Endometrial stem cell medicine and preparation process thereof

Abstract

The present invention relates to the field of biomedicine technology, and more specifically, to an endometrial stem cell drug and its preparation process. The present invention optimizes the culture medium components of endometrial stem cells to obtain a more optimal culture medium; the endometrial stem cell culture medium comprises DMEM, VEGF, and β-ecdysterone. The concentration of the VEGF is 20 ng / ml, and the concentration of the β-ecdysterone is 20 ng / ml. Endometrial stem cells prepared using the culture medium of the present invention have a stronger proliferation ability and better anti-ovarian aging and ovarian repair effects.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an endometrial stem cell drug and a preparation process thereof. Background Art

[0002] Premature ovarian failure (POF) is a condition in which women experience ovarian dysfunction before the age of 40. The incidence of POF is 1-3% among adult women. Currently, there are many treatments for POF and ovarian loss, such as stem cells or active peptides.

[0003] For example, prior art CN118909045 A discloses the use of endometrial stem cells in ovarian repair. Specifically, the combined use of endometrial stem cells and the PDPA-N-5 polypeptide for the treatment of chemotherapy-induced premature ovarian failure has shown significant efficacy in promoting ovarian function recovery and holds great promise for future applications.

[0004] For example, prior art CN118121680A discloses a composition for repairing oocytes in female ovaries, its preparation method, and application. The composition comprises stem cells and / or polypeptides that upregulate P-AMPK expression, wherein the stem cells are endometrial stem cells. Endometrial stem cells were obtained by separation and preparation, and active peptides with anti-aging effects were isolated from Polygonum multiflorum. The stem cells and active peptides were used together to treat an ovarian aging model and were found to effectively promote follicular secretion and E2 secretion, with good anti-ovarian aging and ovarian repair effects.

[0005] For example, prior art CN118453654A discloses a stem cell anti-ovarian aging agent and preparation method, comprising: culturing endometrial mesenchymal stem cells in stem cell culture medium A to a density of 80%-85%, switching to culture medium B for a further 24-36 hours, collecting the supernatant, and extracting the exosomes therefrom; wherein, stem cell culture medium A is composed of a mixture of the following raw materials by mass fraction: 10% FBS, 88-89.5% DMEM / F12 medium by mass fraction, and 0.5-2% vitamin B1 by mass fraction; stem cell culture medium B is composed of a mixture of the following raw materials by mass fraction: 99-99.5% DMEM / F12 medium by mass fraction and 0.5-1% vitamin B1 by mass fraction; the exosomes are then combined with endometrial stem cell membrane sheets to prepare an anti-ovarian aging preparation. The preparation prepared by the present invention can increase the protein content in the exosomes and prevent premature ovarian failure.

[0006] The activity of stem cells is a key factor affecting the efficacy of stem cell therapy. Therefore, it is necessary to optimize the stem cell culture medium to obtain stem cells with stronger proliferation ability and higher activity. Therefore, the present invention will provide a better culture medium and, based on this, provide an endometrial stem cell drug and its preparation process. Summary of the Invention

[0007] The present invention first provides an endometrial stem cell medicine to achieve the above-mentioned purpose, wherein the medicine comprises endometrial stem cells.

[0008] Preferably, the culture medium of the endometrial stem cells comprises DMEM, VEGF and β-ecdysterone.

[0009] Preferably, the concentration of VEGF is 20 ng / ml, and the concentration of β-ecdysterone is 20 ng / ml.

[0010] Secondly, the present invention also provides a preparation process of the above-mentioned drug, which comprises the following steps:

[0011] (1) Obtain endometrial tissue under sterile conditions, enzymatically hydrolyze, and obtain a single-cell suspension;

[0012] (2) Culture was performed using a culture medium containing DMEM, VEGF, and β-ecdysterone.

[0013] Preferably, trypsin I and type I collagenase are used for enzymatic hydrolysis in step (1).

[0014] Finally, the present invention provides an application of the above-mentioned medicine, which is used to prepare a medicine for treating premature ovarian failure or repairing ovarian egg cells.

[0015] The beneficial effects of the above technical solution of the present invention are as follows:

[0016] The present invention optimizes the culture medium components of endometrial stem cells to obtain a more optimal culture medium. Endometrial stem cells prepared by culturing using the culture medium of the present invention have stronger proliferation ability and better anti-ovarian aging and ovarian repair effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the expression level of P-AMPK in different experimental groups. DETAILED DESCRIPTION

[0018] In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0019] Example 1 Isolation and Preparation of Stem Cells

[0020] 1) Obtaining endometrial stem cells: The entire endometrium plus 5 mm of myometrium were harvested under sterile conditions, away from the lesion site;

[0021] 2) Digestion: Cut the endometrium obtained in step 1) into 1mm pieces 3 To the left and right fragments, add 0.2% by weight of trypsin I (100,000 U / g, purchased from Sigma) and 0.2% by weight of collagenase type I (200,000 U / g, purchased from Sigma), mix, and digest in a 37°C water bath for 2 h. After digestion, rinse in physiological saline and centrifuge. The resulting precipitate is washed again with physiological saline, pipetted evenly, and passed through a 40 μm cell sieve. The filtrate is collected, centrifuged, and the supernatant is discarded to obtain a cell pellet.

[0022] 3) Primary culture: Add DMEM + 20 ng / ml VEGF + 20 ng / ml β-ecdysterone culture medium to the cell pellet obtained in step 2) to obtain a single cell suspension. 4 The cells were seeded at a concentration of 100 cells / mL in a culture dish and cultured for 5 h. The non-adherent cells in the culture dish were transferred to a new culture dish, and the culture medium was replaced and cultured for another 5 days. The adherent cells were collected and labeled as P0 cells. P0 endometrial stem cells were collected for surface antigen detection, and the marker antigen detection results are shown in Table 1.

[0023] Table 1 Antigen identification results

[0024] CD73 CD90 CD34 99.3％* 97.0％* 0.018％

[0025] 4) When the cell fusion rate reaches about 85-90%, the P0 cells are subcultured to obtain P3 cells for standby use.

[0026] Example 2

[0027] Except that the culture medium in step 3) was DMEM+40 ng / ml VEGF, the other conditions were the same as those in Example 1.

[0028] Example 3

[0029] Except that the culture medium in step 3) was DMEM+40 ng / ml β-ecdysterone, the other conditions were the same as those in Example 1.

[0030] Example 4

[0031] Except that DMEM medium was used as the culture medium in step 3), the other conditions were the same as those in Example 1.

[0032] Example 5

[0033] The endometrial stem cells isolated from Examples 1, 2, 3 and 4 were concentrated at 2×10 4 pieces / cm 2 The cells were seeded at a density of 100 μg / mL into T25 culture flasks. After 10 days, the number of clones with more than 10 cells was observed and recorded under a microscope. The relative number of cells in each group was calculated based on the cell number in Example 4. The results showed that the cells obtained in Example 1 contained more endometrial stem cells with proliferation ability (P < 0.001), as shown in Table 1.

[0034] Table 1 Relative multiples of endometrial stem cells prepared in different examples

[0035]

[0036]

[0037] Compared with Example 4: **P<0.01, ***P<0.001.

[0038] Example 6 Experiment on premature ovarian failure model in mice

[0039] Referring to the method of CN118121680A, ICR mice with normal estrous cycle were randomly divided into model group, stem cell transplantation group and blank control group, with 30 mice in each group. The model group and stem cell transplantation group were injected with 0.1mL of cyclophosphamide solution (120mg / kg) and busulfan solution (12mg / kg) intraperitoneally once. Vaginal secretion smears were gently taken with cotton swabs every day, and Papanicolaou staining was performed to observe the changes in vaginal exfoliated cells. When continuous estrus was observed for 10 consecutive days, it was considered that premature ovarian failure modeling was successful. On the 14th day of modeling, the stem cell transplantation group was injected with 0.1mL of endometrial stem cell suspension (1x10 8 The modeling group and the blank control group were injected with an equal amount of normal saline via the tail vein each time.

[0040] Reproductive hormone assays: Serum from each group of mice was obtained by tail snipation, and follicle-stimulating hormone (FSH) and estradiol (E2) levels were measured by chemiluminescence. All sera were assayed using the same batch of reagents. The results are shown in Table 2.

[0041] Table 2 Comparison of FSH and E2 in each group of mice

[0042]

[0043]

[0044] Compared with the model group: **P<0.01, ***P<0.001.

[0045] As can be seen from the results in Table 2, compared to the control group, the mouse model group had decreased E2 secretion and increased FSH secretion, indicating ovarian failure in the mice. After the stem cell transplantation treatment of Examples 1-4, the mice had increased E2 secretion and decreased FSH secretion, approaching the hormone secretion levels of normal rats, indicating that the combination group can effectively improve ovarian function. Furthermore, compared to the model group, Example 1 had a better effect (p < 0.001).

[0046] The mice in each group were sacrificed by cervical dislocation and bilateral ovarian tissue was obtained. Ovarian tissue was frozen and sectioned and observed under a fluorescence microscope (x400) for histological changes. The results are shown in Table 3.

[0047] Table 3 Comparison of follicle numbers in each group

[0048]

[0049]

[0050] Compared with the model group: **P<0.01, ***P<0.001.

[0051] As can be seen from the results in Table 3, the total number of follicles in the model group was significantly lower than that in the blank control group. However, after treatment with the combined group, the total number of follicles in the model group was significantly increased, and was basically similar to the number of follicles in normal mice, indicating that it has a good effect in restoring ovarian function. Compared with the model group, Example 1 has a better effect (p < 0.001).

[0052] Example 4 Western blotting assay

[0053] Referring to the method of CN118121680A, Western blotting was used to detect the expression of P-AMPK protein in the model group and the stem cell transplantation group of Example 1-Example 4. The cells of the above groups were taken after 48 hours of action, and the cells were collected by centrifugation after trypsin digestion. After RIPA lysis, the supernatant was collected by centrifugation at 12000r / min at 4℃. After BCA protein quantification, the protein was boiled with protein loading buffer and frozen at -20℃ for later use. A 12% polyacrylamide gel was prepared, and the sample amount was 20μg for SDS-PAGE electrophoresis. After the electrophoresis, the membrane was wet-transferred (PVDF) and blocked with skim milk powder for 2 hours. The primary antibody was P-AMPK (1:500) or anti-GAPDH (1:5000), and the secondary antibody was horseradish peroxide-labeled goat anti-rabbit antibody (1:10000). After adding ECL luminescent liquid, the imaging system was used for analysis. The results are shown in FIG. Figure 1 shown.

[0054] Depend on Figure 1It can be seen that compared with the control group, the P-AMPK level in the injury model group was significantly decreased. The stem cells prepared in Examples 1 to 4 can significantly increase the P-AMPK level after treatment, and the effect of Example 1 is the most significant (P<0.001).

[0055] It should be understood that the above are only some embodiments of the present invention. It should be pointed out that for ordinary technicians in this field, other variations and improvements can be made without departing from the creative concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A method for preparing an endometrial stem cell drug, characterized in that: The preparation method comprises the following steps: (1) Under sterile conditions, endometrial tissue was obtained and enzymatically hydrolyzed to obtain a single-cell suspension of endometrial stem cells; (2) Culture was performed using a medium consisting of DMEM, 20 ng / ml VEGF, and 20 ng / ml β-ecdysterone.

2. The preparation method according to claim 1, characterized in that In the step (1), trypsin I and collagenase type I are used for enzymatic digestion.

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