Atomizing agent containing mesenchymal stem cell factors and application of atomizing agent in treatment of nervous system diseases

Abstract

The invention relates to the technical field of biology, in particular to an atomizing agent containing mesenchymal stem cell factors and application of the atomizing agent to treatment of nervous system diseases. The atomizing agent containing the mesenchymal stem cell factor is characterized in that in the atomizing agent, the median of the particle size of the stem cell factor is 10-20 microns. Furthermore, the invention discloses application of the atomizing agent containing the mesenchymal stem cell factor in treatment of nervous system diseases. According to the atomizing agent containing the mesenchymal stem cell factor, the stem cell factor is combined with an atomizing technology, the atomizing agent directly acts on brain nerves through the nasal cavity, the medicine utilization rate is increased, the treatment time is shortened, the curative effect is more remarkable, the treatment process is painless and noninvasive, and the comfort level of a patient is improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an atomizer containing mesenchymal stem cell factors and application thereof in treating nervous system diseases. Background Art

[0002] Nasal drug delivery system (NDDS) refers to preparations used in the nasal cavity and absorbed through the nasal mucosa to exert local or systemic therapeutic effects. Modern medicine has been studying nasal mucosal drug delivery to treat systemic diseases for several decades. In 1976, VanDyke et al. reported that after 10% cocaine hydrochloride solution was administered through the nasal cavity, the drug was rapidly absorbed, and the peak plasma concentration reached 120-474 μg / L after 15-60 minutes. In June 1984, a special seminar on "Nasal route of systemic medication" was held in the United States. In December 1991, a European academic research conference on "Oral and nasal administration as an alternative to intravenous administration" was held in Paris, marking a more in-depth study of this route of administration in modern pharmaceutical treatment. At present, nasal administration has gradually attracted people's attention and has become one of the hot spots in the field of preparation research.

[0003] The nasal cavity is a complex organ from a dynamic point of view, because three different processes (drug deposition, drug clearance and drug absorption) are all completed in the nasal cavity. The surface area of ​​the nasal mucosa is 150cm 2 The epithelial cells on the surface of the mucosa have many microvilli, which are similar to the villi in the small intestine, which can increase the effective area for drug absorption. In addition, the subepithelial layer of the nasal mucosa is rich in capillaries and lymphatic capillaries, which can enable drugs to be quickly absorbed into the circulation.

[0004] The nasal cavity is part of the skull and has many small holes. The nasal endothelium is like a sieve-like plate. Nerve bundles pass through these small holes and are surrounded by cerebrospinal fluid. At the same time, because the nasal mucosa is close to the brain, the drug concentration in the cerebrospinal fluid can exceed the drug concentration in the plasma. Nasal administration can quickly reach the drug concentration for treating brain neurological diseases, so it can be delivered to the cerebrospinal fluid after being dripped into the nasal cavity. The nasal cavity is easier to access and is rich in vascular plexuses, which allows locally applied drugs to quickly reach effective concentrations. Nasal administration requires the drug solution to be made into a mist rather than a droplet that is easy to lose. Nasal administration is a very good way to directly deliver drugs into the blood system, which avoids the need for intravenous catheterization but achieves rapid and effective drug delivery.

[0005] Generally speaking, the ideal volume of drug applied to one nostril is 0.25 ml / time to 0.3 ml / time. In clinical applications, doctors will use a volume of 1 ml in each nostril, although this results in a certain amount of drug loss. When administering drugs via nasal spray, almost all particles with a size of 10-20 microns are deposited on the nasal mucosa. The spray particle size is very important for nasal administration of drugs to treat neurological diseases. According to literature research results, the optimal particle size for nasal administration of brain treatment is 15 microns, which is conducive to the optimal magnetic conductivity effect of drug particles, and at the same time, they will not be adhered to the inner wall of the respiratory tract because they are too small.

[0006] When taking drugs orally, all drug molecules absorbed through the gastrointestinal tract circulate through the "portal vein", then enter the inferior vena cava through the second hepatic portal after being metabolized by the liver, and then enter the heart to circulate to all parts of the body. One of the important functions of the liver is detoxification, and it is full of proteases that break down drug molecules. Therefore, most oral drugs are broken down in the liver, and these drugs do not enter the blood circulation. This destructive effect of the liver is called "first-pass metabolism of the liver". Drugs administered by other means such as nasal administration and intravenous administration do not circulate through the portal vein, so there is no loss of first-pass metabolism. Drugs administered by nasal administration can contact the olfactory mucosa, which can provide good evidence that molecular transport of the olfactory mucosa can transport drugs to the cerebrospinal fluid. The olfactory mucosa is located in the upper nasal cavity, below the cribriform plate of the skull, and contains olfactory cells, which extend through the cribriform plate into the cranial cavity. When drug molecules come into contact with this special mucosa, they are quickly and directly transported to the brain, bypassing the blood-brain barrier, and the drug level in the cerebrospinal fluid can quickly reach a high concentration (usually faster than intravenous administration). This process of transferring molecules from the nose to the brain is called the nose-brain pathway.

[0007] Several studies have shown that the nose-brain pathway can quickly transport some drugs from the nose to the cerebrospinal fluid, bypassing the blood-brain barrier. According to The Lancet, German researchers used intranasal spray to deliver three peptides (including vasopressin and insulin) to healthy volunteers. A few minutes after administration, the concentration of peptides in the cerebrospinal fluid increased. However, the concentration of peptides in the blood was found to increase, suggesting that it bypassed the blood-brain barrier and could be delivered directly to the brain. Summary of the invention

[0008] The purpose of the present invention is to provide an atomizer containing mesenchymal stem cell factor and its use. The atomizer combines stem cell factor with atomization technology to directly act on brain nerves through the nasal cavity, thereby improving drug utilization, reducing treatment time, and achieving more significant therapeutic effects. The treatment process is painless and non-invasive, thereby improving patient comfort.

[0009] Based on the above purpose, the present invention adopts the following technical solutions:

[0010] A nebulizer containing mesenchymal stem cell factors, wherein the median particle size of the stem cell factors in the nebulizer is 10-20 μm.

[0011] Preferably, the following components are included: 150,000-250,000 stem cell factor / mL, and the balance is physiological saline containing 0.5-2% human blood protein.

[0012] Preferably, the preparation method is as follows:

[0013] (1) Mixing stem cell factor and physiological saline to obtain a stem cell factor suspension;

[0014] (2) The suspension is ultrasonically crushed until the median particle size is 10-20 μm.

[0015] Preferably, the preparation method of the stem cell factor is as follows:

[0016] (1) Explant method for culturing primary mesenchymal stem cells

[0017] The umbilical cord is cleaned until the cleaning solution is bloodless, and the umbilical vein intima and artery are removed to obtain Wharton's jelly; the Wharton's jelly is inoculated into a culture bottle to evenly distribute the tissue blocks, and a culture medium is added for digestion to harvest a primary mesenchymal stem cell (MSC) suspension;

[0018] (2) Subculture

[0019] The harvested primary MSC cells were digested and subcultured;

[0020] (3) Preparation of stem cell factors

[0021] The mesenchymal stem cells (MSC) subcultured in step (2) are added to the culture medium, centrifuged, the supernatant is discarded, the cells are collected, and implanted in physiological saline to complete the preparation of stem cell factors.

[0022] Preferably, the subculture specifically comprises:

[0023] (1) Discard the culture medium in the culture flask, add PBS buffer to wash the cell suspension and then discard it, then add stem cell mild digestion enzyme to digest the cells. When the cells are observed to become round, add PBS buffer to dilute the cell suspension to make the cells completely dispersed;

[0024] (2) Transfer the cell suspension to a centrifuge tube, centrifuge, discard the supernatant, add complete culture medium to resuspend the cells at a density of 8000 cells / cm 2 After inoculation, cells were subcultured.

[0025] Preferably, the speed of ultracentrifugation is 6500-8500 r / min and the time is 20-25 min.

[0026] Preferably, during the preparation of stem cell factors, the median particle size of the primary mesenchymal stem cells obtained is 16-40 μm.

[0027] Application of the above-mentioned aerosol containing mesenchymal stem cell factor in the treatment of nervous system diseases.

[0028] Mesenchymal stem cells (MSC) are an important member of the stem cell family. They are derived from the early mesoderm of development and are pluripotent stem cells. MSC were first discovered in the bone marrow. They are increasingly attracting attention because of their multidirectional differentiation potential, hematopoietic support, promotion of stem cell engraftment, immune regulation and self-replication.

[0029] For example, mesenchymal stem cells can differentiate into various tissue cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, myocardium, and endothelium under specific induction conditions in vivo or in vitro. They still have multidirectional differentiation potential after continuous subculture and cryopreservation, and can be used as ideal seed cells for repairing tissue and organ damage caused by aging and pathology.

[0030] Mesenchymal stem cells have been clinically used to treat a variety of blood system diseases, cardiovascular diseases, liver cirrhosis, nervous system diseases, partial meniscus resection and repair of knee joint injuries, autoimmune diseases, and have made major breakthroughs, saving the lives of more patients. In addition, mesenchymal stem cells have long-term development prospects in the repair of the nervous system and more.

[0031] Mesenchymal stem cells are a branch of stem cell research. Stem cells are a type of cell with the ability to self-replicate and multidirectionally differentiate. They can continuously renew themselves and transform into one or more cells that constitute human tissues or organs under certain conditions. Stem cells are the origin cells of the body and the ancestral cells that form various tissues and organs of the human body.

[0032] Mesenchymal stem cells are a type of adult stem cells that exist in a variety of tissues (such as bone marrow, umbilical cord blood and umbilical cord tissue, placental tissue, adipose tissue, etc.) and have multidirectional differentiation potential. They are non-hematopoietic stem cells. This type of stem cell has the potential to differentiate into a variety of mesenchymal series cells (such as osteoblasts, chondrogenic cells, and adipogenic cells, etc.) or non-mesenchymal series cells, and has a unique cytokine secretion function. The cell model is used in proliferation, transplantation and differentiation research, as well as the identification of in vitro immune responses.

[0033] Cytokines (CK): soluble proteins produced by a variety of cells, which have multiple functions such as regulating innate immunity and adaptive immunity, hematopoiesis, cell growth, and damaged tissue repair. Cytokines can be divided into interleukins, interferons, tumor necrosis factors, chemokines, growth factors, etc.

[0034] Stem cell derivatives are highly active cytokines extracted from mesenchymal stem cells or other tissue stem cells (such as neural stem cells, skin stem cells, etc.) from various sources. The ingredients include: KGF, EGF, bFGF, NGF, HGF, VEGF, IL-6, G-CSF, TGF-β, PDGF, EPO, SDF-1, etc., as well as a variety of active peptides, various enzymes, hyaluronic acid, nucleic acids and other ingredients.

[0035] Human albumin can increase blood volume and maintain plasma colloidal osmotic pressure. Albumin accounts for 80% of plasma colloidal osmotic pressure and mainly regulates the dynamic balance of water between tissues and blood vessels. Due to the high molecular weight of albumin, it passes through the membrane more slowly than salts and water. It can transport and detoxify. Albumin can bind anions and cations, can transport different substances, and can also transport toxic substances to detoxification organs. It can provide nutrients, and tissue proteins and plasma proteins can be converted into each other. When carbon metabolism is impaired, albumin can serve as a carbon source to provide sufficient oxygen for tissues.

[0036] Pulmonary diseases are diseases of the lungs themselves or the pulmonary manifestations of systemic diseases. The nervous system is composed of the respiratory tract (nose, pharynx, larynx, trachea and bronchi at all levels) and alveoli. Stem cell factor atomization can repair lung damage. The present invention prepares an atomizer by mixing stem cell factor with human blood protein and normal saline. The effect of atomization inhalation therapy through the nasal cavity is to inhale drug particles into the airway or lungs through atomization, so that the drug is absorbed through the mucosa and exerts a local effect.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] The atomizer of the present invention can contact the olfactory mucosa with drugs administered by nasal administration, and can provide good evidence that the molecular transport of the olfactory mucosa can transport drugs to the cerebrospinal fluid. The olfactory mucosa is located in the upper nasal cavity, below the cribriform plate of the skull, and contains olfactory cells, which extend through the cribriform plate into the cranial cavity. When drug molecules contact this special mucosa, they are quickly and directly transported to the brain, bypassing the blood-brain barrier, and the drug level in the cerebrospinal fluid can quickly reach a high concentration (usually faster than intravenous administration). This process of transferring molecules from the nose to the brain is called the nose-brain pathway. The atomizer of the present invention is used to directly act on the brain nerves through the nasal cavity, improve the utilization rate of drugs, reduce the treatment time, and have a more significant therapeutic effect. Mild patients generally show an improvement trend in about 30 days; moderate patients generally show an improvement trend in about 90 days, and the therapeutic effect is generally most significant in about 180 days. In addition, compared with traditional treatments, stem cell factor atomization therapy reduces the distribution of drugs throughout the body because the drugs act directly on the brain, thereby reducing side effects. The entire treatment process is non-invasive and painless, and the process is more comfortable. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a microscope photo of the particle size of the atomized stem cell factor in Example 1 of the present invention;

[0040] Figure 2 This is a distribution diagram of the number of stem cell factors with different particle sizes in the primary mesenchymal stem cells obtained in Example 1 of the present invention;

[0041] Figure 3 This is a cell phenotype diagram of the primary mesenchymal stem cells obtained in Example 1 of the present invention;

[0042] Figure 4 This is a diagram showing the results of identifying primary mesenchymal stem cells obtained in Example 1 of the present invention;

[0043] Figure 5 This is a distribution diagram of the number of stem cell factors of different particle sizes in the primary mesenchymal stem cells obtained in Example 2 of the present invention. DETAILED DESCRIPTION

[0044] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention is described in detail below, but the following embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by those skilled in the art without making creative work belong to the scope of protection of the present invention.

[0045] In the description of the present embodiment, it should be understood that the terms "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features, that is, include one or more of the features. In the description of the present invention, the meaning of "multiple" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined. When a feature "includes or contains" one or some of the features it covers, unless otherwise specifically described, this indicates that other features are not excluded and other features may be further included.

[0046] In the description of the present embodiment, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0047] A nebulizer containing mesenchymal stem cell factors, wherein the median particle size of the stem cell factors in the nebulizer is 10-20 μm.

[0048] Preferably, the following components are included: 150,000-250,000 stem cell factor / mL, and the balance is physiological saline containing 0.5-2% human blood protein.

[0049] Preferably, the preparation method is as follows:

[0050] (1) Mixing stem cell factor and physiological saline to obtain a stem cell factor suspension;

[0051] (2) The suspension is ultrasonically crushed until the median particle size is 10-20 μm.

[0052] Preferably, the preparation method of the stem cell factor is as follows:

[0053] (1) Explant method for culturing primary mesenchymal stem cells

[0054] The umbilical cord is cleaned until the cleaning solution is bloodless, and the umbilical vein intima and artery are removed to obtain Wharton's jelly; the Wharton's jelly is inoculated into a culture bottle to evenly distribute the tissue blocks, and a culture medium is added for digestion to harvest a primary mesenchymal stem cell (MSC) suspension;

[0055] (2) Subculture

[0056] The harvested primary MSC cells were digested and subcultured;

[0057] (3) Preparation of stem cell factors

[0058] The mesenchymal stem cells (MSC) subcultured in step (2) are added to the culture medium, centrifuged, the supernatant is discarded, the cells are collected, and implanted in physiological saline to complete the preparation of stem cell factors.

[0059] Preferably, the subculture specifically comprises:

[0060] (1) Discard the culture medium in the culture flask, add PBS buffer to wash the cell suspension and then discard it, then add stem cell mild digestion enzyme to digest the cells. When the cells are observed to become round, add PBS buffer to dilute the cell suspension to make the cells completely dispersed;

[0061] (2) Transfer the cell suspension to a centrifuge tube, centrifuge, discard the supernatant, add complete culture medium to resuspend the cells at a density of 8000 cells / cm 2 After inoculation, cells were subcultured.

[0062] Preferably, the speed of ultracentrifugation is 6500-8500 r / min and the time is 20-25 min.

[0063] Preferably, during the preparation of stem cell factors, the median particle size of the primary mesenchymal stem cells obtained is 16-40 μm.

[0064] Furthermore, the above-mentioned aerosol containing mesenchymal stem cell factor is used in the treatment of nervous system diseases.

[0065] Example 1

[0066] A nebulizer containing mesenchymal stem cell factor, the nebulizer includes the following components: 200,000 / mL of stem cell factor and the balance is physiological saline containing 1% human blood protein, wherein the median particle size of the stem cell factor is 10-20 μm.

[0067] Wherein, the atomizer preparation method is as follows:

[0068] (1) Take out the frozen MSC cells from liquid nitrogen and quickly put the cryotube into a 37°C water bath for rapid thawing; in a biological safety cabinet or clean bench, count and wash the cells with 100 ml of normal saline according to the required number of cells, collect the cells, prepare normal saline containing 1% human albumin, add the collected cells with a syringe, and prepare a stem cell factor suspension (2up5MSC / 100ml);

[0069] (2) subjecting the suspension to ultrasonic grinding at 60% intensity for 3 seconds; or centrifuging at a speed of 7000 r / min for 23 minutes, until the median particle size is 10-20 μm, and observing that there is no visible flocculent precipitation and impurities in the cell suspension;

[0070] (3) Perfusion: The cell suspension is perfused at a dose of 3 ml / vial and then stored at -80 degrees Celsius.

[0071] The steps of preparing stem cell factor are as follows:

[0072] (1) Explant method for culturing primary mesenchymal stem cells

[0073] Step 1, wash the umbilical cord with PBS until the washing solution is bloodless, and remove the umbilical vein intima and artery;

[0074] Step 2, cut Wharton's jelly into small pieces with surgical scissors or a scalpel, and inoculate them into culture bottles or culture dishes to evenly distribute the tissue blocks. The amount of tissue blocks implanted and the amount of culture medium added for different culture vessels are shown in the table below. The culture dishes are placed in a carbon dioxide incubator at 37°C, 5% CO2, and 95% humidity for culture. The medium is changed on the 7th day, the medium is changed on the 10th day, and the primary mesenchymal stem cell (MSC) suspension is harvested on the 14th day;

[0075] (2) The harvested primary MSC cells are digested and subcultured. The harvested primary MSC cells are marked as P0. Each digestion is regarded as one generation. A total of 5 digestions are performed and marked as P5. The specific method is as follows:

[0076] Step 1: After culturing for 72 hours, observe the cultured MSC cells under a microscope. When the cell confluence reaches 90%, they can be passaged.

[0077] Step 2: In a biosafety cabinet, discard the culture medium in the culture flask, add 10 mL of PBS to wash the cells and then discard. Add 2 mL of stem cell mild digestion enzyme to digest the cells at room temperature. When the cells are observed to be round under a microscope (usually 2-5 minutes), immediately add 10 mL of PBS to dilute the cell suspension, use a pipette to gently blow the cells that have not completely detached from the flask wall, and gently blow and mix to make the cells completely dispersed;

[0078] Step 3: Transfer the cell suspension to a 15 mL centrifuge tube and centrifuge at 1300 rpm (250 g) for 5 min. Discard the supernatant, add 2 mL of complete medium to resuspend the cells, and count them with trypan blue staining. The density is 8000 cells / cm 2 Inoculate the cells and place the culture flask at 37°C, 5% CO2, and saturated humidity for 48-72 hours to subculture the mesenchymal stem cells and complete the first generation of culture;

[0079] Step 4, repeat step 2 and step 3 4 times, complete 5 cultures, obtain the fifth generation of cultured cells, mark them as P5, place them in a liquid nitrogen environment, and store them at -80°C.

[0080] Application of the above-mentioned aerosol containing mesenchymal stem cell factor in the treatment of nervous system diseases.

[0081] Example 2

[0082] A nebulizer containing mesenchymal stem cell factor, the nebulizer includes the following components: 150,000 / mL of stem cell factor and the balance is physiological saline containing 0.5% human blood protein, wherein the median particle size of the stem cell factor is 10-20 μm.

[0083] Wherein, the atomizer preparation method is as follows:

[0084] (1) Take out the frozen MSC cells from liquid nitrogen and quickly put the cryotube into a 37°C water bath for rapid thawing; in a biological safety cabinet or clean bench, count and wash the cells with 100 ml of physiological saline according to the required number of cells, collect the cells, prepare physiological saline containing 0.5% human albumin, add the collected cells with a syringe, and prepare a stem cell factor suspension;

[0085] (2) subjecting the suspension to ultrasonic grinding at 60% intensity for 3 seconds; or centrifuging at a speed of 7000 r / min for 23 minutes, until the median particle size is 10-20 μm, and observing that there is no visible flocculent precipitation and impurities in the cell suspension;

[0086] (3) Perfusion: The cell suspension is perfused at a dose of 3 ml / vial and then stored at -80 degrees Celsius.

[0087] The rest is the same as in Example 1.

[0088] Example 3

[0089] A nebulizer containing mesenchymal stem cell factor, the nebulizer includes the following components: 250,000 / mL of stem cell factor and the balance is physiological saline containing 2% human blood protein, wherein the median particle size of the stem cell factor is 10-20 μm.

[0090] Wherein, the atomizer preparation method is as follows:

[0091] (1) Take out the frozen MSC cells from liquid nitrogen and quickly put the cryotube into a 37°C water bath for rapid thawing; in a biological safety cabinet or clean bench, count and wash and collect the cells according to the required number of cells, prepare a physiological saline solution containing 2% human albumin, add the above collected cells with a syringe, and prepare a cytokine suspension;

[0092] (2) subjecting the suspension to ultrasonic grinding at 60% intensity for 2 seconds, and then to centrifuge grinding at a speed of 8000 r / min for 20 minutes until the median particle size is 10-20 μm, and observing that there is no visible flocculent precipitation and impurities in the cell suspension;

[0093] (3) Perfusion: The cell suspension is perfused at a dose of 3 ml / vial and then stored at -80 degrees Celsius.

[0094] In Example 1, during the process of culturing primary mesenchymal stem cells by the explant method, the amount of tissue explants and the amount of culture medium added in different culture vessels are shown in Table 1, and the culture dishes or culture bottles are placed in a carbon dioxide incubator at 37°C, 5% CO2, and 95% humidity. The rest is the same as in Example 1.

[0095] Table 1. The amount of tissue blocks implanted and the amount of culture medium added in different culture vessels

[0096] Culture vessels Approximate bottom area (cm2) Liquid volume (mL) Number of implanted tissue blocks 60mm dish 21 5 5 100mm dish 55 12 12 150mm dish 150 30 30 T25 25 5 6 T75 75 15 15 T175 175 35 40 T225 225 45 50

[0097] Take 0.1 ml of the atomized agent of stem cell factor obtained in Example 1 of the present invention and analyze the particle size of stem cell factor under a microscope. The results are shown in Figure 1 .

[0098] The biological characteristics of the primary mesenchymal stem cells obtained by the explant method in Example 1 were identified as follows:

[0099] (1) Cell counting: Use a cell counting plate or an automatic cell counter. The results are shown in Figure 2 ;

[0100] (2) Cell phenotype, results are shown in Figure 3 ;

[0101] (3) Cell identification: flow cytometry

[0102] 10 5 cells, 100 μl reaction system, 10 μl fluorescent antibody, and isotype control and negative control at the same time; incubate at room temperature for 30 min in the dark, wash cells twice with 1 ml sheath fluid, and then resuspend cells with 300 μl sheath fluid; measure on flow cytometer, measure background parameters with negative control, adjust fluorescence compensation, and obtain the proportion of positive expression cells. The results are shown in Figure 4 .

[0103] The primary mesenchymal stem cells obtained by the explant method in Example 2 were counted and the results are shown in Figure 5 .

[0104] Parkinson's disease (PD), also known as tremor paralysis, is a common neurodegenerative disease in middle-aged and elderly people. Clinically, it is characterized by resting tremor, bradykinesia, muscle rigidity and postural balance disorders.

[0105] The aerosol containing mesenchymal stem cell factors of Example 1 of the present invention was applied to Parkinson's patients. The specific scheme and results are as follows:

[0106] Twenty patients with advanced Parkinson's disease were treated with the aerosol of Example 1 for intranasal transplantation, 3 ml each time, once a day in the first two weeks of the first month, once every two days in the next two weeks, 3 times a week from the second to third months, and twice a week after the fifth month. The specific results were evaluated and counted by the Movement Disorders Society Unified Parkinson's Rating Scale (MDS-UPDRS), and the results were counted once in the second week, fourth week, third month, sixth month, ninth month, and twelfth month.

[0107] Table 1 12-month MDS-UPDRS score data of 20 patients

[0108]

[0109]

[0110] It can be seen from the above table that in the sixth month, the total score decreased by 19.6%, and in the twelfth month, the total score decreased by 28.8%, and the most significant improvement occurred in the sixth month. It can be seen that the atomizer of the present invention is used in the treatment of nervous system diseases, and the therapeutic effect is significant.

[0111] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. An aerosol containing mesenchymal stem cell factor, characterized in that: In the aerosol, the median particle size of the stem cell factor is 10-20 μm.

2. The aerosol containing mesenchymal stem cell factor according to claim 1, characterized in that: The invention comprises the following ingredients: 150,000-250,000 / mL of stem cell factor and the balance of physiological saline containing 0.5-2% human blood protein.

3. The aerosol containing mesenchymal stem cell factor according to claim 1, characterized in that: The preparation method is as follows: (1) Mixing stem cell factor and physiological saline to obtain a stem cell factor suspension; (2) The suspension is ultrasonically crushed until the median particle size is 10-20 μm.

4. The aerosol containing mesenchymal stem cell factor according to claim 1, characterized in that: The preparation method of the stem cell factor is as follows: (1) Explant method for culturing primary mesenchymal stem cells The umbilical cord is cleaned until the cleaning solution is bloodless, and the umbilical vein intima and artery are removed to obtain Wharton's jelly; the Wharton's jelly is inoculated into a culture bottle to evenly distribute the tissue blocks, and a culture medium is added for digestion to harvest a primary mesenchymal stem cell (MSC) suspension; (2) Subculture The harvested primary MSC cells were digested and subcultured; (3) Preparation of stem cell factors The mesenchymal stem cells (MSC) subcultured in step (2) are added to the culture medium, centrifuged, the supernatant is discarded, the cells are collected, and implanted in physiological saline to complete the preparation of stem cell factors.

5. The aerosol containing mesenchymal stem cell factor according to claim 3, characterized in that: The subculture specifically includes: (1) Discard the culture medium in the culture flask, add PBS buffer to wash the cell suspension and then discard it, then add stem cell mild digestion enzyme to digest the cells. When the cells are observed to become round, add PBS buffer to dilute the cell suspension to make the cells completely dispersed; (2) Transfer the cell suspension to a centrifuge tube, centrifuge, discard the supernatant, add complete culture medium to resuspend the cells at a density of 8000 cells / cm 2 After inoculation, cells were subcultured.

6. The aerosol containing mesenchymal stem cell factors according to claim 3, characterized in that: The ultrasonic crushing process is as follows: the speed of ultracentrifugation is 6500-8500 r / min, and the time is 20-25 min.

7. The aerosol containing mesenchymal stem cell factor according to claim 1, characterized in that: During the preparation of stem cell factors, the median particle size of the obtained primary mesenchymal stem cells was 16-40 μm.

8. Use of the aerosol containing mesenchymal stem cell factor according to any one of claims 1 to 7 in treating nervous system diseases.

Images