Use of peginterferon alfa-2b in preparing a model of embryonic cleft palate related products

Abstract

The application of Plerixafor in preparing embryo cleft palate model related products relates to the technical field of molecular biology, and specifically discloses the application of Plerixafor in constructing an embryo cleft palate model and / or jointly using retinoic acid to simulate clinical combined medication to increase the incidence of embryo cleft palate. The present application finds that, during embryonic development, the application of Plerixafor to a mother can cause a certain probability of cleft palate; the combined use of Plerixafor and retinoic acid can significantly increase the incidence of embryo cleft palate caused by the separate use of Plerixafor and retinoic acid to the mother, and thus the application of Plerixafor in constructing embryo cleft palate model related products is provided. The present application provides two new methods for constructing cleft palate embryos, expands the application field of Plerixafor, and further studies the side effects of Plerixafor and the toxic side effects caused by the combination of Plerixafor and retinoic acid, thereby providing a theoretical basis for the occurrence and prevention of cleft palate.

Description

Technical Field

[0001] This invention relates to the field of molecular biology technology, and more specifically, to the application of plexafor in the preparation of embryonic cleft palate models and related products. Background Technology

[0002] Cleft palate is one of the most common birth defects in humans, often caused by abnormalities in palate development. The development of the palate is a complex process involving cell proliferation, migration, and differentiation. Their synergistic effects drive a series of complex biological processes, including the descent, elevation, horizontal growth, and fusion of the palatine processes. This process is influenced by the interaction of environmental and genetic factors, and any failure in this process can lead to cleft palate. Current research on the pathogenesis of cleft palate focuses primarily on the impact of gene alterations on processes such as proliferation. However, recent studies have revealed that cell migration is also a crucial aspect of palate development. Therefore, investigating cleft palate caused by migration abnormalities and their pathogenesis is equally important. In particular, researching how gene alterations trigger migration abnormalities during palate development can provide a basis for clinical genetic counseling, disease prevention, understanding drug side effects and combined drug therapy, and is of great significance for eugenics and improving the quality of my country's newborn population. Chemokine receptor 4 (CXC motif chemokine receptor 4, CXCR4) is a G protein-coupled receptor that participates not only in cell homing and chemotaxis in the hematopoietic and immune systems, but also regulates heart and brain development. Homozygous knockout mice die before birth; therefore, CXCR4 is a key regulatory gene for normal development. The CXCR4 inhibitor plerixafor has seen significant progress in the application of CXCR4 in HIV-1 infection, WHIM syndrome, autoimmune diseases, and stem cell mobilization. In 2008, it was approved by the U.S. Food and Drug Administration (FDA) for use in combination with granulocyte colony-stimulating factor (G-CSF) as a hematopoietic stem cell mobilizing agent for autologous transplantation in patients with non-Hodgkin's lymphoma and multiple myeloma. However, plexafor, a potent CXCR4 inhibitor with an IC50 of 44 nM, has also been found to have some side effects, such as reducing the generation rate of retinal organoids, impairing ganglion cell differentiation, and inducing morphological changes. Due to the difficulty in obtaining maternal or embryonic samples from women with cleft palate, and based on embryological and etiological studies, mice are an readily available experimental animal with high litter size and high gene sequence homology with humans (99%), and are generally considered a suitable animal model for most studies. Therefore, mice are often chosen as an important model for in vivo experimental studies of cleft palate. Currently, there are no reports on the effects of plexafor on palatal development and cleft palate occurrence, including in mice. Summary of the Invention

[0003] The purpose of this invention is to provide the use of praxavir and a method for constructing an animal model of cleft palate; and further, a method for constructing an animal model of cleft palate by using praxavir in combination with retinoic acid, thereby proposing new applications for praxavir.

[0004] The technical solution of the present invention is as follows:

[0005] In a first aspect, the present invention provides a method for inducing embryonic developmental abnormalities resulting in cleft palate using plexafor, and for constructing an embryonic cleft palate model. This model is constructed by administering plexafor to pregnant mice on days 10.5 to 13.5 of gestation, wherein the pregnant mice are those carrying normal fetuses prior to administration.

[0006] The cleft palate in the fetal mice was caused by abnormal migration of the palatine process due to the use of prasafone.

[0007] This invention involves administering pleroxafer to pregnant mice during a specific period of pregnancy, which can effectively induce cleft palate in the embryos, thereby successfully constructing an animal model of cleft palate. The method is simple, has a high success rate, and avoids the wide range of effects and high lethality of gene knockout model construction methods on fetal mice.

[0008] In the construction method of the present invention, the administration method is intraperitoneal injection, and the daily injection dose is 5 mg / kg.

[0009] The intraperitoneal injection method is convenient to operate, the experimental results are highly informative, and the injection dosage of this invention reduces the mortality rate of embryos and greatly improves the survival rate of pregnant mice and fetuses.

[0010] The model constructed in this invention can observe phenotypic changes in the palate of fetal mice after birth and detect the expression of migration-related genes, thereby better explaining the effect of plesafool ​​on the occurrence of cleft palate.

[0011] Secondly, the present invention provides the application of combining plexafor and retinoic acid to exacerbate cleft palate and in the construction of embryonic cleft palate models.

[0012] This invention has found that administering praxavir to the mother in combination with 50 mg / kg of RA during embryonic development can increase the incidence of cleft palate in embryos, leading to this invention.

[0013] Most models of cleft palate in mice induced by chemical drugs focus on cleft palate induced by gavage with retinoic acid (RA). Specifically, on day 10.5 of gestation (E10.5), pregnant mice are gavaged with different concentrations of RA to establish a fetal cleft palate model. RA-induced cleft palate in mice is concentration-dependent; 50 mg / kg RA induces cleft palate in 40% of fetal mice. Since combination therapy is often required in clinical practice, we observed changes in cleft palate in mice by combining plexafor with retinoic acid, providing a new application for studying the effects of plexafor combined with other drugs on cleft palate development. This invention establishes a fetal cleft palate model by using a convenient method of intraperitoneal injection of plexafor combined with RA at a reduced concentration of 50 mg / kg. Intraperitoneal injection is convenient, has a low embryonic mortality rate, allows for in-depth study of the role of migration abnormalities in palatal development, and provides a new application method for the combined use of plexafor with other drugs to induce cleft palate models.

[0014] Thirdly, this invention provides the pharmaceutical application of plexafor in the construction of embryonic cleft palate.

[0015] In the application of this invention, the active ingredient of the drug exists in a free form or in the form of a pharmaceutically acceptable salt.

[0016] The active ingredient or its pharmaceutically usable salt may also be used in the form of a hydrate or other solvent, depending on the application requirements.

[0017] In the application of this invention, the drug contains at least one pharmaceutically acceptable carrier.

[0018] In the application of this invention, the drug is administered orally, intravenously, or via intraperitoneal injection.

[0019] The medicament of the present invention can be prepared using conventional methods, such as conventional mixing, granulation, sugar coating, dissolution, or freeze-drying. It may contain a therapeutically effective amount of a pharmacologically active ingredient or may simultaneously contain one or more pharmaceutically acceptable carriers. The preferred route of administration for the medicament of the present invention is intraperitoneal injection.

[0020] The dosage form of the drug of the present invention can be sugar-coated tablets, tablets, or capsules. The unit content of the active ingredient contained in a single dose of each dosage form needs to constitute an effective amount, i.e., 5 mg / kg.

[0021] When preparing drugs for oral dosage forms, any conventional pharmaceutical medium, such as water, glycols, oils, or alcohols, can be used; or in the case of oral solid dosage forms such as powders, capsules, and tablets, carriers such as starch, sugar, microcrystalline cellulose, diluents, granulators, lubricants, binders, disintegrants, etc., can be used.

[0022] In the application of this invention, the drug is administered during the early stages of palatal development in embryonic formation (when palatal process cells migrate).

[0023] The beneficial effects of this invention are at least as follows:

[0024] This invention provides two novel methods for constructing embryonic cleft palate models using plexafor, expanding the application field of plexafor, deepening the study of the mechanisms of its side effects and the mechanisms of side effects caused by its combination with retinoic acid treatment, and providing a theoretical basis for the study of cleft palate development and treatment mechanisms. The embryonic cleft palate animal models of this invention have a short modeling cycle, are simple to operate, and are inexpensive, with high survival rates in pregnant and fetal mice, improving the success rate of model construction to 75.6%. Attached Figure Description

[0025] Figure 1 These are partial stereomicroscopic observation results for the blank control group (control), experimental group 1 (plerixafor), and experimental group 2 (RA+plerixafor).

[0026] Figure 2 The results of partial H&E staining observations in the blank control group (control), experimental group 1 (plerixafor), and experimental group 2 (RA+plerixafor) are shown.

[0027] Figure 3 The partial immunofluorescence staining results of the blank control group (control), experimental group 1 (plerixafor), and experimental group 2 (RA+plerixafor) show the expression of Cxcr4, Rac1, and Rhoa on E16.5. (Cxcr4 is a target gene of plerixafor, and Rac1 and Rhoa are downstream target genes of Cxcr4 that affect the migration of palatine process cells. Their abnormalities are the main cause of cleft palate.)

[0028] Figure 4 Quantitative results and statistical analysis of the relative expression of Cxcr4, Rac1 and Rhoa on E16.5 in the blank control group (control), experimental group 1 (plerixafor) and experimental group 2 (RA+plerixafor) were obtained by partial immunofluorescence staining. Detailed Implementation

[0029] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0031] The experimental materials and reagents used in the specific embodiments of this invention are as follows:

[0032] C57BL / 6 mice: females: healthy, 6-8 weeks old, 22-25g; males: healthy, 8-10 weeks old, 25-28g.

[0033] Reagents used: retinoic acid (RA, R2625, Sigma-Aldrich), corn oil (C8267, Sigma-Aldrich), pleroxafer (MCE, HY-10046), polyclonal anti-CXCR4 (Cat. No. 11073-2-AP, Proteintech), polyclonal anti-RAC1 (Cat. No. 24072-1-AP, Proteintech), polyclonal anti-RHOA (Cat. No. 10749-1-AP, Proteintech).

[0034] Solution preparation method:

[0035] RA: Dissolve RA powder in corn oil.

[0036] Presalifos: Dissolve according to the instructions in the product manual.

[0037] Example 1

[0038] This embodiment verifies the effectiveness of plexafor in constructing an embryonic cleft palate model.

[0039] The experimental procedure is as follows:

[0040] 1. Select male and female C57BL / 6 mice aged 8-10 weeks and put them together in a 1:2 ratio at 9 pm on the same day. After about 10-12 hours, separate the female and male mice into different cages and observe the formation of vaginal plugs in the female mice. Record the female mice that have observed vaginal plugs as 0.5 days of pregnancy (i.e. E0.5) and record their weight.

[0041] 2. On the morning of day E10.5, the mice were weighed, and mice that gained more than 10% in weight were identified as pregnant mice (18 mice in total). Experimental group 2 (6 mice) was administered RA corn oil solution by gavage. The amount administered was calculated based on the weight of the mice, with 50 mg of RA per kg of body weight (i.e., the RA dosage was 50 mg / kg). The control group (6 mice) was administered corn oil by gavage and intraperitoneal injection of physiological saline. Experimental group 1 (6 mice) was administered corn oil by gavage.

[0042] 3. During E10.5-13.5 days, pregnant mice in experimental group 1 (6 mice) and experimental group 2 (6 mice) were injected intraperitoneally daily. The dosage was calculated based on the weight of the mice, with 5 mg of plexafol per kg of body weight (i.e., 5 mg / kg of plexafol).

[0043] 4. After euthanizing pregnant mice by cervical dislocation in E16.5, palatal tissue samples were collected from the fetuses and palatal development was observed using a stereomicroscope.

[0044] Specifically, stereomicroscopy observation involves directly observing the development of the palate at E16.5 under a stereomicroscope. The observation steps are as follows:

[0045] Pregnant mice were euthanized at E16.5 by cervical dislocation, and the embryos were collected and placed in pre-cooled PBS. The heads of all fetal mice were removed using microsurgical scissors, and the lower jaw and tongue were removed from the corner of the mouth to expose the palate for observation under a stereomicroscope. Some observation photographs are shown below. Figure 1 .

[0046] Figure 1 This image shows partial stereomicroscopic observations of the control group, experimental group 1 (plerixafor), and experimental group 2 (RA+plerixafor). In the image, "12.5×" represents a magnification of 12.5x, and the scale bar represents 1 mm; the arrows and dashed lines indicate the palatal tissue.

[0047] As shown in the figure, at day E16.5, the palate of the control group mice had completely fused with that of the experimental group 1 (plerixafor) and the experimental group 2 (RA+plerixafor) mice, which had obvious cleft palates.

[0048] Based on the above experiments, the statistical results are shown in Table 1.

[0049] Table 1

[0050]

[0051]

[0052] Specifically, the results of this embodiment are as follows: the survival rate of pregnant mice and fetuses is 100%, the cleft palate rate of embryos in experimental group 1 (plerixafor) is 6.7%, and the cleft palate rate of embryos in experimental group 2 (RA+plerixafor) is 75.6%.

[0053] Example 2

[0054] Based on the experimental results of Example 1, this example constructs an experimental group again to further verify the effectiveness of plesavor in constructing an embryonic cleft palate model.

[0055] The specific experimental procedure is the same as in Example 1, with the only difference being:

[0056] In step 4, after euthanizing pregnant mice by cervical dislocation in E16.5, palatal tissue samples from fetal mice were collected and subjected to stereomicroscopic observation, HE staining, and immunofluorescence detection.

[0057] Figure 2 The images show partial optical microscopic observations of the control group, experimental group 1 (plerixafor), and experimental group 2 (RA+plerixafor). They reveal that at day 16.5, the palates of the control mice were completely fused, while the mice in experimental groups 1 (plerixafor) and 2 (RA+plerixafor) developed significant cleft palates. Furthermore, the cleft palate of the mice in experimental group 2 (RA+plerixafor) was more severe, indicating that plerixafor can induce cleft palates in fetal mice, and further demonstrating that combined administration of retinoic acid via gavage exacerbates the cleft palate condition in fetal mice.

[0058] H&E staining involves observing the development of the palate at E16.5 under a microscope after paraffin-embedded sections are stained with H&E. The specific steps are as follows:

[0059] (1) The tissue sections were baked in an oven at 65°C for 3 hours;

[0060] (2) Dewaxing with xylene twice, 10 minutes each time;

[0061] (3) Gradient alcohol hydration: 100% alcohol twice, 2 min each time; 95% alcohol twice, 2 min each time; 80% alcohol - 2 min, 70% alcohol - 2 min, 50% alcohol - 2 min;

[0062] (4) Rinse three times with PBS, 5 min each time. Gently wipe the sections clean and place them in a hematoxylin staining jar for 2-3 min;

[0063] (5) Rinse 3 times with PBS, 5 min each time. Gently clean the sections and place them in an eosin staining jar for 30 s - 1 min;

[0064] (6) Rinse 3 times with PBS, 5 min each time. Dehydrate with a gradient of alcohol: 70% alcohol for 2 min, 80% alcohol for 2 min, 95% alcohol for 2 min, and 100% alcohol twice, 2 min each time;

[0065] (7) Xylene was applied twice, each time for 5 minutes to allow the xylene to become transparent;

[0066] (8) After clearing, mount the slide. Use a pipette to drop neutral resin onto the tissue specimen, remove excess resin, and then cover with a coverslip, being careful not to create air bubbles.

[0067] After H&E staining, the tissue sections were observed under an optical microscope. Some observation results are shown below. Figure 2 .

[0068] Figure 2 The images show partial H&E staining results for the blank control group (control), experimental control group (RA), and experimental group (RA + 100 μg / kg vo-ohpic). In the figures, "10×" represents a magnification of 100x, the scale bar represents 200 μm, "PS" represents the palatine process of the fetal mouse, and "T" represents the tongue of the fetal mouse.

[0069] This embodiment also uses immunofluorescence staining to observe the effect of plerixafor on the palatal migration ability of fetal rats. Details are as follows:

[0070] The specific staining steps are as follows:

[0071] (a) Dewaxing: Dewax in xylene for 10 minutes. Replace with fresh xylene and dewax for another 10 minutes. Dewax in anhydrous ethanol for 5 minutes, then replace with fresh anhydrous ethanol for 3 minutes. Dewax in 95% ethanol for 3 minutes. Dewax in 85% ethanol for 3 minutes. Dewax in 75% ethanol for 3 minutes. Dewax in 50% ethanol for 3 minutes. Rinse three times with PBS, 5 minutes each time.

[0072] (b) Antigen retrieval: Add distilled water to the bath and preheat it to 99°C along with the retrieval solution. Add the slices and boil for 30 minutes. Let it stand to room temperature and rinse with PBS 3 times for 5 minutes each time.

[0073] (c) Blocking: Block with goat serum working solution, 37° or room temperature for 30-60 min; incubate at room temperature in the dark for 30 min.

[0074] (d) Add primary antibody and incubate overnight at 4°C in a humidified chamber.

[0075] (e) Remove at 4°C and allow to return to room temperature (30 minutes). Rinse with PBS 3 times, 5 minutes each time.

[0076] (f) Add secondary antibodies, goat anti-mouse ALexa Fluor 647 (1:300) and goat anti-rabbit ALexa Fluor 488 (1:300) fluorescent secondary antibodies, and incubate at room temperature in the dark.

[0077] (g) The cell nuclei were stained with mounting medium containing DAPI and then mounted.

[0078] Make sure no small air bubbles remain on the tissue area, allow it to air dry naturally, and then take photos.

[0079] Observed under a fluorescence microscope. Some observation results are shown below. Figure 3 .

[0080] from Figure 3 It can be seen that the fluorescence intensity of different migration genes in the palate of mice in experimental group 1 (plerixafor) and experimental group 2 (RA+plerixafor) is lower than that in the control group, indicating that the palate migration ability of mice in experimental group 1 (plerixafor) and experimental group 2 (RA+plerixafor) is significantly weakened.

[0081] Figure 4 To use ImageJ software to Figure 3 The expression levels of Cxcr4, Rac1, and Rhoa were quantified, and statistical analysis was performed using GraphPad Prism 8 software. ** in the figure represent P < 0.01, indicating a statistically significant difference.

[0082] from Figure 4 It was found that the fluorescence intensity of the palatal migration gene in experimental group 1 (plerixafor) and experimental group 2 (RA+plerixafor) was significantly lower than that in the control group, showing a statistically significant difference. This indicates that the palatal migration ability of mice in experimental group 1 (plerixafor) and experimental group 2 (RA+plerixafor) was significantly weakened.

[0083] Furthermore, this invention has also found that when using praxavir to construct a fetal rat cleft palate model, no deaths of pregnant rats or fetuses occurred; when retinoic acid (50 mg / kg) was combined with praxavir to construct a high-success-rate model, no deaths of pregnant rats or fetuses also occurred. This indicates that our invention can significantly reduce the mortality rate of experimental animals caused by drug toxicity and achieve a high success rate.

[0084] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for constructing a cleft palate embryo model, characterized in that, The method was constructed by intraperitoneal injection of plexafor in pregnant mice from day 10.5 to day 13.5 of gestation, and simultaneous administration of retinoic acid on day 10.5 of gestation. The pregnant mice were those carrying normal fetuses before drug administration. The retinoic acid was administered by gavage at a dose of 50 mg / kg, and the plexafor was administered at a dose of 5 mg / kg. The mice were C57BL / 6 mice.

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