Medicine containing endometrial stem cells as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicines, in particular to a medicine containing endometrial stem cells as well as a preparation method and application of the medicine. By optimizing culture medium components of the endometrial stem cells, a better culture medium is obtained. 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. According to the invention, by optimizing a polypeptide sequence, a better active polypeptide is obtained. The active polypeptide provided by the invention has better ovarian aging resistance and ovarian repair effects.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a medicine containing endometrial stem cells and a preparation method and application 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 CN113786474A discloses that ovarian 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 the ovarian aging model, and it was found that the secretion of follicles and E2 could be effectively promoted, and the polypeptide was GLHCESMNEKQAAYKPLPQQ.

[0004] For example, the prior art CN113633754A discloses that ovarian stem cells were obtained by separation and preparation, and active peptides with anti-aging effects were isolated from Polygonum multiflorum. After the stem cells and the active peptides were used together to treat the ovarian aging model, it was found that they can effectively promote the secretion of follicles and promote the secretion of E2, and have a good anti-ovarian aging effect, wherein the active peptide is FQG IVQLPYWPSFAMW.

[0005] 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, wherein the stem cells are endometrial stem cells, and the polypeptide is QFLFQGKFLPYWSSMWVQ.

[0006] The activity of stem cells is a key factor affecting the therapeutic effect of stem cell therapy. Therefore, it is necessary to optimize the culture medium of stem cells to obtain stem cells with stronger proliferation ability and higher activity. At the same time, the above-mentioned prior art involves different Polygonum multiflorum polypeptides, and it is well known in the art that different amino acid sequences have completely different activities. Therefore, by mutating or improving the known polypeptides, polypeptides with better effects can be obtained; based on this, the present invention will provide a better culture medium and mutant polypeptides, and based on this, provide a drug containing endometrial stem cells and a preparation method and application thereof. Summary of the invention

[0007] The present invention first provides a medicine containing endometrial stem cells to achieve the above-mentioned purpose. The medicine comprises endometrial stem cells and an active polypeptide; optionally, the amino acid sequence of the active polypeptide is SEQ ID NO: 9.

[0008] Preferably, the culture medium of the endometrial stem cells comprises DMEM, LI F and total flavonoids from Epimedium.

[0009] Preferably, the concentration of the LIF is 20 ng / ml, and the concentration of the total flavonoids of Epimedium is 20 ng / ml.

[0010] Secondly, the present invention also provides a method for preparing 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 culture medium containing DMEM, LIF and total flavonoids from Epimedium.

[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] (1) The present invention optimizes the culture medium components of endometrial stem cells to obtain a better culture medium. 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.

[0017] (2) The present invention obtains a better active polypeptide by optimizing the polypeptide sequence. The active polypeptide of the present invention has a better effect of resisting ovarian aging and repairing ovaries. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the expression level of P-AMPK in different experimental groups. DETAILED DESCRIPTION

[0019] 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.

[0020] Example 1 Isolation and preparation of stem cells

[0021] 1) Obtaining endometrial stem cells: Obtain the entire endometrial layer + 5 mm of myometrium under sterile conditions, far away from the lesion site;

[0022] 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;

[0023] 3) Primary culture: Add DMEM + 20 ng / ml LI F + 20 ng / ml total flavonoids from Epimedium 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.

[0024] Table 1 Antigen identification results

[0025] CD73 CD90 CD34 98.5％* 95.5％* 0.014％

[0026] 4) When the cell fusion rate reaches about 85-90%, the P0 cells are subcultured to obtain P3 cells for use.

[0027] Example 2

[0028] Except that the culture medium in step 3) is DMEM+40ng / ml LIF culture medium, the other conditions are the same as those in Example 1.

[0029] Example 3

[0030] Except that the culture medium in step 3) is DMEM+40ng / ml of total flavonoids from Epimedium, the other conditions are the same as those in Example 1.

[0031] Example 4

[0032] Except that the culture medium in step 3) is DMEM, the other conditions are the same as those in Example 1.

[0033] Example 5

[0034] 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 2.

[0035] Table 2 Relative multiples of endometrial stem cells prepared in different embodiments

[0036]

[0037]

[0038] Compared with Example 4: **P<0.01, ***P<0.001.

[0039] The endometrial stem cells of Example 1 were used for subsequent experiments.

[0040] Example 6 Synthesis of polypeptide

[0041] Referring to CN118121680A, single-point and multi-point mutants were designed based on QFLFQGKFLPYWSSMWVQ (SEQ ID NO: 1). The results are shown in Table 3. The underlined amino acids are mutation sites.

[0042] Table 3 Mutants of Polygonum multiflorum polypeptide

[0043]

[0044]

[0045] The Polygonum multiflorum polypeptides in Table 3 were synthesized by solid phase synthesis and purified by HPLC to obtain the Polygonum multiflorum polypeptides in Table 3 with a purity of 95%.

[0046] Example 7: Verification of the effect of polypeptide

[0047] Referring to the method of CN118121680A, ICR mice with normal estrous cycle were randomly divided into model group, polypeptide group and blank control group, with 10 mice in each group. The model group and polypeptide group were injected with cyclophosphamide solution (120mg / kg) and busulfan solution (12mg / kg) 0.1mL at one time, 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. The polypeptide group was injected with 0.1mL of P1 polypeptide (10mg / kg) through the tail vein on the 14th day of modeling, and repeated once every 4 days for a total of 5 times. The modeling group and the blank control group were injected with an equal amount of normal saline through the tail vein each time.

[0048] 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 4.

[0049] Table 4 Comparison of FSH and E2 in each group of mice

[0050]

[0051]

[0052] Compared with the model group: **P<0.01, ***P<0.001.

[0053] From the results in Table 4, it can be seen that compared with the control group, the E2 secretion of the mouse model group decreased and the FSH secretion increased, indicating that the mouse ovarian function was failing. After the polypeptide treatment in Table 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 polypeptide can effectively improve the ovarian function. Compared with the model group, the effect of the polypeptide with the amino acid sequence of SEQ ID NO: 9 was better (p<0.001).

[0054] Subsequent experiments were conducted using the polypeptide of SEQ ID NO: 9.

[0055] Example 8: Verification of the effect of combined treatment

[0056] The endometrial stem cells prepared in Example 1 were used as stem cells for the experiment; the polypeptide used was the polypeptide of SEQ ID NO: 9.

[0057] Referring to the method of Example 7, a stem cell transplantation group and a stem cell + polypeptide combination group were constructed. The stem cell transplantation group was injected with 0.1 mL of the endometrial stem cell suspension (1×10 8The stem cell + polypeptide combination group was injected with 0.1 mL of the endometrial stem cell suspension (1x10 8 / mL) and P1 polypeptide (10mg / kg) 0.1mL, repeated once every 4 days, a total of 5 times. The modeling group and the blank control group were injected with an equal amount of normal saline via the tail vein each time.

[0058] 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 5.

[0059] Table 5 Comparison of FSH and E2 in each group of mice

[0060]

[0061]

[0062] Compared with the model group: **P<0.01, ***P<0.001.

[0063] From the results in Table 5, we can see that compared with the control group, the E2 secretion of the mouse model group decreased, and the FSH secretion increased, indicating that the mouse ovarian function was failing. After the combined group treatment, the E2 secretion of the mouse increased, and the FSH secretion decreased, which was better than 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 stem cells + polypeptides was better (p<0.001).

[0064] The mice from each group were killed by cervical dislocation, and the ovarian tissues from both sides were taken and frozen sections were observed under a fluorescence microscope (x400) for histological changes of the ovaries. The results are shown in Table 6.

[0065] Table 6 Comparison of follicle numbers in each group

[0066]

[0067]

[0068] Compared with the model group: **P<0.01, ***P<0.001.

[0069] From the results in Table 6, 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 has been significantly increased, which is higher than the number of follicles in normal mice, indicating that it has a good effect in restoring ovarian function. Compared with the model group, the combined group has a better effect (p<0.001).

[0070] Example 9 Western blot detection

[0071] Referring to the method of CN118121680A, Western blotting was used to detect the expression of P-AMPK protein in the model group, the polypeptide (SEQ ID NO: 9) treatment group, the stem cells prepared in Example 1, and the stem cells prepared in Example 1 + polypeptide (SEQ ID NO: 9) combination group. The cells were collected by centrifugation after 48 hours of the above grouping. After RIPA lysis solution, the supernatant was collected by centrifugation at 4°C 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 to PVDF was performed and blocked with skim milk powder for 2h. 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 solution, the imaging system was used for analysis. Figure 1 shown.

[0072] Depend on Figure 1 It can be seen that compared with the control group, the P-AMPK level in the injury model group was significantly decreased, and the P-AMPK level could be significantly increased after treatment with the peptide treatment group, stem cells, and stem cell + peptide combination group (P<0.01).

[0073] 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. A drug containing endometrial stem cells, characterized in that: The drug comprises endometrial stem cells and an active polypeptide; optionally, the amino acid sequence of the active polypeptide is SEQ ID NO:

9.

2. The drug according to claim 1, characterized in that The culture medium of the endometrial stem cells comprises DMEM, LIF and total flavonoids of epimedium.

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

4. The drug according to claim 2, characterized in that The culture medium included total flavonoids from Epimedium 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 LIF, 20 ng / ml total flavonoids from Epimedium and DMEM.

6. A method for preparing the drug according to any one of claims 1 to 5, the 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 culture medium containing DMEM, LIF and total flavonoids from Epimedium.

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 1-5, characterized in that: The application is to prepare medicine for treating premature ovarian failure.

9. The use of the drug according to claims 1-5, characterized in that: The application is to prepare medicine for treating ovarian oocyte repair.

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