Composition, preparation method thereof and spray preparation for improving sleep

Abstract

The invention relates to the field of stem cell biomedicine, and provides a preparation method of a mesenchymal stem cell factor composition in order to solve the problem that umbilical cord mesenchymal stem cells in the prior art are difficult to be used for acute insomnia symptoms, and the preparation method comprises the following steps: S100, inoculating primary umbilical cord mesenchymal stem cells into a first culture medium to obtain P1-generation mesenchymal stem cells; s200, subculturing the P1-generation mesenchymal stem cells in a second culture medium environment for 5-7 generations to obtain generations of mesenchymal stem cells; s300, subculturing the mesenchymal stem cells for 2-4 generations in a third culture medium environment to obtain a cell suspension; filtering the culture medium supernate of the P6-P7 generation mesenchymal stem cells through a filter membrane, and taking filtrate; and mixing the cell suspension with the filtrate to obtain the mesenchymal stem cell factor composition. According to the method, the culture operation of the umbilical cord mesenchymal stem cells is improved, and factors secreted in the culture period are biased to sedative factors and melatonin precursor factors, so that the supernate in the culture process has the effect of improving acute insomnia.

Description

Technical Field

[0001] The present invention relates to the field of stem cell biomedicine, and in particular to a composition and a preparation method thereof, and a spray preparation for improving sleep. Background Art

[0002] Mesenchymal stem cells are a type of pluripotent stem cells that have all the common properties of stem cells, namely self-renewal and multidirectional differentiation capabilities. Currently, human umbilical cord mesenchymal stem cells can be injected intravenously to improve chronic insomnia.

[0003] Patent publication number CN117736995A discloses transgenic stem cells and their use in the treatment of insomnia, which uses the supernatant of umbilical cord mesenchymal stem cells as an intravenous injection to treat insomnia symptoms. Currently, there is still a lack of research on the use of mesenchymal stem cells in improving or treating acute insomnia. Summary of the invention

[0004] The purpose of the present invention is to provide a mesenchymal stem cell factor composition and a preparation method thereof, so as to solve the problem in the prior art that umbilical cord mesenchymal stem cells are difficult to be used for acute insomnia symptoms.

[0005] The present invention also aims to provide a spray preparation for improving sleep, which can effectively deliver drugs through the nasal cavity and has high bioavailability.

[0006] The embodiments of the present invention are implemented by the following technical solutions:

[0007] A method for preparing a mesenchymal stem cell factor composition, comprising:

[0008] S100, inoculating primary umbilical cord mesenchymal stem cells into the first culture medium, and when the cell confluence reaches 80%-90%, discarding the old culture medium, washing the cells with D-hanks solution, performing enzyme digestion, adding D-hanks solution and centrifuging, resuspending the cell precipitate with the second culture medium to obtain P1 generation mesenchymal stem cells; the culture temperature is 37°C, the carbon dioxide concentration is 5%-6%, the oxygen concentration is 5%-8%, and the remaining gas is nitrogen;

[0009] The first culture medium is DMEM high glucose culture medium; the second culture medium includes DMEM / F12 culture medium matrix, 2-4 ng / ml NGF and 1-1.5 ng / ml IL-10;

[0010] S200 and P1 generation mesenchymal stem cells are subcultured for 5-7 generations in the second culture medium environment to obtain generation mesenchymal stem cells; the culture temperature is 37°C, the oxygen concentration is ≥99.5%, and the pressure is 1.3-1.8 atmospheres;

[0011] S300 and generation mesenchymal stem cells are subcultured for 2-4 generations in the third culture medium environment to obtain a cell suspension; the culture medium supernatant of the P6-P7 generation mesenchymal stem cells is filtered through a filter membrane and the filtrate is obtained; the cell suspension is mixed with the filtrate to obtain a mesenchymal stem cell factor composition; the third culture medium includes DMEM / F12 culture medium matrix, 2-4ng / mlNGF, 1-1.5ng / ml IL-10 and 4-5ng / ml bFGF; the culture temperature is 37°C, the carbon dioxide concentration is 5%-6%, the oxygen concentration is 3%-5%, and the remaining gas is nitrogen.

[0012] At present, the existing technology makes umbilical cord mesenchymal stem cells into intravenous injections for the treatment of insomnia symptoms, which mainly utilizes the neuroprotective effect and neuroregenerative repair effect of mesenchymal stem cells. Among them, the neuroprotective effect mainly reflects that mesenchymal stem cells reduce oxidative stress and protect nerve cells from damage by secreting antioxidant enzymes such as superoxide dismutase and glutathione peroxidase; reduce inflammatory response and protect nerve cells by secreting anti-inflammatory factors (such as IL-10, TGF-β) and inhibiting pro-inflammatory factors (such as TNF-α, IL-1β); reduce apoptosis of nerve cells and protect the survival of nerve cells by activating the PI3K / Akt pathway and inhibiting caspase-3 activity; promote differentiation, growth, survival and functional repair of nerve cells by secreting neurotrophic factors; secrete extracellular matrix components (such as fibronectin and laminin) to provide support and protection for nerve cells. Among them, the regeneration and repair of cells is mainly manifested in the use of their multidirectional differentiation potential, which can be differentiated into nerve cells under specific conditions, participate in the repair and regeneration of neural tissue; secrete neurotrophic factors and support factors to promote the differentiation and maturation of nerve cells; promote the formation and functional recovery of synapses, and improve the connection and communication of neural networks. As can be seen from the above, its target of action is more nerve cells, and the action process is a long-lasting process. When treating chronic insomnia, it has a more significant effect and the effect is maintained for a longer time. However, when treating acute insomnia, the effect is not significant. Therefore, the applicant expects to induce mesenchymal stem cells to produce more factors that are effective for acute insomnia through the improvement of the culture method, such as sedative factors or factors that activate melatonin receptors. In addition, differentiation efficiency and differentiation stability need to be considered in the process of directional induction to improve the therapeutic effect. Because insomnia or staying up late can damage neurons, such as neurons losing synapses. The composition provided by the present invention can not only treat acute insomnia, but also has the effect of protecting and repairing neurons, avoiding the transformation of acute insomnia into chronic insomnia, especially for patients who have had acute insomnia for a long time.

[0013] Preferably, the seeding density of each passage in S200 is 5000-7000 cells / cm 2In parts by weight, the mesenchymal stem cell factor composition includes 6-8 parts of the cell suspension in S300 and 11-15 parts of the filtrate corresponding to the P7 generation mesenchymal stem cells.

[0014] The best ratio of the mesenchymal stem cell factor composition obtained by experiments is 7 parts of cell suspension and 12 parts of the filtrate of P7 mesenchymal stem cells. A 0.22 μm filter membrane can be used for filtration.

[0015] Preferably, the second culture medium further comprises: 2-5 μM GDC-0941.

[0016] The applicant found during the experiment that the culture time of S200 subculture was much longer than that of S100 and S300, so GDC-0941 was added to reduce the culture time. At the same time, secondary directional induction enhanced the factor expression after the administration of the composition.

[0017] Preferably, after the subculture in S300 is completed, the cells are washed with D-hanks solution, enzymatic digestion is performed, D-hanks solution is added and centrifuged, and the cell pellet is resuspended with a preservation solution to obtain a cell suspension; the preservation solution includes 3wt%-5wt% sodium hyaluronate, 0.7wt%-0.9wt% sodium chloride, 5wt%-10wt% glucose, 2vt%-5vt% compound amino acids, and the rest is distilled water.

[0018] Preferably, the S300 further includes:

[0019] D100, adding liposomes to the filtrate to obtain a factor-liposome solution after reaction;

[0020] D200, adding chitosan solution to the factor-liposome solution to obtain a drug solution after reaction;

[0021] D300, mixing the drug solution with the cell suspension to obtain a mesenchymal stem cell factor composition.

[0022] Since the invention of the applicant aims to prepare a drug that can treat acute insomnia, and the cause of acute insomnia may be more due to jet lag, pressure or psychological factors, etc., the drug action time is more on the night of the same day, and it may be necessary to continue to use it every day. If the drug is delivered by intravenous injection, it is relatively cumbersome for the patient, so the applicant hopes to use other delivery methods, such as spraying from the nasal cavity. However, relative to intravenous injection, the performance requirements for drug delivery are higher. In order to improve the bioadhesion and mucosal permeability of the drug, increase the delivery performance of the drug, and improve bioavailability, the present invention adds chitosan and liposomes in the filtrate.

[0023] Preferably, the D100 specifically comprises: by weight, 30-50 parts of DOTAP, 70-90 parts of filtrate and 30-40 parts of ethanol are mixed to obtain a mixed solution, and then the ethanol is removed from the mixed solution by vacuum rotary evaporation, and then 15-20 parts of HEPES buffer is added for hydration, and the factor-liposome solution is obtained after ultrasonic treatment.

[0024] The vacuum rotary evaporation temperature can be selected to be 40°C, the rotary evaporation time can be selected to be 40min, the ultrasonic frequency can be selected to be 260W, and the ultrasonic time can be selected to be 5min. The sedative factor in the present invention is mainly GABA, GABA has a negatively charged carboxyl group, and DOTAP has a positively charged quaternary ammonium salt head group, so that DOTAP can be used to better load the drug factor. The factor that activates the melatonin receptor in the present invention is mainly 5-hydroxytryptamine, whose amino group is alkaline, and DOTAP has a long-chain fatty acid, which can promote the protonation of 5-hydroxytryptamine, increase solubility and chemical stability, and increase its bioavailability. Protonated 5-hydroxytryptamine is more likely to pass through the ion channels on the cell membrane and has enhanced targeting. In addition, 5-hydroxytryptamine is easily oxidized, and the effect of oxidation on its drug efficacy can be reduced by coating loading.

[0025] Preferably, the D200 specifically comprises: gradually injecting 0.2wt%-0.3wt% chitosan solution into the factor-liposome solution at an injection rate of 7-10ml / min, stirring for 2-4h after the injection, to obtain a drug solution; the volume ratio of the factor-liposome solution to the chitosan solution is 1:4-2:5.

[0026] The stirring speed can be selected to be 200r / min. After the liposomes are modified by chitosan, the stability, biocompatibility and receptor recognition performance of the liposomes are increased. Since the duration of the drug action of the present invention is generally less than 10 hours, it is also necessary to control the thickness of the chitosan layer through the process so that the release rate of the drug is adapted to the action time.

[0027] Preferably, the isolation step of primary umbilical cord mesenchymal stem cells comprises:

[0028] A100, wash the umbilical cord with PBS, cut it into 2 cm long segments, peel off the venous intima and artery, and then cut it into pieces to obtain tissue blocks;

[0029] A200, after placing the tissue block in the culture dish, add complete culture medium;

[0030] A300, place the culture dish at 37°C, 5% CO 2 , and 95% humidity in a carbon dioxide incubator. After 7 days, the medium was replaced by 2 / 3 of the volume. When cells crawled out from the tissue block, the crawled cells were collected as primary umbilical cord mesenchymal stem cells; the outer edge of the crawled cells was ≥0.5 cm away from the edge of the tissue block.

[0031] A mesenchymal stem cell factor composition prepared by the preparation method.

[0032] A spray preparation for improving sleep, comprising a mesenchymal stem cell factor composition.

[0033] The present invention has at least the following beneficial effects:

[0034] The present invention improves the culture operation of umbilical cord mesenchymal stem cells and increases their directional differentiation rate. The factors secreted during the culture period are no longer biased towards repair or protection factors, but rather towards sedative factors and melatonin precursor factors, so that the supernatant during the culture process has the effect of improving acute insomnia; at the same time, the mesenchymal stem cells obtained after subculture can not only improve acute insomnia to a certain extent, but also repair and regenerate neuronal damage caused by acute insomnia, thereby preventing acute insomnia from turning into chronic insomnia; after the composition provided by the present invention is made into a spray preparation, not only the bioavailability of the drug can be ensured, but also... BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0036] Figure 1 is the graph of 5-HT expression level;

[0037] Figure 2 is the GABA expression level diagram; DETAILED DESCRIPTION

[0038] In order to make the purpose, method scheme and advantages of the embodiments of the present invention clearer, the method scheme in the embodiments of the present invention is clearly and completely described. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0039] Embodiment 1: A method for preparing a mesenchymal stem cell factor composition, comprising:

[0040] S100, inoculating primary umbilical cord mesenchymal stem cells into the first culture medium, and when the cell confluence reaches 80%, discarding the old culture medium, washing the cells with D-hanks solution, digesting with trypsin, adding D-hanks solution and centrifuging, resuspending the cell precipitate with the second culture medium to obtain P1 generation mesenchymal stem cells; the culture temperature is 37°C, the carbon dioxide concentration is 5%, the oxygen concentration is 5%, and the remaining gas is nitrogen;

[0041] The first culture medium is DMEM high glucose culture medium; the second culture medium includes DMEM / F12 culture medium matrix, 2ng / ml NGF and 1ng / ml IL-10;

[0042] S200 and P1 generation mesenchymal stem cells were subcultured for 7 generations in the second culture medium environment to obtain generation mesenchymal stem cells; each generation of cells was centrifuged and the supernatant was removed, and the cell pellet was resuspended in the second culture medium; the culture temperature was 37°C, the oxygen concentration was ≥99.5%, and the pressure was 1.3 atmospheres; the seeding density of each generation in S200 was 5000 cells / cm 2 ;

[0043] S300, mesenchymal stem cells of the generation were subcultured for 2 generations under the third culture medium environment. After the subculture, the cells were washed with D-hanks solution, digested with enzymes, then added with D-hanks solution and centrifuged, and the cell pellet was resuspended with preservation solution to obtain a cell suspension; the preservation solution included 3wt% sodium hyaluronate, 0.7wt% sodium chloride, 5wt% glucose, 2vt% compound amino acids, and the rest was distilled water; the culture medium supernatant of P6-P7 mesenchymal stem cells was filtered through a filter membrane and the filtrate was taken; the cell suspension was mixed with the filtrate to obtain a mesenchymal stem cell factor composition; the third culture medium included DMEM / F12 culture medium matrix, 2ng / ml NGF, 1ng / ml IL-10 and 4ng / ml bFGF; the culture temperature was 37°C, the carbon dioxide concentration was 5%, the oxygen concentration was 3%, and the remaining gas was nitrogen.

[0044] In parts by weight, the mesenchymal stem cell factor composition includes 6 parts of the cell suspension in S300 and 11 parts of the filtrate corresponding to the P7 mesenchymal stem cells.

[0045] The isolation step of primary umbilical cord mesenchymal stem cells comprises:

[0046] A100, wash the umbilical cord with PBS (pH=7.4), cut it into 2 cm long segments, peel off the venous intima and artery, and then cut it into pieces to obtain tissue blocks;

[0047] A200, after placing the tissue block in the culture dish, add complete culture medium;

[0048] A300, place the culture dish at 37°C, 5% CO 2 , and 95% humidity in a carbon dioxide incubator. After 7 days, the medium was replaced by 2 / 3 of the volume. When cells crawled out from the tissue block, the crawled cells were collected as primary umbilical cord mesenchymal stem cells; the outer edge of the crawled cells was ≥0.5 cm away from the edge of the tissue block.

[0049] Embodiment 2: A method for preparing a mesenchymal stem cell factor composition, comprising:

[0050] S100, inoculating primary umbilical cord mesenchymal stem cells into the first culture medium, and when the cell confluence reaches 90%, discarding the old culture medium, washing the cells with D-hanks solution, digesting with trypsin, adding D-hanks solution and centrifuging, resuspending the cell precipitate with the second culture medium to obtain P1 generation mesenchymal stem cells; the culture temperature is 37°C, the carbon dioxide concentration is 6%, the oxygen concentration is 8%, and the remaining gas is nitrogen;

[0051] The first culture medium is DMEM high glucose culture medium; the second culture medium includes DMEM / F12 culture medium matrix, 4ng / ml NGF and 1.5ng / ml IL-10;

[0052] S200 and P1 generation mesenchymal stem cells were subcultured for 7 generations in the second culture medium environment to obtain generation mesenchymal stem cells; each generation of cells was centrifuged and the supernatant was removed, and the cell pellet was resuspended in the second culture medium; the culture temperature was 37°C, the oxygen concentration was ≥99.5%, and the pressure was 1.8 atmospheres; the seeding density of each generation in S200 was 7000 cells / cm 2 ;

[0053] S300, mesenchymal stem cells of the generation were subcultured for 4 generations under the third culture medium environment. After the subculture, the cells were washed with D-hanks solution, digested with enzymes, then added with D-hanks solution and centrifuged, and the cell pellet was resuspended with preservation solution to obtain a cell suspension; the preservation solution included 5wt% sodium hyaluronate, 0.9wt% sodium chloride, 10wt% glucose, 5vt% compound amino acids, and the rest was distilled water; the culture medium supernatant of P6-P7 mesenchymal stem cells was filtered through a filter membrane and the filtrate was taken; the cell suspension was mixed with the filtrate to obtain a mesenchymal stem cell factor composition; the third culture medium included DMEM / F12 culture medium matrix, 4ng / ml NGF, 1.5ng / ml IL-10 and 5ng / ml bFGF; the culture temperature was 37°C, the carbon dioxide concentration was 6%, the oxygen concentration was 5%, and the remaining gas was nitrogen.

[0054] In parts by weight, the mesenchymal stem cell factor composition includes 8 parts of the cell suspension in S300 and 15 parts of the filtrate corresponding to the P7 mesenchymal stem cells.

[0055] The isolation step of primary umbilical cord mesenchymal stem cells comprises:

[0056] A100, wash the umbilical cord with PBS (pH=7.4), cut it into 2 cm long segments, peel off the venous intima and artery, and then cut it into pieces to obtain tissue blocks;

[0057] A200, after placing the tissue block in the culture dish, add complete culture medium;

[0058] A300, place the culture dish at 37°C, 5% CO 2 , and 95% humidity in a carbon dioxide incubator. After 7 days, the medium was replaced by 2 / 3 of the volume. When cells crawled out from the tissue block, the crawled cells were collected as primary umbilical cord mesenchymal stem cells; the outer edge of the crawled cells was ≥0.5 cm away from the edge of the tissue block.

[0059] Embodiment 3: A method for preparing a mesenchymal stem cell factor composition, comprising:

[0060] S100, inoculating primary umbilical cord mesenchymal stem cells into the first culture medium, and when the cell confluence reaches 85%, discarding the old culture medium, washing the cells with D-hanks solution, digesting with trypsin, adding D-hanks solution and centrifuging, resuspending the cell precipitate with the second culture medium, and obtaining P1 generation mesenchymal stem cells; the culture temperature is 37°C, the carbon dioxide concentration is 5.5%, the oxygen concentration is 6%, and the remaining gas is nitrogen;

[0061] The first culture medium is DMEM high glucose culture medium; the second culture medium includes DMEM / F12 culture medium matrix, 3ng / ml NGF and 1.25ng / ml IL-10;

[0062] S200 and P1 generation mesenchymal stem cells were subcultured for 7 generations in the second culture medium environment to obtain generation mesenchymal stem cells; each generation of cells was centrifuged and the supernatant was removed, and the cell pellet was resuspended in the second culture medium; the culture temperature was 37°C, the oxygen concentration was ≥99.5%, and the pressure was 1.6 atmospheres; the seeding density of each generation in S200 was 6000 cells / cm 2 In parts by weight, the mesenchymal stem cell factor composition includes 7 parts of the cell suspension in S300 and 13 parts of the filtrate corresponding to the P7 generation mesenchymal stem cells.

[0063] S300, mesenchymal stem cells are subcultured for 2-4 generations in the third culture medium environment. After the subculture, the cells are washed with D-hanks solution, digested with enzymes, added with D-hanks solution and centrifuged, and the cell pellet is resuspended with preservation solution to obtain a cell suspension; the preservation solution includes 4wt% sodium hyaluronate, 0.8wt% sodium chloride, 7wt% glucose, 4vt% compound amino acids, and the rest is distilled water; the culture medium supernatant of P6-P7 mesenchymal stem cells is filtered through a filter membrane and the filtrate is taken; the cell suspension is mixed with the filtrate to obtain a mesenchymal stem cell factor composition; the third culture medium includes DMEM / F12 culture medium matrix, 3ng / ml NGF, 1.25ng / ml IL-10 and 4.5ng / ml bFGF; the culture temperature is 37°C, the carbon dioxide concentration is 6%, the oxygen concentration is 4%, and the remaining gas is nitrogen.

[0064] The isolation step of primary umbilical cord mesenchymal stem cells comprises:

[0065] A100, wash the umbilical cord with PBS (pH=7.4), cut it into 2 cm long segments, peel off the venous intima and artery, and then cut it into pieces to obtain tissue blocks;

[0066] A200, after placing the tissue block in the culture dish, add complete culture medium;

[0067] A300, place the culture dish at 37°C, 5% CO 2 , and 95% humidity in a carbon dioxide incubator. After 7 days, the medium was replaced by 2 / 3 of the volume. When cells crawled out from the tissue block, the crawled cells were collected as primary umbilical cord mesenchymal stem cells; the outer edge of the crawled cells was ≥0.5 cm away from the edge of the tissue block.

[0068] Example 4: The difference from Example 3 is that 2 μM GDC-0941 is additionally added to the second culture medium.

[0069] Example 5: The difference from Example 3 is that 5 μM GDC-0941 is additionally added to the second culture medium.

[0070] Example 6: The difference from Example 3 is that 3 μM GDC-0941 is additionally added to the second culture medium.

[0071] Embodiment 7: The difference from Embodiment 3 is that: S300 further includes:

[0072] D100, by weight, 30 parts of DOTAP, 70 parts of filtrate and 30 parts of ethanol were mixed to obtain a mixed solution, and then the mixed solution was subjected to vacuum rotary evaporation at 40°C for 40 minutes to remove the ethanol, and then 15 parts of HEPES buffer was added for hydration, and an ultrasonic probe was used to perform ultrasonic treatment at 260W for 5 minutes to obtain a factor-liposome solution;

[0073] D200, gradually inject 0.2wt% chitosan solution into the factor-liposome solution at a speed of 7ml / min. After the injection, stir at 200r / min for 2h to obtain a drug solution; the volume ratio of the factor-liposome solution to the chitosan solution is 1:4;

[0074] D300, mixing 20 parts of the drug solution with 7 parts of the cell suspension to obtain a mesenchymal stem cell factor composition.

[0075] Embodiment 8: The difference from Embodiment 3 is that: S300 further includes:

[0076] D100, by weight, 50 parts of DOTAP, 90 parts of the filtrate and 40 parts of ethanol were mixed to obtain a mixed solution, and then the mixed solution was subjected to vacuum rotary evaporation at 40°C for 40 minutes to remove the ethanol, and then 20 parts of HEPES buffer was added for hydration, and an ultrasonic probe was used to perform ultrasonic treatment at 260W for 5 minutes to obtain a factor-liposome solution;

[0077] D200, gradually inject 0.3wt% chitosan solution into the factor-liposome solution at a speed of 10ml / min, and stir at 200r / min for 4h after the injection to obtain a drug solution; the volume ratio of the factor-liposome solution to the chitosan solution is 2:5;

[0078] D300, mixing 20 parts of the drug solution with 7 parts of the cell suspension to obtain a mesenchymal stem cell factor composition.

[0079] Embodiment 9: The difference from Embodiment 3 is that: S300 further includes:

[0080] D100, by weight, 40 parts of DOTAP, 80 parts of filtrate and 35 parts of ethanol were mixed to obtain a mixed solution, and then the mixed solution was subjected to vacuum rotary evaporation at 40°C for 40 minutes to remove the ethanol, and then 18 parts of HEPES buffer was added for hydration, and an ultrasonic probe was used to perform ultrasonic treatment at 260W for 5 minutes to obtain a factor-liposome solution;

[0081] D200, gradually inject 0.25wt% chitosan solution into the factor-liposome solution at a speed of 8ml / min, and stir at 200r / min for 3h after the injection to obtain a drug solution; the volume ratio of the factor-liposome solution to the chitosan solution is 1:3;

[0082] D300, mixing 20 parts of the drug solution with 7 parts of the cell suspension to obtain a mesenchymal stem cell factor composition.

[0083] Comparative Example 1: The difference from Example 3 is that there is no NGF in the second culture medium.

[0084] Comparative Example 2: The difference from Example 3 is that: there is no IL-10 in the second culture medium.

[0085] Comparative Example 3: The difference from Example 3 is that the culture temperature in S200 is 37° C., the oxygen concentration is ≥99.5%, and the pressure is 1 atmosphere.

[0086] Comparative Example 4: The difference from Example 3 is that the culture temperature in S200 is 37°C, the carbon dioxide concentration is 6%, the oxygen concentration is 4%, and the remaining gas is nitrogen.

[0087] Comparative Example 5: The difference from Example 3 is that bFGF is not added to the third culture medium.

[0088] Comparative Example 6: Only the filtrate in Example 3 was used.

[0089] Comparative Example 7: Only the cell suspension in Example 3 was used.

[0090] Blank: Normal saline

[0091] Experimental drugs: D-Hanks solution was obtained from Shanghai Lianmai Biotechnology Co., Ltd., D-Hanks balanced salt solution (5×, without phenol red).

[0092] DMEM high-glucose medium (without sodium pyruvate and glutamine) was obtained from Shanghai Xiaopeng Biotechnology Co., Ltd.

[0093] DMEM / F12 culture medium was from Shanghai Xiaopeng Biotechnology Co., Ltd., DMEM / F12 1:1 mixture, with L-Glutamine, without HEPES.

[0094] The complete culture medium used was human umbilical cord mesenchymal stem cell complete culture medium from Wuhan Sean Biotechnology Co., Ltd.

[0095] HEPES buffer was used from Yisheng Biotechnology (Shanghai) Co., Ltd., HEPES Free Acid (1M) Buffer Solution HEPES solution (1M) cell culture grade.

[0096] The size of the culture dish is 60 mm.

[0097] During the experiment, the culture medium in the culture dish was 5 ml, the culture humidity was saturated humidity, and the medium replacement and resuspension operations in S200 and S300 were the same as those in S100.

[0098] Test 1: Effect verification test

[0099] a. Animal model establishment

[0100] Healthy SD rats were selected, weighing 180-220g, and fed normally for one week to adapt to the environment. The feeding conditions were as follows: temperature 25±0.5℃, relative humidity 60±2%, lighting conditions of 12 hours (06:00-18:00) of fluorescent light irradiation and 12 hours of no lighting per day. During the experiment, the rats were placed in a cylindrical glass cylinder with a diameter of 18cm and a height of 28cm. An electric grid was placed at the bottom of the cylinder, and a 6cm high and 6cm diameter wooden stake was placed in the center of the electric grid. The current was turned on once every 30min, with a current of 0.5mA and a frequency of 1Hz. Each electric shock lasted for 30s, and the electric shock was continuous for 3 days. If the circadian rhythm disappears, the activity does not stop during the day, and the rats in the control group are normal, it means that the model is successful.

[0101] b. Administration

[0102] The model rats were divided into 16 groups, each with 10 rats, and the rats in each group were administrated with the compositions prepared by the preparation methods of Examples 1-9 and Comparative Examples 1-7 and the blank. Administration method: nasal spray administration at 6 am, the dosage was 1.2 ml / kg, and it was used for three consecutive days.

[0103] c. Sleep time monitoring

[0104] During the three days of drug administration, the total sleep time (min) of the rats was monitored using the non-invasive sleep activity monitoring system of World Union Research (Beijing) Technology Co., Ltd. During the monitoring period, the rats freely consumed filtered water and sterilized mouse chow. The housing temperature during the monitoring period was between 25±0.5°C, the relative humidity was 60±2%, and the lighting conditions were 12 hours (06:00-18:00) of fluorescent light exposure and 12 hours of no lighting per day. The experimental data took the average of the three-day data, as shown in Table 1 for details.

[0105] Table 1

[0106]

[0107] From the comparison between the test results of Examples 1-9 and the blank examples, it can be seen that the mesenchymal stem cell factor composition prepared by the preparation method of the present invention has a good therapeutic effect on acute insomnia, and the composition prepared by the preparation parameters provided in Example 9 has the best therapeutic effect.

[0108] From the comparison of the data of Example 3 and Examples 4-6, it can be seen that the additional addition of GDC-0941 in the second culture medium can slightly improve the therapeutic effect. At the same time, when monitoring the time, it was found that after adding GDC-0941, the time taken for passage in S200 was reduced by 34%-47%.

[0109] From the comparison of the data of Example 3 and Examples 7-9, it can be seen that after the filtrate is further processed, the therapeutic effect of the composition on acute insomnia can be greatly improved.

[0110] From the data comparison of Comparative Examples 1-2 and Example 3 and Comparative Example 7, it can be seen that when NGF or IL-10 is added alone in the second culture medium, the therapeutic effect of the composition is equivalent to the therapeutic effect of administering only the cell suspension. The applicant speculates that the reason is that the directional induction in S200 is almost ineffective, the content of related factors is greatly reduced, and the therapeutic effect of the final composition is mainly the effect of the cell suspension, and the effect of the filtrate is almost gone.

[0111] From the comparison of the data of Comparative Examples 3-4 and Example 3, it can be seen that specific culture conditions can improve the therapeutic effect of the composition.

[0112] From the comparison of the data of Comparative Examples 6-7 and Example 3, it can be seen that the filtrate plays a major role in the treatment of acute insomnia in the present composition. The cell suspension has a certain enhancement effect, but it is still mainly used to prevent acute insomnia from turning into chronic insomnia.

[0113] Experiment 2: Molecular level detection

[0114] Healthy SD rats weighing 180-220g were selected and raised in the electric shock environment of Experiment 1. Electric shock was given for 9 hours every day. The drug was administered daily according to the operation of Experiment 1. After 7 days, the drug administration was stopped and the rats were moved to a normal breeding environment. The breeding conditions were: temperature 25±0.5℃, relative humidity 60±2%, lighting conditions of 12 hours (06:00-18:00) of fluorescent light exposure and 12 hours of no lighting every day. After 48 hours, the rats were killed by dislocation of the neck, the brain tissue was removed by dissection, about 20mg of cerebral cortex tissue was cut, a certain amount of PBS (pH 7.4) was added, and it was placed on ice and homogenized using a homogenizer; centrifuged at 3000r / min and 4℃ for 20min, and the supernatant was collected; ELISA kits (purchased from R&D Systems, USA) were used to detect the concentrations of factors such as 5-hydroxytryptamine (5-HT) and γ-aminobutyric acid (GABA). The specific steps were carried out according to the instructions of the kit. The experimental results are shown in Figure 1-2 , control group 1 consisted of rats administered with normal saline, and control group 2 consisted of rats raised in a normal environment.

[0115] from Figure 1-2It can be seen that the mesenchymal stem cell factor composition prepared by the preparation method of Examples 1-9 of the present invention has a good effect on the protection and regeneration of nerve cells during acute insomnia, and can reduce the possibility of acute insomnia turning into chronic insomnia.

[0116] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a mesenchymal stem cell factor composition, characterized in that: include: S100, inoculating primary umbilical cord mesenchymal stem cells into the first culture medium, and when the cell confluence reaches 80%-90%, discarding the old culture medium, washing the cells with D-hanks solution, performing enzyme digestion, adding D-hanks solution and centrifuging, resuspending the cell precipitate with the second culture medium to obtain P1 generation mesenchymal stem cells; the culture temperature is 37°C, the carbon dioxide concentration is 5%-6%, the oxygen concentration is 5%-8%, and the remaining gas is nitrogen; The first culture medium is DMEM high glucose culture medium; the second culture medium includes DMEM / F12 culture medium matrix, 2-4 ng / ml NGF and 1-1.5 ng / ml IL-10; S200 and P1 generation mesenchymal stem cells are subcultured for 5-7 generations in the second culture medium environment to obtain generation mesenchymal stem cells; each generation of cells is centrifuged and the supernatant is removed, and the cell pellet is resuspended in the second culture medium; the culture temperature is 37°C, the oxygen concentration is ≥99.5%, and the pressure is 1.3-1.8 atmospheres; S300 and generation mesenchymal stem cells are subcultured for 2-4 generations in the third culture medium environment to obtain a cell suspension; the culture medium supernatant of the P6-P7 generation mesenchymal stem cells is filtered through a filter membrane and the filtrate is obtained; the cell suspension is mixed with the filtrate to obtain a mesenchymal stem cell factor composition; the third culture medium includes DMEM / F12 culture medium matrix, 2-4ng / ml NGF, 1-1.5ng / ml IL-10 and 4-5ng / ml bFGF; the culture temperature is 37°C, the carbon dioxide concentration is 5%-6%, the oxygen concentration is 3%-5%, and the remaining gas is nitrogen.

2. The preparation method according to claim 1, characterized in that: The seeding density of each passage in S200 is 5000-7000 cells / cm 2 In parts by weight, the mesenchymal stem cell factor composition includes 6-8 parts of the cell suspension in S300 and 11-15 parts of the filtrate corresponding to the P7 generation mesenchymal stem cells.

3. The preparation method according to claim 1, characterized in that: The second culture medium further comprises: 2-5 μM GDC-0941.

4. The preparation method according to claim 1, characterized in that: After the subculture in S300 is completed, the cells are washed with D-hanks solution, enzymatic digestion is performed, D-hanks solution is added again and centrifuged, and the cell pellet is resuspended with preservation solution to obtain a cell suspension; the preservation solution includes 3wt%-5wt% sodium hyaluronate, 0.7wt%-0.9wt% sodium chloride, 5wt%-10wt% glucose, 2vt%-5vt% compound amino acids, and the rest is distilled water.

5. The preparation method according to claim 1, characterized in that: The S300 further includes: D100, adding liposomes to the filtrate to obtain a factor-liposome solution after reaction; D200, adding chitosan solution to the factor-liposome solution to obtain a drug solution after reaction; D300, mixing the drug solution with the cell suspension to obtain a mesenchymal stem cell factor composition.

6. The preparation method according to claim 5, characterized in that: The D100 specifically comprises: by weight, 30-50 parts of DOTAP, 70-90 parts of filtrate and 30-40 parts of ethanol are mixed to obtain a mixed solution, and then the ethanol is removed from the mixed solution by vacuum rotary evaporation, and then 15-20 parts of HEPES buffer is added for hydration, and the factor-liposome solution is obtained after ultrasonic treatment.

7. The preparation method according to claim 6, characterized in that: The D200 specifically comprises: gradually injecting 0.2wt%-0.3wt% chitosan solution into the factor-liposome solution at an injection speed of 7-10ml / min, stirring for 2-4h after the injection, to obtain a drug solution; the volume ratio of the factor-liposome solution to the chitosan solution is 1:4-2:

5.

8. The preparation method according to any one of claims 1 to 7, characterized in that: The isolation step of primary umbilical cord mesenchymal stem cells comprises: A100, wash the umbilical cord with PBS, cut it into 2 cm long segments, peel off the venous intima and artery, and then cut it into pieces to obtain tissue blocks; A200, after placing the tissue block in the culture dish, add complete culture medium; A300. Place the culture dish in a carbon dioxide incubator at 37°C, 5% CO2, and 95% humidity. After 7 days, replace the medium by 2 / 3. When cells crawl out from around the tissue block, collect the crawling cells as primary umbilical cord mesenchymal stem cells; the outer edge of the crawling cells is ≥0.5 cm away from the edge of the tissue block.

9. A mesenchymal stem cell factor composition obtained by the preparation method according to any one of claims 1 to 8.

10. A spray preparation for improving sleep, characterized in that: The invention comprises the mesenchymal stem cell factor composition according to claim 9.

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