Endometrial stem cell medicine and preparation process thereof

Abstract

The invention relates to the technical field of biological medicines, in particular to an endometrial stem cell medicine and a preparation process thereof. By optimizing culture medium components of the endometrial stem cells, a better culture medium is obtained; the culture medium of the endometrial stem cells comprises DMEM (Dulbecco Modified Eagle Medium), VEGF (Vascular Endothelial Growth The concentration of the VEGF is 20 ng / m < l >, and the concentration of the beta-ecdysterone is 20 ng / m < l >. The endometrial stem cells cultured and prepared by using the culture medium disclosed by the invention are stronger in multiplication capacity, and have better ovarian aging resistance 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 medicine and a preparation process thereof. Background Art

[0002] The phenomenon of ovarian dysfunction in women before the age of 40 is called premature ovarian failure. The incidence of POF accounts for 1-3% of adult women. Currently, there are many methods for treating premature ovarian failure and ovarian loss, such as using stem cells or active peptides.

[0003] For example, the prior art CN118909045 A discloses an application of endometrial stem cells in ovarian repair. Specifically, it provides a method of using endometrial stem cells in combination with PDPA-N-5 polypeptide to treat premature ovarian failure caused by chemotherapy, which has a significant effect of promoting the recovery of ovarian function and has a good application prospect.

[0004] For example, the prior art CN118121680A discloses a composition for repairing oocytes in female ovaries, and its preparation method and application, wherein the composition comprises stem cells and / or polypeptides that upregulate the expression of P-AMPK, and 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 ovarian aging models, and it was found that the secretion of follicles and E2 secretion could be effectively promoted, and the secretion had good anti-ovarian aging and ovarian repair effects.

[0005] For example, the prior art CN118453654A discloses a stem cell anti-ovarian aging reagent and preparation method, including: when endometrial mesenchymal stem cells are cultured to a density of 80%-85% using stem cell culture medium A, culture medium B is changed to continue culturing for 24-36h, and then the supernatant is collected to extract the exosomes therein; wherein, the stem cell culture medium A is composed of the following raw materials in mass fraction: 10% FBS by mass, 88-89.5% DMEM / F12 culture medium by mass, and 0.5-2% vitamin B1 by mass; the stem cell culture medium B is composed of the following raw materials in mass fraction: 99-99.5% DMEM / F12 culture medium by mass, and 0.5-1% vitamin B1 by mass; the exosomes are compounded 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 exosomes and resist premature ovarian failure.

[0006] The activity of stem cells is a key factor affecting the therapeutic effect of stem cells. Therefore, it is necessary to optimize the culture medium of stem cells 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, perform enzymatic hydrolysis, and obtain a single cell suspension;

[0012] (2) Culture was performed using a 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 the application of the above-mentioned medicine, which is used for preparing medicine for treating premature ovarian failure or repairing ovarian oocytes.

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

[0016] The present invention obtains a better culture medium by optimizing the culture medium components of endometrial stem cells. The endometrial stem cells prepared by culture 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 more clear, 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: Obtain the entire endometrial layer + 5 mm of myometrium under sterile conditions, far away from the lesion site;

[0021] 2) Digestion: Cut the endometrium obtained in step 1) into 1 mm 3 To the left and right fragments, add 0.2% trypsin I (100,000 U / g, purchased from Sigma) and 0.2% type I collagenase (200,000 U / g, purchased from Sigma) by weight, mix, and digest in a 37°C water bath for 2 h. After digestion, rinse in physiological saline, centrifuge, and wash the obtained precipitate with physiological saline, blow and evenly, pass through a 40 μm cell sieve, collect the filtrate, centrifuge and discard the supernatant to obtain a cell precipitate;

[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 inoculated into a culture dish at a concentration of 100 / mL and cultured for 5 h. The non-adherent cells in the culture dish were transferred to a new culture dish. The culture medium was replaced and cultured for another 5 days. The adherent cells were collected and marked as P0 cells. P0 endometrial stem cells were taken for surface antigen detection. 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 use.

[0026] Example 2

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

[0028] Example 3

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

[0030] Example 4

[0031] Except that the culture medium in step 3) is DMEM, the other conditions are 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 to 2×10 4 Pieces / cm 2 The cells were inoculated at a density of 100 μg / mL into a T25 culture flask. 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 number of cells 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 embodiments

[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 0.1 mL of cyclophosphamide solution (120 mg / kg) and busulfan solution (12 mg / kg) intraperitoneally once, and 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 was successfully modeled. On the 14th day of modeling, the stem cell transplantation group was injected with 0.1 mL of the 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 determination: The serum of each group of mice was obtained by tail snip method, and the levels of follicle stimulating hormone (FSH) and estradiol (E2) of mice were detected by chemiluminescence method. All serums were detected by 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] From the results in Table 2, it can be seen that compared with the control, the E2 secretion of the mouse model group decreased and the FSH secretion increased, indicating that the mouse ovarian function was failing. After the stem cell transplantation treatment of Examples 1-4, the E2 secretion of the mouse increased and the FSH secretion decreased, which was close to the hormone secretion level of normal rats, indicating that the combined group can effectively improve the ovarian function. Compared with the model group, the effect of Example 1 was better (p<0.001).

[0046] The mice in each group were killed by cervical dislocation, and the ovarian tissues were taken from both sides. The frozen sections were observed under a fluorescence microscope (x400) to observe the histological changes of the ovaries. 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] From the results in Table 3, it can be seen that the total number of follicles in the model group is significantly less than that in the blank control group, but after the combined treatment, the total number of follicles in the model group is significantly increased, which is basically close to the number of follicles in normal mice, indicating that it has a good effect of restoring ovarian function. Compared with the model group, the effect of Example 1 is better (p<0.001).

[0052] Example 4 Western blot detection

[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 solution, the supernatant was collected by centrifugation at 4°C and 12000r / min. After BCA protein quantification, the protein was boiled with protein loading buffer and stored at -20°C for later use. A 12% polyacrylamide gel was prepared, and the sample amount was 20μg for SDS-PAGE electrophoresis. After the electrophoresis, wet transfer (PVDF) was performed 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 goat anti-rabbit antibody labeled with horseradish peroxide (l:10000), and the imaging system was used for analysis after adding ECL luminescent liquid. The results are shown in 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 modifications and improvements can be made without departing from the creative concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. An endometrial stem cell drug, characterized in that: The medicament comprises endometrial stem cells.

2. The drug according to claim 1, characterized in that The culture medium of the endometrial stem cells contains DMEM, VEGF and β-ecdysterone.

3. The drug according to claim 1, characterized in that VEGF was included in the culture medium at a concentration of 20 ng / ml.

4. The drug according to claim 2, characterized in that The culture medium included β-ecdysterone at a concentration of 20 ng / ml.

5. The drug according to claim 2, characterized in that The culture medium composition includes: 20 ng / ml VEGF, 20 ng / ml β-ecdysterone and DMEM.

6. A process for preparing the drug according to any one of claims 1 to 5, the preparation method comprising the following steps: (1) Obtain endometrial tissue under sterile conditions, perform enzymatic hydrolysis, and obtain a single cell suspension; (2) Culture was performed using a medium containing DMEM, VEGF, and β-ecdysterone.

7. The method according to claim 6, characterized in that In the step (1), trypsin I and type I collagenase are used for enzymatic digestion.

8. The use of the drug according to claims 1-3, characterized in that: The application is to prepare medicine for treating premature ovarian failure.

9. The use of the drug according to claims 1-3, characterized in that: The application is to prepare medicine for treating ovarian oocyte repair.

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