CN111296356A - Method for establishing rat ovarian premature senility model by using cisplatin - Google Patents
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A01K67/02—Breeding vertebrates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A01K2227/105—Murine
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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Abstract
The invention discloses a method for establishing a rat ovarian progeria model by using cisplatin, which comprises the following steps: cisplatin with the dose of 2-3 mg/Kg per day according to the body weight is applied to a rat in an intraperitoneal injection mode, and the application method comprises the following steps: the administration is carried out continuously for 7 days, and 15 days after the last injection, the rat premature ovarian failure model is obtained. Or: cisplatin with the dose of 4-6 mg/Kg according to the body weight is applied to a rat in an intraperitoneal injection mode, and the application method comprises the following steps: administration on day 1, day 8; and 15 days after the last injection, obtaining a rat premature ovarian failure model. The invention establishes the animal model of the chemotherapy-induced damage premature ovarian failure by using different concentrations and different methods, and screens the optimal concentration and administration time of cisplatin by comparing the superiority and inferiority among various methods, thereby providing an experimental method which has simple operation, high success rate and reliable result for establishing the stable animal model of the premature ovarian failure for basic research.
Description
Technical Field
The invention relates to a method for establishing a rat ovarian premature senility model by using cisplatin.
Background
Premature Ovarian Failure (POF) is a disease characterized by amenorrhea, infertility, estrogen deficiency and elevated gonadotropin levels in women before age 40. The incidence rate of the traditional Chinese medicine is 1-3% in general population, 4-8% in secondary amenorrhea women and 0.01% before 20 years old. In recent years, the incidence of POF in women of childbearing age has been increasing year by year and the development of POF towards younger age, and due to the decreased amount of estrogen secreted by the ovaries of premature ovarian failure patients, some other diseases, such as osteoporosis, urogenital atrophy, lipid metabolism disorder and cardiovascular diseases, can be caused.
Cisplatin, also known as cisplatin, DDP, and stannoplatin, is a commonly used metal platinum complex at present, and has important significance for its anti-tumor effect in platinum atoms in molecules. However, only cis is meaningful and trans is not. It can be cross-linked with DNA strand and shows cytotoxic effect. After dissolution, the cell membrane can pass through the charged cell membrane without carrier transportation in vivo. Because the concentration of intracellular chloride ions is low (4mmol/L), the chloride ions are replaced by water, the charge is positive, the double-function group has the function similar to an alkylating agent, the double-function group can be combined with the basic group of DNA in a cell nucleus to form three forms of cross-linking, DNA damage is caused, DNA replication and transcription are damaged, and the synthesis of RNA and protein is inhibited at high concentration. Cisplatin has the advantages of wide anticancer spectrum, effectiveness of hypoxic cells, strong action and the like, is widely used for treating testicular cancer, ovarian cancer, uterine cancer, bladder cancer, neck cancer, prostatic cancer, brain cancer and the like, and has obvious curative effect. However, cisplatin has certain toxicity when being used for treating cancer, can cause side effects, has direct relation with ovarian damage, and researches show that cisplatin can induce ovarian cell apoptosis and cause tissue necrosis, thereby causing premature ovarian failure.
Disclosure of Invention
Aiming at the prior art, the invention provides a method for establishing a rat ovarian premature senility model by using cisplatin. According to the principle that cisplatin can induce ovarian cell apoptosis to cause tissue necrosis and further cause premature ovarian failure, the invention establishes an animal model of chemotherapy-induced damaged premature ovarian failure by using different concentrations and different methods, and screens out the optimal concentration and administration time of cisplatin by comparing the advantages and disadvantages of various methods, thereby providing an experimental method which is simple in operation, high in success rate and reliable in result for screening the optimal dose of the animal model of premature ovarian failure caused by cisplatin.
The invention is realized by the following technical scheme:
a method for establishing a rat premature ovarian failure model by using cisplatin comprises the following steps: cisplatin with the dose of 2-3 mg/Kg per day according to the body weight is applied to a rat in an intraperitoneal injection mode, and the application method comprises the following steps: the administration is carried out continuously for 7 days, and 15 days after the last injection, the rat premature ovarian failure model is obtained. Or: cisplatin with the dose of 4-6 mg/Kg according to the body weight is applied to a rat in an intraperitoneal injection mode, and the application method comprises the following steps: administration on day 1, day 8; and 15 days after the last injection, obtaining a rat premature ovarian failure model.
Further, cisplatin can be dissolved in 0.9% sodium chloride solution to prepare cisplatin injection, and then injected. The cisplatin can be purchased in the market, such as cisplatin produced by Zilu pharmaceutical company.
Further, the rats were weighed daily during the injection and 2 times weekly after injection; 15 days after the last injection, blood is taken for anesthesia to detect the hormone level, the ovary weight is compared with the ovary weight, a vaginal smear is taken every day during the experiment period, and the change of the estrus cycle is observed.
Further, detecting hormone levels means: levels of estrogen (E2), follicle stimulating growth hormone (FSH), and Luteinizing Hormone (LH) were measured.
Further, the test of ovarian weight refers to: the bilateral ovarian organ coefficients of the rats in each group were compared. Further, the vaginal smear refers to: the vagina cast-off cell smear method is adopted, and methylene blue is used as a staining agent.
The invention successfully establishes the rat premature ovarian failure model by using cisplatin, and lays a good foundation for establishing a stable premature ovarian failure animal model for basic research.
The various terms and phrases used herein have the ordinary meaning as is well known to those skilled in the art. To the extent that the terms and phrases are not inconsistent with known meanings, the meaning of the present invention will prevail.
Drawings
FIG. 1: e2 level detection results are shown.
FIG. 2: schematic representation of the results of FSH level detection.
FIG. 3: the results of LH level measurements are shown schematically.
FIG. 4: the detection result of the rat body weight is shown schematically.
FIG. 5: rat ovarian weight statistical representation.
FIG. 6: schematic diagram of estrus cycle detection (10 times under optical mirror), wherein A, B, C, D is sequentially: the estrus early stage, the estrus late stage and the estrus interval.
FIG. 7: HE staining shows the effect of different methods of modeling on follicular morphology (10 times under light microscope), wherein A, B, C, D is: blank, 2mg, 4mg, 6 mg.
Detailed Description
The present invention will be further described with reference to the following examples. However, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention.
The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible.
The instruments, reagents, materials and the like used in the following examples are conventional instruments, reagents, materials and the like in the prior art and are commercially available in a normal manner unless otherwise specified. Unless otherwise specified, the experimental methods, detection methods, and the like described in the following examples are conventional experimental methods, detection methods, and the like in the prior art.
Example 1: establishing rat ovary premature senility model
The method comprises the following steps: 50ml of 0.9% sodium chloride solution is added into 10mg medical cisplatin (Qilu pharmacy, batch number: AA1A8029A) to prepare 2mg/kg cisplatin injection, and the cisplatin injection is injected into the abdominal cavity for 7 days continuously according to 10 ml/kg.
Example 2: establishing rat ovary premature senility model
The method comprises the following steps: 33.33ml of 0.9 percent sodium chloride solution is added into 10mg medical cisplatin (Qilu pharmacy, batch number: AA1A8029A) to prepare 3mg/kg cisplatin injection, and the cisplatin injection is injected into the abdominal cavity for 7 days continuously according to 10 ml/kg.
Example 3 establishment of model of premature ovarian failure in rats
The method comprises the following steps: adding 25ml of 0.9% sodium chloride solution into 10mg medical cisplatin (QILU pharmaceutical, batch number: AA1A8029A), and making into 4mg/kg cisplatin injection, which is administered by intraperitoneal injection on the first day and the eighth day respectively according to 10 ml/kg.
Example 4: establishing rat ovary premature senility model
The method comprises the following steps: adding 0.9% sodium chloride solution 16.67 into medical cisplatin (QILU pharmaceutical, batch number: AA1A8029A) with specification of 10mg to obtain cisplatin injection with concentration of 6mg/kg, and performing intraperitoneal injection on the first day and the eighth day respectively according to the concentration of 10 ml/kg.
Experimental establishment of rat ovarian progeria model
1. Experimental apparatus and materials: as shown in table 1.
TABLE 1
2. The experimental method comprises the following steps:
① animal information including SD rat, female, 6-8 weeks, weight 180-.
② grouping and dosing as shown in Table 2.
TABLE 2
③ the administration route and frequency are intraperitoneal injection, continuous injection for 7 days in 2mg and 3mg groups, injection for the first day and the eighth day in 4mg and 6mg groups, and continuous injection for 7 days in control group.
④ pathological examination, animal anesthesia, blood collection, serum separation, ELISA for measuring the change of E2, FSH, LH level in serum 15 days after the last injection, animal dissection, weighing ovary tissue, and observing the morphology of the rat ovary tissue.
The injection period was weighed daily, the dosing period was twice weekly, and vaginal smear test estrus cycles were performed daily.
3. The statistical method comprises the following steps:
statistical analysis was performed using Graph pad. All the measurement data are expressed by mean value plus or minus standard deviation, and the difference of the mean value among the groups is analyzed by single-factor variance, and P < 0.05 represents that the difference has statistical significance.
4. And (3) test results: (only one survived after completion of the 3mg group injection, not counted) as shown in table 3, table 4, figure 1, figure 2, figure 3:
① hormone levels, E2 levels were all reduced in the 2mg, 4mg, and 6mg groups compared to the blank group, but only the 2mg group was significantly reduced (P < 0.05), as shown in FIG. 1;
compared with the blank group, the FSH level of the 2mg group, the 4mg group and the 6mg group is increased, and only the 2mg group is obviously increased (P is less than 0.001), as shown in figure 2;
compared with the blank group, LH levels in the 2mg group, the 4mg group and the 6mg group were all increased, and similarly, only the 2mg group was significantly increased (P < 0.05), as shown in FIG. 3.
TABLE 3
TABLE 4
② weight change (mean ± SD), as shown in fig. 4:
rats in the 2mg group (7 consecutive days) had a significant body weight loss (P < 0.05, beginning on day 2 of injection) compared to the blank group.
In the 3mg group (7 consecutive days of injection) rats showed a significant weight loss (P < 0.05, starting on day 4 of injection) compared to the blank group, and only 1 rat survived until day 12.
Rats with 4mg and 6mg (injection on days 1 and 8) showed significant weight loss (P < 0.05) after the first and second injections, respectively, compared to the blank group, and the weight of the 4mg group was not different from that of the blank group 4 days after the administration was stopped.
③ ovarian weight As shown in Table 5 and FIG. 5, the ovarian weights of rats in the 2mg and 6mg groups were reduced to different degrees compared with the control group after cisplatin injection, wherein the ovarian weight of the 2mg group was significantly reduced (P < 0.05), and there was no significant difference between the 4mg and 6mg groups.
TABLE 5
④ Observation of estrus cycle (vaginal smear) 3mg of animals died early without a complete estrus cycle.
As shown in the table and fig. 6.
Normal rats had an estrus cycle of 4-5 days. The method is divided into four stages, namely, the prophase of estrus: the rounded nucleated epithelial cells predominate, with very few leukocytes and keratinized epithelial cells (panel a). In the estrus: keratinized epithelial cells are the majority, with scattered proliferation to leukocyte and nucleated epithelial cells being rare (panel B). In the anaphase of estrus: there were 3 kinds of flaky keratinized epithelial cells, nucleated epithelial cells and leukocytes, without much difference (panel C). The estrus interval: leukocytes account for the vast majority, with few nucleated and keratinized epithelial cells (panel D).
TABLE 6
⑤ pathological result is that as shown in figure 7, the blank group of ovaries has complete cell morphology, and it can be seen that all levels of follicles grow actively, and most of them are primordial follicles, more follicular granular layers, and the corpus luteum develops well.4 mg and 6mg groups (first and eight days of administration) have inflammatory cell infiltration, all levels of follicles decrease, atretic follicles increase, 2mg groups (7 days of continuous administration) have few levels of follicles, and mature follicles, most of them are atretic follicles, the follicular granular layers become thinner, and the corpus luteum obviously decreases.
And (4) conclusion: the 2mg/kg cis-platinum injection is prepared by 0.9 percent sodium chloride solution and 10mg medical cis-platinum (zilu pharmacy), and the injection is injected into the abdominal cavity for 7 days continuously according to 10ml/kg, so that the molding effect is optimal.
According to the experimental results, the following results are obtained: the invention can compare different doses of cisplatin and different administration time of premature ovarian failure modeling methods, thereby effectively determining the establishment scheme of the rat ovarian model.
The above examples are provided to those of ordinary skill in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications apparent to those skilled in the art are intended to be within the scope of the appended claims.
Claims (10)
1. A method for establishing a rat ovarian progeria model by using cisplatin is characterized by comprising the following steps: cisplatin with the dose of 2-3 mg/Kg per day according to the body weight is applied to a rat in an intraperitoneal injection mode, and the application method comprises the following steps: the administration is carried out continuously for 7 days, and 15 days after the last injection, the rat premature ovarian failure model is obtained.
2. A method for establishing a rat ovarian progeria model by using cisplatin is characterized by comprising the following steps: cisplatin with the dose of 4-6 mg/Kg according to the body weight is applied to a rat in an intraperitoneal injection mode, and the application method comprises the following steps: administration on day 1, day 8; and 15 days after the last injection, obtaining a rat premature ovarian failure model.
3. A method according to claim 1 or 2, wherein said method comprises the steps of: when in injection, cisplatin is dissolved in 0.9 percent sodium chloride solution to prepare cisplatin injection.
4. The method for establishing a rat premature ovarian failure model by using cisplatin as claimed in claim 1, wherein: cisplatin is dissolved in 0.9% sodium chloride solution to prepare cisplatin injection of 2mg/Kg, and the cisplatin injection with the dosage of 10ml/Kg per day according to body weight is applied to rats in an intraperitoneal injection mode, and the application method comprises the following steps: the administration is carried out continuously for 7 days, and 15 days after the last injection, the rat premature ovarian failure model is obtained.
5. A method according to claim 1 or 2, wherein said method comprises the steps of: 15 days after the last injection, blood was taken under anesthesia to detect hormone levels.
6. A method according to claim 5, wherein said method comprises: the hormone level is detected by: levels of estrogen (E2), follicle stimulating growth hormone (FSH), and Luteinizing Hormone (LH) were measured.
7. A method according to claim 1 or 2, wherein said method comprises the steps of: 15 days after the last injection, ovaries were examined against ovarian weight.
8. The method for establishing a rat premature ovarian failure model by using cisplatin as claimed in claim 7, wherein: the test versus ovarian weight refers to: the bilateral ovarian organ coefficients of the rats in each group were compared.
9. A method according to claim 1 or 2, wherein said method comprises the steps of: vaginal smears were performed daily during the experiment and observed for estrous cycle changes.
10. The method for establishing a rat premature ovarian failure model by using cisplatin as claimed in claim 9, wherein: the vaginal smear is: the vagina cast-off cell smear method is adopted, and methylene blue is used as a staining agent.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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