Construction method of zebrafish osteoporosis model and effectiveness evaluation method of zebrafish osteoporosis model

Abstract

The invention relates to the technical field of biomedicine, in particular to a zebra fish osteoporosis model construction method and a zebra fish osteoporosis model effectiveness evaluation method. The optimal medicine ammonium tetrathiomolybdate for constructing the zebrafish osteoporosis model is obtained through screening and optimization, the medicine is used for constructing the zebrafish osteoporosis model, and deformity and death are not prone to occurring on the basis that zebrafish has the osteoporosis symptom; meanwhile, the head width, the body length, the movement distance, the maximum movement speed, the ingestion fluorescence intensity, the ch-mk interval and the number of spinal mineralized bones of the zebra fish are taken into the effectiveness evaluation parameters of the model, and the model is combined with the evaluation parameters, so that drugs directly influencing skeletal development can be screened more accurately; meanwhile, the research of the osteoporosis model in the aspects of behaviors and sustainable development is facilitated.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a method for constructing a zebrafish osteoporosis model and a method for evaluating its effectiveness. Background Art

[0002] Osteoporosis currently has a high incidence rate among the elderly, which can cause bone pain and fatigue, spinal deformity, accelerated bone and joint degeneration, increased fracture risk, and affect respiratory function and quality of life. The development of drugs to treat osteoporosis has received increasing attention. Zebrafish has a high degree of genetic homology with humans, up to 85%, and has a high degree of similarity with human bone development. Therefore, there are many studies in the fields of bone development genetic diseases, bone growth signaling pathways, osteogenesis imperfecta, scoliosis, etc.

[0003] Currently, prednisone and dexamethasone are mainly used to induce zebrafish osteoporosis models, but they can easily lead to deformities and death in zebrafish, which is not conducive to the study of the behavior and sustained development of osteoporosis models. The existing methods mainly use Alizarin red staining to evaluate whether the zebrafish osteoporosis model is successful and the effect of drugs on osteogenesis, which is a relatively single parameter.

[0004] Therefore, there is a need in the art to propose a method for constructing a zebrafish osteoporosis model and a method for evaluating the effectiveness of the model, which can quickly and with low side effects induce a model for evaluating and screening drugs for treating osteoporosis. Summary of the invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a method for constructing a zebrafish osteoporosis model and a method for evaluating its effectiveness.

[0006] The invention provides application of ammonium tetrathiomolybdate or a preparation thereof in preparing a drug combination for a zebrafish osteoporosis model.

[0007] In the present invention, ammonium tetrathiomolybdate or its preparation can be used to prepare a zebrafish osteoporosis model; at the same time, the present invention does not exclude the use of a drug combination containing ammonium tetrathiomolybdate or its preparation in the construction of a more sophisticated zebrafish osteoporosis model.

[0008] The invention provides a method for preparing a zebrafish osteoporosis model, which comprises treating the zebrafish with ammonium tetrathiomolybdate.

[0009] Further, the concentration of the ammonium tetrathiomolybdate is 20 μmol / L to 50 μmol / L, preferably 50 μmol / L;

[0010] The present invention screens drugs for constructing a zebrafish osteoporosis model, and the test results show that among penicillamine (D-Penicillamine), sodium dimercaptopropane sulfonate (DMPS) and ammonium tetrathiomolybdate (ATTM), penicillamine (D-Penicillamine) can cause a decrease in the feeding desire of zebrafish, and the decrease in the number of mineralized bones of sodium dimercaptopropane sulfonate (DMPS) is smaller than that of ammonium tetrathiomolybdate (ATTM), and ammonium tetrathiomolybdate has the best effect.

[0011] The present invention optimizes the concentration of ammonium tetrathiomolybdate (ATTM). Test results show that a concentration of 100 μmol / L will cause 30% of young fish to die, so this concentration is not selected. Concentrations of 20 and 50 μmol / L can significantly reduce the amount of spinal mineralized bones, and 50 μmol / L has the best effect.

[0012] In the preparation method described in the present invention, the zebrafish includes fry; further, the fry includes fry 24 to 72 hours after fertilization, and further includes fry 48 hours after fertilization.

[0013] In the preparation method of the present invention, the use of ammonium tetrathiomolybdate to treat zebrafish comprises: exposing young fish 24 to 72 hours after fertilization to a culture solution containing ammonium tetrathiomolybdate for culture.

[0014] Furthermore, the culture time is 5 to 7 days, specifically 6 days.

[0015] During the culture period, the culture medium containing ammonium tetrathiomolybdate is replaced every 24 to 72 hours, and in a specific embodiment of the present invention, every 24 hours.

[0016] The culture solution includes E3 culture medium.

[0017] The present invention provides a method for evaluating the effectiveness of a zebrafish osteoporosis model prepared by the preparation method, comprising judging the effectiveness of the zebrafish osteoporosis model according to the head width, body length, movement distance, maximum movement speed, feeding fluorescence intensity, ch-mk interval and / or spinal mineralized bone quantity of zebrafish treated with ammonium tetrathiomolybdate and a control group of zebrafish.

[0018] Furthermore, the criteria for judging the effectiveness are:

[0019] The head width changes in the range of -5% to 5%, the body length changes in the range of -5% to 5%, the movement distance changes in the range of -30% to 30%, the maximum movement speed changes in the range of -50% to 50%, the feeding fluorescence intensity changes in the range of -10% to 10%, the ch-mk interval changes in the range of -20% to 20%, and the amount of mineralized bone decreases in the range of 30% to 100%. It is determined that the zebrafish osteoporosis model prepared by the preparation method described in any one of claims 1 to 8 is effective.

[0020] Among the criteria for judging the effectiveness, the number of mineralized bones is the first criterion, and other criteria are used to assist in judging whether the drug has a negative effect, and to exclude the delayed bone mineralization time caused by the negative effect leading to the overall delayed development of the fish. Therefore, the combination of various parameters in the criteria for judging the effectiveness of the present invention is conducive to the accurate judgment of the effectiveness of the constructed zebrafish osteoporosis model.

[0021] In the present invention, the control is a control group, which is crucial to ensure the scientificity, reliability and effectiveness of the experimental results. In the present invention, those skilled in the art can know that the control is a blank control group without drug treatment according to the experimental design, and the present invention will not elaborate on this.

[0022] Existing drugs and models used to construct osteoporosis models ignore the effects of drugs on the overall growth, morphology, development, and behavior of zebrafish. In this evaluation method, the zebrafish head width, body length, movement distance, maximum movement speed, feeding fluorescence intensity, ch-mk spacing, and the number of mineralized bones in the spine are included in the evaluation parameters, which can exclude false positive results caused by abnormal mineralized bone quantity due to changes in zebrafish feeding desire and growth and development speed caused by drugs. More accurately screen for drugs that directly affect bone development.

[0023] The present invention obtains the most suitable ammonium tetrathiomolybdate, a drug for constructing a zebrafish osteoporosis model, through screening and optimization. The drug is used for constructing a zebrafish osteoporosis model, and is not prone to deformity and death on the basis of the zebrafish having osteoporosis symptoms. At the same time, the present invention incorporates the zebrafish head width, body length, movement distance, maximum movement speed, feeding fluorescence intensity, ch-mk interval, and the number of mineralized bones in the spine into the model effectiveness evaluation parameters. The model combined with the evaluation parameters can more accurately screen drugs that directly affect bone development, and is conducive to the research of the osteoporosis model in terms of behavior and sustained development. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The figure shows the analysis of mineralized bone quantity in zebrafish spine after induction with penicillamine, sodium dimercaptopropanesulfonate and ammonium tetrathiomolybdate;

[0025] Figure 2The figure shows the analysis of the mineralized bone quantity in the zebrafish spine after induction with different concentrations of ammonium tetrathiomolybdate;

[0026] Figure 3 Shows the changes in the amount of mineralized bone in the zebrafish spine after treatment with different drugs;

[0027] Figure 4 Shows the changes in the distance between mk and ch in zebrafish after treatment with different drugs;

[0028] Figure 5 Shows the changes in zebrafish head width after treatment with different drugs;

[0029] Figure 6 Shows the changes in zebrafish body length after treatment with different drugs;

[0030] Figure 7 Shows the changes in the moving distance and maximum speed of zebrafish after treatment with different drugs;

[0031] Figure 8 Shows the changes in fluorescence intensity of zebrafish feeding after treatment with different drugs;

[0032] Fig. 9 Shown is the heat map of skeletal development drug screening evaluation. DETAILED DESCRIPTION

[0033] The present invention provides a method for constructing a zebrafish osteoporosis model and a method for evaluating its effectiveness. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0034] The developmental stages of zebrafish (Danio rerio) can be divided into embryonic stage, larval stage, juvenile stage and adult stage; the embryonic stage of zebrafish is 0~72h, including: fertilized egg stage (0 hours), cleavage stage (0~3 hours), blastocyst stage (3~6 hours), gastrula stage (6~10 hours), segmentation stage (10~24 hours) and hatching stage (48~72 hours); the larval stage of zebrafish (3 days~4 weeks) includes: free-swimming larvae (3 days), organ development (3 days~7 days) and pigmentation (5 days~7 days); the juvenile stage of zebrafish (4 weeks~10 weeks) includes: juvenile stage (4 weeks) and sexual maturity (10 weeks); after zebrafish reaches adult stage (more than 10 weeks), the adult fish continues to grow, but the growth rate gradually slows down. The life span of adult fish is usually 2~3 years.

[0035] In the present invention, dpf in 2dpf and 8dpf is the abbreviation of days post fertilization, which means the number of days after fertilization; in zebrafish research, dpf is often used to indicate the developmental stage of embryos or larvae.

[0036] The standard for determining zebrafish head width is: under an optical microscope, measure the horizontal distance at the widest part of the zebrafish head;

[0037] Zebrafish body length was measured as: the straight line length from the tip of the snout to the base of the caudal fin;

[0038] The zebrafish movement distance is determined by recording the movement trajectory of the zebrafish through a video tracking system within a specific time and calculating its total travel distance; the specific time is the time period from the exposure of the zebrafish to the test drug to the development to 8 dpf;

[0039] The maximum movement speed of zebrafish is determined as follows: after the zebrafish are exposed to the test drug and cultured to 8dpf, the movement trajectory of the zebrafish is recorded by a video tracking system, and the highest instantaneous speed reached during the test is calculated; the specific measurement steps are: zebrafish that have developed to 8dpf are placed in a 96-well plate, with one fry in each well, and the 96-well plate is placed in a dark environment. After acclimating to the environment for 35 minutes, the movement video is recorded with a motion camera for 10 minutes, and the movement distance and maximum movement speed of the zebrafish are analyzed. The movement distance can determine whether the zebrafish's movement ability is abnormal, and the maximum movement speed can determine whether the zebrafish's development process is abnormal.

[0040] The fluorescence intensity of zebrafish feeding is as follows: observe the zebrafish after feeding with fluorescent dye-labeled food under a fluorescence microscope, and use image processing software (such as ImageJ) to measure its fluorescence intensity; the specific steps are as follows: when the zebrafish develops to 5dpf, start feeding it with Paramecium labeled with DiR fluorescent dye, and measure the fluorescence intensity in the intestine of the zebrafish 10-30min after feeding, with the optimal time being 20min; measure the maximum fluorescence intensity at 8dpf as an indicator to evaluate the feeding ability of zebrafish.

[0041] Meckel's cartilage-horn hyoid bone distance (ch-mk): The cartilage of zebrafish exposed to the test drug until 8 dpf was first dyed blue using Alcian blue staining, and the distance between the Meckel's cartilage and the horn hyoid bone in the zebrafish head was measured to determine whether there was obvious head cartilage deformity.

[0042] The standard for determining the amount of mineralized bone in the zebrafish spine is: after staining the mineralized bone with a fluorescent dye (such as Alizarin Red or calcein), the amount of mineralized bone in the spine is observed and counted under a microscope, and the presence of osteoporosis is determined by counting the number of mineralized bones on the spine.

[0043] In the present invention, the culture medium or culture solution is E3 culture medium; E3 culture medium commonly used for zebrafish fry culture, the E3 culture medium includes: 0.001% methylene blue, 5mM NaCl, 0.17mM KCl, 0.33mM CaCl2, 0.33mM MgSO4, and the pH is adjusted to about 7.2.

[0044] In this application, the terms used in the embodiments of this application are only for the purpose of describing specific embodiments, and are not intended to limit this application. The term "and / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " in "and / or" indicates that the associated objects before and after are in an "and" relationship.

[0045] The use of the terms "include", "have" or "contain", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example, not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context. The use of any and all examples or exemplary language herein, such as "for example" or "including", is intended only to better illustrate the present invention and does not limit the scope of the present invention unless claims are presented. No language in this specification should be interpreted as indicating that any non-claimed element is essential to the practice of the present invention.

[0046] The numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0047] The test materials used in the present invention are all common commercial products and can be purchased on the market. The present invention is further described below in conjunction with the embodiments:

[0048] Example 1 Drug screening for zebrafish osteoporosis model construction

[0049] 1. Candidate compound screening

[0050] According to the amount of mineralized bones in zebrafish at 8 dpf, three candidate compounds at 50 μmol / L were selected for the experiment. The three candidate compounds were penicillamine (D-Penicillamine), sodium dimercaptopropane sulfonate (DMPS) and ammonium tetrathiomolybdate (ATTM). The specific treatments were as follows: when the embryos developed to 48 hours (2 dpf), 40 to 60 young fish were cultured in 30 mL of culture medium, and the corresponding three drugs were added to each group of culture medium for culture; the culture medium was replaced once every 24 hours, and the culture was carried out for 6 days, and alizarin red staining was performed to evaluate the bone sclerosis.

[0051] The results are as follows Figure 1 As shown, penicillamine (D-Penicillamine) will cause a decrease in the feeding desire of zebrafish, so this compound is not selected; the decrease in the number of mineralized bones caused by sodium dithiopropane sulfonate (DMPS) is smaller, not as good as ammonium tetrathiomolybdate (ATTM), and ammonium tetrathiomolybdate has the best effect.

[0052] 2. Optimization of ammonium tetrathiomolybdate concentration

[0053] According to the number of mineralized bones in zebrafish at 8 dpf, ammonium tetrathiomolybdate was selected for modeling, and four concentrations of 1, 20, 50, and 100 μmol / L were selected for the experiment. The experimental steps were as above; the experimental results are shown in Figure 2 As shown in the figure, a concentration of 100 μmol / L will cause 30% of the fry to die, so this concentration is not selected. Concentrations of 20 and 50 μmol / L can show a significant decrease in the amount of spinal mineralized bones, and 50 μmol / L has the best effect.

[0054] 3. Construction of zebrafish osteoporosis model and effectiveness evaluation

[0055] 1. The steps for constructing the zebrafish osteoporosis model are as follows:

[0056] Frying fish were selected as experimental subjects. The embryos developed to 48 hours (2 dpf). 40 to 60 fry were cultured per 30 mL of culture medium. 50 μmol / L ammonium tetrathiomolybdate (ATTM) was added to the culture medium for culture. During the culture process, the culture medium was replaced once every 24 hours (the culture medium was replaced once every 24 hours to 72 hours). The zebrafish osteoporosis model was obtained after 6 days of culture.

[0057] 2. Evaluation of the effectiveness of the zebrafish osteoporosis model

[0058] Zebrafish treated with 20 μmol / L prednisone (PDN) were used as the positive control group (PDN group) of the zebrafish osteoporosis model. The treatment steps were the same as "1. Steps for constructing the zebrafish osteoporosis model";

[0059] Zebrafish were treated with calcitriol at a concentration of 20 nmol / L as the positive drug group (Calcitriol group) to increase bone mineralization. The treatment steps were the same as "1. Steps for constructing a zebrafish osteoporosis model";

[0060] Zebrafish were treated with E3 medium as a blank control group (control group) of the zebrafish osteoporosis model. The treatment steps were as described in “1. Steps for constructing a zebrafish osteoporosis model”;

[0061] Zebrafish were treated with 50 μmol / L ammonium tetrathiomolybdate (ATTM) as the zebrafish osteoporosis model group (ATTM group), and the treatment steps were the same as "1. Steps for constructing a zebrafish osteoporosis model";

[0062] The zebrafish head width, body length, movement distance, maximum movement speed, feeding fluorescence intensity, ch-mk distance, and number of mineralized bones in the spine were included in the evaluation parameters.

[0063] (1) Comparison of mineralized bone quantity

[0064] like Figure 3 As shown in the figure, when zebrafish develop normally to 8dpf, the number of mineralized bones is 4-9, with 6-8 accounting for the highest proportion; when the PDN group develops to 8dpf, the number of mineralized bones is 0-3, with 2 accounting for the highest proportion; when the ATTM-treated group develops to 8dpf, the number of mineralized bones is 0-4, with 2-3 accounting for the highest proportion; when the Calcitriol-treated group develops to 8dpf, the number of mineralized bones is 7-10, with 8-9 accounting for the highest proportion.

[0065] (2) Comparison of Meckel's cartilage angle and hyoid bone distance (mk-ch)

[0066] like Figure 4 As shown, when zebrafish developed normally to 8dpf, no obvious craniofacial cartilage deformity was observed in the four groups of zebrafish, and the mk-ch distance in the PDN and ATTM groups was significantly shorter than that in the control group, indicating that the two drugs had a certain effect on the development of zebrafish mandibular cartilage; Figure 4 The yellow line in the middle is the mk-ch spacing.

[0067] (3) Head width comparison

[0068] like Figure 5 As shown, when zebrafish developed normally to 8dpf, there was no obvious morphological deformity in the four groups of zebrafish, and the head width of the PDN and ATTM groups was significantly shorter than that of the control group, indicating that the two drugs had a certain effect on the development of zebrafish; Figure 5 The middle blue line marks the head width.

[0069] (4) Body length comparison

[0070] like Figure 6 As shown, when zebrafish developed normally to 8dpf, there was no obvious morphological deformity in the four groups of zebrafish, and the body length of the PDN group was significantly shorter than that of the control group, indicating that the drug had a certain effect on the development of zebrafish; Figure 6 The blue line in the middle is the body length.

[0071] (5) Comparison of moving distance and maximum speed

[0072] like Figure 7 As shown, when zebrafish developed normally to 8dpf, none of the four groups of zebrafish showed obvious movement disorders, and the moving distance and maximum speed of the PDN group were significantly smaller than those of the control group, indicating that the drug had a certain effect on the activity enthusiasm and activity ability of zebrafish; Figure 7 Shows the movement line graph and statistical analysis results of individual samples.

[0073] (6) Comparison of fluorescence intensity during feeding

[0074] like Figure 8 As shown, when zebrafish develop normally to 8 dpf, all four groups of zebrafish can eat independently. The fluorescence intensity of the intestine is the strongest 20-25 minutes after feeding. The fluorescence intensity of the PDN group is significantly lower than that of the control group after feeding, indicating that the drug has a certain effect on the feeding of zebrafish. Figure 8 Figure 2 shows the changes in intestinal fluorescence intensity and the statistical analysis results of individual samples.

[0075] (7) Parameter heat map comparison

[0076] The data of the seven parameters are standardized using SSMD, and each parameter is processed into a heat map, such as Fig. 9 As shown, the effects of drugs on zebrafish skeletal development can be intuitively compared, and the physiological effects of drugs on zebrafish can be observed, providing a method for screening drugs with small negative effects and direct effects on bones; wherein, NMR: number of mineralized bones in the spine; mk-ch: distance between the Meckel cartilage angle and the hyoid bone; HW: head width; BL: body length; DIS: moving distance; VEL: maximum speed; F: feeding fluorescence intensity.

[0077] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of ammonium tetrathiomolybdate or its preparation in preparing a drug combination for a zebrafish osteoporosis model.

2. A method for preparing a zebrafish osteoporosis model, characterized in that: It involves treating zebrafish with ammonium tetrathiomolybdate.

3. The preparation method according to claim 2, characterized in that: The concentration of the ammonium tetrathiomolybdate is 20 μmol / L to 50 μmol / L.

4. The preparation method according to claim 2, characterized in that: The zebrafish includes juvenile zebrafish.

5. The preparation method according to claim 4, characterized in that: The fry include fry 24 to 72 hours after fertilization.

6. The preparation method according to any one of claims 2 to 5, characterized in that The method of treating zebrafish with ammonium tetrathiomolybdate comprises: exposing young fish 24 to 72 hours after fertilization to a culture solution containing ammonium tetrathiomolybdate for culture.

7. The preparation method according to claim 6, characterized in that: The culture time is 5 to 7 days.

8. The preparation method according to claim 7, characterized in that: During the culture period, the culture solution containing ammonium tetrathiomolybdate is replaced every 24 to 72 hours.

9. A method for evaluating the effectiveness of the zebrafish osteoporosis model prepared by the preparation method according to any one of claims 2 to 8, characterized in that: The method comprises judging the effectiveness of the zebrafish osteoporosis model according to the data change range of the head width, body length, movement distance, maximum movement speed, feeding fluorescence intensity, ch-mk interval and / or spinal mineralized bone quantity of the zebrafish treated with ammonium tetrathiomolybdate and the control group zebrafish.

10. The evaluation method according to claim 9, characterized in that: The criteria for judging effectiveness are: The head width changes in the range of -5% to 5%, the body length changes in the range of -5% to 5%, the movement distance changes in the range of -30% to 30%, the maximum movement speed changes in the range of -50% to 50%, the feeding fluorescence intensity changes in the range of -10% to 10%, the ch-mk interval changes in the range of -20% to 20%, and the amount of mineralized bone decreases in the range of 30% to 100%. It is determined that the zebrafish osteoporosis model prepared by the preparation method described in any one of claims 1 to 8 is effective.

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