Experimental rat hysteromyoma model feed as well as preparation method and application thereof

Abstract

The invention relates to experimental rat hysteromyoma model feed. The experimental rat hysteromyoma model feed comprises 18-22 parts of casein, 0.25-0.4 part of L-cystine, 35-40 parts of corn starch, 8-12 parts of cane sugar, 10-14 parts of maltodextrin, 3-5 parts of cellulose, 5-10 parts of soybean oil, 1-2 parts of a mineral mixture, 1-2 parts of a vitamin mixture and 0.1-0.3 part of choline tartrate. The pharmaceutical composition further comprises the following components in parts by weight: 0.003 to 0.006 part of estradiol valerate and / or 0.05 to 0.2 part of progesterone. The composition can also comprise cyclodextrin, the cyclodextrin and estradiol valerate and / or progesterone are prepared into an inclusion compound, or the cyclodextrin is directly added into the feed, and the total mass ratio of the cyclodextrin to estradiol valerate and / or progesterone is 1: (2-4). The cyclodextrin is selected from at least one of beta-cyclodextrin, hydroxyethyl-beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin and ethidene diamine-beta-cyclodextrin, and the cyclodextrin is at least one of beta-cyclodextrin, hydroxyethyl-beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin and ethidene diamine-beta-cyclodextrin. After a rat is fed with the feed for a period of time, a rat hysteromyoma model can be successfully obtained, the modeling process is milder, the model is more stable, and research on diseases and drugs is facilitated.

Description

Technical Field

[0001] The invention relates to the technical field of animal model feed, and in particular to an experimental rat uterine fibroid model feed and a preparation method and application thereof. Background Art

[0002] Uterine fibroids are benign tumors of the uterine muscle layer, which mainly occur in women of childbearing age and after menopause. The cause of uterine fibroids is not yet fully understood. Genetic changes and abnormal sex hormone levels may lead to the occurrence of uterine fibroids, which are often manifested by abdominal pain, increased menstrual flow, prolonged menstruation, shortened menstrual cycle, anemia, premature birth, miscarriage, infertility, and compression of the rectum or bladder.

[0003] At present, there are many methods for constructing rat uterine fibroid models, such as genetic engineering, which can be used to study uterine fibroid disease by gene insertion or knockout technology. This method has strong specificity and a high construction success rate, but there are defects such as genetic engineering technology difficulty, high cost, cumbersome process, and low efficiency. It is also possible to use immunodeficient mouse xenotransplantation uterine fibroid tissue to construct. Although the xenotransplantation animal model has the advantages of high modeling success rate and better ability to reflect human disease conditions, immunodeficient rats are expensive and require a sterile living environment, so they are demanding on research conditions and researchers. In addition, single estrogen and estrogen progesterone hormone combination are two commonly used methods for establishing induced uterine fibroid animal models, but the current methods are long-term injection of estrogen or progesterone, multiple injections may cause local infection, and long-term intraperitoneal injections may cause abdominal visceral adhesion or nodule formation, animal neuro-endocrine system, circulatory system, and body metabolism will be greatly affected, and even change the physiological state of the animal, thereby obtaining incorrect experimental data. Therefore, there is an urgent need for a method to construct an experimental rat uterine fibroids model with a milder modeling process to facilitate the screening of therapeutic drugs. Summary of the invention

[0004] In order to overcome at least one problem existing in the prior art, the present invention provides an experimental rat uterine fibroids model feed, which optimizes its preparation method and applies it to constructing an experimental rat uterine fibroids model to solve the problems existing in the related technology, such as strong animal stress response and many human intervention factors.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] The first aspect of the present invention is to provide an experimental rat uterine fibroid model feed, which includes the following raw materials in parts by weight: 18 to 22 parts of casein, 0.25 to 0.4 parts of L-cystine, 35 to 40 parts of corn starch, 8 to 12 parts of sucrose, 10 to 14 parts of maltodextrin, 3 to 5 parts of cellulose, 5 to 10 parts of soybean oil, 1 to 2 parts of mineral mixture, 1 to 2 parts of vitamin mixture, and 0.1 to 0.3 parts of choline bitartrate; wherein the feed also includes 0.003 to 0.006 parts of estradiol valerate, and / or 0.05 to 0.2 parts of progesterone.

[0007] Furthermore, the feed comprises the following raw materials in parts by weight: 18-20 parts of casein, 0.3-0.35 parts of L-cystine, 36-39 parts of corn starch, 10-12 parts of sucrose, 12-14 parts of maltodextrin, 4-5 parts of cellulose, 6-8 parts of soybean oil, 1.5-2 parts of mineral mixture, 1.5-2 parts of vitamin mixture, and 0.15-0.252 parts of choline bitartrate;

[0008] Wherein, the feed further includes 0.004-0.006 parts of estradiol valerate; or, the feed further includes 0.1-0.2 parts of progesterone; or, the feed further includes 0.004-0.006 parts of estradiol valerate and 0.1-0.2 parts of progesterone.

[0009] Furthermore, the feed includes the following raw materials in parts by weight: 19 parts of casein, 0.3 parts of L-cystine, 38 parts of corn starch, 11 parts of sucrose, 13 parts of maltodextrin, 5 parts of cellulose, 7 parts of soybean oil, 2 parts of mineral mixture, 2 parts of vitamin mixture, and 0.2 parts of choline bitartrate; wherein the feed also includes 0.005 parts of estradiol valerate, and / or 0.15 parts of progesterone (for example, 0.005 parts of estradiol valerate; or, 0.15 parts of progesterone; or, 0.005 parts of estradiol valerate + 0.15 parts of progesterone).

[0010] Furthermore, the feed also includes cyclodextrin, and the cyclodextrin is selected from at least one of β-cyclodextrin, hydroxyethyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, and ethylenediamine-β-cyclodextrin; preferably, the cyclodextrin is selected from at least one of hydroxyethyl-β-cyclodextrin and ethylenediamine-β-cyclodextrin, and more preferably, the cyclodextrin is composed of hydroxyethyl-β-cyclodextrin and ethylenediamine-β-cyclodextrin. The above-mentioned hydroxyethyl-β-cyclodextrin and ethylenediamine-β-cyclodextrin can be in any ratio, and preferably the mass ratio of hydroxyethyl-β-cyclodextrin to ethylenediamine-β-cyclodextrin is 1:1.

[0011] Furthermore, the cyclodextrin is prepared into an inclusion compound with estradiol valerate and / or progesterone; or, the cyclodextrin is directly added to the feed, and the total mass ratio of the cyclodextrin to estradiol valerate and / or progesterone is 1:2-4, preferably 1:3.

[0012] Furthermore, the particle sizes of the casein, maltodextrin and sucrose can be conventional mesh sizes in the art, for example, the mesh size of casein is 100-200 mesh, the mesh size of maltodextrin is 100-200 mesh, and the mesh size of sucrose is 60-100 mesh.

[0013] Furthermore, the mineral mixture is composed of the following raw materials in parts by weight: 20-25 parts of sucrose, 33-35 parts of calcium carbonate, 18-21 parts of potassium dihydrogen phosphate, 5-9 parts of sodium chloride, 5-8 parts of potassium citrate monohydrate, 3-6 parts of potassium sulfate, 2-4 parts of magnesium oxide, 0.6-0.8 parts of ferric citrate, 0.1-0.3 parts of zinc carbonate, 0.1-0.3 parts of sodium metasilicate, 0.04-0.08 parts of hydrated manganese carbonate, 0.0 2~0.04 parts, potassium chromium sulfate 0.01~0.03 parts, boric acid 0.005~0.01 parts, sodium fluoride 0.005~0.08 parts, nickel carbonate 0.002~0.004 parts, anhydrous lithium chloride 0.001~0.003 parts, sodium selenate 0.001~0.003 parts, potassium iodate 0.001~0.003 parts, ammonium molybdate tetrahydrate 0.001~0.003 parts, ammonium metavanadate 0.001~0.003 parts.

[0014] Furthermore, the vitamin mixture is composed of the following raw materials in parts by weight: 96-98 parts of sucrose, 1-3 parts of niacin, vitamin B 12 0.2-0.4 parts, biotin 0.2-0.4 parts, D-pantothenic acid hemi-calcium salt 0.1-0.3 parts, vitamin D3 0.1-0.2 parts, retinol acetate 0.06-0.09 parts, vitamin B6 0.05-0.09 parts, riboflavin 0.05-0.08 parts, thiamine hydrochloride 0.05-0.08 parts, folic acid 0.01-0.03 parts, vitamin K 0.01-0.02 parts.

[0015] Furthermore, the feed preparation method comprises the steps of: mixing the prepared raw materials, granulating and drying; the mixing temperature is 20°C to 30°C; wherein, when the feed raw materials contain cyclodextrin, it can be directly mixed with other raw materials; or, cyclodextrin is first prepared into an inclusion compound with estradiol valerate and / or progesterone, and then the inclusion compound is mixed with other raw materials.

[0016] Furthermore, the process parameters of granulation and drying after mixing can refer to the conventional methods in the art, for example, the mixing time is 1 to 2 hours, the drying temperature is 20 to 25° C., the humidity during the drying process is 20 to 30%, and the drying time is 24 to 36 hours.

[0017] Further, the preparation step of the inclusion compound includes: dissolving estradiol valerate and / or progesterone in an appropriate amount of anhydrous ethanol at room temperature to obtain a first solution; dissolving equal amounts of hydroxyethyl-β-cyclodextrin and ethylenediamine-β-cyclodextrin in purified water at 40°C to obtain a second solution; slowly adding the first solution to the second solution under light-proof conditions, stirring at a constant temperature of 30-35°C for 2h, stirring at 4°C for 2h, and standing in a refrigerator for 2h, separating the product by conventional methods and freeze-drying to obtain the inclusion compound. It is understandable that the preparation of the above inclusion compound can also be prepared by conventional methods in the art. The use of the inclusion compound can improve the solubility of estrogen and progesterone, improve the stability of estrogen and progesterone, and reduce gastrointestinal irritation, thereby making estrogen and progesterone easier to absorb, so as to facilitate the construction of a uterine fibroids model. The ratio of the above estrogen, progesterone and cyclodextrin is based on the ability to achieve almost complete inclusion of estrogen and progesterone.

[0018] The second aspect of the present invention is to provide an application of any experimental rat uterine fibroids model feed described in the first aspect; wherein the feed is used to construct an experimental rat uterine fibroids model.

[0019] The third aspect of the present invention is to provide a method for constructing an experimental rat uterine fibroids model, which comprises the steps of: using any of the experimental rat uterine fibroids model feeds described in the first aspect, allowing the experimental rats to eat and drink water freely every day, and constructing the experimental rat uterine fibroids model after 5 to 7 weeks; wherein the experimental rats are SD female rats, preferably SD female rats of about 2 months old.

[0020] Furthermore, the experimental rat uterine fibroid model is constructed by feeding a feed containing estradiol valerate for 6 weeks; or, the experimental rat uterine fibroid model is constructed by feeding a feed containing progesterone for 6 weeks; or, the experimental rat uterine fibroid model is constructed by feeding a feed containing estradiol valerate for 5 weeks and a feed containing estradiol valerate and progesterone for 1 week; wherein the feed contains or does not contain cyclodextrin.

[0021] Furthermore, when the pigs were fed with a combination of estrogen and progesterone and feed containing cyclodextrin, the modeling success rate was 80-90%; when the pigs were fed with a combination of estrogen and progesterone but feed without cyclodextrin, the modeling success rate was 50-70%; when the pigs were fed with estrogen and progesterone alone but feed without cyclodextrin, the modeling success rate was 15-50%.

[0022] The fifth aspect of the present invention is to provide an experimental rat uterine fibroids model prepared by any construction method described in the fourth aspect for use in screening drugs for treating uterine fibroids.

[0023] Compared with the prior art, the present invention adopts the above technical solution to have the following beneficial effects:

[0024] The feed components adopted by the present invention are as follows: casein is a phosphorus-calcium binding protein, which is a high-quality protein source, is easy to digest and absorb, can supplement the calcium required in the body, and promote the bone growth, muscle development and weight gain of animals; sucrose can be hydrolyzed to form glucose and fructose, thereby participating in metabolism, being absorbed, transformed and energy-supplying by the body; cellulose can promote gastrointestinal motility, keep the intestines unobstructed and maintain a normal food intake; maltodextrin has a thickening effect and can plasticize the feed, maintain the hardness and shape of the feed, and contains a certain amount of vitamins and trace elements to promote the normal material metabolism and metabolism of the body; L-cystine is used as a feed nutrition enhancer, which is beneficial to the development of animals, increases body weight and liver and kidney functions; the mineral mixture provides the mineral elements required for the normal growth and development of animals; the vitamin mixture provides the vitamins required for the normal growth and development of animals; choline bitartrate can be used as a dietary supplement; choline is a precursor for synthesizing acetylcholine and is a substance necessary for the synthesis of various phospholipids; soybean oil is rich in unsaturated fatty acids, which can enhance lipid metabolism, prevent cardiovascular and cerebrovascular diseases, and have anti-oxidation and anti-aging effects, and can improve the negative effects of rats caused by eating too much high-fat and high-calorie feed. The above-mentioned feed components of the present invention ensure that rats are supplied with sufficient protein, amino acids, cellulose, minerals, vitamins and choline, which is of great significance to the normal growth and development of experimental rats. The addition of soybean oil improves the palatability of the feed. The above-mentioned feed components well meet the daily nutritional needs of experimental rats and will not affect the modeling of uterine fibroids.

[0025] The feed of the present invention constructs a uterine fibroid model in a gentler way by combining the use of estrogen and progesterone, wherein the main component of estradiol valerate is estradiol, which is secreted by mature ovarian follicles, can promote the proliferation and hypertrophy of uterine muscle cells, increase the sensitivity of uterine smooth muscle to oxytocin, and can also promote endometrial growth, so that endometrial glandular stroma hyperplasia and repair; progesterone is a natural progesterone, which is mainly used for the treatment of miscarriage and can also be combined with estrogen to treat menopausal syndrome. The present invention promotes the formation of uterine fibroids by adding a certain amount of estradiol valerate and progesterone to the feed, causing abnormal increase in estrogen and progesterone levels in female rats.

[0026] In order to further improve the absorption of estradiol valerate and progesterone, the present invention also adds cyclodextrin to the feed to increase the stability of estrogen and progesterone drugs, so as to improve the bioavailability of estrogen and progesterone, and can be used to mask the odor of estrogen and progesterone to ensure the normal daily feed intake of experimental rats, thereby improving the success rate of modeling.

[0027] After female rats are fed with the feed of the present invention for a period of time, a rat uterine fibroid model can be successfully constructed. The uterine fibroid model is constructed by adding estrogen and progesterone: estradiol valerate and progesterone to the feed. This method is more in line with the growth and development process of human uterine fibroids and the physiological and pathological mechanism of the occurrence and development of the disease. It can simulate the occurrence process of human uterine fibroids, the modeling process is gentler, the model is more stable, and it is more conducive to the study of diseases and drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are only used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0029] Figure 1 It is a diagram showing the mechanism of action of the experimental rat uterine fibroid model feed of the present invention. DETAILED DESCRIPTION

[0030] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the scope of protection of the present invention. The experimental methods without specific conditions in the following embodiments are usually measured according to national standards. The experimental materials without the source indicated in the following embodiments are all commercially available raw materials. The equipment used in each step in the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to the general international standards, conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise specified, all parts are weight parts and all percentages are mass percentages. Unless otherwise defined or specified, all professional and scientific terms used in the present invention have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equal to the recorded content can be applied to the method of the present invention.

[0031] Reference to "embodiments" in the present invention means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present invention. The appearance of the phrase in various places in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those of ordinary skill in the art that the embodiments described in the present invention may be combined with other embodiments without conflict.

[0032] Unless otherwise defined, the technical terms or scientific terms involved in the present invention shall have the usual meaning understood by persons with ordinary skills in the technical field to which the present invention belongs. The words "one", "a", "a", "the" and the like involved in the present invention do not indicate a quantitative limitation and may indicate the singular or the plural. The terms "include", "comprise", "have" and any of their variations involved in the present invention are intended to cover non-exclusive inclusions; for example, a process, method, system, product or device that includes a series of steps or units (units) is not limited to the listed steps or units, but may also include steps or units that are not listed, or may also include other steps or units that are inherent to these processes, methods, products or devices. The terms "first", "second", "third" and the like involved in the present invention are merely to distinguish similar objects and do not represent a specific ordering of objects.

[0033] In the description of the present invention, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0034] In certain embodiments, the feed of the present invention comprises the following raw materials in parts by weight: 18 to 22 parts of casein, 0.25 to 0.4 parts of L-cystine, 35 to 40 parts of corn starch, 8 to 12 parts of sucrose, 10 to 14 parts of maltodextrin, 3 to 5 parts of cellulose, 5 to 10 parts of soybean oil, 1 to 2 parts of mineral mixture, 1 to 2 parts of vitamin mixture, and 0.1 to 0.3 parts of choline bitartrate; wherein the feed also comprises 0.003 to 0.006 parts of estradiol valerate, and / or 0.05 to 0.2 parts of progesterone. The above-mentioned feed also includes cyclodextrin, and the cyclodextrin is selected from at least one of β-cyclodextrin, hydroxyethyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, and ethylenediamine-β-cyclodextrin; wherein the cyclodextrin is prepared into an inclusion compound with estradiol valerate and / or progesterone, or the cyclodextrin is directly added to the feed, and the total mass ratio of the cyclodextrin to estradiol valerate and / or progesterone is 1:2-4. The above-mentioned raw materials are mixed at 20-30°C, granulated, and dried. The feeds prepared within the above-mentioned individual component ratio range can well meet the daily nutritional needs of experimental rats, and can successfully construct animal models of uterine fibroids.

[0035] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The present invention is further described below in conjunction with the drawings and specific embodiments, but is not intended to be a limitation of the present invention.

[0036] Example 1 - Preparation of feed

[0037] This embodiment is some preferred feed composition and preparation thereof, and the feed specifically includes:

[0038] (1) Feed composition 1: Casein (mesh number 150): 190g, L-cystine: 3g, sucrose (mesh number 60 mesh): 110g, corn starch: 380g, cellulose: 50g, maltodextrin (mesh number 150 mesh): 130g, mineral mixture: 20g, vitamin mixture: 20g, soybean oil: 70g, water: 25g, choline bitartrate: 2g, estradiol valerate: 0.05g.

[0039] (2) Feed composition 2: Casein (mesh number 150): 190g, L-cystine: 3g, sucrose (mesh number 60 mesh): 110g, corn starch: 380g, cellulose: 50g, maltodextrin (mesh number 150 mesh): 130g, mineral mixture: 20g, vitamin mixture: 20g, soybean oil: 70g, water: 25g, choline bitartrate: 2g, progesterone: 1.5g.

[0040] (3) Feed composition 3: Casein (mesh number 150): 190g, L-cystine: 3g, sucrose (mesh number 60 mesh): 110g, corn starch: 380g, cellulose: 50g, maltodextrin (mesh number 150 mesh): 130g, mineral mixture: 20g, vitamin mixture: 20g, soybean oil: 70g, water: 25g, choline bitartrate: 2g, estradiol valerate: 0.05g, progesterone: 1.5g.

[0041] (4) Feed composition 4: Casein (mesh number 150): 190g, L-cystine: 3g, sucrose (mesh number 60 mesh): 110g, corn starch: 380g, cellulose: 50g, maltodextrin (mesh number 150 mesh): 130g, mineral mixture: 20g, vitamin mixture: 20g, soybean oil: 70g, water: 25g, choline bitartrate: 2g, estradiol valerate: 0.05g; hydroxyethyl-β-cyclodextrin 0.075g; ethylenediamine-β-cyclodextrin 0.075g.

[0042] (5) Feed composition 5:Casein (mesh number 150): 190g, L-cystine: 3g, sucrose (mesh number 60 mesh): 110g, corn starch: 380g, cellulose: 50g, maltodextrin (mesh number 150 mesh): 130g, mineral mixture: 20g, vitamin mixture: 20g, soybean oil: 70g, water: 25g, choline bitartrate: 2g, estradiol valerate: 0.05g; progesterone: 1.5g; hydroxyethyl-β-cyclodextrin 2.32g; ethylenediamine-β-cyclodextrin 2.32g.

[0043] (6) Comparative feed composition 1: Casein (mesh number is 150 mesh): 190g, L-cystine: 3g, sucrose (mesh number is 60 mesh): 110g, corn starch: 380g, cellulose: 50g, maltodextrin (mesh number is 150 mesh): 130g, mineral mixture: 20g, vitamin mixture: 20g, soybean oil: 70g, choline bitartrate: 2g, water: 25g, estradiol valerate: 0.005g;

[0044] (7) Comparative feed composition 2: Casein (mesh number is 150 mesh): 190g, L-cystine: 3g, sucrose (mesh number is 60 mesh): 110g, corn starch: 380g, cellulose: 50g, maltodextrin (mesh number is 150 mesh): 130g, mineral mixture: 20g, vitamin mixture: 20g, soybean oil: 70g, choline bitartrate: 2g, water: 25g, estradiol valerate: 0.005g, progesterone: 0.15g;

[0045] (8) Blank feed composition: Casein (mesh number 150): 190g, L-cystine: 3g, sucrose (mesh number 60 mesh): 110g, corn starch: 380g, cellulose: 50g, maltodextrin (mesh number 150 mesh): 130g, mineral mixture: 20g, vitamin mixture: 20g, soybean oil: 70g, water: 25g, choline bitartrate: 2g.

[0046] In the above feed, the mineral mixture is composed of the following raw materials in parts by weight: 22 parts of sucrose, 35 parts of calcium carbonate, 20 parts of potassium dihydrogen phosphate, 8 parts of sodium chloride, 6 parts of potassium citrate monohydrate, 4 parts of potassium sulfate, 2 parts of magnesium oxide, 0.6 parts of ferric citrate, 0.2 parts of zinc carbonate, 0.2 parts of sodium metasilicate, 0.05 parts of hydrated manganese carbonate, 0.04 parts of copper carbonate, 0.01 parts of potassium chromium sulfate, 0.008 parts of boric acid, 0.01 parts of sodium fluoride, 0.004 parts of nickel carbonate, 0.003 parts of anhydrous lithium chloride, 0.002 parts of sodium selenate, 0.002 parts of potassium iodate, 0.002 parts of ammonium molybdate tetrahydrate, and 0.002 parts of ammonium metavanadate. The vitamin mixture is composed of the following raw materials in parts by weight: 96 parts of sucrose, 2 parts of niacin, vitamin B 120.4 parts, biotin 0.3 parts, D-pantothenic acid hemi-calcium salt 0.2 parts, vitamin D3 0.1 parts, retinol acetate 0.08 parts, vitamin B6 0.06 parts, riboflavin 0.06 parts, thiamine hydrochloride 0.06 parts, folic acid 0.02 parts, vitamin K 0.02 parts.

[0047] The preparation methods of the above feeds are the same, specifically: premix the above raw materials with a small mixing mill, mix them as a whole with a stainless steel trough mixer, granulate them with a single screw extruder, and dry them in a hot air circulation oven; wherein the mixing temperature is 25°C and the mixing time is 1.5h; the drying temperature is 20°C, the humidity during the drying process is 20%, and the drying time is 24h. The first feed, the second feed, the third feed, the fourth feed, the fifth feed, the first comparison feed, the second comparison feed, and the blank feed are obtained by the above preparation method.

[0048] Example 2 - Preparation of feed 2

[0049] This embodiment is some preferred feed composition and preparation thereof, and the feed specifically includes:

[0050] (1) Feed composition 6: Casein (mesh number 150): 190g, L-cystine: 3g, sucrose (mesh number 60 mesh): 110g, corn starch: 380g, cellulose: 50g, maltodextrin (mesh number 150 mesh): 130g, mineral mixture: 20g, vitamin mixture: 20g, soybean oil: 70g, water: 25g, choline bitartrate: 2g, cyclodextrin inclusion compound containing 0.05g of estradiol valerate.

[0051] (2) Feed composition 7: Casein (mesh number 150): 190g, L-cystine: 3g, sucrose (mesh number 60 mesh): 110g, corn starch: 380g, cellulose: 50g, maltodextrin (mesh number 150 mesh): 130g, mineral mixture: 20g, vitamin mixture: 20g, soybean oil: 70g, water: 25g, choline bitartrate: 2g, cyclodextrin inclusion compound containing 0.05g estradiol valerate and 1.5g progesterone.

[0052] In the above feed, the composition of the mineral mixture and the vitamin mixture is the same as that in Example 1.

[0053] The preparation method of the above-mentioned cyclodextrin inclusion compound is specifically as follows: dissolving estradiol valerate, or estradiol valerate and progesterone (the quantitative ratio of estradiol valerate to progesterone is 1:30) in an appropriate amount of anhydrous ethanol at room temperature to obtain a first solution; dissolving equal amounts of hydroxyethyl-β-cyclodextrin and ethylenediamine-β-cyclodextrin (whose total mass is 50 times the total mass of the hormone) in purified water (the volume ratio of water to ethanol is 10:1) at 40°C to obtain a second solution; slowly adding the first solution to the second solution under light-proof conditions, stirring at a constant temperature of 30-35°C for 2h, Stir at 4°C for 2 hours, and place in a refrigerator for 2 hours. Separate the product by conventional methods and freeze-dry. During the above stirring and freeze-drying process, almost all of the solvent ethanol can be removed to obtain a cyclodextrin-hormone inclusion complex (the drug inclusion rate of the two inclusion complexes is about 40%, so the amount of inclusion complex added in the feed can be obtained, that is, the amount of cyclodextrin inclusion complex containing 0.05g of estradiol valerate is about 0.125g, and the amount of cyclodextrin inclusion complex containing 0.05g of estradiol valerate and 1.5g of progesterone is about 3.875g). Premix the other raw materials with the inclusion complex using a small stirring pulverizer, mix them as a whole using a stainless steel trough mixer, granulate them using a single screw extruder dough making machine, and dry them in a hot air circulation oven; wherein, the mixing temperature is 25°C and the mixing time is 1.5h; the drying temperature is 20°C, the humidity during the drying process is 20%, and the drying time is 24h. The sixth feed and the seventh feed are obtained by the above preparation method.

[0054] Example 3-Verification of stability and solubility of cyclodextrin-hormone inclusion complex

[0055] This example verifies the stability and solubility of the cyclodextrin-hormone inclusion complex prepared in Example 2, and specifically includes the following experimental steps:

[0056] (1) Stability verification;

[0057] The content of estradiol valerate in the newly prepared cyclodextrin-estradiol valerate inclusion complex and the content of estradiol valerate + progesterone in the newly prepared cyclodextrin-estradiol valerate / progesterone inclusion complex were immediately detected, and the contents were stored in plastic bottles at room temperature, non-dark conditions, and ambient humidity for 1 month and 3 months, and the loss of estrogen and progesterone was calculated. The results are shown in the following table:

[0058] Table 1 - Loss of estrogen and progesterone

[0059] Hormone loss in 1 month Hormone loss in 3 months Cyclodextrin directly mixed with estradiol valerate 0.52％ 1.15％ Cyclodextrin mixed directly with estradiol valerate / progesterone 0.49％ 1.21％ Cyclodextrin-estradiol valerate inclusion complex 0.15％ 0.48％ Cyclodextrin-estradiol valerate / progesterone inclusion complex 0.13％ 0.45％

[0060] From the above experimental results, it can be seen that the stability of cyclodextrin-hormone inclusion complex is better than that of cyclodextrin and hormone directly mixed, which can improve the storage environment of estrogen and progesterone. Based on the properties of estrogen and progesterone, they should be stored away from light. If there are no conditions for storage away from light, they can be stored in a non-dark environment for a short period of time.

[0061] (2) Solubility verification;

[0062] The solubility of cyclodextrin-estradiol valerate inclusion complex and cyclodextrin-estradiol valerate / progesterone inclusion complex in room temperature water was compared with the solubility of estradiol valerate and estradiol valerate / progesterone in water, respectively. It was found that the solubility of cyclodextrin-estradiol valerate inclusion complex was about 13.5 times that of estradiol valerate, and the solubility of cyclodextrin-estradiol valerate / progesterone inclusion complex was about 14.2 times that of estradiol valerate / progesterone. From the experimental results, it can be seen that the cyclodextrin-hormone inclusion complex significantly improves the solubility of estradiol valerate / progesterone, thereby further improving the utilization rate of estradiol valerate / progesterone.

[0063] Example 4-Construction of uterine fibroid model

[0064] This embodiment is a construction of some preferred experimental rat uterine fibroid models, which uses the feed prepared in Example 1 and Example 2, and specifically includes the following steps:

[0065] (1) Experimental animals: 84 8-week-old female SD rats weighing approximately 210.00 g were purchased and selected, with 12 rats in each group.

[0066] (2) Experimental group settings:

[0067]

[0068] (3) Experimental rat feeding process: Each group of rats were allowed to eat and drink freely every day, the room temperature was maintained at 20℃~22℃, the humidity was 60%±2%, and the light duration was 12h±1h; the feeding time was 6 weeks. The average food intake of the rats was counted, and the body weight and uterine weight of each rat were measured after 6 weeks. The morphology of the uterus and fibroids was observed by dissection, and the uterine coefficient (i.e. uterine weight / rat body weight×100%), serum estrogen and progesterone levels, uterine smooth muscle fiber thickness, and uterine fibroid formation rate of each group of rats were calculated; the specific test data are shown in Tables 3 to 5.

[0069] The naked eye can see that the uterus is enlarged, dark in color, and has hyperplasia of the uterine layer. Some animals have 1 to 3 myoma-like nodules in the uterine horns. The uterine coefficient and estrogen and progesterone are greater than those of the control group, and the uterine myometrium is significantly thickened, indicating that the model was successfully constructed.

[0070] Table 3 - Average food intake, body weight, uterine weight and uterine fibroid formation rate of rats

[0071]

[0072]

[0073] Table 4 - Data on serum estrogen and progesterone levels in rats in the test examples of the present invention

[0074] - Estradiol (pmol / L) Progesterone (ng / ml) Experimental Group 1 28.62±5.41 1.65±0.26 Experimental Group 2 26.77±6.23 1.13±0.32 Experimental Group 3 22.05±3.74 1.94±0.31 Experimental Group 4 30.05±4.98 1.68±0.30 Experimental Group 5 31.25±5.64 1.74±0.28 Comparison group 23.88±3.52 0.93±0.25 Control group 21.33±3.89 0.75±0.35

[0075] Table 5 - Influence data of rat uterine smooth muscle fiber thickness in the test examples of the present invention

[0076] - Average thickness of myometrium Maximum thickness of myometrium Experimental Group 1 225.26±45.26 396.44±56.08 Experimental Group 2 205.27±33.32 352.96±40.56 Experimental Group 3 224.57±43.74 345.28±55.46 Experimental Group 4 234.54±36.28 399.87±44.37 Experimental Group 5 242.38±37.24 405.24±45.37 Comparison group 171.87±20.28 334.78±29.72 Control group 168.25±18.35 310.05±30.04

[0077] From the above experimental results, it can be seen that there is no significant difference in the average feed intake of rats in each group, and no rat death occurred in each group, indicating that the above feeds are all safe; at the end of the 6th week, the wet weight of the uterus and the uterine coefficient of the experimental groups 1, 2, 4 and 5 were significantly higher than those of the control group and the control group, among which the experimental groups 4-5 were more significant; the serum estradiol of the experimental groups 1, 2, 4 and 5 and the progesterone levels of the experimental groups 1, 3, 4 and 5 were significantly higher than those of the control group and the control group, among which the experimental groups 4-5 were more significant, which shows that the modeling effect is better when the hormone is prepared into an inclusion compound with cyclodextrin in advance; compared with the experimental group 2 (only estrogen is used) and the experimental group 3 (only progesterone is used), after the experimental group 1 added a certain dose of progesterone in the last week, the uterine coefficient, estrogen and progesterone levels and uterine myometrium thickness were significantly improved, indicating that the experimental group using estrogen and progesterone in combination has a better modeling effect. Compared with a single estrogen or progesterone; after adding a certain amount of cyclodextrin to test group 4 and test group 5, and after adding a certain dose of progesterone in the last week, the uterine coefficient and estrogen and progesterone levels were more significantly improved, indicating that the use of cyclodextrin can better promote the absorption of estrogen and progesterone; the uterine smooth muscle layer of rats in the uterine fibroid model was mainly manifested in obvious thickening of the uterine smooth muscle layer, disordered and irregular arrangement of smooth muscle fibers, and hyperplasia and hypertrophy of smooth muscle fibers. Compared with the other groups, the average thickness and maximum thickness of the uterine muscle layer of rats in test groups 4 and test groups 5 were significantly increased, followed by test group 1, test group 2, and test group 3; combined with the above experimental results, it is shown that the feeds in test groups 1 to 5 of the present invention are all easy to promote the formation of uterine fibroids in rats, among which test groups 5 and test group 4 have better modeling effects, and their uterine fibroid formation rates are all above 80%, and the uterine fibroid formation rates of test groups 1 to 2 are all above 50%.

[0078] As can be seen from the above examples, the feed of the present invention, under the premise of ensuring sufficient nutritional supply of rats, constructs a uterine fibroid model by adding estrogen and progesterone, and adopts cyclodextrin to promote the absorption of estrogen and progesterone, comprehensively simulates the pathogenesis of uterine fibroids in humans, and estrogen is the main precipitating factor and growth factor of the occurrence and development of uterine fibroids. Studies have found that the local estrogen concentration, estrogen receptor concentration and estrogen receptor of uterine fibroid tissue are also high, and the detection of estrogen and progesterone receptor content in uterine muscle tissue, fibroids and endometrial tissue shows that the estrogen and progesterone receptor content in fibroid tissue is higher than that in uterine muscle tissue, but lower than that in endometrial tissue. The estrogen and progesterone receptor content significantly increases in the proliferation phase compared with the secretion phase. Hormones promote cell proliferation of uterine fibroids through certain mechanisms on the one hand, and the abnormal expression of various growth factors and their receptor-related genes and proteins in the local fibroids on the other hand are involved in the occurrence and development of uterine fibroids, and estrogen, progesterone, especially progesterone, inhibit apoptosis by regulating apoptosis gene bcl-2 or bcl-2 / bax, thereby indirectly stimulating the increase in the number of fibroid cells. The sex hormone dependence of uterine fibroids reveals the biological characteristics of uterine fibroids to a large extent. The mutual synergistic effect of estrogen and progesterone promotes the occurrence of fibroids and makes it possible for normal uterine smooth muscle cells to differentiate into smooth muscle cells and decidual cells, eventually producing multiple nodules. Experimental rats ingest feed containing estrogen and progesterone, simulate various pathogenic factors in the pathogenesis, actively study the mechanism of occurrence of uterine fibroids, and explore the role of various factors in the pathogenesis of fibroids. It is undoubtedly very important to find a reliable and effective treatment method. After feeding rats with the feed containing estrogen and progesterone of the present invention for 6 weeks, a rat uterine fibroid model can be obtained. The modeling process is more gentle, the model is more stable, and it is more conducive to the study of diseases and drugs. The present invention can scientifically feed experimental rats in an automated and customized feeding method according to the needs of model indicators, avoid artificial manipulation of feeding time and quantity non-compliance, and thus be conducive to the integrity and consistency of the model, and improve the success rate and efficiency of modeling.

[0079] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention are also within the scope of the present invention. Therefore, the equalization changes and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. An experimental rat uterine fibroid model feed, characterized in that: The feed comprises the following raw materials in parts by weight: 18 to 22 parts of casein, 0.25 to 0.4 parts of L-cystine, 35 to 40 parts of corn starch, 8 to 12 parts of sucrose, 10 to 14 parts of maltodextrin, 3 to 5 parts of cellulose, 5 to 10 parts of soybean oil, 1 to 2 parts of mineral mixture, 1 to 2 parts of vitamin mixture, and 0.1 to 0.3 parts of choline bitartrate; wherein the feed also comprises 0.003 to 0.006 parts of estradiol valerate, and / or 0.05 to 0.2 parts of progesterone.

2. The experimental rat uterine fibroid model feed according to claim 1, characterized in that: The feed comprises the following raw materials in parts by weight: 19 parts of casein, 0.3 parts of L-cystine, 38 parts of corn starch, 11 parts of sucrose, 13 parts of maltodextrin, 5 parts of cellulose, 7 parts of soybean oil, 2 parts of mineral mixture, 2 parts of vitamin mixture, and 0.2 parts of choline bitartrate; wherein the feed also comprises 0.005 parts of estradiol valerate and / or 0.15 parts of progesterone.

3. The experimental rat uterine fibroid model feed according to claim 1, characterized in that: The feed further comprises cyclodextrin, and the cyclodextrin is selected from at least one of β-cyclodextrin, hydroxyethyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin and ethylenediamine-β-cyclodextrin.

4. The experimental rat uterine fibroid model feed according to claim 3, characterized in that: The cyclodextrin is hydroxyethyl-β-cyclodextrin and ethylenediamine-β-cyclodextrin; wherein the cyclodextrin is prepared into an inclusion compound with estradiol valerate and / or progesterone; or, the cyclodextrin is directly added to the feed, and the total mass ratio of the cyclodextrin to estradiol valerate and / or progesterone is 1:2-4.

5. The experimental rat uterine fibroid model feed according to claim 1, characterized in that: The mineral mixture is composed of the following raw materials in parts by weight: 20-25 parts of sucrose, 33-35 parts of calcium carbonate, 18-21 parts of potassium dihydrogen phosphate, 5-9 parts of sodium chloride, 5-8 parts of potassium citrate monohydrate, 3-6 parts of potassium sulfate, 2-4 parts of magnesium oxide, 0.6-0.8 parts of ferric citrate, 0.1-0.3 parts of zinc carbonate, 0.1-0.3 parts of sodium metasilicate, 0.04-0.08 parts of hydrated manganese carbonate, 0.02-0.06 parts of copper carbonate, 0.03-0.07 parts of sodium hydrated manganese carbonate, 0.01-0.06 parts of sodium hydrated manganese carbonate, 0.02-0.06 parts of sodium hydrated manganese carbonate, 0.01-0.06 parts of sodium hydrated manganese carbonate, 0.01-0.06 parts of sodium hydrated manganese carbonate, 0.01-0.06 parts of sodium hydrated manganese carbonate, 0.01-0.06 parts of sodium hydrated manganese carbonate, 0.01-0.06 parts of sodium hydrated manganese carbonate, 0.01-0.06 parts of potassium ... 0.04 parts, potassium chromium sulfate 0.01-0.03 parts, boric acid 0.005-0.01 parts, sodium fluoride 0.005-0.08 parts, nickel carbonate 0.002-0.004 parts, anhydrous lithium chloride 0.001-0.003 parts, sodium selenate 0.001-0.003 parts, potassium iodate 0.001-0.003 parts, ammonium molybdate tetrahydrate 0.001-0.003 parts, ammonium metavanadate 0.001-0.003 parts.

6. The experimental rat uterine fibroid model feed according to claim 1, characterized in that: The vitamin mixture is composed of the following raw materials in parts by weight: 96-98 parts of sucrose, 1-3 parts of niacin, vitamin B 12 0.2-0.4 parts, biotin 0.2-0.4 parts, D-pantothenic acid hemi-calcium salt 0.1-0.3 parts, vitamin D3 0.1-0.2 parts, retinol acetate 0.06-0.09 parts, vitamin B6 0.05-0.09 parts, riboflavin 0.05-0.08 parts, thiamine hydrochloride 0.05-0.08 parts, folic acid 0.01-0.03 parts, vitamin K 0.01-0.02 parts.

7. The experimental rat uterine fibroid model feed according to any one of claims 1 to 6, characterized in that: The feed preparation method comprises the steps of: mixing the raw materials, granulating and drying; the mixing temperature is 20°C to 30°C; When the feed raw material contains cyclodextrin, it can be directly mixed with other raw materials; or, cyclodextrin and estradiol valerate and / or progesterone are first prepared into an inclusion compound, and then the inclusion compound is mixed with other raw materials.

8. A use of the experimental rat uterine fibroid model feed as claimed in any one of claims 1 to 7, characterized in that: The feed is used to construct an experimental rat uterine fibroid model.

9. A method for constructing an experimental rat uterine fibroid model, characterized in that: The construction method comprises the steps of: using the experimental rat uterine fibroids model feed as described in any one of claims 1 to 7, allowing the experimental rats to freely eat and drink water every day, and constructing the experimental rat uterine fibroids model after 5 to 7 weeks; wherein the experimental rats are SD female rats.

10. The construction method according to claim 9, characterized in that: Feeding the rats with a feed containing estradiol valerate for 6 weeks to construct the experimental rat uterine fibroids model; or, feeding the rats with a feed containing progesterone for 6 weeks to construct the experimental rat uterine fibroids model; or, feeding the rats with a feed containing estradiol valerate for 5 weeks and a feed containing estradiol valerate and progesterone for 1 week to construct the experimental rat uterine fibroids model; Wherein, the feed contains or does not contain cyclodextrin.

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