Application of C646 in preparation of in-vitro activator for primordial follicles

Abstract

The invention relates to the technical field of biology, and discloses application of C646 in preparation of a primordial follicle in-vitro activator. The invention particularly relates to application of C646 in preparation of an mTOR agonist and / or an AKT signal channel key protein agonist. The C646 promotes the growth and differentiation of precursor granulosa cells into granulosa cells by improving the phosphorylation level of key protein on an mTOR / PI3K signal channel. In addition, C646 can also improve expression of AKT and further promote activation of primordial follicles, so that the number of primary follicles is increased. When the concentration of the C646 in the culture medium is 4 mu M, the primary follicles are obviously increased, which indicates that the 4 mu M C646 can obviously promote the activation of the original follicles. Meanwhile, in-vivo renal envelope transplantation experiments find that the ovary can stimulate activation of primordial follicles after being subjected to C646 short treatment.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of C646 in preparing an in vitro primordial follicle activator. Background Art

[0002] Assisted reproductive technology is the most effective means of assisting pregnancy at this stage. Currently, assisted reproductive technology must have a prerequisite for female patients: there must be follicles in the ovaries that can respond to gonadotropin. However, patients with premature ovarian failure do not have enough follicles in their ovaries that can respond to gonadotropin. Patients with special groups such as those who have undergone radiotherapy / chemotherapy after cancer and those who have cryopreserved or removed ovaries also face the problem of infertility.

[0003] In order to solve the fertility problems of this type of patients, new assisted reproductive technologies and new means of utilizing follicle resources must be developed. In fact, patients with premature ovarian failure still have a certain number of primordial follicles in their ovaries, so whether these primordial follicles can be used to help patients with premature ovarian failure give birth to offspring has attracted attention.

[0004] Multiple studies have confirmed the key role of the mTOR signaling pathway in the activation of primordial follicles, that is, when the mTOR in the single layer of precursor granulosa cells located outside the oocyte in the primordial follicle is upregulated, the precursor granulosa cells grow and differentiate into granulosa cells. At the same time, the level of the substance KITL secreted by it increases, which activates the PI3K signaling pathway in the oocyte by binding to the receptor (KIT) on the oocyte membrane, thereby promoting the activation of primordial follicles.

[0005] According to the above-mentioned known primordial follicle activation regulation mechanism, after clinical transformation and application research, the in vitro activation of primordial follicles (abbreviated as in vitro activation, IVA) technology was invented, which successfully helped patients with premature ovarian failure to give birth to healthy next generations, bringing hope to the fertility problems of patients with similar problems mentioned above. IV A uses effective means to activate primordial follicles in vitro with drugs to form primary follicles, and then waits for them to grow to a stage that can respond to hormones, and then performs traditional assisted reproduction. This technology has successfully helped some infertile patients successfully give birth to the next generation. However, in the existing IVA clinical strategy, the safety and success rate of related drugs are still a major hidden danger. At the same time, patients need to undergo multiple laparoscopic surgeries, which take a long time and increase the pain of patients. Therefore, it is extremely important to develop a more efficient in vitro activation system for primordial follicles.

[0006] Based on this, the present invention rationally develops an application of C646 in preparing an in vitro activator for primordial follicles. Summary of the invention

[0007] The purpose of the present invention is to provide the use of C646 in the preparation of an in vitro primordial follicle activator. C646 can increase the phosphorylation level of key proteins in the PI3K / mTOR signaling pathway and enhance the expression of VEGFA / VEGFR1, further promoting the activation of primordial follicles.

[0008] To achieve the above purpose, the following technical solutions are used:

[0009] The invention discloses an application of C646 in preparing an in vitro activator for primordial follicles.

[0010] C646 of the present invention activates primordial follicles by increasing the mTOR level and / or activating mTOR.

[0011] The mTOR described in the present invention is the mTOR protein in the ovary.

[0012] The C646 of the present invention activates primordial follicles by increasing the AKT level and / or activating AKT.

[0013] The AKT described in the present invention is AKT in the ovary.

[0014] The invention discloses an application of C646 in the preparation of a drug for promoting the transformation of primordial follicles into primary follicles.

[0015] The application of C646 described in the present invention in preparing drugs for assisted reproduction.

[0016] The drug for activating primordial follicles in vitro of the present invention comprises C646 and acceptable adjuvants and / or auxiliary agents.

[0017] In the use or preparation provided by the present invention, the concentration of C646 is 1-5 μM.

[0018] In the use or preparation provided by the present invention, the optimal concentration of C646 is 4 μM.

[0019] The beneficial effects of the present invention are as follows:

[0020] 1. The present invention adds C646 to culture mouse ovaries in vitro to promote the activation of primordial follicles present therein. Studies have found that the total protein levels and phosphorylated forms of key proteins in the mTOR and PI3K / AKT signaling pathways in mouse ovarian tissues are increased. In the process of adding C646 to culture newborn mouse ovaries, the total number of follicles remains unchanged but the number of activated follicles increases. The experiment also found that when the concentration of C646 was 1 μM, there was no significant change in the total number of follicles (P=0.8605), the number of primordial follicles (P=0.4748), and the number of primary follicles (P=0.1568) compared with the control group; when the concentration of C646 was 2 μM, 3 μM, and 5 μM, there was no significant change in the total number of follicles (2 μM: P=0.6948; 3 μM: P=0.9212; 5 μM: P=0.3283), the number of primordial follicles did not change significantly (2 μM: P=0.4748; 3 μM: P=0.0614; 5 μM: P=0.0717), and the number of primary follicles increased significantly (2 μM: P=0.0074; 3 μM: P=0.0025; 5 μM: P=0.0034). When the concentration of C646 was 4 μM, there was no significant difference in the total number of follicles between the control group and the C646 group (P=0.9013). Compared with the control group, the number of primordial follicles in the 4 μM C646 group was significantly reduced (P=0.0334), and the number of primary follicles was significantly increased (P=0.0009), indicating that 4 μM C646 significantly promoted the activation of primordial follicles. P<0.05 was considered significant.

[0021] 2. The addition of 4 μM C646 to culture mouse ovaries in vitro can promote the increase in the levels of key proteins in the VEGFA / VEGFR1 signaling pathway in mouse ovarian tissue, and the activation of primordial follicles is inhibited after inhibiting VEGFR1.

[0022] 3. The present invention provides the use of a small molecule drug C646 in the preparation of a preparation for primordial follicle activation. Experiments show that C646 promotes the activation of primordial follicles by increasing the level of mTOR or by increasing the level of VEGFA or VEGFR1, thereby increasing the number of primary follicles. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 :HE staining of 2dpp ovaries cultured in vitro for 3 days. (Upper left: whole ovary of control group, lower left: partial magnification of control group; upper right: whole ovary of 4μM C646 group, lower right: partial magnification of ovary of 4μM C646 group.)

[0024] Figure 2 :Results of whole ovary counting after 2dpp ovaries were cultured in 1μM C646 medium for 3 days. (Left: Total follicle count; Middle: Primordial follicle count; Right: Primary follicle count.)

[0025] Figure 3 :Results of whole ovary counting after 2dpp ovaries were cultured in 2μM C646 medium for 3 days. (Left: Total follicle count; Middle: Primordial follicle count; Right: Primary follicle count.)

[0026] Figure 4 :Results of whole ovary counting after 2dpp ovaries were cultured in 3μM C646 medium for 3 days. (Left: Total follicle count; Middle: Primordial follicle count; Right: Primary follicle count.)

[0027] Figure 5 :Results of whole ovary counting after 2dpp ovaries were cultured in 4μM C646 medium for 3 days. (Left: Total follicle count; Middle: Primordial follicle count; Right: Primary follicle count.)

[0028] Figure 6 :Results of whole ovary counting after 2dpp ovaries were cultured in 5μM C646 medium for 3 days. (Left: Total follicle count; Middle: Primordial follicle count; Right: Primary follicle count.)

[0029] Figure 7 :2dpp ovarian culture for 2 days to detect changes in mTOR and PI3K signaling pathway proteins in mouse ovarian tissue. (Left: changes in mTOR and p-mTOR protein levels; middle: changes in Foxo3a and p-Foxo3a protein levels; right: changes in AKT and p-AKT protein levels.)

[0030] Figure 8 :2dpp ovarian culture 2 days after the detection of p-FOXO3a nuclear export in mouse ovarian tissue. From left to right: DAPI staining (labeling cell nuclei), DDX4 staining (labeling oocytes), Foxo3a staining, three-channel merge image, and local magnification image.

[0031] Fig. 9 : Changes in transcription and protein expression of the VEGFA / VEGFR signaling pathway were detected after 2 days of ovarian culture at 2 dpp (A: changes in Vegfa transcription levels; B: changes in Vegfr transcription levels; C and D: changes in VEGFA / VEGFR protein levels).

[0032] Fig.10 : HE staining of ovarian sections 2 weeks after renal capsule transplantation after 2 dpp ovaries were cultured in vitro for 30 min (upper left: whole ovary of control group, lower left: local magnified image of control group; upper right: whole ovary of 4 μM C646 group, lower right: local magnified image of ovary of 4 μM C646 group). DETAILED DESCRIPTION

[0033] The present invention discloses the application of C646 in the preparation of an in vitro activator of primordial follicles. The present invention is further described in detail below in conjunction with examples and drawings, but the embodiments of the present invention are not limited thereto. Unless otherwise defined, all scientific and technical terms used in the present invention have the same meanings as those commonly understood by technicians in the technical field to which the present invention relates. In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it cannot be understood as a limitation on the scope of implementation of the present invention.

[0034] The C646 described in the present invention is a histone acetyltransferase inhibitor.

[0035] Example 1

[0036] Application of C646 in the preparation of in vitro activators of primordial follicles.

[0037] (1) Preparation of ovarian basal culture medium:

[0038] Add 0.5 g DMEM, 0.53 g F12, 0.357 g HEPES, 0.2438 g NaHCO3, 0.006 g penicillin, and 0.005 g streptomycin to a volume of 100 mL with ultrapure water.

[0039] (2) Preparation of 4 mM C646:

[0040] Take 2 mg of C646 and add 1.1225 mL of dimethyl sulfoxide solvent to obtain the product.

[0041] (3) 1.2 mL of ovarian basal medium and 1.2 μL of 4 mM C646 reagent were added to a 6-well cell culture plate, and the plate was preheated at 37° C. in a cell culture incubator to obtain a culture medium with a concentration of 4 μM C646;

[0042] (4) The ovaries of 2-day-old mice were separated and cleaned in sterile PBS and then transferred to the culture medium of step (3) for culture. After 3 days of in vitro culture of the ovaries of 2-day-old mice, the ovarian tissues were collected, sliced ​​and observed for the development of follicles at various levels and the number of follicles was counted.

[0043] Example 2

[0044] Application of C646 in the preparation of drugs for promoting the transformation of primordial follicles to primary follicles.

[0045] (1) Preparation of ovarian basal culture medium:

[0046] Add 0.5 g DMEM, 0.53 g F12, 0.357 g HEPES, 0.2438 g NaHCO3, 0.006 g penicillin, and 0.005 g streptomycin to a volume of 100 mL with ultrapure water.

[0047] (2) Preparation of 4 mM C646:

[0048] Take 0.2 mg of C646 and add 1.225 mL of dimethyl sulfoxide solvent to obtain the product.

[0049] (3) 1.2 mL of ovarian basal medium and 1.2 μL of 4 mM C646 reagent were added to a 6-well cell culture plate, and the plate was preheated at 37° C. in a cell culture incubator to obtain a culture medium with a concentration of 4 μM C646;

[0050] (4) The ovaries of 2-day-old mice were separated and cleaned in sterile PBS and then transferred to the culture medium of step (3) for culture. After culturing the ovaries of 2-day-old mice in vitro for 30 minutes, the ovaries activated in vitro were transplanted under the renal capsule of immunodeficient mice, and the ovarian tissue was allowed to continue to grow under the renal capsule for 14 days. The ovarian tissue was collected and sliced ​​to observe the development of follicles at all levels.

[0051] Example 3

[0052] Use of C646 in the preparation of mTOR agonists and / or AKT signaling pathway key protein agonists.

[0053] (1) Preparation of ovarian basal culture medium:

[0054] Add 0.5 g DMEM, 0.53 g F12, 0.357 g HEPES, 0.2438 g NaHCO3, 0.006 g penicillin, and 0.005 g streptomycin to a volume of 100 mL with ultrapure water.

[0055] (2) Preparation of 4 mM C646:

[0056] Take 2 mg of C646 and add 1.225 mL of dimethyl sulfoxide solvent to obtain the product.

[0057] (3) 1.2 mL of ovarian basal medium and 1.2 μL of 4 mM C646 reagent were added to a 6-well cell culture plate, and the plate was preheated at 37° C. in a cell culture incubator to obtain a culture medium with a concentration of 4 μM C646;

[0058] (4) The ovaries of 2-day-old mice were separated and cleaned in sterile PBS and then transferred to the culture medium of step (3) for culture. After culturing the ovaries of 2-day-old mice in vitro for 2 days, the ovaries were collected for detecting changes in signaling pathways such as mTOR / AKT.

[0059] (5) Extract proteins and use protein immunoblotting to detect the levels of mTOR, p-mTOR, AKT, p-AKT, FOXO3a, and p-FOXO3a proteins in mouse ovarian tissue; and collect and detect mouse ovarian tissue, and detect the nuclear export of p-FOXO3a in oocytes by tissue sectioning and immunofluorescence staining.

[0060] Example 4

[0061] Application of C646 in the preparation of drugs for assisted reproduction.

[0062] C646 is prepared into a conventional pharmaceutical preparation according to conventional pharmaceutical excipients and used in assisted reproductive applications.

[0063] In order to further verify the effectiveness of the present invention, the inventors conducted a series of verification experiments, as follows:

[0064] 1. C646 promotes primordial follicle activation in newborn mouse ovaries

[0065] 1. Experimental Materials

[0066] 2-day-old ICR female mice;

[0067] 2. Experimental reagents

[0068] (1) Reagent A: Ovarian basal culture medium. The specific formula is as follows. The preparation process must be performed aseptically.

[0069] DMEM 0.5g F12 0.53g HEPES 0.357g <![CDATA[NaHCO3]]> 0.2438g penicillin 0.006g Streptomycin 0.005g Ultrapure water Make up to 100mL

[0070] (2) Reagent B: 4 mM C646 (solvent is dimethyl sulfoxide);

[0071] Take 2 mg of C646 and add 1.1225 mL of dimethyl sulfoxide solvent to obtain the product.

[0072] (3) Reagent C: sterile PBS.

[0073] 3. Experimental methods

[0074] (1) 1.2 mL of reagent A and 1.2 μL of reagent B were added to a 6-well plate in sequence, and the plate was placed in a cell culture incubator and preheated at 37° C. to obtain a culture medium with a concentration of 4 μM C646; according to the above method, culture medium with concentrations of 1 μM, 2 μM, 3 μM, and 5 μM was also prepared;

[0075] (2) After the ovaries of 2-day-old mice were separated in reagent C, they were transferred to the culture medium of step 1 for culture. The control group was 1.2 mL DMEM / F12 culture medium supplemented with 1.2 μL DMSO; the experimental group was 1.2 mL DMEM / F12 culture medium supplemented with 1.2 μL of 1 mM, 2 mM, 3 mM, and 5 mM C646 (solvent: DMSO). After culturing the ovaries of 2-day-old mice in vitro for 3 days, the ovarian tissues were collected, sliced, and the development of follicles at all levels was observed and the number of follicles was counted.

[0076] Table 1 Effect of C646 culture concentration on total follicles, primary follicles and primordial follicles

[0077]

[0078]

[0079] (3) Two days later, the levels of mTOR, p-mTOR, AKT, p-AKT, FOXO3a, and p-FOXO3a proteins in mouse ovarian tissues were collected and detected;

[0080] (4) Two days later, the p-FOXO3a nuclear export in the mouse ovarian tissue was collected and detected;

[0081] (5) The ovaries of 2-day-old mice were separated in reagent C and transferred to the culture medium of step 1 for culture. After culturing the ovaries of 2-day-old mice in vitro for 30 minutes, the ovaries activated in vitro were transplanted under the renal capsule of immunodeficient mice and the ovarian tissue was allowed to continue to grow under the renal capsule for 14 days. The ovarian tissue was collected and sliced ​​to observe the development of follicles at all levels.

[0082] 4. Experimental results:

[0083] (1) After culturing the ovaries of 2-day-old mice in vitro for 3 days, the ovarian tissues were collected and sliced ​​to observe the development of follicles at all levels and to count the number of follicles. Figure 1 The results of the statistical follicle numbers are shown in Tables 1 and Figure 2-Figure 6Hematoxylin staining showed that after adding different concentrations of C646 for culture, when the concentration of C646 was 1μM, there was no significant change in the total number of follicles (P=0.8605), the number of primordial follicles (P=0.4748), and the number of primary follicles (P=0.1568) compared with the control group; when the concentration of C646 was 2μM, 3μM, and 5μM, there was no significant change in the total number of follicles (2μM: P=0.6948; 3μM: P=0.9212; 5μM: P=0.3283), the number of primordial follicles did not change significantly (2μM: P=0.4748; 3μM: P=0.0614; 5μM: P=0.0717), and the number of primary follicles increased significantly (2μM: P=0.0074; 3μM: P=0.0025; 5μM: P=0.0034). When the concentration of C646 was 4 μM, there was no significant difference in the total number of follicles between the control group and the C646 group (P=0.9013). Compared with the control group, the number of primordial follicles in the 4 μM C646 group was significantly reduced (P=0.0334), and the number of primary follicles was significantly increased (P=0.0009), indicating that 4 μM C646 significantly promoted the activation of primordial follicles. P<0.05 was considered significant.

[0084] (2) Two days later, the levels of mTOR, p-mTOR, AKT, p-AKT, FOXO3a, and p-FOXO3a proteins in mouse ovarian tissues were collected and detected. Figure 7 As shown, mTOR and p-mTOR increased significantly, and AKT and p-AKT also increased significantly, indicating that C646 promoted the expression of PI3K / mTOR signaling pathway.

[0085] (3) Two days later, the p-FOXO3a nuclear export in the mouse ovarian tissue was collected and detected. Figure 8 As shown, after the addition of C646, the number of oocytes with p-FOXO3a exuclear increased in the 4 μM C646 group, indicating that the primordial follicles were activated.

[0086] (4) Two days later, the VEGFA and VEGFR transcription and protein levels in the mouse ovarian tissues were collected and detected. Fig. 9 As shown in the results, the transcription level of VEGFA and VEGFR increased significantly (P<0.0001) and the protein level increased significantly (P<0.0001), indicating that 4 μM C646 promoted the expression of VEGFA / VEGFR signaling pathway. P<0.05 was considered significant.

[0087] (5) After culturing the ovaries of two-day-old mice in vitro for 30 minutes, the activated ovaries were transplanted under the renal capsule of immunodeficient mice and the ovarian tissue was allowed to continue to grow under the renal capsule for 14 days. The ovarian tissue was collected and sliced ​​to observe the development of follicles at all levels. The results were as follows: Fig.10As shown, 30 min of C646 treatment significantly promoted follicle development, scale bar: 200 μm, indicating that 4 μM C646 can activate primordial follicles in vitro.

[0088] Although the present invention has been described in detail above by general explanation and specific implementation methods, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, these modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. Application of C646 in the preparation of in vitro activator for primordial follicles.

2. The use according to claim 1, characterized in that: Use of C646 in the preparation of mTOR agonists and / or AKT signaling pathway key protein agonists.

3. The use according to claim 2, characterized in that: The mTOR is the mTOR protein in the ovary.

4. The use according to claim 1, characterized in that: Activation of primordial follicles by increasing AKT levels and / or activating AKT.

5. The use according to claim 2, characterized in that: The AKT is AKT in the ovary.

6. Use of C646 as claimed in claim 1 in the preparation of a drug for promoting the transformation of primordial follicles to primary follicles.

7. A use of C646 as claimed in claim 1 in the preparation of drugs for assisted reproduction.

8. A drug for activating primordial follicles in vitro, characterized in that: Comprising C646 and acceptable excipients and / or adjuvants.

9. The use according to any one of claims 1 or 2 or 6 or 7 or the medicine according to claim 8, characterized in that: The concentration of C646 was 4 μM.

10. The use according to any one of claims 6 or 7 or the medicine according to claim 8, characterized in that: The dosage form of the drug includes but is not limited to injection.

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