流产动物模型 z-bio 2021-12-23 463

　　Abortion is a common disease in gynecology, and its etiology is more complicated. Commonly used animal models of abortion include drug-induced abortion, bromocriptine-induced abortion, kidney-deficiency abortion and repeated spontaneous abortion models.

　　(1) Animal model of drug-induced abortion

　　[Modeling mechanism] Medical abortion refers to the method of using drugs to terminate the pregnancy in the early pregnancy. At present, the effective drug is mifepristone combined with misoprostol, and the complete abortion rate can reach 95% to 98%.

　　Mifepristone has a very strong affinity with progesterone receptors, causing decidual degeneration, necrosis and bleeding, anti-implantation, preventing embryonic development, and terminating early pregnancy. Misoprostol can promote the contraction of the uterus, and the villous bulbs are discharged during the gradual contraction process to terminate pregnancy.

　　【Method of Modeling】

　　1. Select SD rats, female rats weighing 220-250g, male rats 250-300g. The rats were caged at a ratio of 2:1 male to female, and vaginal smears were performed the next morning to find sperm on the first day of pregnancy.

　　2. On the 7th day after conception of an animal, mifepristone (8:00am) + misoprostol (6:00pm) each time (approximately 10 hours between the two drugs), the rats were completely aborted with 16.6mg/kg ( Body weight) + 100 μg/kg (body weight); 8.3 mg/kg (body weight) + 100 μg/kg (body weight) for incomplete abortion. At the same time, a human quantitative cotton ball is placed in the vagina (the cotton ball weighs 85-90 mg, and half of it is wrapped with a plastic film to prevent blood leakage and urine reflux). Take out the cotton ball at 8:00 and 18:00 the next day, put it in a plastic bag and keep it under airtight refrigeration. At the same time, replace a new cotton ball in the vagina. Observe the bleeding from the vagina. Continue to the 14th day. The pathological changes of the uterus, the bleeding volume of each mouse vagina cotton ball collected.

　　【Model Features】

　　1. Mifepristone + misoprostol induced abortion method is easy to observe the change of uterine bleeding.

　　2. Uterine bleeding calculation method: 0.02ml blood was collected from the tail vein of the rat with a hemoglobin pipette, diluted with 4ml sodium hydroxide solution at a concentration of 1.25mol/L as the standard, and then the old vaginal bleeding cotton ball was collected by the rat itself. Wash the cotton ball several times with a small amount of concentrated sodium hydroxide solution. The spectrophotometer measures the respective absorbance (A value) at a wavelength of 546nm, and substitutes the formula to calculate the amount of uterine bleeding in the rat.

　　Uterine bleeding (ml) = tail vein blood (0.02ml) × intrauterine extract A × V2/(tail vein blood A value × V1)

　　where: V1 = 1.25mol/L sodium hydroxide amount (4ml) used to dilute tail vein blood.

　　V2 = 1.25mol/L sodium hydroxide amount (ml) used to extract uterine blood (cotton ball).

　　3. The modeling time is short, and it can be successfully copied in 1 to 2 weeks.

　　【Model evaluation and application】

　　The application of mifepristone and misoprostol for medical abortion is currently one of the main methods for terminating early pregnancy. It has the advantages of easy oral administration, fast action, light side effects, avoiding surgical pain, and reducing complications. This method is simple and reliable, and the drug efficacy evaluation index is objective and accurate. This model has certain advantages in observing the change of uterine bleeding. It can be used as a drug screening and drug efficacy evaluation model for the prevention and treatment of uterine bleeding caused by drug termination of early pregnancy. .

　　(2) Bromocriptine-induced abortion animal model

　　[Modeling mechanism] Early pregnancy is in a physiologically high prolactin (PRL) state. PRL plays an important role in the regulation of the corpus luteum function and the maintenance of pregnancy. Subcutaneous injection of bromocriptine in mice during early pregnancy can significantly reduce serum PRL, reduce the expression of LH/CG receptors and mRNA in the corpus luteum of pregnancy, decrease the function of the corpus luteum and even dissolve the corpus luteum, and decrease the secretion of progesterone and estradiol, which affects Decidualization of the endometrium, causing animal abortion.

　　[Modeling method] Establishment of bromocriptine-induced abortion model in SD rats: The rats used were kept in the laboratory for 2 weeks to adapt to the environment. Males and females were caged at 1:1 ratio, and those who saw vaginal suppository or vagina in the next morning Sperm found on smear is counted as the first day of pregnancy. Two dose groups of 0.125 mg/d and 0.25 mg/d of bromocriptine were injected subcutaneously on 6-8 days of pregnancy. Peripheral blood was collected by tail-cutting method at 4, 7, 10, and 12 days of gestation, and serum was extracted and placed at -20°C for later use. The PRL level was determined by radioimmunoassay. They were executed on the 12th day of pregnancy and the uterus was removed. Count the number of live embryos. Those without embryos are counted as miscarriage.

　　Abortion rate = number of aborted mice/number of pregnant mice×100%.

　　【Model Features】

　　1. Bromocriptine-induced Sprague-Dawley rat abortion model serum PRL and progesterone are lower than the control group, the endocrine function is insufficient and the pathophysiological characteristics of Th1-type immune response are in line with the clinical characteristics of low PRL and low immune recognition function abortion.

　　2. The abortion rate in the 0.25 mg/d dose group was 100%, and the abortion rate in the 0.125 mg/d dose group was 75%.

　　[Model evaluation and application] The bromocriptine induced abortion model in rats is for in-depth study of the pathogenesis of abortion caused by abnormal immune-endocrine function at the maternal-fetal interface and the pathological mechanism of drug-induced abortion, as well as the prevention and treatment measures, and the study of drug precautions An ideal model for curative effect and pathogenesis of spontaneous abortion.

　　(3) Kidney Deficiency Abortion Animal Model

　　【Modeling mechanism】Hydroxycarbamide can cause animals to have slow weight gain, fluffy coat, less movement of the arched back and other similar symptoms of kidney deficiency. Mifepristone can antagonize progesterone receptors and terminate early pregnancy, resist implantation, and cause miscarriage. The selection of hydroxyurea plus mifepristone can successfully prepare a rat kidney deficiency abortion model.

　　【Method of Modeling】

　　1. Sprague-Dawley rats, weighing (220±20) g, were adaptively reared for one week and the experiment was started. The male and female 1:1 cages were used for single cage mating. The sperm seen in the vaginal smear was regarded as the first day of pregnancy. take out.

　　2. From the 3rd day to the 12th day of pregnancy, they were given hydroxyurea distilled water solution. The dosage for animals is 20 mg per 100 grams of body weight per day. The ground powder is dissolved in distilled water for later use. Mifepristone was administered once on the 12th day of pregnancy. Mifepristone, 25mg per tablet. The amount of animal administered is 3.75 mg per 100 grams of body weight per day. The ground powder is dissolved in distilled water for later use. Observe the general conditions of all modeled rats, such as: weight, coat, activity, etc.

　　3. All female mice were sacrificed on the 15th day of pregnancy. Observe the changes in the number of live births, the number of abortions, the abortion rate, and the effect on serum anti-trophoblast antibodies and IL-2.

　　4. Standards for determining the number of normal embryos and aborted embryos Normal embryos: there is no congestion in the uterus, and the embryos are pale red. Aborted embryos: The embryos in the uterus were dark brown or had vaginal bleeding. The uterus was "bamboo-shaped" during dissection. There were no congested or necrotic embryos in the official cavity. Abortion rate = number of aborted embryos/(number of aborted embryos + number of normal embryos ).

　　【Model Features】

　　1. About 1 week after the administration of hydroxyurea, rats showed signs of slow weight gain, fluffy coat, less movement of the arched back, and other similar symptoms of kidney deficiency; the model control group also saw symptoms such as weight loss, chills, and cold limbs; mifes was administered on the 12th day of pregnancy After ketone, vaginal bleeding was seen on the 12th or 14th day of pregnancy.

　　2. The abortion rate of the abortion model of kidney deficiency rats caused by hydroxyurea plus mifepristone can reach 80%.

　　3. Modeling can reduce the weight of decidua in pregnant rats compared with normal pregnant rats.

　　4. The miscarriage uterus is dark and black under the naked eye, and under light microscope, hemorrhage in the amniotic sac, placental hemorrhage may be seen, or hemorrhage and necrosis of decidual cells may be seen. There are more lipid droplets in the corpus luteum of pregnancy, or with vascular congestion, nuclear pyknosis, and focal necrosis.

　　5. The progesterone and progesterone receptor mRNAs of the model pregnant rats were lower than those of normal pregnant rats during the same period, suggesting that the termination of pregnancy by mifepristone mimics the process of spontaneous abortion due to corpus luteum insufficiency.

　　[Model evaluation and application] A rat kidney deficiency abortion model prepared by the method of hydroxyurea plus mifepristone was used. About 1 week after hydroxyurea administration, the rats showed slow weight gain, fluffy coat, less movement of the arched back, and similar symptoms of kidney deficiency. After the administration of mifepristone on the 12th day of gestation, vaginal bleeding was seen on the 13th or 14th day of gestation; miscarriage was found during anatomy; this suggested that the model was successfully prepared. Hydroxyurea and mifepristone establish a kidney-deficiency abortion model that simulates the basic pathological changes of clinical patients with kidney-deficiency spontaneous abortion, and is a reproducible combined disease and syndrome model.

　　(4) Recurrent spontaneous abortion animal model

　　[Modeling mechanism] CBA/J and DBA/2 are two commonly used inbred strains of mice. The CBA/J(female)×DBA/2(male) mating combination is prone to recurrent spontaneous abortion (RSA). The mating combination of DBA/2 male mice and CBA/J female mice (named CBA/J×DBA/2 in experimental zoology), as an animal model of repeated spontaneous abortion, has received extensive attention in the world.

　　[Method of Modeling] Female CBA/J mice and male DBA/2 mice, both reaching the age of 10 weeks, have matured sexually. According to the same cage as male and female in a 1:1 manner, as a 60-day observation period, the occurrence of vaginal thrombus is defined as 1 day of pregnancy. Observe the average number of litters per litter of mice, the average time from the birth of hermaphrodite to the first litter, and the growth curve of the mice after birth.

　　【Model Features】

　　1. CBA/J and DBA/2 are two commonly used mouse inbred lines second only to BALB/c and C57BL/6. CBA/J×DBA/2 mice have the characteristics of repeated spontaneous abortions, and the embryo absorption rate is constant at 30%-45%. The miscarriage of mice due to chromosomal abnormalities only accounts for about 4%.

　　2. The abortion rate is relatively constant.

　　3. Compared with immunodeficient animals such as nude mice, it has strong resistance to infection and low breeding cost.

　　【Model Evaluation and Application】The advantages of this model are: ①The source of experimental animals is relatively sufficient and reliable. ②Abortion is characterized by abortion during peri-implantation period. Human recurrent spontaneous abortions of unknown reasons also occurred during this period, so it is more valuable for research. ③The experiment is highly repeatable.