High-adhesion mesenchymal stem cells and application thereof in preparation of medicine for treating intrauterine adhesion

Abstract

The application discloses high-adhesion modified mesenchymal stem cells and application thereof in preparation of a medicine for treating intrauterine adhesion. The modified mesenchymal stem cells are genetically engineered to overexpress integrin alpha 2 subunit and / or integrin alpha 5 subunit, thereby enhancing the adhesion capacity to the extracellular matrix (collagen and fibronectin) of the endometrium. In vitro experiments show that the initial adhesion rate, liquid flushing resistance and anoikis resistance of the modified MSCs are significantly better than those of common MSCs. When used in combination with the "dynamic culture and drug delivery device for intrauterine adhesion treatment" applied by the inventor on the same day, the retention rate of the modified MSCs in the simulated intrauterine dynamic culture environment is significantly improved. Animal experiments show that the modified MSCs in combination with the device can significantly promote endometrial regeneration, inhibit fibrosis and improve the pregnancy rate. The application solves the core bottleneck of low cell retention rate and short survival time in the MSC transplantation treatment of intrauterine adhesion by enhancing the adhesion capacity of the MSCs, and forms a complete treatment scheme in cooperation with the device, thereby having a good clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the fields of cell biology and regenerative medicine, specifically relating to a highly adhesive modified mesenchymal stem cell, its preparation method, and its application in the preparation of drugs for treating intrauterine adhesions. Background Technology

[0002] Intrauterine adhesions are a common and difficult-to-treat gynecological disease. Damage to the basal layer of the endometrium leads to fibrous tissue hyperplasia and partial or complete closure of the uterine cavity, clinically manifesting as menstrual abnormalities, infertility, and recurrent miscarriages. Currently, the main treatment is hysteroscopic adhesiolysis, but the re-adhesion rate after surgery is as high as 30%-60%, and repeated surgeries further damage the endometrium.

[0003] Mesenchymal stem cell (MSC) transplantation has brought new hope to the treatment of intrauterine adhesions. MSCs possess multi-lineage differentiation potential, immunomodulatory functions, and tissue repair-promoting capabilities, which can promote the regeneration of damaged endometrium and inhibit fibrosis. However, current MSC transplantation treatments for intrauterine adhesions face a core bottleneck: low cell retention rate and short survival time.

[0004] The existing technology has the following technical defects: Low cell retention rate: After a single intrauterine injection of MSC suspension, due to gravity, most cells are deposited at the lowest point of the uterine cavity, with insufficient cell coverage in areas such as the anterior and lateral walls. Furthermore, because MSCs have limited adhesion to the endometrium, even deposited cells are easily washed away by intrauterine fluid.

[0005] Short cell survival time: MSCs are anchorage-dependent cells, requiring adhesion to the extracellular matrix to survive (anorepopulation). MSCs that fail to adhere or adhere poorly will die rapidly through the anorepopulation mechanism.

[0006] Unstable treatment outcomes: Due to individual differences in cell retention and survival rates, the clinical outcomes of MSC transplantation vary, making it difficult to establish a stable treatment plan.

[0007] Incompatible with dynamic culture devices: Patent 1 filed by the applicant on the same day (A dynamic culture and drug delivery device for the treatment of intrauterine adhesions) achieves "dynamic culture" of MSCs in the uterine cavity by sealing the uterine cavity with a double-lumen tube, guiding adhesion by changing the body position, and maintaining activity by changing the medium regularly. The efficacy of this device is highly dependent on the adhesion ability of MSCs themselves.

[0008] Therefore, there is an urgent need to develop a modified mesenchymal stem cell with high adhesion, which can firmly bind to the endometrial matrix, resist fluid flushing, and survive for a long time in a dynamic culture environment. Summary of the Invention

[0009] Purpose of the invention The purpose of this invention is to provide a highly adhesive modified mesenchymal stem cell, which enhances the expression of integrins on the surface of MSCs (especially integrins that bind to the extracellular matrix of endometrial cells) through genetic engineering, thereby improving the adhesion ability of MSCs to the endometrium, enabling them to remain efficiently and survive for a long time in the dynamic culture environment of the uterine cavity, and ultimately improving the treatment effect of intrauterine adhesions. Technical solution

[0010] This invention provides a highly adhesive modified mesenchymal stem cell, wherein the mesenchymal stem cell is genetically engineered to overexpress one or more integrin subunits, wherein the integrin subunits are selected from the integrin family that binds to the extracellular matrix of endometrial cells.

[0011] (a) Integral target selection The main components of the extracellular matrix of the endometrium include type I collagen, type III collagen, fibronectin, and laminin. Integrins that mediate the binding of MSCs to these matrix components include: α2β1: Composed of α2 and β1 subunits, its main ligands are type I and type III collagen, and it is the main integrin for collagen binding. α11β1: Composed of α11 and β1 subunits, with type I collagen as the main ligand. α5β1: Composed of α5 and β1 subunits, its main ligand is fibronectin. αvβ3: Composed of αv and β3 subunits, its main ligands are fibronectin and hyalin. α6β1: Composed of α6 and β1 subunits, its main ligand is laminin. The preferred targets of this invention are α2β1 (collagen binding) and α5β1 (fibronectin binding), because they are abundant in the endometrial ECM.

[0012] (II) Genetic Modification Strategies This invention employs one or a combination of the following strategies to achieve integrin overexpression: Strategy A: Overexpression of integrin α2 subunit Using lentiviral vectors or CRISPR activation systems, MSCs stably and highly express the integrin α2 subunit (ITGA2 gene). The α2 subunit pairs with the endogenous β1 subunit to form more functional α2β1 integrins, enhancing their binding ability to endometrial collagen.

[0013] Strategy B: Overexpression of integrin α5 subunit Using lentiviral vectors or CRISPR activation systems, MSCs stably and highly express the integrin α5 subunit (ITGA5 gene). The α5 subunit pairs with the endogenous β1 subunit to form more functional α5β1 integrins, enhancing their binding ability to endometrial fibronectin.

[0014] Strategy C: Dual overexpression of α2 and α5 subunits Simultaneous overexpression of ITGA2 and ITGA5 genes enables high expression of α2β1 and α5β1 on the surface of MSCs, achieving dual high adhesion to collagen and fibronectin.

[0015] Strategy D: Overexpression of integrin β1 subunit (auxiliary strategy) Since both α2 and α5 require pairing with β1, overexpression of the β1 subunit (ITGB1 gene) can increase pairing efficiency and can be used in conjunction with the α subunit as an auxiliary strategy.

[0016] (III) Construction of gene modification vectors Taking lentiviral vectors as an example, the vector structure includes: 5'LTR and 3'LTR: Long terminal repeat sequences that mediate viral integration ψ: Viral packaging signal RRE: Rev Response Element cPPT: Central polypurine tracts enhance nuclear transport. Promoters: Selected from strong promoters such as CMV, EF1α, and PGK, driving integrin gene expression. Integrin genes: coding sequences of ITGA2, ITGA5, or ITGB1 WPRE: Post-transcriptional regulatory element of marmot hepatitis virus, enhancing expression Marker genes: Optional, such as GFP or puromycin resistance genes, used for screening and tracking. (iv) Improved MSC preparation method Includes the following steps: MSC isolation and culture: MSCs are isolated from human umbilical cord, placenta, fat, or bone marrow tissue and expanded and cultured to the appropriate passage number. Lentiviral packaging: Lentiviral vectors carrying integrin genes were co-transfected with packaging plasmids into 293T cells, and viral supernatants were collected. MSC infection: MSCs were co-cultured with lentiviral supernatant, and polybrene was added to improve infection efficiency. Selection and amplification: 48-72 hours post-infection, selection reagents were added to obtain stable expression cell lines, which were then amplified and cultured. Quality control: Flow cytometry was used to detect integrin expression levels, confirming successful modification. (v) Synergistic application with the device of Patent 1 The modified MSCs of this invention are applicable for use in conjunction with Patent 1 (a dynamic stem cell culture and drug delivery device for the treatment of intrauterine adhesions) filed on the same day by the applicant, forming a complete intrauterine adhesion treatment system. Patent 1's device provides a closed uterine cavity environment, guides adhesion through changes in body position, and maintains activity through regular fluid changes; the modified MSCs of this invention provide high adhesion capacity, and the two work synergistically to achieve the best therapeutic effect.

[0017] Mechanism of action The mechanism of this invention in improving MSC-enhanced treatment of intrauterine adhesions includes: Enhanced initial adhesion: Overexpression of integrins increases the density of functional integrins on the surface of MSCs, significantly improving the binding rate and binding strength with endometrial ECM, and enabling faster initial adhesion during the position-guided adhesion phase.

[0018] Resistance to liquid erosion: Due to the enhanced adhesion mediated by integrin, the detachment rate of modified MSCs is significantly reduced under the liquid shear force generated by regular liquid changes.

[0019] Inhibition of anopyroptosis: MSCs are anchorage-dependent cells and must adhere to ECMs to survive. Modified MSCs, due to enhanced adhesion, inhibit anopyroptosis and improve cell survival.

[0020] Promotes survival signal transduction: After integrins bind to ECM, they activate the downstream FAK-PI3K-Akt signaling pathway, promoting cell survival, proliferation and anti-apoptosis.

[0021] Synergistic enhancement of repair function: Modified MSCs with extended survival time continuously secrete repair factors such as KGF-2, HGF, and IL-10, which promote endometrial epithelial proliferation, angiogenesis, and inhibit fibrosis, ultimately achieving endometrial regeneration. Beneficial effects

[0022] Compared with unmodified conventional MSCs, the improved MSCs of this invention are expected to have the following beneficial effects: Integrin expression levels were significantly increased, laying the foundation for enhanced adhesion. Initial adhesion rate: Significantly improved, achieving wall adhesion faster. Erosion resistance: Significantly enhanced, more resistant to liquid erosion. Abortion rate was significantly reduced, and survival rate was increased. In vivo survival time: significantly prolonged, allowing for long-term efficacy. In vivo retention rate: significantly increased, with more cells remaining at the lesion site. Endometrial repair effect: Significant improvement, enhanced therapeutic effect When the modified MSCs of this invention are used in conjunction with the device of Patent 1, a synergistic enhancement effect is expected: the device of Patent 1 provides the best adhesion environment for the modified MSCs, and the modified MSCs make full use of the advantages of the device to maximize their adhesion potential. The two work together to achieve a therapeutic effect of "1+1>2". Detailed Implementation

[0023] (The following examples describe the expected implementation and results of the present invention. Specific experimental data will be provided in the form of a supplementary experimental report within 12 months from the priority date.) Example 1: Preparation of MSCs modified by integrin α2 overexpression 1.1 Construction of Lentiviral Vector The human ITGA2 gene coding sequence (GenBank: NM_002203.4) was synthesized, cloned into a lentiviral expression vector, and the pLVX-EF1α-ITGA2-IRES-Puro recombinant plasmid was constructed. The sequence was verified to be correct by enzyme digestion and sequencing.

[0024] 1.2 Lentiviral Packaging 293T cells were seeded in culture dishes at a density of 70-80%. pLVX-EF1α-ITGA2-IRES-Puro was co-transfected with packaging plasmids psPAX2 and pMD2.G using liposome transfection reagent. The culture medium was replaced with fresh medium 6 hours after transfection. Viral supernatant was collected at 48 and 72 hours post-transfection, filtered through a 0.45 μm filter, and concentrated by ultracentrifugation. Viral titer was determined using qPCR.

[0025] 1.3 Isolation and culture of human umbilical cord MSCs Umbilical cords were aseptically collected from healthy full-term cesarean section fetuses (with informed consent). Arteries and veins were removed, and Wharton's jelly was cut into 1 mm³ pieces and placed in α-MEM medium containing 10% FBS, incubated at 37°C and 5% CO2. Cells emerged after 7-10 days and were passaged to P3 for further development. Flow cytometry analysis confirmed that the cells met the MSC surface marker criteria: CD73⁺, CD90⁺, CD105⁺, CD34⁻, CD45⁻, and HLA-DR⁻.

[0026] 1.4 MSC Infection and Screening P3 generation MSCs were seeded in 6-well plates at a density of 5 × 10⁶. 4 / well. Add lentiviral supernatant (MOI=10) and add 8 μg / mL of polybrene. Replace with fresh medium 24 hours post-infection. Add 1 μg / mL of puromycin for selection 48 hours post-infection. After 7 days of selection, surviving cells are considered stable expression lines and are then amplified.

[0027] 1.5 Integrin Expression Validation Collect modified MSCs and ordinary MSCs (control), and adjust the concentration to 1×10⁻⁶. 6 / mL. Add fluorescently labeled anti-human integrin α2 antibody and incubate at 4°C for 30 minutes. Wash twice with PBS and analyze by flow cytometry.

[0028] Expected results: The integrin α2 positivity rate and average fluorescence intensity of modified MSCs were significantly higher than those of ordinary MSCs.

[0029] Example 2: Preparation of MSCs modified by integrin α5 overexpression Following the method in Example 1, the ITGA2 gene was replaced with the ITGA5 gene (GenBank: NM_002205.5), and the pLVX-EF1α-ITGA5-IRES-Puro vector was constructed. Lentiviral virus was packaged, infected MSCs, and screened to obtain integrin α5 overexpressing modified MSCs.

[0030] Expected results: The integrin α5 positivity rate and average fluorescence intensity of the modified MSCs were significantly higher than those of the ordinary MSCs.

[0031] Example 3: Preparation of modified MSCs with integrin α2 / α5 double overexpression A dual-gene lentiviral vector simultaneously expressing ITGA2 and ITGA5 was constructed using an IRES or 2A linker peptide strategy, such as pLVX-EF1α-ITGA2-IRES-ITGA5-Puro. Lentiviral vectors were packaged according to the method in Example 1, infecting MSCs, and screening to obtain modified MSCs with dual overexpression of α2 / α5.

[0032] Expected results: The double positivity rate of integrin α2 and α5 in modified MSCs was significantly higher than that in ordinary MSCs, and the expression levels of both integrins were significantly increased.

[0033] Example 4: Validation of in vitro adhesion function (expected plan) 4.1 Static Adhesion Experiment Endometrial ECM components (fibronectin 10 μg / mL, type I collagen 20 μg / mL) were coated onto 96-well plates and incubated overnight at 4°C. After washing with PBS, the plates were blocked with 1% BSA. 100 μL of cell suspension (containing 2 × 10⁶ cells / mL) was added to each well. 4 MSCs were divided into four groups: normal MSCs, α2-overexpressing MSCs, α5-overexpressing MSCs, and α2 / α5 double-overexpressing MSCs. After incubation at 37°C for 15, 30, 60, and 120 minutes, respectively, unattached cells were gently washed three times with PBS. CCK-8 working solution was added to each well, and the cells were incubated at 37°C for 2 hours. The OD value at 450 nm was measured using a microplate reader. Adhesion rate = (OD value of experimental groups / OD value of total cells) × 100%.

[0034] Expected results: The adhesion rate of modified MSCs was significantly higher than that of ordinary MSCs at all time points, with the α2 / α5 double overexpression group showing the highest adhesion rate and the fastest adhesion kinetics.

[0035] 4.2 Flow Shear Force Experiment A microfluidic channel system was used, with channels coated with a fibronectin-collagen mixed matrix (simulating endometrial ECM). MSCs from each group were seeded into the channels at a density of 2 × 10⁶. 5 / mL, let stand for 2 hours to allow cells to adhere. Connect the syringe pump and perfuse the culture medium for 30 minutes at shear forces of 0.5, 1.0, 2.0, and 5.0 dyn / cm². Count the number of cells before and after applying the shear force under a microscope and calculate the detachment rate.

[0036] Expected results: The shedding rate of modified MSCs under various shear stress conditions was significantly lower than that of ordinary MSCs. Among them, the α2 / α5 double overexpression group had the lowest shedding rate and showed the strongest anti-erosion ability.

[0037] 4.3 Apoplexy Assay MSCs from each group were cultured in suspension in low-adsorption culture plates (without adherence). Cells were collected at 0, 6, 12, and 24 hours after suspension, and Annexin V / PI double staining was performed. Apoptosis rate was detected by flow cytometry.

[0038] Expected results: The apoptosis rate of the modified MSCs at all time points in suspension culture was significantly lower than that of ordinary MSCs. Among them, the α2 / α5 double overexpression group had the lowest apoptosis rate and showed the strongest anti-anodic apoptosis ability.

[0039] Example 5: In vitro simulation in conjunction with the device of Patent 1 (expected solution) A miniaturized version of the device from Patent 1 (for rats) was used to simulate the dynamic uterine cavity culture environment in vitro. The uterine cavity simulation chamber had a volume of 0.3 mL, and the walls were coated with a mixture of endometrial ECM matrix. Each group's MSC suspension was perfused (5 × 10⁻⁶). 6 Simulated cell position changes (rotating the device 90° every 30 minutes for 4 hours) and simulated medium changes (twice daily, 0.3 mL each time, for 7 consecutive days) were performed. Cell counts were observed daily under a fluorescence microscope, and cell counts were performed on day 7 to calculate the retention rate.

[0040] Expected results: In a simulated dynamic culture environment, the 7-day retention rate of the modified MSCs was significantly higher than that of ordinary MSCs, with the α2 / α5 double overexpression group showing the highest retention rate and exhibiting the best synergistic effect with the device of Patent 1.

[0041] Example 6: In vivo validation of a rat model of intrauterine adhesions (expected protocol) Establishment of an intrauterine adhesion model in SD rats: After anesthesia, a hysteroscope was inserted vaginally, and the endometrium on the right side was mechanically scraped down to the superficial myometrium. Lipopolysaccharide was then instilled into the uterine cavity to induce an inflammatory response. The left uterus served as a normal control. Successful model establishment was defined as follows: HE staining of the right uterus showed thinning of the endometrium, reduction of glands, and fibrosis area >50%.

[0042] The model rats were randomly divided into 6 groups, with 10 rats in each group: Group 1: Model control (no treatment) Group 2: Single injection of standard MSCs Group 3: Conventional MSC + Patent 1 device Group 4: Single injection of α2 / α5 dual-overexpression modified MSCs Group 5: α2 / α5 dual overexpression modified MSCs + Patent 1 device Group 6: Sham surgery control After treatment, the following indicators will be tested: Stem cell retention rate: Some animals were sacrificed on days 1, 3, and 7, and uterine tissue homogenates were collected for flow cytometry analysis of GFP-labeled cells. Endometrial thickness: measured on day 14 using HE staining. Fibrosis area: Percentage of collagen deposition area calculated by Masson staining on day 14. Inflammatory factors: TNF-α, IL-6, and IL-10 levels were measured by ELISA on day 7. Pregnancy rate: On day 30, rats were mated with male rats to observe the conception rate and number of embryos. Expected results: The stem cell retention rate in the modified MSC + patented device group was significantly higher than that in other treatment groups. The improved MSC+ patent 1 device group showed the closest endometrial thickness recovery to the sham surgery control group. The improved MSC+ patent 1 device group has the smallest fibrous area. The modified MSC+ patent 1 device group had the highest IL-10 levels and the lowest TNF-α and IL-6 levels. The modified MSC + patented device group had the highest pregnancy rate, significantly better than other treatment groups. The improved MSC+Patent 1 device set exhibits synergistic effects across all detection indicators. Example 7: Safety Evaluation (Expected Solution) 7.1 In vitro safety Proliferative activity: The proliferation curves of modified MSCs and ordinary MSCs were detected by CCK-8 assay, and no significant difference was expected. Karyotype analysis: G-banding analysis of modified MSC karyotype, expected to be normal. Tumorigenicity: Soft agar colony formation assay showed no expected colony formation; subcutaneous injection of modified MSCs into nude mice and observation for 3 months showed no expected tumor formation. 7.2 In vivo safety In the animal experiments of Example 6, the following indicators were monitored: General condition: Weight, activity level, food intake, etc., expected to be normal. Hematology: Complete blood count, liver and kidney function tests, expected to be normal. Histology: HE staining of liver, spleen, lung, and kidney showed no abnormal cell infiltration or tumor formation as expected.

Claims

1. A highly adhesive modified mesenchymal stem cell, characterized in that, The mesenchymal stem cells are genetically engineered to overexpress one or more integrin subunits, which are selected from the integrin family that bind to the extracellular matrix of endometrial cells.

2. The improved mesenchymal stem cells according to claim 1, characterized in that, The integrin subunit is selected from the integrin α2 subunit (ITGA2), the integrin α5 subunit (ITGA5), the integrin β1 subunit (ITGB1), or a combination thereof.

3. The improved mesenchymal stem cells according to claim 2, characterized in that, The modified mesenchymal stem cells simultaneously overexpress integrin α2 subunit and integrin α5 subunit.

4. The improved mesenchymal stem cells according to claim 1, characterized in that, The genetic engineering modifications are achieved through lentiviral vectors, retroviral vectors, adenoviral vectors, CRISPR activation systems, or transposon systems.

5. The improved mesenchymal stem cells according to claim 4, characterized in that, The lentiviral vector contains a promoter, an integrin gene coding sequence, and a selection marker gene, wherein the promoter is selected from CMV, EF1α, or PGK.

6. The improved mesenchymal stem cells according to claim 1, characterized in that, The mesenchymal stem cells are derived from the umbilical cord, placenta, adipose tissue, or bone marrow.

7. A method for preparing the modified mesenchymal stem cells according to any one of claims 1-6, characterized in that, Includes the following steps: (1) Isolate and culture mesenchymal stem cells; (2) Construct a recombinant vector carrying an integrin gene; (3) Introduce the recombinant vector into mesenchymal stem cells; (4) Screen to obtain modified mesenchymal stem cells that stably overexpress integrin.

8. The method according to claim 7, characterized in that, The recombinant vector is a lentiviral vector, and step (3) is achieved through lentiviral infection.

9. A cell preparation, characterized in that, It comprises the modified mesenchymal stem cells as described in any one of claims 1-6 and a pharmaceutically acceptable carrier.

10. The cell preparation according to claim 9, characterized in that, The pharmaceutically acceptable carrier is physiological saline, culture medium, or medical fluid that meets the requirements for intrauterine perfusion.

11. A system for treating intrauterine adhesions, characterized in that, include: (1) The modified mesenchymal stem cells according to any one of claims 1-6 or the cell preparation according to claim 9; (2) A dynamic intrauterine culture and drug delivery device, the device comprising a dual-lumen tube body, a liquid inlet system, and a liquid outlet system, for achieving intrauterine cavity closure, guided wall adhesion by changes in body position, and periodic liquid replacement.

12. The treatment system according to claim 11, characterized in that, The intrauterine dynamic culture and drug delivery device is a medical device capable of achieving intrauterine cavity closure and fluid exchange.

13. The use of the modified mesenchymal stem cells according to any one of claims 1-6 in the preparation of a drug for treating intrauterine adhesions.

14. The application according to claim 13, characterized in that, The modified mesenchymal stem cells are used in combination with a dynamic intrauterine culture and drug delivery device, which is used to achieve intrauterine cavity closure, guided adhesion by changes in body position, and regular fluid changes.

15. A method for treating intrauterine adhesions, characterized in that, The procedure includes the following steps: (1) inserting the intrauterine dynamic culture and drug delivery device into the patient's uterine cavity and sealing the internal cervical os; (2) infusing the modified mesenchymal stem cell suspension according to any one of claims 1-6 into the uterine cavity through the device; (3) guiding the stem cells to adhere evenly to the wall of the uterine cavity by changing the body position; (4) changing the culture medium periodically through the device to maintain the activity and function of the stem cells; and (5) removing the device after the treatment is completed.