A stem cell preparation for treating premature ovarian failure and a preparation method and application thereof

Abstract

This invention provides a stem cell preparation for treating premature ovarian failure, comprising modified mesenchymal stem cells and a pharmaceutically acceptable carrier, wherein the modified mesenchymal stem cells express more than twice the amount of TFPI protein compared to unmodified mesenchymal stem cells. This invention also provides a method for preparing this stem cell preparation and its application in the treatment of premature ovarian failure.

Description

Technical Field

[0001] This invention relates to a modified mesenchymal stem cell that highly expresses TFPI protein, stem cell preparations comprising the modified stem cell, methods for preparing the same, and applications thereof. Background Technology

[0002] Premature ovarian failure (POF) is an endocrine disorder characterized by ovarian insufficiency in women before the age of 40. It is characterized by amenorrhea, elevated gonadotropin levels, decreased estrogen levels, and a lack of mature follicles. Epidemiological surveys show that POF affects approximately 1%-5% of women of reproductive age worldwide, and its incidence is showing a significant upward trend. The causes of POF involve multiple factors, including genetic defects, autoimmune diseases, iatrogenic injuries (surgery, chemotherapy, radiotherapy), and infections, but the cause remains unknown in 50%-90% of cases. Currently, prevention and treatment options for POF are very limited. Hormone replacement therapy is the most commonly used treatment in clinical practice, effectively alleviating symptoms caused by low estrogen levels. However, this therapy only improves symptoms and cannot fundamentally reverse ovarian function or restore fertility. Furthermore, long-term use may increase the risk of breast cancer and endometrial cancer. Therefore, developing novel treatment strategies that can effectively repair damaged ovarian tissue and restore ovarian reserve function is of significant clinical importance. Summary of the Invention

[0003] On one hand, the present invention provides a stem cell preparation for treating premature ovarian failure, comprising modified mesenchymal stem cells and a pharmaceutically acceptable carrier, wherein the modified mesenchymal stem cells express more than twice the amount of TFPI protein compared with unmodified mesenchymal stem cells.

[0004] In some embodiments, the modified mesenchymal stem cells express TFPI protein at a level that is 2, 3, 4, 5, 6, 7, or 8 times higher than that of unmodified mesenchymal stem cells.

[0005] In some embodiments, the mesenchymal stem cells are hematogenous mesenchymal stem cells.

[0006] In some implementations, the TFPI protein is a human TFPI protein.

[0007] In some embodiments, the amino acid sequence of the TFPI protein is shown in SEQ ID NO: 1.

[0008] On the other hand, the present invention provides a method for preparing a stem cell preparation for treating premature ovarian failure, comprising preparing stem cells contained in the stem cell preparation through the following steps: 1) Collect menstrual blood, and isolate and culture mesenchymal stem cells from it; 2) Introducing a foreign gene expressing the TFPI protein into the mesenchymal stem cells; and 3) Isolate and culture mesenchymal stem cells expressing the exogenous gene.

[0009] In some implementations, the TFPI protein is a human TFPI protein.

[0010] In some embodiments, the exogenous gene comprises a nucleotide sequence as shown in SEQ ID NO: 2.

[0011] On the other hand, the present invention provides the use of the above-mentioned stem cell preparation in the preparation of a drug for treating premature ovarian failure.

[0012] The hematogenous mesenchymal stem cells with high expression of TFPI protein provided by this invention have a significant function in improving premature ovarian failure. Detailed Implementation

[0013] Unless otherwise stated, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art.

[0014] The term "comprising" means including the listed components, steps, or features, but does not exclude the inclusion of other unlisted components, steps, or additional features; it also covers situations where the components, steps, or features are constituted.

[0015] Unless otherwise stated, the term "above" refers to a range of numbers including the stated number and above.

[0016] The term "tissue factor pathway inhibitor (TFPI)" refers to a natural anticoagulant protein that regulates the extrinsic coagulation pathway and belongs to the Kunitz-type serine protease inhibitor family. The amino acid sequence of the human TFPI protein is shown in SEQ ID NO: 1.

[0017] The term "exogenous gene" refers to a nucleic acid fragment that is artificially introduced into a host cell through transfection, transduction, vector delivery, or other means, relative to the host cell's natural genome. It can be transcribed and translated within the cell to achieve overexpression of the target protein and usually contains the coding gene and its expression regulatory sequence.

[0018] The term "stem cell preparation" refers to a cell-based biological composition prepared with stem cells as the core active ingredient, combined with pharmaceutically acceptable excipients, buffers, protectants, or carriers.

[0019] The term "menstrual blood-derived mesenchymal stem cells (MenSC)," also known as menstrual blood-derived endometrial stem cells, refers to a type of adult mesenchymal stem cells that can be obtained non-invasively from the endometrial tissue shed from a woman's menstrual blood.

[0020] The following specific embodiments illustrate the present invention.

[0021] Example 1: Preparation of human hematogenous mesenchymal stem cells (MenSC) With informed consent from volunteers and approval from the hospital's ethics committee, approximately 10 mL of menstrual blood was collected from healthy women of childbearing age (20-35 years old) using a menstrual blood collection device on days 2-4 of their menstrual cycle. The blood sample was diluted with an equal volume of PBS buffer and slowly added to the supernatant of Ficoll-Paque lymphocyte separation medium (blood sample to separation medium volume ratio 2:1). The mixture was centrifuged at 400 g at room temperature for 30 min. The middle white flocculent layer of mononuclear cells was carefully aspirated and transferred to a new centrifuge tube. The cells were centrifuged at 400 g at room temperature for 5 min and washed twice with PBS buffer. The separated cell pellet was resuspended in DMEM / F12 complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics, seeded in T25 culture flasks, and incubated at 37°C with 5% CO2. After 48 hours of culture, the medium was replaced with fresh medium to remove non-adherent cells. The medium was then replaced every 3 days thereafter.

[0022] When the adherent cells reach approximately 80% confluence, discard the culture medium, wash the cells twice with PBS buffer, add 0.25% trypsin-EDTA digestion solution, and digest at room temperature for 3 minutes. Observe under a microscope until the cells become rounded and partially detached. Add an equal volume of DMEM / F12 medium containing 10% fetal bovine serum to terminate the digestion. Gently pipette to prepare a single-cell suspension, passage at a 1:3 ratio in new culture flasks, and continue culturing and amplification.

[0023] Third-generation MenSCs in good growth condition were collected by digestion with 0.25% trypsin, washed twice with PBS, and the cell density was adjusted to 1×10⁻⁶. 6 Cells / mL. FITC- or PE-labeled mouse anti-human monoclonal antibodies CD44, CD90, CD105, CD34, and CD45 were added separately and incubated at 4°C in the dark for 30 minutes. After washing twice with PBS, the expression of cell surface markers was detected by flow cytometry. The results were: CD44 positivity rate >96%, CD90 positivity rate >90%, CD105 positivity rate >92%; CD34 positivity rate <1%, CD45 positivity rate <1%. These results met the identification criteria for mesenchymal stem cells.

[0024] Example 2: Construction of Lentiviral Expression Vector Based on the human TFPI protein sequence (P10646·TFPI1_HUMAN, SEQ ID NO: 1) from the Uniprot protein database, its encoding nucleotide sequence (SEQ ID NO: 2) was synthesized according to human codon preference. The TFPI coding sequence was cloned into the MCS site of the lentiviral expression vector pLVX-EF1α-MCS-PGK-Puro via enzyme digestion and ligation. The recombinant lentiviral vector pLVX-EF1α-TFPI-PGK-Puro was constructed. In this vector, the TFPI gene is transcribed and expressed under the EF1α promoter, and the PGK promoter drives the expression of the puromycin resistance gene, which is used for screening stably transfected cells.

[0025] Human TFPI protein amino acid sequence (SEQ ID NO: 1): MIYTMKKVHALWASVCLLLNLAPAPLNADSEEDEEHTIITDTELPPLKLMHSFCAFKADDGPCKAIMKRFFFNIFTRQCEEFIYGGCEGNQNRFESLEECKKMCTRDNANRIIKTTLQQEKPDFCFLEEDPGICRGYITRYFYNNQTKQ CERFKYGGCLGNMNNFETLEECKNICEDGPNGFQVDNYGTQLNAVNNSLTPQSTKVPSLFEFHGPSWCLTPADRGLCRANENRFYYNSVIGKCRPFKYSGCGGNENNFTSKQECLRACKKGFIQRISKGGLIKTKRKRKKQRVKIAYEEIFVKNM Human TFPI protein-encoding nucleotide sequence (SEQ ID NO: 2): ATGATCTACACTATGAAGAAAGTGCACGCCCTCTGGGCTTCTGTTTGTCTGTTGCTGAACCTTGCCCCCGCCCCTTTGAATGCCGATTCTGAGGAAGACGAAGAACACACCATAATTACAGACACCGAGCTGCCACCACTGAAGCTTATGCATTCTTTCTGTGCCTTCAAAGCTGATGACGGTCCCTGTAAAGCCATTATGAAGAGGTTCTTTTTCAACATCTTTACACGACAATGCGAAGAATTCATCTATGGAGGCTGTGAGGGTAATCAGAACCGATTCGAGAGCCTGGAGGAATGCAAAAAGATGTGCACGCGAGACAACGCTAACCGGATAATCAAAACCACACTGCAACAGGAGAAGCCGGACTTCTGCTTCCTGGAAGAGGATCCCGGGATCTGTAGGGGCTACATCACGCGCTACTTCTATAACAACCAAACGAAACAGTGCGAGAGGTTCAAATACGGCGGATGTCTGGGAAATATGAATAACTTCGAGACGTTGGAGGAGTGTAAAAACATCTGCGAAGACGGTCCTAATGGGTTCCAAGTGGACAACTACGGAACACAGCTGAATGCCGTGAATAACAGCCTCACTCCACAGAGCACCAAAGTCCCGAGCCTGTTTGAGTTCCACGGACCTTCTTGGTGTTTGACTCCAGCAGATCGGGGGCTGTGCAGAGCAAACGAGAACAGGTTTTATTACAACTCCGTCATTGGGAAGTGCCGCCCGTTCAAGTACTCTGGTTGCGGTGGCAACGAGAACAATTTCACCTCTAAACAGGAGTGTCTGCGAGCCTGCAAAAAGGGCTTCATCCAACGCATTAGCAAGGGAGGTCTGATCAAAACCAAGCGCAAAAGAAAGAAACAGAGGGTCAAAATCGCATACGAGGAGATATTTGTGAAGAATATG The recombinant lentiviral vector pLVX-EF1α-TFPI-PGK-Puro was mixed with helper packaging plasmids psPAX2 and pMD2.G at a mass ratio of 4:3:1 and co-transfected into HEK293T cells using liposome transfection. Cell supernatants were collected at 48 and 72 hours post-transfection, filtered through a 0.45 μm filter, and concentrated using PEG precipitation. The viral pellet was resuspended in PBS buffer, and the viral titer was determined by qRT-PCR before storage at -80°C for later use.

[0026] Example 3: Construction of MenSC with high TFPI expression Take the third generation MenSC cultured in Example 1, and use 5 × 10 5 Cells were seeded at a density of 100 cells / well in 6-well plates and incubated in serum-free DMEM / F12 medium at 37°C with 5% CO2. 2 The culture was incubated overnight. The next day, the culture medium was discarded, and serum-free DMEM / F12 medium containing TFPI lentivirus (MOI=50) prepared in Example 2 and polybrene (final concentration 8 μg / mL) was added. After mixing, the medium was incubated at 37°C in a 5% CO2 incubator for 12 hours. After 12 hours, the medium was replaced with DMEM / F12 complete medium containing 10% fetal bovine serum, and the culture was continued for 48 hours. 48 hours after lentivirus transfection, puromycin (final concentration 2 μg / mL) was added to the culture system for selection, and the medium was replaced with fresh puromycin every 3 days. After 10 days of selection and culture, resistant cell clones were obtained. Single-cell clones were picked and expanded, and the culture supernatant of TFPI-MenSC and untransfected control MenSC was collected. The expression level of TFPI protein was detected by Western blot. The primary antibody was rabbit anti-human TFPI polyclonal antibody (1:1000 dilution), and the secondary antibody was HRP-labeled goat anti-rabbit IgG (1:5000 dilution). GAPDH was used as an internal control. The results showed that the TFPI protein content in the control MenSC culture supernatant was approximately 35.7 ng / mL, while the TFPI protein content in the TFPI-MenSC culture supernatant was approximately 284.3 ng / mL, an increase of approximately 8-fold.

[0027] Example 4: Evaluation of the therapeutic effect of TFPI-MenSC on a cyclophosphamide-induced POF rat model Fifty 8-week-old SPF-grade female SD rats, weighing 180-220 g, were randomly divided into 6 groups (n=10 per group) after one week of acclimatization: normal control group (NC group), model control group (POF group), TFPI treatment group, MenSC treatment group (MenSC group), low-dose TFPI-MenSC group (TFPI-MenSC-L group), and high-dose TFPI-MenSC group (TFPI-MenSC-H group). Except for the normal control group, rats in the other groups underwent intraperitoneal injection of cyclophosphamide (CTX) to establish the POF model: 50 mg / kg intraperitoneally on the first day, followed by 15 mg / kg / day for 14 consecutive days, with a total dose of approximately 260 mg / kg. Treatment began on the second day after model establishment. Rats in the MenSC group received unmodified MenSCs (1×10⁻⁶) via tail vein injection. TFPI-MenSCs-L group received TFPI-MenSC (5 × 10 cells / time) via tail vein injection. 6 TFPI-MenSC (5× cells / time) was injected via tail vein into the TFPI-MenSC-H group. Cells / dose). The normal control group and the model control group were injected with an equal volume of normal saline (300µL), while the TFPI treatment group was injected with an equal volume of normal saline containing 20µg TFPI (CUSABIO, CSB-MP023437HU). Each group was administered the medication once a week for 5 consecutive weeks.

[0028] The day after treatment, blood was collected from the orbital venous plexus of rats in each group, serum was separated, and serum estradiol (E2) and follicle-stimulating hormone (FSH) levels were detected by ELISA. After another week of feeding, the rats were sacrificed, and the left ovary was harvested, fixed in 10% formaldehyde, dehydrated with graded ethanol, embedded in paraffin, and sectioned (5µm). Hematoxylin and eosin (HE) staining was performed, and the number of follicles at each stage was counted under a light microscope. The results are shown in Tables 1 and 2.

[0029] Table 1. Serum estradiol (E2) and follicle-stimulating hormone (FSH) levels in rats.

[0030] The same lowercase letter in the same column indicates that the difference between groups is not significant. P > 0.05); different lowercase letters in the same column indicate significant differences between groups ( P < 0.05).

[0031] Compared with the normal control group (NC), the serum E2 level of the model group (POF) rats was significantly decreased, and the FSH level was significantly increased, indicating that the model was successfully established. Compared with the model group, only the TFPI-MenSC-L treatment group and the TFPI-MenSC-H treatment group showed significantly increased E2 levels, and there was also a significant difference between the TFPI-MenSC-L treatment group and the TFPI-MenSC-H treatment group, indicating that the E2 level is dose-dependent at this MenSC dosage. Compared with other treatment groups, the FSH level of the TFPI-MenSC-L treatment group and the TFPI-MenSC-H treatment group was significantly decreased. This indicates that MenSC with high TFPI expression is significantly superior to unmodified MenSC and TFPI reagent alone in improving endocrine function in POF rats.

[0032] Table 2 Number of follicles at each stage in rats

[0033] The same lowercase letter in the same column indicates that the difference between groups is not significant. P > 0.05); different lowercase letters in the same column indicate significant differences between groups ( P < 0.05).

[0034] The results of follicle count showed that the total number of follicles at each stage in the model group (POF) was significantly reduced compared with the normal control group (NC); the TFPI-MenSC-L treatment group and the TFPI-MenSC-H treatment group showed a significant increase compared with the model group; the unmodified MenSC group and the TFPI group basically did not show any effect of increasing the number of follicles at each stage.

[0035] At the experimental doses of TFPI and modified MenSC, no significant anticoagulation-related side effects (absence of condition and behavioral abnormalities) were observed. In later experiments, the dose of TFPI in the group was further increased to 60 µg TFPI per administration. POF model rats exhibited anticoagulation-related symptoms such as bleeding at the injection site and scattered petechiae on the abdominal skin, but no significant improvement in E2 levels, FSH levels, or follicle count was observed. This suggests that the ability of TFPI-modified MenSC to improve ovarian function does not originate from the TFPI secreted by MenSC, but rather may be due to changes in MenSC function caused by high TFPI expression (e.g., the synthesis and secretion of new cytokines or small molecule compounds), thereby possessing the ability to improve premature ovarian failure.

Claims

1. A stem cell preparation for treating premature ovarian failure, comprising modified mesenchymal stem cells and a pharmaceutically acceptable carrier, wherein the modified mesenchymal stem cells express more than twice the amount of TFPI protein compared to unmodified mesenchymal stem cells.

2. The stem cell preparation of claim 1, wherein the modified mesenchymal stem cells express more than 8 times more TFPI protein compared with unmodified mesenchymal stem cells.

3. The stem cell preparation of claim 2, wherein the mesenchymal stem cells are hematogenous mesenchymal stem cells.

4. The stem cell preparation of claim 2, wherein the TFPI protein is a human TFPI protein.

5. The stem cell preparation according to any one of claims 2-4, wherein the amino acid sequence of the TFPI protein is as shown in SEQ ID NO:

1.

6. A method for preparing a stem cell preparation for treating premature ovarian failure, comprising preparing stem cells contained in the stem cell preparation by the following steps: 1) Collect menstrual blood, and isolate and culture mesenchymal stem cells from it; 2) Introducing a foreign gene expressing the TFPI protein into the mesenchymal stem cells; and 3) Isolate and culture mesenchymal stem cells expressing the exogenous gene.

7. The method of claim 6, wherein the TFPI protein is a human TFPI protein.

8. The method of claim 6, wherein the exogenous gene comprises a nucleotide sequence as shown in SEQ ID NO:

2.

9. The use of the stem cell preparation according to any one of claims 1-5 in the preparation of a medicament for treating premature ovarian failure.

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