CN109266679A - A kind of preparation method and application of BMPR2 gene mutation rat - Google Patents
A kind of preparation method and application of BMPR2 gene mutation rat Download PDFInfo
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- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The present invention provides a kind of preparation method and applications of BMPR2 gene mutation rat, belong to biomedicine technical field.The identification sequence that rat BMPR2 gene mutation provided by the invention is knocked in is under the auxiliary of gene editing carrier, the nucleic acid sequence of BMPR2 gene can be accurately identified, by being injected into fertilized eggs after transcribing vector in vitro, pass through zygote transplation again, obtain the Offspring rat of gene editing, then by mating mating, second filial rat is obtained.The rat model is induced by hypobaric hypoxia, shows close to human diseases phenotype, serious pulmonary hypertension, application value with higher.
Description
Technical field
The present invention relates to biomedicine technical fields, in particular to a kind of preparation side of BMPR2 gene mutation rat
Method and application.
Background technique
Pulmonary hypertension (pulmonary arterial hypertension, PAH) pathomechanism is complicated, poor prognosis,
5 years survival rates are only 57% (Chest 2012,142:448-456).Heredity pulmonary hypertension (heritable
Pulmonary arterial hypertension, HPAH) refer to that the pulmonary artery caused by being mutated by specific Disease-causing gene is high
Pressure.At present, it has been found that 7 PAH Disease-causing genes are (the Bone Morphogenetic of Bone Morphogenetic Protein receptor 2 respectively
Protein Receptor 2, BMPR2), Bone Morphogenetic Protein receptor 1B (BMPR1B), 1 (ACVRL1/ of activin acceptor class kinases
ALK1), vascular cell adhesion molecule (Endoglin, ENG), Smad albumen 9 (SMAD9), caveolin-1 (CAV1) are with potassium ion
Channel protein 3 (KCNK3).BMPR2 is the most important HPAH Disease-causing gene being currently known, and is accounted in idiopathic pulmonary hypertension
Than 20-40%, the accounting 50-80% in familial pulmonary hypertension.Compared to the PAH patient for not carrying BMPR2 gene mutation,
The patient clinical phenotype for carrying mutation is heavier, and treatment is more difficult, and patient's prognosis is worse, and mortality risk is higher.Therefore, HPAH has been
As the important diseases for seriously threatening human health, the burden of load-bearing is brought to family and society.
One can real simulation HPAH disease process, replicate disease phenotype animal model be explore HPAH disease machine
The important prerequisite of system, research and development newtype drug, but existing pulmonary hypertension animal model can not achieve object above, it is existing
Main problem is as follows:
1) Rats of Pulmonary Hypertension model, either monocrotaline (monocrotaline, MCT) and anoxic are commonly used at present
Induce pulmonary hypertension or anoxic joint VEGF inhibitor SU5416 induce pulmonary hypertension, be by specific environment or
The pulmonary hypertension that medicine irritation is induced is unable to pulmonary hypertension disease process and disease caused by real simulation gene mutation
Phenotype.
2) (including whole body knocks out existing mouse model, endothelial cell specific strikes based on BMPR2 gene knockout mostly
Except), some animals are that transgenosis is overexpressed saltant type BMPR2 gene, this carries BMPR2 gene with PAH patient in real world
The case where heterozygous mutant, simultaneously misfits, and can not also reflect the case where carrying BMPR2 missense mutation more than more than half patient.
3) existing BMPR2 gene modification mouse cannot simulate the Clinicopathologic phenotype of patient well.It is most small
Without spontaneous PAH, individual models (BMPR2+/-) mouse shows slight PAH in baseline level and pulmonary artery occurs mouse model
It thickens, but the lesser extent of Pulmonary Vascular pressure rise, right ventricle plumpness phenotype is also unobvious.Even if most mouse models exist
Under anoxic stimulation, pulmonary vascular resistance is also moderate raising.None mouse model shows PAH Patients with Lung plexus vasculosus
The characteristic of sample lesion.
Summary of the invention
The first object of the present invention is to provide a kind of preparation method of BMPR2 gene mutation rat, and this method can be prepared
Altitude simulation and duplication pulmonary hypertension disease model out.
The second object of the present invention is to provide the BMPR2 that the preparation method of BMPR2 gene mutation rat is prepared
Application of the gene mutation rat in screening treatment heredity pulmonary hypertension drug.
The third object of the present invention is what the preparation method for providing above-mentioned BMPR2 gene mutation rat was prepared
Application of the BMPR2 gene mutation rat in screening treatment induction property pulmonary hypertension drug.
In order to realize above-mentioned purpose of the invention, using following technical scheme:
A kind of preparation method of BMPR2 gene mutation rat is mutated rat BMPR2 gene, mutational site c.C1471T.
The BMPR2 gene mutation rat that the preparation method of above-mentioned BMPR2 gene mutation rat is prepared is controlled in screening
Treat the application in heredity pulmonary hypertension drug.
The BMPR2 gene mutation rat that the preparation method of above-mentioned BMPR2 gene mutation rat is prepared is controlled in screening
Treat the application in induction property pulmonary hypertension drug.
Compared with prior art, the beneficial effect comprise that
1) BMPR2 gene mutation knocks in rat and carries BMPR2 heterozygous mutation, is suffered to human inheritance's property pulmonary hypertension
The complete simulation of person's pathogenesis basis；
2) under hypobaric hypoxia induction, BMPR2 gene mutation knocks in rat and shows more serious pulmonary hypertension table
Type and closer to mankind's pulmonary artery remodeling Pathology, be to human inheritance's property patients with pulmonary hypertension disease phenotype more
Good simulation；
3) disease phenotype that rat is knocked in BMPR2 gene mutation is stablized, and is that research heredity and induction property pulmonary artery are high
The good model pressed pathomechanism, screen newtype drug.
Detailed description of the invention
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached
Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair
The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this
A little attached drawings obtain other relevant attached drawings.
Fig. 1 is that the rat model that experimental example of the present invention provides knocks out gene sequencing result schematic diagram；
Fig. 2 is the rat right ventricular systolic pressure result schematic diagram that experimental example of the present invention provides；
Fig. 3 is the rat right ventricular free wall heights Experiment result figure that experimental example of the present invention provides；
Fig. 4 is the rat tricuspid annulus displacement diagram that experimental example of the present invention provides；
Fig. 5 is the rat Right ventricular hypertrophy index results figure that experimental example of the present invention provides；
Fig. 6 is the lung tissue flesh thin vessels coloration result schematic diagram that experimental example of the present invention provides；
Fig. 7 is the lung tissue thin vessels schematic diagram that experimental example of the present invention provides.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art should
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
A kind of preparation method and application of BMPR2 gene mutation rat of the embodiment of the present invention is carried out specifically below
It is bright.
A kind of preparation method of BMPR2 gene mutation rat is mutated rat BMPR2 gene, mutational site c.C1471T.
Further, in preferred embodiments of the present invention, comprising the following steps:
Synthesis base sequence identifies sequence as shown in SEQ ID No.1-2 respectively, and by annealing renaturation at identification piece
Section；
It will identify that segment is connect with the linearized vector by digestion processing, obtain Cas9-BMPR2-RNA recombinant vector,
Fertilized eggs are transcribed and injected to Cas9-BMPR2-RNA recombinant vector, obtains injection fertilized eggs；
SD female mice is mated with the SD hero mouse ligatured, and chooses the SD female mice for seeing bolt, and injection zygote transplation is arrived
The ampulla for seeing the SD female mice of bolt, obtains Founder rat；
Founder rat is mated to obtain F with wild type SD rat1For rat and screening obtains F1 generation Bmpr2+/R491WRat；
By F1 generation Bmpr2+/R491WRat is divided into male heterozygosis mutation mouse and female heterozygous mutation mouse, and male heterozygosis is mutated
Mouse and female heterozygous mutation mouse mate in proportion with wild-type rats mate respectively, obtain BMPR2 mutation and knock in F2 for mouse；
By BMPR2 gene knock-in model mouse hypobaric hypoxia induce obtain within 2-3 weeks it is close to human diseases phenotype, serious
Pulmonary hypertension model mouse.
The Genetic Detection that 670 PAH patients have been completed over nearly 6 years finds that 144 patients carry BMPR2 gene mutation,
Wherein c.C1471T (p.R491W) is Chinese's hot spot mutation, and the frequency of mutation is up to 9.3%.Further, we are in French state
Discovery is verified in the database at family's Pulmonary Vascular disease center, R491W is also French hot spot mutation, accounting 6%.Therefore, we
Determine that R491W is the hot spot mutation that Chinese and western shares；The identification sequence is directed to the site BMPR2 gene c.C1471T (p.R491W)
It is modified, obtains pulmonary hypertension model mouse.
We have made BMPR2 gene p.R491W mutation using CRISPR/Cas9 technology and have knocked in rat model.The rat
Model is induced by hypobaric hypoxia, shows close to human disease conditions, serious pulmonary hypertension phenotype.Our invention
Important model mouse will be provided for the pathogenic pathomechanism of research and development BMPR2 mutation, research and development newtype drug, it is with higher to apply valence
Value.
Identify that sequence includes SEQ ID NO.1, i.e. sgRNA:R-bmpr2-gRNA up35 '
TAGGgaccaggatgcagaggct；
SEQ ID NO.2, i.e. R-bmpr2-gRNA down35 ' aaacAGCCTCTGCATCCTGGTC.
Further, in preferred embodiments of the present invention, BSAI is selected in digestion processing.
Further, in preferred embodiments of the present invention, SD female mice is 7-8 week old, and the SD female mice is big by SD female
Mouse injection hormone carries out super row and obtains.
Further, in preferred embodiments of the present invention, SD female rats are 3-4 week old.
Further, in preferred embodiments of the present invention, male heterozygosis mutation mouse and female heterozygous mutation mouse are pressed
It is mated according to the ratio of 1-3:1.
In an experiment, the week old breeding range of male mouse and female mice is wider, and preferably SD female mice is 7-8 week old, SD in the present invention
Female rats are 3-4 week old.
Further, in preferred embodiments of the present invention, hypobaric hypoxia condition is 40-65KPa, the O of 9%-12%2。
It further, further include that PCR identification is carried out to pulmonary hypertension model mouse in preferred embodiments of the present invention,
The base sequence of the primer of PCR identification is as shown in SEQ ID NO.3-4.
The upstream sequence of primer is as shown in SEQ ID NO.3:
R-BMPR2-MUT-S, SEQ ID NO.1:5 '-CACACATCTTTAATCCCAGCACTAG-3 '；
The downstream sequence of primer is as shown in SEQ ID NO.4:
R-BMPR2-MUT-A, SEQ ID NO.2:5 '-GAATTCAGATCACAGCACCAGAG.
The BMPR2 gene mutation rat that the preparation method of above-mentioned BMPR2 gene mutation rat is prepared is controlled in screening
Treat the application in heredity pulmonary hypertension drug.
The BMPR2 gene mutation rat that the preparation method of above-mentioned BMPR2 gene mutation rat is prepared is controlled in screening
Treat the application in induction property pulmonary hypertension drug.
The BMPR2 gene mutation rat that the above method is prepared, application and research treat the drug of pulmonary hypertension
In, which has preferable effect.
Feature and performance of the invention are described in further detail with reference to embodiments, and be repeated several times real
It tests.
Embodiment 1
The present embodiment provides a kind of preparation methods of BMPR2 gene mutation rat, pass through mutation rat BMPR2 gene
The site c.C1471T obtains the rat model that can preferably simulate pulmonary hypertension；Including synthesis base sequence respectively such as SEQ ID
Sequence is identified shown in No.1-2；Identify that sequence includes SEQ ID NO.1, i.e. sgRNA:R-bmpr2-gRNA up35 '
TAGGgaccaggatgcagaggct；
SEQ ID NO.2, i.e. R-bmpr2-gRNA down35 ' aaacAGCCTCTGCATCCTGGTC.
Include the following steps；
1.1 select the site (p.R491W) c.C1471T of rat BMPR2 gene as identification target spot, design identification sequence
The base sequence of sgRNA synthesizes as shown in SEQ ID NO.1-2 and identifies sequence as shown in SEQ ID NO.1-2；
1.2 by the single-stranded annealing renaturation of the base sequence of the sgRNA of synthesis at identification segment；Will identification segment be inserted by
On the carrier framework of BSAI linearization for enzyme restriction processing；Obtain Cas9-BMPR2-RNA recombinant vector；
1.3 carry the Cas9-BMPR2-RNA recombination that injectable is transcribed in vitro into Cas9-BMPR2-RNA recombinant vector
Body；
1.4 ligation SD rat hero mouse, and hormone ovulation rate is injected to the SD rat female mice of 3 week old；
1.5 take about 150 pieces of rat fertilized eggs and inject the Cas9-BMPR2-RNA recombinant vector through transcribing, obtain recombinating by
Smart ovum；
1.6 mate the SD female mice of 7 week old with the SD hero mouse ligatured, the SD female mice for seeing bolt are chosen, as receptor female mice；
1.7 will recombinate zygote transplation to receptor female mice ampulla of uterine tube, obtain Founder rat；
1.8 by the Founder rat of acquisition be F0It mates to obtain F with wild type SD rat for rat1In generation, passes through PCR amplification
F is obtained with sanger sequencing screening1For Bmpr2+/R491WRat, F1For Bmpr2+/R491WRat be divided into male heterozygosis mutation mouse and
Female heterozygous is mutated mouse, by F1The male heterozygosis mutation mouse and female heterozygous mutation mouse in generation mate mating according to 1:1 ratio and obtain F2
For Bmpr2+/R491WBMPR2 gene knockout model mouse, F2It is as shown in Figure 1 for the sequencing result of rat；
1.9 40kPa pressure, 9% O2Hypobaric hypoxia under the conditions of induce F2For Bmpr2+/R491WBMPR2 gene
It knocks out model mouse 2 weeks, obtains pulmonary hypertension model mouse.
Obtain pulmonary hypertension model mouse can stablize heredity carry out pass on.
The primer sequence that wherein PCR amplification detection uses is as shown in SEQ ID NO.3-4.
The upstream sequence of primer is as shown in SEQ ID NO.3:
R-BMPR2-MUT-S, SEQ ID NO.1:5 '-CACACATCTTTAATCCCAGCACTAG-3 '；
The downstream sequence of primer is as shown in SEQ ID NO.4:
R-BMPR2-MUT-A, SEQ ID NO.2:5 '-GAATTCAGATCACAGCACCAGAG.
Embodiment 2
The present embodiment provides a kind of preparation methods of BMPR2 gene mutation rat, pass through mutation rat BMPR2 gene
The site C1471T obtains the rat model that can preferably simulate pulmonary hypertension；Including synthesis base sequence respectively such as SEQ ID
Sequence is identified shown in No.1-2；Identify that sequence includes SEQ ID NO.1, i.e. sgRNA:R-bmpr2-gRNA up35 '
TAGGgaccaggatgcagaggct；
SEQ ID NO.2, i.e. R-bmpr2-gRNA down35 ' aaacAGCCTCTGCATCCTGGTC.
Include the following steps；
1.1 select the site (p.R491W) c.C1471T of rat BMPR2 gene as identification target spot, design identification sequence
The base sequence of sgRNA synthesizes as shown in SEQ ID NO.1-2 and identifies sequence as shown in SEQ ID NO.1-2；
1.2 by the single-stranded annealing renaturation of the base sequence of the sgRNA of synthesis at identification segment；Will identification segment be inserted by
On the carrier framework of BSAI linearization for enzyme restriction processing；Obtain Cas9-BMPR2-RNA recombinant vector；
1.3 carry the Cas9-BMPR2-RNA recombination that injectable is transcribed in vitro into Cas9-BMPR2-RNA recombinant vector
Body；
1.4 ligation SD rat hero mouse, and hormone ovulation rate is injected to the SD rat female mice of 4 week old；
1.5 take about 150 pieces of rat fertilized eggs and inject the Cas9-BMPR2-RNA recombinant vector through transcribing, obtain recombinating by
Smart ovum；
1.6 mate the SD female mice of 8 week old with the SD hero mouse ligatured, the SD female mice for seeing bolt are chosen, as receptor female mice；
1.7 will recombinate zygote transplation to receptor female mice ampulla of uterine tube, obtain Founder rat；
1.8 by the Founder rat of acquisition be F0It mates to obtain F with wild type SD rat for rat1In generation, passes through PCR amplification
F is obtained with sanger sequencing screening1For Bmpr2+/R491WRat, F1For Bmpr2+/R491WRat be divided into male heterozygosis mutation mouse and
Female heterozygous is mutated mouse, by F1The male heterozygosis mutation mouse and female heterozygous mutation mouse in generation mate mating according to 3:1 ratio and obtain F2
For Bmpr2+/R491WBMPR2 gene knockout model mouse, F2It is as shown in Figure 1 for the sequencing result of rat；
1.9 65kPa pressure, 12% O2Hypobaric hypoxia under the conditions of induce F2For Bmpr2+/R491WBMPR2 gene
It knocks out model mouse 3 weeks, obtains pulmonary hypertension model mouse.
Obtain pulmonary hypertension model mouse can stablize heredity carry out pass on.
The primer sequence that wherein PCR amplification detection uses is as shown in SEQ ID NO.3-4.
The upstream sequence of primer is as shown in SEQ ID NO.3:
R-BMPR2-MUT-S, SEQ ID NO.1:5 '-CACACATCTTTAATCCCAGCACTAG-3 '；
The downstream sequence of primer is as shown in SEQ ID NO.4:
R-BMPR2-MUT-A, SEQ ID NO.2:5 '-GAATTCAGATCACAGCACCAGAG.
Embodiment 3
The present embodiment provides a kind of preparation methods of BMPR2 gene mutation rat, pass through mutation rat BMPR2 gene
The site c.C1471T obtains the rat model that can preferably simulate pulmonary hypertension；Including synthesis base sequence respectively such as SEQ ID
Sequence is identified shown in No.1-2；Identify that sequence includes SEQ ID NO.1, i.e. sgRNA:R-bmpr2-gRNA up3 5 '
TAGGgaccaggatgcagaggct；
SEQ ID NO.2, i.e. 5 ' aaacAGCCTCTGCATCCTGGTC of R-bmpr2-gRNA down3., including following step
Suddenly；
1.1 select the site (p.R491W) c.C1471T of rat BMPR2 gene as identification target spot, design identification sequence
The base sequence of sgRNA synthesizes as shown in SEQ ID NO.1-2 and identifies sequence as shown in SEQ ID NO.1-2；
1.2 by the single-stranded annealing renaturation of the base sequence of the sgRNA of synthesis at identification segment；Will identification segment be inserted by
On the carrier framework of BSAI linearization for enzyme restriction processing；Obtain Cas9-BMPR2-RNA recombinant vector；
1.3 carry the Cas9-BMPR2-RNA recombination that injectable is transcribed in vitro into Cas9-BMPR2-RNA recombinant vector
Body；
1.4 ligation SD rat hero mouse, and hormone ovulation rate is injected to the SD rat female mice of 3 week old；
1.5 take about 150 pieces of rat fertilized eggs and inject the Cas9-BMPR2-RNA recombinant vector through transcribing, obtain recombinating by
Smart ovum；
1.6 mate the SD female mice of 8 week old with the SD hero mouse ligatured, the SD female mice for seeing bolt are chosen, as receptor female mice；
1.7 will recombinate zygote transplation to receptor female mice ampulla of uterine tube, obtain Founder rat；
1.8 by the Founder rat of acquisition be F0It mates to obtain F with wild type SD rat for rat1In generation, passes through PCR amplification
F is obtained with sanger sequencing screening1For Bmpr2+/R491WRat, F1For Bmpr2+/R491WRat be divided into male heterozygosis mutation mouse and
Female heterozygous is mutated mouse, by F1The male heterozygosis mutation mouse and female heterozygous mutation mouse in generation mate mating according to 2:1 ratio and obtain F2
For Bmpr2+/R491WBMPR2 gene knockout model mouse, F2It is as shown in Figure 1 for the sequencing result of rat；
1.9 50kPa pressure, 10% O2Hypobaric hypoxia under the conditions of induce F2For Bmpr2+/R491WBMPR2 gene
It knocks out model mouse 3 weeks, obtains pulmonary hypertension model mouse.
Obtain pulmonary hypertension model mouse can stablize heredity carry out pass on.
The primer sequence that wherein PCR amplification detection uses is as shown in SEQ ID NO.3-4.
The upstream sequence of primer is as shown in SEQ ID NO.3:
R-BMPR2-MUT-S, SEQ ID NO.1:5 '-CACACATCTTTAATCCCAGCACTAG-3 '；
The downstream sequence of primer is as shown in SEQ ID NO.4:
R-BMPR2-MUT-A, SEQ ID NO.2:5 '-GAATTCAGATCACAGCACCAGAG.
Experimental example
The pulmonary artery that this experimental example knocks out the knockout technique for the rat BMPR2 gene knockout that embodiment 3 provides is high
Pressure model mouse is evaluated.
By the F2 of 8 week old for Bmpr2+/R491WPressure of the BMPR2 gene knockout model mouse in 50kPa, 10% O2It is low
It forces down and induces 3 weeks under the conditions of oxygen, obtain pulmonary hypertension model mouse, while normal oxygen Bmpr is set2+/R491WRat and brood wild
Type rat is as control.
After hypobaric hypoxia stimulates 3 weeks, to anoxic and normal oxygen group Bmpr2+/R491WRat and the wild-type rats row ultrasound heart
Cardon and right heart catheter determine disease phenotype.After execution, coring is dirty to be separated left ventricle, right ventricle and weighs respectively, with the right heart
Left ventricle includes that interventricular septum (LV+S) weight obtains Right ventricular hypertrophy index (Fulton ' s Ratio) in room (RV) weight ratio.Take a left side
Upper lobe is fixed with formalin, does pathological study, is taken lower right lobe tissue liquid nitrogen flash freezer, is stored in -80 DEG C of refrigerators
It is analyzed for molecular level.
The right heart of ultrasonic cardiography graph evaluation reconstructs, and after rat weight, is anaesthetized using isoflurane-oxygen mixed gas (1.5%),
Net chesk hair is shaved, is placed in echocardiogram instrument detection platform (vevo2100, VisualSonics).Probe is placed in a breastbone left side
Side shows the left room long axis view of breastbone with midsternal line at 10 °~30 °.Probe rotates clockwise 90 ° of left room short axles of display and cuts
Face.M type curve is taken by two dimensional image guidance and is measured.Popping one's head in, how general carry out to the mobile display pulmonary artery short axis view in head
Strangle blood flow detection.Rat head is downwardly inclined, probe shows the anxious face of apical four-chamber to the apex of the heart at xiphoid-process, and it is right to record left room
Room size, the displacement of M type curved measurement tricuspid annulus.Body tilts to the left after rat is returned to left room long axis view, probe from
Right ventricle's short axis view is obtained on the right side of breastbone guides M type curved measurement right ventricular free wall thickness.Selection clearly image video recording to
With post analysis.The standard that ultrasonic measurement is formulated according to U.S.'s echocardiogram association, analysis indexes include heart rate, diastole and contraction
Each chamber chamber transverse diameter of latter stage heart and vertical diameter, right ventricular free wall thickness, chamber interval thickness, right ventricle shortening fraction, tricuspid
The displacement of annulus systole phase, ejection fraction, cardiac output, stroke output etc..
Right heart catheter assesses haemodynamics
Right heart catheter detection pulmonary artery blood hydromechanics and right ventricle cardiac output are the gold for assessing pulmonary hypertension model
Standard.Rat is weighed, gives yellow Jackets (1%) anesthesia by 40mg/kg.Connect polygraph (Powerlab/
8sp) and its matched pressure transducer, pressure transducer and conduit are full of with heparinized saline.Rat is lain on the back, four limbs
And head is fixed on operating table.Art portion is wiped with cotton ball soaked in alcohol, cuts off skin above the manubrium at 1cm, blunt separation is right
Side subcutaneous tissue, exposure right side vena jugularis externa, blunt separation blood vessel, distal end ligature, and proximal end surgical thread beats section.Conduit is placed on
With heart the same horizontal position, instrument zeroing.The vena jugularis externa of separator well is cut off into an osculum with eye scissors, PE-50 is led
Pipe is sent into, and is tensed surgical thread and is fixed.Conduit is gently sent into the chambers of the heart, in case (2cm or so enters superior vena cava to pneumothorax, and 4cm is left
It is right to enter right ventricle), steering nozzle, observes and records waveform to left down.
As a result as shown in Fig. 2, right heart catheter detection is shown under normal oxygen bar line, R491W is mutated rat and wild-type rats are right
It is not significantly different between ventricular systolic pressure, does not observe the mutation spontaneous pulmonary hypertension of rat temporarily.Through 10% hypoxia inducible
Afterwards, wild-type rats and when R491W mutation rat right ventricular systolic pressure more normal oxygen increase respectively 39.0% (P=0.001) and
74% (P < 0.001) shows the success of hypoxia inducible pulmonary hypertension model.And right ventricular systolic pressure after R491 mutation Rats Exposed To Hypoxia
The highest in three groups increases 25% (P=0.002) compared with wild type Hypoxic Rats, the results showed that R491W mutation causes big
Mouse pulmonary hypertension is heavier.
As shown in Fig. 3, Fig. 4 and Fig. 5, the left side is wild-type rats in figure, and the right is saltant type rat；Rat is super by row
Sound cardiogram takes M type curve in right ventricle short axis view, measures right ventricular free wall thickness (RVWT)；It is anxious in apical four-chamber
Face shows tricuspid valve, and M type curve, measurement tricuspid annulus displacement (TAPSE) are taken at tricuspid annulus.
Under normoxic condition, R491W is mutated rat and wild-type rats right ventricular free wall thickness, tricuspid annulus displacement without bright
Significant difference is different.
After anoxic, the right ventricular free wall of wild-type rats and R491W mutation rat right ventricular is significantly increased, and
The right free wall thickening of R491W mutation rat becomes apparent compared with wild type, is 1.2 times (P < 0.001) of wild-type rats.And
Wild-type rats and R491W mutation rat tricuspid annulus displacement significantly reduce after anoxic, and more normal oxygen rat reduces respectively
19% (P=0.005) and 25% (P=0.001).
Therefore, R491W mutation rat is even more serious compared to the right heart reconstruct of wild-type rats after anoxic, is mainly shown as the right side
Room free wall is thicker, and tricuspid annulus displacement is smaller.The left ventricle of rat heart and right ventricle are separated and weighed along interventricular septum, is counted
Calculate the right heart/left heart+interventricular septum ratio (Fulton ' s ratio).Hypoxia inducible makes R491W mutation rat and wild as the result is shown
Type rat right ventricular reconstructs, and the right heart/left heart+interventricular septum ratio significantly increases.And R491W is mutated the Rat Right heart after anoxic
It is high compared with wild-type rats by 32.0% (P=0.004) that room accounts for heart specific gravity, shows that right ventricle plumpness degree is even more serious.
Pathologic state colony assesses pulmonary artery remodeling
Dehydration embedding is at paraffin mass after lung tissue of rats is fixed, row HE dyeing, elastic fibers dyeing after paraffin section, with flat
Alpha Actinin (α-SMA) antibody of sliding flesh specificity does immunohistochemical staining.Count lung tissue flesh thin vessels (<
75um) ratio and media thickness.
Take lung tissue row histopathology HE dyeing, elastic fibers dyeing and SMA immunohistochemical staining.Each tissue
Slice chooses about 30 visuals field and takes pictures, and chooses the lung parteriole of diameter≤75 μm, whether has smooth muscle layer according to parteriole blood vessel
And whether smooth muscle layer forms complete lumen of vessels and lung parteriole is divided into non-flesh, part flesh and complete flesh respectively
Thin vessels.
As a result as shown in Figure 6 and Figure 7, A indicates lung tissue flesh thin vessels ratio in Fig. 6, and B indicates that lung tissue α-SMA exempts from
Flesh thin vessels are shown in epidemic disease histochemical stain, Normoxia: normal oxygen；Hypoxia: anoxic, WT: brood wild-type rats；
R491W:Bmpr2+/R491WRat is knocked in mutation；A indicates lung tissue vessel wall thickness, lung tissue HE, α SMA immuning tissue in Fig. 7
Flesh thin vessels wall thickness is shown in chemical staining and elastic fibers dyeing, H.E.: haematoxylin eosin stains；SMA: α smooth muscle flesh of α is dynamic
Albumen；VEG: elastic fibers dyeing.
Count results show that R491W mutation rat flesh thin vessels (< 75um) quantity accounting is significantly higher than wild type,
In the thin vessels ratio of flesh completely 67.0% (P=0.001) is significantly increased compared with wild type.With Image Pro Plus software
The percentage (media thickness/(media thickness+intravascular space diameter) x 100%) of analysis measurement lung parteriole media thickness, knot
Fruit shows Bmpr2+/R491WPulmonary Vessels in Rats wall middle layer is more plump compared with wild type.The above structure shows that R491W is mutated induced lung
Vascular remodeling is more serious.
In conclusion the identification sequence of rat BMPR2 gene knockout provided in an embodiment of the present invention can be identified accurately
Gene knockout site, by constructing the carrier of gene knockout and being injected into rat fertilized eggs, and by the zygote transplation of injection
Rat obtains the rat of gene knockout, and induces to obtain pulmonary hypertension model mouse by hypobaric hypoxia, the pulmonary hypertension mould
Type mouse shows typical symptom, illustrates modeling success, and effect is preferable.
Embodiments described above is a part of the embodiment of the present invention, instead of all the embodiments.The present invention is implemented
The detailed description of example is not intended to limit the range of claimed invention, but is merely representative of selected implementation of the invention
Example.Based on the embodiments of the present invention, obtained by those of ordinary skill in the art without making creative efforts
Every other embodiment, shall fall within the protection scope of the present invention.
SEQUENCE LISTING
<110>Fu Wai Hospital, Chinese Academy of Medical Sciences, Baoding Long March cardiopulmonary angiosis research institute
<120>a kind of preparation method and application of BMPR2 gene mutation rat
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> DNA
<213>artificial sequence
<400> 1
tagggaccag gatgcagagg ct 22
<210> 2
<211> 22
<212> DNA
<213>artificial sequence
<400> 2
aaacagcctc tgcatcctgg tc 22
<210> 3
<211> 25
<212> DNA
<213>artificial sequence
<400> 3
cacacatctt taatcccagc actag 25
<210> 4
<211> 23
<212> DNA
<213>artificial sequence
<400> 4
gaattcagat cacagcacca gag 23
Claims (10)
1. a kind of preparation method of BMPR2 gene mutation rat, which is characterized in that mutation rat BMPR2 gene, mutational site is
c.C1471T。
2. the preparation method of BMPR2 gene mutation rat according to claim 1, which comprises the following steps:
Synthesis base sequence identifies sequence as shown in SEQ ID No.1-2 respectively, and by annealing renaturation at identification segment；
The identification segment is connect with the linearized vector by digestion processing, obtains Cas9-BMPR2-RNA recombinant vector,
Fertilized eggs are transcribed and injected to the Cas9-BMPR2-RNA recombinant vector, obtains injection fertilized eggs；
SD female mice is mated with the SD hero mouse ligatured, and chooses the SD female mice for seeing bolt, and the injection fertilized eggs are moved
The ampulla for seeing the SD female mice of bolt is planted, Founder rat is obtained；
The Founder rat is mated with wild type SD rat obtain F1 generation rat and screening obtain F1 generation Bmpr2+/
R491W rat；
The F1 generation Bmpr2+/R491W rat is divided into male heterozygosis mutation mouse and female heterozygous mutation mouse, the male is miscellaneous
It closes mutation mouse and female heterozygous mutation mouse mates in proportion with wild-type rats mate respectively, obtain BMPR2 gene mutation
F2 is knocked in for mouse；
By the BMPR2 gene mutation knock in model mouse hypobaric hypoxia induce obtain within 2-3 weeks it is close to human diseases phenotype, tight
The pulmonary hypertension model mouse of weight.
3. the preparation method of BMPR2 gene mutation rat according to claim 2, which is characterized in that the digestion processing
Select BSAI.
4. the preparation method of BMPR2 gene mutation rat according to claim 2, which is characterized in that the SD female mice is
7-8 week old, the SD female mice carry out super row by SD female rats injection hormone and obtain.
5. the preparation method of BMPR2 gene mutation rat according to claim 4, which is characterized in that the SD female is big
Mouse is 3-4 week old.
6. the preparation method of BMPR2 gene mutation rat according to claim 2, which is characterized in that the male heterozygosis
Mutation mouse and female heterozygous mutation mouse mate according to the ratio of 1-3:1.
7. the preparation method of BMPR2 gene mutation rat according to claim 2, which is characterized in that the hypobaric hypoxia
Condition is 40-65KPa, the O of 9%-12%2。
8. the preparation method of BMPR2 gene mutation rat according to claim 2, which is characterized in that further include to described
Pulmonary hypertension model mouse carries out PCR identification, and the base sequence of the primer of the PCR identification is as shown in SEQ ID NO.3-4.
9. the BMPR2 gene mutation rat that the preparation method of BMPR2 gene mutation rat as described in claim 1 is prepared
Application in screening treatment heredity pulmonary hypertension drug.
10. the BMPR2 gene mutation that the preparation method of BMPR2 gene mutation rat as described in claim 1 is prepared is big
Application of the mouse in screening treatment induction property pulmonary hypertension drug.
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| CN112342241A (en) * | 2020-04-08 | 2021-02-09 | 中国人民解放军总医院第四医学中心 | Light-operated bone morphogenetic protein receptor system OptoBMPR and construction method and application thereof |
| WO2021256647A1 (en) * | 2020-06-19 | 2021-12-23 | 숙명여자대학교산학협력단 | Mutation complex including gain-of-function mutant of bmpr2 gene, induced pluripotent stem cells and mesenchymal stem cells derived from mutant, and use thereof |
| WO2021256646A1 (en) * | 2020-06-19 | 2021-12-23 | 숙명여자대학교산학협력단 | Novel gain-of-function mutant of bmpr2 gene and use thereof |
| CN113913434A (en) * | 2021-10-11 | 2022-01-11 | 湖北省妇幼保健院 | BMPR2 gene mutant and application thereof |
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| CN112342241B (en) * | 2020-04-08 | 2021-11-12 | 中国人民解放军总医院第四医学中心 | Light-operated bone morphogenetic protein receptor system OptoBMPR and construction method and application thereof |
| WO2021256647A1 (en) * | 2020-06-19 | 2021-12-23 | 숙명여자대학교산학협력단 | Mutation complex including gain-of-function mutant of bmpr2 gene, induced pluripotent stem cells and mesenchymal stem cells derived from mutant, and use thereof |
| WO2021256646A1 (en) * | 2020-06-19 | 2021-12-23 | 숙명여자대학교산학협력단 | Novel gain-of-function mutant of bmpr2 gene and use thereof |
| CN113913434A (en) * | 2021-10-11 | 2022-01-11 | 湖北省妇幼保健院 | BMPR2 gene mutant and application thereof |
| CN116267798A (en) * | 2022-12-19 | 2023-06-23 | 中国医学科学院阜外医院 | Establishment and application of spontaneous pulmonary artery high-pressure model |
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