A cytokine composition and its use in combating age-related frailty

Abstract

The application discloses a cytokine composition and application thereof in anti-aging weakness, and relates to the technical field of biological medicine. A specific preparation method comprises the following steps: using bone marrow mesenchymal stem cells, culturing, filtering and ultrafiltration concentration to obtain mesenchymal stem cell liquid; adding tyrosine methyl ester derivative, caffeic acid methyl ester and surfactin into a PBS buffer solution to obtain an auxiliary solution; and then mixing the mesenchymal stem cell liquid and the auxiliary solution to obtain the cytokine composition. The prepared cytokine composition can effectively reduce the aging rate of the bone marrow mesenchymal stem cells, can improve the immunosuppressive capacity, and has certain potential in the aspect of aging weakness.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a cytokine composition and its application in resisting aging and frailty. Background Art

[0002] Frailty is a complex geriatric syndrome. With advancing age, multiple factors contribute to frailty, including genomic instability, DNA damage, epigenetic alterations, and stem cell exhaustion. These interrelated factors collectively lead to a loss of tissue homeostasis and a reduction in reserve capacity during frailty. Consequently, the aging body gradually exhibits symptoms of frailty and organ function declines. Due to its complex pathophysiology, effective strategies are needed to intervene in the pathogenic processes and improve the health of patients with frailty. Mesenchymal stem cells (MSCs) possess regenerative and anti-inflammatory properties. Bone marrow MSC transplantation is a promising therapeutic strategy to address the pathophysiology of frailty syndrome. Currently, MSCs therapy has been tested in human subjects in Phase I and II trials, which have demonstrated the efficacy of MSCs in treating frailty. Stem cells primarily secrete multiple growth factors and cytokines, promoting mesenchymal cell regeneration and regulating immune processes and systemic inflammation, thereby providing a comprehensive treatment for frailty. However, existing technologies using cytokines to combat frailty have limited efficacy.

[0003] Therefore, there is an urgent need for a cytokine composition that can effectively resist aging and frailty. Summary of the Invention

[0004] The purpose of the present invention is to provide a cytokine composition so as to better combat aging and frailty.

[0005] In order to solve the above technical problems, the present invention specifically provides the following technical solutions:

[0006] The present invention discloses a cytokine composition comprising a mesenchymal stem cell fluid and an auxiliary material solution; the auxiliary material solution is obtained by adding a tyrosine methyl ester derivative, caffeic acid methyl ester, and surfactin to a PBS buffer; the tyrosine ester derivative is obtained by reacting tyrosine, methanol, and lauroyl chloride. The use of the tyrosine methyl ester derivative and caffeic acid methyl ester can promote the action of the mesenchymal stem cell fluid, further effectively slowing down oxidative damage to the mesenchymal stem cells and reducing aging-related β-galactosidase activity, thereby reversing mesenchymal stem cell exhaustion, and can effectively reduce the proliferation of CD3+ total T cells and enhance immunosuppression.

[0007] Preferably, the volume ratio of the mesenchymal stem cell solution to the auxiliary material solution is 1:0.8-1.5.

[0008] The present invention discloses a method for preparing a cytokine composition, comprising:

[0009] S1: First, bone marrow mesenchymal stem cells were inoculated into DMEM culture medium and subcultured to P4-P6. Then, P4-P6 bone marrow mesenchymal stem cells were inoculated into serum-free DMEM / F12 culture medium and cultured. After the culture was completed, the supernatant was collected and subjected to microporous filtration and ultrafiltration centrifugation to obtain mesenchymal stem cell fluid.

[0010] S2: adding the mesenchymal stem cell solution to the auxiliary material solution and mixing to obtain a cytokine composition.

[0011] Preferably, the subculture temperature in S1 is 35-37° C., the culture conditions are 4%-6% CO 2 , and the subculture time is after the cells grow to 70-85% confluence.

[0012] Preferably, the culture time of serum-free DMEM / F12 culture medium in S1 is 36-60 hours.

[0013] Preferably, the temperature of the ultrafiltration centrifugation in S1 is 0-12°C, the speed is 1500-3500 rpm, and the ultrafiltration membrane used for ultrafiltration is 5-10 kD.

[0014] Preferably, the excipient solution in S2 is obtained by adding tyrosine methyl ester derivative, caffeic acid methyl ester and surfactin into PBS buffer.

[0015] More preferably, the amount of tyrosine methyl ester derivative added to PBS buffer is 0.08-0.3 mg / ml, the amount of caffeic acid methyl ester added to PBS buffer is 0.05-0.25 mg / ml, and the amount of surfactin added to PBS buffer is 0.009-0.017 mg / ml.

[0016] Preferably, the volume ratio of the mesenchymal stem cell solution to the auxiliary material solution is 1:0.8-1.5.

[0017] The present invention also discloses an application of a cytokine composition in resisting aging and frailty, comprising the above cytokine composition or the cytokine composition prepared by the above method.

[0018] The present invention discloses a method for preparing a cytokine composition, comprising:

[0019] S1: Preparation of mesenchymal stem cell fluid: Bone marrow mesenchymal stem cells were inoculated in DMEM culture medium and cultured to P4-P6. P4-P6 bone marrow mesenchymal stem cells were inoculated in serum-free DMEM / F12 culture medium. After the culture was completed, the supernatant was collected and subjected to microfiltration and ultrafiltration centrifugation to obtain the mesenchymal stem cell fluid.

[0020] S2: Preparation of cytokine composition: adding mesenchymal stem cell solution to the auxiliary material solution and mixing to obtain a cytokine composition.

[0021] Preferably, in the above S1, the DMEM culture medium contains 8-12% fetal bovine serum, 90-110 U / mL penicillin and 90-110 U / mL streptomycin in a volume fraction.

[0022] Preferably, in the above S1, the subculture temperature is 35-37°C, the culture conditions are 4-6% CO2, and the subculture time is after the cells grow to 70-85% fusion.

[0023] Preferably, in the above S1, the culture time in serum-free DMEM / F12 culture medium is 36-60 hours.

[0024] Preferably, in the above S1, the temperature of ultrafiltration centrifugation is 0-12°C, the speed is 1500-3500 rpm, and the ultrafiltration membrane used for ultrafiltration is 5-10 kD.

[0025] Preferably, in the above S2, the auxiliary material solution is obtained by adding tyrosine methyl ester derivative, caffeic acid methyl ester and surfactin into PBS buffer.

[0026] More preferably, the amount of the tyrosine methyl ester derivative added to the PBS buffer is 0.08-0.3 mg / ml.

[0027] More preferably, the amount of caffeic acid methyl ester added to the PBS buffer is 0.05-0.25 mg / ml.

[0028] More preferably, the amount of surfactin added to the PBS buffer is 0.009-0.017 mg / ml.

[0029] Preferably, in the above S2, the volume ratio of the amount of the mesenchymal stem cell solution used to the amount of the auxiliary material solution used is 1:0.8-1.5.

[0030] The present invention discloses a method for preparing a tyrosine methyl ester derivative, which is specifically as follows:

[0031] Tyrosine is added to a hydrochloric acid-methanol mixture for reaction, followed by vacuum separation and recrystallization to obtain tyrosine methyl ester. Lauroyl chloride is added to benzene to obtain a lauroyl chloride-benzene solution. Tyrosine methyl ester is then added to pyridine and benzene to obtain a dissolving solution, to which the lauroyl chloride-benzene solution is added for reaction. After the reaction is completed, the solution is allowed to stand, washed, and dried to obtain a crude product, which is then recrystallized to obtain a tyrosine methyl ester derivative.

[0032] Preferably, the amount of tyrosine added to the hydrochloric acid-methanol mixture is 0.1-0.2 g / ml.

[0033] Preferably, the volume ratio of hydrochloric acid to methanol in the hydrochloric acid-methanol mixture is 1:8-12.

[0034] Preferably, the amount of lauroyl chloride added to benzene is 0.08-0.15 g / ml.

[0035] Preferably, the usage amount of tyrosine methyl ester is 0.5-0.8 g / ml.

[0036] Preferably, the volume ratio of the amount of benzene used to the amount of pyridine used in the dissolving solution is 1:0.2-0.4.

[0037] Preferably, the mass ratio of the amount of tyrosine methyl ester used to the amount of lauroyl chloride used is 1:0.5-0.6.

[0038] Preferably, when preparing tyrosine methyl ester, the reaction temperature is 55-65° C. and the reaction time is 6-10 h.

[0039] Preferably, when preparing tyrosine methyl ester, the recrystallization solvent used is 75-85% ethanol, and the number of crystallizations is 2-4 times.

[0040] Preferably, when preparing the tyrosine methyl ester derivative, the reaction temperature is 65-75° C., the reaction time is 3-5 h, and after the reaction is completed, the temperature is cooled to 23-27° C. and the reaction is continued for 0.8-1.2 h.

[0041] More preferably, in the preparation of the cytokine composition, mannitol hexaacetate can be added in addition to the tyrosine methyl ester derivative, caffeic acid methyl ester, and surfactin. Mannitol hexaacetate can interact with the tyrosine methyl ester derivative and caffeic acid methyl ester to exert a synergistic effect, thereby further reducing cell senescence and enhancing immunosuppression.

[0042] More preferably, the amount of mannitol hexaacetate added to the PBS buffer is 0.015-0.08 mg / ml.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] This invention proposes a cytokine composition and its use in combating aging-related frailty. Bone marrow mesenchymal stem cells are cultured, filtered, and concentrated by ultrafiltration to obtain a mesenchymal stem cell fluid. A tyrosine methyl ester derivative, caffeic acid methyl ester, and surfactin are added to a PBS buffer to obtain an auxiliary solution. The mesenchymal stem cell fluid and auxiliary solution are then mixed to obtain the cytokine composition. This composition can effectively reduce the aging rate of bone marrow mesenchymal stem cells and enhance their immunosuppressive capacity, demonstrating its potential for combating aging-related frailty. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the embodiments or the description of the prior art. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other implementation drawings based on the provided drawings without inventive effort.

[0046] Figure 1 is the infrared spectrum of tyrosine methyl ester derivative;

[0047] Figure 2 This is a graph showing the aging rate of bone marrow mesenchymal stem cells under the intervention of a cytokine composition;

[0048] Figure 3 Figure 2 is a graph showing in vitro immunosuppression by cytokine combinations. DETAILED DESCRIPTION

[0049] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0050] The following first describes the concepts involved in this application with reference to the accompanying drawings. It should be noted that the following description of each concept is intended only to make the content of this application easier to understand and does not limit the scope of protection of this application. At the same time, the embodiments and features in the embodiments of this application can be combined with each other unless there is a conflict. The following detailed description of this application will be made with reference to the accompanying drawings and in conjunction with the embodiments.

[0051] Example 1:

[0052] Preparation of mesenchymal stem cell fluid: First, bone marrow mesenchymal stem cells (MSCs) were isolated and extracted from iliac bone marrow. These cells were then seeded in Dulbecco's Modified Eagle's Medium (DMEM) and cultured at 37°C in 5% CO2. Cells were passaged after reaching 80% confluency and cultured to passage 5. P5 logarithmically growing MSCs were inoculated in serum-free DMEM / F12 medium. After 48 hours of culture, the supernatant was collected and filtered through a 0.22 μm microfiltration filter. The supernatant was then subjected to low-temperature ultrafiltration centrifugation at 2500 rpm at 4°C to obtain a concentrated MSC fluid. The DMEM medium contained 10% fetal bovine serum, 100 U / mL penicillin, and 100 U / mL streptomycin. The ultrafiltration membrane used for ultrafiltration was 10 kD.

[0053] Preparation of tyrosine methyl ester derivatives: Add tyrosine to a mixture of hydrochloric acid and methanol and react at 60°C for 8 hours. After the reaction, remove the precipitate under reduced pressure and recrystallize twice from 80% ethanol to obtain tyrosine methyl ester. Add lauroyl chloride to benzene to obtain a lauroyl chloride-benzene solution. Then, add tyrosine methyl ester to pyridine and benzene to obtain a solution. Add the lauroyl chloride-benzene solution to the solution and react at 70°C for 4 hours. Cool to 25°C and continue the reaction for 1 hour. After the reaction, allow to stand for 48 hours, wash with water, and dry to obtain a crude product. The crude product is recrystallized twice from methanol to obtain a tyrosine methyl ester derivative. The amount of tyrosine added to the hydrochloric acid-methanol mixture is 0.167 g / ml, the volume ratio of hydrochloric acid and methanol in the hydrochloric acid-methanol mixture is 1:10, the amount of lauroyl chloride added to benzene is 0.11 g / ml, the amount of tyrosine methyl ester used is 0.67 g / ml, the volume ratio of benzene used to pyridine used in the dissolving solution is 1:0.3, and the mass ratio of tyrosine methyl ester used to lauroyl chloride used is 1:0.55.

[0054] Preparation of the cytokine composition: Add a tyrosine methyl ester derivative, caffeic acid methyl ester, and surfactin to a PBS buffer solution to obtain an excipient solution. Then, add a mesenchymal stem cell solution to the excipient solution and mix to obtain the cytokine composition. The tyrosine methyl ester derivative is added to the PBS buffer at a concentration of 0.1 mg / ml, the caffeic acid methyl ester is added to the PBS buffer at a concentration of 0.08 mg / ml, and the surfactin is added to the PBS buffer at a concentration of 0.015 mg / ml. The volume ratio of the mesenchymal stem cell solution to the excipient solution is 1:1.

[0055] Example 2:

[0056] The preparation of mesenchymal stem cell solution was the same as in Example 1.

[0057] The preparation of tyrosine methyl ester derivatives is the same as in Example 1.

[0058] Preparation of cytokine composition: The preparation of the cytokine composition in this example is compared with that in Example 1, except that the amount of tyrosine methyl ester derivative added to PBS buffer is 0.15 mg / ml, and other conditions and parameters are the same as in Example 1.

[0059] Example 3:

[0060] The preparation of mesenchymal stem cell solution was the same as in Example 1.

[0061] The preparation of tyrosine methyl ester derivatives is the same as in Example 1.

[0062] Preparation of cytokine composition: The preparation of the cytokine composition in this example is compared with that in Example 1, except that the amount of caffeic acid methyl ester added to the PBS buffer is 0.14 mg / ml, and the other conditions and parameters are the same as in Example 1.

[0063] Example 4:

[0064] The preparation of mesenchymal stem cell solution was the same as in Example 1.

[0065] The preparation of tyrosine methyl ester derivatives is the same as in Example 1.

[0066] Preparation of the cytokine composition: Tyrosine methyl ester derivative, caffeic acid methyl ester, surfactin, and mannitol hexaacetate are added to PBS buffer to obtain an excipient solution. Mesenchymal stem cell fluid is then added to the excipient solution and mixed to obtain the cytokine composition. The tyrosine methyl ester derivative is added to the PBS buffer at a concentration of 0.1 mg / ml, caffeic acid methyl ester is added to the PBS buffer at a concentration of 0.08 mg / ml, surfactin is added to the PBS buffer at a concentration of 0.015 mg / ml, and mannitol hexaacetate is added to the PBS buffer at a concentration of 0.05 mg / ml. The volume ratio of the mesenchymal stem cell fluid to the excipient solution is 1:1.

[0067] Example 5:

[0068] The preparation of mesenchymal stem cell solution was the same as in Example 1.

[0069] The preparation of tyrosine methyl ester derivatives is the same as in Example 1.

[0070] Preparation of cytokine composition: The preparation of the cytokine composition in this example is compared with that in Example 4, except that the amount of mannitol hexaacetate added is 0.03 mg / ml, and the other conditions and parameters are the same as in Example 1.

[0071] Example 6:

[0072] The preparation of mesenchymal stem cell solution was the same as in Example 1.

[0073] The preparation of tyrosine methyl ester derivatives is the same as in Example 1.

[0074] Preparation of cytokine composition: The preparation of the cytokine composition in this example is compared with that in Example 4, except that the amount of mannitol hexaacetate added is 0.07 mg / ml, and the other conditions and parameters are the same as in Example 1.

[0075] Comparative Example 1:

[0076] The preparation of mesenchymal stem cell solution was the same as in Example 1.

[0077] The preparation of tyrosine methyl ester derivatives is the same as in Example 1.

[0078] Preparation of cytokine composition: The preparation of the cytokine composition in this example is different from that in Example 1, except that no tyrosine methyl ester derivative and caffeic acid methyl ester are used. Other conditions and parameters are the same as in Example 1.

[0079] Comparative Example 2:

[0080] The preparation of mesenchymal stem cell solution was the same as in Example 1.

[0081] The preparation of tyrosine methyl ester derivatives is the same as in Example 1.

[0082] Preparation of cytokine composition: The preparation of the cytokine composition in this example is different from that in Example 1, except that no tyrosine methyl ester derivative is used. Other conditions and parameters are the same as those in Example 1.

[0083] Comparative Example 3:

[0084] The preparation of mesenchymal stem cell solution was the same as in Example 1.

[0085] The preparation of tyrosine methyl ester derivatives is the same as in Example 1.

[0086] Preparation of cytokine composition: The preparation of the cytokine composition in this example is different from that in Example 1, except that no caffeic acid methyl ester is used. Other conditions and parameters are the same as in Example 1.

[0087] Comparative Example 4:

[0088] The preparation of mesenchymal stem cell solution was the same as in Example 1.

[0089] The preparation of tyrosine methyl ester derivatives is the same as in Example 1.

[0090] Preparation of cytokine composition: The preparation of the cytokine composition in this example is compared with that in Example 1, except that the amount of surfactin added to PBS buffer is 0.005 mg / ml, and other conditions and parameters are the same as in Example 1.

[0091] Experimental Example 1:

[0092] The tyrosine methyl ester derivatives prepared in Example 1 were characterized by infrared spectroscopy analysis in the scanning range of 500-4000 cm -1 The results are as follows Figure 1 As shown, at 1600cm -1 and 1500cm -1 Nearby is the characteristic absorption peak of benzene ring C=C, at 700-800cm -1 It is the characteristic absorption peak of benzene ring CH, 1600-1800cm -1It is the characteristic absorption peak of C=O, 1500-1600cm -1 is the characteristic absorption peak of CN, 1300-1500 cm -1 and 2800-3000cm -1 They are all characteristic absorption peaks of -CH3 and -CH2, which are consistent with the structural characteristics of tyrosine methyl ester derivatives.

[0093] Experimental Example 2:

[0094] Observation of aging of bone marrow mesenchymal stem cells using the β-galactosidase method: Bone marrow mesenchymal stem cells were seeded in DMEM / F12 culture medium. When the cells reached 50% confluence, the medium was replaced with serum-free DMEM / F12 culture medium. No serum was added to the control group, while the remaining groups were treated with the cytokine compositions prepared in Examples 1-6 and Comparative Examples 1-3. After 72 hours of treatment, the cells were stained using a senescence-associated β-galactosidase staining kit and observed under an inverted optical microscope. Blue cells were senescent cells. Five fields of view were randomly read in each group, and the percentage of senescent cells to the total number of observed cells, i.e., the cell senescence rate, was calculated. The volume ratio of the cytokine composition to the serum-free DMEM / F12 culture medium was 1:4.5.

[0095] The results are as follows Figure 2 As shown, after the cytokine composition prepared in Example 1 of the present invention intervened in bone marrow mesenchymal stem cells, the aging rate of the bone marrow mesenchymal stem cells was lower than that of Comparative Example 1, indicating that the use of tyrosine methyl ester derivatives and caffeic acid methyl ester in the cytokine composition can further reverse the aging of bone marrow mesenchymal stem cells and reduce the aging rate; Example 1 is compared with Examples 2 and 3, indicating that an appropriate increase in the amount of tyrosine methyl ester derivatives and caffeic acid methyl ester in the cytokine composition can reduce the aging rate of bone marrow mesenchymal stem cells; Example 4 is compared with Example 1, and the aging rate of the bone marrow mesenchymal stem cells in Example 4 is lower than that in Example 1, indicating that mannitol hexaacetate can be used on the basis of using tyrosine methyl ester derivatives and caffeic acid methyl ester. Synergistically reduce the aging rate of bone marrow mesenchymal stem cells; Example 4 is compared with Examples 5 and 6, indicating that the amount of mannitol hexaacetate used is inversely proportional to the reduction of the aging rate of bone marrow mesenchymal stem cells within a certain range; Example 1 is compared with Comparative Examples 2 and 3, and the aging rate of bone marrow mesenchymal stem cells in Example 1 is lower than that in Comparative Examples 2 and 3, indicating that tyrosine methyl ester derivatives and caffeic acid methyl ester need to be used together, and the use of either tyrosine methyl ester derivatives or caffeic acid methyl ester alone has no obvious effect on reducing the aging rate of bone marrow mesenchymal stem cells; Example 1 is compared with Comparative Example 4, when tyrosine methyl ester derivatives and caffeic acid methyl ester are used, the amount of surfactin used is too low, and has no obvious effect on reducing the aging rate of bone marrow mesenchymal stem cells.

[0096] Experimental Example 3:

[0097] In vitro immunosuppression by cytokine combination: Human peripheral blood mononuclear cells were labeled with CFSE and activated with CD3 and CD28 monoclonal antibodies. They were then co-cultured with bone marrow mesenchymal stem cells and the cytokine combination for 72 hours. The control group was co-cultured with bone marrow mesenchymal stem cells only. The proliferation of PBMCs was detected by flow cytometry, and the inhibition rate of total CD3+ T cells was calculated.

[0098] The results are as follows Figure 3 As shown, the proliferation of CD3+ total T cells co-cultured with the cytokine composition prepared in Example 1 of the present invention was significantly reduced, and the inhibition rate was higher than that of Comparative Example 1, indicating that the use of tyrosine methyl ester derivatives and caffeic acid methyl ester in the cytokine composition can further inhibit the proliferation of CD3+ total T cells and improve the immunosuppressive effect; Example 1 is compared with Examples 2 and 3, indicating that an appropriate increase in the amount of tyrosine methyl ester derivatives and caffeic acid methyl ester in the cytokine composition can improve the inhibition rate of CD3+ total T cells; Example 4 is compared with Example 1, and the cytokine composition prepared in Example 4 is co-cultured, and the inhibition rate of CD3+ total T cells is higher than that of Example 1, indicating that mannitol hexaacetate can be used on the basis of tyrosine methyl ester derivatives and caffeic acid methyl ester. , and further improve the inhibition rate of total CD3+ T cells, that is, improve immunosuppression; Example 4, compared with Examples 5 and 6, shows that the usage of mannitol hexaacetate is proportional to the inhibition rate of total CD3+ T cells within a certain range; Example 1, compared with Comparative Examples 2 and 3, has a higher inhibition rate on total CD3+ T cells than Comparative Examples 2 and 3, indicating that tyrosine methyl ester derivatives and caffeic acid methyl ester need to be used together, and the use of any one of tyrosine methyl ester derivatives and caffeic acid methyl ester alone has no obvious effect on improving the inhibition rate of total CD3+ T cells; Example 1, compared with Comparative Example 4, shows that when tyrosine methyl ester derivatives and caffeic acid methyl ester are used in the cytokine composition, the usage of surfactin is too low and has no obvious effect on improving the inhibition rate of total CD3+ T cells.

[0099] The embodiments and / or implementation methods described above are only used to illustrate the preferred embodiments and / or implementation methods for realizing the technology of the present invention, and do not impose any form of limitation on the implementation methods of the technology of the present invention. Any person skilled in the art may make slight changes or modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as technologies or embodiments that are essentially the same as the present invention.

[0100] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and core ideas of this application. The above is only the preferred implementation method of this application. It should be pointed out that due to the limitations of textual expression, there are objectively infinite specific structures. For ordinary technicians in this technical field, without departing from the principles of this application, they can also make several improvements, modifications or changes, and can also combine the above technical features in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the inventive concept and technical solution to other occasions without improvement, should be regarded as the scope of protection of this application.

Claims

1. A cytokine composition comprising a mesenchymal stem cell solution and an auxiliary material solution; the auxiliary material solution is prepared by adding a tyrosine methyl ester derivative, caffeic acid methyl ester, and surfactin to a PBS buffer solution; the tyrosine methyl ester derivative is prepared by reacting tyrosine, methanol, and lauroyl chloride; the volume ratio of the mesenchymal stem cell solution to the auxiliary material solution is 1:0.8-1.5; In the preparation of the tyrosine methyl ester derivative, tyrosine is added to a hydrochloric acid and methanol mixture for reaction, and tyrosine methyl ester is obtained through vacuum separation and recrystallization; lauroyl chloride is added to benzene to obtain a lauroyl chloride-benzene solution; tyrosine methyl ester is then added to pyridine and benzene to obtain a dissolving solution, and the lauroyl chloride-benzene solution is added to the dissolving solution for reaction. After the reaction is completed, the solution is allowed to stand, washed, and dried to obtain a crude product, and the crude product is recrystallized to obtain the tyrosine methyl ester derivative; wherein the amount of tyrosine added to the hydrochloric acid and methanol mixture is 0.1-0.2 g / ml, the volume ratio of hydrochloric acid to methanol in the hydrochloric acid and methanol mixture is 1:8-12, and the amount of lauroyl chloride added to benzene is 0.08-0.

15. g / ml, the amount of tyrosine methyl ester used is 0.5-0.8g / ml, the volume ratio of the amount of benzene used to the amount of pyridine used in the dissolving solution is 1:0.2-0.4, and the mass ratio of the amount of tyrosine methyl ester used to the amount of lauroyl chloride used is 1:0.5-0.6; when preparing tyrosine methyl ester, the reaction temperature is 55-65°C, the reaction time is 6-10h, the recrystallization solvent used is 75-85% ethanol, and the number of crystallizations is 2-4; when preparing tyrosine methyl ester derivatives, the reaction temperature is 65-75°C, the reaction time is 3-5h, and after the reaction is completed, the reaction is cooled to 23-27°C and the reaction is continued for 0.8-1.2h. In the preparation of mesenchymal stem cell fluid, bone marrow mesenchymal stem cells are first inoculated into DMEM culture medium and subcultured to P4-P6. Then, P4-P6 bone marrow mesenchymal stem cells are inoculated into serum-free DMEM / F12 culture medium and cultured. After the culture is completed, the supernatant is collected and subjected to microfiltration and ultrafiltration centrifugation to obtain the mesenchymal stem cell fluid. The amount of the tyrosine methyl ester derivative added to the PBS buffer is 0.1-0.15 mg / ml, the amount of caffeic acid methyl ester added to the PBS buffer is 0.08-0.14 mg / ml, and the amount of surfactin added to the PBS buffer is 0.015 mg / ml.

2. A method for preparing the cytokine composition according to claim 1, comprising: S1: First, bone marrow mesenchymal stem cells were inoculated into DMEM culture medium and subcultured to P4-P6. Then, P4-P6 bone marrow mesenchymal stem cells were inoculated into serum-free DMEM / F12 culture medium and cultured. After the culture, the supernatant was collected and subjected to microfiltration and ultrafiltration centrifugation to obtain mesenchymal stem cell fluid. S2: adding the mesenchymal stem cell solution to the auxiliary material solution and mixing to obtain a cytokine composition.

3. The preparation method according to claim 2, wherein: The subculture temperature in S1 is 35-37° C., the culture conditions are 4%-6% CO 2 , and the subculture time is after the cells grow to 70-85% fusion.

4. The preparation method according to claim 2, wherein: The culture time of the serum-free DMEM / F12 culture medium in the S1 is 36-60 hours.

5. The preparation method according to claim 2, wherein: The temperature of the ultrafiltration centrifugation in S1 is 0-12°C, the speed is 1500-3500 rpm, and the ultrafiltration membrane used for ultrafiltration is 5-10 kD.

6. Use of the cytokine composition according to claim 1 in the preparation of an agent for resisting the aging of bone marrow mesenchymal stem cells.

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