Construction method and application of a fetal myocardial noncompaction animal model

Abstract

The present invention relates to a method for constructing an animal model of fetal myocardial noncompaction, comprising the step of intrauterine perfusion of mid-gestational mice with oleoylethanolamine. By intrauterine perfusion of OEA into gestational day E9 mice, NVM fetal mice were created, exhibiting the characteristic of NVM, characterized by the failure of ventricular trabeculae to compact. This method provides new insights and approaches for the etiology, diagnosis, prevention, and treatment of NVM.

Description

Technical Field

[0001] The present invention relates to the technical field of animal model construction, and in particular to a construction method and application of a fetal myocardial noncompaction animal model. Background Art

[0002] Noncompaction ventricular myocardium (NVM), also known as spongy myocardium, is a rare congenital cardiomyopathy. During early human embryonic development, the myocardium exhibits a loose reticular fiber structure. This "spongy" structure gradually compacts between 5 and 8 weeks of gestation. Myocardial compaction progresses from the epicardium to the endocardium, and from the base to the apex. NVM is caused by genetic or chromosomal abnormalities, resulting in arrested endocardial development, failure to close the intermyocardial sinusoids and reticular spaces, and failure of reticular fiber compaction. Multiple trabecular protrusions and deep intertrabecular recesses are observed on the myocardial surface. NVM can occur in isolation or in conjunction with other congenital heart anomalies, including right or left ventricular outflow tract obstruction, cyanotic congenital heart disease, and coronary artery anomalies. Clinically detected genetic mutations that cause NVM include G4.5 gene mutations located in the Xq28 region of chromosome X, α-dystrophin mutations, CSX mutations, and FKBP12 mutations.

[0003] Due to medical ethical restrictions, animal models are crucial tools for studying the pathogenesis of NVM and exploring treatments. Primates share many similarities with humans in gestation and cardiac development, offering unparalleled advantages as models for human heart disease research. However, due to their high cost, complex procedures, and long experimental cycles, mice and rats remain the preferred experimental animals for constructing heart disease models.

[0004] Existing research on NVM primarily consists of clinical case reports and treatments. However, the clinical incidence of NVM is low, and research and clinical resources are very limited. The etiology of NVM remains unclear, and there are no definitive treatment options or effective prevention or early screening methods, resulting in slow research progress.

[0005] Currently, there are few animal models for NVM. Most of the NVM models reported in research papers are genetically altered NVM models, or gene-deficient models. These models utilize gene targeting to knock out a single gene and then examine whether the offspring of these animals display NVM symptoms. For example, a mouse model specifically knocks out the YAP1 gene in cardiomyocytes (Hey2-CreER; Yap1flox / flox) has been developed. However, NVM involves a wide range of gene mutations, and a single gene can only alter the downstream signaling pathways it regulates and cannot accurately mimic all NVM phenotypes. Some studies have induced NVM in fetal mice by administering 70 mg / kg of all-trans retinoic acid to pregnant mice. However, retinoic acid is essential for maintaining normal embryonic development and adult tissue differentiation, and no studies have reported the clinical basis and pathological mechanisms underlying the induction of NVM in fetuses with excessive retinoic acid use. Therefore, retinoic acid-induced fetal NVM models rarely mimic the true causes of clinical NVM. Other studies have established a model of cardiac noncompaction in offspring by feeding pregnant mice a high-fat diet combined with streptozotocin (STZ). However, STZ itself acts by selectively destroying pancreatic β cells and is commonly used to establish animal models of diabetes. STZ-induced NVM also induces type 2 diabetes in pregnant mice. However, the mechanisms of cardiac dysplasia under diabetic pathology are completely different from those of clinical NVM and cannot simulate the pathogenesis of clinical NVM. Summary of the Invention

[0006] The purpose of the present invention is to overcome the shortcomings of the existing technology and provide a method for constructing and applying a fetal myocardial noncompaction animal model. The animal model established by this method has obvious symptoms of NVM.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] In a first aspect, the present invention provides a method for constructing an animal model of fetal myocardial noncompaction, comprising the step of using oleoylethanolamine to perform intrauterine perfusion on mid-gestational mice.

[0009] Oleoylethanolamine (OEA) is an endogenous cannabinoid produced in humans from fatty acids. Through the cannabinoid receptors CB1 and CB2, OEA inhibits downstream cAMP, p38, and ERK1 / 2 signaling pathways, regulating gamete formation, early embryo implantation, and placental development. OEA levels are elevated in the follicular fluid of infertile patients and in the serum of patients with ectopic pregnancies. OEA regulates the fiber oscillation of fallopian tube epithelial cells, leading to ectopic pregnancies and infertility.

[0010] The present invention perfuses oleoylethanolamine into the uterine cavity of mid-pregnant mice to cause the fetuses of the mice to have obvious NVM symptoms. The method is simple to operate and takes a short time to establish the NVM disease model.

[0011] As a preferred embodiment of the first aspect, the specific operation of the intrauterine perfusion is as follows: the needle of a 1 ml syringe is polished to a blunt shape, the syringe with the polished needle is filled with oleoylethanolamine solution and inserted into the mouse cervix to inject the solution 0.5 cm away.

[0012] As a preferred embodiment of the first aspect, the dosage of oleoylethanolamine is 10 mg / kg, and the volume is about 30 μl.

[0013] As a preferred embodiment of the first aspect, the second trimester is gestational day 9. Since days E9 to E9.5 are the critical period for myocardial compaction, during which myocardial cells form trabeculae and compact, OEA is intrauterine perfused into pregnant mice on gestational day E9.

[0014] As a preferred embodiment of the first aspect, before performing the intrauterine perfusion, the method further includes placing the female mouse and the male mouse in the same cage, observing the vaginal plug on the next day to confirm that the female mouse is pregnant and setting the pregnancy as 0.5 days.

[0015] As a preferred embodiment of the first aspect, it also includes detecting the thickness of the non-compacted layer myocardium and the compacted layer myocardium of the fetal rat heart sample and staining the paraffin sections of the fetal rat heart sample to observe morphological changes.

[0016] As a preferred embodiment of the first aspect, the ratio of female mice to male mice is 2:1.

[0017] In a second aspect, the present invention provides an animal model of fetal myocardial noncompaction constructed by the construction method described in the first aspect.

[0018] In a third aspect, the present invention provides a use of the fetal myocardial noncompaction animal model as described in the second aspect in screening or preparing candidate drugs for treating fetal myocardial noncompaction or the occurrence and development mechanism of fetal myocardial noncompaction.

[0019] In a fourth aspect, the present invention provides the use of oleoylethanolamine in constructing an animal model of fetal myocardial noncompaction.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention provides a method for constructing a NVM mouse animal model. NVM fetal mice were constructed by intrauterine perfusion of OEA into mice at gestational day E9. The results showed that the fetal mice perfused with OEA in the uterine cavity exhibited NVM characteristics of inability to compact the ventricular myocardial trabeculae, which provides new ideas and new means for the etiology research, disease diagnosis, prevention and treatment of NVM. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of H&E staining results of heart tissue sections from E17.5 fetal mice after intrauterine perfusion of OEA or normal saline (the upper figure is a 100x magnified photograph of the fetal heart, and the lower figure is a 400x magnified photograph of the fetal heart). DETAILED DESCRIPTION

[0023] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0024] Experimental Materials:

[0025] (1) C57BL / 6 mice: purchased from Uoda (Guangzhou) Biotechnology Co., Ltd.

[0026] (2) Preparation of OEA solution: Dissolve OEA in 10% DMSO-containing saline to prepare a 100 mg / mL stock solution, which can be stored at -20°C. Dilute the 100 mg / mL OEA stock solution with saline to a concentration of 10 mg / mL as the working solution.

[0027] Example 1

[0028] This example provides a method for constructing an animal model of fetal myocardial noncompaction, which includes the following steps:

[0029] (1) Modeling:

[0030] a: 10-12 week old, 20-26 g C57BL / 6 female and male mice were selected and housed in a 2:1 ratio. The vaginal plug was examined the next morning and recorded as day E0.5 of pregnancy. Confirmed pregnant female mice were divided into a control group and an OEA group, with 4 mice in each group.

[0031] b: Pregnant mice in the OEA group received one intrauterine perfusion of 10 mg / kg OEA (volume approximately 30 μL) at gestational age of 9; pregnant mice in the control group received one intrauterine perfusion of 30 μL normal saline at gestational age of 9.

[0032] The specific operation of intrauterine perfusion in pregnant mice is as follows:

[0033] The needle of a 1 ml syringe was polished to a blunt shape, and the syringe with the polished needle was filled with oleoylethanolamine solution and inserted into the mouse cervix to inject the solution 0.5 cm away.

[0034] (2) NVM feature detection:

[0035] Fetal heart samples were collected at gestational day E17.5, dehydrated, and paraffin-embedded. Paraffin sections of the fetal hearts were stained with hematoxylin-eosin (H&E) to observe morphological changes.

[0036] The results are shown in Table 1 and Figure 1 As shown, Figure 1 There was no significant difference in the overall size of the hearts between the two groups, but the loose myocardial trabeculae in the OEA group almost filled the ventricular cavity. Under high-power microscopy, it can be observed that the interstitial space between the myocardial cells in the OEA group was significantly larger than that in the control group. Table 1 measured the thickness of the non-compacted myocardium layer (N) and the compacted myocardium layer (C) of the fetal heart, and calculated the ratio of the two thicknesses (N / C). According to the reference report [1], N / C>2 is consistent with the diagnosis of NVM. Therefore, it can be clearly determined that the OEA group significantly caused NVM symptoms.

[0037] Table 1: Left ventricular N, C and N / C values ​​of fetal rats in the normal group and OEA group

[0038]

[0039]

[0040] References

[0041] 【1】Jenni R, Oechslin E, Schneider J, et al. Echocardiographic and pathoanatomical characteristics of isolated left ventricular non-compaction: a step towards

[0042] classification as a distinct cardiomyopathy.Heart.2001;86:666-671.

[0043] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for constructing an animal model of fetal myocardial noncompaction, characterized in that: The method comprises the steps of using oleoylethanolamine to perform intrauterine perfusion on mid-pregnant mice; the intrauterine perfusion comprises grinding the needle of a syringe into a rounded shape, aspirating the oleoylethanolamine solution with the syringe after the grinding needle, inserting the syringe into the mouse cervix at a position 0.5 cm, and then injecting the oleoylethanolamine solution; the dosage of the oleoylethanolamine is 10 mg / kg.

2. The construction method according to claim 1, wherein The second trimester is the 9th day of pregnancy.

3. The construction method according to claim 1, wherein Before the intrauterine perfusion, the female mouse and the male mouse were placed in the same cage, and the vaginal plug was observed on the next day to confirm that the female mouse was pregnant and the pregnancy was determined to be 0.5 days.

4. The construction method according to claim 3, wherein: The ratio of female to male mice was 2:

1.

5. The construction method according to claim 1, wherein: It also includes detecting the thickness of the non-compacted layer of the fetal heart myocardium and the thickness of the compacted layer of the fetal heart sample, and observing the morphological changes of the paraffin sections of the fetal heart sample.

6. Use of the method for constructing an animal model of fetal myocardial noncompaction according to any one of claims 1 to 5 in screening or preparing candidate drugs for treating fetal myocardial noncompaction or the occurrence and development mechanism of fetal myocardial noncompaction.

Images