Construction method of ovarian radiation injury animal model

Abstract

The invention relates to a construction method of an ovary radiation injury animal model, which is characterized in that gamma rays are used for irradiating a female mouse to establish the ovary radiation injury animal model, and the mouse part irradiated by the gamma rays is the pelvic cavity of the mouse. And the total dose of gamma ray irradiation is 2-8 Gy. The ovarian radiation injury animal model shows that the mouse ovarian hormone level is disordered, the number of embryos is reduced, the number of effector follicles and the number of total follicles in the mouse ovarian tissue are reduced, and the number of atresia follicles is increased. The construction of the mouse ovary radiation injury model through pelvic partial illumination is more stable.

Description

Technical Field

[0001] The present invention relates to the field of medical biotechnology, and more particularly, to a method for constructing an animal model of ovarian radiation injury. Background Art

[0002] In recent years, with the continuous improvement of tumor screening and diagnosis technologies, the number of patients with malignant tumors has been increasing. A large proportion of them are pelvic and abdominal malignant tumors, including colorectal cancer, cervical cancer, endometrial cancer, etc. Coupled with the progress of various treatment technologies, more and more patients with malignant tumors have survived. However, the related side effects caused by tumor treatment measures cannot be ignored (1, 2).

[0003] As a key part of malignant tumor treatment measures, radiotherapy can kill tumor cells while also causing damage to normal tissues. In view of this, more and more researchers are committed to studying the damage to normal tissues caused by radiotherapy. However, the vast majority of researchers focus on the prevention and treatment of radiation-induced intestinal injury, radiation-induced lung injury, and radiation-induced hematological system injury. The research on the prevention and treatment of radiation-induced ovarian injury only accounts for a small part. The ovary, as an important organ maintaining normal physiological functions of women, is very sensitive to radiation. A low dose of radiation can cause serious adverse outcomes, including gonadal hypofunction and subsequent reduction or even loss of fertility. Therefore, the research on the prevention and treatment of radiation-induced ovarian injury is of great significance.

[0004] In the previous research process on the prevention and treatment of ovarian radiation injury, most researchers used the whole-body irradiation method for modeling (3 - 6), which is greatly inconsistent with the clinical radiotherapy process. The lack of an animal model relatively close to the clinical radiotherapy process has also become an important factor hindering the research on the prevention and treatment of ovarian radiation injury.

[0005] Chinese patent document CN108990906 A discloses a method for establishing an animal model of irritable bowel syndrome in rats. The method is to irradiate rats with Co60 rays and then combine with restraint stress to establish the animal model of irritable bowel syndrome in rats. Using the method for establishing an animal model of irritable bowel syndrome in rats of the present invention, an animal model of irritable bowel syndrome in rats can be established with a short cycle, low cost, simple operation, and good reproducibility, and it can reduce or avoid rectal irritation and damage to other systems of the body caused during the establishment of the animal model.

[0006] However, there has been no report on a method for constructing an animal model of ovarian radiation injury. Summary of the Invention

[0007] The object of the present invention is to provide a method for constructing an animal model of ovarian radiation injury in view of the deficiencies in the prior art. The mouse ovarian radiation injury model constructed by this method is more stable.

[0008] In the first aspect, the present invention provides a method for constructing an animal model of ovarian radiation injury. The method is to establish the animal model of ovarian radiation injury after irradiating female mice with γ-rays. The part of the mouse irradiated with γ-rays is the pelvic cavity of the mouse.

[0009] As a preferred example, the total dose of γ-ray irradiation is 2-8 Gy.

[0010] As another preferred example, the genetic background of the mouse is: C57BL / 6J.

[0011] As another preferred example, the animal model of ovarian radiation injury is manifested as disordered ovarian hormone levels in mice, reduced embryo numbers, decreased numbers of effective follicles and total follicles in mouse ovarian tissues, and increased numbers of atretic follicles.

[0012] As another preferred example, the total dose of γ-ray irradiation is 2-4 Gy.

[0013] As another preferred example, the total dose of γ-ray irradiation is 2 Gy, 4 Gy, 6 Gy, 8 Gy.

[0014] The advantages of the present invention are as follows:

[0015] 1. Radiation can cause disordered ovarian hormone levels in mice. The greater the total irradiation dose, the more obvious the disorder of mouse hormone levels. Between the total irradiation dose of 2 Gy and 8 Gy, compared with the control group, there are statistical differences in AMH / LH / FSH / E2 in the ovaries of irradiated mice, indicating that the mouse model is successfully established at the ovarian endocrine level after receiving pelvic local irradiation of 2 Gy to 8 Gy.

[0016] 2. Radiation can cause a decrease in the number of mouse embryos. As the irradiation dose increases, the number of mouse embryos decreases significantly. When the total irradiation dose is 2 Gy and 4 Gy, there are obvious statistical differences compared with the control group, and when the total irradiation dose is 8 Gy, all mice are not pregnant, indicating that the mouse model is successfully established at the ovarian reproductive level after receiving pelvic local irradiation of 2 Gy to 4 Gy.

[0017] 3. Follicle counting is performed on the ovarian tissues of mice receiving different doses of pelvic local irradiation. Compared with the control group, the numbers of effective follicles and total follicles in the ovarian tissues of each irradiated group of mice are both reduced, and the numbers of atretic follicles are both increased. The mouse model is successfully established at the ovarian endocrine and reproductive levels after receiving pelvic local irradiation of 2 Gy to 4 Gy.

[0018] 4; The disorder levels of AMH / LH / FSH / E2 in the whole-body irradiation group were more obvious than those in the pelvic local irradiation group, suggesting that the construction of a mouse ovarian radiation injury model by pelvic local irradiation was more stable. Description of the Drawings

[0019] Figure 1 : Radiation can cause disorders in the hormone levels of mouse ovaries. The greater the total irradiation dose, the more obvious the disorder of mouse hormone levels. Between the total irradiation doses of 2 Gy and 8 Gy, compared with the control group, there were statistically significant differences in AMH / LH / FSH / E2 in the ovaries of irradiated mice, suggesting that successful modeling was achieved in terms of ovarian endocrine levels when mice received pelvic local irradiation of 2 Gy to 8 Gy. (Con: Control group; IR: Irradiated group) (N.S.: No statistical difference; **: P < 0.01; ***: P < 0.001).

[0020] Figure 2 : Mice received different doses of pelvic irradiation, were mated with sexually mature mice after superovulation, and the number of embryos in the uteri of all pregnant mice was counted on the 15th day after pregnancy. As the irradiation dose increased, the number of mouse embryos decreased significantly. When the total irradiation doses were 2 Gy and 4 Gy, there were obvious statistical differences compared with the control group. And when the total irradiation dose was 8 Gy, all mice were not pregnant, suggesting that successful modeling was achieved in terms of ovarian reproductive levels when mice received pelvic local irradiation of 2 Gy to 4 Gy. (Con: Control group; IR: Irradiated group) (N.S.: No statistical difference; **: P < 0.01; ***: P < 0.001).

[0021] Figure 3 : Follicle counting was performed on the ovarian groups of mice receiving different doses of pelvic local irradiation. Compared with the control group, the number of effective follicles and the total number of follicles in the ovarian tissues of irradiated mice in each group decreased, and the number of atretic follicles increased ( Figure 3 A). Further, the follicle numbers in the 2 Gy group and the 4 Gy group were counted separately. In the 2 Gy group, the number of atretic follicles accounted for about 40% of the total number of follicles ( Figure 3 B), and in the 4 Gy group, the number of atretic follicles accounted for more than 60% of the total number of follicles ( Figure 3 C), further suggesting that successful modeling was achieved in terms of ovarian endocrine and reproductive levels when mice received pelvic local irradiation of 2 Gy to 4 Gy. (Con: Control group; IR: Irradiated group) (N.S.: No statistical difference, ***: P < 0.001).

[0022] Figure 4:Two groups of mice were respectively given pelvic local irradiation or whole-body irradiation at 2 Gy, and the hormone levels were detected at different time points after the irradiation ended. The results showed that in the first week after the irradiation ended, the levels of ovarian AMH / LH / FSH / E2 in the two groups of mice were basically the same. However, as time went on, the damage to the hypothalamic-pituitary-gonadal axis caused by whole-body irradiation in mice led to further aggravation of the disorder of ovarian hormone levels. It was manifested that at the 4th week, 12th week, and 28th week after the irradiation ended, the disorder levels of AMH / LH / FSH / E2 in the whole-body irradiation group of mice were more obvious than those in the pelvic local irradiation group, suggesting that the construction of a mouse ovarian radiation injury model by pelvic local irradiation was more stable. (TBI: whole-body irradiation, ABI: pelvic local irradiation). Specific implementation manners

[0023] The present invention will be further described below in conjunction with specific implementation manners. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content recorded in the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

[0024] Example 1

[0025] 1. Experimental materials:

[0026] Experimental animals: The genetic background of the mice selected in this experiment was C57BL / 6J. They were purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd., with a weekly age of 6 - 8 weeks, and were raised in an animal room with day-night alternating lighting and a constant temperature of 20 °C.

[0027] Main reagents: ELISA kits for detecting mouse estrogen (E2), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and anti-Müllerian hormone (AMH) (Shanghai Zhuocai Biotechnology Co., Ltd.); hematoxylin dye, eosin dye, Giemsa dye (Zhuhai Baisuo Biotechnology Co., Ltd.).

[0028] The present invention uses γ-rays. (It should be noted that the radiation sources also include: X-rays, neutrons, protons, etc., but the present invention only involves γ-rays).

[0029] 2. Experimental methods:

[0030] (a) Modeling of experimental animals: Female mice were divided into a blank control group and an irradiation group. According to different irradiation doses and irradiation methods, each group of mice received pelvic local irradiation or whole-body irradiation with different irradiation doses.

[0031] (b) Experimental arrangement:

[0032] i) Forty female mice were evenly divided into five groups and received pelvic local irradiation with total doses of 0 Gy (as the control group), 1 Gy, 2 Gy, 4 Gy, and 8 Gy respectively according to the radiation dose gradient design. Twenty-four hours after the modeling was completed, hormone level detection and follicle counting were performed.

[0033] ii) Forty female mice were evenly divided into five groups and received pelvic local irradiation with total doses of 0 Gy (as the control group), 1 Gy, 2 Gy, 4 Gy, and 8 Gy respectively according to the radiation dose gradient design. Twenty-four hours after the modeling was completed, all female mice were superovulated and then caged with male mice at sexual maturity in a 1:1 ratio. Whether pregnancy occurred was judged by monitoring the formation of vaginal mucus plugs, and the number of embryos in the uteri of all pregnant mice was counted on the 15th day of pregnancy.

[0034] iii) Sixteen female mice were evenly divided into two groups and received pelvic local irradiation with a total dose of 2 Gy or 2 Gy pelvic local irradiation and. At different time points after the modeling was completed, hormone level detection was performed respectively.

[0035] (c) Hormone levels: Twenty-four hours after the modeling was completed, the mice were anesthetized, and blood was collected from the eyeballs to obtain serum. The levels of AMH, E2, FSH, and LH in the mice were detected by ELISA method.

[0036] (d) Follicle counting: The ovarian tissues of the mice were taken to make paraffin specimens, and the specimens were subjected to and follicle counting to evaluate the changes in the number of effective follicles, atretic follicles, and total follicles.

[0037] (e) Superovulation: After the modeling was completed, all mice received intraperitoneal injection of the same dose of PMSG + HCG.

[0038] 3. Statistical analysis

[0039] Analysis was performed using GraphPad Prism 9.0 and SPSS 22.0 software, and all data were expressed as mean ± standard deviation (Means ± SD). Unpaired t-tests were used for the differential analysis between measurement data, and P < 0.05 was considered statistically significant.

[0040] 4. Experimental results

[0041] (a) After pelvic irradiation with different doses, the changes in the sex hormone levels of the mice are shown in Figure 1 .

[0042] Radiation can cause disorders in the ovarian hormone levels of mice. The greater the total irradiation dose, the more obvious the disorder in the hormone levels of mice. Between the total irradiation doses of 2 Gy and 8 Gy, compared with the control group, there were statistically significant differences in AMH / LH / FSH / E2 in the ovaries of irradiated mice, suggesting that successful modeling was achieved in terms of ovarian endocrine levels when mice received pelvic local irradiation of 2 Gy to 8 Gy. (Con: Control group; IR: Irradiated group) (N.S.: No statistical difference; **: P < 0.01; ***: P < 0.001)

[0043] (b) Comparison of the number of embryos in each group of mice after receiving different doses of pelvic irradiation, see Figure 2 .

[0044] Mice received different doses of pelvic irradiation, were mated with sexually mature mice after superovulation, and the number of embryos in the uteri of all pregnant mice was counted on the 15th day after pregnancy. As the irradiation dose increased, the number of mouse embryos decreased significantly. When the total irradiation doses were 2 Gy and 4 Gy, there were obvious statistical differences compared with the control group. And when the total irradiation dose was 8 Gy, all mice did not become pregnant, suggesting that successful modeling was achieved in terms of ovarian reproductive levels when mice received pelvic local irradiation of 2 Gy to 4 Gy. (Con: Control group; IR: Irradiated group) (N.S.: No statistical difference; **: P < 0.01; ***: P < 0.001).

[0045] (c) Comparison of follicle counts in mice after different doses of pelvic irradiation, see Figure 3 .

[0046] Follicle counts were performed on the ovarian tissues of mice receiving different doses of pelvic local irradiation. Compared with the control group, the number of effective follicles and the total number of follicles in the ovarian tissues of irradiated mice decreased, and the number of atretic follicles increased ( Figure 3 A). Further, the follicle counts of mice in the 2 Gy group and the 4 Gy group were counted separately. In the 2 Gy group, the number of atretic follicles accounted for about 40% of the total number of follicles ( Figure 3 B), and in the 4 Gy group, the number of atretic follicles accounted for more than 60% of the total number of follicles ( Figure 3 C), further suggesting that successful modeling was achieved in terms of ovarian endocrine and reproductive levels when mice received pelvic local irradiation of 2 Gy to 4 Gy. (Con: Control group; IR: Irradiated group) (N.S.: No statistical difference, ***: P < 0.001).

[0047] (d) Changes in hormone levels in mice receiving different irradiation methods at different time points, see Figure 4 .

[0048] Two groups of mice were respectively given pelvic local irradiation or whole-body irradiation at 2 Gy, and the hormone levels were detected at different time points after the irradiation ended. The results showed that at the 1st week after the irradiation ended, the levels of AMH / LH / FSH / E2 in the ovaries of the two groups of mice were basically the same. However, as time went on, the damage to the hypothalamic-pituitary-gonadal axis caused by whole-body irradiation in mice, and then the disorder of ovarian hormone levels was further aggravated. It was manifested that at the 4th week, 12th week, and 28th week after the irradiation ended, the disorder levels of AMH / LH / FSH / E2 in the whole-body irradiation group of mice were more obvious than those in the pelvic local irradiation group, suggesting that the construction of a mouse ovarian radiation injury model by pelvic local irradiation was more stable.

[0049] (TBI: total body irradiation, ABI: abdominal pelvic irradiation).

[0050] References:

[0051] 1. Siegel RL, Miller KD, Fuchs HE, Jemal A. Cancer statistics, 2022. CA Cancer J Clin. 2022;72(1):7 - 33.

[0052] 2. Miller KD, Nogueira L, Devasia T, Mariotto AB, Yabroff KR, Jemal A, et al. Cancer treatment and survivorship statistics, 2022. CA Cancer J Clin. 2022;72(5):409 - 36.

[0053] 3. Simsek Y, Gurocak S, Turkoz Y, Akpolat N, Celik O, Ozer A, et al. Ameliorative effects of resveratrol on acute ovarian toxicity induced by total body irradiation in young adult rats. J Pediatr Adolesc Gynecol. 2012;25(4):262-6. 4. Gao W, Liang JX, Ma C, Dong JY, Yan Q. The Protective Effect of N-Acetylcysteine on Ionizing Radiation Induced Ovarian Failure and Loss of Ovarian Reserve in Female Mouse. Biomed Res Int. 2017;2017:4176170. 5. Kuang ZK, Yang YS, Zhao X, Yu L, Zou MY, Du L, et al. Protective effect of Bushen Jiedu formula on reproductive system injury induced by nuclear radiation in female rats. Zhonghua Zhongyiyao Zazhi. 2018;33(07):2816-9.

[0054] 6. Zhao FQ, An MX, Ding XN, Liu JY, Zhao Y, Xie ZH, et al. Exploration of the regulatory effect of Zuogui Pills on follicular apoptosis in rats damaged by ~(60)Co-γ rays based on the PI3K / Akt / mTOR signaling pathway. Zhongguo Shiyan Fangji Xue Zazhi. 2022;28(18):12-9.

[0055] ~(60)Co-γ rays damaged rat follicle apoptosis regulation. Chinese Journal of Experimental Traditional Medical Formulae. 2022;28(18):12-9.

[0056] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the method of the present invention, several improvements and supplements can be made, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. A method for constructing an animal model of ovarian radiation damage, characterized in that: The method is to establish the ovarian radiation damage animal model after irradiating female mice with gamma rays, and the gamma ray irradiated mice are located in the pelvic cavity of the mice.

2. The construction method according to claim 1, characterized in that: The total dose of the gamma-ray irradiation is 2-8Gy.

3. The construction method according to claim 1, characterized in that: The genetic background of the mice is: C57BL / 6J.

4. The construction method according to claim 1, characterized in that: The animal model of ovarian radiation damage is characterized by disordered ovarian hormone levels in mice, reduced number of embryos, reduced number of effector follicles and total follicles in mouse ovarian tissue, and increased number of atretic follicles.

5. The construction method according to claim 2, characterized in that: The total dose of the gamma-ray irradiation is 2-4 Gy.

6. The construction method according to claim 2, characterized in that: The total dose of the gamma-ray irradiation is 2Gy, 4Gy, 6Gy, and 8Gy.

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