Application of inhibitor in improvement of placenta hypertrophy of cloned animals

Abstract

The invention belongs to the technical field of animal models, and particularly relates to application of an inhibitor in improvement of placenta hypertrophy of cloned animals. According to the method, mice are subjected to grouping, superovulation and caged fertilization to obtain pregnant mice, the placenta hypertrophy mouse model is constructed by replacing alpha-ketoglutaric acid drinking water and sodium crotonate drinking water and adding a gene Idh2 inhibitor and an IDH1 inhibitor, on the 16th day of the pregnancy of the mice, fetuses and placentas of the mice are dissected and separated, and the placenta hypertrophy mouse model is constructed. And observing and recording the size of the fetus, the size of the placenta and the weight of the placenta of the mouse. According to the method, on the basis of not applying molecular biology means, a mouse model with placenta hypertrophy is constructed through drug induction, the purpose of reducing the hypertrophy placenta is achieved by adding an inhibitor of a key gene, and the problem that at present, a gene editing means is adopted to reduce the hypertrophy placenta of a cloned animal, so that the placenta hypertrophy is reduced is solved. And the problems of long period, low efficiency and high cost are solved.

Description

Technical Field

[0001] The invention belongs to the technical field of animal models, and particularly relates to the application of an inhibitor in improving placental hypertrophy of cloned animals. Background Art

[0002] Somatic cell nuclear transfer (SCNT) is a technology that reprograms somatic cells to a totipotent state, and has great application potential in many fields such as animal production and regenerative medicine. However, epigenetic reprogramming barriers during somatic cell reprogramming greatly reduce the efficiency of SCNT, and SCNT embryos are prone to various abnormalities that hinder embryo development. Among them, large offspring syndrome is one of the most common abnormalities in cloned cattle, sheep, and mice, including the production of giant placentas and fetuses. Studies have found that regardless of the source and type of donor cells for somatic cell nuclear transfer, giant placentas can be generally observed during the pregnancy of cloned animals. As a key organ in the pregnancy process, the placenta maintains pregnancy and supports fetal development by providing a large amount of oxygen and nutrients, promoting the production of hormones during pregnancy, and exercising immune function. In cloned animals, abnormal hypertrophy of the placenta is also a major factor restricting the success rate of cloning. Therefore, how to effectively improve the phenomenon of placental hypertrophy in cloned animals is one of the important methods to improve the success rate of cloned animal pregnancy and promote in-depth research and application of cloning technology.

[0003] Previous studies mainly used molecular biological methods to edit genes related to epigenetic modifications in donor cells. By knocking in recombinant genes or knocking out target genes, the purpose of reducing placental hypertrophy in cloned animals can be achieved, thereby improving the pregnancy success rate of cloned animals. However, gene editing methods have a long overall cycle, are expensive, and have low efficiency. Therefore, how to use a more convenient method to construct a model of placental hypertrophy for research is an effective way to solve the problem of placental hypertrophy in cloned animals. Summary of the invention

[0004] The present invention proposes the use of inhibitors in improving placental hypertrophy in cloned animals. This method constructs a mouse model of placental hypertrophy by drug induction without using molecular biological methods, and achieves the purpose of reducing hypertrophic placentas by adding inhibitors of key genes, thereby solving the problems of long cycle, low efficiency and high cost in the current use of gene editing methods to reduce hypertrophic placentas in cloned animals.

[0005] The technical solution of the present invention is achieved in this way:

[0006] A substance for enlarging animal placenta, which is one or both of alpha-ketoglutaric acid and sodium crotonate.

[0007] Use of an inhibitor in improving placental hypertrophy in cloned animals, wherein the inhibitor is one or both of an inhibitor of gene Idh2 or an inhibitor of IDH1.

[0008] Optionally, the inhibitor is used to reduce the hypertrophy of the animal placenta caused by α-ketoglutarate.

[0009] Optionally, the inhibitor is used to reduce the hypertrophy of the animal placenta caused by sodium crotonate.

[0010] Optionally, the inhibitor of gene Idh2 is ensidipine mesylate.

[0011] Optionally, the IDH1 inhibitor is IDH1 Inhibitor 2 (compound 13).

[0012] After adopting the above technical solution, the beneficial effects of the present invention are:

[0013] The present invention can construct a mouse model of placental hypertrophy by only using α-ketoglutaric acid or sodium crotonate for drug induction without applying molecular biological methods, and can achieve the purpose of reducing hypertrophic placenta by adding gene Idh2 inhibitor or IDH1 inhibitor, thus solving the problems of long cycle, low efficiency and high cost in reducing hypertrophic placenta of cloned animals by current gene editing methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0015] Figure 1A The placenta weight of pregnant mice after drinking α-KG and ensidipine mesylate in drinking water was counted;

[0016] Figure 1B The fetal weights of pregnant mice after drinking α-KG and ensidipine mesylate in drinking water were counted;

[0017] Figure 1C To calculate the length of the placenta of pregnant mice after drinking α-KG and ensidipine mesylate in drinking water;

[0018] Figure 1D The statistics of the placental width of pregnant mice after drinking α-KG and ensidipine mesylate in drinking water;

[0019] Figure 2AThe placenta weight of pregnant mice was counted after drinking Nacr and IDH1 inhibitor drinking water;

[0020] Figure 2B The fetal weight of pregnant mice after drinking Nacr and IDH1 inhibitor drinking water was counted;

[0021] Figure 2C The length of the placenta of mice was counted after drinking Nacr and IDH1 inhibitor drinking water;

[0022] Figure 2D The width of the placenta of pregnant mice was counted after drinking Nacr and IDH1 inhibitor drinking water;

[0023] Figure 3A The ELISA was used to detect the content of α-KG in the placenta of pregnant mice after drinking α-KG and ensidipine mesylate in drinking water;

[0024] Figure 3B ELISA was used to detect the content of crotonyl-CoA in the placenta of pregnant mice after drinking Nacr and IDH1 inhibitor drinking water;

[0025] Figure 4A This is the HE staining picture of the placenta of pregnant mice after drinking α-KG and ensidipine mesylate in drinking water;

[0026] Figure 4B This is the histological HE staining of the placenta of pregnant mice after drinking drinking water containing Nacr and IDH1 inhibitor. DETAILED DESCRIPTION

[0027] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only 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 creative work are within the scope of protection of the present invention.

[0028] The examples of the present application disclose the use of an inhibitor in improving placental hypertrophy in cloned animals.

[0029] Example

[0030] The application of inhibitors in improving placental hypertrophy in cloned animals includes the following steps:

[0031] (1) Animal preparation: Female Kunming white mice aged 7-8 weeks were selected and randomly divided into 8 groups, with 5 mice in each group;

[0032] (2) Superovulation and cage combination: pregnant mare serum gonadotropin (PMSG) was injected subcutaneously into mice. Human chorionic gonadotropin (hCG) was injected intraperitoneally 23.5 hours later. The mice were then caged with male Kunming white mice. The formation of plugs was checked 12-13 hours later.

[0033] (3) When embolus was detected, the drinking water used by 3 groups of mice was replaced with drinking water supplemented with 0.75% α-KG (α-ketoglutaric acid), and the drinking water of another 3 groups of mice was replaced with drinking water supplemented with 10 mM Nacr (sodium crotonate), and it was recorded as the first day of pregnancy;

[0034] (4) On the 10th day of gestation, two groups of mice were randomly selected to drink water supplemented with 0.75% α-KG (α-ketoglutaric acid). One group of mice was fed with drinking water supplemented with 0.75% α-KG (α-ketoglutaric acid) and replaced with ordinary drinking water. The drinking water used by the other group was supplemented with 0.75% α-KG (α-ketoglutaric acid) and 10 mg / kg of ensidipine mesylate (an inhibitor of gene Idh2) was added to inhibit the expression of α-ketoglutarate synthase. In the other three groups of mice that drank 10 mM Nacr, one group was randomly selected to be replaced with ordinary drinking water, and the drinking water of the other group was supplemented with 10 mM Nacr and an IDH1 inhibitor (IDH1Inhibitor2 (compound 13)) was added to inhibit the expression of Idh1.

[0035] (5) Feeding was continued until the 16th day of pregnancy, during which time all mice were allowed to freely consume standard mouse chow. The mice were killed by cervical dislocation and the fetuses and placentas were separated by dissection. The size, weight, and related indices of the fetuses and placentas were observed and photographed.

[0036] α-ketoglutarate is an important metabolic intermediate in the tricarboxylic acid cycle of microorganisms and a key node connecting carbon-nitrogen metabolism in cells. An appropriate amount of α-ketoglutarate helps metabolism, can provide energy for cells, help stimulate collagen synthesis, and affect age-related processes including stem cell proliferation. It can also inhibit protein degradation in muscles. Excessive α-ketoglutarate will increase the body's metabolic burden, affect the body's liver, and affect the body's health. Experiments have verified that 0.75% α-ketoglutarate has no other adverse effects on the mouse body, so 0.75% α-ketoglutarate was selected for the experiment.

[0037] The present invention observes pregnant mice and finds that no matter what kind of drinking water is drunk, it will not have much impact on pregnant mice. Through dissection, it is found that the volume of the placenta of mice drinking drinking water added with α-KG and Nacr on the 16th day of pregnancy and mice drinking drinking water added with α-KG and Nacr replaced with ordinary drinking water on the 10th day of pregnancy are significantly increased, but the fetus is not greatly affected. However, when ensidipine mesylate is added to the drinking water added with α-KG on the 10th day of pregnancy, and when IDH1 inhibitor is added to the drinking water added with Nacr on the 10th day of pregnancy, the size of the placenta is reduced, and the fetus is also not greatly affected.

[0038] The present invention further conducted statistics on the weight of the fetuses and placentas of 8 groups of mice, and the length and width of the placentas. The results showed that the addition of α-KG and Nacr increased the weight of the placenta, and its length and width were also significantly increased compared to the length and width of the placenta of mice drinking ordinary water, but it did not have much effect on the weight of the fetus. The addition of ensidipine mesylate weakened the effect of α-KG, and the weight, length and width of the placenta were significantly reduced, and it also did not have much effect on the fetus. Similarly, the addition of IDH1 inhibitors also weakened the effect of Nacr, and the weight, length and width of the placenta were significantly reduced, but it did not have much effect on the fetus ( Figure 1A-Figure 1D ; Figure 2A-2D ).

[0039] ELISA test found that the content of α-KG in the placenta increased after drinking water supplemented with α-KG, while the addition of ensidipine mesylate reduced the content of α-KG in the placenta; the addition of Nacr in drinking water increased the content of crotonyl-CoA in the placenta, while the addition of IDH1 inhibitor reduced the content of crotonyl-CoA in the placenta, indicating that the enlargement of the placenta is indeed caused by excessive α-KG and crotonyl-CoA ( Figure 3A-3B ). Figure 1A , Figure 1C , Figure 1D , Figure 2A , Figure 2C , Figure 2D , Figure 3A , Figure 3B The "abbc" marked in the bar graph represents the significant difference between different groups, where the same letters represent no significant difference between the two groups (P>0.05), and different letters represent a significant difference between the two groups (P<0.05).

[0040] In order to compare with the cloned mouse placenta, the present invention performed histological HE staining on 8 groups of mouse placentas for observation, showing that the placental maze layer area of ​​mice drinking α-KG-added drinking water and Nacr-added drinking water increased and was damaged, which was consistent with the phenotype of cloned mouse placenta. When ensidipine mesylate or IDH1 inhibitor was added, the maze layer area was reduced and the damage was improved ( Figure 4A-4B ), indicating that the placenta of mice drinking drinking water supplemented with α-KG or Nacr can achieve the same effect as the placenta of cloned mice.

[0041] The present invention generates a mouse model of placental hypertrophy by drug induction, and achieves the purpose of reducing the size of the placenta by feeding the inhibitor drug. The present invention also provides a new solution to the common phenomenon of placental hypertrophy in the pregnancy process of cloned animals, that is, by adding an inhibitor, the size of the placenta during the pregnancy of cloned animals can be reduced, which can improve the pregnancy success rate of cloned animals.

[0042] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A substance for enlarging the placenta of an animal, characterized in that: The substance is one or both of α-ketoglutaric acid and sodium crotonate.

2. Use of an inhibitor in improving placental hypertrophy in cloned animals, characterized in that: The inhibitor is one or both of an inhibitor of gene Idh2 or an inhibitor of IDH1.

3. The use according to claim 2, characterized in that: The inhibitor is useful for reducing the hypertrophy of the animal placenta caused by alpha-ketoglutarate.

4. The use according to claim 2, characterized in that: The inhibitor is used to reduce the hypertrophy of the animal placenta caused by sodium crotonate.

5. The use according to claim 2, characterized in that: The inhibitor of gene Idh2 is ensidipine mesylate.

6. The use according to claim 2, characterized in that: The IDH1 inhibitor is IDH1Inhibitor 2 (compound 13).

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