疾病动物模型,围绝经期综合征动物模型 z-bio 2021-03-11 407

　　Perimenopausal period (perimenopausal period) refers to the period from the endocrine, biological and clinical features related to menopause appearing close to menopause to within one year of menopause, that is, the transition period of menopause to one year after menopause. Perimenopausal syndrome is a syndrome in which ovarian function declines, leading to decreased estrogen secretion, causing endocrine disorders, low immunity, and autonomic dysfunction. Common clinical manifestations such as hot flashes and night sweats, menstrual disorders, changes in mental state, cardiovascular and genitourinary system diseases, and osteoporosis. Commonly used animal models of perimenopausal syndrome include ovarian removal model, natural aging model, X-ray damaged ovarian model and so on.

　　1. Ovarian removal animal model

　　[Modeling mechanism] The ovariecto-mized animal model is also called the castration model. 90% of female estrogen is secreted by the ovaries. Removal of the ovaries and cutting off its source can artificially cause an environment where estrogen is suddenly reduced. Simulate perimenopausal syndrome.

　　[Modeling method] Generally, female Sprague Dawley rats or Wistar rats (mouse are also available) of 3 to 6 months old are commonly used. After intraperitoneal anesthesia with sodium pentobarbital or chloral hydrate, the last ribs and mid-axillary line are taken Intersection with about 2cm from the outside of the spine. Cut the long hairs, disinfect the surgical field, cut the skin and back muscles about 1.5 to 2.0 cm, the ovaries are located below the kidneys, ligate the fallopian tubes (including fat) under the ovaries with silk thread, remove the ovaries, and remove the other ovaries in the same way. After verifying the integrity of the ovary and no bleeding from the incision, the uterus is returned to its original place, the abdomen is closed, and the skin is sutured. Apply 0.5% iodophor and sprinkle an appropriate amount of penicillin sodium powder on the sutured wound, and continue to inject 1.5ml penicillin normal saline (40,000 U/ml) into the intraperitoneal cavity of each rat on the 1st to 3rd day after the operation. One week after the ovariectomy, vaginal exfoliated cell smears were performed one by one every day, once a day, for 5 consecutive days, and the model was successful with no estrus reaction.

　　【Model Features】

　　1. The rats in the model group showed no changes in estrus in vaginal smears for two consecutive weeks, but only changes in estrus interval. The smears showed a few nuclear cells and a large number of white blood cells.

　　2. After 1 month of castration, compared with the blank control group, the serum hormone level of rats in the model group was lower than that of the blank control group. The ratio to body weight) also decreased, and the difference was statistically significant (P<0.05).

　　3. After 6 weeks of castration, the liver cells were slightly swollen, with blurred boundaries, a large number of lipid droplets were seen in the cells, and individual cells were filled with lipid droplets; the liver sinusoids were chaotic, slender, and mildly congested. The content of cholesterol and triglyceride in liver tissue homogenate increased significantly, and lipid accumulation changes in the liver of rats, which can be used as a model of perimenopausal lipid metabolism disorder.

　　4. After 3 months of castration, serum calcitonin concentration decreased successively, bone resorption increased, bone formation rate slowed down, cortical bone thinned, bone marrow cavity enlarged, bone trabecula number decreased, missing or broken, and cells in marrow cavity The number is reduced, showing the pathological characteristics of perimenopausal osteoporosis, which is an ideal model for perimenopausal osteoporosis.

　　5. Appeared perimenopausal syndrome manifestations, alternative application of exogenous estrogen can alleviate these symptoms.

　　[Model Evaluation and Application] Model mice have no natural aging process. All other systems are young, and there is still a certain gap between the aging of the body during the perimenopausal period, and the complete removal of the ovary is different from the failure of the ovarian function of the perimenopausal mice. However, the vaginal exfoliated cell smear and serum sex hormone levels of this model have perimenopausal changes, so the establishment of this model is successful. In addition, this model has a short modeling time and a high model success rate. The animals used are cheaper and easier to obtain than old rats, cost-saving, and convenient to use. It is widely used by scientific researchers and is often used for the etiology, pathogenesis and mechanism of perimenopausal syndrome. Treatment research, screening and development of effective therapeutic drugs.

　　2. Natural aging animal model

　　[Modeling mechanism] Female rats around 12 months of age (11 to 13 months of age) began to have estrous cycle disorders. Serology and corresponding biochemical indicators of the ovary showed that the reproductive function of the rat's ovary was progressive at this stage. The change of decline is very similar to the perimenopausal performance of women, and it can be used as an animal model for perimenopausal ovarian aging research.

　　[Modeling method] Several female Sprague-Dawley rats or Wistar rats around the age of 12 months were observed for 15 days of continuous vaginal cytology. The model group was selected as the model group with prolonged estrus cycle and disorder, including prolonged estrus cycle. After that, estrus continued and repeated false pregnancies. After the rats are sexually mature, the estrus cycle is repeated every 4 to 5 days and nights. During this process, the follicles of the ovary, the uterus and the vaginal epithelium all undergo periodic changes. Large and lightly stained nucleus can be seen in the estrus. Keratinocytes, vaginal epithelium gradually thickens during the interestrous period, and there are many white blood cells between the cells. Vaginal exfoliated cell smear method: fix the rat in the dorsal position of the left hand, hold the straw in the right hand, and inhale 0.1～0.2ml of normal saline in it. Gently insert 0.5 to 1 cm into the vagina of the rat, suction 2 to 3 times, and drop a drop of the suction liquid on the glass slide. Then drop a drop of 95% ethanol solution, dry and dye with 1% methylene blue for 10 minutes, and rinse the excess dye solution with water. After drying, place it under a microscope for cytological examination. Vaginal smears were examined one by one, once a day, for 15 consecutive days.

　　【Model Features】

　　1. Morphological observation of ovarian tissue

　　(1) Observation results under light microscope: The ovaries of the young control rats are mature, with distinct layers of cortex and medulla, and there are more follicles and corpus corpus luteum at all levels. The follicular fluid is abundant, and a small amount of atresia follicles and interstitial glands can be seen. The ovaries of the rats in the model group were atrophied, developed poorly, and increased in medulla. Both primordial follicles and growing follicles were significantly reduced. The granular cells were arranged irregularly. The disintegration of granular cells was seen. No typical mature follicles were seen, and the number of atretic follicles increased; The number of corpus luteum is also small, while the number of degenerated corpus luteum is increased, the interstitial glands are few and loose, the proliferation or disintegration of fibrous tissue can be seen, the number of blood vessels is reduced, and the lumen of small arteries is narrow and blocked.

　　(2) Electron microscope observation results: the young control ovarian primordial follicles are abundant, the oocytes are larger, the nucleus is uniform, the nucleolus is obvious, the surrounding monolayer granular cells have irregular nuclei, and there are thin layers of heterochromosomes in the nuclear membrane; growing follicles It can be seen that the zona pellucida is widened, granular cells are distributed in multiple layers, the nucleus is lightly stained, the nucleolus is obvious, the chromatin is uniform, the organelles are intact, the outline is clear, the cytoplasm is rich in organelles, and secretory granules can be seen; there are sinusoid spaces between the cells. In the model group, there were more vacuoles in ovarian granulosa cells and decreased secretory granules. Dense matter was seen in them. The endoplasmic reticulum was not abundant on the slippery surface, and the sinusoid spaces between cells were enlarged. Fibres and matrix dissolve in the interstitium.

　　2. Compared with the young control group, the serum follicle-stimulating hormone FSH, luteinizing hormone LH, and gonadotropin-releasing hormone GnRH levels increased in the model group, while the serum estrogen E2 level decreased; compared with the young control group, the ovarian index and uterine index of the model group Significantly reduced; the above are all significantly different (P<0.05).

　　3. In perimenopausal women, due to the decline of ovarian function, the level of estrogen decreases, and the total cholesterol, triglycerides, and low-density lipoprotein cholesterol in the blood increase. Abnormal blood lipid metabolism is the main cause of atheroscbrasis and coronary heart disease. The levels of serum total cholesterol, serum triglycerides, serum high-density lipoprotein cholesterol, serum low-density lipoprotein cholesterol, etc. of this model animal are significantly different from those of the young group, which is very consistent with the characteristics of perimenopausal period. This model is successful.

　　[Model evaluation and application] The natural aging model is similar to the biological characteristics of human perimenopausal period. It has a natural aging process. It is the failure of ovarian function rather than ovarian removal (castration), so it is closer to the fact itself, which is more ideal. model. The disadvantage is that the cycle is long and the cost is high.

　　3. X-ray damaged ovarian animal model

　　【Modeling mechanism】The animal model of ovary hurt by X-ray is the main clinical manifestation of perimenopausal by using X-ray irradiation to destroy the symptoms of the ovary.

　　[Modeling method] Wistar rat, 150～180g, fix the rat on a flat plate, expose the "ovarian area" of the lower abdomen, and place it under the 2100C irradiation field of the electron linear accelerator, the field width is 4cm, the dose rate is 4Gy/min, High-energy X-rays (6Mev) were irradiated for 2.5 minutes, and the total dose was 9Gy.

　　【Model Features】

　　1. Three to five days after X-ray irradiation, the rats showed obvious symptoms of radiation enteritis, and a few animals died.

　　2. On the 10th day after irradiation, the body weight was significantly reduced. On 21 days after irradiation, the weight of the rat’s uterus and ovaries decreased significantly, and the weight of the pituitary gland increased significantly, suggesting that the pituitary gland proliferates after the ovaries are injured.

　　3. Autonomous activities increased significantly, and the levels of triglycerides and cholesterol increased significantly.

　　[Model Evaluation and Application] This method has reduced its application range due to the limitations of laboratory equipment and conditions, as well as the poor control of the irradiation time and intensity.