CN114317604A

CN114317604A - Spontaneous pulmonary hypertension model and construction method

Info

Publication number: CN114317604A
Application number: CN202210205304.6A
Authority: CN
Inventors: 王晓建; 马铭婕; 潘慧; 李天骐; 俞莉萍
Original assignee: Fuwai Hospital of CAMS and PUMC
Current assignee: Fuwai Hospital of CAMS and PUMC
Priority date: 2022-03-04
Filing date: 2022-03-04
Publication date: 2022-04-12
Anticipated expiration: 2042-03-04
Also published as: CN114317604B

Abstract

The invention discloses a spontaneous pulmonary hypertension model and a construction method thereof, wherein the pulmonary hypertension model is a non-human animal model, and the spontaneous pulmonary hypertension non-human animal model is constructed by introducing BMP9 gene. The invention also provides application of the spontaneous pulmonary hypertension non-human animal model in screening drugs for treating pulmonary hypertension or evaluating the treatment/prevention effect of pulmonary hypertension.

Description

Spontaneous pulmonary hypertension model and construction method

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a spontaneous pulmonary hypertension model and a construction method thereof.

Background

Pulmonary Arterial Hypertension (PAH) refers to a clinical and pathophysiological syndrome in which the structure or function of pulmonary vessels is altered by various causes, resulting in pulmonary vascular resistance and increased pulmonary artery pressure. PAH is a type of malignant, lethal disease. Early PAH is not available, with a median survival time of only 2.8 years for untreated PAH patients [1], and a survival rate of only 21% for 5 years [2 ]. In recent years, a plurality of PAH targeting drugs are available, the short-term survival rate of patients is obviously improved, but the long-term survival rate is still lower than 50%, the average survival time is only 66.2 months [3-4], and the death rate of PAH patients who live in ICU due to acute right heart failure is more as high as 41% [5 ]. Therefore, the disease burden of PAH is very heavy, and it is urgently needed to deeply research the pathological mechanism of pulmonary vascular remodeling and develop effective and effective therapeutic drugs and means to prolong the life of patients and improve prognosis.

The establishment of a disease model truly simulating the characteristics of the PAH patient at the whole level of animals is the basis and key for exploring the occurrence and development mechanism of the PAH disease, discovering new targets of medicaments and carrying out preclinical pharmacodynamic evaluation, and has very important scientific significance and clinical significance. Subcutaneous injection of monocrotaline and long-term continuous hypoxia are two most widely used animal models of PAH at present, and both rat models can show the increase of pulmonary artery pressure, but are greatly different from the actual conditions of clinical patients. First, monocrotaline is a toxin that induces pulmonary hypertension by damaging the pulmonary vascular endothelium. In the real world, PAH patients do not contact monocrotaline and do not take in toxin, so that pulmonary hypertension of the patients is not induced by monocrotaline. Secondly, the pulmonary hypertension caused by hypoxia is transient and mild pulmonary vascular pressure rise, and the pulmonary vascular pressure of the animal is quickly and automatically restored after the animal is taken out from the hypoxia chamber and put in a normal oxygen environment, which is far away from the clinical characteristics of continuous and progressive pulmonary vascular pressure rise of clinical patients. In addition, from the pathological aspect, pulmonary vascular remodeling of the two animal models is mainly thickening of the tunica media, and the pathological characteristics of malignant pulmonary vascular remodeling of clinical patients such as intimal hyperplasia and intimal fibrosis are rarely seen. Therefore, the existing PAH animal model can not simulate the development and outcome of human diseases, the research on the diseases by using the animal model is not looking at the human diseases but looking at the disease of the animal model, and the pathological knowledge and new drug targets obtained based on the animal model are difficult to be converted into the clinic. Therefore, the establishment of new animal models of diseases more closely related to the pathological features of pulmonary hypertension in clinical application is urgent.

Reference to the literature

[1] G E D'Alonzo, R J Barst, S M Ayres, et al. Survival in patients with primary pulmonary hypertension. Results from a national prospective registry. Annals of Internal Medicine, 1991. 115(5): p. 343-349.

[2] Zhi-Cheng Jing, Xi-Qi Xu, Zhi-Yan Han, et al. Registry and survival study in chinese patients with idiopathic and familial pulmonary arterial hypertension. Chest. 2007 Aug;132:373-379.

[3] Xuchiqi, Sun Mingli, Jiangxin, et al, comparison of clinical characteristics and survival rates of idiopathic and familial pulmonary hypertension in different treatment times, J. China cardiovascular disease, 2014.42 (6): p.465-.

[4] Rui Zhang, Li-Zhi Dai, Wei-Ping Xie, et al. Survival of Chinese patients with pulmonary arterial hypertension in the modern treatment era. Chest. 2011 Aug;140(2):301-309.

[5] Harrison W Farber, Joseph Loscalzo. Pulmonary arterial hypertension. N Engl J Med,2004.351(16):p.1655-65。

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a spontaneous pulmonary hypertension animal model and a construction method thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a construction method of a non-human animal model of pulmonary hypertension, which modifies the genome of a non-human animal, so that the modified animal genome comprises BMP9 gene; and obtaining a non-human animal comprising the modified genome.

Further, the genome of the non-human animal is modified by introducing the BMP9 nucleic acid sequence into the genome of a single cell embryo or embryonic stem cell of the non-human animal.

Further, the nucleic acid sequence is operably linked to an expression vector.

Further, the animal is a rodent.

Further, the rodent is a rat.

Further, the animal model exhibits one or more symptoms of pulmonary vascular remodeling, right ventricular hypertrophy, or elevated pulmonary artery pressure.

In a second aspect, the invention provides a cell line derived from a pulmonary hypertension non-human animal prepared by the method of construction according to the first aspect of the invention.

In a third aspect, the invention provides embryonic stem cells derived from a pulmonary hypertension non-human animal prepared by the method of construction according to the first aspect of the invention.

A fourth aspect of the invention provides a method as defined in any one of the following:

1) a method for breeding a pulmonary hypertension non-human animal comprises the steps of breeding the non-human animal modified with BMP9 and a wild animal, and screening BMP9 positive animals;

2) a method of identifying a therapeutic agent for treating pulmonary arterial hypertension, the method comprising:

administering a pharmaceutical agent to a pulmonary hypertension non-human animal prepared by the method of construction according to the first aspect of the invention;

performing one or more assays to determine whether the agent has an effect on one or more abnormalities associated with pulmonary hypertension; and

identifying the agent as a therapeutic agent when the agent has a therapeutic effect on one or more abnormalities associated with pulmonary arterial hypertension.

A fifth aspect of the invention provides a use as claimed in any one of:

1) the application of the non-human animal model of pulmonary hypertension, the cell line of the second aspect of the invention or the embryonic stem cell of the third aspect of the invention, which is prepared by the construction method of the first aspect of the invention, in screening drugs for treating pulmonary hypertension;

2) the use of a non-human animal model of pulmonary hypertension prepared by the construction method of the first aspect of the invention, the cell line of the second aspect of the invention, or the embryonic stem cell of the third aspect of the invention, in the assessment of the therapeutic/prophylactic effect of pulmonary hypertension;

3) application of BMP9 in preparing spontaneous pulmonary hypertension non-human animal model.

The invention has the beneficial effects that:

the invention provides a non-human animal model of pulmonary hypertension, which has clear genetic background and is closer to the real symptoms of diseases, solves the problem that the existing animal model has larger difference with clinical diseases, and has important significance for the clinical research of the pulmonary hypertension.

Drawings

FIG. 1 is a schematic representation of pcDNA3.1-ALB-BMP9-WT transgenic plasmid.

FIG. 2 is a diagram of the PCR identification of TG-BMP9-WT rat genotypes; wherein, N: negative control; p: a positive control; m: nucleic acid marker; 1-7: TG-BMP9-WT rats. No. 1-4 are positive mice, and No. 5-7 are negative mice.

Figure 3 is a doppler echocardiogram of a rat.

FIG. 4 is a graph of right heart catheter measuring rat pulmonary artery pressure; wherein 4A and 4B are rat mean pulmonary artery pressure measurements and statistical plots; 4C and 4D are rat right ventricular systolic pressure measurements and statistical plots.

FIG. 5 is a graph of Elasticity VG (EVG) staining for detecting the nature of reconstructed lesions of pulmonary vessels.

FIG. 6 is a graph of the statistical percentage of the layer thickness in pulmonary vessels, wherein 6A is a graph of the percentage of layer thickness in small vessels of 50-100 μm; 6B is a graph showing the percentage of the thickness of the middle layer in the blood vessel having a diameter of 50 μm or less.

Detailed Description

The invention will be described in further detail below with the understanding that the terminology is intended to be in the nature of words of description rather than of limitation.

The terms "nucleic acid sequence", "nucleotide sequence" or "isolated nucleotide sequence" or "polynucleotide" or "isolated polynucleotide sequence" are used interchangeably herein and refer to a nucleic acid molecule, DNA or RNA containing deoxyribonucleotides or ribonucleotides, respectively. The nucleic acid may be double-stranded, single-stranded, or contain portions of double-stranded or single-stranded sequence.

"expression" refers to the process of transcribing a polynucleic acid into mRNA and translating into a peptide, polypeptide or protein. If the polynucleic acid is derived from genomic DNA and a suitable eukaryotic host cell or organism is selected, expression may include splicing of the mRNA. "nucleic acid" includes ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), which may be complementary DNA (cDNA) or genomic DNA.

As used herein, the term "treatment" refers to the administration of a compound or composition to control the progression of a disease. Control of disease progression is understood to achieve beneficial or desired clinical results, including but not limited to alleviation of symptoms, reduction of disease duration, stabilization of the pathological state (in particular avoidance of additional exacerbations), delay of disease progression, amelioration and remission (both partial and total) of the pathological state. Control of disease progression also involves prolongation of survival compared to expected survival without treatment.

The present invention provides a novel non-human animal model of pulmonary hypertension. In fact, the inventors have discovered a new animal model of pulmonary hypertension that reproduces the core features of human disease.

The gene for bone morphogenetic protein 9 (BMP 9, also known as growth differentiation factor 2, GDF 2) is located on chromosome 10q11.22 with gene ID 2658, and includes genes and their encoded proteins and homologs, mutations, and isoforms. The term encompasses full-length, unprocessed genes or proteins, as well as any form of gene or protein that results 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/.

As used herein, the term "non-human animal" includes non-human vertebrates, more preferably mammals, such as domesticated livestock (e.g., cattle, horses, pigs), pets (e.g., dogs, cats), or rodents. The term "rodent" refers to any and all members of a phylogenetic rodent (e.g., mouse, rat, squirrel, beaver, woodchuck, hamster, guinea pig, and guinea pig), including any progeny of all progeny derived therefrom.

As used herein, the term "non-human animal model" refers to a non-human animal that has or displays the characteristics of a disease or condition. By used as an animal model is meant any use of the animal for studying a disease or condition, e.g., for studying progression or development or response to a new or existing therapy.

The invention provides a construction method of a non-human animal model of pulmonary hypertension, which modifies the genome of a non-human animal to ensure that the modified animal genome contains BMP9 gene; and obtaining a non-human animal comprising the modified genome.

In some embodiments, the genome of the non-human animal is modified by introducing the BMP9 nucleic acid sequence into the genome of a single cell embryo or embryonic stem cell of the non-human animal.

In a specific embodiment of the invention, the genome of the non-human animal is modified by introducing the BMP9 nucleic acid sequence into the genome of a single cell embryo (zygote) of the non-human animal.

In some embodiments, the nucleic acid sequence is operably linked to an expression vector. The expression vectors are completely commercially available at present, such as some viral vectors, plasmids, and phages.

In a preferred embodiment, the expression vector is a plasmid selected from the group of plasmids conventionally used in the art for constructing transgenic constructs, typically with a "spacer sequence" and multiple cloning sites or alternate sequences located on either side of the "spacer sequence", such that one can insert the corresponding DNA sequence of the gene into the multiple cloning site or replace the alternate sequence thereon in both forward and reverse directions. The expression vector usually further contains a promoter, an origin of replication, and/or a marker gene.

In a specific embodiment of the invention, the plasmid is pcDNA3.1-ALB.

As an alternative embodiment, the expression vector can be introduced into cells by a known method such as electroporation, calcium phosphate method, liposome method, DEAE dextran method, microinjection, viral infection, lipofection, or binding to a cell membrane-permeable peptide.

In a particular embodiment of the invention, the introduction of the expression vector into the cell uses microinjection.

As an alternative embodiment, the non-human animal is a rodent.

In some embodiments, rodents of the disclosure include mice, rats, and hamsters, as non-limiting examples. In some embodiments, rodents of the disclosure include, as non-limiting examples, mice and rats. In some embodiments, the rodent is selected from the superfamily murinus (Muroidea). In some embodiments, the rodents of the disclosure are from a family selected from the group consisting of: calomyidae (e.g. mouse-like hamsters), cricotidae (Cricetidae) (e.g. hamsters, new world rats and mice, voles), Muridae (Muridae) (true mice and rats, gerbils, acanthos, coronaries), Nesomyidae (Nesomyidae) (climbing rats, rock rats, tailed rats, madagassah rats and mice), cephalomyidae (placathoideae) (e.g. spiny mice) and Spalacidae (Spalacidae) (e.g. mole rats, bamboo rats and zokors). In some embodiments, the rodent of the present disclosure is selected from a true mouse or rat (muridae), gerbil, acanthomys, and corolla. In some embodiments, the mice of the present disclosure are from a member of the murine family (Muridae).

In a particular embodiment of the invention, the rodent is a rat.

In some embodiments, the non-human animal model of pulmonary arterial hypertension exhibits symptoms of one or more of pulmonary vascular remodeling, right ventricular hypertrophy, or pulmonary arterial pressure elevation.

In a specific embodiment of the invention, the pulmonary hypertension non-human animal spontaneously develops pulmonary hypertension after a period of growth.

The non-human animals of the invention may be used for in vivo testing. In addition, the non-human animals of the invention may be used as a source of somatic, fetal or embryonic cells, which once isolated and cultured, may be used for in vitro testing. In addition, if desired, immortalized cell lines can be prepared from the cells using conventional techniques. Thus, in another aspect, the invention provides an isolated cell line derived from a non-human animal of the invention.

In some embodiments, the invention provides embryonic stem cells derived from the pulmonary arterial hypertension non-human animal described above.

In some embodiments, the invention provides progeny of the non-human animal. Progeny of the non-human animals of the invention may be obtained by conventional methods, e.g. by conventional crossing techniques between non-human animals of the invention, or by in vitro fertilization of eggs and/or sperm of non-human animals of the invention. As used herein, the term "progeny" refers to each progeny of each generation after the originally transformed non-human animal.

In some embodiments, the BMP9 modified non-human animal is bred with a wild-type animal to obtain progeny of the BMP9 positive animal.

In some embodiments, the invention provides a method of identifying a therapeutic agent for treating pulmonary hypertension, the method comprising:

administering an agent to the pulmonary arterial hypertension non-human animal as described above;

The agents of the invention are preferably administered in a pharmaceutically acceptable vehicle. Suitable pharmaceutical carriers are known to those skilled in the art. For parenteral administration, the compounds are generally dissolved or suspended in sterile water or saline. For enteral administration, the compounds are incorporated into inert carriers in the form of tablets, liquids or capsules. Suitable carriers may be starches or sugars and include lubricants, flavoring agents, binders and other materials of the same nature. The compounds may also be administered topically by means of a topically applied solution, cream, gel, or polymeric material (e.g., pluronic (tm), BASF).

Alternatively, the compounds may be administered in liposomes or microspheres (or microparticles). Methods of preparing liposomes and microspheres for administration to a patient are known to those skilled in the art. Essentially, the material is dissolved in an aqueous solution, if necessary, the appropriate phospholipids and lipids are added together with a surfactant, and the material is dialyzed or sonicated as necessary. Microspheres formed from polymers or proteins are well known to those skilled in the art and can be tailored to pass through the gastrointestinal tract directly into the bloodstream. Alternatively, the compound may be incorporated and the microspheres or a composite of microspheres implanted for slow release over a period of days to months.

The methods of the invention are preferably used to identify agents that alleviate such symptoms or signs.

In another embodiment, the invention relates to a method of assessing the effectiveness of a pulmonary hypertension treatment, comprising the steps of:

i) providing a non-human animal model according to the invention with a pharmaceutical composition or compound to be tested,

ii) evaluating the effect observed on said model of treatment with the pharmaceutical composition or compound.

According to a preferred embodiment of the invention, said effect to be observed is a physiopathological change. Said physiopathological changes to be detected in the animal model of the invention are any improvement of the physiopathological changes present in the animal model as previously described, for example a decrease in pulmonary artery pressure and a decrease in the thickness of the pulmonary blood vessels after treatment, as compared to the control (untreated animal).

In another embodiment, the invention provides the use of an animal model according to the invention or a cell line according to the invention or an embryonic stem cell according to the invention for screening a medicament for the treatment or prevention of pulmonary hypertension.

Candidate compounds or drugs for use in the methods of the invention may include all different types of organic or inorganic molecules, including peptides, oligosaccharides or polysaccharides, fatty acids, steroids, and the like. Moreover, possible compounds to be screened include, for example, hematopoietic stem cells, enzymes, and gene therapy products, e.g., recombinant vectors, and the like. These compounds may be administered alone or in combination with each other.

Screening using animal models, candidate compounds can be administered before, during, or after the appearance of a particular disease phenotype. Diagnostic tests known to those skilled in the art can be used to monitor the progression or regression of the disease. Methods of monitoring disease progression or resolution symptoms are well known to those skilled in the art. Such as doppler ultrasound, pathology detection, etc.

In another embodiment, the invention provides the use of an animal model according to the invention or a cell line according to the invention or an embryonic stem cell according to the invention for assessing the effect of a treatment for pulmonary hypertension.

In another embodiment, the invention provides the use of BMP9 in the preparation of a non-human animal model of idiopathic pulmonary hypertension. As an alternative embodiment, the non-human animal is a rodent. In some embodiments, rodents of the disclosure include mice, rats, and hamsters, as non-limiting examples. In some embodiments, rodents of the disclosure include, as non-limiting examples, mice and rats. In some embodiments, the rodent is selected from the superfamily murinus (Muroidea). In some embodiments, the rodents of the disclosure are from a family selected from the group consisting of: calomyidae (e.g. mouse-like hamsters), cricotidae (Cricetidae) (e.g. hamsters, new world rats and mice, voles), Muridae (Muridae) (true mice and rats, gerbils, acanthos, coronaries), Nesomyidae (Nesomyidae) (climbing rats, rock rats, tailed rats, madagassah rats and mice), cephalomyidae (placathoideae) (e.g. spiny mice) and Spalacidae (Spalacidae) (e.g. mole rats, bamboo rats and zokors). In some embodiments, the rodent of the present disclosure is selected from a true mouse or rat (muridae), gerbil, acanthomys, and corolla. In some embodiments, the mice of the present disclosure are from a member of the murine family (Muridae).

In some embodiments, the non-human animal model of idiopathic pulmonary hypertension is obtained by modifying the genome of the non-human animal by introducing the BMP9 nucleic acid sequence into the genome of a single cell embryo (fertilized egg) or embryonic stem cell of the non-human animal.

The invention is further illustrated below with reference to specific examples. It should be understood that the particular embodiments described herein are presented by way of example and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention.

Example construction and detection of spontaneous pulmonary hypertension model

First, construct the vector

1. Artificially synthesizing human BMP9 full-length CDS (Gene ID: 2658);

2. a transgenic plasmid pcDNA3.1-ALB-BMP9-WT (FIG. 1) containing the full-length CDS of the human wild-type BMP9 gene was constructed. The plasmid is independently constructed in a laboratory, and the ALB gene promoter sequence (2460 bp, the green highlight part in figure 1) drives the human wild type BMP9 gene (1290 bp, the red highlight part in figure 1) to highly express in the liver.

3. The plasmid was 8574 bp in length and confirmed to be correct by Sanger sequencing.

Second, microinjection

1. Ligation of male mice: vasectomized SD male mice.

2. Superovulation: 10 SD mice of 3-4 weeks old were injected with hormone for superovulation.

3. Injecting the fertilized eggs: about 100 fertilized eggs are injected.

4. Preparation of receptor mice: after 8-week-old SD female mice and the ligated male mice are mated, female mice with emboli are selected.

5. Embryo transplantation: transplanting the fertilized eggs after injection to the ampulla of the oviduct of the receptor mouse.

Third, genotype identification

1. And (4) tail shearing numbering: rats born 7-10 days were cut to number the toes and tip of the tail.

2. Extracting genome DNA: rat genomic DNA was extracted using the genomic DNA extraction kit (EE 101-12) of all-round gold (Transgen).

3. And (3) PCR detection: PCR genotyping primers (Table 1) were synthesized, and the genotypes of rats were identified using the kit RR042A from TaKaRa under the reagent ratios shown in Table 2 and the reaction conditions shown in Table 3.

TABLE 1 primer sequences

TABLE 2 PCR reaction System

TABLE 3 PCR amplification procedure

4. And (4) analyzing results:

after completion of PCR, the PCR reaction was terminated with 6X loading buffer and electrophoresed on 1% agarose gel. Only the rats carrying the BMP9 transgenic fragment amplified a 552bp positive band, and the littermate negative rats did not have a band (FIG. 2).

Fourthly, breeding offspring:

1. the obtained fountain rats were F0 generation rats and wild type SD rats to obtain F1 generation rats, and the F1 generation TG-BMP9-WT rats were identified by PCR.

2. Each generation of heterozygous positive male mice is divided into 1 male and female: mating with wild female mouse in cage at the ratio of 2 to obtain the next generation of rats.

3. By 12 months at 2021, the rats have been passaged to F5 generation, the genotype is stable, the proportion of positive rats in each generation is 42-55%, and the Mendelian law of inheritance is met.

Fifthly, a TG-BMP9-WT spontaneous pulmonary hypertension experimental method:

1. right heart catheter for pulmonary artery pressure:

a multi-lead physiological recorder (Powerlab/8sp) and a matched pressure transducer are utilized to detect the pulmonary artery blood flow parameters of the rat. Rats were anesthetized with isoflurane-oxygen mixture (1.5%) on their back, their limbs were fixed, the skin was cut 1cm above the right sternum of the rat, the right external jugular vein was exposed, and the perivascular connective tissue was isolated. Two ligatures are passed under the blood vessel, the far end of the external jugular vein is ligated, a loose knot is tied at the proximal end, and the ligature at the proximal end is lifted to block the blood flow. The catheter is advanced into the vessel and the proximal loose knot is tightened to secure the catheter. The catheter enters the right ventricle via the pulmonary vein and then the pulmonary artery. And observing the waveform, confirming the position of the catheter, and recording the pressure after the catheter enters the right ventricle and the pulmonary artery respectively. And after the waveform is stabilized, taking three sections of waveforms, wherein each section of waveform comprises 10 cardiac cycles, recording data of average pulmonary artery pressure and right ventricular systolic pressure, and taking the average value of three groups of data as a final detection result.

2. Echocardiography for detecting rat heart structure and function

Rats are anesthetized by isoflurane-oxygen mixed gas (1.5 percent), lie on the back on a measuring board, the chest is coated with a couplant, a small animal ultrasonic instrument (Vevo 2100 imaging system) is used for measuring the thickness of the anterior wall of the left ventricle, the thickness of the posterior wall of the left ventricle and the size of the lumen of the left ventricle in the systolic period and the diastolic period respectively in the same cardiac cycle of the short-axis section of the left ventricle beside the sternum, the width of the outflow tract of the right ventricle is detected in the short-axis section of the aorta, the blood flow in the pulmonary artery is measured in the short-axis section of the aorta by using a Doppler mode, the displacement of the systolic phase of the tricuspid valve annulus is detected in the four-lumen cardiac section, and the free wall thickness of the right ventricle and the size of the lumen diameter in the diastolic period are measured in the short-axis section of the right ventricle beside the sternum.

3. Pathology of disease

Rat lung tissue is taken, dehydrated after being fixed for 14 days at normal temperature by paraformaldehyde, embedded in paraffin, stained by HE and EVG, the vascular reconstruction property is evaluated, the percentage of thickening of the membrane (thickness of muscular layer/outer diameter of blood vessel) in rat pulmonary arterioles (< 50 μm and 50-100 μm) is measured, and at least 5 rats in each group are counted, and 15-20 blood vessels are counted in each rat.

4. Statistical analysis

The data were plotted using GraphPad Prism 8.0 software

The mean value comparison between the groups adopts one-factor variance analysis, the mean value comparison between the two groups adopts independent sample t test, P<0.05 indicated that the difference was statistically significant.

5. As a result:

1) TG-BMP9-WT rats were phenotypically normal at 2 months of age, with no apparent abnormalities in pulmonary artery pressure and right ventricle.

2) TG-BMP9-WT rats developed spontaneous pulmonary hypertension at 6 months of age. Echocardiography showed that there was a bimodal trend in pulmonary artery blood flow seen in TG-BMP9-WT rats (n = 5) in the aortic short axis section doppler mode compared to WT rats (n = 8) and the peak pulmonary artery blood flow was much lower than in WT rats (P = 0.005) (fig. 3). Right heart catheter detection rat pulmonary circulation hemodynamics shows that 5 TG-BMP9-WT rats with the age of 6 months all show spontaneous pulmonary hypertension, and the incidence rate is 100%. The wild type rats had an average mPAP of 18mmHg and an average RVSP of 19 mmHg. The mean pulmonary artery pressure (mPAP) of the transgenic rats is 43mmHg (P < 0.001), and the maximum value can reach 59 mmHg; the mean Right Ventricular Systolic Pressure (RVSP) was 57mmHg (P < 0.001) up to 90mmHg (FIG. 4).

Pathological examination shows that the generation of the tiny pulmonary vessels of the TG-BMP9-WT rats at the age of 6 months is obviously reconstructed, the intima is proliferated and fibrillated, and the tiny pulmonary vessels are almost completely blocked (figure 5). Furthermore, the proportion of the thickness of small blood vessels of 50-100 mu m and the thickness of the middle layer of the micro blood vessels below 50 mu m in the diameter of the blood vessels is counted by using the pathological picture of the lung tissue. As shown in FIG. 6, TG-BMP9-WT rats showed significant thickening of media in small blood vessels of 50-100 μm (P < 0.01; 6A), and the thickening of media in fine blood vessels of 50 μm or less was more significant (P < 0.01; 6B).

Sequence listing

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Claims

1. A method of constructing a non-human animal model of pulmonary arterial hypertension, comprising modifying the genome of a non-human animal such that the modified animal genome comprises the BMP9 gene; and obtaining a non-human animal comprising the modified genome.

2. The method of claim 1, wherein the genome of the non-human animal is modified by introducing the BMP9 nucleic acid sequence into the genome of a single cell embryo or embryonic stem cell of the non-human animal.

3. The method of claim 1, wherein the nucleic acid sequence is operably linked to an expression vector.

4. The method of construction of any one of claims 1-3 wherein the animal is a rodent.

5. The method of claim 4, wherein the rodent is a rat.

6. The method of construction of any one of claims 1-3, wherein the pulmonary hypertension animal model exhibits one or more symptoms of pulmonary vascular remodeling, right ventricular hypertrophy, or pulmonary arterial pressure elevation.

7. A cell line derived from a pulmonary hypertension non-human animal prepared by the method of construction of any one of claims 1 to 6.

8. Embryonic stem cells derived from a pulmonary hypertension non-human animal prepared by the method of construction of any one of claims 1 to 6.

9. The method of any one of:

1) a method for breeding pulmonary hypertension animals is characterized in that nonhuman animals modified with BMP9 are bred with wild animals, and BMP9 positive animals are screened;

2) a method of identifying a therapeutic agent for treating pulmonary hypertension, the method comprising:

administering a pharmaceutical agent to a pulmonary hypertension non-human animal prepared by the construction method of any one of claims 1-6;

10. Use according to any one of the following:

1) use of the non-human animal model of pulmonary hypertension, the cell line of claim 7 or the embryonic stem cell of claim 8, prepared by the construction method of any one of claims 1 to 6, in screening a drug for treating/preventing pulmonary hypertension;

2) use of the non-human animal model of pulmonary hypertension prepared by the construction method according to any one of claims 1 to 6, the cell line according to claim 7 or the embryonic stem cell according to claim 8 for evaluating the therapeutic effect of pulmonary hypertension;

3) application of BMP9 in constructing spontaneous pulmonary hypertension non-human animal model.