Construction method of idiopathic central precocious puberty animal model

Abstract

The invention discloses a construction method of an idiopathic central precocious puberty animal model. The construction method comprises the following steps: setting an illumination period in which the ratio of illumination time to dark time is (16-19) hours: (5-8) hours, feeding rodents under the condition of the illumination period until sexual maturity, and constructing the idiopathic central precocious puberty animal model. According to the construction method, only the factor'illumination 'necessary for growth and development is adopted as a regulation and control object, the idiopathic central precocious puberty animal model is successfully constructed by setting the specific illumination and darkness ratio, no new interference factor needs to be brought in, and the method is low in economic cost, effective, reliable, easy to operate, high in repeatability and wide in application prospect.

Description

Technical Field

[0001] This application relates to the technical field of animal models, and particularly to a method for constructing an animal model of idiopathic central precocious puberty. Background Art

[0002] Central precocious puberty refers to the appearance of puberty-related physiological and reproductive characteristics before the age of 8. Among them, idiopathic central precocious puberty refers to precocious puberty without organic lesions, and its incidence accounts for 80% of female central precocious puberty. In the past, studies on idiopathic central precocious puberty mostly used gene mutation methods to explore the pathogenesis of idiopathic central precocious puberty from a genetic perspective. However, in recent years, the incidence of idiopathic central precocious puberty has increased rapidly, which suggests that in addition to genetic factors, the occurrence of idiopathic central precocious puberty may also involve changes in environmental factors. Idiopathic central precocious puberty can lead to short stature, social phobia, increased depressive mood, and the risk of breast cancer in children. Therefore, constructing a suitable animal model for exploring the pathogenesis and prevention mechanism of idiopathic central precocious puberty is crucial for the treatment of this disease. Currently, animal models of idiopathic central precocious puberty are mainly constructed by chemical / hormone induction, gene editing, or nutritional regulation, but there are research limitations. For example, the use of exogenous drugs or hormone stimulation methods reduces the scientificity and representativeness of the model and cannot reproduce the disease conditions under natural conditions. Moreover, due to the androgenic properties of danazol, it may cause adverse side effects such as masculinization in women, resulting in unpredictable interfering factors. At the same time, the stimulation of exogenous substances cannot simulate the increased incidence caused by environmental factors. Gene editing methods may lead to non-physiological developmental changes, making it difficult to truly simulate the natural puberty process and unable to simulate the increased incidence caused by current environmental factors. Studies have shown that a high-fat diet may lead to early puberty, but dietary factors are often accompanied by various metabolic changes, such as obesity, and obesity can clearly cause precocious puberty. The models constructed by nutritional conditions can cause various systemic factor changes leading to abnormal sexual development, so they are not suitable for single-factor analysis.

[0003] Therefore, there is an urgent need to develop a method for constructing an animal model of idiopathic central precocious puberty that is simpler and has fewer interfering factors. Summary of the Invention

[0004] Based on this, it is necessary to provide a method for constructing an animal model of idiopathic central precocious puberty.

[0005] In the first aspect of this application, a method for constructing an animal model of idiopathic central precocious puberty is provided. The method for constructing the animal model of idiopathic central precocious puberty includes setting a light cycle with a ratio of light time to dark time of (16 - 19) hours : (5 - 8) hours, and raising rodent animals under the conditions of the light cycle until sexual maturity to construct the animal model of idiopathic central precocious puberty.

[0006] In some embodiments, the light in the light cycle is continuous light, and the darkness in the light cycle is continuous darkness.

[0007] In some embodiments, the intensity of the light in the long light cycle is 20 lux to 30 lux.

[0008] In some embodiments, the rodent selected is an animal at 2 weeks old to 4 weeks old.

[0009] In some embodiments, the rodent includes a mouse.

[0010] In some embodiments, the mouse includes any one of a mouse, a rat, and a hamster.

[0011] In some embodiments, the rodent is selected from C57 BL / 6 mice.

[0012] In some embodiments, the feeding time is 4 days to 20 days.

[0013] In some embodiments, the rodent is fed in a soundproof box.

[0014] In some embodiments, it further includes determining the onset of sexual development by observing the opening of the vaginal orifice and / or the separation of the prepuce of the glans penis of the rodent.

[0015] The method for constructing an animal model of idiopathic central precocious puberty provided by the embodiments of the present application only uses the essential factor for growth and development, "light", as the regulation object, and successfully constructs an animal model of idiopathic central precocious puberty by setting a specific light-to-dark ratio, and there is no need to introduce new interfering factors. This construction method has low economic cost, is effective and reliable, is easy to operate, has strong repeatability, and has broad application prospects. Description of the Drawings

[0016] In order to more clearly illustrate the technical solutions in the embodiments and examples of the present application and more completely understand the present application and its beneficial effects, the following will briefly introduce the drawings required to be used in the description of the embodiments or examples. Obviously, the drawings in the following description are only some embodiments of the present application. For those skilled in the art, without creative efforts, other drawings can be obtained according to these drawings.

[0017] Figure 1This is the verification result of the idiopathic central precocious puberty animal model in an embodiment of the present application. Among them, Figure A shows the time of vaginal opening; Figure B shows the estrus time; Figure C shows the body weight growth curve; Figure D shows the serum corticosterone level; Figure E shows the immunofluorescence intensity of hypothalamic GnRH neurons, where GnRH is gonadotropin-releasing hormone; Figure F shows the level of luteinizing hormone secreted by the pituitary gland; Figure G shows the percentage of mature follicles in the gonadal ovary.

[0018] Among them, * indicates p < 0.05, and ** indicates p < 0.01. Detailed implementation manners

[0019] To facilitate the understanding of the present application, the present application will be described more comprehensively below with reference to the relevant drawings. The preferred embodiments of the present application are given in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure content of the present application more thorough and comprehensive.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this application belongs. The terms used in the description of this application herein are only for the purpose of describing specific embodiments and are not intended to limit this application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0021] In the present application, "optionally", "optional", "option" mean optional, that is, it means any one of the two parallel options of "yes" or "no". If "optional" appears multiple times in a technical solution, without special instructions and without contradictions or mutual restrictions, each "optional" is independent of each other.

[0022] In the present application, "preferred", "better", "more preferable", "preferably" are only used to describe the embodiments or examples with better effects, and it should be understood that they do not constitute a limitation on the protection scope of the present application.

[0023] The terms "having", "containing", "including" and "comprising" used in the present application are synonyms, which are inclusive or open-ended and do not exclude additional, unrecited members or features. Members or features such as materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features also include actions, conditions for the occurrence of actions, timing, states, etc.

[0024] In the present application, in the technical features or technical solutions described in an open language, the closed technical features or technical solutions composed of the listed contents are also included, and the open technical features or technical solutions containing the listed contents are also included.

[0025] In this application, for units related to data ranges, if a unit is only attached after the right endpoint, it means that the units of the left endpoint and the right endpoint are the same.

[0026] In this application, for method processes involving multiple steps, unless there are clear different descriptions in this article, the execution of these steps has no strict order restriction, and they can be executed in other orders than the described one. Moreover, any step can include multiple sub-steps or multiple stages. These sub-steps or stages do not necessarily need to be completed at the same moment, but can be executed at different moments, and their execution order does not necessarily have to be sequential, but can be executed alternately or simultaneously with other steps or a part of the sub-steps or stages of other steps.

[0027] In this application, for exemplary descriptions such as "in some embodiments" or "in one embodiment", etc., they can cover but are not limited to the following meanings: These solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0028] In this application, in "the first aspect", "the second aspect", "the third aspect", etc., the terms "first", "second", "third", etc. are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", etc. only serve the purpose of non-exhaustive listing and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0029] In this application, for numerical intervals (i.e., numerical ranges), unless otherwise specified, the distribution of the optional numerical values within this numerical interval is regarded as continuous, and includes the two numerical endpoints of this numerical interval (i.e., the minimum value and the maximum value), as well as each numerical value between these two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within this numerical interval, it includes the two endpoint integers of this numerical range, as well as each integer between the two endpoints, which is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be combined. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein. The "numerical values" in this numerical interval can be any quantitative values, such as numbers, percentages, ratios, etc. "Numerical intervals" are allowed to broadly include numerical interval types such as percentage intervals, ratio intervals, and ratio value intervals.

[0030] Currently, the establishment of animal models for idiopathic central precocious puberty mainly follows the following several approaches:

[0031] 1. Chemical / hormone-induced models: Current research includes methods of injecting danazol (chemical) and leptin (hormone).

[0032] 2. Gene editing model: Use the CRISPR / Cas9 technology to knockout or activate genes related to sexual development (such as Kiss1, Gpr54) to construct animal models of idiopathic central precocious puberty or delayed sexual development.

[0033] 3. Nutritional regulation: High-fat diet.

[0034] However, there are some research limitations in the above-mentioned methods. For example:

[0035] 1. Chemical / hormone-induced model: Using exogenous drugs or hormone stimulation methods reduces the scientific nature and representativeness of the model and cannot simulate the disease conditions under natural conditions. And due to the androgenic properties of danazol, it may cause adverse side effects such as virilization in women, resulting in unpredictable interfering factors. At the same time, exogenous substance stimulation cannot simulate the increased incidence caused by environmental factors.

[0036] 2. Gene editing model: Gene editing methods may lead to non-physiological developmental changes and it is difficult to truly simulate the natural puberty process. The epidemiology of the sharp increase in idiopathic central precocious puberty in the past 5 years does suggest the possibility of precocious puberty caused by non-genetic factors, and gene editing models cannot simulate the increased incidence caused by current environmental factors.

[0037] 3. Nutritional regulation: Studies have shown that a high-fat diet may lead to early puberty, but dietary factors are often accompanied by various metabolic changes, such as obesity, and obesity is clearly known to cause precocious puberty. Since this model can cause changes in multiple systemic factors leading to abnormal sexual development, it is not suitable for single-factor analysis.

[0038] Based on this, the embodiments of the present application at least provide a method for constructing an animal model of idiopathic central precocious puberty.

[0039] In some embodiments, based on the current epidemiological situation, the present application takes "light", an essential factor for growth and development, as the regulation object, without introducing new interfering factors, and adjusts the time and intensity of light within the scope of animal ethics regulations from the perspective of day length.

[0040] In some embodiments, it is generally considered that both mice and humans are non-seasonal mating animals, and the fact that the present application selects light as an intervention factor can still achieve the effect of intervening in sexual development, which is the novelty of the animal model constructed in the present application.

[0041] In some embodiments, the animal model constructed in the present application can also be used to screen drugs for preventing or treating central precocious puberty.

[0042] In the first aspect of the present application, a method for constructing an animal model of idiopathic central precocious puberty is provided. The method for constructing the animal model of idiopathic central precocious puberty includes setting a light cycle with a ratio of light time to dark time of (16 - 19) hours : (5 - 8) hours, and raising a rodent animal under the condition of this light cycle until sexual maturity to construct an animal model of idiopathic central precocious puberty.

[0043] In the present application, unless otherwise specified, "light cycle" refers to the relative lengths of day and night in a day, with a 24 - hour cycle.

[0044] In some embodiments, the ratio of light time to dark time in the light cycle can be, but is not limited to, 16 hours : 8 hours, 17 hours : 7 hours, 18 hours : 6 hours, 19 hours : 5 hours, or the ratio or range between any two of the above ratios.

[0045] In the method for constructing an animal model of idiopathic central precocious puberty provided in the embodiments of the present application, only the essential factor for growth and development, "light", is used as the regulation object. By setting a specific light - to - dark ratio, an animal model of idiopathic central precocious puberty is successfully constructed without the need to incorporate new interfering factors. This construction method has low economic cost, is effective and reliable, easy to operate, and highly repeatable, and has broad application prospects.

[0046] In some embodiments, the light in the light cycle is continuous light, and the dark in the light cycle is continuous dark.

[0047] It should be noted that when raising a rodent animal under the set light cycle condition in the embodiments of the present application, the time for the rodent animal to enter light or dark every day is the same.

[0048] In some embodiments, the intensity of the light in the light cycle is 20 lux - 30 lux, and this light intensity conforms to the ethics of experimental animals. Non - restrictively, the intensity of the light in the light cycle can be, but is not limited to, 20 lux, 22 lux, 24 lux, 26 lux, 28 lux, 30 lux, or the value or range between any two of the above values.

[0049] In some embodiments, the rodent animal selected is a weaned rodent animal.

[0050] In some embodiments, the rodent animal selected is an animal at 2 - week - old to 4 - week - old. Non - restrictively, the rodent animal can be selected as, but is not limited to, an animal at 2 - week - old, 3 - week - old, 4 - week - old, or an animal within the age or range between any two of the above ages.

[0051] In some embodiments, the rodent animal selected is an animal at 21 - day - old to 24 - day - old.

[0052] In some embodiments, the rodent includes a mouse; optionally, the mouse includes any one of a mouse, a rat, and a hamster.

[0053] In some embodiments, the rodent is selected from C57 BL / 6 mice or transgenic C57 BL / 6 mice produced by gene editing means.

[0054] In some embodiments, the time for raising the rodent is 4 days to 20 days. Without limitation, the time for raising the rodent can be, but is not limited to, 4 days, 6 days, 8 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, or the time or range between any two of the above durations.

[0055] In some embodiments, the rodent is raised in a soundproof box.

[0056] In some embodiments, the rodent is female.

[0057] In some embodiments, the rodent is male.

[0058] In some embodiments, the method for constructing an idiopathic central precocious puberty animal model further includes judging the sexual development time of female rodents by observing the opening of the vulva and the morphology of vaginal exfoliated cells during the breeding process. Exemplarily, the opening time of the vulva and the time when vaginal exfoliated cells start to keratinize in female rodents are significantly advanced.

[0059] In some embodiments, the method for constructing an idiopathic central precocious puberty animal model further includes judging the sexual development time of male rodents by observing the separation of the prepuce of the glans penis during the breeding process. Exemplarily, the separation time of the prepuce of the glans penis in male rodents is significantly advanced.

[0060] In some embodiments, it is further judged whether the rodent is sexually mature by hormone levels and histological detection. Exemplarily, when the levels of luteinizing hormone and gonadotropin-releasing hormone in female rodents are significantly increased, and the proportion of mature follicles is significantly increased, it indicates that the female rodent has reached sexual maturity; when the levels of luteinizing hormone and gonadotropin-releasing hormone in male rodents are significantly increased, and the sperm count is significantly increased, it indicates that the male rodent has reached sexual maturity.

[0061] In the second aspect of the present application, there is provided an idiopathic central precocious puberty animal model constructed by the method for constructing an idiopathic central precocious puberty animal model in the first aspect of the present application.

[0062] In the third aspect of the present application, there is provided the use of the idiopathic central precocious puberty animal model of the second aspect of the present application in screening drugs for preventing and / or treating idiopathic central precocious puberty.

[0063] Some embodiments are provided below.

[0064] The embodiments of the present application will be described in detail below in conjunction with the embodiments. It should be understood that these embodiments are only used to illustrate the present application and not to limit the scope of the present application. For the experimental methods without specified conditions in the following embodiments, the guidance given in the present application is preferably referred to, and it can also be carried out according to the experimental manuals or conventional conditions in the art, or according to the conditions recommended by the manufacturer, or referring to the experimental methods known in the art.

[0065] In the following embodiments, for the measurement parameters of the raw material components, if not otherwise specified, there may be slight deviations within the weighing accuracy range. For the temperature and time parameters, acceptable deviations caused by the instrument test accuracy or operation accuracy are allowed.

[0066] Example 1

[0067] As the wild-type mice most commonly used in medical scientific research, C57 BL / 6 mice cannot produce melatonin due to the specificity of their species. The release of melatonin can inhibit the hypothalamic-pituitary-gonadal axis and delay sexual development. The reasons for constructing the model using this strain of mice in the present application are as follows: 1) C57 BL / 6 mice are the strain adopted by the vast majority of medical research; 2) This strain of mice does not secrete melatonin, excluding the influence of melatonin on sexual development.

[0068] I. Construction of the central precocious puberty animal model

[0069] 1. Grouping of experimental animals: In the embodiments of the present application, 21-day-old (equivalent to human juveniles) C57 BL / 6 mice are used. The mice born in the same cage, of the same age and the same sex are arranged in descending order of body weight, and two mice with similar body weights are divided into one group (block). The two mice with similar body weights are randomly grouped into the treatment group and the control group by the method of block randomization.

[0070] 2. Construction of precocious puberty model: After grouping, the mice were placed in a breeding cage lined with 0.5 cm thick corncob bedding and having a bottom area of 25 cm × 15 cm (3 - 5 mice per cage). The cage was placed in the center of a soundproof box (length × width × height = 60 cm × 35 cm × 40 cm), and a white light source with a length of 15 cm was attached to the top of the soundproof box, and the light intensity at the bottom of the cage was 20 - 30 lux. An exhaust fan was attached to the side wall at the top of the soundproof box for ventilation purposes. The light cycle of the soundproof box was set to 12 hours of light: 12 hours of darkness (12:12, i.e., the normal light group, the control group), or 19 hours of light: 5 hours of darkness (19:5, i.e., the long light group, the treatment group). Sufficient food and water were provided for the mice, and the cage was changed every 7 days.

[0071] The advantages of this model are as follows: 1. Mice are nocturnal animals, and the 19 - hour light environment will not cause sleep deprivation in mice, so the influence of sleep on sexual development can be excluded. 2. No exogenous chemical agents are used to intervene in the mice in this model, excluding the physiological and pathological effects caused by drugs. 3. A construction method for an idiopathic central precocious puberty model of C57 BL / 6 mice with low economic cost, effectiveness, reliability, simplicity in operation, and strong repeatability.

[0072] II. Verification of the central precocious puberty animal model

[0073] The model was verified using "observation of sexual development phenotype", "cytological analysis", "hormone determination", and "histology of sexual organs", and the required central precocious puberty model could be obtained after verification. Note: The following verification methods selected different batches and different offspring, so there was good sample diversity. The experimental techniques used to verify the model constructed in the embodiments of this application have been verified and accepted many times in the biomedical field, and all the verification results are as Figure 1 shown.

[0074] Observation of sexual development phenotype and cytological analysis: In this application, female mice born in the same cage and 21 - day - old young mice of the same age were weighed, and after random grouping according to their weights, the mice were respectively placed in breeding cages with 12:12 and 19:5 breeding cycles. The opening condition of the vulva was observed at 9:00 every morning, and the observation situation was as Figure 1As shown in Figure A, the results indicate that long light exposure (19 hours of light: 5 hours of darkness) can significantly advance the time of vaginal opening. After the vaginal opening, the vagina was flushed with 0.2 mL of ultrapure water. After aspirating the flushing fluid, the flushing fluid was dropped onto a glass slide and air-dried naturally. After air-drying, anhydrous ethanol was dropped to cover it. After the anhydrous ethanol was air-dried, 0.1% crystal violet staining was performed for 5 minutes. Then, the glass slide was soaked in ultrapure water 3 times for 3 minutes each time. After the washing was completed, the glass slide was air-dried naturally, and a bright-field microscope (Olympus CKX53) was used to analyze the estrus situation according to cell morphology under a 10-fold magnification. This technique is a common method for judging the estrus period. Using this method, the sexual development progress of the model constructed in the embodiments of the present application was evaluated. The results are as Figure 1 shown in Figure B, showing that the first estrus time of the mice in the long light exposure group was significantly advanced. At the same time, to exclude the influence of body weight on sexual development, the body weight of the mice was weighed every day. The results are as Figure 1 shown in Figure C, and the results show that there was no change in the body weight of the mice in the model of the present application, indicating that the model of the present application avoided the influence of body weight on sexual development.

[0075] Others: When the mice were 25 days old, the mice were anesthetized. After anesthesia, 0.6 - 1 mL of apical blood was drawn with a 1 mL sterile syringe, and the serum was taken after standing for serum corticosterone determination and luteinizing hormone enzyme-linked immunosorbent assay of serum hormones. The results of serum corticosterone determination are as Figure 1 shown in Figure D, and the results show that there was no difference in the serum corticosterone levels between the normal group mice and the long light exposure group mice, indicating that the model of the present application did not affect the stress of the mice; the results of luteinizing hormone enzyme-linked immunosorbent assay of serum hormones are as Figure 1 shown in Figure F, and it can be seen that the luteinizing hormone level of the mice in the long light exposure group was significantly increased. Then, the mice were perfused with 1×PBS and 4% formalin. After perfusion, the brains of the mice were taken for gonadotropin-releasing hormone neuron immunofluorescence determination. The results are as Figure 1 shown in Figure E, showing that the immunofluorescence area of GnRH (gonadotropin-releasing hormone) neurons in the long light exposure group mice was significantly increased, indicating that the excitability of GnRH neurons was significantly enhanced; at the same time, the ovaries of the mice were taken out, and the ovaries were fixed and dehydrated for the analysis of the number of mature follicles. The results are as Figure 1 shown in Figure G, and it can be seen that the proportion of mature follicles in the long light exposure group mice was significantly increased.

[0076] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope recorded in this specification.

[0077] The above-described embodiments merely represent several implementation manners of the present application. The description thereof is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, and these all fall within the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the appended claims.

Claims

1. A method for constructing an animal model of idiopathic central precocious puberty, characterized in that: The method for constructing the idiopathic central precocious puberty animal model includes setting a light cycle in which the ratio of light time to dark time is (16-19) hours: (5-8) hours, raising rodents under the conditions of the light cycle until sexual maturity, and constructing the idiopathic central precocious puberty animal model.

2. The method for constructing an animal model of idiopathic central precocious puberty according to claim 1, characterized in that: The illumination in the illumination cycle is continuous illumination, and the darkness in the illumination cycle is continuous darkness.

3. The method for constructing an animal model of idiopathic central precocious puberty according to claim 2, characterized in that: The intensity of light in the light cycle is 20 lux to 30 lux.

4. The method for constructing an animal model of idiopathic central precocious puberty according to claim 1, characterized in that: The rodents selected are animals between 2 and 4 weeks old.

5. The method for constructing an animal model of idiopathic central precocious puberty according to claim 4, characterized in that: The rodents include mice.

6. The method for constructing an animal model of idiopathic central precocious puberty according to claim 5, characterized in that: The rat includes any one of a mouse, a rat and a hamster.

7. The method for constructing an animal model of idiopathic central precocious puberty according to claim 6, characterized in that: The rodent is selected from C57 BL / 6 mice.

8. The method for constructing an animal model of idiopathic central precocious puberty according to claim 1, characterized in that: The feeding time is 4 to 20 days.

9. The method for constructing an animal model of idiopathic central precocious puberty according to claim 8, characterized in that: The rodents were housed in a soundproof box.

10. The method for constructing an animal model of idiopathic central precocious puberty according to any one of claims 1 to 9, characterized in that: It also includes observing the opening of the vulva and / or the separation of the glans and foreskin in rodents to determine the onset of sexual development.

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