CN102199625A - Construction method for miRNA (micro Ribonucleic Acid) transgenic mouse model - Google Patents

Abstract

The invention provides a construction method for a miRNA (micro Ribonucleic Acid) transgenic mice model. In the construction method, a transgenic mouse with overexpression miR-27b in a myocardial cell is established by using a transgenic technology; the systematic phenotyping on the transgenic mouse finds that the mouse possibly has myocardial hypertrophy and heart function damage; the basic pathological changes of the mouse are similar to those of human hearts, therefore, with the establishment of the transgenic mouse model, a favorable animal model for researching the heart disease nosogenesis and researching and developing new drugs is provided.

Description

A kind of construction process of miRNA transgene mouse model

Technical field

The invention belongs to field of transgenic technology, specifically, relate to a kind of construction process of miRNA transgene mouse model.

Background technology

Heart is the vitals of human body, as a kind of important function organ, heart promotes blood flow, provide ample amount of blood flow to organ, tissue, with supply oxygen and various nutritive substance, and take away metabolic end product (as carbonic acid gas, urea and uric acid etc.), make cell keep normal metabolism and function.In recent years, the sickness rate of heart disease was trend of rising gradually, and at present, China has 3,000,000 people to die from cardiovascular disorder every year, has become the No.1 killer of harm humans health.Most of heart disease patients finally can the death because form heart failure.Heart disease be the process of a complexity, the molecular mechanism that disease takes place is still indeterminate, might be a plurality of signal path results of interaction.Heart disease is formed Study on Mechanism will help people 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.

To form mechanism be very necessary to the research heart disease to set up good heart disease animal model, and transgenic animal are importances that animal model is set up.Transgenic technology (transgenic technology) is that foreign DNA is imported in fertilised non-human eggs or the embryonic stem cell, inserting at random or the mode of homologous recombination is incorporated into and is subjected in the Autosome, and entails offspring's technology with the division of cell.Animal by this technology produces is called " transgenic animal (transgenic animal) ".Provide multiple disease model for life science and medical science by transgenic technology, in the cardiovascular research field, transgenic animal have also obtained using very widely.

In recent years, a large focal spot of microRNA research becoming biological study.MiRNA is the strand microRNA that a kind of size is about 21-23 base, is to process the back by the single stranded RNA precursor of about 70-90 base size with hairpin structure through the Dicer enzyme to generate.Studies show that at present, behind 3 ' end non-coding region (3 ' UTR) complementary pairing of these non-coding small molecule RNAs and target gene mRNA molecule, can pass through to reduce mRNA stability of molecule and translation and suppress the regulation and control of dual mode participation expression of target gene.In the heart disease process, miRNAs has brought into play important function.Along with to miRNA the going deep into of functional study in heart, heart tissue specificity miRNA transgenic animal have also progressed into people's sight.2007, people such as Eva set up myocardial cell's specificity miR-195 transgenic mice, and myocardial hypertrophy and heart failure appear in this mouse.Another research group has set up myocardial cell's specificity miR-208 transgenic mice at report in 2010, finds that myocardial hypertrophy and irregular pulse appear in this mouse.Heart tissue specificity miRNA transgenic animal model is just becoming the strong instrument of miRNA functional study, will provide good animal model for studying human heart disease mechanism and new drug development and therapeutic evaluation.

The miR-27b expression level in the human sample 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 construction process that the purpose of this invention is to provide a kind of miRNA transgene mouse model.

The objective of the invention is to adopt following technical scheme to realize.According to a kind of miRNA expression carrier that contains provided by the invention, it comprises the miRNA gene of myocardial cell's specificity promoter α-MHC and one or more copies.

Aforesaid expression vector, described miRNA are miR-27b.

Aforesaid expression vector, the carrier that sets out are plasmid pRCH.

Preferably, described expression vector is to comprise the miR-27b gene of α-MHC promotor, 2 copies and the people hGH gene α of totally three elements-MHC-miR-27b-hGH carrier.

The present invention also provides a kind of construction process of miRNA transgene mouse model, and it is that aforesaid expression vector is imported in the mouse fertilized egg, the zygote that obtains is moved in the mouse uterine tube carry out gestation then, obtains transgenic mice.

The present invention further provides and make up the transgenic mice obtain by preceding method and be used for the treatment of application in the medicine of heart disease in screening.

By technique scheme, the present invention has following advantage and beneficial effect at least:

The present invention utilizes transgenic technology to set up and cross the transgenic mice of expressing miR-27b in the myocardial cell, transgenic mice is carried out the phenotype analytical of system and find that this mouse can myocardial hypertrophy occur and heart function is impaired, basic pathological change is similar to the human heart disease, so be established as research heart disease pathogenesis and the new drug development of this transgene mouse model provide good animal model.

Description of drawings

Fig. 1 makes up synoptic diagram for preferred embodiment myocardial cell specificity miR-27b transgene carrier of the present invention.

Fig. 2 identifies synoptic diagram for preferred embodiment myocardial cell specificity miR-27b transgenic mice PCR of the present invention, and wherein, 1 is Marker, and 2-4 is the transgenic positive mouse, and 5-6 is the negative mouse of transgenosis, 7 positive contrasts.

Fig. 3 A and Fig. 3 B have shown the expression level of identifying miR-27b in the transgenic mice heart tissue with Northern blot and in situ hybridization respectively.

Fig. 4 A and Fig. 4 B have shown the morphological analysis result of myocardial cell's specificity miR-27b transgenic mice heart and the measurement result of mouse heart weight/body weight and cardiac weight/shin bone length ratio respectively, Fig. 4 C is presented at loose marker gene ANF in the transgenic mice heart tissue, β-MHC, the expression level of SKA.

Fig. 5 be 3 the monthly age mouse H﹠amp; E, Laminin colored graph and adult myocardial cell aspect graph, A wherein, B: the H﹠amp of control mice and transgenic mice heart tissue; The E coloration result; C, D: the Laminin coloration result of control mice and transgenic mice heart tissue; E, F: the control mice of growing up and transgenic mice myocardial cell's morphological observation result.

Fig. 6 be 3 monthly ages and 6 the monthly age mouse Masson detection figure, A wherein, B:3 monthly age control mice and transgenic mice heart tissue Masson coloration result; C, D: monthly age control mice and transgenic mice heart tissue Masson coloration result.

Fig. 7 be 3 monthly ages and 6 the monthly age mouse ultrastructure detection figure, A wherein, B:3 monthly age control mice and transgenic mice heart tissue Electronic Speculum detected result; C, D: monthly age control mice and transgenic mice heart tissue Electronic Speculum detected result.

Fig. 8 is 3 monthly ages and 6 monthly age mouse heart Function detection results, the wherein representative M type ultrasonic cardiogram of A:3 monthly age control mice and transgenic mice; B, C: be respectively wall thickness (LVPWs) and left ventricular end diastolic heavy wall (LVPWd) observed value behind 3 monthly ages and the 6 monthly age mouse left ventricle end-systoles; D, E: be respectively 3 monthly ages and 6 monthly age mouse left ventricular end-systolic dimensions (Lvids) and left ventricular end diastolic dimension (Lvidd); The ratio (LVM/BW) of F:3 monthly age and 6 monthly age mouse left ventricular masses and body weight; G, the H:3 monthly age and 6 the monthly age mouse ejection fraction (EF) and shorten mark (FS); The I:3 monthly age and 6 the monthly age mouse left ventricular end-diastolic pressure (LVEDP); The J:3 monthly age and 6 the monthly age mouse mean arterial pressure (MAP).

Embodiment

A first aspect of the present invention is to make up heart tissue specific transgenic carrier, may further comprise the steps:

Have the plasmid of α-MHC with Apa I and Sal I double digestion, can obtain the fragment of 5.5kb, insulin gene promotor in the pRCH plasmid is removed with KpnI and Sal I double digestion, α-MHC promoter fragment is connected with promoterless pRCH fragment.With the miR-27b genome sequence with having added Sal 1 respectively, Mlu1 and Mlu 1, the primer of EcoR 1 increases and connects, obtain the miR-27b fragment of 2 copies, be connected into the above-mentioned carrier of cutting with Sal 1 and EcoR 1 enzyme, obtained containing the miR-27b gene of α-MHC promotor, 2 copies and the people hGH gene transgene carrier of the α-MHC-miR-27b-hGH of totally three elements (Fig. 1).

A second aspect of the present invention is that mouse is carried out gene type assay, comprising:

The 10 days mouse that will be born are cut the about 0.5cm of tail point and put into the Eppendorf pipe, add the lysis buffer lysing cell, add the protein that salts out of high density, centrifugal collection contains the supernatant of DNA, supernatant is separated out genomic dna behind ethanol sedimentation, genomic dna is dissolved in the TE solution for standby again.

With the template of genomic dna, be used to identify wild-type and transgenic positive mouse with primer 1 (5 '-ATGACAGACAGATCCCTCCTATCTCC-3 ') and primer 2 (5 '-TCAGCACGCTGTTTGCACTCTT-3 ') as PCR reaction.This can only amplify the band of size for 482bp and/or 864bp to primer from transgenic mice.Thereby distinguish the generation mice of different genotype.

A third aspect of the present invention is that expression of exogenous gene in the transgenic mice heart tissue is detected, and comprising:

With total RNA of Trizol extraction mouse tissue, get the total RNA of 25 μ g and carry out microRNANorthern hybridization, carry out Northern blot hybridization with the miR-27b probe, find that miR-27b gene expression level in the heart tissue of transgenic mice obviously raises.

Get the heart tissue section of transgenosis and control mice and carry out the in situ hybridization detection, find that miR-27b distributes and expression level obviously raises in the transgenic mice heart tissue.

A fourth aspect of the present invention is the phenotype analytical that transgenic mice is carried out system, comprising:

Morphological observation finds that the miR-27b transgenic mice begins to occur cardiac enlargement about three monthly ages, and the ratio of the cardiac weight of measurement and body weight or shin bone length contrasts mouse obviously to be increased; Histology is passed through H﹠amp; E and Laminin dyeing show that transgenic mice myocardial cell size increases; Myocardial cell's measuring result of in-vitro separation adult mice has also been verified this point; The Masson coloration result shows that tangible fibrosis appears in the transgenic mice heart tissue; Electronic Speculum result shows and organizes plastosome to occur obviously by 6 monthly age mouse hearts damage changes; Mouse is carried out Function detection, and to find that heart function appears in transgenic mice obviously impaired.

The present invention has made up one and has crossed the transgene carrier of expressing miR-27b in myocardial cell's specificity, and it is imported in the zygote of animal, obtains stable animal filial generation of carrying the external source goal gene by zygote transplation.Expression to miR-27b in the transgenic mice heart tissue detects successfully expression excessively in the transgenic mice heart of discovery miR-27b.Transgenic mice is carried out the phenotype analytical of system, it is impaired to find that myocardial hypertrophy, myocardial fibrosis and heart function appear in transgenic mice, the pathological change of its appearance and human heart disease are similar, so establishing of this myocardial cell's specificity miR-27b transgenic mice may provide ideal animal model for cardiac disease treatment and drug screening.

Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.

The reagent and the material that use in following examples are the commercial goods.

The structure of embodiment 1 myocardial cell's specificity miR-27b transgene carrier

Have the plasmid of α-MHC with Apa I and Sal I double digestion, can obtain the fragment of 5.5kb, insulin gene promotor in the pRCH plasmid is removed with Kpn I and Sal I double digestion, α-MHC promoter fragment is connected with promoterless pRCH fragment.With the miR-27b genome sequence with having added Sal 1 respectively, Mlu1 and Mlu 1, the primer of EcoR 1 increases and connects, obtain the miR-27b fragment of 2 copies, be connected into the above-mentioned carrier of cutting with Sal 1 and EcoR 1 enzyme, obtained containing the miR-27b gene of α-MHC promotor, 2 copies and the people hGH gene transgene carrier of the α-MHC-miR-27b-hGH of totally three elements (Fig. 1).

The importing of embodiment 2 transgene carriers and the screening of transgenic mice

Transgene carrier after Kpn I and Sac II linearizing, by zygote protokaryon microinjection, is injected 210 pieces in zygote altogether, transplant 200 pieces on ovum, the female mouse pregnancy of 3 false pregnancys is arranged, obtain 13 of generation mices.The results are shown in Table 1.

Table 1

Injection ovum number	Transplant the ovum number	Transplant the mouse number	Conceived mouse number	Birth mouse number	Positive mouse number
						210	200	8	3	13	4

The transgenic mice that obtains is carried out genotype identification:

(1) preparation of mouse gene group DNA

1, cuts 15 days about 0.5cm of mousetail point and put into the Eppendorf pipe.

2, every pipe adds mouse tail lysis buffer (0.5%SDS, 0.1M NaCl, 0.05MEDTA, 0.01M Tris-Cl pH8.0, Proteinase K, 200 μ g/ml) 400 μ l.

3,55 ℃ of incubated overnight.

4, every pipe adds the saturated NaCl (6M) of 200 μ l.

5, the Eppendorf pipe is placed carton acutely rock 200 times.

6, the Eppendorf pipe is placed on ice 10min.

7, room temperature 14, and 000rpm is centrifugal, 10min.

8, supernatant 500 μ l are transferred in the clean Eppendorf pipe, every pipe adds 0.8ml ethanol, mixing.

9,14,000rpm is centrifugal, 5min.Abandon supernatant.

10, down, drying at room temperature with the Eppendorf mouth of pipe.DNA is resuspended among 50～100 μ lTE, 37 ℃ be incubated to DNA dissolve fully the back (24-48hr) can be used for PCR or Southern hybridization analysis.

(2) PCR identifies the genotype of mouse

Primer 1 (5 '-ATGACAGACAGATCCCTCCTATCTCC-3 ') and primer 2 (5 '-TCAGCACGCTGTTTGCACTCTT-3 ') are used to identify wild-type and transgenic positive mouse.This can amplify size to primer and be the band of 482bp from the transgenic positive mouse.The results are shown in Figure 2.

The detection of miR-27b expression level in the embodiment 3 transgenic mice heart tissues

(1) Northern blot detects the expression of miR-27b

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 transition to the new centrifuge tube, add the 0.5ml Virahol, 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, dry air 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 the expression of miR-27b is identified in Northern hybridization

(1) material is prepared: 180 ℃ of roasting vessel: erlenmeyer flask, graduated cylinder, tweezers etc. were greater than 6 hours; Electrophoresis chamber: clean comb and electrophoresis chamber, 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) the total RNA of sample preparation: 25ug adds loading beffer (1: 1), boiled 5 minutes, and ice bath 2 minutes, 12000 left the heart 30 seconds.

(3) electrophoresis: 15%PAGE/8M urea/0.5 * TBE; Electric current: 30mA; Time: about 1 hour, blueness on earth.(note rifle flushing well during application of sample, RNA is added on the straight line) electrophoretic buffer: 0.5 * TBE with 200ul.

(4) dye glue: add EB with 0.5 * TBE and dye glue (special-purpose ware), RT 10min.

(5) change film: semidrying, 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.(annotate: film need not be caught up with bubble.Other have caught up with bubble to add some points at every turn again changes film liquid).

(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 purifying: the G-25 chromatography 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 purifying 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) compressing tablet develops.

Annotate: dosing

1. PAGE mix (300ml prescription):

2. urea PAGE glue (every glue prescription):

PAGE mixture: 6ml

10％AP：30μl

TEMED：6μl

Annotate: guarantee that no RNAase enzyme pollutes; Sheet glass ddH ₂The O flushing dries up.

3. 20 * SSC (1L prescription):

NaCl：175.3g

Trisodium Citrate: 88.2g

HCL adjust pH to 7.0, water is settled to 1L.

The results are shown in Figure 3A.The result shows with control mice and compares that miR-27b expression level in the transgenic mice heart tissue obviously raises.

(2) the miR-27b transgenic mice is identified in miRNA in situ hybridization

1, gets mouse heart tissue fixedly 1h of 4%PFA room temperature.Tissue after fixing is put into 30% sucrose, 4 ℃ spend the night (to reduce the frost damage of tissue).

2, next day tissue is carried out frozen section, thickness is 10 μ m.

3, the frozen section room temperature that the cuts 10min that dries in the air.

4, PBS washes 3min.(during the 1.5ml diacetyl oxide is added in the acetylize solution)

5, section is immersed in the acetylize solution and is rocked 10min gently.

6, acetylize step is washed 5min through PBS after finishing.

7, section moves into and contains in the container of 75ml, adds 37.5 μ l Proteinase Ks (DEPC water preparation, concentration is 10mg/ml), room temperature treatment 5min.

8, section is washed 3 times through PBS, each 3min.(in this process, preparing the wet box of hybridization)

9, section level is put into wet box, drips 700 μ l hybridization solutions in every section, and room temperature is carried out prehybridization 4-8h.

10, the hybridization solution of 150 μ l sex change and the probe of 0.1ul LNA digoxigenin labeled are prepared in every section.

11, rapid cooled on ice behind 80 ℃ of heating of above-mentioned probe 5min.

12, the probe mixed solution is added to tissue and covers slide, 50-60 ℃ of hybridization is spent the night.

13, after hybridization finishes, the careful slide of removing among the 5 * SSC that is immersed in prior preheating that cuts into slices.

14, will cut into slices and move among 0.2 * SSC, hatch 1h for 60 ℃.

15, section room temperature in B1 solution is placed 10min.

16, prepare a wet box, the 3MM filter paper of one deck water-soaked is put in the bottom.

17, remove section and go up unnecessary B1 solution, the section level is placed in the wet box.Every section adds 500 μ l confining liquids, and room temperature is placed 1h.

18, be added in the section after the alkaline phosphatase enzyme antibody that will dilute good anti-digoxigenin labeled diluted by 1: 2000,4 ℃ are spent the night.

19, section is through the NBT/BCIP colour developing, and it is blue that positive findings is.

The results are shown in Figure 3B.The expression signal (blueness) that the result shows miR-27b distribution range and intensity in the transgenic mice heart tissue all have enhancing.

The phenotype analytical of embodiment 4miR-27b transgenic mice

(1) mensuration of heart weight, body weight and shin bone length

Disconnected neck was put to death after mouse was weighed, take out heart and cut off great vessels on every side earlier, clean blood with physiological saline, filter paper blots residual liquid, take by weighing cardiac weight with electronic balance, vernier caliper measurement mouse shin bone length, the ratio of calculating cardiac weight and body weight or shin bone length, with the degree of these two index expression myocardial hypertrophies (Fig. 4 A, B).Fig. 4 A result shows that observing the heart of finding transgenic mice from the general form aspect obviously increases than control mice; Fig. 4 B result shows that the cardiac weight of transgenic mice and the ratio of body weight or shin bone length obviously increase than control mice.

(2) detection of molecular marker expression level

1. reverse transcription reaction

MRNA Selective PCR 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 conditions 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 conditions: 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 LightCyclerSoftware software and analyzes.The results are shown in Figure 4C.Fig. 4 C shows loose marker gene in the transgenic mice heart tissue, and as ANF, the expression level of β-MHC and SKA obviously raises than control group mice.

(3) Hematorylin-Yihong dyeing

1, fixing

Be organized in the neutral formalin damping fluid (40% formaldehyde 100ml, water 900ml, Sodium phosphate dibasic 4g, SODIUM PHOSPHATE, MONOBASIC 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 tissue block, 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 slide glass, add an amount of water again, sample is floated on the water surface.Slide glass is placed on about 40 ℃ film dryer platform, treats that sample launches, water can be outwelled gently, slide glass is closed in the Riker mount in 37 ℃ of dried overnight.

5, hematoxylin-eosin staining

Dimethylbenzene, 15 minutes;

Dimethylbenzene, 15 minutes;

Dehydrated alcohol, 15 minutes;

95% ethanol, 5 minutes;

70% ethanol, 5 minutes;

Water, 5 minutes;

Harris phenodin dye liquor (Hematorylin 2.5g, dehydrated alcohol 25ml, potassium alum 50g, red precipitate 1.25g, Glacial acetic acid 20ml, water 500ml), 5 minutes;

The flowing water flushing, 5-10 minute;

70% ethanol-hydrochloric acid (200ml70% ethanol+10 hydrochloric acid), 15-30 second;

70% ethanol-ammoniacal liquor (200ml70% ethanol+10 ammoniacal liquor), 1 minute;

Water, 5 minutes;

70% ethanol, 3 minutes;

Eosin Y dye liquor (the 0.5g Eosin Y is dissolved in the red B of 50mg flame of 5ml water, 50ml water, 2ml Glacial acetic acid, 390ml 95% ethanol), 1 minute;

95% ethanol, 5 minutes;

Dehydrated alcohol, 5 minutes;

Dimethylbenzene, 5 minutes;

Dimethylbenzene, 5 minutes.

6, neutral gum mounting

The result is shown in Fig. 5 A and B, and from heart tissue transverse section coloration result, the transgenic mice heart is than the control mice cardiac enlargement.

(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 suitable 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 suitable proportion dilution, hatched 30 minutes for 37 ℃.

7, PBS flushing, 5 minutes * 3 times.

8, drip the horseradish enzyme labelling strepto-avidin (PBS dilution) of suitable proportion dilution, hatched 30 minutes for 37 ℃.

9, PBS flushing, 5 minutes * 3 times.

10, chromogenic reagent.

11, water fully washes, and redyes mounting.

The result is shown in Fig. 5 C and D, and the result shows that transgenic mice myocardial cell size obviously increases than control mice.

(4) adult mice myocardial cell's separation and Ce Liangtaishi liquid formula (adjusting pH value to 7.4):

No calcium perfusate: tyrode's solution+10mM taurine

Enzyme liquid: tyrode's solution+10mM taurine+1mg/mL bovine serum albumin+0.6mg/mlII Collagen Type VI enzyme+calcium liquid (50 μ M)

II Collagen Type VI enzyme (warthington biochemical corporation): 394U/mg

Separation process:

1, open thermostat, temperature remains in 37 ℃, with no calcium perfusate flushing perfusion device, emptying bubble.

2, get mouse, disconnected neck is put to death, and opens chest and cores, and heart is placed ice-cold tyrode's solution.

3, heart is hung on the perfusion device about 5 minutes of the retrograde perfusion of row aorta.

4, use enzyme liquid perfusion about 15 minutes, then to the heart softness.

5, take off heart, cut ventricle, vibration is 5 minutes in the good enzyme liquid of incubation, supernatant discarded.

6, add enzyme liquid and continue vibration, per 5 minutes taking-up supernatants.

7, with the supernatant set, filter to centrifuge tube with filter screen, 500 rev/mins centrifugal, supernatant discarded.

8, add the cell culture fluid re-suspended cell, drop to slide, microscopic examination.

The results are shown in Figure 5E and F.The result shows that transgenic mice myocardial cell size obviously increases than control mice.

(5) Masson three-color process

1, tissue slice takes off cured to water.

2, ambient temperature overnight in Bouin ' s stationary liquid.

3, the color of stationary liquid 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% acetate 2 minutes.

12, Qie Pian differentiation, dehydration and mounting

The results are shown in Figure 6, result's demonstration is compared with control mice, and the transgenosis heart tissue at 3 monthly ages and 6 monthly ages tangible fibrosis occurs and changes (indigo plant is dyed the zone).

(6) transmission electron microscope observation of mouse heart tissue

1, tissue is cut into 1mm ³Fritter.

2,3% glutaraldehyde (1/15mol/L PBS, pH7.4) in, fix 2 hours before 4 ℃.

3, under 4 ℃, rinsing repeatedly in 1/15mol/L PBS+0.19mol/L sucrose damping fluid.

4,1% osmic acid (0.24mol/L PBS, pH7.4) in, fix 1 hour after 4 ℃.

5, in the 1/15mol/L PBS+0.19mol/L sucrose damping fluid, 4 ℃ of rinsings 15 minutes.

6, tissue dewatering:

50% ethanol 5-10 minute

70% ethanol 5-10 minute

90% ethanol 5-10 minute

90% ethanol+90% acetone (1: 1) 5-10 minute

90% acetone 5-10 minute

90% acetone 5-10 minute

100% acetone 10-15 minute

(above step is all carried out at 4 ℃)

100% acetone 10-15 minute (room temperature).

7,100% acetone: embedding medium (1: 1) room temperature was soaked 30 minutes, and embedding medium soaks into and spends the night.

8,35 ℃, 12 hours; 45 ℃, 12 hours; 60 ℃, carried out polymerization in 24 hours.

9, cut into slices on ultramicrotome, slice thickness is 60-70nm.

10, the dyeing of acetic acid uranium dye liquor lucifuge is 10 minutes, lead citrate dyeing 10 minutes.

11, electron microscopic observation.

The results are shown in Figure 7, the result shows and to compare with control mice, 3 the monthly age mouse the heart ultrastructure do not have obvious change, and 6 monthly ages transgenic mice heart ultrastructures take place obviously to change, showing that mainly myocardial mitochondria the cavity sample occurs and changes, is a kind of embodiment of injury of mitochondria.

(7) detection of mouse heart function

1. the ultransonic detection of mouse heart

After the mouse of different genotype is anaesthetized with tribromoethyl alcohol amine, remove the quilt hair of anterior pectorial region, 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 8A-H, the result shows that wall thickness (LVPWs) and left ventricular end diastolic heavy wall (LVPWd) are than the control mice attenuation behind the transgenic mice left ventricle end-systole; Transgenic mice left ventricular end-systolic dimension (Lvids) and left ventricular end diastolic dimension (Lvidd) obviously increase than control mice; The ratio of transgenic mice left ventricular mass and body weight (LVM/BW) obviously increases than control mice; Ejection fraction of transgenic mice (EF) and shortening mark (FS) obviously reduce than control mice.The cardiac systolic function of above results suggest myocardial cell's specificity miR-27b transgenic mice is impaired.

2. the mouse carotid arterial cannulation detects haemodynamics

(1) mouse anesthesia, four limbs open and are fixed on the operating table.

(2) chest median incision is separated subcutis, exposes right carotid.

(3) with the ligation of right carotid distal end, the proximal part folder closes.

(4) on right carotid, do an opening, millar conduit (1.4F) is inserted.

(5) BIOPAC16 road physiograph record arterial blood pressure signal.

(6) deeply, will pop one's head in and insert left ventricle, BIOPAC16 road physiograph record ventricular pressure signal through aorta with the millar conduit.

(7) AcqKnowledge software analysis processing data

The results are shown in Figure 8I, J, the result shows that the left ventricular end-diastolic pressure (LVEDP) of transgenic mice increases than control mice; The mean arterial pressure of transgenic mice mouse (MAP) has the trend of increase than control mice.This result has reflected that from another angle the heart function of transgenic mice is impaired.

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.

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Claims (6)

1. one kind contains the miRNA expression carrier, it is characterized in that, it comprises the miRNA gene of myocardial cell's specificity promoter α-MHC and one or more copies.

2. expression vector according to claim 1 is characterized in that, described miRNA is miR-27b.

3. expression vector according to claim 1 and 2 is characterized in that, the carrier that sets out is plasmid pRCH.

4. expression vector according to claim 3 is characterized in that, it comprises the miR-27b gene of people hGH gene and 2 copies.

5. the construction process of a miRNA transgene mouse model is characterized in that, it is that each described expression vector of claim 1-4 is imported mouse fertilized egg, then the zygote that obtains is moved into the mouse uterine tube and carries out gestation, obtains transgenic mice.

6. make up the transgenic mice that obtains by claim 5 or 6 described methods and be used for the treatment of application in the medicine of heart disease in screening.

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