Use of mesenchymal stem cells in the preparation of a composition for treating the ultra-rare disease plasminogen deficiency

Abstract

The application provides application of mesenchymal stem cells in preparation of a composition for treating the ultra-rare disease plasminogen deficiency (PLGD). The application discloses a novel treatment method for the ultra-rare disease plasminogen deficiency, which comprises treatment by using mesenchymal stem cells. The mesenchymal stem cells can significantly improve the symptoms of infertility caused by plasminogen deficiency, improve intrauterine adhesion, treat endometrial inflammation, repair endometrium, significantly improve the pregnancy rate and the number of offspring.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and more specifically, this invention relates to the application of mesenchymal stem cells in the preparation of compositions for treating plasminogen deficiency, an extremely rare disease. Background Technology

[0002] Plasminogen (PLG) deficiency is an extremely rare multisystem disease, with an estimated prevalence of approximately 1.6 cases per million people. It is characterized by the formation of fibrin-rich pseudomembranes on mucous membranes and is a lifelong, systemic, multisystemic disorder. Common symptoms include ligneous conjunctivitis, which can lead to visual impairment or loss; other affected systems include the respiratory tract, oropharynx, female reproductive tract, gums, middle ear, kidneys, skin, and central nervous system. Due to the lack of specific treatment options, patients with plasminogen deficiency face a significantly reduced quality of life and even life-threatening risks.

[0003] The fibrinolytic system plays a crucial role in the female menstrual cycle, and fertility is significantly affected in women with PLG deficiency. The inventors previously treated patients with PLG deficiency who presented with intractable abnormal uterine bleeding and infertility. Repeated hysteroscopic examinations revealed no endometrial proliferative lesions pathologically, but rather extensive inflammatory cell infiltration and tissue necrosis. There is currently no effective treatment for PLG deficiency-related abnormal uterine bleeding and infertility. How to effectively restore normal uterine function in patients with PLG deficiency, enabling them to have regular menstruation and pregnancy, is a significant clinical challenge.

[0004] For PLG deficiency, the main treatment is symptomatic surgical excision (removal of cellulose deposits or lignosy lesions in tissues and organs), but there is a very high recurrence rate and a risk that the recurrence will be more severe.

[0005] On June 4, 2021, Liminal BioSciences announced that the U.S. Food and Drug Administration (FDA) approved Ryplazim (plasminogen, human-TVMH) for the treatment of patients with plasminogen type 1 deficiency (hypoplasminemia). Ryplazim is currently the only FDA-approved treatment for plasminogen deficiency. However, this orphan drug has not yet been marketed and has obvious limitations: (1) It requires long-term and frequent intravenous injection: According to the Ryplazim drug instructions, Ryplazim requires long-term intravenous injection, and the recommended dose is 6.6 mg / kg body weight intravenously every 2 to 4 days (Q2D to Q4D); the drug needs to be prepared on-site before each injection and the intravenous injection should be completed within 3 hours; if the lesion does not resolve within 12 weeks, or if there is a new or recurrent lesion, the dosing frequency should be increased by one day every 4-8 weeks until Q2D dosing, and the clinical effect should be reassessed at the same time; if the lesion resolves before 12 weeks, continue to dosing at the same dosing frequency, and monitor for new or recurrent lesions every 12 weeks. (2) Higher risks: including (i) bleeding: Patients with type 1 plasminogen deficiency may experience bleeding due to active mucosal disease-related lesions during Ryplazim treatment; depending on the lesion site, this may manifest as gastrointestinal bleeding, hemoptysis, epistaxis, vaginal bleeding, or hematuria; Ryplazim may prolong or worsen bleeding in patients with bleeding disorders or those using anticoagulants and / or antiplatelet drugs and other medications that may interfere with normal coagulation; and uncontrollable bleeding may occur, requiring emergency treatment and discontinuation of Ryplazim; (ii) tissue shedding: Tissue shedding may occur at the mucosal site after Ryplazim treatment when plasminogen activity levels return to physiological levels and fibrinolysis occurs; after treatment, respiratory... (iii) Lesions of the gastrointestinal and genitourinary systems may detach, leading to bleeding or organ obstruction; patients with tracheobronchial lesions may experience airway obstruction or hemoptysis; patients with gastrointestinal and genitourinary lesions may experience tissue detachment, resulting in pain and tissue bleeding in the affected organ systems; (iv) Monitoring of neutralizing antibodies is required: monitor patients for loss of clinical efficacy due to the occurrence of new or recurrent lesions during Ryplazim treatment and obtain plasminogen activity trough values ​​to confirm that adequate plasminogen activity levels have been reached and maintained; (v) Abnormal laboratory indicators: D-dimer levels in the blood of patients using Ryplazim may be elevated; plasma D-dimer levels in patients undergoing venous thromboembolism (VTE) screening should be approached with caution.

[0006] In summary, there is an urgent need in this field to develop drugs with higher safety profiles for plasminogen deficiency, particularly those that can help improve female infertility. Summary of the Invention

[0007] The purpose of this invention is to provide the use of mesenchymal stem cells in the preparation of compositions for the treatment of plasminogen deficiency, an extremely rare disease.

[0008] In a first aspect of the invention, the use of mesenchymal stem cells in the preparation of compositions for treating plasminogen deficiency is provided; wherein plasminogen deficiency is characterized by low expression or low activity of plasminogen PLG (including no expression or no activity) and manifests as infertility.

[0009] In one or more embodiments, the plasminogen genotype of the patient with plasminogen deficiency is PLG. - / - or PLG + / - .

[0010] In one or more embodiments, the mesenchymal stem cells include those selected from: umbilical cord mesenchymal stem cells (UcMSC), adipose-derived mesenchymal stem cells (AdMSC), and endometrial mesenchymal stem cells (EnMSC).

[0011] In one or more embodiments, the mesenchymal stem cells are human cells (hUcMSC, hAdMSC, hEnMSC).

[0012] In one or more embodiments, the plasminogen deficiency also manifests as abnormal uterine bleeding and symptoms of impaired endometrial shedding.

[0013] In one or more embodiments, the patient with plasminogen deficiency meets the following criteria: (1) not postmenopausal; (2) has plasminogen glycosides (PLGs). - / - (3) FSH < 10 mIU / mL and AMH > 1.2 ng / mL on day 5 of menstruation; (4) Regular ovulation is confirmed by ultrasound monitoring or basal body temperature monitoring; (5) No organic lesions of the endometrium; (6) No endometrial or systemic infectious lesions.

[0014] In one or more embodiments, the organic lesions include: septate uterus, uterine fibroids, severe adenomyosis, endometriosis, endometrial cancer and precancerous lesions, cervical cancer and precancerous lesions, etc.

[0015] In one or more embodiments, the mesenchymal stem cells include their cultures, their lysis products, or mixtures containing them.

[0016] In one or more embodiments, the composition includes mesenchymal stem cells mounted in a hydrogel.

[0017] In one or more embodiments, the mesenchymal stem cells are loaded onto cross-linked sodium hyaluronate gel (HA-GEL).

[0018] In one or more embodiments, the composition includes mesenchymal stem cells dissolved in an electrolyte injection solution.

[0019] In one or more embodiments, the electrolyte injection solution includes an injection solution containing 1 to 5% (e.g., 2 to 3%) human hemoglobin.

[0020] In one or more embodiments, the concentration of mesenchymal stem cells in the composition is 5 × 10⁻⁶. 4 ~5×10 9 5 × 10⁵ cells / mL; preferably 5 × 10⁵ cells / mL. 5 ~5×10 8 5 × 10⁵ cells / mL; more preferably 5 × 10⁵ cells / mL. 6 ~5×10 7 .

[0021] In one or more embodiments, the concentration of mesenchymal stem cells in the composition is 1 × 10⁻⁶. 6 ~4×10 7 Cells / mL.

[0022] In one or more embodiments, the concentration of mesenchymal stem cells in the composition is 1.5 × 10⁻⁶. 6 ~3.5×10 7 Cells / mL.

[0023] In one or more embodiments, the concentration of mesenchymal stem cells in the composition is 2.5 × 10⁻⁶. 6 4×10 6 5×10 7 1.5×10 7 2×10 7 2.5×10 7 4×10 7 5×10 7 1.5×10 8 2.5×10 8 Cells / mL.

[0024] In one or more embodiments, the treatment of plasminogen deficiency for symptom relief or treatment is to (significantly) increase pregnancy rates (i.e., the composition is also used to increase pregnancy rates).

[0025] In one or more embodiments, the treatment of plasminogen deficiency for symptom relief or treatment is as follows: (significantly) increasing the number of offspring, improving menstruation, repairing the endometrium, treating intrauterine adhesions, and improving intrauterine inflammation (i.e., the composition is also used to increase the number of offspring, improve menstruation, repair the endometrium, treat intrauterine adhesions, and improve intrauterine inflammation).

[0026] In one or more embodiments, the composition includes a pharmaceutical composition.

[0027] In one or more embodiments, the pharmaceutical composition is placed in a medicine box or kit.

[0028] In one or more embodiments, the composition is prepared in a unit dose (unit dose) manner.

[0029] In another aspect of the invention, a method for treating plasminogen deficiency is provided, comprising: administering mesenchymal stem cells to a subject requiring treatment, wherein the plasminogen PLG for treating plasminogen deficiency is poorly expressed or poorly active; preferably, the plasminogen deficiency manifests as infertility.

[0030] In one or more embodiments, in the method for treating plasminogen deficiency, the dose of the mesenchymal stem cells is 1 × 10⁻⁶. 4 ~1×10 9 1 cell / dose (human); preferably 1×10 5 ~1×10 8 Cells / dose (human); preferably 1×10 6 ~5×10 7 Cells / dose (human).

[0031] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0032] Figure 1 PLG - / - Pregnancy rate (percentage) of female mice treated with mesenchymal stem cells or PLG.

[0033] Figure 2 PLG - / - Number of offspring born to female mice after treatment with mesenchymal stem cells or PLG (vertical axis: number of offspring).

[0034] Figure 3 PLG + / - Pregnancy rate (percentage) of female mice treated with mesenchymal stem cells or PLG.

[0035] Figure 4 PLG + / - Number of offspring born to female mice after treatment with mesenchymal stem cells or PLG (vertical axis: number of offspring).

[0036] Figure 5Analysis of the effects of UcMSCs transplantation on endometrial repair and improved menstruation in patients with PLGD complex compound mutations (AC).

[0037] Figure 6 PLG - / - A schematic diagram illustrating the genetic modification performed during the creation of female mice.

[0038] Figure 7 1. Evaluation and screening of suitable hydrogel materials for carrying mesenchymal stem cells (MSCs). A. Live / dead staining showed that different hydrogel materials had different effects on MSC survival when co-cultured with MSCs. Compared with the control group, HA-GEL co-culture had no effect on MSC survival, while collagen scaffold inhibited MSC survival (inhibition rate 16.1%), 2.5% CPAA gel inhibited MSC survival (inhibition rate 30.6%), and 15% PLGA-PEG-PLGA gel inhibited MSC survival (inhibition rate 81.6%). B. Transwell migration assays showed that HA-GEL and endometrial cancer cell lines ECC-1 or Ishikawa had different effects on MSC migration. The number of migrating cells was counted in five fields of view under any 10x microscope: HA-GEL-coated bottom of the Transwell chamber inhibited mesenchymal stem cell (MSC) migration; when HA-GEL-coated bottom of the Transwell chamber was used for co-culturing with ECC-1 in the lower chamber, MSC migration was unaffected; when HA-GEL-coated bottom of the Transwell chamber was used for co-culturing with Ishikawa in the lower chamber, MSC migration was promoted. RT-qPCR showed that HA-GEL co-culturing had no effect on the expression levels of stem cell genes and cytokine mRNA in MSCs. MSCs were derived from the endometrium; HA-GEL was cross-linked sodium hyaluronate gel; collagen scaffold was a collagen membrane; CPAA was cross-linked polyacrylamide gel; and PLGA-PEG-PLGA was a polylactic acid-polyethylene glycol-polylactic acid thermosensitive hydrogel. *: P < 0.05, **: P < 0.01, ***: P < 0.001, ns showed no statistical significance. Detailed Implementation

[0039] Through in-depth research and experimentation, the inventors have for the first time disclosed a novel treatment method for the extremely rare disease plasminogen deficiency, which includes the application of mesenchymal stem cells. These mesenchymal stem cells can significantly improve infertility symptoms caused by plasminogen deficiency, and significantly increase pregnancy rates and the number of offspring.

[0040] As used in this article, “mesenchymal stem cell(s)” and “MSC(s)” can be used interchangeably.

[0041] As used in this article, the gene / protein sequence of the human “PLG (Plasminogen)” can be found in Gene ID:5340.

[0042] The information shown; the gene / protein sequence of the mouse (mouse) source “PLG (Plasminogen)” can be found in the information shown in Gene ID:18815.

[0043] In this invention, the term "containing" indicates that various ingredients may be used together in the mixtures or compositions of this invention. Therefore, the terms "consistent with..." and "composed of..." are included in the term "containing". As used herein, the terms "effective amount" or "effective dose" refer to an amount that is functional or active in a subject and acceptable to humans and / or animals.

[0044] As used in this article, a “pharmaceuticalally acceptable” ingredient is a substance that is suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio.

[0045] As used herein, a "pharmaceutically acceptable carrier" is a solvent, suspending agent, or excipient that is controllable in terms of toxicity and side effects, environmentally friendly, or harmless to humans and animals, for delivering the mesenchymal stem cells or cell cultures, cell lysis products, cell culture supernatants, or cell lysis products of the present invention to the object requiring treatment. The carrier may be liquid or solid, preferably a carrier capable of maintaining the activity of the mesenchymal stem cells or cell cultures, cell lysis products, cell culture supernatants, or cell lysis products to a high degree.

[0046] As used herein, the term "effective amount" or "effective dose" means an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals, as used herein.

[0047] Typically, the cells can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5-8, preferably about 6-8.

[0048] Each maximum numerical limit given in this specification includes each lower numerical limit, as if those lower numerical limits were explicitly stated herein. Each minimum numerical limit given throughout this specification includes each higher numerical limit, as if such higher numerical limits were explicitly stated herein. Each numerical range given in this specification includes each narrower numerical range falling within such a wider range, as if those narrower numerical ranges were explicitly stated herein.

[0049] Mesenchymal stem cells are a type of adult pluripotent stem cell that naturally exists in various tissues of the human body and can be isolated from tissues such as bone marrow, peripheral blood, umbilical cord blood, and adipose tissue.

[0050] In this invention, the cells may be isolated cells, their cultures, their lysis products, or mixtures containing the like. The cells may be naturally isolated cells, cells cultured in vitro, or cells that have been recombined and modified from natural or in vitro cultured cells while maintaining their therapeutic function / activity.

[0051] In a specific embodiment of the present invention, the inventors' experimental research showed that, targeting the PLG (Plasminogen) gene, PLG... - / - Female mice were unable to conceive normally when housed with wild-type male mice. Compared to the control group, mice injected via tail vein with human umbilical cord mesenchymal stem cells (PLG) were more fertile. - / - Pregnancy rates were significantly improved in female mice. Furthermore, intrauterine transplantation of MSCs can effectively improve the uterine environment and increase pregnancy rates, and could be used to treat plasminogen deficiency (manifested as infertility).

[0052] The mesenchymal stem cells of this invention are living cells. After obtaining these cells, they can be prepared / proliferated in large quantities in vitro through methods such as passage and regeneration. This is typically done by seeding them into solid agar plates or liquid culture media for cell expansion culture. The obtained living cells can be further subjected to laboratory domestication, genetic breeding, and molecular genetic manipulation to obtain mutants and transformants. In addition, the mesenchymal stem cells can also be used as host cells for heterologous expression in bioengineering.

[0053] In addition to the isolated, naturally occurring mesenchymal stem cells, this invention also includes variants, domesticated cells, or genetically engineered cells. The variants are functionally equivalent or functionally enhanced variants; preferably, the functionally equivalent or functionally enhanced variants include those with properties that improve infertility.

[0054] The variant can be a variant formed by mutagenesis of the cells. Cell mutagenesis modification refers to inducing mutations in the genetic characteristics of plants and animals using physical and chemical factors, then selecting individual plants / individuals from the mutant population that meet certain requirements, and subsequently cultivating them into modified cells. Based on focusing on the main activities of the mesenchymal stem cells, mutagenesis can be used to screen for mesenchymal stem cell variants with stronger effects or functions; alternatively, mutagenesis can be used to screen for cells whose function or function remains unchanged but exhibit superior growth performance, more ideal environmental compatibility (e.g., modification through mutagenesis when toxic side effects occur), and more significant resistance to adversity. Mutagenesis can be performed using some mutagenesis methods known to those skilled in the art, including: starting cell culture, mutagenesis treatment, observation of a large number of cells, comparison of the properties of interest, and finally screening for cells whose properties of interest have been enhanced and modified. This mutagenesis can usually be performed in a high-throughput manner, and preferably can be repeated multiple times or through multiple generations.

[0055] Cell domestication differs from cell mutation. It generally refers to the method of selectively adapting cells to specific conditions through artificial means. It typically involves the following steps: starting cell culture with alterations to various conditions / experimental parameters; observation of the cultured cells at scale; comparison of the properties of interest; and finally, selection of cells / cell populations whose properties have been enhanced or modified. Domestication can yield cells with higher tolerance and activity. Cell domestication usually requires two or more passages to allow cells to adapt to certain conditions or achieve improvements in the properties of interest.

[0056] Cells modified by bioengineering / genetic engineering based on the natural MSC cells of this invention are also included in this invention. For example, exogenous genes can be introduced and endogenous genes can be deleted / knocked out in the mesenchymal stem cells, with the basic condition being that the cells retain their function or activity. The purpose of the modification may include: making the cells more viable in the body of the patient requiring treatment, or making the cells, while retaining their function or activity, further develop at least one other superior property. In some common cell modification work, one or more metabolic fluxes / signaling pathways can be enhanced / deactivated by analyzing intracellular metabolic fluxes / signaling pathways, thereby further optimizing cell performance.

[0057] Methods for introducing exogenous genes and deleting / knocking out endogenous genes are known in the art. For example, viral or non-viral constructs / vectors are used to carry expression cassettes of the exogenous gene to be introduced (including exogenous genes for overexpression, gene knockout, and gene editing). This construct / vector is then introduced into cells for targeted modification. The exogenous gene may include, but is not limited to, functional genes, enzyme genes, structural genes, miRNAs, siRNAs, shRNAs, etc.

[0058] In addition, cells can be modified by directly introducing exogenous proteins into the cells. For example, some membrane-penetrating peptides known in the art can be used to directly introduce exogenous proteins into the cells.

[0059] Based on the clear explanation of the cells, their biological characteristics, functions, and culture methods described in this invention, the methods for culturing the cells described in this invention in large quantities to obtain their cultures and lysis products are familiar to those skilled in the art. That is, the cultures or lysis products of mesenchymal stem cells obtained by these methods, as well as compositions containing them or their applications, are also included in this invention.

[0060] The culture medium suitable for culturing the mesenchymal stem cells described in this invention can be diverse. It should be understood that any culture medium capable of providing the necessary nutrients (such as carbon sources, nitrogen sources, etc.) for the growth and reproduction of the mesenchymal stem cells can be included in this invention. The culture medium can be solid or liquid; it can be commercially available or laboratory-prepared.

[0061] Based on obtaining the aforementioned mesenchymal stem cells, the present invention also provides cell cultures, cell lysis products, cell culture supernatants, or cell lysis products of the aforementioned mesenchymal stem cells, which have the function of treating plasminogen deficiency (manifested as infertility).

[0062] After obtaining the mesenchymal stem cells, those skilled in the art can easily obtain their culture products. For example, they can refer to some culture media or culture processes provided in the specific embodiments of the present invention, or use culture media or culture processes that are appropriately modified from the embodiments of the present invention but can still produce culture products, thereby obtaining cell cultures. The cell cultures contain active cells or substances secreted by them, and thus also have functions or activities similar to those of living cells.

[0063] The cell lysis product is a mixture formed by lysing cells using a cell-lysing reagent during or after the culture of the mesenchymal stem cells. The cell lysis product may be the product after removing solid impurities following lysis. It may be a purified or concentrated product, depending on the requirements.

[0064] The cell culture supernatant of the mesenchymal stem cells of the present invention is the culture medium remaining after removing cells and solid impurities during or after the culture of the mesenchymal stem cells. It may be unconcentrated or concentrated. Typically, cells and solid impurities can be removed by methods such as centrifugation or filtration.

[0065] The mesenchymal stem cells described in this invention have low immunogenicity and multi-directional differentiation capacity in the human body, making them ideal therapeutic cells.

[0066] The present invention also provides the application of the mesenchymal stem cells described herein for the preparation of compositions for treating plasminogen deficiency; wherein plasminogen deficiency is characterized by low expression or low activity of plasminogen (PLG) (including no expression or no activity) and manifests as infertility.

[0067] To better utilize the mesenchymal stem cells described in this invention, their culture or production process can be optimized. This optimization may include various culture conditions and the optimization of different components of the culture medium. Factors that significantly influence the culture and activity of these cells can be identified. It should be understood that these improvements are also encompassed within this invention.

[0068] The mesenchymal stem cells described herein can achieve long-term growth and stable passage. The culture media and methods used to culture these mesenchymal stem cells are not limited to those disclosed in this invention.

[0069] The present invention also provides a method for treating plasminogen deficiency, comprising: administering mesenchymal stem cells, their cultures, their lysate products, or mixtures thereof to the subject requiring treatment.

[0070] The present invention also provides a composition comprising the mesenchymal stem cells or cell cultures described herein, cell lysis products, cell culture supernatants, or cell lysis products. The composition includes, but is not limited to, pharmaceutical compositions, health product compositions, etc.

[0071] In addition to the mesenchymal stem cells of the present invention or the cell cultures, cell lysis products, cell culture supernatants or cell lysis products described herein, the composition may also include: a physiologically acceptable carrier or a microbiologically acceptable carrier.

[0072] Typically, the effective dose of the mesenchymal stem cells or the cell cultures, cell lysis products, cell culture supernatants or cell lysis products of the present invention may vary depending on the mode of application or the condition of the object to be treated.

[0073] Although specific embodiments of the present invention provide dosing regimens for animals such as mice, it should be understood that converting animal dosages to human dosages is readily achievable by those skilled in the art, for example, using the Meeh-Rubner formula: Meeh-Rubner formula: A = k × (W 2 / 3 ) / 10,000. Where A is the body surface area, expressed in meters. 2 Calculations: W represents body weight in grams; K is a constant that varies depending on the animal species, generally 9.1 for mice and rats, 9.8 for guinea pigs, 10.1 for rabbits, 9.9 for cats, 11.2 for dogs, 11.8 for monkeys, and 10.6 for humans. It should be understood that dosage conversions can vary depending on the drug and clinical circumstances, and are based on the assessment of an experienced pharmacist.

[0074] The dosage form of the composition can be diverse, including but not limited to: lyophilized agents, wettable powders, emulsifiable concentrates, aqueous solutions, emulsions, oily or aqueous dispersions, suspensions, powders, granules, or microcapsules. It should be understood that any dosage form that allows the composition of the present invention to be delivered to the object to be treated while retaining all or part of its activity is desirable. Preferred dosage forms are those that are easy to administer, such as suspensions, solutions, capsules, powders, lyophilized agents, etc.

[0075] Concentrated compositions contain higher levels of active ingredients, such as 20-90 wt%, while diluted compositions and those actually used contain lower levels of active ingredients, typically 0.00005-0.5 wt%. In addition, other suitable chemical agents, synergists, trace elements, stabilizers, binders, wetting agents, dispersants, emulsifiers, penetrants, solvents, fillers, and other commonly used components may be included. The compositions of this invention may also contain other active ingredients, such as digestive aids.

[0076] The positive advancements of this invention include at least:

[0077] They were the first to propose the idea of ​​using mesenchymal stem cells to treat the rare disease PLG deficiency. Compared with existing surgical symptomatic treatments and orphan drugs that may be marketed in the future, this approach has better treatment effects, a more convenient treatment method, more lasting treatment effects, and fewer side effects.

[0078] Compared with surgical treatment, the treatment of plasminogen deficiency by mesenchymal stem cell therapy can overcome at least the following disadvantages: (1) high risk of surgical trauma or complications; (2) poor efficacy of symptomatic treatment; and (3) high recurrence rate.

[0079] Compared with Ryplazim, mesenchymal stem cell therapy for plasminogen deficiency can overcome at least the following disadvantages: (1) it requires long-term and frequent injections, and each freshly prepared drug must be administered intravenously within 3 hours; (2) it has a short duration of action and requires re-injection every 2-4 days; and (3) it has many side effects.

[0080] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Science Press, or according to the manufacturer's recommendations.

[0081] Mesenchymal stem cell extraction reagents and equipment

[0082] The reagents and equipment required for preparing mesenchymal stem cells are shown in Table 1.

[0083] Table 1

[0084]

[0085] Example 1: Preparation of mesenchymal stem cells

[0086] I. Preparation of Umbilical Cord Mesenchymal Stem Cells (UcMSCs)

[0087] 1. Acquisition and culture of adipose-derived mesenchymal stem cells

[0088] Perinatal tissues such as umbilical cord, placenta, or amnion must be taken from tissues (waste products) after vaginal delivery or cesarean section (or cesarean section) at least 34 weeks of gestation. The umbilical cord donor (mother) must be free of factors that could lead to infectious diseases and pass all required examinations, including routine blood tests and biochemical tests. The preparation method is as follows:

[0089] (1) Take the umbilical cord out of the umbilical cord tissue collection bottle, put the umbilical cord tissue into a 15cm culture dish with sterile surgical forceps, cut the umbilical cord into 2cm segments with sterile surgical scissors, and rinse 3 times with physiological saline.

[0090] (2) Using ophthalmic scissors and forceps, remove veins and arteries from the umbilical cord tissue, peel off the umbilical cord epidermis, and place the obtained Wharton's jelly into a petri dish and immerse it in physiological saline to prevent the tissue from drying out; cut the Wharton's jelly into small pieces, each 2 mm in size. 3 Evenly place the tissue blocks into a 15cm culture dish, spacing them 1cm apart. After filling the dish, place it in a biosafety cabinet and observe until the edges of the tissue blocks become transparent. Then, slowly add the complete culture medium.

[0091] (3) Use a pipette to draw up the complete culture medium and add 25 mL to each culture dish. After completion, move it slowly and gently place it in a cell culture incubator at 37°C and 5% CO2 concentration for culture.

[0092] (4) Change medium: Replace the complete culture medium completely on the 7th and 11th days after inoculation. Tilt the culture dish to remove the old culture medium and add the new complete culture medium with a pipette. Add 25 mL of complete culture medium to each culture dish. After completion, carefully move the culture dish and place it in a cell culture incubator at 37°C and 5% CO2 concentration for culture.

[0093] 2. Passaging of umbilical cord mesenchymal stem cells

[0094] (1) Subculture was performed on the 14th day after the tissue block adhered to the wall.

[0095] (2) Transfer the culture dish to the biosafety cabinet and remove any tissue blocks from which no cells have migrated. Aspirate the old culture medium and wash the tissue blocks with 10–15 mL of physiological saline.

[0096] (3) Add 2-3 mL of stem cell digestive enzyme to each culture dish and digest for 2-5 minutes at room temperature. The digestion is complete when the cells shrink into a round shape under a microscope.

[0097] (4) Add 10 mL of complete culture medium to each dish to stop digestion. Gently pipette the cells to detach them. Collect the tissue blocks and cell suspension. Filter the suspension through a 100 μm cell filter and collect the filtrate. Centrifuge at 1500 rpm for 5 minutes.

[0098] (5) After centrifugation, discard the supernatant, add 2 mL of complete culture medium to each tube to resuspend the cell pellet, and take 50 μl of cell suspension to perform cell counting and survival rate detection using trypan blue staining solution.

[0099] (6) According to the cell count results, perform proportional passage, adding 25 mL of complete culture medium to each T150 culture flask.

[0100] (7) Transfer the culture flask to a cell culture incubator at 37°C and 5% CO2 concentration for culture.

[0101] 3. Cryopreservation of umbilical cord mesenchymal stem cells

[0102] (1) Observe the cell growth status under a microscope. When the cell culture reaches 80-90% confluence, it can be cryopreserved.

[0103] (2) Keep 10-15 mL of culture medium supernatant in each bottle for later use, and keep 30 mL of mixed culture medium for QC testing and sample retention.

[0104] (3) Wash the cells once with physiological saline, add 2-3 mL of mild digestive enzyme to each culture flask, digest at room temperature for 2-5 minutes, observe the cell morphology under a microscope, when the cell adhesions break and the cells shrink and become round, add 10-15 mL of the remaining complete culture medium to stop the digestion.

[0105] (4) Gently pipette the cells to detach them, collect the cell suspension into a 50mL centrifuge tube, adjust the cell suspension volume to 45mL / tube, and centrifuge at 1500rpm for 5 minutes.

[0106] (5) After centrifugation, discard the supernatant, resuspend the cell pellet in 2 mL of physiological saline in each tube, transfer it to a new tube, add physiological saline to 45 mL, and centrifuge at 1500 rpm for 5 minutes.

[0107] (6) After centrifugation, discard the supernatant, add 1 mL of cell cryopreservation solution to each tube to resuspend the cell pellet once, gently pipette, and add the corresponding amount of cryopreservation solution according to the standard of 1 mL of cryopreservation solution per flask of cells. Take 50 μL of cell suspension for cell counting and cell viability detection. Based on the cell counting results, determine the cell density according to 1 × 10⁻⁶ cells / mL. 7 Add cells / mL to stem cell cryopreservation medium. Retain 2 × 10⁶ cells. 7 For QC testing. Add 1.1 mL of cell suspension to each cryovial / vial.

[0108] (7) Tighten the tube / bottle cap, pass it out of the GMP room through the transfer window, quickly transfer it to the cryopreservation room, put the cells into the programmed cooling instrument, take one vial as a cryopreservation sample, and perform programmed cooling.

[0109] (8) Place the frozen cells in a -80℃ freezer for short-term storage, and transfer them to a liquid nitrogen tank for long-term storage after QC testing is qualified.

[0110] II. Preparation of Adipose-Derived Mesenchymal Stem Cells (AdMSCs)

[0111] 1. Acquisition and culture of adipose-derived mesenchymal stem cells

[0112] Adipose tissue donors must provide a negative infectious disease test report from within the past three months, confirming the absence of risk factors for infectious diseases, and pass all required examinations, including routine blood tests and biochemical tests. The preparation method is as follows:

[0113] Aspirate the upper layer of adipose tissue from the adipose tissue collection bottle and transfer it into a 50mL centrifuge tube. Add physiological saline, rinse twice, and centrifuge at 1500rpm for 5 minutes.

[0114] Pipette 25 mL of the upper fat layer and transfer it to a 50 mL centrifuge tube (add no more than 25 mL of fat to each tube). Calculate the total adipose tissue volume. Add an equal volume of 25 mL of 0.3% collagenase II working solution (2X), mix well, and incubate at 37°C for 1000 rpm for 30–50 minutes. After digestion, centrifuge at 800 rpm for 3 minutes. Remove the fat and fascia after centrifugation, add physiological saline to a final volume of 45 mL per tube, and centrifuge at 1500 rpm for 10 minutes.

[0115] Remove the supernatant and resuspend the cells in the tubes using 2 mL of physiological saline. Combine the cells from every four centrifuge tubes into one centrifuge tube, wash the cell pellet once with 45 mL of physiological saline, and centrifuge at 1500 rpm for 5 minutes at room temperature. After centrifugation, discard the supernatant and repeat the washing process once.

[0116] Inoculation: Inoculate one T150 culture flask with cells contained in 50 mL of adipose tissue, add 25 mL of serum-free culture medium to each flask, and incubate the cell culture flasks in a 37°C, 5% CO2 incubator.

[0117] 2. Passaging of adipose-derived mesenchymal stem cells

[0118] Observe the cell growth status under a microscope. When the cell culture reaches 80-90% confluence, it can be passaged.

[0119] 10-15 mL of culture medium supernatant should be retained from each bottle for later use, and 30 mL of mixed culture medium should be sent for QC testing and retained as a sample.

[0120] Wash the cells once with physiological saline, add 2-3 mL of mild digestive enzyme to each culture flask, digest at room temperature for 2-5 minutes, observe the cell morphology under a microscope, when the cell adhesions break and the cells shrink and become round, add 10-15 mL of the remaining complete culture medium to stop the digestion.

[0121] Gently pipette the cells to detach them, collect the cell suspension into a 50mL centrifuge tube, adjust the cell suspension volume to 45mL / tube, and centrifuge at 1500rpm for 5 minutes.

[0122] After centrifugation, discard the supernatant, add 2 mL of complete culture medium to each tube to resuspend the cell pellet, and take 50 μl of cell suspension to perform cell counting and viability detection using trypan blue staining solution.

[0123] Based on the cell count results, perform proportional passage, adding 25 mL of complete culture medium to each T150 culture flask.

[0124] The culture flasks were transferred to a cell culture incubator at 37°C and 5% CO2 concentration for further culture.

[0125] 3. Cryopreservation of adipose-derived mesenchymal stem cells

[0126] The procedure is the same as for cryopreservation of umbilical cord mesenchymal stem cells.

[0127] III. Preparation of Endometrial Mesenchymal Stem Cells (EnMSCs)

[0128] 1. Extraction and culture of endometrial mesenchymal stem cells (EnMSC)

[0129] (1) Tissue digestion

[0130] The collected fresh, normal endometrial tissue was placed in a 6cm dish;

[0131] Use ophthalmic scissors to cut the tissue into small pieces until it can be drawn up with a 1ml pipette tip;

[0132] Place the chopped tissue into a 15ml centrifuge tube and add 5ml of pre-prepared digestion solution;

[0133] Digest on a shaker at 37°C for 50 minutes;

[0134] Repeatedly blow the suspension in the centrifuge tube and centrifuge at 1000 rpm for 3 minutes;

[0135] Discard the supernatant, resuspend the cells in sterile PBS, and filter the cell suspension through a 40µm pore size filter to obtain the filtrate.

[0136] Collect the filtrate into a 15ml centrifuge tube and centrifuge at 1000rpm for 3 minutes;

[0137] Discard the supernatant, resuspend the cells in complete culture medium, and plate them in large dishes;

[0138] The next day, the culture medium was aspirated, washed twice with sterile PBS, and then fresh complete culture medium was added again.

[0139] (2) Flow cytometry cell sorting

[0140] Endometrial mesenchymal stem cells need to be extracted from endometrial tissue through primary digestion and then separated into SUSD2+ positive cell populations by flow cytometry.

[0141] The filtrate after digesting the endometrial tissue was filtered through a 40µm filter to obtain the filtrate.

[0142] The filtrate was collected by centrifugation into a centrifuge tube and centrifuged at 1000 rpm for 3 minutes.

[0143] Remove the supernatant and resuspend the cells in 1 ml of PBS;

[0144] Add 5 μL of PE-SUSD2 flow cytometry antibody and incubate for 30 minutes in the dark;

[0145] The stained cell suspension was washed twice with PBS;

[0146] Adjust the cell density to 1×10⁻⁶ 7 Positive cell populations were sorted by flow cytometry at a density of cells per ml.

[0147] After sorting, the SUSD2+ cell population was obtained and distributed into 12-well plates;

[0148] The complete culture medium for mesenchymal stem cells was changed the next day.

[0149] 2. Digestion and passaging

[0150] The same cell line should be digested and passaged, and it needs to be passaged to the third generation before use.

[0151] 3. Cell cryopreservation

[0152] After cell digestion, the precipitated cells were resuspended in a cryopreservation solution of 10% DMSO and 90% FBS. The suspended cells were aliquoted into labeled cryovials and placed in pre-arranged cryopreservation boxes. After incubating the cryopreservation boxes overnight at -80°C, the cryovials were transferred to liquid nitrogen.

[0153] Example 2: Human umbilical cord mesenchymal stem cells improve pregnancy rate in female animals

[0154] 1. Laboratory animals

[0155] Wild-type female mice: C57BL / 6 female mice.

[0156] Wild-type male mice: C57BL / 6 male mice.

[0157] PLG - / - Establishment of female mice: Mice were prepared using the CRISPR / Cas9 method, with sgRNA targeting exons of the plasminogen PLG gene (knockout regions from Exon 2 to Exon 10) in patients with plasminogen deficiency, thereby inactivating the encoded protein. Figure 6 ).

[0158] The sequence of the gRNA is shown in Table 2.

[0159] Table 2. gRNA Design

[0160]

[0161]

[0162] PLG - / -Fertility of female mice: such as Figure 1 The control group (con) was infertile, according to experimental analysis.

[0163] 2. Experimental Grouping

[0164] Control group (Con): PLG - / - Female mice (or, single knockout PLG) + / - Female mice and male C57BL / 6 mice were housed together.

[0165] UcMSC Group: PLG - / - Female mice (or, PLG) + / - Female mice and C57BL / 6 male mice were housed together. Female mice were given UcMSCs. One dose of 1 x 10^6 umbilical cord mesenchymal stem cells (UcMSCs) per mouse was administered via tail vein injection. The mice were kept together for 48 hours after the injection and observed.

[0166] AdMSC Group: PLG - / - Female mice (or, PLG) + / - Female mice and C57BL / 6 male mice were housed together. Female mice were given AdMSCs. A single dose of 1 x 10^6 AdMSCs per mouse was administered via tail vein injection. The mice were kept together for 48 hours after the injection and observed.

[0167] EnMSC Group: PLG - / - Female mice (or, PLG) + / - Female mice and C57BL / 6 male mice were housed together. Female mice were given EnMSCs. One dose of 1 x 10^6 endometrial mesenchymal stem cells (EnMSCs) per mouse was administered via tail vein injection. The mice were kept together for 48 hours after the injection and observed.

[0168] PLG Group: PLG - / - Female mice (or, PLG) + / - Female mice were injected with PLG research reagent (Sigma) via the tail vein, and the injected female mice were housed together with C57BL / 6 male mice.

[0169] PLG+EnMSC group: In addition to the PLG group, EnMSCs were administered; 1 x 10^6 endometrial mesenchymal stem cells (EnMSCs) per mouse were administered once via tail vein injection and the mice were kept in cages and observed for 48 hours after the injection.

[0170] 3. Delivery rate and offspring analysis

[0171] (1) PLG lacks homozygosity (PLG) - / - ) and hybrid (PLG) + / -Pregnancy analysis after mouse mesenchymal stem cell therapy

[0172] Following the aforementioned grouping, 8-week-old female mice and 8-week-old male mice were placed in culture cages and coupled 48 hours after tail vein injection of stem cells. They were fed a normal diet, and mating and conception rates in the female mice were observed during the period.

[0173] PLG - / - Pregnancy rates in female mice treated with mesenchymal stem cells or PLG are as follows: Figure 1 In the control group (Con) without mesenchymal stem cell treatment, all mice were unable to become pregnant; PLG mice treated with mesenchymal stem cells... - / - In female mice, the pregnancy rate was significantly improved, with umbilical cord mesenchymal stem cells (UcMSC) and adipose mesenchymal stem cells (AdMSC) showing the best results and higher pregnancy rates than female mice injected with PLG via the tail vein.

[0174] according to Figure 1 For PLG - / - In female mice, the therapeutic effect of mesenchymal stem cells (EnMSC) in the endometrial tissue was relatively lower than that of the other two types of mesenchymal cells (UcMSC and AdMSC); however, combining this with further tail vein injection of PLG could further improve and increase the pregnancy rate.

[0175] PLG + / - Pregnancy rates in female mice treated with mesenchymal stem cells or PLG are as follows: Figure 3 The control group (Con) that did not receive mesenchymal stem cell treatment had a pregnancy rate higher than 50%, while PLG treated with mesenchymal stem cells had a pregnancy rate higher than 50%. + / - In female mice, the pregnancy rate was significantly improved, with umbilical cord mesenchymal stem cells (UcMSCs) showing the best results, and mesenchymal stem cells (EnMSCs) in endometrial tissue were also significantly improved.

[0176] according to Figure 3 PLG + / - In female mice, there was no significant effect from tail vein injection of PLG, but the combination of tail vein injection of PLG with the use of mesenchymal stem cells resulted in a pregnancy rate that unexpectedly reached 100%.

[0177] (2) Analysis of progeny of PLG-deficient homozygous and heterozygous mice after mesenchymal stem cell therapy

[0178] Analyze the specific number of pups (number of pups per litter) in each female mouse after stem cell treatment.

[0179] PLG - / - The number of offspring in female mice treated with mesenchymal stem cells or PLG is as follows: Figure 2The control group (Con) that did not receive mesenchymal stem cell treatment had no offspring; the PLG mice treated with mesenchymal stem cells... - / - Female mice were able to produce offspring in large numbers, with umbilical cord mesenchymal stem cells (UcMSCs) and adipose-derived mesenchymal stem cells (AdMSCs) showing relatively superior results and producing significantly more offspring than female mice injected with PLG via the tail vein.

[0180] according to Figure 2 The therapeutic effect of mesenchymal stem cells (EnMSC) in endometrial tissue is actually lower than that of the former two (UcMSC, AdMSC); combined with further tail vein injection of PLG, the pregnancy rate can be further improved and increased, although it is still lower than that of the former two (UcMSC, AdMSC).

[0181] PLG + / - The number of offspring in female mice treated with mesenchymal stem cells or PLG is as follows: Figure 4 The control group (Con) that did not receive mesenchymal stem cell treatment had a certain number of progeny cells, while PLG treated with mesenchymal stem cells... + / - In female mice, the number of offspring was significantly increased, with umbilical cord mesenchymal stem cells (UcMSCs) showing the best effect. The other two types of mesenchymal cells also significantly increased the number of offspring. Tail vein injection of PLG increased the number of offspring to some extent.

[0182] according to Figure 3 PLG + / - In female mice, there was no significant difference between tail vein injection of PLG and the control group. However, the combination of tail vein injection of PLG and the use of mesenchymal stem cells resulted in a pregnancy rate of 100% unexpectedly.

[0183] Therefore, co-cage animal experiments show that PLG - / - Female mice were unable to conceive normally when housed with wild-type male mice. Compared with the control group, mice that underwent tail vein injection of human umbilical cord mesenchymal stem cells (UcMSCs) PLG were significantly more fertile. - / - The pregnancy rate of female mice was significantly improved afterward.

[0184] Example 3: Hydrogel materials suitable for carrying mesenchymal stem cells

[0185] Four hydrogel materials with different properties and morphologies were prepared: cross-linked sodium hyaluronate gel (HA-GEL); collagen scaffold; cross-linked polyacrylamide gel (CPAA); and polylactic acid-polyethylene glycol-polylactic acid thermosensitive hydrogel (PLGA-PEG-PLGA). After co-culturing the four hydrogel materials with endometrial-derived mesenchymal stem cells for 48 hours, cell viability was assessed by Live / dead staining. Membrane-permeable, non-fluorescent calcein AM, upon entering the cells, was converted by esterases in living cells into calcein AM emitting strong green fluorescence. EthD-1 entered cells with damaged cell membranes, and after binding to nucleic acids, its fluorescence increased 40-fold, thus producing bright red fluorescence in dead cells. The results showed that the survival rate of mesenchymal stem cells co-cultured with HA-GEL hydrogel was not different from that of the control group. Collagen membrane (16.1%), 2.5% CPAA gel (30.6%), and 15% PLGA-PEG-PLGA gel (81.6%) all inhibited cell survival to varying degrees when co-cultured with mesenchymal stem cells. Figure 7 A). The above results suggest that HA-GEL hydrogel does not affect the survival rate of mesenchymal stem cells, while the other three hydrogels inhibit mesenchymal stem cell survival to some extent. HA-GEL hydrogel is the preferred choice as a carrier for carrying mesenchymal stem cells for subsequent in vitro evaluation and intrauterine transplantation.

[0186] The effects of HA-GEL and endometrial cancer cells on mesenchymal stem cell (MSC) migration were assessed using a transwell assay. In a transwell model, the bottom of the upper chamber was coated with HA-GEL hydrogel, followed by seeding of MSCs. The lower chamber was seeded with either ECC-1 or Ishikawa endometrial cancer cells for indirect co-culture. After 48 hours of culture, the number of migrating MSCs was counted by crystal violet staining. Results showed that the number of migrating cells in the HA-GEL group was lower than that in the control group. The number of migrating cells in the HA-GEL+ECC-1 group was not significantly different from that in the control group, while the number of migrating cells in the HA-GEL+Ishikawa group was higher than that in the control group. These results suggest that HA-GEL hydrogel alone can inhibit the migration ability of MSCs; compared with the control group, co-culture of MSCs with ECC-1 combined with HA-GEL hydrogel did not affect MSC migration, while co-culture of MSCs with Ishikawa combined with HA-GEL hydrogel promoted MSC migration. Figure 7 B).

[0187] Furthermore, the effects of HA-GEL on the normal function of mesenchymal stem cells were evaluated. Mesenchymal stem cells were co-cultured with HA-GEL hydrogel, and compared with the control group of endometrial-derived mesenchymal stem cells cultured alone, the mRNA expression changes of stem cell genes and cytokines closely related to cell proliferation and angiogenesis were detected by RT-qPCR. The results confirmed that the HA-GEL hydrogel system carrying mesenchymal stem cells did not affect the expression of cytokines and stem cell genes in mesenchymal stem cells. Figure 7 C).

[0188] The results show that HA-GEL-loaded mesenchymal stem cells can effectively maintain the viability of mesenchymal stem cells without affecting the expression of stem cell genes and cytokines.

[0189] Example 4: Mesenchymal stem cells improve infertility-related symptoms in patients with PLG deficiency

[0190] 1. PLG deficiency patients

[0191] Inclusion criteria for patients with PLG deficiency (informed consent):

[0192] (1) Age between 18 and 45, not yet menopausal, and with fertility requirements;

[0193] (2) PLG was confirmed by whole exome sequencing. - / - The patient presents with abnormal uterine bleeding and infertility.

[0194] (3) On day 5 of menstruation, FSH < 10 mIU / mL and AMH > 1.2 ng / mL were measured;

[0195] (4) Regular ovulation has been confirmed by ultrasound monitoring or basal body temperature monitoring;

[0196] (5) No organic lesions of the endometrium: Before treatment, the patient was assessed clinically and by imaging, and the endometrial curettage or biopsy pathology was determined to be free of lesions;

[0197] (6) No evidence of endometrial or systemic infectious lesions: Before treatment, routine vaginal secretion, cervical secretion culture (bacteria, chlamydia, mycoplasma), blood routine, and blood infectious markers (CRP, PCT) should be performed to rule out infectious diseases.

[0198] (7) Those who have the means to be followed up and are willing to continue to be followed up.

[0199] Exclusion criteria for participants:

[0200] (1) Exclude the following organic lesions: septate uterus, uterine fibroids, severe adenomyosis, endometriosis, endometrial cancer and precancerous lesions, cervical cancer and precancerous lesions, etc.

[0201] (2) Those who are currently or have been taking immunosuppressants for the past month;

[0202] (3) Those who have a history of severe adverse reactions to stem cell therapy or a known history of allergy to stem cells or stem cell culture medium components;

[0203] (4) Patients with serious internal medical conditions, severely impaired liver and kidney function, deep vein thrombosis, or stroke;

[0204] (5) Patients with concurrent malignant tumors in other systems of the body;

[0205] (6) Those who have a history of uterine surgery (excluding hysteroscopic surgery / diagnostic curettage) or uterine perforation within the past three months;

[0206] (7) Acute or subacute reproductive tract infection, reproductive tract tuberculosis, those who have not received anti-tuberculosis treatment; those with reproductive tract malformations.

[0207] (8) Other gynecological or non-gynecological conditions that are clinically deemed unsuitable for pregnancy.

[0208] This is a multicenter clinical study. Eligible participants, after obtaining informed consent, were given 1×10⁻⁶ doses. 6 / kg UcMSCs were administered intravenously once a month for 6 consecutive months.

[0209] 2. The ameliorative effect of mesenchymal stem cells on patients with PLG deficiency

[0210] (1) Dosage forms of stem cell preparations

[0211] The stem cell preparation used in this invention is a suspension of human umbilical cord mesenchymal stem cells, loaded into a sterile disposable syringe.

[0212] (2) How to use

[0213] In this invention, the stem cell preparation is used via intrauterine perfusion.

[0214] (3) Dosage and course of treatment

[0215] After obtaining informed consent, eligible participants were administered the vaccine at a dose of 2×10-1. 7 The dosage of UcMSCs is once a month for 3 consecutive months.

[0216] 3. Treatment outcomes of mesenchymal stem cells

[0217] (1) UcMSCs transplantation in patients with complex compound PLGD mutations promotes endometrial repair and improves menstruation.

[0218] PLGD patients were treated with UcMSCs combined with intrauterine instillation of sodium hyaluronate (HA-GEL), at a dose of 2×10⁻⁶. 7Each time, 2ml of UcMSCs and 3ml of HA-GEL are thoroughly mixed and then instilled into the uterine cavity under hysteroscopy. This is done once a month for three consecutive months.

[0219] like Figure 5 After treatment, the patient's intrauterine adhesions improved significantly, and the AFS score gradually decreased (Table 3).

[0220] Ultrasound scans showed that after UcMSCs treatment on days 5-7 of the menstrual cycle, and follow-up ultrasounds on days 19-21 of the menstrual cycle, the endometrial thickness increased; uterine blood supply improved, and blood flow resistance decreased. The patient's menstrual cycle significantly improved, from a previous 20+ / 30 days to 6-7 / 30 days after treatment. However, there were no significant changes in the patient's peripheral blood PLG, PLGA, and fibrinolysis-related data.

[0221] Pre-treatment endometrial pathology showed diluted glands, thin endometrium with abundant fibrous adhesions, and negative PLG expression. Figure 5 After three treatments with UcMSCs, the endometrial glands showed secretory changes, with PLG infiltrating and interspersed in the stroma with positive expression.

[0222] Table 3. Hysteroscopic AFS scores of PLGD patients before and after UcMSCs treatment

[0223]

[0224] Based on the above, mesenchymal stem cells can significantly improve infertility-related symptoms in patients with PLG deficiency.

[0225] This invention prepares a formulation for intrauterine administration by combining UcMSCs with cross-linked sodium hyaluronate gel for intrauterine use. The latter serves as a carrier for intrauterine stem cell transplantation, prolonging the in vivo survival time of stem cells and improving their in vivo environment.

[0226] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. The use of mesenchymal stem cells in the preparation of compositions for treating plasminogen deficiency; wherein plasminogen deficiency is characterized by low expression or low activity of plasminogen PLG and manifests as infertility.

2. The application as described in claim 1, characterized in that, The plasminogen genotype of the patients with plasminogen deficiency is PLG. - / - or PLG + / - .

3. The application as described in claim 1, characterized in that, The mesenchymal stem cells include those selected from: umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, and endometrial mesenchymal stem cells; preferably, the mesenchymal stem cells are human cells.

4. The application as described in claim 1, characterized in that, The composition includes mesenchymal stem cells, which are mounted in a hydrogel; preferably, the mesenchymal stem cells are mounted in a cross-linked sodium hyaluronate gel; or The composition includes mesenchymal stem cells dissolved in an electrolyte injection solution; preferably, the electrolyte injection solution includes an injection solution containing 1-5% human hemoglobin.

5. The application as described in claim 1 or 4, characterized in that, The concentration of mesenchymal stem cells in the composition is 5 × 10⁻⁶. 4 ~5×10 9 5 × 10⁵ cells / mL; preferably 5 × 10⁵ cells / mL. 5 ~5×10 8 5 × 10⁵ cells / mL; more preferably 5 × 10⁵ cells / mL. 6 ~5×10 7 .

6. The application as described in claim 1, characterized in that, The treatment of plasminogen deficiency aims to alleviate or treat symptoms by increasing pregnancy rates.

7. The application as described in claim 1, characterized in that, The treatment of plasminogen deficiency aims to alleviate or treat symptoms by: increasing the number of offspring, improving menstruation, repairing the endometrium, treating intrauterine adhesions, and improving intrauterine inflammation.

8. The application as described in claim 1, characterized in that, The composition includes pharmaceutical compositions.

9. The application as described in claim 8, characterized in that, The pharmaceutical composition is placed in a medicine box or kit; or The composition was prepared in a unit dosage manner.

10. A method for treating plasminogen deficiency, comprising: The recipient is given mesenchymal stem cells for treatment of plasminogen deficiency, wherein plasminogen PLG is expressed at low levels or has low activity; preferably, the plasminogen deficiency is manifested as infertility.

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