Use of CD146+ mesenchymal stem cell in preparation of drug for preventing and / or treating cerebral apoplexy

Abstract

A use of a CD146+ mesenchymal stem cell in preparation of a drug for preventing and / or treating cerebral apoplexy. The drug can significantly alleviate neurological impairment symptoms of a middle cerebral artery occlusion (MCAO) model of a mouse, improves the exercise behavior capability, and decreases the degree of cerebral infarction. The umbilical cord mesenchymal stem cell subpopulation of a specific surface marker has been confirmed for the first time to have an effect in treatment of cerebral apoplexy, has broad prospects in the treatment of cerebral apoplexy, and has high clinical value.

Description

Use of CD146+ mesenchymal stem cells in the preparation of drugs for preventing and / or treating stroke

[0001] The present invention claims priority to a Chinese patent application filed with the Patent Office of China on October 16, 2023, with application number 202311337166.8 and invention name “Use of CD146+ mesenchymal stem cells in the preparation of drugs for preventing and / or treating stroke”, the entire contents of which are incorporated by reference into the application. Technical Field

[0002] The present invention belongs to the field of biomedicine, and specifically relates to use of CD146+ mesenchymal stem cells in preparing a drug for preventing and / or treating stroke. Background Art

[0003] Stroke, commonly known as stroke, includes ischemic stroke (cerebral infarction) and hemorrhagic stroke (intracerebral hemorrhage, intraventricular hemorrhage, subarachnoid hemorrhage). According to the World Health Organization definition, stroke refers to damage to cerebral blood vessels and focal (or overall) brain tissue damage caused by various reasons, causing clinical symptoms for more than 24 hours or death. It is characterized by high morbidity, disability, recurrence and mortality. Current diagnosis and treatment methods mainly focus on restoring cerebral perfusion in the acute phase of stroke, but this is not conducive to the regeneration of brain tissue after damage. Therefore, there is an urgent need for a wider range of brain tissue repair therapies to supplement current treatment options, extend the treatment time window, and help reduce the disability and burden caused by stroke (de Celis-Ruiz E, Fuentes B, Alonso de M, et al. Final Results of Allogeneic Adipose Tissue-Derived Mesenchymal Stem Cells in Acute Ischemic Stroke (AMASCIS): A Phase II, Randomized, Double-Blind, Placebo-Controlled, Single-Center, Pilot Clinical Trial. Cell Transplant. 2022; 31:9636897221083863).

[0004] Many cell types (such as monocytes, neural stem cells, embryonic stem cells, induced pluripotent stem cells, and mesenchymal cells) have been used in preclinical research and clinical trials for stroke. Among them, mesenchymal stem cells (MSCs) are easy to obtain, have relatively low immunogenicity, rarely involve bioethical issues, and have advantages in clinical application (Lalu MM, Montroy J, Dowlatshahi D, Hutton B, Juneau P, Wesch N, Y Zhang S, McGinn R, Corbett D, Stewart DJ, A Fergusson D. From the lab to patients: a systematic review and meta-analysis of mesenchymal stem cell therapy for stroke. Transl Stroke Res. 2020; 11(3): 345-64.; Sarmah D, Agrawal V, Rane P, Bhute S, Watanabe M, Kalia K, Ghosh Z, Dave KR, Yavagal DR, Bhattacharya P. Mesenchymal stem cell therapy in ischemic stroke: a meta-analysis of preclinical studies. Clin Pharmacol Ther. 2018;103(6):990-98).

[0005] Chinese patent application 202310550692.6 discloses a cell composition for treating acute stroke and a preparation method thereof. The cell composition is prepared from the following components in parts by mass: 5-9 parts of astrocytes, 4-6 parts of umbilical cord mesenchymal stem cells, 2-4 parts of neural stem cells, 1-3 parts of ginsenosides, 2-4 parts of baicalin, 2-4 parts of ligustrazine, 0.4-0.8 parts of mannitol, 0.06-0.10 parts of calcium sodium edetate, 0.1-0.3 parts of phosphatidylserine, 0.4-0.6 parts of sodium chloride, and 40-60 parts of water.

[0006] Chinese patent application 202111367038.9 discloses a human umbilical cord mesenchymal stem cell culture medium, which includes a basal culture medium, vitamin C, insulin, platelet-derived growth factor, epidermal growth factor, basic fibroblast growth factor, β-mercaptoethanol, SAG, folic acid, hypoxia-inducible factor-1, protoporphyrin cobalt and simvastatin; the human umbilical cord mesenchymal stem cell injection obtained by culture with the culture medium has a strong proliferation ability and can be directly used for the treatment of stroke after resuscitation, and has important application value.

[0007] Some studies have used mesenchymal stem cells (MSCs) in stroke treatment. However, the heterogeneity of MSCs not only affects treatment effectiveness but also poses a safety challenge. Therefore, there is an urgent need to accurately distinguish stem cell subpopulations and identify cells that are therapeutically effective without safety risks. This will significantly advance the clinical application of stem cells.

[0008] Summary of the Invention

[0009] CD146 is a Ca 2+ CD146 is a membrane glycoprotein that is independent of cell adhesion molecules. Studies have shown that it is active in a variety of cell physiological processes, including development, signal transduction, cell migration, differentiation, angiogenesis, and immune response (Yang YK, Ogando CR, Wang See C, Chang TY, Barabino GA. Changes in phenotype and differentiation potential of human mesenchymal stem cells aging in vitro. Stem Cell Res Ther. 2018; 9(1): 131; Bikorimana JP, Saad W, Abusarah J, et al. CD146 Defines a Mesenchymal Stromal Cell Subpopulation with Enhanced Suppressive Properties. Cells. 2022; 11(15): 2263.).

[0010] The inventors isolated CD146+ mesenchymal stem cells from the umbilical cord and creatively applied them to the preparation of drugs for preventing and / or treating stroke, achieving unexpected technical results.

[0011] In order to achieve the above technical objectives, the present invention hereby proposes the following technical solutions:

[0012] In one aspect, the present invention provides use of CD146+ mesenchymal stem cells in the preparation of a medicament for preventing and / or treating stroke.

[0013] In some embodiments, the drug prevents and / or treats stroke by at least one of the following:

[0014] (1) Alleviate the neurological deficit symptoms of the subjects;

[0015] (2) Improve the subjects' motor performance;

[0016] (3) Improve the degree of cerebral infarction in subjects.

[0017] In some embodiments, the subject is a human or an animal, preferably a human.

[0018] In some embodiments, the stroke includes ischemic stroke and hemorrhagic stroke.

[0019] In some preferred embodiments, the stroke is ischemic stroke.

[0020] In some embodiments, the drug further comprises a pharmaceutically acceptable excipient or carrier, which includes, but is not limited to, a solvent, a diluent, a disintegrant, a precipitation inhibitor, a surfactant, a glidant, a binder, a lubricant, a dispersant, a suspending agent, an isotonic agent, a thickener, an emulsifier, a preservative, a stabilizer, a hydrating agent, an emulsification accelerator, a buffer, an absorbent, a colorant, a flavoring agent, a sweetener, an ion exchanger, a release agent, a coating agent, a flavoring agent or an antioxidant.

[0021] In some embodiments, the CD146+ expression rate of the mesenchymal stem cells is greater than 85%.

[0022] In some embodiments, the mesenchymal stem cells are isolated from bone marrow, umbilical cord, adipose tissue, cord blood, placenta, amnion, chorion, dental pulp, thymus, synovium, fetal blood, or liver.

[0023] In some preferred embodiments, the mesenchymal stem cells are isolated from umbilical cord.

[0024] In some preferred embodiments, the mesenchymal stem cells are isolated from umbilical cord Whartons jelly, and the isolation is preferably performed by trypsin digestion.

[0025] In some embodiments, the mesenchymal stem cells may be a subpopulation of mesenchymal stem cells.

[0026] In some preferred embodiments, the mesenchymal stem cells are a subpopulation of mesenchymal stem cells expressing CD146.

[0027] In some embodiments, the drug comprises at least 1×10 6 CD146+ mesenchymal stem cells.

[0028] In some preferred embodiments, the drug comprises at least 4×10 6 CD146+ mesenchymal stem cells.

[0029] In some embodiments, the drug may be an injection or a gel, preferably an injection.

[0030] In some embodiments, the injection solution may further include at least one of a vitamin solution, an amino acid solution, a 0.9% sodium chloride solution, and a lyoprotectant.

[0031] In some embodiments, the lyoprotectant comprises sucrose or trehalose.

[0032] In the present invention, unless otherwise specified, the administration method of the drug is not particularly limited and can be by injection, topical administration, etc. Doctors can select the administration method according to actual clinical needs.

[0033] In some embodiments, the drug is administered by injection, including but not limited to intravenous injection, arterial injection, intrathecal injection of the spinal cord, intraarticular injection, myocardial injection, intramuscular injection or tracheal injection.

[0034] The use of the CD146+ umbilical cord mesenchymal stem cell subset provided by this invention in the preparation of a drug for the prevention, treatment, and / or treatment of stroke has been shown to significantly improve neurological symptoms, enhance motor function, and reduce the severity of cerebral infarction in mice induced by middle cerebral artery occlusion (MCAO). This study, the first to demonstrate the role of a specific surface marker-expressing umbilical cord mesenchymal stem cell subset in the treatment of stroke, demonstrates promising clinical applications in stroke treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIG1 shows the morphological characteristics of umbilical cord CD146+ mesenchymal stem cells (4×), with a scale bar of 1000 μm.

[0036] FIG2 shows the expression of human umbilical cord mesenchymal stem cell-related markers.

[0037] FIG3 shows the neurological deficit symptom scores of mice in each experimental group.

[0038] FIG4 shows the results of the pole climbing test of mice in each experimental group.

[0039] FIG5 shows the balance beam test results of mice in each experimental group.

[0040] Figures 6 and 7 show the cerebral infarction conditions of mice in each experimental group. DETAILED DESCRIPTION

[0041] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0042] Unless the context clearly dictates otherwise, as used herein, the expressions "a" and "an" include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0043] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0044] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the examples. If the specific conditions are not specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for all reagents or instruments, they are all conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are provided in the following specific embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention. Such structures and technologies are also described in many publications, such as "Molecular Cloning Laboratory Manual (4th Edition)" (Cold Spring Harbor Laboratory Science Press), Ausubel, FM et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience.

[0045] Example 1 Preparation and Passaging Method of CD146+ Mesenchymal Stem Cell Subpopulation

[0046] 1.1 Materials and Methods

[0047] 1) First-pass human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) were prepared and provided by our company (Beijing Sanlikang Cell Technology Co., Ltd., Beijing). After further passage (P) with 90% minimum essential medium (αMEM, BI) and 10% fetal bovine serum (Gibco, Australia) at 37°C in 5% CO₂, MSCs were harvested using 0.5% trypsin-EDTA (Gibco, Australia), counted, and reseeded in 100 mm cell culture dishes at a density of 20,000 cells / cm².

[0048] 2) Phenotypic characterization

[0049] The percentage of CD146 positive cells was detected by flow cytometry (Beckman, cytoflexs). The cells were divided into two tubes, 4×10 5 Antibodies: CD45-FITC, CD34-FITC, CD90-PerCP, CD73-PE, CD105-APC (Sino Biological), and CD146-FITC (BioLegend) were added to each tube and incubated at 4°C for 30 min. After washing twice with PBS, the cell percentage was determined by flow cytometry.

[0050] 3) Preparation of the preparation

[0051] The digested hUC-MSCs cells were resuspended in 0.9% normal saline (Shijiazhuang Siyao Group).

[0052] 1.2 Experimental Results

[0053] When the growth status was observed under an inverted microscope, the CD146+ subpopulation cells all grew in a monolayer adherent to the wall with irregular morphology and a long spindle shape (Figure 1).

[0054] The CD146+ subpopulation highly expressed human umbilical cord mesenchymal stem cell-related markers CD73, CD90, and CD105; they did not express hematopoietic stem cell-related markers CD34 and CD45, and the CD146+ expression rate was 97.4% (Figure 2).

[0055] Example 2 Construction of mouse ischemic stroke model and cell therapy

[0056] Ten-week-old male C57BL / 6 mice in the experimental group underwent MCAO surgery under anesthesia to occlude the origin of the MCA with a suture. Blood flow and reperfusion were restored about 1-1.5 hours after occlusion. Within 1 hour after reperfusion, mice in the cell therapy group were administered the drug via the tail vein.

[0057] The experimental groups are as follows:

[0058] Sham operation group, model control group, CD146+MSC low-dose treatment group (4×10 4 / mouse), CD146+MSC medium-dose treatment group (5×10 5 / mouse) and CD146+MSC high-dose treatment group (1×10 6 / mouse), 15 mice in each group.

[0059] Example 3 Evaluation of the effect of hUC-MSCs in treating ischemic stroke in mice

[0060] 24 hours after MCAO surgery, the neurological symptoms of the mice were evaluated, the time they spent climbing poles and balancing beams was counted, and the infarct area on the affected side of the brain detected by MRI was counted.

[0061] Neurological deficit symptom scoring criteria:

[0062] ①0 points: no symptoms of nerve damage;

[0063] ②1 point: Unable to fully extend the contralateral forepaw;

[0064] ③2 points: Turn toward the paralyzed side;

[0065] ④3 points: falling to the opposite side;

[0066] ⑤4 points: Unable to walk spontaneously and loss of consciousness.

[0067] The neurological symptoms of mice were scored according to the Longa scoring method. The results showed that the neurological deficit symptom scores of ischemic stroke model mice (medium-dose group and high-dose group) on the 5th day after cell treatment were significantly different from those of the model group mice (p﹤0.05) (Figure 3).

[0068] According to the Beam Balance Test (BBT) and Pole Test (PT) experiments, the total time for mice to pass the balance beam and the total time to complete the pole climbing were recorded and analyzed. The results showed that on the 14th day after cell therapy, the motor ability of mice with ischemic stroke model was significantly improved. Among them, the total time for mice in the medium-dose group and the high-dose group to pass the balance beam was shortened by 51%; the total time to complete the pole climbing was shortened by 64% (medium-dose group) and 56% (high-dose group), respectively (Figures 4 and 5).

[0069] Image analysis software (Image J) was used to calculate the infarct area of ​​the mouse brain as shown by MRI. The results showed that the CD146+MSC medium-dose group had the smallest brain damage percentage of 0.53%, which was a 53% improvement compared with the model group (1.14%), with a significant difference (p < 0.05) (Figures 6 and 7).

[0070] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. Use of CD146+ mesenchymal stem cells in the preparation of a drug for treating stroke, characterized in that: The CD146+ mesenchymal stem cells are a subpopulation of mesenchymal stem cells expressing CD146; the CD146+ mesenchymal stem cells all highly express markers CD73, CD90, and CD105; and do not express markers CD34 and CD45; the mesenchymal stem cells are isolated from umbilical cord; The drug prevents and / or treats stroke by at least one of the following means: (1) Alleviate the neurological deficit symptoms of the subjects; (2) Improve the subjects' motor behavior ability; (3) Improve the degree of cerebral infarction in subjects.

2. The use according to claim 1, characterized in that The stroke includes ischemic stroke and hemorrhagic stroke.

3. The use according to claim 2, characterized in that: The drug also includes pharmaceutically acceptable excipients or carriers, which include solvents, diluents, disintegrants, precipitation inhibitors, surfactants, glidants, binders, lubricants, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, flavors, sweeteners, ion exchangers, release agents, coating agents, flavoring agents or antioxidants.

4. The use according to claim 3, characterized in that The drug contains at least 1×10 6 CD146+ mesenchymal stem cells.

5. The use according to claim 4, characterized in that The medicine is an injection or a gel.

6. The use according to claim 5, characterized in that The injection solution also includes at least one of a vitamin solution, an amino acid solution, a 0.9% sodium chloride solution and a freeze-drying protective agent.

7. The use according to claim 6, characterized in that The lyoprotectant includes sucrose or trehalose.

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