Use of an inhibitor in improving placental hypertrophy in cloned animals

Abstract

The present invention belongs to the technical field of animal models, and particularly relates to the application of inhibitors in improving placental hypertrophy in cloned animals. This method involves grouping mice, superovulating, and co-housing them for pregnancy to obtain pregnant mice. By replacing the drinking water with α-ketoglutaric acid and sodium crotonate, and adding gene Idh2 inhibitors and IDH1 inhibitors, a mouse model of placental hypertrophy is constructed. On the 16th day of mouse pregnancy, the mouse fetuses and placentas are dissected and separated, and the sizes of the mouse fetuses, the sizes of the placentas, and the weights of the placentas are observed and recorded. Without using molecular biology methods, this method constructs a mouse model of placental hypertrophy through drug induction, and by adding inhibitors of key genes, the purpose of reducing the hypertrophic placenta is achieved, solving the problems of long cycle, low efficiency, and high cost in reducing the hypertrophic placenta of cloned animals by using gene editing methods at present.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal models, and particularly relates to the application of inhibitors in improving placental hypertrophy in 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 multiple fields such as animal production and regenerative medicine. However, due to epigenetic reprogramming barriers during the somatic cell reprogramming process, the efficiency of SCNT is greatly reduced, and SCNT embryos are extremely prone to various abnormal phenomena 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 a huge placenta and fetus. It has been found that regardless of the source and type of donor cells for somatic cell nuclear transfer, a huge placenta can generally be observed during the gestation period of cloned animals. As a key organ during pregnancy, 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 performing immune functions. In cloned animals, abnormal placental hypertrophy is also a major factor restricting the cloning success rate. Therefore, how to efficiently improve the placental hypertrophy phenomenon in cloned animals is one of the important methods to improve the pregnancy success rate of cloned animals and promote the in-depth research and application of cloning technology.

[0003] Previous studies mainly used molecular biology methods to perform gene editing on genes related to epigenetic modifications in donor cells. By knocking in recombinant genes or knocking out target genes, the purpose of reducing the hypertrophic placenta in cloned animals can be achieved, thereby improving the pregnancy success rate of cloned animals. However, gene editing methods have an overall long cycle, high cost, and 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 application of inhibitors in improving placental hypertrophy in cloned animals. This method constructs a mouse model of placental hypertrophy through drug induction without applying molecular biology methods, and by adding inhibitors of key genes, the purpose of reducing the hypertrophic placenta is achieved, solving the problems of long cycle, low efficiency, and high cost existing in reducing the hypertrophic placenta in cloned animals by using gene editing methods.

[0005] The technical solution of the present invention is realized as follows:

[0006] A substance for increasing the placenta of an animal, which is one or both of α-ketoglutaric acid or 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 and an IDH1 inhibitor.

[0008] Optionally, the inhibitor is used to reduce the hypertrophied animal placenta caused by α-ketoglutaric acid.

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

[0010] Optionally, the inhibitor of gene Idh2 is enasidenib 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 as follows:

[0013] Based on the non-application of molecular biology means, the present invention can construct a mouse model with placental hypertrophy only by using α-ketoglutaric acid or sodium crotonate for drug induction, and by adding an inhibitor of gene Idh2 or an IDH1 inhibitor, the purpose of reducing the hypertrophied placenta can be achieved, solving the problems of long cycle, low efficiency and high cost existing in reducing the hypertrophied placenta of cloned animals by using gene editing means at present. Description of the Drawings

[0014] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention, and those of ordinary skill in the art can also obtain other drawings without creative efforts based on these drawings.

[0015] Figure 1A Statistical chart of the placental weight of pregnant mice after drinking α-KG and enasidenib mesylate drinking water;

[0016] Figure 1B Statistical chart of the fetal weight of pregnant mice after drinking α-KG and enasidenib mesylate drinking water;

[0017] Figure 1C Statistical chart of the placental length of pregnant mice after drinking α-KG and enasidenib mesylate drinking water;

[0018] Figure 1D Statistical chart of the placental width of pregnant mice after drinking α-KG and enasidenib mesylate drinking water;

[0019] Figure 2AStatistical analysis of placental weight of pregnant mice after drinking water containing Nacr and IDH1 inhibitor;

[0020] Figure 2B Statistical analysis of fetal weight of pregnant mice after drinking water containing Nacr and IDH1 inhibitor;

[0021] Figure 2C Statistical analysis of placental length of mice after drinking water containing Nacr and IDH1 inhibitor;

[0022] Figure 2D Statistical analysis of placental width of pregnant mice after drinking water containing Nacr and IDH1 inhibitor;

[0023] Figure 3A ELISA detection of the content of α-KG in the placenta of pregnant mice after drinking water containing α-KG and enasidenib mesylate;

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

[0025] Figure 4A HE staining map of placental tissue of pregnant mice after drinking water containing α-KG and enasidenib mesylate;

[0026] Figure 4B HE staining map of placental tissue of pregnant mice after drinking water containing Nacr and IDH1 inhibitor. Detailed implementation mode

[0027] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0028] The embodiments of the present application disclose the application of inhibitors in improving placental hypertrophy in cloned animals.

[0029] Embodiment

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

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

[0032] (2) Superovulation and co-housing: Inject pregnant mare serum gonadotropin (PMSG) into mice subcutaneously. 23.5 h later, inject human chorionic gonadotropin (hCG) into the abdominal cavity of the mice. Then co-house them with male Kunming mice. Check for the formation of vaginal plugs 12 - 13 h later;

[0033] (3) When vaginal plugs are detected, change the ordinary drinking water of 3 groups of mice to drinking water supplemented with 0.75% α-KG (α-ketoglutaric acid), and change the drinking water of another 3 groups of mice to drinking water supplemented with 10 mM Nacr (sodium crotonate), and mark it as the first day of pregnancy;

[0034] (4) When feeding until the 10th day of pregnancy, randomly select 2 groups of mice drinking water supplemented with 0.75% α-KG (α-ketoglutaric acid). Change the drinking water of one group supplemented with 0.75% α-KG (α-ketoglutaric acid) to ordinary drinking water, and add 10 mg / kg of enasidenib mesylate (inhibitor of gene Idh2) to the drinking water of the other group on the basis of adding 0.75% α-KG (α-ketoglutaric acid) to inhibit the expression of α-ketoglutaric acid synthase. For another 3 groups of mice drinking 10 mM Nacr, randomly select one group and change it to ordinary drinking water, and add IDH1 inhibitor (IDH1 Inhibitor2 (compound 13)) to the drinking water of the other group on the basis of adding 10 mM Nacr to inhibit the expression of Idh1.

[0035] (5) Continue to feed until the 16th day of pregnancy. During this period, all mice freely ingest standard mouse food. Sacrifice the mice by cervical dislocation, dissect to separate the fetal mice and placentas, and observe and photograph to record the size, weight and related indicators of the fetal mice and placentas.

[0036] α-Ketoglutaric acid is an important metabolic intermediate in the tricarboxylic acid cycle of microorganisms and a key node connecting carbon-nitrogen metabolism in cells. Appropriate α-ketoglutaric acid helps metabolism, can provide energy for cells, helps stimulate the synthesis of collagen, and affects age-related processes including stem cell proliferation. At the same time, it can also inhibit protein degradation in muscles. Excessive α-ketoglutaric acid will increase the metabolic burden of the body, affect the liver of the body, and affect the health of the body. After experimental verification, 0.75% α-ketoglutaric acid has no other adverse effects on the mouse body. Therefore, 0.75% α-ketoglutaric acid is selected for the experiment.

[0037] Observation of pregnant mice in the present invention found that no matter what kind of drinking water was consumed, it had little impact on pregnant female mice. Through dissection, it was found that on the 16th day of pregnancy, the volume of the placenta in mice drinking water supplemented with α-KG and Nacr, and on the 10th day of pregnancy, when the drinking water supplemented with α-KG and Nacr was changed to ordinary drinking water, all showed a significant increase. However, it had little impact on the fetus. But when enasidenib mesylate was added to the drinking water supplemented with α-KG on the 10th day of pregnancy, and when an IDH1 inhibitor was added to the drinking water supplemented with Nacr on the 10th day of pregnancy, the size of the placenta was reduced, and again, it had little impact on the fetus.

[0038] The present invention further counted the weights of the fetuses and placentas, and the lengths and widths of the placentas of 8 groups of mice. The results showed that the addition of both α-KG and Nacr increased the weight of the placenta, and its length and width were also significantly increased compared with those of the placentas of mice drinking ordinary water, but it had little impact on the fetal weight. The addition of enasidenib mesylate weakened the effect of α-KG, and the weight, length, and width of the placenta were all significantly reduced, and again, it had little impact on the fetus. Similarly, the addition of the IDH1 inhibitor also weakened the effect of Nacr, and the weight, length, and width of the placenta were all significantly reduced, but it had little impact on the fetus ( Figure 1A - Figure 1D ; Figure 2A - Figure 2D ).

[0039] ELISA detection found that the content of α-KG in the placenta increased in mice drinking water supplemented with α-KG, and the addition of enasidenib mesylate reduced the content of α-KG in the placenta; the addition of Nacr in the drinking water increased the content of crotonyl-CoA in the placenta, and the addition of the IDH1 inhibitor reduced the content of crotonyl-CoA in the placenta, indicating that the increase in the size of the placenta was indeed caused by excessive α-KG and crotonyl-CoA ( Figure 3A - Figure 3B ). Figure 1A 、 Figure 1C 、 Figure 1D 、 Figure 2A 、 Figure 2C 、 Figure 2D 、 Figure 3A 、 Figure 3B In the bar charts in , the "abbc" marked represents the significant differences between different groups. Among them, the same letter represents no significant difference between two groups (P>0.05), and different letters represent significant differences between two groups (P<0.05).

[0040] For comparison with cloned mouse placentas, the present invention observed histological HE-stained sections of 8 groups of mouse placentas, showing that in mice drinking water supplemented with α-KG or Nacr, the area of the placental labyrinth layer increased and was damaged, which was consistent with the phenotype of cloned mouse placentas. When enasidenib mesylate or an IDH1 inhibitor was added, the area of the labyrinth layer could be reduced and the damage improved( Figure 4A - Figure 4B ), indicating that the placentas of mice drinking water supplemented with α-KG or Nacr could achieve the same effects as those of cloned mouse placentas.

[0041] The present invention generates a mouse model with placental hypertrophy by means of drug induction and achieves the purpose of reducing placental size by feeding inhibitor drugs. The present invention also provides a new solution to the common phenomenon of placental hypertrophy during the pregnancy of cloned animals, that is, by adding inhibitors to reduce the size of the placenta during the pregnancy of cloned animals, the pregnancy success rate of cloned animals can be improved.

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

Claims

1. Use of an inhibitor in improving placental hypertrophy in cloned animals, characterized in that, The inhibitor is an IDH1 inhibitor, and the IDH1 inhibitor is used to reduce the enlarged animal placenta caused by sodium crotonate; the steps for constructing the enlarged animal placenta model are as follows: (1) Animal preparation: Female Kunming white mice at 7-8 weeks of age were selected; (2) Superovulation and co-housing: Pregnant mare serum gonadotropin was injected into the mice by subcutaneous injection. 23.5 h later, human chorionic gonadotropin was injected into the abdominal cavity of the mice, and then they were co-housed with male Kunming white mice. The formation of vaginal plugs was checked 12-13 h later; (3) When vaginal plugs were detected, the drinking water of the mice was changed to water supplemented with 10 mM sodium crotonate, and this was recorded as day 1 of pregnancy; (4) On the 10th day of pregnancy, for the mice drinking 10 mM sodium crotonate, one group was randomly selected and changed to normal drinking water, and the drinking water of the other group was supplemented with an IDH1 inhibitor on the basis of adding 10 mM sodium crotonate to inhibit the expression of Idh1; (5) Feeding was continued until the 16th day of pregnancy. During this period, all mice freely consumed standard rodent food. The mice were sacrificed by cervical dislocation, and the fetal and placental tissues of the mice were dissected. The sizes, weights and related indicators of the fetal and placental tissues of the mice were observed and photographed.

2. The application according to claim 1, wherein The IDH1 inhibitor is IDH1 Inhibitor 2 (compound 13).

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