Application of vitamin D in preparation of protective medicine for avoiding ovarian injury caused by chemotherapy

Abstract

The invention discloses application of vitamin D in preparation of a protection medicine for avoiding ovarian injury caused by chemotherapy, and belongs to the technical field of biology. It is found for the first time that in an experiment of intraperitoneal injection of calcitriol (the active form of vitamin D) and retinoic acid in newborn mice, DNA damage caused by chemotherapeutic drugs cyclophosphamide and adriamycin can be partially reversed when calcitriol and retinoic acid are independently used; the number of primordial follicle apoptosis caused by chemotherapeutic drugs (including cyclophosphamide and adriamycin) is remarkably reduced, and the effect of combined medication (calcitriol and retinoic acid) is better. The discovery expands the application of vitamins in the female reproductive system, provides a new thought for preventing ovarian follicle development from being damaged by chemotherapy drugs and in-vivo protection of fertility, and also has important significance for protecting the life quality of female patients after chemotherapy.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to application of vitamin D in preparation of a protective drug for avoiding ovary damage caused by chemotherapy. BACKGROUND

[0002] Cancer is a recognized global public health problem, and its treatment often adopts chemotherapy. Chemotherapy drugs can be roughly divided into six categories: alkylating agents, antimetabolites, antitumor antibiotics, plant anticancer drugs, hormones and other categories. 1) Alkylating agents such as cyclophosphamide, ifosfamide; ifosfamide belongs to antitumor drugs, which mainly inhibits DNA synthesis to achieve the purpose of anti-tumor. Cyclophosphamide mainly inhibits phosphodiesterase to produce anti-tumor effect. 2) Antimetabolites: this kind of drugs have mutual competition effect on the binding reaction of nucleic acid metabolites and enzymes, such as gemcitabine, methotrexate, pemetrexed, urofϊnate, hydroxyurea, etc. 3) Antibiotics: antibiotics with anti-tumor effect, such as anthracyclines like doxorubicin, dactinomycin like more mycosis, and pingyangmycin like bleomycin. 4) Antitumor plant drugs: vincristine and paclitaxel acting on microtubules and tubulin, interfering with intracellular spindle formation, making cells stay in mitotic metaphase; camptothecin and podophyllotoxin acting on topoisomerase; harringtonine and indigo carmine inhibiting DNA synthesis of tumor cells. 5) Hormones: can change the internal environment, and then affect tumor growth, such as tamoxifen, toremifene, flutamide, letrozole, etc. 6) Other categories: such as platinum like cisplatin, carboplatin and oxaliplatin, etc. Among them, cyclophosphamide is the most widely used alkylating agent, doxorubicin, oxaliplatin and vincristine are commonly used chemotherapy drugs. However, chemotherapy drugs often cause the death of a large number of ovarian follicles in female patients while treating cancer patients.

[0003] Women establish a certain number of primary follicle banks at birth as a reserve of female fertility. Irreversible follicle apoptosis caused by chemotherapy drugs will lead to more than 90% of female patients to have a sharp decline in fertility, premature ovarian failure and infertility. With the younger cancer patients and the rising survival rate, especially young female patients with hematological diseases, they are eager to restore ovarian function and fertility after cure in order to improve their own quality of life, maintain family stability and social harmony. At present, the commonly used method of fertility preservation at home and abroad is to freeze the ovarian tissue or oocyte before chemotherapy, and to restore fertility by transplanting the ovarian tissue in situ after the end of chemotherapy. However, this method needs surgery, and has the risk of low success rate and high cost of tissue preservation.

[0004] Vitamin D (VD) is a steroid hormone that is primarily synthesized in the skin upon ultraviolet irradiation and to a lesser extent from the diet. VD is converted by liver-secreted enzymes to 25-hydroxyvitamin D and then by kidney-secreted enzymes to the active form, 1,25-dihydroxyvitamin D (also known as calcitriol). The important functions of VD are to maintain calcium homeostasis and bone formation, but it also regulates energy metabolism, innate and acquired immunity, and cell growth, differentiation, and apoptosis. VD activates the vitamin D receptor (VDR) and tightly binds to retinoic acid receptor (RXR) to form the active signal transduction complex VD-VDR-RXR, which enters the nucleus to regulate the transcription of about 1000 downstream target genes. VD can also act through rapid nongenomic effects; VDR can act on the cell membrane in response to VD. VDR is not only present in calcium-regulating tissues such as the intestine, bone, and parathyroid gland, but also in reproductive-related organs such as the ovary, uterus, and placenta. However, whether vitamin D can protect primordial follicles from chemotherapy drugs remains to be further studied. SUMMARY

[0005] In order to overcome the shortcomings and deficiencies existing in the prior art, the primary purpose of the present application is to provide the application of vitamin D in the preparation of a protective drug for avoiding ovarian damage caused by chemotherapy.

[0006] The application of vitamin D in the preparation of a protective drug for avoiding ovarian damage caused by chemotherapy.

[0007] The purpose of the present application is achieved by the following technical solutions:

[0008] The application of vitamin D is any one of the following applications (1) and (2):

[0009] (1) The application of vitamin D in the preparation of a protective drug for avoiding ovarian damage caused by chemotherapy.

[0010] (2) The application of vitamin D in combination with vitamin A in the preparation of a protective drug for avoiding ovarian damage caused by chemotherapy.

[0011] Further, the drug for chemotherapy includes at least one of cyclophosphamide (Cy), doxorubicin (DOX), oxaliplatin (OXA), and vincristine (VIN).

[0012] Further, the ovarian damage includes at least one of DNA damage and primordial follicle apoptosis.

[0013] Further, the vitamin D includes vitamin D and derivatives thereof; and the vitamin A includes vitamin A and derivatives thereof.

[0014] Further, the vitamin D derivative includes calcitriol. Calcitriol is the active form of vitamin D; the vitamin A derivative includes retinoic acid (RA). Retinoic acid is a ligand for retinoic acid receptors (RAR) and retinoid X receptors (RXR).

[0015] Further, the ovary is a mammalian ovary; the mammal includes human and mouse.

[0016] Further, when the mammal is human, the effective dose of the drug is calculated according to the individual body weight, and the dose of calcitriol is 0.1-5000 μg / kg, preferably 2.50-50 μg / kg; the drug is administered once every other day; the dose of retinoic acid is 0.1-2500 mg / kg, preferably 1.50-25 mg / kg; the drug is administered once every other day.

[0017] Further, when the mammal is human, the effective dose of the drug is calculated according to the individual body weight, and the dose of calcitriol is 0.1-5000 μg / kg, preferably 2.50-50 μg / kg; the drug is administered once every other day; the dose of retinoic acid is 0.1-2500 mg / kg, preferably 1.50-25 mg / kg; the drug is administered once every other day.

[0018] Further, the ovary function protecting drug further comprises a pharmaceutically acceptable carrier.

[0019] Further, the ovary function protecting drug is administered orally or by intramuscular injection.

[0020] The inventor of the present application has found that the chemotherapy drugs cyclophosphamide and doxorubicin can increase DNA damage of oocytes and cause apoptosis of primordial follicles. In the experiment of intraperitoneal injection of calcitriol and retinoic acid in newborn mice, both calcitriol and retinoic acid can partially reverse the DNA damage caused by the chemotherapy drugs cyclophosphamide and doxorubicin, and significantly reduce the number of primordial follicles caused by chemotherapy drugs. This finding expands the application of VD and VA in the female reproductive system, provides a new idea for preventing chemotherapy drugs from damaging the development of ovarian follicles and protecting fertility in vivo, and also has important significance for protecting the quality of life of female patients after chemotherapy.

[0021] Compared with the prior art, the present application has the following advantages and beneficial effects:

[0022] The use of chemotherapy improves the overall survival rate and disease-free survival rate of cancer patients, but chemotherapy drugs can damage the follicle and ovarian blood vessels and interstitium, ultimately leading to loss of ovarian reserve, which seriously damages the ovarian function of women and has a major impact on women with fertility needs. However, the current fertility preservation treatment for female cancer patients will delay cancer treatment or cause further damage to the patient's body. Therefore, it is necessary to find an oral drug to protect the ovarian reserve of young female cancer patients and prevent chemotherapy drugs from damaging ovarian function.

[0023] In the present application, we found that retinoic acid and calcitriol can partially reverse the DNA damage caused by chemotherapy drugs (cyclophosphamide and doxorubicin) and reduce the number of primordial follicle apoptosis caused by chemotherapy. As a necessary nutrient element for the body, VD and VA can be taken orally and have good application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 PBS, cyclophosphamide (Cy), cyclophosphamide (Cy) + different concentrations of calcitriol (Calcitriol) were injected intraperitoneally into 3dpp newborn mice, and the ovarian morphology of the mice at 8dpp was observed (a) and the number of primordial follicles was counted (b) graph (scale: 50μm).

[0025] Figure 2 PBS, doxorubicin (DOX), doxorubicin (DOX) + different concentrations of calcitriol (Calcitriol) were injected intraperitoneally into 3dpp newborn mice, and the ovarian morphology of the mice at 8dpp was observed (a) and the number of primordial follicles was counted (b) graph (scale: 50μm).

[0026] Figure 3 PBS, cyclophosphamide (Cy), cyclophosphamide (Cy) + calcitriol (50μg / kg Calcitriol), cyclophosphamide (Cy) + calcitriol (50μg / kg Calcitriol) and retinoic acid (25mg / kg RA) were injected intraperitoneally into 3dpp newborn mice, and the ovarian morphology of the mice at 8dpp was observed (a) and the number of primordial follicles was counted (b) graph (scale: 50μm).

[0027] Figure 4 PBS, doxorubicin (DOX), doxorubicin (DOX) + calcitriol (50μg / kg Calcitriol), doxorubicin (DOX) + calcitriol (50μg / kg Calcitriol) and retinoic acid (25mg / kg RA) were injected intraperitoneally into 3dpp newborn mice, and the ovarian morphology of the mice at 8dpp was observed (a) and the number of primordial follicles was counted (b) graph (scale: 50μm). DETAILED DESCRIPTION

[0028] The application will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the application are not limited thereto.

[0029] The test methods in the following examples, unless otherwise indicated, were carried out under conventional experimental conditions or under the experimental conditions recommended by the manufacturer. The materials, reagents, etc. used, unless otherwise specified, were reagents and materials obtained from commercial sources.

[0030] Example 1

[0031] I. Preparation of reagents related to in vitro culture

[0032] 1.1 Preparation of drug solution

[0033] 1 mg calcitriol powder was added to 1 mL DMSO to prepare a concentrated stock solution under light-proof conditions; 30 mg retinoic acid powder was added to 1 mL DMSO to prepare a concentrated stock solution. 40 mg cyclophosphamide powder was added to 1 mL PBS to prepare a cyclophosphamide solution; 10 mg doxorubicin powder was added to 1 mL PBS to prepare a doxorubicin solution; 0.1 mg vincristine powder was added to 1 mL PBS to prepare a vincristine solution; 2 mg oxaliplatin powder was added to 1 mL PBS to prepare an oxaliplatin solution. After the solids were completely dissolved, the solution was filtered with a 0.22 μm filter, sealed with a sealing film and stored at -20°C.

[0034] 1.2 Preparation of DMEM / F12 medium (Table 1)

[0035] Table 1. DMEM / F12 medium

[0036]

[0037] After the solids were completely dissolved, the solution was filtered with a 0.22 μm filter, operated in a clean bench, sealed with a sealing film and stored at 4°C.

[0038] 1.3 Preparation of phosphate buffered solution (PBS) (Table 2)

[0039] Table 2. PBS buffer

[0040]

[0041]

[0042] Autoclaved and stored at 4°C.

[0043] 2. Preparation of reagents related to immunofluorescence

[0044] 2.1 Preparation of 10% blocking serum

[0045] Donkey serum was prepared with PBS at a ratio of 1:10 as needed.

[0046] 2.2 Preparation of primary antibody

[0047] Corresponding primary antibody: 10% blocking serum was prepared with PBS at a ratio of 1:200 as needed.

[0048] 2.3 Preparation of secondary antibody

[0049] Corresponding secondary antibody: PBS was prepared at a ratio of 1:200 as needed according to the species of primary antibody.

[0050] 2.4 Preparation of DAPI working solution

[0051] DAPI stock solution: PBS was prepared at a ratio of 1:200 as needed.

[0052] 2.5 Preparation of citric acid buffer working solution (Table 3, Table 4, Table 5)

[0053] Table 3. 0.1M citric acid (A solution)

[0054]

[0055] Table 4. 0.1M sodium citrate (B solution)

[0056]

[0057] Table 5. Citric acid working solution

[0058]

[0059] II. Intraperitoneal injection of mice and isolation of ovaries

[0060] 1. Intraperitoneal injection of mice

[0061] 1.1 Preparation of intraperitoneal injection solution: 1 mg / ml calcitriol concentrated stock solution (dissolved in DMSO) was diluted with PBS to the corresponding different concentrations (25 μg / kg or 50 μg / kg of drug administration) to prepare calcitriol injection solution for intraperitoneal injection; 30 mg / ml retinoic acid concentrated stock solution (dissolved in DMSO) was diluted with corn oil to the corresponding concentration (25 mg / kg of drug administration) to prepare retinoic acid injection solution for intraperitoneal injection.

[0062] 1.2 Intraperitoneal injection scheme:

[0063] The experimental animals were randomly divided into a control (Control) group, a chemotherapy drug group (Cy / DOX), and a chemotherapy drug and treatment drug group (including Calcitriol / Calcitriol+RA combination). From 3 dpp mice, the injection scheme of each group was as follows:

[0064] Chemotherapy drug + therapeutic drug group: 3 times of intraperitoneal injection, once every 48 h; 3 dpp, the first intraperitoneal injection of therapeutic drug (Calcitriol / Calcitriol+RA combination) in mice, 1 h after the second intraperitoneal injection of therapeutic drug (Calcitriol / Calcitriol+RA combination) in mice, chemotherapy drug (Cy 75 mg / kg or DOX 10 mg / kg) was given, and the third injection of therapeutic drug (Calcitriol / Calcitriol+RA combination) in mice was performed at 7 dpp;

[0065] Chemotherapy drug group (Cy / DOX): the above-mentioned group was injected with chemotherapy drug, and Cy (75 mg / kg) or DOX (10 mg / kg) was injected intraperitoneally at the same time; the above-mentioned group was injected with therapeutic drug group (including Calcitriol / Calcitriol+RA combination), and the same amount of PBS was injected intraperitoneally at the same time.

[0066] Control group: the above-mentioned group was injected with chemotherapy drug or therapeutic drug, and the same amount of PBS or corn oil was injected intraperitoneally at the same time.

[0067] The ovary was taken 24 h after the last administration of Calcitriol or PBS. The ovary of mice at 8 dpp was taken for the next experiment.

[0068] 1.3 Intraperitoneal injection method: weigh the mice and use a microsyringe to suck the corresponding volume of drug. The mouse's head is facing down, the index finger and ring finger are clamped at the root of the lower limbs, the thumb and index finger are fixed on the upper limbs and head and gently stretched, so that the mouse's abdominal skin is tense. The needle tip of the microsyringe is flat upward, and the needle is slightly inserted subcutaneously, and then the syringe is inserted into the abdominal cavity at an angle of 45°. After feeling the empty feeling, the corresponding drug is injected.

[0069] 2. Isolation of mouse ovary

[0070] 2.1 Surgical instruments (ophthalmic straight scissors, 10 cm toothed forceps, sharp forceps, 1 mL syringe), 90 mm culture dishes, six-well plates, etc. were placed in the ultraclean bench and ultraviolet irradiated for 30 min, and the ultraviolet lamp was irradiated for 30 min between cells.

[0071] 2.2 The mouse was sacrificed by cervical dislocation, the abdomen was disinfected with 70% alcohol, a V-shaped incision was made at the umbilical part to open the abdominal cavity, and the mouse was divided into head and tail parts along the upper part of the kidneys. The tail part was placed in pre-cooled PBS.

[0072] 2.3 Under the operation of a stereomicroscope, the mouse ovary was taken out together with the surrounding connective tissue, and the connective tissue around the ovary was stripped clean with a 1 mL injection needle.

[0073] III. Hematoxylin staining

[0074] 1. Ovary paraffin embedding

[0075] 1.1 Fixation of sample: The separated mouse ovaries were fixed in 4% paraformaldehyde at 4°C for 12-16 hours.

[0076] 1.2 Alcohol concentration gradient dehydration and transparency: 70% alcohol, 80% alcohol, 95% alcohol / Eosin staining solution, 95% alcohol, anhydrous alcohol, anhydrous ethanol / dimethylbenzene (volume 1:1), dimethylbenzene, each gradient for 5 minutes, and the last dimethylbenzene was absorbed with filter paper.

[0077] 1.3 Wax immersion and embedding: The tissue was immersed in wax at 60°C water bath for 3 hours, transferred to an iron paraffin box, covered with an embedding box, and taken out to room temperature for solidification.

[0078] 2. Sectioning

[0079] 2.1 Open the microtome, adjust the section thickness to 5 μm, and start continuous sectioning after fixing the wax block.

[0080] 2.2 Stretching the section: After fully stretching the wax tape in a 42°C water bath, use a glass slide to pull it up.

[0081] 2.3 Baking the section: 42°C overnight.

[0082] 3. Hematoxylin staining

[0083] 3.1 De-waxing and rehydration: The glass slide with attached tissue was sequentially passed through dimethylbenzene, dimethylbenzene, anhydrous alcohol, anhydrous alcohol, 95% alcohol, 80% alcohol, and 70% alcohol, each for 5 minutes, and finally rinsed with deionized water.

[0084] 3.2 Staining: Stained with hematoxylin staining solution for 1 minute and 30 seconds, and rinsed with deionized water.

[0085] 3.3 Dehydration and mounting: The glass slide was sequentially passed through 70% alcohol, 80% alcohol, 95% alcohol, anhydrous alcohol, anhydrous alcohol, dimethylbenzene, dimethylbenzene, each for 3 minutes, and mounted with neutral resin.

[0086] 4. Slide scanning: Count the stained continuous sections, take one out of every 5 continuous sections for counting, and the final statistical result is obtained by adding the counting results and multiplying by 5. Abnormal follicles are manifested as: apoptotic oocytes and / or ≥2 apoptotic granulosa cells, and follicles showing asymmetric morphology.

[0087] Four, immunofluorescence staining

[0088] 1. Sectioning of ovarian tissue wax block, de-waxing, and rehydration.

[0089] 2. Antigen retrieval: put the slices into citrate buffer working solution, microwave oven for 4 min at high heat, 4 min at low heat x 3, and cool to room temperature naturally.

[0090] 3. Wash the slices: PBS 5 min x 2.

[0091] 4. Blocking: 10% donkey serum blocking at room temperature for 1 hour.

[0092] 5. Incubate the first antibody: overnight at 4°C.

[0093] 6. Wash the slices: PBS 5 min x 6.

[0094] 7. Incubate the second antibody: 1 hour at 37°C. From this step, all operations need to be carried out in the dark.

[0095] 8. Wash the slices: PBS 5 min x 6.

[0096] 9. DAPI: incubate for 1 min.

[0097] 10. Wash the slices: PBS 5 min x 3.

[0098] 11. Mounting: 20 μL of anti-fluorescence quencher, cover with a cover glass.

[0099] 12. Take pictures with a laser confocal microscope. Take a fluorescent photo every 3 sections, count and calculate the average of 5 sections for one ovary.

[0100] Five, immunoblotting

[0101] 1. Protein extraction and concentration determination.

[0102] 2. Preparation of polyacrylamide gel, electrophoresis solution, and membrane transfer solution.

[0103] 3. Protein loading 20 μg, electrophoresis 60 V for 40 min, 100 V for 1 hour.

[0104] 4. Membrane transfer 100 V for 1 hour.

[0105] 5. 5% skim milk blocking at room temperature for 1 hour.

[0106] 6. Incubate the corresponding first antibody overnight at 4°C.

[0107] 7. Incubate the corresponding species second antibody at room temperature for 1 hour.

[0108] 8. Exposure and imaging, calculate the relative protein expression.

[0109] Six, results

[0110] Figure 1For 3dpp newborn mice, intraperitoneal injection of PBS, cyclophosphamide (Cy), cyclophosphamide (Cy) + different concentrations of calcitriol (Calcitriol) to 8dpp mice, ovarian morphology observation (a) and follicle count statistics (b) chart (scale: 50 μm). Among them: a is the comparison of ovarian morphology between the control group, the cyclophosphamide group, and the different concentrations (25 μg / kg, 50 μg / kg) of calcitriol + cyclophosphamide group. The arrow points to the surviving primordial oocytes; b is the total ovarian follicle count statistics. As shown in the figure, compared with the control group, the number of surviving primordial follicles in the cyclophosphamide group was significantly reduced (4257±156.95 vs 1092.2±91.80, p<0.001). Compared with the cyclophosphamide group, the number of surviving primordial follicles in the 25 μg / kg and 50 μg / kg calcitriol + cyclophosphamide group was significantly increased (1092.2±91.80 vs 2198±560.79, p<0.01; 1092.2±91.80 vs 2291.2±154.40, p<0.001).

[0111] Figure 2 For 3dpp newborn mice, intraperitoneal injection of PBS, doxorubicin (DOX), doxorubicin (DOX) + different concentrations of calcitriol (Calcitriol) to 8dpp mice, ovarian morphology observation (a) and primordial follicle count statistics (b) chart (scale: 50 μm). Among them: a is the comparison of ovarian morphology between the control group, the doxorubicin group, and the different concentrations (25 μg / kg, 50 μg / kg) of calcitriol + doxorubicin group. The arrow points to the surviving primordial oocytes; b is the total ovarian follicle count statistics. As shown in the figure, compared with the control group, the number of surviving primordial follicles in the doxorubicin group was significantly reduced (4257±156.95 vs 209.4±65.18, p<0.001). Compared with the doxorubicin group, the number of surviving primordial follicles in the 25 μg / kg and 50 μg / kg calcitriol + doxorubicin group was significantly increased (209.4±65.18 vs 420±46.47, p<0.001; 209.4±65.18 vs 967.4±104.83, p<0.001).

[0112] Figure 3Figure 6: Ovarian morphology observation (a) and primordial follicle count (b) of 8 dpp mice injected with PBS, Cy, Cy+Calcitriol, Cy+Calcitriol+RA. (Scale bar: 50 μm). a: Comparison of ovarian morphology between control, Cy, Cy+Calcitriol, Cy+Calcitriol+RA groups. Arrow points to surviving primordial oocytes; b: Total ovarian follicle count. Compared with Cy group, Cy+Calcitriol+RA has a very significant rescue effect on Cy (1092.2±91.80 vs 3717.4±606.6, p<0.001), and the number of rescued primordial follicles is close to that of the control group; compared with Cy+Calcitriol group, the effect is better (2291.2±154.40 vs 3717.4±606.6, p<0.001).

[0113] Figure 4 Figure 7: Ovarian morphology observation (a) and primordial follicle count (b) of 8 dpp mice injected with PBS, DOX, DOX+Calcitriol, DOX+Calcitriol+RA. (Scale bar: 50 μm). a: Comparison of ovarian morphology between control, DOX, DOX+Calcitriol, DOX+Calcitriol+RA groups. Arrow points to surviving primordial oocytes; b: Total ovarian follicle count. Compared with DOX group, DOX+Calcitriol+RA has a very significant rescue effect on DOX (209.4±65.18 vs 2012.5±410.87, p<0.001); compared with DOX+Calcitriol group, the effect is better (967.4±104.83 vs 2012.5±410.87, p<0.001).

[0114] The above results show that chemotherapy drug treatment leads to a decrease in the number of primordial follicles in the ovary, and that the use of calcitriol and retinoic acid alone can significantly increase the number of surviving primordial follicles after chemotherapy drug treatment, and that the combined use of calcitriol and retinoic acid has a better effect than the use of either drug alone, indicating that both retinoic acid and calcitriol can prevent chemotherapy drugs (including cyclophosphamide and doxorubicin) from causing damage to the ovary and protect the primordial follicles in the ovary of mice.

[0115] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications, etc. made without departing from the spirit and principles of the present application should be equivalent replacement manners and should be included in the protection scope of the present application.

Claims

1. Use of vitamin D, characterized in that: For any one of the following applications (1), (2): (1) Application of vitamin D in the preparation of a protective drug for avoiding ovary damage caused by chemotherapy. (2) Application of vitamin D combined with vitamin A in the preparation of a protective drug for avoiding ovary damage caused by chemotherapy.

2. Use according to claim 1, characterized in that: The vitamin D includes vitamin D and derivatives thereof; the vitamin D derivatives include calcitriol; the vitamin A includes vitamin A and derivatives thereof; the vitamin A derivatives include retinoic acid.

3. Use according to claim 1 or 2, characterized in that: The drug for chemotherapy includes at least one of cyclophosphamide, doxorubicin, vincristine and oxaliplatin.

4. Use according to claim 1 or 2, characterized in that: The ovary damage includes at least one of DNA damage and primordial follicle apoptosis.

5. Use according to claim 1 or 2, characterized in that: The ovary is a mammalian ovary.

6. Use according to claim 5, characterized in that: The mammal includes human and mouse.

7. Use according to claim 6, characterized in that: When the mammal is human, the effective dose of the drug is calculated according to individual body weight, and the administration of calcitriol is 0.1-5000 μg / kg, and the administration of retinoic acid is 0.1-2500 mg / kg, once every other day; When the mammal is mouse, the effective dose of the drug is calculated according to individual body weight, and the administration of calcitriol is 0.1-5000 μg / kg, and the administration of retinoic acid is 0.1-2500 mg / kg, once every other day.

8. Use according to claim 7, characterized in that: When the mammal is human, the effective dose of the drug is calculated according to individual body weight, and the administration of calcitriol is 2.5-50 μg / kg, and the administration of retinoic acid is 1.5-25 mg / kg, once every other day; When the mammal is mouse, the effective dose of the drug is calculated according to individual body weight, and the administration of calcitriol is 25-50 μg / kg, and the administration of retinoic acid is 15-25 mg / kg, once every other day.

9. Use according to claim 1 or 2, characterized in that: The ovary function protective drug further comprises a pharmaceutically acceptable carrier.

10. Use according to claim 1 or 2, characterized in that: The administration mode of the ovary function protective drug is oral administration or intramuscular injection.

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