Application of umbilical cord mesenchymal stem cells in treatment of plateau hypoxia sleep disorder

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses application of umbilical cord mesenchymal stem cells in treatment of sleep disorder caused by high altitude hypoxia. The sleep disorder is induced by a low-oxygen environment with the oxygen concentration of 14%-18%, and is represented as difficulty in falling asleep, reduction of rapid eye movement sleep (REM), increase of awakening times and the like. The third-generation to fifth-generation high-purity umbilical cord mesenchymal stem cells are obtained through in-vitro culture, and nasal spray intervention is performed through intravenous injection or by using a source exosome. The invention provides a non-drug biological intervention means which is safe, efficient and capable of being popularized, and has a good clinical transformation prospect. The cell and the exosome thereof can improve the sleep structure and neurological function of an animal model and a clinical object, and obviously improve the sleep quality in a plateau low-oxygen environment.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of stem cell therapy technology in sleep disorders induced by plateau hypoxia. Background Art

[0002] Sleep disorders are a common health issue for humans in the hypoxic environment of high altitudes, manifesting primarily as difficulty falling asleep, reduced sleep efficiency, and decreased rapid eye movement (REM) sleep. These sleep disorders not only severely impact a person's physical and mental health and quality of life, but can also reduce work efficiency and even trigger other neurological or cardiovascular diseases.

[0003] Traditional approaches to improving sleep disorders, such as medication (sedatives and hypnotics) and oxygen therapy, have significant drawbacks, including drug dependency, significant side effects, short-lived efficacy, and an inability to fundamentally address nerve damage. Therefore, there is an urgent need to develop a new treatment option that is safe, non-toxic, and highly effective.

[0004] In recent years, mesenchymal stem cells (MSCs), with their remarkable properties of tissue repair, antioxidant, anti-inflammatory, and neuroregeneration, have been gradually applied to the treatment of various neurological diseases. Among them, umbilical cord-derived mesenchymal stem cells (UC-MSCs) have become a hot topic in stem cell clinical research due to their wide availability, convenient collection, no ethical issues, and low immunogenicity.

[0005] However, there is currently no research on the application of UC-MSCs in the treatment of high-altitude hypoxic sleep disorders, so there is a lot of room for research and practical application prospects. Summary of the Invention

[0006] The purpose of the present invention is to provide an application of umbilical cord mesenchymal stem cells in the treatment of sleep disorders caused by plateau hypoxia, so as to solve the problems of existing treatment methods such as short-term efficacy, obvious drug side effects, inability to effectively repair nerve damage, and inability to continuously improve sleep quality in the long term. The technical solution provided by the present invention is specifically as follows: the umbilical cord tissue of a healthy person obtained after approval by the ethics committee is repeatedly washed with sterile PBS, chopped and added with 0.2% collagenase to digest at 37°C for 2 hours, and then centrifuged and filtered to obtain the primary cell precipitate, which is inoculated in DMEM / F12 culture medium containing fetal bovine serum and cultured until the cell fusion reaches about 80%, and then passaged and expanded, and flow cytometry is used to detect specific markers on the surface of umbilical cord mesenchymal stem cells such as CD29, CD44, CD73, CD90 and CD105 to ensure that the cell purity and viability meet the requirements of stem cell preparations before use; the established animal model of plateau hypoxia sleep disorder or clinically diagnosed patients with plateau hypoxia sleep disorder are used as treatment objects, and the cells are cultured through the tail vein or peripheral blood. The prepared umbilical cord mesenchymal stem cell suspension (5×10^6 cells / time, suspended in 1 mL of normal saline) was transplanted by intravenous injection, once every other day for 3 consecutive treatments. After treatment, the patients' or animals' sleep rhythm, rapid eye movement sleep duration, number of awakenings, total sleep duration and other sleep quality indicators were monitored and evaluated by electroencephalogram (EEG). At the same time, a comprehensive evaluation was conducted based on molecular biological and pathological indicators such as serum inflammatory factors (IL-6, TNF-α), oxidative stress indicators (MDA, SOD) and brain tissue pathological changes (HIF-1α, VEGF, BDNF expression) to determine the actual effect and safety of umbilical cord mesenchymal stem cell transplantation in improving sleep disorders caused by the hypoxic environment at high altitude. The present invention fully utilizes the immunomodulatory effects, anti-inflammatory and antioxidant capabilities, and neuroprotective and repair effects of umbilical cord mesenchymal stem cells to achieve long-term and effective improvement of sleep disorders caused by plateau hypoxia, avoiding the side effects and short-term therapeutic effects of traditional drug treatments. It has the advantages of stable efficacy, high safety, and sustainable repair of neural function, and is suitable for future clinical transformation and application promotion. DETAILED DESCRIPTION

[0007] The technical solution of the present invention will be further explained below with reference to specific embodiments.

[0008] Example 1:

[0009] Fresh healthy human umbilical cord tissue was obtained with the approval of the ethics committee. Under sterile conditions, umbilical cord segments of about 3-5 cm in length were cut and repeatedly washed with sterile PBS solution until the tissue was clean and free of contamination. Then, the tissue fragments (about 1-2 mm) were cut. 3Size); add 0.2% type I collagenase and digest at 37°C for 2 hours, gently shaking every 30 minutes. After digestion, filter the cell suspension through a 200-mesh sterile stainless steel mesh and centrifuge (1500 rpm for 5 minutes). Collect the precipitate and resuspend it in DMEM / F12 medium supplemented with 10% fetal bovine serum to prepare a cell suspension. Inoculate the cells into culture flasks and culture them in a 37°C, 5% CO2 incubator. Change the culture medium for the first time after 48 hours of culture, and then change the culture medium every 2-3 days. When the cells reach approximately 80% confluency, digest them with trypsin-EDTA solution for subculture and expansion.

[0010] At passage 3, a small number of cells were collected and flow cytometry was used to analyze the expression of surface markers of umbilical cord mesenchymal stem cells. The results showed a positive rate of ≥95% for CD29, CD44, CD73, CD90, and CD105, while CD34 and CD45 were negative, confirming that the obtained cells were highly pure umbilical cord-derived mesenchymal stem cells (UC-MSCs). The cells were then passaged to passages 3-5 for subsequent experiments.

[0011] Furthermore, healthy male SD rats weighing approximately 220-250g were selected and randomly divided into a normal control group (normoxia environment), a model group (hypoxia environment) and a treatment group (hypoxia environment), with 10 rats in each group. The animals in the model and treatment groups were placed in a hypoxia chamber with an oxygen concentration controlled at 14%-18%, with an exposure time of 10 hours per day for 14 days; the control group animals were kept in a normoxia (21%) environment. Before and after modeling, an electroencephalogram (EEG) monitor was used to record the animals' sleep duration, REM sleep cycles and number of awakenings to determine whether the model was successfully established. The criteria for successful model establishment were: compared with the normal control group, the total sleep duration of the model group animals was reduced by more than 20%, the REM sleep stage duration was significantly reduced (reduced by more than 30%), and the number of awakenings was significantly increased.

[0012] Furthermore, the 3rd-5th passage umbilical cord mesenchymal stem cells (UC-MSCs) prepared in Example 1 were centrifuged and then prepared with physiological saline to form a cell suspension at a concentration of 5 × 10^6 cells / mL. During the preparation process, the sterility and cell viability of the cell suspension were ensured to be ≥ 95%. The prepared cell suspension was injected into the tail vein of the treatment group rats every other day at a dose of 5 × 10^6 cells per injection for three consecutive treatments. The model control group was treated with an equal amount of physiological saline; the normal control group received no treatment.

[0013] For 7-14 days after the end of treatment, the electroencephalogram (EEG) of each group of rats was continuously recorded to assess changes in REM sleep duration, total sleep duration, and number of awakenings, thereby evaluating the sleep improvement effect. Serum was also collected from the animals, and serum inflammatory factors (such as IL-6 and TNF-α) and oxidative stress indicators (MDA and SOD activity) were measured using ELISA. In addition, brain tissue was obtained from the animals for HE staining and immunohistochemical staining to observe neuronal damage and repair, and to detect changes in the expression levels of HIF-1α, VEGF, and BDNF proteins in brain tissue to determine the effect of treatment on neural repair and regeneration.

[0014] Using multi-omics methods such as RNA-seq, qRT-PCR, and Western blot, we comprehensively analyzed the specific mechanisms of action of umbilical cord mesenchymal stem cells in intervening in high-altitude hypoxic sleep disorders. The results showed that after UC-MSC transplantation, the expression of neuroprotective factors such as HIF-1α, VEGF, and BDNF in the animal brain tissue was significantly increased, serum inflammatory factors (IL-6 and TNF-α) were significantly reduced, the oxidative stress marker (MDA) was reduced, and the activity of the antioxidant enzyme (SOD) was enhanced. These results suggest that UC-MSCs can effectively improve the symptoms of sleep disorders caused by the high-altitude hypoxic environment through multiple pathways, including improving neurovascular regeneration, reducing inflammatory responses, and alleviating oxidative stress damage.

[0015] Furthermore, to enhance the operability and cell-independence of umbilical cord mesenchymal stem cell therapy, the present invention also proposes a drug delivery method based on exosomes, the source of umbilical cord mesenchymal stem cells. The exosomes are membranous nanovesicles extracted from the supernatant of UC-MSCs culture in vitro, with a particle size of approximately 30–150 nm. They are purified by differential ultracentrifugation, membrane filtration, or commercial extraction kits, and verified by NTA particle size analysis, TEM morphology observation, and marker protein verification such as CD63 and TSG101. After being formulated into an atomized spray formulation, it can be delivered into the body via nasal spray or oral atomization, reaching the central nervous system directly through the nose-brain pathway, making it suitable for regulating the sleep center, thereby achieving the purpose of improving sleep disorders caused by high-altitude hypoxia.

[0016] In specific applications, the exosome preparation is freeze-dried with a protective agent such as trehalose, reconstituted, and then loaded into a metered nasal spray bottle or ultrasonic nebulizer inhaler. Each spray dose of 0.2–0.3 mL is administered daily at a fixed time (such as before bed) for 7–14 consecutive days. This approach avoids the inconvenience of cell injections and reduces immune risks, making it suitable for long-term use in remote training, high-altitude military deployments, and field work.

[0017] The above examples demonstrate that the preparation process of umbilical cord mesenchymal stem cells of the present invention is accurate and reliable, the animal model is stable, the administration method is accurate and effective, the efficacy evaluation is comprehensive and objective, the treatment mechanism is clear and reasonable, and it is suitable for clinical promotion and application. Although the embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for treating high-altitude hypoxic sleep disorders using umbilical cord mesenchymal stem cells, characterized by: The sleep disorder is induced by being in a low oxygen environment with an oxygen concentration of 14% to 18%.

2. The use of umbilical cord mesenchymal stem cells in the treatment of high-altitude hypoxic sleep disorders according to claim 1, characterized in that: The umbilical cord mesenchymal stem cells are third to fifth generation cells obtained by digesting umbilical cord tissue with 0.2% collagenase I at 37° C., culturing with DMEM / F12 medium containing 10% fetal bovine serum, and amplifying in vitro.

3. The use of umbilical cord mesenchymal stem cells in the treatment of high altitude hypoxia sleep disorder according to claim 1, characterized in that: The umbilical cord mesenchymal stem cells are cells that express CD29, CD44, CD73, CD90, and CD105 positively, and are CD34 and CD45 negatively.

4. The use of umbilical cord mesenchymal stem cells in the treatment of high altitude hypoxia sleep disorder according to claim 1, characterized in that: The drug was administered by intravenous injection, with a dose of 5×10 6 The cells were suspended in 1 mL of normal saline and injected once every other day for 3 times.

5. The use of umbilical cord mesenchymal stem cells in the treatment of high altitude hypoxia sleep disorder according to claim 1, characterized in that: The administration method is exosome freeze-dried powder solution or buffer solution, which is administered through nasal spray or atomized inhalation.