invention is struck the packing of low miR-27b adenovirus, may further comprise the steps:
Bunchiness and sequence miR-27b mature sequence reverse complemental (anti-miR-27b) (5 '-GCAGAACTTAGCCACTGTGAA-3 ') are connected into the pAd-Track carrier, after Pme I linearisation, in the BJ5183 bacterium, recombinate, obtain pAd-anti-miR-27b with the pAd-easy carrier.The carrier transfection 293A cell after Pme I linearisation is reclaimed that obtains is obtained to strike the adenovirus (Ad-anti-miR-27b) of low miR-27b.
A third aspect of the present invention is that the TAC surgery models is set up the myocardial hypertrophy mouse model and determined the time that treatment begins, and comprising:
The myocardial hypertrophy mouse model (control group mice is carried out same operation, but does not carry out the ascending aorta ligation) that utilizes ligation heart ascending aorta operation build-up pressure load to cause.Before the operation and operation back 1w, 2w carries out ultrasound detection respectively, determines to take place the time of the time of obvious myocardial hypertrophy for treatment experiment beginning.Utilizing northern-blot checking miR-27b also is obviously to raise at TAC postoperative 1w and 2w expression.
A fourth aspect of the present invention be with Ad-anti-miR-27b and Antagomir-27b (its contained nucleotides sequence is classified 5 '-GCAGAACUUAGCCACUGUGAA-3 ' as) in intravenous injection mode lead-in portion TAC mice body, matched group and another part TAC operation mice are through the normal saline of intravenous injection equal volume.
A fifth aspect of the present invention is the phenotype analytical that the mice for the treatment of 3 weeks of back is carried out system, comprising:
Extract total RNA of mouse tissue with Trizol, get the total RNA of 25 μ g and carry out microRNA Northern hybridization, carry out Northern blot hybridization with the miR-27b probe, find that Ad-anti-miR-27b and Antagomir-27b can reduce effectively because the rising of the miR-27b expression that the TAC operation causes.
Get the Sham group, TAC group and TAC treatment group are carried out morphological observation and are found, TAC operation group mouse heart obviously increases than Sham group heart, and the heart of TAC treatment group obviously reduces than TAC group heart, and the heart body weight ratio of measurement and heart tibia length ratio have also reflected this trend.These several groups of mices have been carried out H﹠amp; E, Masson and Laminin dyeing, the result shows that the myocardial cell size of TAC operation back mice increases, fibrosis obviously increases, and the TAC group mice of using after Ad-anti-miR-27b or the Antagomir-27b treatment is compared than TAC group mice, the myocardial cell size reduces, and myocardial fibrosis also obviously reduces.Mice is carried out Function detection also find similar trend.
The inventor is by Ad-anti-miR-27b and Antagomir-27b, the myocardial hypertrophy mouse model that the pressure load that is caused by the TAC operation causes is treated experiment, find that these two kinds of inhibitor can reduce the miR-27b that is caused by TAC operation effectively and express to raise and can alleviate effectively by the pressure load and increase the cardiac enlargement that causes, the myocardial cell size increases, fibrosis increases and cardiac function impaired, the result shows that this miRNA might become the new target molecules of cardiac disease treatment, and it is produced the newtype drug that inhibiting adenovirus or inhibitor might become cardiac disease treatment.
Further describe the present invention by the following examples.But these embodiment do not constitute any qualification to protection domain of the present invention.
Embodiment 1 crosses packing and the cell infection experiment of expressing the miR-27b adenovirus
The gene order that will comprise the miR-27b precursor is connected into the pAd-Track carrier, recombinates in the BJ5183 bacterium with the pAd-easy carrier after Pme I linearisation, obtains pAd-miR-27b.The carrier transfection 293A cell after Pac I linearisation is reclaimed that obtains was obtained to express the adenovirus (pAd-miR-27b) of miR-27b.Infect myocardial cell 48h with pressing 100moi after the virus amplification, (simultaneously with Ad-GFP as negative control, with Ad-GFP infect add simultaneously that phenylephrine (PE) handles as positive control), measure by immunofluorescence dyeing and measuring software, analyze miR-27b and cross the influence of expression the myocardial cell size, extract cell RNA simultaneously, detect the variation of loose marker gene after the reverse transcription.The results are shown in Figure 1, shown that miR-27b is at external promotion myocardial hypertrophy.
To comprise bunchiness with miR-27b mature sequence reverse complementary sequence, be connected into the pAd-Track carrier, after Pme I linearisation, in the BJ5183 bacterium, recombinate with the pAd-easy carrier, obtain pAd-anti-miR-27b.The carrier transfection 293A cell after Pac I linearisation is reclaimed that obtains is obtained to strike the adenovirus Ad-anti-miR-27b of low miR-27b.With the adenovirus Ad-anti-miR-27b amplification postoperative infection that obtains myocardial cell of isolating former generation, carry RNA behind the 48h, carry out the detection of miRNA northern blot, see Fig. 3, find that this adenovirus can successfully strike the expression of low miR-27b.
The foundation of embodiment 3TAC surgery models and the detection of miR-27b expression
(1) foundation of surgery models
1, uses 0.125% tribromoethanol with Animal Anesthesia, and mice is carried out tracheal intubation;
2, mice is moved on to dissecting table and undergo surgery, fixing limbs and mouse tail;
3, under microscope instructs, cut off the thoracic cavity, use the machine for chest-opening expansion and fix a little otch, be convenient to the ligation operation of back along the mice breastbone;
4, isolate the heart ascending aorta, use No. 26 standard marker, around ascending aorta, carry out ligation, cause that the pressure load increases; Close the thoracic cavity, mice is placed on the warm stage until reviving;
5, control group mice is carried out same operation technique, but does not carry out the ascending aorta ligation.
(2) detection of miR-27b expression
1. the extraction of total tissue RNA
(1) mouse heart is organized 100mg put into 2ml Trizol, carried out homogenate, in incubated at room temperature 5 minutes with homogenizer.
(2) add the 0.4ml chloroform, covered tight bottle cap thermal agitation 15 seconds, placed 2-3 minute.2-8 ℃, centrifugal 15 minutes of 10000-12000g.
(3) with in upper water phase transfer to the new centrifuge tube, add the 0.5ml isopropyl alcohol, mixing, room temperature was placed 10 minutes, and 2-8 ℃, centrifugal 10 minutes of 10000-12000g.
(4) abandon supernatant, add the washed with isopropyl alcohol of 2ml 75% at least, 2-8 ℃, be not more than 7500g centrifugal 5 minutes.
(5) abandon supernatant, air drying 5-10 minute, the water 500 μ l of the no RNase of adding, suction makes the RNA dissolving repeatedly.
(6) 55-60 ℃ is incubated 10 minutes, and-70 ℃ of refrigerator storage are standby.
2.miRNA Northern hybridization detects the expression of miR-27b
(1) material is prepared: 180 ℃ of roasting vessel: erlenmeyer flask, graduated cylinder, tweezers etc. were greater than 6 hours; Electrophoresis tank: clean comb and electrophoresis tank, and spend the night, with the flushing of DEPC water, drying for standby with 3% hydrogen peroxide dipping; It is standby to handle DEPC water (2L).
(2) sample treatment: the total RNA of 25 μ g adds sample buffer (1: 1), boiled 5 minutes, and ice bath 2 minutes, 12000 left the heart 30 seconds.
(3) electrophoresis: 15%PAGE/8M carbamide/0.5 * TBE; Electric current: 30mA; Time: about 1 hour, blueness on earth.Electrophoretic buffer: 0.5 * TBE.
(4) dye glue: add EB with 0.5 * TBE and dye glue (special-purpose ware), RT 10min.
(5) change film: semidry method, electric current: 200mA; Time: 30 minutes; In proper order: wipe clean, from following three metafiltration paper+film+glue+three metafiltration paper; Catch up with bubble.
(6) fixing: be placed on the filter paper behind the commentaries on classics film and dry, UV-crosslinked 2 times, hybrid heater was baked 1 hour for 80 ℃, and preservative film is sealed up for safekeeping.
(7) prehybridization: prehybridization solution is in 65 ℃ of thawings; Film places TBE, puts into the hybridization bottle of pouring half bottle of TBE into then, pours out TBE then, and film is close on the tube wall; Put into hybridization solution 2ml (minimum 2ml, every 10cm
2/ ml); 37 ℃ of hybrid heaters, prehybridization is more than 2 hours.
Label probe:
After centrifugal, 37 ℃ were reacted 1 hour
(8) probe purification: the G-25 chromatographic column, the jolting pillar makes medium fall into pipe, and the tail that fractures is uncapped gently, centrifugal 1 minute of 3800rpm (1000g); Probe mixture is added in the middle of the pillar centrifugal 4 minutes of 3300rpm (1000g).
(9) hybridization: the probe behind the purification is put into the hybridization bottle, and 37 ℃ of hybridization are spent the night.
(10) wash film: film washing liquid (2 * SSC/0.1%SDS), 37 ℃ in hybrid heater, 2 times, each 20-30 minute.
(11) tabletting develops.
1. PAGE mixture (300ml prescription):
2. carbamide PAGE glue (every glue prescription):
PAGE mixture: 6ml
10％AP： 30μl
TEMED： 6μl
3. 20 * SSC (1L prescription):
NaCl： 175.3g
Sodium citrate: 88.2g
HCL adjust pH to 7.0, water is settled to 1L.
The results are shown in Figure 4 and table 1.Table 1 statistical result shows TAC operation back 2w, and tangible myocardial hypertrophy appears in mice, and Fig. 4 presentation of results is compared with Sham operation group, and when TAC postoperative 1w and 2w, the miR-27b expression obviously raises.
Table 1TAC postoperative 2w mouse heart ultrasound detection result
*P＜0.05 (representative is compared with the Sham group).
Embodiment 4 imports back detection miR-27b changes of expression level in the mice body of TAC operation back respectively with Ad-anti-miR-27b and Antagomir-27b
Concrete experimental design is as follows: get 28 of mices altogether, wherein 11 mices are Sham operation groups, and all the other 17 mices are TAC operation group.TAC operation group mice is divided into Saline (normal saline group, 8) at random, and Ad-anti-miR-27b group (4) and Antagomir-27b group (5) are handled according to the experimental strategy among Fig. 2.(Sham operation group also gives normal saline simultaneously and handles).
(1) mouse tail vein injection
1, mice is fixed, tail is stretching, tighten;
2, with cotton ball soaked in alcohol wiping tail, distend the blood vessels; Perhaps warm up heat, make venectasia with hot water or hot towel; Select suitable syringe needle for use, thin more good more; Inject (blood vessel is thicker) herein at afterbody near the place of epimere.With ethanol or hot water wiping, in the time of wiping, can firmly pull tail on the table.Inject state is that tail turns white, near the high-visible two red veins in the coccyx both sides of white;
3, left-handed forefinger, middle finger, the third finger and thumb are fixed mousetail;
Left hand pulls tail when 4, injecting, and makes tail be close to desktop, and it is the inserting needle position that tail is close to the turning with a table limit, the general selection apart from the sharp 1/4 or 1/3 place's inserting needle of tail, and skin is thinner herein, and blood vessel is clear, and inserting needle is easy;
5, have or not blood back to come testing needle by seeing whether at intravenous;
6, injection Ad-anti-miR-27b and Antagomir-27b.Adenovirus dosage is 2.5 * 10
10Pfu only needs injection once, and the dosage of RNA inhibitor is 80mg/kg/d and injected in continuous three days.
(2) Northern blot detects the miR-27b expression
Method the results are shown in Figure 5 with embodiment 3, illustrates that Ad-anti-miR-27b and Antagomir-27b all can reduce effectively because the rising of the miR-27b expression that the TAC operation causes.
Embodiment 5 utilizes adenovirus and inhibitor that TAC operation mice is treated the back cardiac function and detects
To several groups of mices of embodiment 4 with the anesthesia of tribromoethanol amine after, remove the anterior pectorial region by hair, utilize Vivid 7 ultrasound measuring instruments (GE company) that have the 12-MHz miniature probe to carry out the ultransonic detection of heart M type.
The results are shown in Figure 6,7 and table 2.The result of Fig. 6 and Fig. 7 shows that tangible myocardial hypertrophy appears in TAC operation back 2w mice and cardiac function is impaired, and the several typical data: increase as left ventricular end diastolic thickness (LVPWd), shorten mark (FS) and reduce, left ventricular mass (LVM) increases.3w detects discovery again after handling through different factors subsequently, further increase the weight of (seeing LVPWd and LVM value) to the plump degree of normal saline group mouse cardiac muscle, cardiac systolic function further impaired (seeing the FS value), and after Ad-anti-27b or Antagomir-27b treatment, can reduce LVPWd and LVM effectively, and the reduction of FS value has also obtained alleviating effectively.Other measurement index sees Table 2.
Table 2 mice treatment back cardiac ultrasonic testing result
*P＜0.05 (representative is compared with the Sham group),
aP＜0.05 (representative adds the normal saline group with the TAC group and compares).
Embodiment 6 utilizes adenovirus and inhibitor that TAC operation mice is treated back cardiomorphology and histological observation
(1) the mensuration mice of heavy, body weight of the heart and the tibia length disconnected neck in back of weighing is put to death, take out heart and cut off trunk on every side earlier, clean blood with normal saline, filter paper blots residual liquid, take by weighing cardiac weight with electronic balance, vernier caliper measurement mice tibia length, the ratio of calculating cardiac weight and body weight or tibia length is with the degree of these two index expression myocardial hypertrophies.
The result is shown in Fig. 8 and 9.The result of Fig. 8 shows that TAC organizes mice and compares with Sham group mice, the ratio of cardiac weight and body weight or tibia length obviously raises, tangible myocardial hypertrophy has taken place in prompting really, and TAC operation group is compared through the normal saline group with TAC through the Ad-anti-miR-27b processed group, obvious reduction all appears in these two indexs, the Antagomir-27b therapeutic effect of Fig. 9 has also reflected identical trend, and these two kinds of inhibitor of these results suggest have better therapeutic effect.
(2) detection of molecular marker expression
1. reverse transcription reaction
MRNA reverse transcription test kit with Takara company carries out reverse transcription reaction, and method is:
According to following mixed sample:
Wherein RNA gets 2 μ g, with the water polishing of no Rnase totally 7 μ l.
Behind the mixing, reaction condition is as follows: 30 ℃ of 10min; 45 ℃ of 30min; 5 ℃ of 5min
Reaction obtains the cDNA masterplate after finishing.
2.Real-time?PCR
The Realtime-PCR instrument (Light Cycler2.0) of real-time fluorescence quantitative PCR employing Roche company detects the cDNA of above-mentioned acquisition.
Reaction system:
Reaction condition: 95 ℃ of 2min; 95 ℃ of 2sec, 60 ℃ of 3sec, 72 ℃ of 11sec, 80 ℃ of 3sec, totally 45 circulations; 65 ℃ of 30sec.The result uses Roche LightCycler Software software and analyzes.The results are shown in Figure 8 and 9.The result of Fig. 8 shows that TAC organizes mice and compares with Sham group mice, loose marker gene (ANF in the heart tissue, BNP, β-MHC) the mRNA expression obviously raises, and TAC operation group is compared through the normal saline group with TAC through the Ad-anti-miR-27b processed group, obvious reduction all appears in these molecular level marker expression levels, and the Antagomir-27b therapeutic effect of Fig. 9 has also reflected identical trend, and these two kinds of inhibitor of these results suggest have better therapeutic effect.
(3) haematoxylin-Yihong dyeing
1, fixing
Be organized in the neutral formalin buffer (40% formaldehyde 100ml, water 900ml, sodium hydrogen phosphate 4g, sodium dihydrogen phosphate 6.5g) and fix more than 20 hours.
2, dehydration
70% ethanol 15 minutes, 80% ethanol 15 minutes, 90% ethanol 15 minutes, 95% ethanol 15 minutes, dehydrated alcohol 15 minutes, dehydrated alcohol 15 minutes, dimethylbenzene 15 minutes, dimethylbenzene 15 minutes.
3, embedding
The tissue that will take off water goes in the paraffin of handling well (melted paraffin wax spends the night in 60 ℃ of placements), saturating wax 2 hours, and wax is changed twice in the centre.Tissue is forwarded in the embedding frame of preheating, impouring dewaxing rapidly with the position of warm tweezers adjustment piece of tissue, is put base plate, and is gone up some dewaxings onboard, carefully removes the bubble in the base plate, and room temperature is placed, and dewaxing is solidified.
4, section
The finishing wax stone is to required size.Go up section at rotary microtome (MICROM HM340E), adjusting the blade angle is 10 ℃, and serial section moves to the wax band that forms on the clean aluminium foil on one side.With knife blade the wax band is cut into required length, moves to respectively on the microscope slide, add an amount of water again, sample is floated on the water surface.Microscope slide is placed on about 40 ℃ film dryer platform, treats that sample launches, water can be outwelled gently, microscope slide is closed in the Riker mount in 37 ℃ of dried overnight.
5, hematoxylin-eosin staining
6, neutral gum mounting
(4) immunohistochemical analysis
1, paraffin section, routine dewaxes to water.
2,3% hydrogen peroxide at room temperature was hatched 10 minutes, to eliminate the activity of endogenous peroxydase.
3, the distillation washing 2 minutes * 3.The container that fills 0.01M citrate buffer (pH6.0) is put in section, put the microwave oven internal heating and the liquid in container temperature is remained between 92-98 ℃ and continue 10-15 minute, take out container, antigen is repaired in room temperature cooling 10-20 minute.PBS soaked 5 minutes.
4,5-10% normal goats serum (PBS dilution) sealing, incubated at room 10 minutes.The serum deprivation that inclines drips one anti-(1%BSA-PBS dilution) of proper proportion dilution, hatches for 37 ℃ to spend the night in 1-2 hour or 4 ℃.
5, PBS flushing, 5 minutes * 3 times.
6, drip the biotin labeling two anti-(PBS dilution) of proper proportion dilution, hatched 30 minutes for 37 ℃.
7, PBS flushing, 5 minutes * 3 times.
8, drip the Radix Cochleariae officinalis enzyme labelling strepto-avidin (PBS dilution) of proper proportion dilution, hatched 30 minutes for 37 ℃.
9, PBS flushing, 5 minutes * 3 times.
10, chromogenic reagent.
11, water fully washes, and redyes mounting.
(4) Masson three-color process
1, tissue slice takes off cured to water.
2, ambient temperature overnight in Bouin ' s fixative.
3, the color of fixative is gone up in the section of the abundant rinsing of distilled water place to go.
4, haematoxylin dyeing is 5 minutes.
5, distilled water flushing is 5 minutes.
6, deionized water soaked into 5-10 minute.
7, Biebrich Scalet-Acid Fucshin dyeing is 5 minutes.
8, deionized water soaked into 5-10 minute.
9, section places Salkowski's solution to place 5 minutes.
10, use Annilline Blue solution-dyed 5 minutes.
11, section placed 1% acetic acid 2 minutes.
12, Qie Pian differentiation, dehydration and mounting
The result is shown in Figure 10 and 11.The result of Figure 10 shows that TAC operation group mice compares ventricle wall thickness and obviously increase and tangible myocardial fibrosis (indigo plant is dyed the zone) occurred with Sham operation group mice, and TAC operation group is compared through the normal saline group with TAC through the Ad-anti-miR-27b processed group, the obvious attenuation of ventricle wall thickness, myocardial fibrosis obviously alleviates.The Antagomir-27b treatment experimental result of Figure 11 also reflects identical trend.
(1) extraction of tissue protein
1, gets mouse heart tissue adding cell pyrolysis liquid (100mM Tris-HCl, 300mM NaCl, 2%Tween-20,0.4%NP-40,20% glycerol) adding protease inhibitor and the inhibitors of phosphatases of embodiment 4, place on ice, smash tissue with homogenizer.
2, place 20 minutes on ice.
3,4 ℃, centrifugal 20 minutes of 12000g collects supernatant, and-70 ℃ of preservations are standby after the packing.
(2) BCA determination of protein concentration
1, in the every hole of elisa plate, adds the good testing protein sample of 25 μ l BSA protein standard substances or dilution.
2, add 200 μ l working solutions in every hole, vibrated 37 ℃ of incubation 30min 30 seconds.
3, be cooled to after the room temperature and measure OD with enzyme connection instrument
562Nm.
4, BSA standard sample bioassay standard curve, protein concentration and OD within the specific limits
562Be directly proportional.Sample protein concentration is calculated through the standard curve fit equation.
(3) protein blot (western blot)
1, gets histone 50 μ g and add equal-volume 2 * loading buffer, in boiling water, boil after 3-5 minute and carry out the SDS-PAGE electrophoresis.
2, after electrophoresis finishes, utilize electroporation (BIO-RAD) that albumen is forwarded on the pvdf membrane.
3, add confining liquid (TBST solution adds 5% defatted milk powder), 37 ℃ were sealed 1-2 hour.
4, add the anti-of dilution on request, 37 ℃, spent the night in 1-2 hour or 4 ℃.
5, TBST solution is washed film 3 times, each 5 minutes.
6, add two anti-(goat-anti rabbits/Mus IgG), room temperature 1 hour.
7, TBST solution is washed film 3 times, each 10 minutes.
8, add ECL system (Chemicon, ChemiLucent Detection System Kit, 2600) colour developing 3 minutes, tabletting, wash film, observed result.
The results are shown in Figure 12, show that TAC operation back PPAR-γ expression obviously reduces, and after Ad-anti-miR-27b or Antagomir-27b processing, can alleviate the reduction of PPAR-γ expression effectively.This result can be alleviated the reduction of the miR-27b expression that the TAC operation causes really effectively from the application of another angle reflection Ad-anti-miR-27b or Antagomir-27b.
Though above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. specificity suppresses the application of chemical compound in the medicine of preparation treatment heart disease that miR-27b expresses.
2. application according to claim 1 is characterized in that, described heart disease is myocardial hypertrophy, myocardial fibrosis and the core function abnormality that causes thereof.
3. medicine for the treatment of heart disease, it comprises that specificity suppresses the chemical compound that miR-27b expresses.
4. medicine according to claim 3 is characterized in that, described chemical compound is small molecule disturbance ribonucleic acid, small molecule disturbance ribonucleic acid chemical modification object or the carrier that produces small molecule disturbance ribonucleic acid in vivo.
5. medicine according to claim 4 is characterized in that, described small molecule disturbance ribonucleic acid is siRNA or shRNA.
6. medicine according to claim 5 is characterized in that the nucleotides sequence of described siRNA is classified 5 '-GCAGAACUUAGCCACUGUGAA-3 ' as.
7. medicine according to claim 4 is characterized in that, described carrier is the viral vector of expression of exogenous gene box for containing with 5 '-GCAGAACTTAGCCACTGTGAA-3 '.
8. medicine according to claim 7 is characterized in that, described viral vector is an adenovirus vector.
9. linear dsdna molecule, it is the viral vector that linearizing claim 7 or 8 described inhibition miR-27b express.
10. suppress the virus that miR-27b expresses, its genomic DNA is the described linear dsdna molecule of claim 9.
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| CN112638475A (en) * | 2018-06-28 | 2021-04-09 | 英国研究与创新基金会 | MicroRNA targeting agents for the treatment of cardiac diseases |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008042231A2 (en) * | 2006-09-29 | 2008-04-10 | Children's Medical Center Corporation | Compositions and methods for evaluating and treating heart failure |
| US20090202493A1 (en) * | 2008-01-30 | 2009-08-13 | The General Hospital Corporation | Mir-150 for the treatment of blood disorders |
| WO2009114681A2 (en) * | 2008-03-13 | 2009-09-17 | Dharmacon, Inc. | Identification of mirna profiles that are diagnostic of hypertrophic cardiomyopathy |
-
2011
- 2011-04-02 CN CN2011100831548A patent/CN102205124A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008042231A2 (en) * | 2006-09-29 | 2008-04-10 | Children's Medical Center Corporation | Compositions and methods for evaluating and treating heart failure |
| US20090202493A1 (en) * | 2008-01-30 | 2009-08-13 | The General Hospital Corporation | Mir-150 for the treatment of blood disorders |
| WO2009114681A2 (en) * | 2008-03-13 | 2009-09-17 | Dharmacon, Inc. | Identification of mirna profiles that are diagnostic of hypertrophic cardiomyopathy |
Non-Patent Citations (2)
| Title |
|---|
| 20070531 Angelika Kuehbacher等 Role of Dicer and Drosha for Endothelial MicroRNA Expression and Angiogenesis 59-68 1-10 第101卷, * |
| ANGELIKA KUEHBACHER等: "Role of Dicer and Drosha for Endothelial MicroRNA Expression and Angiogenesis", <CIRCULATION RESEARCH>, vol. 101, 31 May 2007 (2007-05-31), pages 59 - 68, XP002664447, DOI: doi:10.1161/CIRCRESAHA.107.153916 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112638475A (en) * | 2018-06-28 | 2021-04-09 | 英国研究与创新基金会 | MicroRNA targeting agents for the treatment of cardiac diseases |
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