CN116267798A - Establishment and application of spontaneous pulmonary artery high-pressure model - Google Patents
Establishment and application of spontaneous pulmonary artery high-pressure model Download PDFInfo
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Abstract
The invention discloses establishment and application of a spontaneous pulmonary artery high pressure model, which constructs a spontaneous pulmonary artery high pressure non-human animal model by expressing BMPR2 muteins. The invention also provides a method for identifying a therapeutic agent for treating pulmonary hypertension for non-therapeutic and non-diagnostic purposes, and also provides application of the BMPR2 mutant protein or a nucleotide sequence for synthesizing the same in constructing a spontaneous pulmonary hypertension non-human animal model. The invention provides a research tool for the research work of pulmonary hypertension and a research model for continuously exploring the pathogenesis related to pulmonary hypertension.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to establishment and application of a spontaneous pulmonary artery high-pressure rat model.
Background
Pulmonary arterial hypertension (Pulmonary Arterial Hypertension, PAH) is a type of malignant pulmonary vascular disease characterized by progressive increases in pulmonary vascular resistance, ultimately leading to patient death from severe right ventricular failure. PAH prognosis is extremely poor, with early untreated patients with median survival of 2.8 years after diagnosis and survival rate of only 21% 5 years.
Gene mutation is an important cause of pulmonary hypertension. Hereditary pulmonary arterial hypertension (heritable pulmonary arterial hypertension, HPAH) is a monogenic autosomal dominant inherited disease. BMPR2 is the first discovered HPAH causative gene, and is also the most prominent HPAH causative gene known at present, accounting for 14-30% of idiopathic pulmonary hypertension etiology and 60-70% of familial pulmonary hypertension etiology. PAH patients carrying BMPR2 mutations had an average 7 years earlier onset than non-carriers, had worse clinical phenotypes and had a higher risk of mortality. Therefore, the BMPR2 mutation plays a very important role in pulmonary hypertension occurrence, development and prognosis, and the molecular mechanism of the pathogenic BMPR2 mutation is very important for understanding pulmonary artery vascular remodeling disease mechanism and developing novel treatment means, and is also a hotspot and a difficulty in the current global pulmonary hypertension research field.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, it is an object of the present invention to provide a method for creating and applying a spontaneous pulmonary arterial hypertension model for solving the problems of the prior art.
The first aspect of the invention provides a method for constructing a spontaneous pulmonary artery high pressure non-human animal model, wherein the spontaneous pulmonary artery high pressure non-human animal model expresses BMPR2 mutant protein.
Further, the amino acid sequence of the BMPR2 mutant protein is shown as SEQ ID NO. 1.
BMPR2 is called bone morphogenic protein receptor type 2, and human BMPR2 gene ID is 659. The BMPR2 gene is highly conserved in evolution, and the amino acid sequence has high homology among various species. The rat Bmpr2 has a gene ID of 140590 and comprises a gene, a protein coded by the gene, a homolog of the gene, mutation and the like. The term "BMPR2" encompasses the full length, unprocessed genes or proteins of different species, as well as any form of genes or proteins derived from processing in a cell. The term encompasses naturally occurring variants of the biomarker. The gene ID is available at https:// www.ncbi.nlm.nih.gov/gene.
The BMPR2 mutant protein is protein expressing an amino acid sequence shown in SEQ ID NO. 1, a nucleic acid molecule for synthesizing the BMPR2 mutant protein is obtained by carrying out nucleotide mutation modification on a BMPR2 gene, the BMPR2 protein after mutation contains p.R491W mutation, the BMPR2 mutation is of a murine mutant type, and the BMPR2 mutation is artificially synthesized.
The term "expression" refers to determining the amount or presence of an RNA transcript of an intrinsic gene or its expression product. Including any steps associated with the manufacture of polypeptides, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
Further, the non-human animal model is selected from the group consisting of mammals.
Further, the mammal includes rodents, carnivores, winged animals, hedgehog animals, and vermin animals.
In some embodiments, the non-human animal is a mammal. The non-human animals include any of those that can be genetically modified to express nucleotide sequences as disclosed herein, e.g., mammals, e.g., mice, rats, rabbits, pigs, cows (e.g., cows, bulls, buffalo), deer, sheep, goats, chickens, cats, dogs, ferrets, primates (e.g., wool, macaque), and the like. For example, for those non-human animals for which suitable genetically modifiable ES cells are not readily available, other methods are employed to make non-human animals comprising the genetic modification. The methods include, for example, modifying a non-ES cell genome (e.g., a fibroblast or induced pluripotent cell) and transferring the genetically modified genome to a suitable cell, such as an enucleated oocyte, using Somatic Cell Nuclear Transfer (SCNT), and seeding the modified cell (e.g., modified oocyte) in a non-human animal under suitable conditions to form an embryo.
The term "non-human animal model" refers to a non-human animal that has or exhibits characteristics of a disease or condition. By animal model is meant any use of the animal for studying a disease or condition, for example for studying progression or development or response to a new therapy or an existing therapy.
Further, the rodent includes a species of the hamster, murine, equine island murine, spiny murine, mole murine, spiny murine, and rock murine families.
Further, the murine species include black rats, brown rats, gerbils, new world rats, old world rats, SD rats, bolnesia rats, tree rats, wood rats, stick rats, rice rats, kangaroo rats, and climbing rats.
In some embodiments, rodents of the present disclosure include, as non-limiting examples, mice, rats, and hamsters. In some embodiments, rodents of the present disclosure include, as non-limiting examples, mice and rats. In some embodiments, the rodent is selected from the general family of rats (muroide). In some embodiments, the rodents of the present disclosure are from a family selected from the group consisting of: the species of hamster (Calomyscidae) (e.g., hamster, baryotus hamster, gu Shili hamster, cricetrimus, hao Shili hamster, hamster bearded, chu Shili hamster, glatiramer hamster), the species of hamster (Cricetidae) (e.g., black line hamster, field mice, crown mice), the species of murine (Muridae) (black mice, brown mice, gerbil, new world rats, old world rats, norway species of rats, borilysia rats, tree rats, cotton rats, wood rats, stick rats, rice rats, kangarter rats, chinaroot), the species of masomidae (Nesomyidae) (long tail rats, south africa rats, african rats, ma Daobai tail rats), the species of acanthaceae (plaatacantomyidae) (e.g., acanthus, pigtail rats), and the species of spaalactaridae (e.g., mole, bamboo rats and zokorea). In some embodiments, the rodents of the present disclosure are selected from the group consisting of mice or rats (murine), gerbils, spines, and coronaries. In some embodiments, the rats of the present disclosure are from members of the murine family (Muridae).
In a specific embodiment of the invention, the rodent is a rat.
Further, the construction method comprises the following steps: and (3) carrying out nucleotide mutation modification on the BMPR2 gene in the single embryo cell or the embryo stem cell by utilizing a gene locus knock-in method so that the mutated BMPR2 protein contains p.R491W mutation.
The term "single embryo cell" refers to a cell that has cell totipotency and is capable of growing into an intact individual after differentiation, including fertilized eggs, embryonic cells, and the like.
The term "embryonic stem cell" refers to a stem cell that can differentiate into embryonic cells having different functions, at different sites and at different times. Embryonic stem cells are derived from a class of cells isolated from early embryos or primitive gonads, have a high proliferative capacity and a multipotent differentiation potential, and can differentiate into primitive cells of all cell types of the three germ layers. The ES cells are classified into ES cells according to the source of the embryonic stem cells, and the ES cells are derived from blastula stage; EC cells, cells with proliferation and self-renewal capacity isolated from teratocarcinoma; EG cells isolated from primordial germ cells.
Further, the method adopted by the BMPR2 gene for carrying out nucleotide mutation modification is a gene knock-in technology.
Further, the gene knock-in technique is a CRISPR/Cas9 gene editing method.
Further, the construction method comprises the following steps:
1) Designing a gene knock-in strategy aiming at a BMPR2 gene, wherein a knock-in site selects a nucleotide site corresponding to 491 amino acid site of a BMPR2 protein;
2) sgRNA targeting vectors and donor oligonucleotides were designed;
3) The p.r491w mutation site in the donor oligonucleotide will introduce the corresponding exon by homology-directed repair; for the p.R491W mutation, silent mutation needs to be introduced to prevent sgRNA binding and re-cleavage of sequences after homology-directed repair;
4) Mixing and introducing the Cas9 mixed sample system into single embryo cells or embryonic stem cells to obtain transformed cell embryos;
5) Culturing the transformed cell embryo to obtain the spontaneous pulmonary artery high-pressure non-human animal model.
The term "vector" refers to a composition that facilitates transduction of a cell by a selected nucleic acid or expression of the nucleic acid in a cell. Vectors include, for example, plasmids, cosmids, viruses, YACs, bacteria, polylysines, chromosomal integration vectors, episomal vectors, and the like.
Further, the Cas 9-like system includes sgrnas and mrnas.
Further, the spontaneous pulmonary arterial hypertension non-human animal model appears at the F3 generation.
Further, the specific sequence of the sgRNA is shown as SEQ ID NO: 2. SEQ ID NO: 3.
Further, the nucleotide mutation modification of the BMPR2 gene comprises the modification of 1471 nucleotide on the sequence of the wild BMPR2 gene from C mutation to T.
Further, the introduction method includes electroporation, calcium phosphate method, liposome method, DEAE dextran method, microinjection, and virus infection.
Further, the vectors include retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, poxviruses, baculoviruses, papillomaviruses, papovaviruses, phages, plasmids.
The term "sgRNA" refers to guide RNA (gRNA), also known as small guide RNA (sgRNA), as a post-transcriptional modification process called RNA editing in the plastid. SgRNA is a small non-coding RNA that can be paired with pre-mRNA and into which some uracil is inserted, resulting in mRNA that has an effect. The guide RNA edited RNA molecule, which is approximately 60-80 nucleotides in length, is transcribed from a single gene, has a 3 'oligo U tail, has a sequence in the middle that is precisely complementary to the mRNA being edited, and has an anchor sequence at the 5' end that is complementary to the sequence of the non-edited mRNA.
The method for constructing the spontaneous pulmonary artery high-pressure non-human animal model provided by the invention further comprises the following steps:
1) Constructing an over-expression vector of the BMPR2 mutant protein, and introducing the vector into a single embryo cell or an embryonic stem cell to obtain a transformed cell embryo;
2) Culturing the transformed cell embryo to obtain the spontaneous pulmonary artery high-pressure non-human animal model.
Further, the BMPR2 mutant protein comprises an amino acid sequence formed by mutating an R into a W at 491 amino acid on a wild type BMPR2 protein sequence.
Further, the nucleic acid overexpression vector of the BMPR2 mutant protein comprises a nucleotide sequence formed by mutating 1471 st nucleotide from C to T on the sequence of a wild type BMPR2 gene.
The terms "nucleic acid", "nucleotide" or "polynucleotide" as used herein are interchangeable and refer to any length of nucleic acid polymer, including DNA and RNA. The nucleotide may be a deoxyribonucleotide, a ribonucleotide, a modified nucleotide or base, and/or an analog thereof, or any substrate that can be incorporated into a polymer by a DNA or RNA polymerase, or by a synthetic reaction.
Further, the spontaneous pulmonary arterial hypertension non-human animal model appears at the F3 generation.
Further, the vectors include retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, poxviruses, baculoviruses, papillomaviruses, papovaviruses, phages, plasmids.
The term "vector" refers to a vector into which a polynucleotide encoding a protein may be operably inserted to cause expression of the protein. Vectors may be used to transform, transduce or transfect host cells such that they express the carried genetic element within the host cells. Examples of vectors include plasmids; phagemid; cosmids and artificial chromosomes, such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC) or P1 derived artificial chromosome (PAC); phage, such as lambda phage or M13 phage; and animal viruses. Classes of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV 40). The vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
Further, the introduction method includes electroporation, calcium phosphate method, liposome method, DEAE dextran method, microinjection, and virus infection.
Further, the spontaneous pulmonary arterial hypertension non-human animal model exhibits one or more symptoms of pulmonary arterial distension, right heart hypertrophy, elevated pulmonary arterial pressure, severe pulmonary vascular remodeling.
In a second aspect, the invention provides a spontaneous pulmonary arterial hypertension non-human animal model prepared by the construction method described above.
In a third aspect the present invention provides a method for identifying a therapeutic agent for the treatment of pulmonary hypertension for non-therapeutic non-diagnostic purposes, the method comprising the steps of:
1) Administering an agent to the aforementioned spontaneous pulmonary arterial hypertension non-human animal model animal;
2) Detecting whether the model animal has a change of pulmonary arterial hypertension related abnormal symptoms after administration of the medicament;
3) An agent is identified as a therapeutic agent when administration of the agent has a therapeutic effect on pulmonary arterial hypertension-related abnormal symptoms.
The term "treatment" refers to clinical intervention in an attempt to alter the natural course of the individual being treated, either for prophylaxis or in the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of a disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating a disease state, and eliminating or improving prognosis.
In a fourth aspect the invention provides the use of a spontaneous pulmonary hypertension non-human animal model derived from the spontaneous pulmonary hypertension non-human animal model described hereinbefore for screening for a medicament for the treatment or prophylaxis of pulmonary hypertension for non-therapeutic purposes.
In a fifth aspect, the invention provides application of a BMPR2 mutant protein or a nucleotide sequence for synthesizing the same in constructing a spontaneous pulmonary hypertension non-human animal model, wherein the amino acid sequence of the BMPR2 mutant protein is shown as SEQ ID NO. 1.
Further, the BMPR2 mutant protein comprises an amino acid sequence formed by mutating an R into a W at 491 amino acid on a wild type BMPR2 protein sequence.
Further, the coding sequence of the BMPR2 mutein comprises the following sequences: the 1471 nucleotide of the wild BMPR2 gene is a nucleotide sequence formed by mutating C into T.
A sixth aspect of the invention provides the use of a spontaneous pulmonary arterial hypertension non-human animal model for assessing the therapeutic effect of a product for treating pulmonary arterial hypertension using the spontaneous pulmonary arterial hypertension non-human animal model described hereinbefore.
Drawings
FIG. 1 is a diagram of construction and genotyping of BMPR2 mutant rats, wherein A is a schematic diagram of sgRNA for constructing BMPR2 point mutation, B is a PCR amplification result of a rat genomic DNA template, C is a sequencing peak diagram of positive rat genotyping PCR products, and red arrows show point mutation sites;
FIG. 2 is Bmpr2 +/R491W Schematic passaging of mutant rats;
FIG. 3 is a test of Bmpr2 +/R491W Echocardiography of mutant rat right ventricle structure and function, wherein a.bmpr2 +/R491W Rats did not spontaneously PAH; bmpr2 +/R491W Spontaneous generation of PAH; BMPR2+ & gt R491W Group rats were subjected to ultrasonic testing for right ventricular pre-diastole wall thickness (RVAW; d) at 3 months; bmpr2 +/R491W Rats did not spontaneously PAH; E.Bmpr2 +/R491W Spontaneous generation of PAH; BMPR2+ & gt R491W Group rats (red) were sonicated at 3 months for right ventricular outflow tract width;
FIG. 4 is a graph of right heart catheter detection BMPR2 mutant rat pulmonary artery pressure, wherein A.Bmpr2 +/R491W Rats have no spontaneous PAH, and the mPAP of the non-diseased group rats does not exceed 25mmHg; bmpr2 +/R491W Spontaneous PAH production, mPAP in rats from the affected group all exceeded 25mmHg; C. bmpr2 at elevated pressure in three spontaneous pulmonary arteries +/R491W 2 mutant rats mPAP was significantly higher than non-diseased rats (P<0.001)；
FIG. 5 is Bmpr2 +/R491W Mutant rat histopathological staining pattern, wherein A.Bmpr2 +/R491W Rats did not spontaneously PAH; bmpr2 +/R491W Spontaneous generation of PAH; statistical graph of percentage of thickness of blood vessel medium film below 50 μm; d, a statistical chart of the percentage of the thickness of the blood vessel medium film of 50-100 mu m; * P (P)<0.05，**P<0.01。
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it should be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials similar or identical to those described in the present specification may be used in the practice or testing of the present invention.
Example 1 Gene editing, rat breeding and genotyping
1. Experimental method and procedure
1. Gene editing site-directed knock-in BMPR2 mutations
1) The c.C1471T (p.R491W) locus of the rat BMPR2 gene is selected as a recognition target, and a recognition sequence sgRNA is designed, wherein the sgRNA sequence is as follows: R-Bmpr2-gRNA up35' TAGGgaccaggatgcagaggct (SEQ ID NO: 2); R-Bmpr2-gRNA Down35' aaacAGCCTCTGCATCCTGGTC (SEQ ID NO: 3).
2) Annealing and renaturating the clipping sequence single chain of the synthesized sgRNA into an identification fragment, inserting the identification fragment into a BSA I linearized carrier framework to obtain a Cas9-Bmpr2-RNA recombinant carrier, and carrying out in vitro transcription on the constructed sgRNA carrier to obtain a Cas9 mixed sample system.
3) Seminiferous ligation of SD rats Male mice 10 SD mice 3-4 weeks old were injected with hormones to promote superovulation.
4) Taking about 150 rat fertilized eggs, and injecting a transcribed Cas9-Bmpr2-RNA recombinant vector mixed sample system into the rat fertilized egg pronucleus to obtain the recombinant fertilized eggs.
5) SD female mice with the age of 8 weeks are mated with ligature male mice, and SD female mice with thrombus are selected as receptor female mice.
6) Transplanting the fertilized eggs after injection to the ampulla of the oviduct of the recipient rat to obtain the postnatal identification genotype of the F0-generation rat.
2. Rat breeding and genotyping
1) Bmpr2+/R491W transgenic SD rats were supplied by animal institute of China medical science sciences; all experimental rats are fed in SPF-class animal houses, the growth condition of the rats is closely observed in the feeding process, the padding is changed once a week, and the feed and the drinking water are supplemented every day.
2) Bmpr2+/R491W transgenic male heterozygous rats and wild female rats were treated with 1:1 proportion mating, post-experiments were performed using Bmpr2+/R491W transgenic male heterozygous rats.
3) Rats were grown to 5 week old with tail cuts of about 2-3mm and rat tail genome DNAPCR amplification was extracted using the dna extraction detection kit (day root, KG 203).
4) Designing a detection primer according to the sequence information, wherein the upstream sequence of the BMPR2 primer is 5'-CACACATCTTTAATCCCAGCACTAG-3' (SEQ ID NO: 4); the downstream sequence was 5'-GAATTCAGATCACACAGACCAGAG-3' (SEQ ID NO: 5).
5) TaKaRa RR042A PCR reaction system is shown in Table 1
TABLE 1PCR reaction System
6) The PCR amplification procedure is shown in Table 2
TABLE 2PCR amplification procedure
7) After the PCR reaction was terminated, a 5 Xloading buffer was added.
8) Electrophoresis was performed on a 1% agarose gel.
9) A band with the same size as the wild rat was selected for Bmpr2+/R491W transgenic heterozygous mutant rat as the PCR result. The PCR products were recovered and sequenced, after which sequence alignment was performed by software Vector NTI, and the sequencing result was that c.C1471T was bimodal, and Bmpr2+/R491W transgenic rats were identified.
2. Experimental results
Bmpr2 +/R491W Mutant rat construction and genotyping
We synthesized Bmpr2 gene sgRNA vector (as in FIG. 1A), made Bmpr2 gene p.R491W mutation knock-in rat model by CRISPR/CASE 9 technique, amplified by PCR with rat genome DNA template (FIG. 1B), positive rat and wild type band size consistent (116#), and sequenced Bmpr2 by one generation +/R491W Mutant rats were identified (fig. 1C). The presence of a bimodal Bmpr2 at c.C1471T +/R491W Heterozygous mutant rats were bred to find that Bmpr2 homozygous mutant rats died, and heterozygous mutant male rats were used in subsequent studies.
Example 2 rat phenotyping
1. Experimental method and procedure
1. Ultrasound evaluation of degree of cardiac remodeling in rats
1) Rats were induced with 3% -4% isoflurane concentration and the isoflurane concentration was converted to 1% -2% to maintain the rat anesthetized state. The hair from the rat collarbone to the xiphoid process was removed using depilatory cream and placed on a small animal ultrasonic testing instrument platform (visualsonic, canada model Vevo 770).
2) Long axis section beside sternum: the probe is vertically arranged above the sternum, is inclined at an angle of about 30-45 degrees to the right side of the rat, and the image shows the long axis of the heart, and if the apex and the outflow tract are both clearly positioned on the same horizontal plane, the long axis section of the left room of the sternum is a qualified long axis section. A map is kept at this section BMode.
3) Pulmonary artery long axis section: on the basis of the short axis of the left ventricle beside the sternum, the Y axis is adjusted to enable the probe to move towards the head of the rat, and the middle annular aorta, the pulmonary artery wrapping the outer side of the aorta and the branches of the left and right pulmonary arteries form a Y-shaped fork, and the Y-shaped fork is a long axis section of the pulmonary artery at the moment. A map of the long axis of the pulmonary artery remains at B Mode. The doppler mode is turned on.
4) Parasternal right ventricle short axis section: on the basis of the short axis section of the left chamber beside the sternum, the probe is shifted from the right sternum to the left, the Y axis is moved to find the maximum section of the heart chamber of the right ventricle, and the free wall and the chamber diameter of the right ventricle are measured by using M Mode.
5) Measurement and analysis: after obtaining images of each section, ultrasonic measurement is carried out according to the standard established by the American echocardiography society, and the pulmonary artery diastole is searched in a pulmonary artery long axis section B Mode diagram and the right ventricular outflow tract width (RVOT) of the diastole is measured; the diastolic right ventricular free wall thickness (RVAW; d) and the cavity diameter size (RVID; d) were measured on the parasternal right-heart short axis section. All ultrasonic measurements were averaged over 3 measurements.
2. Pressure measurement of right heart catheter
The detection of pulmonary artery hemodynamics and right ventricular cardiac output by right heart catheters is a gold standard for assessing pulmonary artery high pressure models, and is also a key technology for determining BMPR2 mutation effect at in vivo levels in this project.
1) Isoflurane at a concentration of 3% -4% induces rats and maintains the anesthetized state of the rats at a concentration of 1% -2%.
2) A multi-guide physiological recorder (Powerlab 8/30) and its associated pressure transducer were connected, the pressure transducer and catheter were filled with heparinized saline (125U/ml), and no bubbles were determined therein (bubbles could lead to deviations in the pressure measurements).
3) The rat was supinated with his limbs and his head fixed to the operating table. The neck skin of the rat was exposed and the skin of the target site was sterilized with 75% alcohol. The left hand is provided with tooth bending forceps, the right hand is used for cutting the skin from the position above the sternum handle and about 1cm far to the right in the middle, and a gland is arranged near the throat of the rat. Avoiding glands, passively separating subcutaneous muscle and connective tissue, exposing right jugular vein, and cleaning connective tissue around jugular vein. The jugular vein is ligated distally, a loose knot is made proximally, and the knot is lifted with a hemostat to stop blood flow.
4) The catheter port and the transducer are flush with the heart, and the three-way pipe is opened to circulate the whole device. Clicking the 'start' to detect the blood pressure, if the blood pressure is not 0, zeroing the bridge amplifier, and stopping recording the blood pressure and closing the three-way pipe after the blood pressure detection value is continuously stabilized at the '0'.
5) A V-shaped small opening is cut on the external jugular vein by using Venus scissors, a proximal ligature is loosened, a section of bent PE-50 catheter with the front end is sent in, and the ligature is fast fastened after the catheter is sent in, so that blood can be prevented from exuding from the opening to disturb vision. If the jugular vein waveform is observed, if the waveform is not present or the waveform is not in the right shape, the opening of the catheter is possibly blocked, and the catheter is flushed with a small amount of heparinized physiological saline.
6) The catheter was gently fed into the heart chamber, the rat right heart waveform was observed, and after stabilizing for 2min, the right heart pressure waveform was recorded for 1-2min.
7) Continuing to forward the catheter and rotating the front end to search for the pulmonary artery, if a rise in diastolic pressure is observed and systolic pressure is similar to that of the right heart, the catheter has entered the pulmonary artery. The catheter was kept stationary and the pressure was allowed to stabilize for 1min and waveforms were recorded for 1-2min. After the pulmonary artery pressure is measured, the catheter is withdrawn from the blood vessel, and the proximal end of the jugular vein incision is ligated.
8) Detection and analysis of right heart and pulmonary artery data: the average value of the waveforms of the pulmonary artery pressures of more than 10 circles is calculated and output as an average pulmonary artery pressure (mPAP).
3. Pathological examination
1) Fixing: after removal of the lung tissue, the lung tissue was fixed in about 10ml of paraformaldehyde at room temperature for 14 days.
2) Flushing with running water: the fixed tissue is taken out from the paraformaldehyde, put into a dehydration box, soaked in flowing water for 1 hour, and if no flowing water exists, the water is replaced every 10 minutes. If the next operation cannot be performed immediately after rinsing, the tissue is immersed in 50% ethanol and stored at 4 ℃.
3) Dehydrating: and placing the dehydration box into a full-automatic dehydrator, setting a program, and running.
4) Embedding: and taking the dehydration box out of the full-automatic dehydrator, and putting the dehydration box into an embedding machine. The dehydration box is opened, an embedding box with proper size is taken, melted wax is put in, a sample is put in a mould by forceps, and the mould is moved to a small ice plate of an operation table to enable the wax to be coagulated and fixed on the sample. The dehydration box is broken off, the lower cover with the sample information is covered on the embedding box, and melted wax is added to enable the melted wax to overflow the dehydration box. Finally, the whole embedding box is moved to an ice plate beside, and the embedding box is kept stand for more than 20 minutes until wax is solidified.
5) And (5) preserving wax blocks: the dehydration box is taken out from the mould together with the whole sample, the embedding box is put back into the embedding machine, the samples are classified and put into a wax block cabinet, and the samples can be stored for a long time at normal temperature.
6) Slicing: the 3um slices were cut using a paraffin microtome, dried overnight at room temperature, and then stored in a slice box.
7) Baking slices: the flakes were baked on a flaker for 45 minutes at 68 ℃ before staining to allow paraffin to melt.
8) The sections were stained with Verhoeff's hematoxylin for 10min and rinsed rapidly with tap water.
9) The 2% ferric trichloride solution differentiated until the visible fiber appeared black under the microscope and the background appeared grey, and was rinsed with tap water. This step was performed under a microscope while observing differentiation to prevent excessive differentiation.
10 Iodine was removed with 5% sodium thiosulfate for 1min and rinsed with tap water.
11 Van Gieson's dye liquor for 3-5min.
12 80%, 90% and 100% ethanol, and soaking in xylene I, II for 5min to make it transparent, and sealing with neutral resin.
13 After EVG staining is carried out on rat pulmonary tissue sections, the pulmonary artery is seen to be formed by an inner elastic plate and an outer elastic plate, the middle membrane of the pulmonary artery is arranged between the two elastic plates, and the diameter of the outermost elastic plate is the external diameter of the pulmonary arteriole. The media thickening and percent midfilm thickness (myolayer thickness/outside vessel diameter x 100%) were measured and calculated in each rat pulmonary arteriole (< 50 μm) in EVG stained sections of lung tissue using CaseViewer software, respectively, and at least 5 rat lung tissue samples were counted per experimental group, with 15-20 vessels counted per rat sample.
4. Statistical method
GraphPad Prism 8.0 software was used for mapping analysis and continuous variables were plotted using mean ± standard deviation
The mean value comparison between multiple groups adopts single factor analysis of variance, and the mean value comparison between two groups adopts independent sample t test to obtain P<A difference of 0.05 is statistically significant.
2. Experimental results
1、Bmpr2 +/R491W Mutant rat spontaneous pulmonary artery high pressure phenotype
The first mutant rat was designated as F0, and the progeny of its pair with the wild type rat was designated as F1, and then passaged down in sequence. We performed echocardiographic measurements at 2, 3, 6 months of age for each generation of mutant rats, with continuous observations of the cardiopulmonary phenotype of the rats. At the F3 generation, we found 30 Bmpr2 +/R491W One rat was found to have spontaneous PAH at 3 months of age at a ratio of 3% (1/30). The rats were not bred on a large scale for the F4-F9 generation, and only maintained population.
From the F10 generation we re-useExpansion of the breeding scale, the mutant rats were found to be Bmpr2 when passaged to F11-F12 generation, three independent batches of breeding +/R491W One rat showed spontaneous PAH phenotype at 3 months of age, 3 out of 20 mutant rats showed spontaneous pulmonary hypertension with an overall incidence of 15% (3/20), and the remaining mutant rats were observed to have no spontaneous PAH phenotype at 4 months of age for a long period of time (fig. 2), where B096 was the F9 mutant rat and two F10 mutant rats (B106, B107) were bred after mating with wild type rat (WT). B106 and B107 were mated with Wild Type (WT) rats, respectively, to obtain F11-generation rats. One of six F11 rats bred in B106 (B113, red highlighting) spontaneously developed pulmonary hypertension at 3 months of age, two offspring rats B117, B118 of B107, which had not spontaneously developed PAH, were mated with wild type rats to obtain F12 rats, one each (B123 and B128) spontaneously developed pulmonary hypertension at 3 months of age, respectively. In the real world, the lifetime incidence of BMPR2 mutation carriers is 14-42%, and the extinguishment rate of a rat model is consistent with the genetic characteristics of the disease.
2. Pulmonary artery bulge and right heart hypertrophy
The echocardiography test shows that three spontaneous PAH mutant rats have obvious pulmonary artery bulge. Bmpr2 compared to non-littermates mutant rats +/R491W Spontaneous PAH rats increased in right precordial anterior wall thickness (RVAW; d) by 50% (1.6.+ -. 0.3mm vs. 0.8.+ -. 0.1mm, P)<0.001 (as in fig. 3A-C); right Ventricular Outflow Tract (RVOT) of three spontaneous PAH mutant rats was widened 55% (6.8.+ -. 0.01mm vs 3.0.+ -. 0.4mm, P)<0.001 (as in fig. 3D-F), where the right cardiac hypertrophy phenotype of B128 was particularly pronounced, the right cardiac chamber diameter increased by 57% (7.1 mm vs 3.0 mm), and the right heart remodeling was severe, suggesting that rat B388 has progressed to the end of PAH disease.
3. Pulmonary artery pressure elevation in BMPR2 mutant rats
Echocardiography clearly suggests that three mutant rats undergo right ventricular remodeling, and to further clarify whether these rats develop pulmonary hypertension, we performed right heart catheter detection. Compared with mutant rats with normal littermate phenotype, the average pulmonary arterial hypertension (mean pulmonary arterial hypertension, mPAP) of the three diseased rats is significantly increased (37.7+ -8.5 mmHg vs 17.8+ -1.2 mmHg, p < 0.001) (fig. 4A-C), and the mPAP of the three rats exceeds 25mmHg, thereby reaching the pulmonary arterial hypertension diagnosis standard.
4、Bmpr2 +/R491W Mutant rats produced severe pulmonary vascular remodeling
In the definition of three Bmpr2 +/R491W After spontaneous pulmonary hypertension in mutant rats, we further assessed the extent of pulmonary vascular remodeling in rats by pathology staining. The pulmonary blood vessel is divided into small pulmonary arteries with diameters of 50-100um and small pulmonary arteries with diameters of less than 50um for statistical analysis, and the results show that compared with non-diseased rats, three Bmpr2 disease is generated +/R491W Rats were either pulmonary arterioles (14.77.+ -. 3.2% vs. 12.+ -. 0.56%, p<0.05 Also the micro-arteries (18.3.+ -. 2.0% vs 14.09.+ -. 0.3%, p)<0.01 The intima thickness was significantly elevated, suggesting that the primary pathological mechanism of BMPR2 mutation pathogenesis was closely related to intima remodeling (fig. 5A-5D).
The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention.
Claims (10)
1. A method for constructing a spontaneous pulmonary artery high-pressure non-human animal model, which is characterized in that the spontaneous pulmonary artery high-pressure non-human animal model expresses BMPR2 mutant protein;
preferably, the amino acid sequence of the BMPR2 mutant protein is shown as SEQ ID NO. 1.
2. The method of claim 1, wherein the non-human animal model is selected from the group consisting of mammals;
preferably, the mammal comprises a rodent, carnivorous animal, winged animal, hedgehog animal, insect animal;
preferably, the rodent comprises a species of the hamster, murine, equine island murine, spiny murine, mole murine, spiny murine, petromurine;
preferably, the murine species include black rats, brown rats, gerbils, new world rats, old world rats, SD rats, bolnesia rats, tree rats, wood rats, stick rats, rice rats, kangaroo rats, climbing rats.
3. The construction method according to claim 1, characterized in that the construction method comprises the steps of: carrying out nucleotide mutation modification on BMPR2 genes in single embryo cells or embryo stem cells by utilizing a gene locus knock-in method so that p.R491W mutation is contained on the mutated BMPR2 protein;
preferably, the method adopted by the BMPR2 gene for carrying out nucleotide mutation modification is a gene knock-in technology;
preferably, the gene knock-in technique is a CRISPR/Cas9 gene editing method;
preferably, the construction method comprises the steps of:
1) Designing a gene knock-in strategy aiming at a BMPR2 gene, wherein a knock-in site selects a nucleotide site corresponding to 491 amino acid site of a BMPR2 protein;
2) sgRNA targeting vectors and donor oligonucleotides were designed;
3) The p.r491w mutation site in the donor oligonucleotide will introduce the corresponding exon by homology-directed repair; for the p.R491W mutation, silent mutation needs to be introduced to prevent sgRNA binding and re-cleavage of sequences after homology-directed repair;
4) Mixing and introducing the Cas9 mixed sample system into single embryo cells or embryonic stem cells to obtain transformed cell embryos;
5) Culturing the transformed cell embryo to obtain a spontaneous pulmonary artery high-pressure non-human animal model;
preferably, the Cas 9-like system comprises sgrnas and mrnas;
preferably, the spontaneous pulmonary arterial hypertension non-human animal model occurs at the F3 generation;
preferably, the specific sequence of the sgRNA is shown in SEQ ID NO: 2. SEQ ID NO:3 is shown in the figure;
preferably, the modification of the BMPR2 gene by nucleotide mutation comprises modification of the 1471 st nucleotide on the sequence of the wild-type BMPR2 gene from C mutation to T;
preferably, the introduction mode comprises electroporation, calcium phosphate method, liposome method, DEAE dextran method, microinjection and virus infection;
preferably, the vector comprises a retrovirus, adenovirus, adeno-associated virus, herpes virus, poxvirus, baculovirus, papillomavirus, papovavirus, phage, plasmid.
4. The construction method according to claim 1, characterized in that the construction method further comprises the steps of:
1) Constructing an over-expression vector of the BMPR2 mutant protein, and introducing the vector into a single embryo cell or an embryonic stem cell to obtain a transformed cell embryo;
2) Culturing the transformed cell embryo to obtain a spontaneous pulmonary artery high-pressure non-human animal model;
preferably, the BMPR2 mutant protein comprises an amino acid sequence formed by mutation of R to W at 491 amino acid on a wild type BMPR2 protein sequence;
preferably, the nucleic acid overexpression vector of the BMPR2 mutant protein comprises a nucleotide sequence formed by mutating 1471 st nucleotide from C to T on the sequence of a wild type BMPR2 gene;
preferably, the spontaneous pulmonary arterial hypertension non-human animal model occurs at the F3 generation;
preferably, the vector comprises a retrovirus, adenovirus, adeno-associated virus, herpes virus, poxvirus, baculovirus, papilloma virus, papovavirus, phage, plasmid;
preferably, the introduction means includes electroporation, calcium phosphate method, liposome method, DEAE dextran method, microinjection, and viral infection.
5. The method of claim 1, wherein the spontaneous pulmonary hypertension non-human animal model exhibits one or more symptoms of pulmonary artery distension, right heart hypertrophy, elevated pulmonary artery pressure, severe pulmonary vascular remodeling.
6. A spontaneous pulmonary artery high pressure non-human animal model prepared by the construction method of claim 1.
7. A method for identifying a therapeutic agent for treating pulmonary arterial hypertension for non-therapeutic non-diagnostic purposes, the method comprising the steps of:
1) Administering an agent to the spontaneous pulmonary arterial hypertension non-human animal model animal of claim 6;
2) Detecting whether the model animal has a change of pulmonary arterial hypertension related abnormal symptoms after administration of the medicament;
3) An agent is identified as a therapeutic agent when administration of the agent has a therapeutic effect on pulmonary arterial hypertension-related abnormal symptoms.
8. Use of a spontaneous pulmonary hypertension non-human animal model for screening for a medicament for treating or preventing pulmonary hypertension for non-therapeutic purposes, wherein the pulmonary hypertension non-human animal model is from the spontaneous pulmonary hypertension non-human animal model of claim 6.
The application of BMPR2 mutant protein or a nucleotide sequence for synthesizing the same in constructing a spontaneous pulmonary artery high-pressure non-human animal model is characterized in that the amino acid sequence of the BMPR2 mutant protein is shown as SEQ ID NO. 1;
preferably, the BMPR2 mutant protein comprises an amino acid sequence formed by mutation of R to W at 491 amino acid on a wild type BMPR2 protein sequence;
preferably, the coding sequence of the BMPR2 mutein comprises the following sequence: the 1471 nucleotide of the wild BMPR2 gene is a nucleotide sequence formed by mutating C into T.
10. Use of a spontaneous pulmonary hypertension non-human animal model for assessing the effect of a treatment of a product of treating pulmonary hypertension, characterized in that said spontaneous pulmonary hypertension non-human animal model is a spontaneous pulmonary hypertension non-human animal model as defined in claim 6.
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| CN109266679A (en) * | 2018-10-12 | 2019-01-25 | 中国医学科学院阜外医院 | A kind of preparation method and application of BMPR2 gene mutation rat |
| CN114317604A (en) * | 2022-03-04 | 2022-04-12 | 中国医学科学院阜外医院 | Spontaneous pulmonary hypertension model and construction method |
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| US20020022229A1 (en) * | 2000-07-12 | 2002-02-21 | Morse Jane H. | Role of PPH1 gene in pulmonary hypertension |
| CN109266679A (en) * | 2018-10-12 | 2019-01-25 | 中国医学科学院阜外医院 | A kind of preparation method and application of BMPR2 gene mutation rat |
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