Construction method and application of fetal cardiac insufficiency densification animal model

Abstract

The invention relates to a construction method of an animal model for fetal cardiac insufficiency in densification. The construction method comprises the step of performing uterine cavity perfusion on a pregnant mouse in the middle stage of pregnancy by using oleoyl ethanolamine. The NVM fetal mouse is constructed by perfusing OEA into the uterine cavity of the mouse with the pregnancy E9 days, the fetal mouse has the NVM characteristic that ventricular muscle trabecula cannot be densified, and a new thought and a new means are provided for NVM etiological research and disease diagnosis, prevention and treatment.

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 incomplete compaction animal model. Background Art

[0002] Noncompaction ventricular myocardium (NVM), also known as spongy myocardium, is a rare congenital cardiomyopathy. In the early stages of human embryonic development, the myocardium presents a loose reticular fiber structure. During the next 5 to 8 weeks of pregnancy, this myocardial "spongy" structure gradually becomes compact. The process of myocardial compaction is from the epicardium to the endocardium, from the base of the heart to the apex of the heart. The onset of NVM is caused by genetic or chromosomal abnormalities, which lead to endocardial development arrest, failure to close the intermyocardial sinusoids and reticular spaces, failure of reticular fiber compaction, and multiple trabecular protrusions and deep recesses between trabeculae on the surface of the myocardium. NVM can exist in isolation or in combination with other congenital heart abnormalities, including right ventricular or left ventricular outflow tract obstruction, cyanotic congenital heart disease, and coronary artery abnormalities. Clinically detected genetic mutations that cause NVM include G4.5 gene mutation located in the Xq28 region of chromosome X, α-dystrophin mutation, CSX mutation, and FKBP12 mutation.

[0003] Due to medical ethical restrictions, animal models are important tools for studying the pathogenesis of NVM and exploring treatment measures. Primates have many similarities with humans in terms of pregnancy and heart development, and have incomparable advantages as models for studying human heart disease. However, due to their high price, complex operation, and long experimental cycle, mice and rats are still the preferred experimental animals for constructing heart disease models.

[0004] In the prior art, most of the research on NVM is clinical NVM case reports and treatment processes. However, the clinical incidence of NVM is low, and scientific research and clinical resources are very limited. The etiology of NVM is still unclear, there is no definite treatment plan and effective means of prevention and early screening, and research progress is slow.

[0005] There are few animal models of NVM currently available. Most of the NVM animal models that appear in research papers are gene-modified NVM animal models, that is, gene defect models, which use gene targeting technology to knock out a single gene and detect whether the offspring of gene-defective animals have NVM symptoms. For example, a mouse model that specifically knocks out the YAP1 gene in cardiomyocytes (Hey2-CreER; Yap1flox / flox). However, there are many types of gene mutations involved in NVM. A single gene can only change the downstream signaling pathways it regulates and cannot accurately simulate all phenotypes of NVM. Some studies have induced fetal mouse NVM models by giving pregnant mice 70 mg / kg all-trans retinoic acid, but retinoic acid is an important element that is essential for maintaining normal embryonic development and maintaining normal differentiation of adult tissues, and no studies have reported the clinical basis and pathological mechanism of excessive use of retinoic acid to induce fetal NVM. Therefore, the fetal mouse NVM model induced by retinoic acid is difficult to simulate the true cause of clinical NVM. There are also studies that have established a model of incomplete cardiac compaction in offspring by using a high-fat diet combined with streptozotocin (STZ) in pregnant mice. However, the mechanism of action of STZ itself is to selectively destroy pancreatic β cells, and it is usually used to establish animal diabetes models. STZ induces NVM and also induces type 2 diabetes in pregnant mice. However, the mechanism of cardiac dysplasia under diabetic pathological conditions is completely different from the mechanism 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 prior art 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-pregnant mice.

[0009] N-oleoylethanolamine (OEA) is an endogenous cannabinoid substance in the human body that is converted from fatty acids as substrates. OEA inhibits the downstream cAMP, p38, and ERK1 / 2 signaling pathways through cannabinoid receptors CB1 and CB2, regulating gamete formation, early embryo implantation, and placental development. The level of OEA is elevated in the follicular fluid of infertile patients and the serum of patients with ectopic pregnancy, and participates in regulating the fiber swing of fallopian tube epithelial cells, resulting in ectopic pregnancy and infertility.

[0010] The invention perfuses oleoylethanolamine into the uterine cavity of mid-pregnant mice, so that the fetus mice have obvious NVM symptoms. The method is simple to operate and takes a short time to construct 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 be round and blunt, 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 the 9th day of pregnancy. Since E9 to E9.5 days are the critical period for myocardial compaction, myocardial cells form trabeculae and compact. Therefore, OEA is perfused into the uterine cavity of pregnant mice on the 9th day of pregnancy.

[0014] As a preferred embodiment of the first aspect, before the intrauterine perfusion, the method further includes placing the female mouse and the male mouse together in a cage, and observing the vaginal plug on the second day to confirm that the female mouse is pregnant and set 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 the 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 an NVM mouse animal model. NVM fetal mice are constructed by intrauterine perfusion of OEA into mice at gestational day E9. The results show that the fetal mice perfused with OEA in the uterine cavity exhibit NVM characteristics of inability to compact ventricular 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 of E17.5 fetal mice after intrauterine perfusion of OEA or normal saline (the upper picture is a 100-fold magnified photo of the fetal heart, and the lower picture is a 400-fold magnified photo 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 Biotechnology Co., Ltd. (Guangzhou).

[0026] (2) Preparation of N-oleoylethanolamine (OEA) solution: Dissolve OEA in physiological saline containing 10% DMSO to prepare a 100 mg / mL stock solution, which can be stored at -20°C. Dilute the 100 mg / mL OEA stock solution with physiological 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 incomplete compaction, which comprises the following steps:

[0029] (1) Modeling:

[0030] a: Select 10-12 week old C57BL / 6 female and male mice weighing 20-26 g, and cage them in a ratio of 2:1. The vaginal plug is checked the next morning and recorded as the E0.5 day of pregnancy. The confirmed pregnant female mice are 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 infusion of 10 mg / kg OEA (volume about 30 μL) at gestational age of 9; pregnant mice in the control group received one intrauterine infusion of 30 μL normal saline at gestational age of 9;

[0032] Among them, the specific operation of pregnant mice receiving intrauterine perfusion 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 cervix of the mouse to inject the solution 0.5 cm away.

[0034] (2) NVM feature detection:

[0035] Fetal heart samples were collected at gestational day E17.5, and the fetal heart tissues were dehydrated and embedded in paraffin. The fetal heart paraffin sections 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 heart between the two groups, but the loose trabeculae of the ventricular wall in the OEA group almost filled the ventricular cavity. Under a high-power microscope, it can be observed that the interstitial space of 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 thickness of the two (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 obviously caused NVM symptoms.

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

[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 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 solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution 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 intrauterine perfusion of mid-gestation pregnant mice with oleylethanolamine.

2. The construction method according to claim 1, characterized in that: The dosage of the oleoylethanolamine is 10 mg / kg.

3. The construction method according to claim 1, characterized in that: The second trimester is the 9th day of pregnancy.

4. The construction method according to claim 1, characterized in that: Prior to the perfusion, the female mouse and the male mouse were placed in the same cage, and the vaginal plug was observed on the second day to confirm that the female mouse was pregnant and the pregnancy was set at 0.5 days.

5. The construction method according to claim 4, characterized in that: The ratio of female mice to male mice was 2:

1.

6. The construction method according to claim 1, characterized in that: It also includes detecting the thickness of the non-compacted layer of myocardium and the thickness of the compacted layer of myocardium in the fetal rat heart sample, and observing the morphological changes of the staining of the paraffin sections of the fetal rat heart sample.

7. An animal model of fetal myocardial noncompaction constructed by the construction method according to any one of claims 1 to 6.

8. Use of the fetal myocardial noncompaction animal model as claimed in claim 7 in screening or preparing candidate drugs for treating fetal myocardial noncompaction or the occurrence and development mechanism of fetal myocardial noncompaction.

9. Application of oleoylethanolamine in constructing an animal model of fetal myocardial noncompaction.

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