A medicine containing endometrial stem cells and its preparation method and application

Abstract

The present invention relates to the field of biomedicine technology, and more specifically, to a drug containing endometrial stem cells, its preparation method, and its use. The present invention optimizes the components of the culture medium for endometrial stem cells to obtain a more optimal culture medium. Endometrial stem cells cultured using the culture medium of the present invention exhibit enhanced proliferation capacity and improved efficacy in resisting ovarian aging and repairing ovaries. The present invention optimizes the polypeptide sequence to obtain a more optimal active polypeptide. The active polypeptide of the present invention exhibits enhanced efficacy in resisting ovarian aging and repairing ovaries.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a medicine containing endometrial stem cells, and a preparation method and application 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, 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. After the stem cells and 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. The polypeptide is 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 active peptides were used together to treat the ovarian aging model, it was found that they can effectively promote the secretion of follicles and E2 secretion, and have a good anti-ovarian aging effect, wherein the active peptide is FQG IVQLPYWPSFAMW.

[0005] For example, prior art CN118121680A discloses a composition for repairing oocytes in female ovaries, its preparation method, and application, wherein the composition comprises stem cells and / or polypeptides that upregulate P-AMPK expression, 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 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. 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 known polypeptides, more effective polypeptides can be obtained. Based on this, the present invention will provide a more optimal culture medium and mutant polypeptides, and based on this, provide a drug containing endometrial stem cells and its preparation method and application. Summary of the Invention

[0007] The present invention first provides a drug containing endometrial stem cells to achieve the above-mentioned purpose, wherein the drug 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 LI F 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, enzymatically hydrolyze, and obtain a single-cell suspension;

[0012] (2) Culture was performed using a culture medium containing DMEM, LI F, 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 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] (1) The present invention optimizes the culture medium components for endometrial stem cells to obtain a more optimal culture medium. Endometrial stem cells cultured using the culture medium of the present invention have a stronger proliferation capacity 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 better anti-ovarian aging and ovarian repair effects. 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 clearer, 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: The entire endometrium plus 5 mm of myometrium were harvested under sterile conditions, away from the lesion site;

[0022] 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.

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

[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 standby use.

[0027] Example 2

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

[0029] Example 3

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

[0031] Example 4

[0032] Except that DMEM medium was used as the culture medium in step 3), the other conditions were 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 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 2.

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

[0036]

[0037]

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

[0039] Subsequently, experiments were conducted using the endometrial stem cells of Example 1.

[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 the 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 cycles were randomly divided into a model group, a polypeptide group, and a blank control group, with 10 mice in each group. The model group and the polypeptide group received a single intraperitoneal injection of cyclophosphamide solution (120 mg / kg) and busulfan solution (12 mg / kg) 0.1 mL. Vaginal secretion smears were gently taken with a cotton swab daily, and Papanicolaou staining was performed to observe changes in vaginal exfoliated cells. When a continuous diestrus period was observed for 10 consecutive days, the modeling of premature ovarian failure was considered successful. On the 14th day of modeling, the polypeptide group received a tail vein injection of 0.1 mL of P1 polypeptide (10 mg / kg), repeated every 4 days for a total of 5 times. The modeling group and the blank control group received an equal amount of normal saline injection each time through the tail vein.

[0048] 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 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] As shown in Table 4, compared to the control group, the mouse model group had decreased E2 secretion and increased FSH secretion, indicating ovarian failure in the mice. After treatment with the peptides described in Table 4, E2 secretion increased and FSH secretion decreased, approaching the hormone secretion levels of normal rats, indicating that the peptides can effectively improve ovarian function. Furthermore, compared to the model group, the peptide with the amino acid sequence of SEQ ID NO: 9 had a greater effect (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 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 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 each time through the tail vein.

[0058] 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 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] The results in Table 5 show that compared to the control group, the model group showed decreased E2 secretion and increased FSH secretion, indicating ovarian failure in the mice. After treatment with the combination therapy, E2 secretion increased and FSH secretion decreased, surpassing the hormone secretion levels of normal rats, indicating that the combination therapy effectively improved ovarian function. Furthermore, the stem cell + peptide combination showed a more pronounced effect than the model group (p<0.001).

[0064] The mice in each group were sacrificed by cervical dislocation and bilateral ovarian tissue was obtained. Ovarian tissue was frozen and sliced ​​and observed under a fluorescence microscope (x400) for histological changes. 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] As shown in Table 6, 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 increased significantly, exceeding the number of follicles in normal mice, indicating a good effect in restoring ovarian function. The combined group showed a better effect than the model group (p < 0.001).

[0070] Example 9 Western blotting detection

[0071] Referring to the method described in CN118121680A, Western blot was performed to examine P-AMPK protein expression in the model group, the peptide (SEQ ID NO: 9) treatment group, the stem cells prepared in Example 1, and the stem cells prepared in Example 1 + peptide (SEQ ID NO: 9) combination group. Cells were collected after 48 hours of treatment from the above groups, trypsinized, and centrifuged. After lysis with RIPA buffer, the supernatant was centrifuged at 12,000 rpm at 4°C and collected. After BCA protein quantification, the protein was boiled with protein loading buffer and stored frozen at -20°C until further use. A 12% polyacrylamide gel was prepared, and 20 μg of sample was loaded for SDS-PAGE electrophoresis. After electrophoresis, the cells were wet-transferred to a PVDF membrane 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 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 was significantly increased after treatment with the peptide treatment group, stem cell, 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 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 drug containing endometrial stem cells, characterized in that: The drug contains endometrial stem cells and an active polypeptide; the amino acid sequence of the active polypeptide is SEQ ID NO:

9.

2. The use of the drug according to claim 1, characterized in that The application is to prepare medicine for treating premature ovarian failure.

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