A method for constructing an animal model of patent foramen ovale

Abstract

The present invention relates to the field of animal model construction, and specifically to a method for constructing a patent foramen ovale animal model. The present invention provides a use of PolyI:C in constructing a patent foramen ovale animal model, characterized in that the animal includes a rodent. The positive rate of patent foramen ovale in rats is 0%. The present invention creatively finds that injection of PolyI:C during pregnancy can induce patent foramen ovale in the heart of rat offspring, and the induced patent foramen ovale will not close as the offspring age. Therefore, PolyI:C can be used as a patent foramen ovale inducing reagent to construct a patent foramen ovale animal model. The present invention also provides a method for constructing a patent foramen ovale animal model, the model establishment process is simple and fast, the method is simple and easy to operate, and has a high incidence of PFO.

Description

[0001] Priority application

[0002] This application claims priority to the Chinese invention patent application [2023114283039] "A method for constructing an animal model of patent foramen ovale" filed on October 31, 2023, which is incorporated by reference in its entirety. Technical Field

[0003] The invention relates to the field of animal model construction, and in particular to a method for constructing a patent foramen ovale animal model. Background Art

[0004] The foramen ovale is a channel that directly connects the two sides of the atrium circulation during the fetal period. After birth, the primary and secondary septums of most people come close to each other, adhere, and fuse, gradually forming a permanent atrial septum. If it is not completely fused after 3 years of age, the remaining slit-like channel is called patent foramen ovale (PFO).

[0005] PFO allows microemboli, air and inflammatory mediators, choline compounds, small molecules such as glutamine, and some oxygen-free venous blood to avoid pulmonary circulation metabolism and directly enter the systemic circulation, leading to cerebrovascular endothelial cell dysfunction or changes in cortical excitability. Studies have shown that patent foramen ovale (PFO) is an independent risk factor for neurological diseases such as stroke, migraine, and epilepsy, and epilepsy patients with PFO are mostly drug-resistant epilepsy, which is easy to relapse and difficult to cure. Therefore, understanding the mechanism of PFO-induced neurological diseases has great clinical significance and scientific value for developing new treatment strategies, improving patient prognosis, and increasing disease cure rates.

[0006] In current scientific research around the world, rodents are the most common experimental animals because of their easy breeding, fast reproduction and high reproduction rate, low price, and similar physiology and pathology to humans. In addition, with the development and maturity of genetic engineering and gene editing technology, the success rate of gene editing and the survival rate of offspring in rodents are significantly higher than those in other animals; and the construction of stable transgenic animals is crucial for exploring the pathogenesis of diseases, drug target mining and new drug development. In summary, these characteristics have led to the widespread use of rodents in research in the fields of medicine, biology, chemistry, etc. Although PFO has been reported to cause the occurrence of a variety of neurological diseases, there is currently no rodent model of patent foramen ovale because the natural positive rate of patent foramen ovale in rodents is 0. This greatly limits the research on the mechanism of PFO and treatment options based on rodents. Summary of the invention

[0007] In a first aspect, the present invention provides the use of PolyI:C in constructing an animal model of patent foramen ovale, characterized in that the animal includes a rodent.

[0008] In some embodiments, the use comprises: injecting PolyI:C into the pregnant rodent to obtain offspring of the rodent, wherein the offspring of the rodent is the patent foramen ovale animal model.

[0009] In some embodiments, the dosage of PolyI:C comprises 10 - 20 mg / kg.

[0010] In some embodiments, the dose of PolyI:C comprises 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg or 20 mg / kg.

[0011] In some embodiments, the rodent comprises a rat.

[0012] In some embodiments, the rodent is 8-15 days pregnant.

[0013] In some embodiments, the rodent is 8, 9, 10, 11, 12, 13, 14, or 15 days pregnant.

[0014] In some embodiments, the rodents are housed at a constant temperature of 20-25° C. and a relative humidity of 50%-65%.

[0015] In some embodiments, the injection times of PolyI:C include 3 times.

[0016] In a second aspect, the present invention provides a use of a YAP inhibitor in constructing an animal model of patent foramen ovale, characterized in that the animal comprises a rodent.

[0017] In some embodiments, the use comprises: injecting the YAP inhibitor into a newborn rodent.

[0018] In some embodiments, the newborn rodent is 1-3 days old.

[0019] In some embodiments, the newborn rodent is 1 day old.

[0020] In some embodiments, the injection site is the heart.

[0021] In some embodiments, the YAP inhibitor comprises verteporfin.

[0022] In some embodiments, the dosage of the YAP inhibitor comprises 90 - 110 mg / kg.

[0023] In some embodiments, the dosage of the YAP inhibitor comprises 90 mg / kg, 91 mg / kg, 92 mg / kg, 93 mg / kg, 94 mg / kg, 95 mg / kg, 96 mg / kg, 97 mg / kg, 98 mg / kg, 99 mg / kg, 100 mg / kg, 101 mg / kg, 102 mg / kg, 103 mg / kg, 104 mg / kg, 105 mg / kg, 106 mg / kg, 107 mg / kg, 108 mg / kg, 109 mg / kg or 110 mg / kg.

[0024] In some embodiments, the number of injections of the YAP inhibitor comprises 3 times.

[0025] In some embodiments, the YAP inhibitor is injected once a day.

[0026] In a third aspect, the present invention provides a method for constructing an animal model of patent foramen ovale, characterized in that the method comprises:

[0027] S101 First injection of PolyI:C into pregnant rodents;

[0028] S102 screening the pregnant rodents to select the pregnant rodents whose body weight does not increase or decreases on the first day after the first injection and whose body weight returns to a normal level on the second day after the first injection;

[0029] S103: continuing to inject PolyI:C into the pregnant rodent to obtain offspring of the rodent, thereby obtaining the patent foramen ovale animal model;

[0030] Wherein, the S103 also includes the step of detecting whether the patent foramen ovale of the offspring of the rodent is positive, and the detection method includes one or more of cardiac pathology detection, cardiac RNA detection, brain tissue marker detection and ultrasound detection.

[0031] In some embodiments, the dosage of PolyI:C comprises 10 - 20 mg / kg.

[0032] In some embodiments, the dose of PolyI:C comprises 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg or 20 mg / kg.

[0033] In some embodiments, the rodent comprises a rat.

[0034] In some embodiments, the rodent is 8-15 days pregnant.

[0035] In some embodiments, the rodent is 8, 9, 10, 11, 12, 13, 14, or 15 days pregnant.

[0036] In some embodiments, the rodents are housed at a constant temperature of 20-25° C. and a relative humidity of 50%-65%.

[0037] In some embodiments, the number of injections of the continued injection of PolyI:C includes 2 times.

[0038] In some embodiments, the cardiac pathology detection comprises paraffin embedding, sectioning and HE staining of a heart sample of the offspring of the rodent to detect whether the foramen ovale is closed.

[0039] In some embodiments, the brain tissue marker detection comprises detecting the level of one or more of glutamate, glutamine, glutamate receptors, and postsynaptic density proteins in the hippocampus of a brain tissue sample of the offspring of the rodent.

[0040] In some embodiments, the offspring of the rodent are assessed as positive for foramen ovale closure when the level of one or more of glutamate, glutamine, glutamate receptors, and postsynaptic density proteins in the hippocampus of the brain tissue sample is elevated.

[0041] In some embodiments, the cardiac RNA detection comprises detecting RNA from a cardiac sample of the offspring of the rodent, wherein the RNA is selected from one or more of Cdh1, Mpp5, Ccne1, Ppp2ca, Ppp2cb, Ppp2r2d, Mst1, Lix1l, Pard6a, Pard6g, Wtip, Fjx1, Taok1, Taok3, Stk4, Crb3, Tjb1, Gpc5, Smad1, Taz, Ywhab, Ywhaq, Ywhae and Dvl2.

[0042] In some embodiments, when the level of one or more of said RNAs in said heart sample is elevated, the offspring of said rodent are assessed as positive for closure of the foramen ovale.

[0043] In some embodiments, the ultrasonic detection includes applying pressure to the abdomen of the offspring of the rodent so that the chest pressure of the offspring of the rodent increases, injecting bubbled saline into the offspring of the rodent and releasing the pressure, and assessing whether the closure of the foramen ovale of the offspring of the rodent is positive by determining whether there is a right-to-left shunt at the foramen ovale.

[0044] In some embodiments, the offspring of the rodent are assessed as positive for foramen ovale closure when a right-to-left shunt is present at the foramen ovale.

[0045] In some embodiments, the method further comprises: injecting the YAP inhibitor into offspring of the rodent.

[0046] In some embodiments, the rodent's offspring comprise 1-3 days old.

[0047] In some embodiments, the offspring of the rodent are 1 day old.

[0048] In some embodiments, the injection site is the heart.

[0049] In some embodiments, the YAP inhibitor comprises verteporfin.

[0050] In some embodiments, the dosage of the YAP inhibitor comprises 90 - 110 mg / kg.

[0051] In some embodiments, the dosage of the YAP inhibitor comprises 90 mg / kg, 91 mg / kg, 92 mg / kg, 93 mg / kg, 94 mg / kg, 95 mg / kg, 96 mg / kg, 97 mg / kg, 98 mg / kg, 99 mg / kg, 100 mg / kg, 101 mg / kg, 102 mg / kg, 103 mg / kg, 104 mg / kg, 105 mg / kg, 106 mg / kg, 107 mg / kg, 108 mg / kg, 109 mg / kg or 110 mg / kg.

[0052] In some embodiments, the number of injections of the YAP inhibitor comprises 3 times.

[0053] In some embodiments, the YAP inhibitor is injected once a day.

[0054] In some embodiments, S104 injects pentylenetetrazol into the offspring of the rodent to construct an animal model of patent foramen ovale and epilepsy co-morbidity.

[0055] In some embodiments, the rodent offspring are comprised between 30 and 40 days old.

[0056] In some embodiments, the age of the offspring of the rodent comprises 35 days of age.

[0057] In some embodiments, the offspring of the rodent are male.

[0058] In some embodiments, the single injection dose of pentylenetetrazol comprises 35 mg / kg, and the injection method of pentylenetetrazol comprises injecting once every other day.

[0059] In some embodiments, the number of injections of pentylenetetrazol is at most 10 times.

[0060] Compared with the prior art, the beneficial effects of the present invention include at least the following aspects:

[0061] The foramen ovale of rats will close 2-7 days after birth, that is, the positive rate of patent foramen ovale in rats is 0%. The prior art only reports that injection of PolyI:C during pregnancy will affect the nervous system of rat offspring. There is no report on how to construct a patent foramen ovale rodent model. The present invention creatively discovered that injection of PolyI:C during pregnancy can induce patent foramen ovale in the hearts of rat offspring, and the induced patent foramen ovale will not close as the offspring age. Further, the present invention discovered that injection of YAP inhibitors into newborn rats can also induce patent foramen ovale in rat hearts, and the induced patent foramen ovale will not close as the offspring age. Therefore, both PolyI:C and YAP inhibitors (such as verteporfin) can be used as patent foramen ovale inducing agents for constructing a patent foramen ovale animal model.

[0062] Based on this, the present invention also provides a method for constructing an animal model of patent foramen ovale. The model establishment process is simple and fast, the method is simple and easy to operate, and has a high incidence of PFO. The construction method provided by the present invention only uses a single influencing factor in the development process, and excludes changes caused by gene mutations. Compared with the traditional transgenic mouse model, the patent foramen ovale animal model constructed by the present invention has more operability, such as in vivo diagnosis, interventional treatment, etc., and is suitable for exploring the relevant pathological mechanisms and changes of patent foramen ovale. In addition, the method for constructing an animal model of patent foramen ovale of the present invention uses relatively cheap rats and does not require genetic identification, which not only improves efficiency but also reduces costs. It can be widely used to explore the pathogenesis of PFO and develop new intervention measures.

[0063] Furthermore, based on the method for constructing a patent foramen ovale animal model provided by the present invention, a patent foramen ovale comorbidity model can also be constructed, such as a patent foramen ovale and epilepsy comorbidity animal model. Without pentylenetetrazol treatment, the patent foramen ovale animal model constructed by the present invention will not have epileptic seizures, and the number of injections for the first spontaneous epileptic seizure in the patent foramen ovale and epilepsy comorbidity animal model constructed by the present invention is significantly shortened, the duration of epileptic seizures is also longer, and the time required for modeling is also significantly shortened (when the modeling is completed, the age of the rat is only about 2 months old). In summary, the construction method provided by the present invention has a high modeling success rate and a low mortality rate, and the epilepsy susceptibility of the constructed patent foramen ovale and epilepsy comorbidity animal model is significantly improved, which can simulate the epilepsy process of epilepsy patients, and can be better applied to the qualitative and quantitative research of targeted therapeutic and / or preventive schemes for patients with patent foramen ovale and epilepsy comorbidity. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without paying creative labor.

[0065] Figure 1 The diagnostic criteria for the closure of the foramen ovale in offspring mice are shown;

[0066] Figure 2 The figure shows the experimental results that MIA mice exhibit a lowered epileptic seizure threshold;

[0067] Figure 3 The result graphs show the expression levels of glutamate, glutamine, glutamate receptors and postsynaptic density proteins in MIA offspring mice;

[0068] Figure 4 Ultrasound images of PFO-negative and -positive rats before and after abdominal pressure application are shown;

[0069] Figure 5 The result graph shows the up-regulated genes of Hippo pathway in rats with positive foramen ovale closure;

[0070] Figure 6 HE staining images of rats showing positive verteporfin-induced foramen ovale closure are shown. DETAILED DESCRIPTION

[0071] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0072] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0073] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0074] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0075] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0076] In this specification, some embodiments may be disclosed in a format of being in a range. It should be understood that this description of "being in a range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the description of the range 1-6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, the above rules apply.

[0077] Embodiment 1

[0078] Animal preparation: 3-month-old Sprague Dawley female and male rats were selected and kept at a constant temperature of 20-25°C and a relative humidity of 50%-65%. Female rats were placed in the same cage with male rats for mating, and then kept in a single cage after mating.

[0079] Pregnant mice were randomly divided into 3 groups, including a control group (5 mg / kg tail vein injection of 0.9% saline), a low-dose model group (4 mg / kg tail vein injection of double-chain poly I:C (dissolved in saline)) and a high-dose model group (10 mg / kg tail vein injection of double-chain poly I:C (dissolved in saline)). On the 15th day of pregnancy, double-chain poly I:C was injected into the pregnant mice through the tail vein under isoflurane gas anesthesia according to the weight of the pregnant mice, and the weight changes were observed for 4 consecutive days. On the first day after the first injection of PolyI:C, the weight did not increase or decreased (the weight of normal pregnant mice is linearly related to the pregnancy time, such as 10±2g per day), and on the second day after the first injection of PolyI:C, the pregnant mice whose weight returned to the expected weight level were included in the subsequent experiments and continued to be injected twice (once a day).

[0080] The offspring were weaned and separated into cages 21 days after birth, and then killed after anesthesia. The hearts were collected and embedded in paraffin, sliced, and stained with HE. The closure of the foramen ovale of the offspring was examined under a microscope. The diagnostic criteria were as follows: Figure 1 As shown (L represents the left atrium and R represents the right atrium).

[0081] Embodiment 2

[0082] 0.9% saline, low-dose PolyI:C, and high-dose PolyI:C were injected into the tail vein of 15-day-old pregnant rats. Figure 1 The cardiac pathological detection method shown is used to detect the closure of the foramen ovale in offspring mice to evaluate the stability of the animal model and the positive rate of detecting patent foramen ovale in offspring mice.

[0083] Table 1

[0084]

[0085] It is reported that the foramen ovale of rats will close 2-7 days after birth. The results are shown in Table 1. The above experimental data prove that the injection of high doses of PolyI:C into the tail vein of 15-day pregnant rats can successfully induce patent foramen ovale in offspring (i.e., the foramen ovale will not close as the offspring age), and the construction method provided by the present invention can successfully construct a patent foramen ovale model in rodents.

[0086] Embodiment 3

[0087] Evaluation of epilepsy susceptibility by chemical kindling

[0088] The PTZ group was evaluated for the rate of kindling of epilepsy using pentylenetetrazol (PTZ, Sigma-Aldrich, USA) solution. PTZ was dissolved in 0.9% saline at a concentration of 35 mg / ml and injected intraperitoneally according to body weight (1 ml / kg) or an equal amount of saline. The animals were observed for 30 min after administration, and the Racine score (stage 1-5) was used to evaluate the severity of epileptic seizures and different systemic epilepsy phenotypes. Each animal was kindled every other day until three consecutive stage 4-5 epileptic seizures occurred, with a maximum of 15 kindlings. The epilepsy susceptibility was scored according to the success rate of kindling in each group.

[0089] Electroencephalogram (EEG) recording

[0090] Rats were anesthetized with isoflurane and fixed in a stereotaxic apparatus in a prone position. The skin and fascia were cut to expose the entire skull, with the bregma as the zero point. Four positions were marked, 3 mm before and after the bregma and 3 mm to the left and right of the midline. Holes were drilled to just penetrate the skull without causing damage. The electrode screws were firmly implanted in the holes with forceps, and a layer of dental cement was used to insulate the screws. After drying, the silver wires on the screws were connected to the silver wires on the head-mounted device, and another layer of dental cement was used to cover all the silver wires for insulation.

[0091] Animals were acclimated one hour before observation. Rats were placed in observation cages and the EEG amplifier was connected to the head-mounted device. After adjusting the camera, 5 minutes of baseline video and EEG were recorded in awake and resting state. The amplifier was disconnected, PTZ was injected, and the EEG was immediately reconnected to record activity for 30 minutes. The software (Pinnacle, USA) recorded EEG from the motor cortex and somatosensory cortex.

[0092] Pregnant mice were injected with 10 mg / kg PolyI:C through the tail vein on day 15 of pregnancy, and PTZ (35 mg / kg) was injected into the offspring 35 days after birth to induce chronic epileptic seizures (MIA group). The control group was injected with normal saline through the tail vein on day 15 of pregnancy, and PTZ (35 mg / kg) was injected into the offspring 35 days after birth to induce chronic epileptic seizures (control group). Heart pathological examination of patent foramen ovale was performed at least one week after the last epileptic seizure. All animals were killed under anesthesia (inhalation of isoflurane), and the heart specimens were paraffin-embedded, sectioned and HE-stained. The closure of the foramen ovale of the offspring was examined under a microscope to evaluate the positive rate of patent foramen ovale in the offspring.

[0093] Brain sample collection: After isoflurane anesthesia, rats received cardiac perfusion with ice-cold saline. The brain and both ovaries were quickly removed and placed in cold saline for at least 45 seconds. For brains used for LC / MS or Western blotting, the hippocampus was dissected from each hemisphere on ice, snap-frozen with liquid nitrogen, and stored at -80°C.

[0094] Heart collection: For pathological diagnosis, the collected hearts were fixed in 10% neutral formalin for 24 h and then embedded in paraffin. For rats undergoing in vivo ultrasound examination of PFO, the rats received cardiac perfusion with ice-cold saline. The hearts were collected and the atrial septum was dissected, snap-frozen with liquid nitrogen, and stored at -80°C until use.

[0095] The success rate of epileptic seizures in the MIA group was significantly higher than that in the control group ( Figure 2 A), especially in male offspring, the success rate was over 90%. The injection time to the first spontaneous epileptic seizure in MIA offspring was also shorter than that in the control group ( Figure 2 B) Spontaneous seizures occurred within 10 injections of PTZ and the duration of seizures was longer ( Figure 2 C), which suggests that the epileptic seizure threshold of the MIA group mice is lowered, and the animal model of patent foramen ovale and epilepsy comorbidity has been successfully established. The EEG recording of the MIA group showed an increase in frequency and a higher amplitude ( Figure 2 D) Without PTZ treatment, the MIA group mice did not develop epileptic seizures.

[0096] This example also measured the glutamate and glutamine levels in the hippocampus of the brain tissue of each experimental group. The glutamate and glutamine concentrations of the MIA group mice increased significantly ( Figure 3 A and Figure 3 B). In addition, the levels of glutamate receptor (GluR1) and postsynaptic density protein (PSD95) in the MIA group mice were increased compared with those in the control group ( Figure 3 C and Figure 3 D). The above results indicate that maternal immune activation (MIA) leads to neuronal hyperexcitability.

[0097] Embodiment 4

[0098] This example develops a method for detecting patent foramen ovale in rodents by ultrasound, which can identify PFO-positive rodents in vivo rather than performing pathological diagnosis after sacrifice.

[0099] In vivo PFO ultrasound examination: Rats inhale the anesthetic isoflurane and lie in a supine position on a warming pad to maintain body temperature. They can be lightly restrained or fixed with tape to minimize limb movement. An indwelling needle is inserted into the rat's tail vein and fixed with 5% sodium heparin. The chest to be examined is shaved or coated with depilatory cream. The ultrasound transducer is placed on the left chest, ensuring the atrial septum and measuring the heart rate amplitude. A 5ml syringe with 0.5ml saline, a 5ml syringe with 0.5ml air and the indwelling needle are connected in a T shape. Alternately inject the two syringes to create small bubbles in the saline, and then slowly inject into the tail vein. If there are no bubbles in the left atrium within the first 5 seconds, an atrial septal defect can be ruled out. Then apply pressure to the rat's abdomen and move up to the chest cavity to make the chest pressure higher. When the bubbled saline is injected again, the pressure is released. The right-to-left shunt through the foramen ovale is recorded by ultrasound to diagnose PFO. Its high accuracy was verified using the pathological diagnosis of PFO.

[0100] PFO was examined by ultrasound in MIA mice, and the right-to-left shunt was observed after the abdominal force was released. PFO-positive animals were selected ( Figure 4 ).

[0101] RNA-seq was performed using hippocampal tissue to look for differences between PFO-positive and -negative animals. 915 upregulated genes and 703 downregulated genes were detected. KEGG was used to calculate and enrich the regulated genes, and the results showed that the hippo pathway was significantly upregulated in MIA offspring.

[0102] To determine whether overactivation of the hippo pathway is observed in PFO-positive hearts, this example evaluates the PCR array of the hippo signaling pathway based on fresh cardiac atrial septal tissue from PFO-positive and -negative rats. In PFO-positive rats, RNA expression in this pathway (including Cdh1, Mpp5, Ccne1, Ppp2ca, Ppp2cb, Ppp2r2d, Mst1, Lix1l, Pard6a, Pard6g, Wtip, Fjx1, Taok1, Taok3, Stk4, Crb3, Tjb1, Gpc5, Smad1, Taz, Ywhab, Ywhaq, Ywhae, Dvl2) was significantly increased ( Figure 5 ).

[0103] Embodiment 5

[0104] Based on the results of the above examples, in this example, the YAP1 inhibitor Verteporfin was injected into the hearts of newborn rats (1 day old) at 100 mg / kg for 3 consecutive days to ensure the role of YAP1 in this process. After pathological diagnosis of heart tissue at 14 days old, PFO was observed in animals treated with Verteporfin. Figure 6), the modeling success rate was 100%. The above experiments showed that YAP1 inhibitors can induce positive patent foramen ovale in rats, and overactivated hippo pathway is the key mechanism of PFO.

[0105] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the enlightenment of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the purpose of the present invention and the claims, which all fall within the protection of the present invention.

Claims

1. Use of PolyI:C in constructing an animal model of patent foramen ovale, characterized in that: The animals include rodents; the use includes: injecting PolyI:C into the pregnant rodents to obtain offspring of the rodents, wherein the offspring of the rodents are the patent foramen ovale animal model; wherein the dosage of the PolyI:C includes 10-20 mg / kg.

2. Use of a YAP inhibitor in constructing an animal model of patent foramen ovale, characterized in that: The animal includes a rodent, and the use includes: injecting the YAP inhibitor into a newborn rodent; wherein the dosage of the YAP inhibitor includes 90-110 mg / kg, and the injection site of the YAP inhibitor is the heart.

3. The use according to claim 2, characterized in that The YAP inhibitors include verteporfin.

4. A method for constructing an animal model of patent foramen ovale, characterized in that: The method comprises: S101 injecting PolyI:C into a pregnant rodent for the first time; wherein the dose of PolyI:C comprises 10-20 mg / kg; S102 screening the pregnant rodents to select the pregnant rodents whose body weight does not increase or decreases on the first day after the first injection and whose body weight returns to a normal level on the second day after the first injection; S103: continuing to inject PolyI:C into the pregnant rodent to obtain offspring of the rodent, thereby obtaining the patent foramen ovale animal model; Wherein, the S103 also includes the step of detecting whether the patent foramen ovale of the offspring of the rodent is positive, and the detection includes one or more of cardiac pathology detection, cardiac RNA detection, brain tissue marker detection and ultrasound detection.

5. The method according to claim 4, characterized in that The method further comprises S104 injecting pentylenetetrazol into the offspring of the rodent to construct an animal model of patent foramen ovale and epilepsy co-morbidity.

6. The method according to claim 4, characterized in that The rodent offspring range in age from 30 to 40 days.

7. The method according to claim 5, characterized in that The number of injections of pentylenetetrazol is at most 10 times.

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