A steady-state magnetic field generating device for regulating ovarian function

Abstract

The invention discloses a steady-state magnetic field generating device for regulating ovarian function, which belongs to the field of biomedicine technology. The device comprises a magnetic field generator and a base for supporting and fixing the magnetic field generator, wherein a placing table for placing a mouse cage is arranged in the middle of the base; the magnetic field generator comprises 4 layers of main magnets and edge compensation magnets arranged in a circular array, and horizontal magnet rings are arranged above the uppermost main magnet and below the lowermost main magnet; the main magnet, the edge compensation magnet and the horizontal magnet ring are fixed on a mounting frame by epoxy resin; the steady-state magnetic field generating device of the invention treats POI mice by a magnetic field strength of 100-400mT, and the results show that the steady-state magnetic field generating device has important potential in repairing ovarian damage or improving ovarian function of POI mice, and has good clinical application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a steady-state magnetic field generating device for regulating ovarian function. Background Art

[0002] Premature ovarian insufficiency (POI) refers to a syndrome of amenorrhea, decreased estrogen levels, and loss of fertility due to ovarian failure in women before the age of 40. The incidence rate is about 1%-3%. Among women under 30 years old, the incidence rate is about 0.1%, but it increases significantly with age. POI is not only a reproductive system disease, but also a systemic health problem. The pathological characteristics of this disease are a sharp decrease in follicular reserves, impaired activation of primordial follicles, and accelerated apoptosis of granulosa cells, which lead to a sharp decline in fertility or even infertility; decreased estrogen levels lead to endocrine disorders and increase long-term health risks, such as osteoporosis and fractures, cardiovascular disease, diabetes, neurodegenerative diseases, etc. Therefore, POI is a disease with a high incidence, wide impact, and heavy burden.

[0003] The pathogenesis of POI involves multiple levels of pathological links, including oxidative stress (such as excessive accumulation of reactive oxygen species (ROS) in the follicular microenvironment can directly damage mitochondrial DNA, leading to apoptosis of granulosa cells, and thus affecting ovarian function), abnormal signaling pathways (such as excessive activation of the PI3K / Akt / mTOR pathway will accelerate the exhaustion of primordial follicles), genetic and epigenetic regulation, immune inflammation (such as activation of NLRP3 inflammasomes), extracellular matrix (ECM) hardening, and environmental toxins (such as bisphenol A and phthalates). These mechanisms are intertwined and together lead to follicular pool exhaustion and ovarian function decline. Understanding the mechanism of POI is very important for formulating strategies to prevent and treat POI.

[0004] At present, the clinical treatment principles of POI are mainly to relieve symptoms, prevent complications, improve fertility, correct metabolic disorders and improve the quality of life. The specific methods of treatment usually depend on the specific conditions of the patients, mainly including hormone replacement therapy (HRT), assisted reproductive technology (ART), etc. Although these treatment methods are very mature, they still face many problems, such as the inability to fundamentally improve the ovarian reserve function, the side effects of long-term use of drugs (breast cancer, thrombosis, etc.), etc.; emerging technologies such as stem cell transplantation and mitochondrial transplantation have certain potential, but there are problems such as complex technology, high cost, safety and ethical controversy. Therefore, it is necessary to develop a non-invasive, safe and individualized new treatment plan to improve the impaired ovarian reserve function and endocrine homeostasis of POI patients and improve the quality of life of POI patients.

[0005] Steady-state magnetic field (SMF) refers to a magnetic field whose intensity and direction do not change with time. When studying its biological effects, the steady-state magnetic field can be further divided into weak magnetic field (<1mT), medium magnetic field (1mT-1T), strong magnetic field (1T-20T) and ultra-strong magnetic field (>20T) according to its intensity. SMF may act on charged particles (such as Ca 2+ , Fe 3+ ) and free radicals, regulate cell behavior, and show unique advantages in disease treatment. There are patent applications for devices and methods for lowering blood lipids with electromagnetic fields and patent applications for magnetic field generators and their applications for regulating blood glucose levels. Studies have reported that SMF can protect mice from cisplatin-induced renal toxicity and reduce acute liver damage caused by acetaminophen overdose in mice, and also show significant therapeutic effects in orthopedic, neurological, and tumor diseases. Other studies have shown that long-term exposure to moderate static magnetic fields has a positive effect on the lifespan and health of mice, indicating that SMF may be used as an adjunct to physical therapy to reduce health risks caused by aging, thereby benefiting animals and even humans. The 1.5-3T SMF used in magnetic resonance imaging (MRI) has been proven to be safe for humans and has been used clinically for more than 40 years. Medical staff who have been exposed to <8T SMF for a long time have no significant health problems. These special properties make steady-state magnetic fields a potentially safer clinical treatment tool. The World Health Organization and the International Commission on Non-Ionizing Radiation Protection (ICNIRP) stipulate that the safe upper limit of human exposure to steady-state magnetic fields is 0.4T (long-term exposure), 2T (whole-body short-term exposure), and 8T (trunk short-term exposure). However, the role and molecular mechanism of steady-state magnetic field in ovarian aging have not been reported yet. Summary of the invention

[0006] The purpose of the present invention is to provide a steady-state magnetic field generating device for regulating ovarian function, to perform steady-state magnetic field treatment on POI mice through the steady-state magnetic field generating device, to study the relationship between the steady-state magnetic field and the ovarian function of POI mice, and to explore the clinical application potential of the steady-state magnetic field in repairing ovarian damage.

[0007] The purpose of the present invention can be achieved through the following technical solutions:

[0008] A steady-state magnetic field generating device for regulating ovarian function comprises a magnetic field generator and a base. A placing table for placing a mouse cage is arranged in the middle of the base, and the placing table is driven by a lifting mechanism.

[0009] The magnetic field generator includes four layers of main magnets and edge compensation magnets arranged in a circular array, and the edge compensation magnets are arranged outside the circumference of the main magnets. Horizontal magnet rings are arranged above the top main magnet and below the bottom main magnet.

[0010] The main magnet, edge compensation magnets, and horizontal magnet ring are fixed to the mounting frame with epoxy resin.

[0011] Furthermore, the mounting frame includes several layers of aluminum alloy annular brackets fixed by non-magnetic bolts, and the main magnet, edge compensation magnet and horizontal magnet ring are fixed on the aluminum alloy annular brackets at corresponding positions by epoxy resin.

[0012] Furthermore, 32 main magnets and 16 edge compensation magnets are arranged in each layer between the horizontal magnet rings.

[0013] Furthermore, the upper surface of the main magnet is an N pole, the lower surface is an S pole, and the magnetic field directions of the edge compensation magnet, the horizontal magnet ring and the main magnet are consistent.

[0014] Furthermore, a plurality of evenly distributed drop holes are provided on the placement platform, and the drop holes are used for dropping the excrement of the mice.

[0015] A method for using a steady-state magnetic field generating device for regulating ovarian function, comprising the following steps: placing a mouse suffering from POI on a placing table of the steady-state magnetic field generating device, adjusting the position of the mouse to the center of the magnetic field generator and performing steady-state magnetic field treatment, the magnetic field strength of the magnetic field generator is 100-400mT, the treatment time is 8-24h / day, and the treatment lasts for 1-3 weeks in total.

[0016] Furthermore, steady-state magnetic field treatment was used to improve the weight loss rate, estrous cycle, ovarian size, ovarian organ index, follicle development, in vitro oocyte development, MII stage oocyte ROS level, MII stage oocyte mitochondrial membrane potential level and MII stage oocyte Ca2+ level in POI mice. 2+ level.

[0017] Beneficial effects of the present invention:

[0018] The steady-state magnetic field generating device of the present invention treats POI mice with a magnetic field strength of 100-400mT, significantly improving the ovarian reserve function of POI mice, promoting the development of primordial follicles, primary follicles and antral follicles, and reducing atretic follicles. In addition, the steady-state magnetic field also significantly improves the in vitro oocyte maturation rate of POI mice, and also has a positive effect on the estrous cycle of POI mice. From a mechanistic point of view, the ROS (oxidative stress) level in the MII stage oocytes of POI mice is significantly increased, and the mitochondrial membrane potential is also significantly reduced, and the steady-state magnetic field can reverse these abnormal phenomena. These results show that the steady-state magnetic field generating device and its related tool devices have important potential in repairing ovarian damage or improving the ovarian function of POI mice, and have good clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The present invention will be further described below in conjunction with the accompanying drawings.

[0020] Figure 1 It is a structural schematic diagram of the steady-state magnetic field generating device of the present invention;

[0021] Figure 2 is a cross-sectional view of the steady-state magnetic field generating device of the present invention;

[0022] Figure 3 It is a structural schematic diagram of the main magnet of the present invention;

[0023] Figure 4 It is a schematic diagram of the structure of the edge compensation magnet of the present invention;

[0024] Figure 5 It is a structural schematic diagram of the horizontal magnet ring of the present invention;

[0025] Figure 6 It is a top view of the first layer structure of the steady-state magnetic field generating device of the present invention;

[0026] Figure 7 The present invention Figure 2 A partial enlarged view of the middle A;

[0027] Figure 8 It is a structural schematic diagram of the base portion of the steady-state magnetic field generating device of the present invention;

[0028] Fig. 9 It is a schematic diagram of the structure of the gear and the annular rack of the steady-state magnetic field generating device of the present invention;

[0029] Fig.10 is a statistical graph of weight changes of all groups of mice in Example 2 of the present invention;

[0030] Fig.11 These are representative images of estrous cycle smears of mice in each group in Example 2 of the present invention for four consecutive days;

[0031] Fig.12 This is a statistical chart of the estrous cycle results of all groups of mice in Example 2 of the present invention;

[0032] Fig.13 This is a comparison diagram of the ovarian tissue sizes of all groups of mice in Example 2 of the present invention;

[0033] Fig.14 is the statistics of the ovarian organ index of all groups of mice in Example 2 of the present invention;

[0034] Fig.15 It is a micrograph of HE staining analysis of ovarian tissue sections of all groups of mice in Example 2 of the present invention;

[0035] Fig.16It is a statistical diagram of the development of follicles at various levels in the ovarian tissue sections of all groups of mice in Example 2 of the present invention;

[0036] Fig.17 It is a micrograph of the in vitro oocyte culture and development of all groups of mice in Example 2 of the present invention;

[0037] Fig.18 This is a statistical chart of the results of in vitro oocyte culture and development of all groups of mice in Example 2 of the present invention;

[0038] Fig.19 It is a fluorescence micrograph of the ROS level determination of the MII stage oocytes of all groups of mice in Example 2 of the present invention;

[0039] Fig. 20 This is a statistical graph showing the ROS levels in the MII stage oocytes of all groups of mice in Example 2 of the present invention;

[0040] Fig.21 It is a fluorescence micrograph of the measurement of mitochondrial membrane potential level of MII stage oocytes of all groups of mice in Example 2 of the present invention;

[0041] Fig. 22 This is a statistical graph of the measurement of mitochondrial membrane potential levels of MII stage oocytes of all groups of mice in Example 2 of the present invention;

[0042] Fig.23 is the Ca in the MII stage oocytes of all groups of mice in Example 2 of the present invention. 2+ Fluorescence micrographs of the assays;

[0043] Fig.24 is the Ca in the MII stage oocytes of all groups of mice in Example 2 of the present invention. 2+ Statistical chart of the measurement;

[0044] Fig.25 It is a GO analysis bubble chart obtained from transcriptomic sequencing analysis of ovarian tissues of the CP group and the magnetic field treatment group in Example 3 of the present invention. DETAILED DESCRIPTION

[0045] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0046] Example 1: This example provides a steady-state magnetic field generating device for regulating ovarian function. Figure 1-Figure 2, including a magnetic field generator and a base 2, wherein a placement table 22 for placing a squirrel cage is arranged in the middle of the base 2, and the placement table 22 is driven by a lifting mechanism.

[0047] The magnetic field generator includes a main magnet 1 and an edge compensation magnet 11, which are arranged in 4 layers, and each layer is arranged in a circular array with 32 main magnets 1 and 16 edge compensation magnets 11, and the edge compensation magnets 11 are arranged outside the circumference of the main magnet 1. Horizontal magnet rings 12 are arranged above the uppermost main magnet 1 and below the lowermost main magnet 1.

[0048] The magnetic field generator also includes a mounting frame, which includes several layers of aluminum alloy annular brackets 13 fixed by non-magnetic bolts; the main magnet 1, the edge compensation magnet 11 and the horizontal magnet ring 12 are fixed on the aluminum alloy annular bracket 13 at corresponding positions by epoxy resin.

[0049] Among them, see Figure 3-Figure 5 The specifications of the main magnet 1 are 50mm (length) × 20mm (width) × 10mm (thickness), the specifications of the edge compensation magnet 11 are 25mm (length) × 10mm (width) × 5mm (thickness), and the specifications of the horizontal magnet ring 12 are 290mm (outer diameter) × 20mm (width) × 10mm (thickness); the upper surface of the main magnet 1 is the N pole (or S pole), and the lower surface is the S pole (or N pole). The magnetic field direction of the edge compensation magnet 11 and the horizontal magnet ring 12 is consistent with that of the main magnet 1, which suppresses axial magnetic field leakage.

[0050] The shortest distance from the inner side surface of each layer of edge compensation magnets 11 to the outer side surface of the corresponding main magnet 1 is 15 mm, the interlayer spacing of each layer of main magnets 1 (for example, the distance from the bottom surface of the first layer of main magnets 1 to the upper surface of the second layer of main magnets 1) is 50 mm, and the interlayer spacing of each layer of edge compensation magnets 11 (for example, the distance from the bottom surface of the first layer of edge compensation magnets 11 to the upper surface of the second layer of edge compensation magnets 11) is 60 mm.

[0051] See also Figure 6 The circumferential radius of each layer of main magnet 1 (the distance from the geometric center of the main magnet 1 to the center of the annular array) is 290 mm, and the circumferential radius of each layer of edge compensation magnet 11 (the distance from the geometric center of the edge compensation magnet 11 to the center of the annular array) is 320 mm.

[0052] See also Figure 2 and Figure 7 The base 2 is arranged below the lowest aluminum alloy annular bracket 13 and is used to support and fix the magnetic field generator.

[0053] See also Figure 7-Figure 8A plug ring 201 is connected to the position of the base 2 on the lower surface of the bottom aluminum alloy ring bracket 13, and a slot 202 is provided above the base 2 at a position corresponding to the plug ring 201. The plug ring 201 is inserted into the slot 202. The position of the aluminum alloy ring bracket 13 can be fixed by the cooperation between the plug ring 201 and the slot 202.

[0054] See also Figure 2 and Figure 7-Figure 9 The lifting mechanism includes a supporting platform 21 , and a plurality of evenly arranged positioning plates 211 are connected between the base 2 and the supporting platform 21 , and the positioning plates 211 are used to fix the position of the supporting platform 21 .

[0055] The placement table 22 is rectangular and is disposed above the supporting platform 21 .

[0056] The placement platform 22 is provided with a plurality of evenly distributed drop holes 221 for dropping the excrement of the mice. A rectangular through hole 222 is provided in the middle of the support platform 21 corresponding to the placement platform 22, and the through hole 222 allows the excrement of the mice to fall into the collection box 4.

[0057] An annular rack 23 is provided above the support platform 21 near the periphery, and the annular rack 23 is used to make the four gears 24 rotate synchronously. An annular slide bar 231 is connected to the position of the lower surface of the annular rack 23 corresponding to the support platform 21, and a slide groove 232 is provided above the support platform 21 corresponding to the position of the annular slide bar 231. The annular slide bar 231 is slidably connected in the slide groove 232. Through the cooperation between the annular slide bar 231 and the slide groove 232, the position of the annular rack 23 can be limited, and the annular rack 23 can be rotated.

[0058] Gears 24 are meshed respectively at the middle positions of the four sides of the placement platform 22 above the annular rack 23 , and a rotating rod 25 is connected to the middle of the gear 24 near the placement platform 22 . The rotating rod 25 is used to drive the first lifting arm 26 to rotate.

[0059] A first lifting arm 26 is provided at the end of the rotating rod 25 away from the gear 24, and the rotating rod 25 is connected to one end of the first lifting arm 26. A second lifting arm 27 is provided on the side of the first lifting arm 26 close to the placing table 22. The end of the first lifting arm 26 away from the rotating rod 25 is connected to one end of the second lifting arm 27, and the other end of the second lifting arm 27 is connected to the middle part of the side of the placing table 22. Through the cooperation of the first lifting arm 26 and the second lifting arm 27, the placing table 22 can be lifted, so that the squirrel cage can be adjusted to a specified position.

[0060] The upper surface of the support platform 21 is connected with fixed blocks 251 at positions corresponding to the rotating rods 25. The rotating rods 25 are rotatably connected in the fixed blocks 251. The fixed blocks 251 are used to fix the positions of the rotating rods 25. A motor 28 is connected to the side of one of the gears 24 away from the placement platform 22. The motor 28 is fixed to the positioning plate 211 and is used to drive one of the gears 24 to rotate.

[0061] In specific use, the mouse cage is placed on the placement table 22, and the motor 28 is started after placement. The motor 28 drives one of the gears 24 to rotate, and one of the gears 24 will drive the annular rack 23 to rotate when rotating. The annular rack 23 will drive the other three gears 24 to rotate when rotating, so that the four gears 24 rotate synchronously and the rotation directions of the four gears 24 are consistent. When the four gears 24 rotate, they will drive the rotating rod 25 to rotate to rotate the first lifting arm 26. During the rotation process, the first lifting arm 26 will lift the second lifting arm 27 and the placement table 22, and make the second lifting arm 27 rotate synchronously. Under the rotation action of the first lifting arm 26 and the second lifting arm 27, the height of the placement table 22 can be adjusted. After the mouse cage is adjusted to the specified position, the mouse is placed in the center of the magnetic field generator and the operation experiment can be carried out.

[0062] The device occupies a relatively small volume in actual use, which can save space. At the same time, the first lifting arm 26 and the second lifting arm 27 are connected to the middle of the four sides of the placement table 22, which can ensure the stability of the placement table 22.

[0063] The four corners of the placing platform 22 are connected to symmetrically arranged external connecting plates 3, and the ends of the external connecting plates 3 are rotatably connected to clamping plates 31, which are used to clamp and fix the lower part of the squirrel cage.

[0064] A spring 32 is connected between the clamping plate 31 and the inner wall of the placement platform 22 , and the spring 32 is used to provide a clamping force to the clamping plate 31 .

[0065] The upper surface of the clamping plate 31 is connected with an arc plate 33 , and the arc plate 33 cooperates with each other to facilitate placing the mouse cage into the placement table 22 .

[0066] In specific use, when placing the mouse cage inside the placement table 22, the bottom of the mouse cage will contact the arc plate 33, and the mouse cage will be pressed down after the contact. Under the action of the arc plate 33, the clamping plate 31 will rotate to a position close to the inner wall of the placement table 22, thereby enlarging the space in the middle of the placement table 22. After the space in the middle of the placement table 22 is enlarged, the mouse cage can continue to fall, so that the lower surface of the mouse cage contacts the upper surface of the placement table 22. During the rotation of the clamping plate 31, the spring 32 will contract and store the restoring force after being squeezed. The restoring force acts on the clamping plate 31 and is transmitted to the sides of the four corners of the lower part of the mouse cage through the clamping plate 31, so that the mouse cage can be clamped and fixed, and the position of the mouse cage can be limited to prevent the mouse cage from shifting and tipping over due to collision and other reasons during the activity of the mouse, thereby avoiding affecting the experiment.

[0067] A guard plate 29 is connected to the position of the gear 24 and the annular rack 23 on the upper surface of the support platform 21. The gear 24, the annular rack 23 and the motor 28 are located between the base 2 and the guard plate 29. The guard plate 29 is used to protect the gear 24, the annular rack 23 and the motor 28 to prevent debris from affecting the gear 24, the annular rack 23 and the motor 28, while ensuring the cleanliness of the device.

[0068] A collecting box 4 is slidably connected to the lower side of the base 2 corresponding to the lower side of the supporting platform 21. The collecting box 4 is corresponding to the lower side of the through hole 222. The collecting box 4 is used to collect the excrement of the mice. A pull rod 41 is connected to the middle of the side of the collecting box 4 away from the base 2. The pull rod 41 is used to take the collecting box 4 out of the base 2.

[0069] Example 2: This example provides a method for using a steady-state magnetic field generating device for regulating ovarian function and functional verification:

[0070] 1. Construction of POI mouse model: Three-week-old mice (Kunming mice KM) were used as research subjects. After adaptive feeding for 1 week, POI was induced in mice by subcutaneous and intraperitoneal injection of cisplatin for 14 consecutive days (cisplatin was weighed and dissolved in physiological saline at a dose of 1.5 mg / g body weight for each mouse, and 0.1 mL was administered).

[0071] During the experiment, mice with POI were divided into two groups. One group was placed in a cage and the cage was placed as follows. Figure 1The steady-state magnetic field treatment was carried out in the steady-state magnetic field generator shown in the figure. The magnetic field strength of the magnetic field generator was 200mT (100-400mT is acceptable), and the treatment time was 8h / day (8-24h / day is acceptable), for 3 consecutive weeks (1-3 weeks is acceptable); another group was placed in a device with the same structure as the steady-state magnetic field generator but without magnetization, as a sham exposure group. Another group of mice was adaptively fed for 1 week, and then injected with normal saline (NaCl, 0.1mL / mouse) through the abdominal cavity for 14 consecutive days as a control group, and raised normally.

[0072] At the end of the experiment, the mice were killed, and the observation indexes and terminal indexes of mice in each group were tested.

[0073] 2. Observation indicators: POI mice may suffer from metabolic disorders (such as changes in fat distribution and abnormal glucose and lipid metabolism) due to decreased estrogen levels, which in turn causes abnormal body weight fluctuations. Weight loss may indicate systemic metabolic abnormalities associated with ovarian dysfunction, so the weight of each group of mice is monitored. The core feature of POI is reproductive endocrine disorders caused by ovarian dysfunction, which manifests as irregular estrous cycles (such as prolonged cycles, irregularity) or stagnation in the estrus period. The estrous cycle disorder directly reflects the abnormal secretion of ovarian hormones (estrogen, progesterone), so continuous vaginal smears were performed on each group of mice in the last 10 days of steady-state magnetic field treatment to observe whether the estrous cycles of each group of mice are regular.

[0074] 3. Terminal indicators: Ovarian size and weight are intuitive indicators for evaluating ovarian reserve function. The ovaries of POI mice shrink significantly due to follicle exhaustion or development stagnation, resulting in a reduction in ovarian volume and a decrease in organ index. POI is manifested by a decrease in primordial follicles, indicating a decrease in ovarian reserve function; a decrease in the number of growing follicles (primary, secondary, and antral follicles) and an increase in the proportion of atretic follicles, indicating follicular maturation disorders. A large number of atretic follicles are typical pathological features of premature ovarian failure; ovarian interstitial fibrosis, thinning or disintegration of the granulosa cell layer, and reduced or no corpus luteum formation indicate ovulation disorders. Therefore, it is necessary to take the ovarian tissues of each group of mice for HE staining (hematoxylin-eosin staining), count follicles, and evaluate the follicle development of each group of mice. The in vitro maturation rate of oocytes in POI mice is reduced, indicating a decrease in oocyte quality and maturation disorders, so mouse oocytes are cultured in vitro. The decline in oocyte quality and maturation disorders may be related to various factors, including but not limited to increased oxidative stress and mitochondrial dysfunction. Therefore, the quality of MII stage oocytes was evaluated by detecting the level of reactive oxygen species (ROS) and mitochondrial membrane potential in MII stage oocytes. 2+ Abnormal levels of Ca2+ can usually cause mitochondrial dysfunction or endoplasmic reticulum stress, which in turn affects oocyte quality. Therefore, Ca2+ in MII oocytes can also be detected. 2+Finally, differential gene expression analysis can be used to identify key signaling pathways related to POI (such as follicle development, apoptosis, oxidative stress, DNA repair pathways, etc.), revealing the molecular mechanism of ovarian function decline.

[0075] In summary, the ovarian function of each group of mice was evaluated overall according to the weight change rate, estrous cycle and other indicators; the ovarian function of each group of mice was evaluated morphologically according to the ovarian size, pathological sections, the number of follicles at all levels and other indicators; the ovarian function of each group of mice was evaluated according to the in vitro maturation rate of oocytes, the level of ROS in MII oocytes, the level of mitochondrial membrane potential in MII oocytes, and the Ca 2+ The ovarian function of each group of mice was functionally evaluated at the level; finally, transcriptomics sequencing was used to reveal abnormal gene expression and explore the molecular mechanism.

[0076] The specific experimental process is as follows:

[0077] a. Maintain the same temperature (22±2℃), humidity (50±10%) and light cycle (12h light / 12h dark) in the breeding environment, provide adequate food and drinking water, and weigh the mice at 9:00 am every day starting from the first day of the experiment using an electronic balance and record the weight to the nearest 0.1g, and draw a curve of weight change over time, such as Fig.10 As shown, steady-state magnetic field treatment can improve the body weight loss rate of POI mice.

[0078] b. Estrus cycle smear: During the last 10 days of the magnetic plate treatment experiment, the estrus of each group of mice was continuously recorded. Vaginal smears were performed on mice at 9:00 am every day. The specific steps are as follows: First, 20 µL of saline was sucked up with a gun tip, and it was gently blown twice at the vaginal opening of the mouse. Then the saline was sucked out and evenly applied to the marked slide, and it was allowed to air dry naturally. After air drying, the slide was fixed in a 95% Vol alcohol solution for 10 minutes, then stained in a 0.23wt% alkaline methylene blue dye solution for 5 minutes, then washed with tap water, and observed after air drying again. In the smear, three types of cells can be observed, namely white blood cells, nucleated epithelial cells, and anucleated keratinocytes. The estrus cycle of mice can be determined by analyzing the cell types in the smear. Fig.11Representative images of estrous cycle smears of each group of mice for four consecutive days are shown, including diestrus, proestrus, estrus, and metestrus. The main cell type in proestrus (Proestrus) is nucleated epithelial cells, which are round or oval in shape, with obvious nuclei. A small number of cornified epithelial cells may appear, and white blood cells are few or absent; the main cell type in estrus (Estrus) is cornified epithelial cells, which are anucleated and polygonal in shape, with irregular edges. There are very few or no nucleated epithelial cells and white blood cells, a clean background, and uniform cell distribution; the main cell types in metestrus (Metestrus) are white blood cells and cornified epithelial cells, among which the number of white blood cells increases, the number of cornified epithelial cells decreases, and there are a small number of nucleated epithelial cells. The cell types are chaotic and the background is relatively messy; the main cell type in diestrus (Diestrus) is white blood cells, which are numerous and dense. Cornified epithelial cells and nucleated epithelial cells are very few or absent, and the background is dark and the cells are densely distributed. The results of 10 consecutive days of vaginal smears were analyzed and statistically analyzed by Fig.12 It can be seen that the mice in the control group have a complete estrous cycle; the estrous cycle of the mice in the POI sham-exposure group is not complete, and most of them are in the estrus period; the estrous cycle of the mice in the POI plus magnetization group after steady-state magnetic field treatment shows regular changes. Steady-state magnetic field treatment is used to improve the estrous cycle of POI mice.

[0079] c. Collect blood samples: Anesthetize the mouse. After the anesthesia takes effect, use your left hand to firmly fix the mouse, and at the same time, moderately press the mouse's eyeball to make the eyeball protrude as much as possible. Then, use your right hand to cut the mouse's whiskers, and then use elbow forceps to quickly remove the mouse's eyeball. At this time, let the blood drip naturally into the 1.5mL EP tube. After the blood has drained out, use the cervical dislocation method to kill the mouse to complete the entire blood sample collection process.

[0080] d. Obtaining ovarian tissue: After completing the blood sample collection and killing the mice, you can proceed to obtain ovarian tissue. First, dissect the mice and accurately locate the "Y"-shaped uterus of the mice during the dissection process. Then, explore upward along the uterus to find the ovarian tissue. Once found, use forceps to carefully remove the fat around the ovarian tissue in a blunt separation manner. After successfully separating the fat, take photos of the removed ovarian tissue for preservation. The relevant photos are as follows: Fig.13 and Fig.14 As shown, it can be seen that steady-state magnetic field treatment can improve the ovarian size and ovarian organ index of POI mice.

[0081] e. Section HE staining: The ovarian tissue fixed in 4wt% formaldehyde solution for 24h was dehydrated with gradient ethanol (70%Vol, 80%Vol, 90%Vol, 95%Vol, 100%Vol), transparentized with xylene, and embedded in paraffin. Continuous sections with a thickness of 5μm were prepared and dried at 60℃ for 2h. The staining process included: xylene dewaxing (10min×2 times), gradient ethanol rehydration (100%Vol, 95%Vol, 85%Vol, 75%Vol), hematoxylin staining for 5min, running water washing for 10min, 1wt% hydrochloric acid ethanol solution differentiation for 30s, tap water washing for 15min, and eosin staining for 2min. Finally, it was dehydrated with gradient ethanol (75%Vol, 85%Vol, 95%Vol, 100%Vol, 2min each), dehydrated with xylene, and permeabilized. Drop neutral resin near the tissue, then slowly cover it with a coverslip. If the resin overflows onto the coverslip, use xylene to clean it. After sealing, bake the slide in a 65°C oven for 2 hours, then take it out and store it at room temperature. Use a fully automatic high-resolution panoramic imaging analysis system to obtain HE staining images of the sections, such as Fig.15 , count the number of follicles at each level, including primordial follicles, primary follicles, secondary follicles, antral follicles and atretic follicles, such as Fig.16 . The primordial follicle is surrounded by a single layer of flat granulosa cells. The oocyte nucleus is large and round, the chromatin is sparse, and the granulosa cells are flat and few in number. The primary follicle is surrounded by a single layer of cuboidal granulosa cells. The granulosa cells change from flat to cubic, and the oocyte volume increases slightly. The secondary follicle consists of a primary oocyte surrounded by multiple layers of granulosa cells. The zona pellucida begins to appear, the number of granulosa cell layers increases, and the oocyte volume increases further. The follicular cavity appears in the granulosa cell layer of the antral follicle. The oocyte is located in the cumulus ovary. The follicular cavity is obvious, and the granulosa cells are divided into cumulus cells and parietal granulosa cells. The follicles of the atretic follicles degenerate, the granulosa cell layer disintegrates, the oocyte nucleus condenses or fragments, the follicle structure is disordered, the cell nucleus is darkly stained, and the follicular cavity collapses. Counting the number and proportion of follicles at each level in the mouse ovary can provide an important basis for studying ovarian function and related diseases. Fig.15 and Fig.16 The results showed that steady-state magnetic field promoted the development of primordial follicles, primary follicles and antral follicles, reduced atretic follicles, and significantly improved the ovarian reserve function of POI mice. Steady-state magnetic field treatment can improve the follicular development of POI mice.

[0082] f. Oocyte in vitro culture: The maturation process of mouse oocytes includes the following key stages: In the GV stage, the oocyte nucleus (germinal vesicle, GV) is clearly visible, the nuclear membrane is intact, and the chromatin is not condensed; in the GVBD stage, the nuclear membrane ruptures, the chromatin begins to condense, and enters the meiotic I stage. GVBD is a sign that the oocyte resumes meiosis; in the MI stage, the chromosomes are arranged on the equatorial plate, the spindle is formed, and it enters the metaphase of meiosis I; in the MII stage, the first meiotic division is completed, the first polar body (PB1) is expelled, and it enters the metaphase of meiosis II. The MII stage oocyte has the ability to be fertilized and is a mature oocyte. Use DMEM-F12 culture medium and M16 culture medium to make oocyte culture dishes, cover them with mineral oil, and place them at 37°C and 5% Vol CO 2 After the ovaries were removed from the mice by dissection, the ovarian tissue was placed in a DMEM-F12 droplet and minced with a blade until the ovaries were in a minced state. Then 1 mL of DMEM-F12 culture medium was added to dilute the tissue suspension. GV stage oocytes were collected under a stereomicroscope using a 150 μm-caliber glass oviduct. The collected oocytes were transferred to fresh DMEM-F12 culture medium and washed repeatedly to remove granulosa cells and tissue fragments. The washed oocytes were transferred to a pre-equilibrated DMEM-F12 culture droplet and incubated at 37°C and 5% VolCO 2 After 30 min of equilibration under the same conditions, the oocytes were transferred to M16 culture microdrops. After 2 h (GVBD stage) and 14 h (MII stage) of culture, Fig.17 , observe and count the maturation of oocytes, and count the GVBD rate of oocytes ((the number of oocytes in the GVBD stage / the total number of oocytes) × 100%), which reflects the ability of oocytes to resume meiosis. A decrease in the GVBD rate may indicate oocyte developmental arrest; the polar body extrusion situation, namely the PB1 rate ((the number of oocytes in the MII stage / the total number of oocytes) × 100%), reflects the in vitro maturation ability of oocytes. A decrease may indicate a decrease in oocyte quality. Fig.18 According to the experimental needs, oocytes at different stages are collected for subsequent functional testing and statistics. Fig.17 and Fig.18 The results showed that steady-state magnetic field treatment significantly increased the in vitro oocyte maturation rate of POI mice and improved the in vitro oocyte development of POI mice.

[0083] g. Determination of ROS levels in MII stage oocytes: Judging oocyte quality based on the fluorescence level of reactive oxygen species (ROS) in mouse oocytes is an important method for assessing the oxidative stress state and developmental potential of oocytes. ROS is a byproduct of cell metabolism. An appropriate amount of ROS is necessary for cell function, but excessive ROS can cause oxidative stress, damage the DNA, proteins and lipids of oocytes, and thus affect their quality and fertilization ability. High ROS levels are a sign of oocyte aging, premature ovarian failure (POI) and exposure to environmental toxins. H2DCFDA (2',7'-dichlorodihydrofluorescein diacetate, a universal oxidative stress indicator) powder was dissolved in DMSO to prepare a 1mM storage solution, which was stored at -20°C in the dark after aliquoting. Dilute with M16 culture medium to a final concentration of 1μM before use, transfer oocytes to M16 culture droplets containing 10μM H2DCFDA working solution, and place at 37°C, 5% Vol CO 2 Incubate in the incubator in the dark for 30 minutes. Dissolve Hoechst 33342 (fluorescent dye for labeling DNA) powder in PBS to prepare a 1 mg / mL storage solution, and store it in the dark at -20°C after aliquoting. Dilute it with M16 culture medium to a final concentration of 5 μg / mL before use. Transfer the H2DCFDA-stained oocytes to M16 culture microdrops containing 5 μg / mL Hoechst 33342 working solution and place them at 37°C and 5% Vol CO 2 Incubate in the incubator in the dark for 10 minutes. After staining, wash the oocytes repeatedly with preheated M16 culture medium to remove unbound probes. Transfer the stained oocytes to a 35 mm glass culture dish, add an appropriate amount of M16 culture medium, and observe the fluorescence intensity of the oocytes (green fluorescence) using a 488 nm excitation wavelength and a 525 nm emission wavelength under a laser confocal microscope (Zeiss LSM 980), and observe the cell nucleus (blue fluorescence) using a 350 nm excitation wavelength and a 461 nm emission wavelength. Fig.19 The fluorescence intensity of the fluorescence images was analyzed using ImageJ software. Fig. 20 , ROS levels are expressed as mean fluorescence values, and brighter fluorescence intensity indicates higher ROS levels. Fig.19 and Fig. 20 The results showed that steady-state magnetic field treatment reduced the elevated ROS level in MII stage oocytes of POI mice.

[0084] h. Determination of mitochondrial membrane potential level of MII stage oocytes: Mitochondrial membrane potential is an important method to evaluate the energy metabolism status and developmental potential of oocytes. Mitochondria are the energy factories of oocytes, and their membrane potential is the core indicator of mitochondrial function. Normal MMP is the basis of oocyte maturation, fertilization and embryonic development, while decreased MMP is usually associated with oocyte aging, oxidative stress and apoptosis. Low mitochondrial membrane potential level is a manifestation of impaired mitochondrial function, insufficient energy supply and poor quality of oocytes. Dissolve TMRE (tetramethylrhodamine ethyl ester) powder with DMSO to prepare a 1mM storage solution, and store it at -20℃ in the dark after aliquoting. Dilute with M16 culture medium to a final concentration of 1μM before use, transfer the oocytes to M16 culture droplets containing 1μM TMRE working solution, and place them at 37℃, 5%Vol CO 2 Incubate in the incubator in the dark for 30 minutes. Dissolve Hoechst 33342 powder in PBS to prepare a 1 mg / mL storage solution, store it in aliquots at -20°C in the dark. Dilute it with M16 culture medium to a final concentration of 5 μg / mL before use, transfer the TMRE-stained oocytes to M16 culture microdrops containing 5 μg / mL Hoechst 33342 working solution, and place them at 37°C, 5% Vol CO 2 Incubate in the incubator in the dark for 10 min. After staining, wash the oocytes repeatedly with preheated M16 culture medium to remove unbound probes. Transfer the stained oocytes to a 35 mm glass culture dish, add an appropriate amount of M16 culture medium, and observe the TMRE fluorescence intensity (red fluorescence) using a 549 nm excitation wavelength and a 574 nm emission wavelength under a laser confocal microscope (Zeiss LSM 980), and use a 350 nm excitation wavelength and a 461 nm emission wavelength to observe the cell nucleus (blue fluorescence). Fig.21 The fluorescence intensity of the fluorescence images was analyzed using ImageJ software. Fig. 22 The mitochondrial membrane potential level is expressed as the average fluorescence value, and the brighter the fluorescence intensity, the higher the mitochondrial membrane potential level. Fig.21 and Fig. 22 The results showed that steady-state magnetic field treatment improved the decreased mitochondrial membrane potential level of MII stage oocytes in POI mice.

[0085] i. MII stage oocyte Ca 2+ Level determination: Calcium ions are key signaling molecules for oocyte maturation, fertilization, and embryonic development, and their dynamic changes are crucial to the function of oocytes. 2+ Participates in regulating the resumption and completion of meiosis. High-quality oocytes have normal Ca 2+ Homeostatic and dynamic changes that respond to fertilization signals and support embryonic development, low-quality oocyte Ca 2+Abnormal homeostasis, basal Ca 2+ The level is too high. Fluo-3AM (Ca 2+ Fluorescent chelator) powder was dissolved in DMSO to prepare a 1mM stock solution, which was stored at -20°C in the dark after aliquoting. Before use, it was diluted with M16 culture medium to a final concentration of 1μM, and the oocytes were transferred to M16 culture microdrops containing 1μM Fluo-3AM working solution and placed at 37°C and 5% Vol CO 2 Incubate in the incubator in the dark for 30 minutes. Dissolve Hoechst 33342 powder in PBS to prepare a 1 mg / mL storage solution, store it in aliquots at -20°C in the dark. Dilute it with M16 culture medium to a final concentration of 5 μg / mL before use, transfer the oocytes stained with Fluo-3AM to M16 culture microdrops containing 5 μg / mL Hoechst 33342 working solution, and place it at 37°C, 5% Vol CO 2 Incubate in the incubator in the dark for 10 minutes. After staining, wash the oocytes repeatedly with preheated M16 culture medium to remove unbound probes. Transfer the stained oocytes to a 35 mm glass culture dish, add an appropriate amount of M16 culture medium, and observe the Fluo-3 fluorescence intensity (green fluorescence) using a 488 nm excitation wavelength and a 525 nm emission wavelength under a laser confocal microscope (Zeiss LSM 980), and observe the cell nucleus (blue fluorescence) using a 350 nm excitation wavelength and a 461 nm emission wavelength. Fig.23 The fluorescence intensity of the fluorescence images was analyzed using ImageJ software, as shown in Fig.24 As shown, Ca 2+ The level is expressed as the average fluorescence value. The brighter the fluorescence intensity, the higher the Ca content in the oocyte. 2+ Higher level. Fig.23 and Fig.24 The results showed that steady-state magnetic field treatment improved Ca2+ in oocytes at the MII stage of POI mice. 2+ level.

[0086] Example 3: RNA-Seq analysis and GO analysis were performed on the ovarian tissues of each group of mice:

[0087] GO analysis classified the differentially expressed genes into three categories: biological process (BP), molecular function (MF) and cellular component (CC), and identified significantly related functional entries through enrichment analysis.

[0088] Bubble charts are a common method for visualizing enrichment results, which can intuitively display the significance of enriched pathways, the number of genes, and functional distribution. The horizontal axis is the enrichment multiple or gene ratio; the vertical axis is the GO entry name; the bubble size represents the number of genes, and the larger the bubble, the more genes are enriched in the entry; the bubble color represents the p-value, and the darker the color (such as red), the more significant the enrichment (the smaller the p-value).

[0089] like Fig.25 As shown in the figure, through the analysis of the GO map, it was found that the differentially expressed genes between the POI sham magnetic exposure group and the POI plus magnetic group were significantly enriched in biological processes such as inflammatory response and lipid metabolism. The steady-state magnetic field may improve the impaired ovarian function of POI mice through related pathways such as inflammatory response and lipid metabolism.

[0090] It should be noted that, in this article, terms such as "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or apparatus.

[0091] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A steady-state magnetic field generating device for regulating ovarian function, comprising a magnetic field generator and a base for supporting and fixing the magnetic field generator, characterized in that: A placement table for placing a squirrel cage is provided in the middle of the base; the magnetic field generator includes 4 layers of main magnets and edge compensation magnets arranged in a circular array, and horizontal magnet rings are provided above the top main magnet and below the bottom main magnet; the main magnet, edge compensation magnet and horizontal magnet ring are fixed on the mounting frame by epoxy resin; the magnetic field strength of the magnetic field generator is 100-400mT; The upper surface of the main magnet is an N pole or an S pole, and the lower surface is an S pole or an N pole. The magnetic field directions of the edge compensation magnet and the horizontal magnet ring are consistent with those of the main magnet, thereby suppressing axial magnetic field leakage. The mouse cage is placed in a steady-state magnetic field generating device for steady-state magnetic field treatment, the treatment time is 8-24h / day, and it continues for 1-3 weeks.

2. A steady-state magnetic field generating device for regulating ovarian function according to claim 1, characterized in that: 32 main magnets and 16 edge compensation magnets are arranged in each layer between the two horizontal magnet rings, and the edge compensation magnets are arranged outside the circumference of the main magnets.

3. A steady-state magnetic field generating device for regulating ovarian function according to claim 1, characterized in that: The upper surface of the main magnet is an N pole, and the lower surface is an S pole. The magnetic field directions of the edge compensation magnet, the horizontal magnet ring and the main magnet are consistent.

4. A steady-state magnetic field generating device for regulating ovarian function according to claim 1, characterized in that: The mounting frame comprises several layers of aluminum alloy annular brackets fixed by non-magnetic bolts, and the main magnet, edge compensation magnet and horizontal magnet ring are fixed on the aluminum alloy annular brackets at corresponding positions by epoxy resin.

5. The steady-state magnetic field generating device for regulating ovarian function according to claim 1, characterized in that: The placing platform is provided with a plurality of evenly distributed dropping holes for dropping mouse excrement.

6. A steady-state magnetic field generating device for regulating ovarian function according to claim 1, characterized in that: The placement table is driven by a lifting mechanism, which includes a supporting platform. A plurality of evenly arranged positioning plates are connected between the base and the supporting platform, and the positioning plates are used to fix the supporting platform.

7. A steady-state magnetic field generating device for regulating ovarian function according to claim 6, characterized in that: An annular rack is arranged above the support platform near the periphery, gears are meshed at the positions above the annular rack corresponding to the middle parts of the four sides of the placement platform, and a rotating rod is connected to the middle part of the gear near the placement platform; A first lifting arm is provided at the end of the rotating rod away from the gear, and the rotating rod is connected to one end of the first lifting arm. A second lifting arm is provided on the side of the first lifting arm close to the placing table, and the end of the first lifting arm away from the rotating rod is connected to one end of the second lifting arm, and the other end of the second lifting arm is connected to the middle part of the side of the placing table.

8. The steady-state magnetic field generating device for regulating ovarian function according to claim 7, characterized in that: A motor is connected to one side of the gears away from the placement platform, and the motor is fixed to the positioning plate.

9. A steady-state magnetic field generating device for regulating ovarian function according to claim 8, characterized in that: The four corners of the placing table are connected to symmetrically arranged external connecting plates, the ends of the external connecting plates are rotatably connected to clamping plates, a spring is connected between the clamping plates and the inner wall of the placing table, and an arc-shaped plate is connected to the upper surface of the clamping plates; When placing the squirrel cage inside the placing table, the bottom of the squirrel cage will contact the arc plate, and the squirrel cage will be pressed down after the contact. Under the action of the arc plate, the clamping plate will rotate to a position close to the inner wall of the placing table, thereby enlarging the space in the middle of the placing table. After the space in the middle of the placing table is enlarged, the squirrel cage can continue to fall, so that the lower surface of the squirrel cage contacts the upper surface of the placing table. During the rotation of the clamping plate, the spring will be squeezed. After being squeezed, the spring will contract and store restoring force. The restoring force acts on the clamping plate and is transmitted to the sides of the four corners below the squirrel cage through the clamping plate, so that the squirrel cage can be clamped and fixed.

10. A steady-state magnetic field generating device for regulating ovarian function according to claim 9, characterized in that: Place the mouse cage on the placing table. The first lifting arm will lift the second lifting arm and the placing table during the rotation process, and make the second lifting arm rotate synchronously. Under the rotation action of the first lifting arm and the second lifting arm, the height of the placing table where the mouse cage is placed is adjusted. After the mouse cage is adjusted to the specified position, the mouse is located in the center of the magnetic field generator.

Images