Construction method of pituitary prolactinoma animal model

Abstract

The invention discloses a construction method of a pituitary prolactinoma animal model. According to the method, the pituitary prolactinoma animal model constructed by subcutaneously injecting the 17B-Estradiol preparation can better simulate the pathophysiological process of human prolactinoma, and the modeling method is simple and convenient, short in time consumption and is an animal model construction method with an excellent effect.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for constructing a pituitary prolactinoma animal model. Background Art

[0002] Pituitary prolactinomas account for 30%-40% of pituitary adenomas, are more common in women aged 20 to 30, and are often microadenomas. The typical clinical manifestations are the triad of amenorrhea, galactorrhea, and infertility. Male prolactinomas are larger, often develop suprasellarly, and are relatively rare. The main clinical manifestations are complete or partial sexual dysfunction and male infertility. If a fast-growing pituitary prolactinoma suffers intratumoral bleeding, acute pituitary apoplexy may occur, manifested by sudden severe headache, nausea, vomiting, decreased vision, and other symptoms of cranial nerve compression, and even coma and exophthalmos, requiring emergency rescue. Patients may develop hypopituitarism after successful rescue.

[0003] Clinically, there are multiple treatment options for prolactinomas, including surgery, radiotherapy, and drug therapy. Neuroendoscopic transsphenoidal pituitary tumor resection has become the main treatment for pituitary adenomas due to its proven efficacy, minimal trauma, clear field of view, wider exposure, precise surgical technique, and rapid postoperative recovery. Improving the prognosis of patients with prolactinomas remains a major concern in neurosurgery. However, adenomas are difficult to completely resect, and residual tumor tissue is highly susceptible to recurrence. Some prolactinomas also develop drug resistance, resulting in a poor prognosis, presenting a clinical challenge that urgently needs to be addressed.

[0004] Currently, there is no stable experimental animal model for pituitary prolactinoma, which seriously hinders the study of the pathogenesis of pituitary prolactinoma and the development of new drugs. Therefore, providing a stable method for constructing a pituitary prolactinoma animal model is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the present invention provides a method for constructing an animal model of pituitary prolactinoma, wherein the animal model is constructed by subcutaneous injection of a 17B-Estradiol preparation. The present invention can effectively simulate the pathophysiological process of human pituitary prolactinoma, and has the advantages of good repeatability, significant effect, low mortality rate, simple and safe operation method, low experimental cost, and suitability for large-scale animal experiments. It overcomes the shortcomings of the poor effect of existing animal model construction, and can be used for the study of the pathogenesis of pituitary prolactinoma and the development of new drugs, and has good application prospects.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] A first aspect of the present invention provides a method for constructing a pituitary prolactinoma animal model, the method comprising: subcutaneously injecting a 17B-Estradiol preparation into the back of male and female animals.

[0008] In the present invention, 17B-Estradiol, also known as β-estradiol, with a CAS number of 50-28-2, is an estrogen secreted by the ovaries. 17B-Estradiol is widely present in rivers, soil, water sources, the atmosphere, and agricultural products. It primarily originates from pesticides, waste gases, food additives, human and animal excrement, and domestic sewage. Even at extremely low concentrations, it can have significant effects on organisms. Its concentration is significantly correlated with prostate cancer, breast cancer, ovarian cancer, uterine cancer, and male reproductive disorders.

[0009] Furthermore, the construction method comprises the following steps: subcutaneously injecting 0.1 ml of the 17B-Estradiol preparation into the back of female animals every other day; and subcutaneously injecting 0.2 ml of the 17B-Estradiol preparation into the back of male animals every other day. The total duration of the construction method is 28 days.

[0010] Furthermore, the preparation method of the 17B-Estradiol preparation is: adding 17B-Estradiol to a solvent; the composition of the solvent is 20% DMSO, 30% anhydrous ethanol, and 50% glycerol.

[0011] Furthermore, the final concentration of 17B-Estradiol in the 17B-Estradiol preparation is 5.5 mg / ml.

[0012] Furthermore, the animals include one or more of mice, rabbits, dogs, and monkeys.

[0013] In the present invention, the animal is preferably a mouse, more preferably a rat.

[0014] Furthermore, the animals are 8-week-old rats.

[0015] Furthermore, the animals are Wistar rats.

[0016] The second aspect of the present invention provides the use of 17B-Estradiol and / or the 17B-Estradiol preparation described in the first aspect of the present invention in constructing an animal model of pituitary prolactinoma.

[0017] The third aspect of the present invention provides the use of an animal model obtained based on the construction method described in the first aspect of the present invention in screening therapeutic drugs for pituitary prolactinomas.

[0018] A fourth aspect of the present invention provides a method for screening a drug for treating pituitary prolactinoma, the method comprising the following steps:

[0019] administering the drug to be tested to the animal model obtained by the construction method described in the first aspect of the present invention;

[0020] The therapeutic effects of the drugs to be tested are analyzed and evaluated, and the test drugs that can significantly improve the animal model of pituitary prolactin adenoma are candidate therapeutic drugs for pituitary prolactin adenoma.

[0021] In some embodiments, the drugs to be tested include natural medicines, traditional Chinese medicine monomers, small molecule compounds, and antibodies.

[0022] In some embodiments, the sources of the natural medicines, Chinese medicine monomers, and small molecule compounds are selected from: newly synthesized or existing databases; wherein the existing databases include but are not limited to general natural product databases (COCONUT, SuperNatural II, NPASS), plant natural product databases (KNApSaCK, CMAUP, TriForC, Alkamid, NPACT DB, BioPhytMol), Chinese medicine natural product databases (TCM@Taiwan, CEMTDD, CHDD, ETCM, TM-MC, TCMID, YaTCM), microbial natural product databases (StreptomeDB, NP Altas, ProCarDB, PAMDB, LichenDatabase), marine natural product databases (MNPD, SWMD), natural product databases of different countries and regions (IMPPAT, NeMedPlant, MedPServer, TlPdb, AfroDB, ANPDB, BIOFACQUIM, NUBBEDB), food natural product databases (FooDB, BitterDB, Phenol-Explorer, PhytoHub, SuperSweet database), toxic natural product databases (Exposome-Explorer, T3DB, Snake Neurotoxin Database, TPPT), natural product industry catalogs (Greenpharma, AnalytiCon Discovery, InterBioScreen, Indofine Chemical Company, Pi Chemicals Systems\Specs, TargetMol), databases for deduplication using MS data (MoNA, MassBank, METLIN, HMDB, YMDB, ReSpect, GNPS), databases for deduplication using NMR data (NMRShiftDB, NAPROC-13), etc.

[0023] In some embodiments, antibodies are used in the broadest sense in the present invention and specifically cover single domain antibodies, monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies, and multispecific antibodies (e.g., bivalent antibodies) formed from at least two intact antibodies, so long as they exhibit the desired biological activity (e.g., have a therapeutic or alleviating effect on pituitary prolactinoma).

[0024] Advantages and beneficial effects of the present invention: The animal model construction method provided by the present invention can better simulate the pathophysiological process of human pituitary prolactin adenoma, has repeatability, significant effect, low mortality rate, simple and safe operation method, low experimental cost and is suitable for large-scale animal experiments. It makes up for the defects of poor effect of existing animal model construction, fills the gap in domestic and foreign research on this subject, can be used for the study of the pathogenesis of pituitary prolactin adenoma and the development of new drugs, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Representative sagittal and coronal MRI images of the completed animal model.

[0026] Figure 2 This is a statistical chart of pituitary volume changes in the completed animal model rats.

[0027] Figure 3 This is a statistical chart showing the changes in pituitary weight of the completed animal model rats.

[0028] Figure 4 This is a statistical chart showing changes in serum prolactin (PRL) levels in rats with the completed animal model.

[0029] Figure 5 Representative images of HE staining of adenoma in the established animal model rats. DETAILED DESCRIPTION

[0030] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making any creative efforts shall fall within the scope of protection of the present invention.

[0031] The various reagents involved in the technical solutions and experimental procedures described in this invention are all commonly used or commercially available reagents that are well known and readily available to those skilled in the art based on their professional knowledge and routine practice. The description of the reagents in this invention is intended to clearly illustrate the material basis involved in the technical solutions, and those skilled in the art, based on their professional qualities and industry common sense, will be able to successfully obtain and correctly use these reagents to achieve the technical objectives of this invention.

[0032] Example

[0033] 1. Experimental materials:

[0034] Eight-week-old Wistar male and female rats were used, with female rats weighing 130-180±10g and male rats weighing 180-230±10g. They were housed in a barrier environment with a temperature of 22°C, a humidity of 50%, a 12-h day / night rhythm, and fed with sterile feed and sterilized water.

[0035] Sterile feed formula: Casein: 180-250 g / kg, soybean oil: 40-60 g / kg, corn starch: 300-400 g / kg, cellulose: 30-50 g / kg. Mineral premix: 30-40 g / kg, vitamin premix: 10-20 g / kg, containing vitamins A, D, E, K, and B vitamins.

[0036] 2. Model construction method:

[0037] 1) Eight male and eight female rats, aged 8 weeks, were randomly divided into a control group (n=3) and a model group (n=5).

[0038] 2) Model group rats were administered a 17B-estradiol preparation: 20% DMSO + 30% anhydrous ethanol + 50% glycerol + 5.5 mg / ml 17B-estradiol (solid). Control group rats were administered a solvent: 20% DMSO + 30% anhydrous ethanol + 50% glycerol. 0.1 ml (female) / 0.2 ml (male) of the 17B-estradiol preparation / solvent were administered subcutaneously to the back every 48 hours.

[0039] 3) Give medication every other day, provide sterile feed and sterilized water, and feed for a total of 28 days.

[0040] 3. Testing of the establishment of animal model of pituitary prolactinoma:

[0041] 1) MRI sagittal and coronal scans were performed on the first and 28th day (the results were as follows Figure 1 ), RadiAnt2025 version software was used to calculate the rat pituitary volume (the results are shown in Figure 2 After 28 days, the pituitary glands of the rats were taken out, weighed and counted (the results are shown in Figure 3 Detailed data are shown in Table 1.

[0042] 2) On days 1 and 28, rats were anesthetized with isoflurane, and blood was collected from the inner canthus. The blood was placed in the center of a centrifuge tube and serum (100 μl) was collected. The serum was added with the labeled antibody and incubated at 4°C for 20 h. The separation agent was added for 15 min, and then centrifuged at 3800 rpm for 15 min. Serum prolactin (PRL) levels were measured using an iodine [125I] prolactin radioimmunoassay kit (results are shown in Table 2). Figure 4 As shown in Figure 3 ), the PRL level in the modeling group was significantly increased compared with the control group, which was considered a successful modeling.

[0043] 3) Take the pituitary sections of rats on day 28 and perform HE staining. If pathological examination shows pituitary prolactin adenoma, the model is considered successful. The results are as follows Figure 5 shown.

[0044] Table 1. Detailed data of rats in the model group and control group

[0045] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention can be implemented over a wide range under equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides embodiments, it will be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any variations, uses, or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the disclosed scope of this application.

Claims

1. A method for constructing an animal model of pituitary prolactinoma, characterized in that: The construction method comprises the steps of: injecting 17B-Estradiol preparation subcutaneously into the back of male and female animals.

2. The construction method according to claim 1, characterized in that The steps of the construction method include: 17B-Estradiol preparation (0.1 ml) was injected subcutaneously into the back of female animals every other day; Male animals were injected subcutaneously with 0.2 ml of 17B-Estradiol preparation on the back every other day; The total duration of the construction method was 28 days.

3. The construction method according to claim 2, characterized in that The 17B-Estradiol preparation is prepared by adding 17B-Estradiol to a solvent; The solvent consists of 20% DMSO, 30% anhydrous ethanol, and 50% glycerol.

4. The construction method according to claim 2, characterized in that The final concentration of 17B-Estradiol in the 17B-Estradiol preparation is 5.5 mg / ml.

5. The construction method according to claim 2, characterized in that The animals include one or more of mice, rabbits, dogs and monkeys.

6. The construction method according to claim 2, characterized in that The animals were 8-week-old rats.

7. The construction method according to claim 6, characterized in that: The animals are Wistar rats.

8. Use of 17B-Estradiol and / or the 17B-Estradiol preparation according to any one of claims 3 to 4 in constructing an animal model of pituitary prolactinoma.

9. Use of an animal model obtained by the construction method according to any one of claims 1 to 7 in screening therapeutic drugs for pituitary prolactinoma.

10. A method for screening drugs for treating pituitary prolactinoma, characterized in that: The method comprises the following steps: administering the drug to be tested to the animal model obtained by the construction method according to any one of claims 1 to 7; The therapeutic effects of the drugs to be tested are analyzed and evaluated, and the test drugs that can significantly improve the animal model of pituitary prolactin adenoma are candidate therapeutic drugs for pituitary prolactin adenoma.

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