Cytokine composition and application thereof in resisting aging weakness

Abstract

The invention discloses a cytokine composition and application thereof in resisting aging and weakness, and relates to the technical field of biological medicines. The specific preparation method comprises the following steps: culturing bone marrow mesenchymal stem cells, filtering, and carrying out ultrafiltration concentration to obtain mesenchymal stem cell liquid; adding a tyrosine methyl ester derivative, methyl caffeate and surfactin into a PBS (Phosphate Buffer Solution) to obtain an auxiliary material solution; and mixing the mesenchymal stem cell solution and the auxiliary material solution to obtain the cell factor composition. The prepared cell factor composition can effectively reduce the aging rate of bone marrow mesenchymal stem cells, can improve the immunosuppression capability, and has certain potential in the aspect of aging weakness.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, 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 the increase of age, multiple factors lead to physical weakness, including genomic instability, DNA damage, epigenetic changes, stem cell exhaustion, etc. These interrelated factors lead to the loss of tissue homeostasis and the reduction of reserve capacity during frailty. Therefore, the aging body gradually shows symptoms of frailty and the physiological function of organs declines. Due to its complex pathophysiology, effective strategies are needed to intervene in the pathogenic process to improve the health of frail patients. Mesenchymal stem cells (MSCs) have regenerative and anti-inflammatory effects. Bone marrow mesenchymal stem cell transplantation is a promising therapeutic strategy to address the pathophysiological problems of frailty syndrome. At present, MSCs treatment has been tested in human subjects in phase I and II trials, which have confirmed the effectiveness of MSCs in treating frailty in aging. Stem cells mainly rely on the secretion of multiple growth factors and cytokines to promote mesenchymal cell regeneration and regulate immune processes and systemic inflammation, etc., to comprehensively treat frailty in aging. However, the use of cytokines in the existing technology to fight frailty in aging is limited.

[0003] Therefore, there is an urgent need for a cytokine composition that can resist aging and weakness to exert an effective effect. Summary of the invention

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

[0005] In order to solve the above technical problems, the present invention specifically provides the following technical solutions: The invention discloses a cytokine composition, comprising a mesenchymal stem cell solution and an auxiliary material solution; the auxiliary material solution is obtained by adding tyrosine methyl ester derivatives, caffeic acid methyl ester and surfactin into a PBS buffer; the tyrosine ester derivative is obtained by reacting tyrosine, methanol and lauroyl chloride. The use of tyrosine methyl ester derivatives and caffeic acid methyl ester can promote the action of the mesenchymal stem cell solution, further effectively slow down the oxidative damage of the mesenchymal stem cells and reduce the activity of β-galactosidase associated with aging, thereby reversing the exhaustion of the mesenchymal stem cells, and can effectively reduce the proliferation of CD3+ total T cells and improve immunosuppression.

[0006] Preferably, the volume ratio of the amount of mesenchymal stem cell solution used to the amount of auxiliary material solution used is 1:0.8-1.5.

[0007] The present invention discloses a method for preparing a cytokine composition, comprising: S1: First, bone marrow mesenchymal stem cells are inoculated into DMEM culture medium and subcultured to P4-P6 generations. Then, P4-P6 bone marrow mesenchymal stem cells are inoculated into serum-free DMEM / F12 culture medium for culture. After the culture is completed, the supernatant is collected and subjected to microporous filtration and ultrafiltration centrifugation to obtain mesenchymal stem cell fluid.

[0008] S2: adding mesenchymal stem cell solution to the auxiliary material solution and mixing to obtain a cytokine composition.

[0009] 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.

[0010] Preferably, the culture time of serum-free DMEM / F12 culture medium in S1 is 36-60 hours.

[0011] 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.

[0012] Preferably, the auxiliary material solution in S2 is obtained by adding tyrosine methyl ester derivative, caffeic acid methyl ester and surfactin into PBS buffer.

[0013] 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.

[0014] Preferably, the volume ratio of the amount of mesenchymal stem cell solution used to the amount of auxiliary material solution used is 1:0.8-1.5.

[0015] 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.

[0016] The present invention discloses a method for preparing a cytokine composition, comprising: S1: Preparation of mesenchymal stem cell fluid: bone marrow mesenchymal stem cells were inoculated in DMEM culture medium and cultured to P4-P6 generations. P4-P6 bone marrow mesenchymal stem cells were inoculated in serum-free DMEM / F12 culture medium for culture. After the culture was completed, the supernatant was collected and subjected to microfiltration and ultrafiltration centrifugation to obtain mesenchymal stem cell fluid.

[0017] S2: Preparation of cytokine composition: adding mesenchymal stem cell solution to the auxiliary material solution and mixing to obtain the cytokine composition.

[0018] 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.

[0019] 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.

[0020] Preferably, in the above S1, the culturing time in serum-free DMEM / F12 culture medium is 36-60 h.

[0021] 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.

[0022] 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.

[0023] More preferably, the amount of tyrosine methyl ester derivative added to PBS buffer is 0.08-0.3 mg / ml. More preferably, the amount of caffeic acid methyl ester added to the PBS buffer is 0.05-0.25 mg / ml.

[0024] More preferably, the amount of surfactin added to PBS buffer is 0.009-0.017 mg / ml.

[0025] 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.

[0026] The present invention discloses a method for preparing a tyrosine methyl ester derivative, which specifically comprises: Tyrosine is added to a hydrochloric acid-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 a tyrosine methyl ester derivative.

[0027] Preferably, the amount of tyrosine added to the hydrochloric acid-methanol mixture is 0.1-0.2 g / ml.

[0028] Preferably, the volume ratio of hydrochloric acid to methanol in the hydrochloric acid-methanol mixture is 1:8-12.

[0029] Preferably, the amount of lauroyl chloride added to benzene is 0.08-0.15 g / ml.

[0030] Preferably, the usage amount of tyrosine methyl ester is 0.5-0.8 g / ml.

[0031] 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.

[0032] 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.

[0033] Preferably, when preparing tyrosine methyl ester, the reaction temperature is 55-65° C. and the reaction time is 6-10 h.

[0034] Preferably, when preparing tyrosine methyl ester, the recrystallization solvent used is 75-85% ethanol, and the number of crystallizations is 2-4 times.

[0035] 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.

[0036] More preferably, in the preparation of the cytokine composition, mannitol hexaacetate can be added on the basis of using tyrosine methyl ester derivatives, caffeic acid methyl ester and surfactin. Mannitol hexaacetate can interact with tyrosine methyl ester derivatives and caffeic acid methyl ester to exert a synergistic effect, thereby further reducing cell senescence and improving immunosuppression.

[0037] More preferably, the amount of mannitol hexaacetate added to the PBS buffer is 0.015-0.08 mg / ml.

[0038] Compared with the prior art, the present invention has the following beneficial effects: The present invention proposes a cytokine composition and its application in anti-aging and frailty. Bone marrow mesenchymal stem cells are cultured, filtered and ultrafiltered to obtain a mesenchymal stem cell solution; tyrosine methyl ester derivatives, caffeic acid methyl ester and surfactin are added to a PBS buffer to obtain an auxiliary solution; then the mesenchymal stem cell solution and the auxiliary solution are mixed to obtain a cytokine composition. The aging rate of bone marrow mesenchymal stem cells can be effectively reduced, and the immunosuppressive ability can be improved, and it has certain potential in the aspect of aging and frailty. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the implementation of the present invention or the technical solution in the prior art, the following briefly introduces the drawings required for the implementation or the prior art description. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0040] Figure 1 is the infrared spectrum of tyrosine methyl ester derivative; Figure 2 This is a graph showing the aging rate of bone marrow mesenchymal stem cells under the intervention of a cytokine combination; Figure 3 Figure 2 is a graph of in vitro immunosuppression by cytokine combinations. DETAILED DESCRIPTION

[0041] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. 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 creative work are within the scope of protection of the present invention.

[0042] The following first describes the concepts involved in the present application in conjunction with the accompanying drawings. It should be noted that the following description of each concept is only to make the content of the present application easier to understand, and does not limit the scope of protection of the present application; at the same time, the embodiments and features in the embodiments of the present application can be combined with each other in the absence of conflict. The present application will be described in detail with reference to the accompanying drawings and in conjunction with the embodiments.

[0043] Embodiment 1: Preparation of mesenchymal stem cell fluid: First, bone marrow mesenchymal stem cells were isolated and extracted from iliac bone marrow, then the bone marrow mesenchymal stem cells were inoculated in DMEM culture medium, cultured at 37°C, 5% CO2, and subcultured after the cells grew to 80% confluence, cultured to P5 generation, and the bone marrow mesenchymal stem cells in the logarithmic growth phase of P5 generation were inoculated in serum-free DMEM / F12 culture medium, and the supernatant was collected after 48 hours of culture, and the supernatant was filtered through 0.22μm micropores and subjected to low-temperature ultrafiltration centrifugation at 4°C and 2500rpm to obtain concentrated mesenchymal stem cell fluid. The DMEM culture medium contains 10% fetal bovine serum, 100U / mL penicillin and 100U / mL streptomycin by volume, and the ultrafiltration membrane used for ultrafiltration is 10kD.

[0044] Preparation of tyrosine methyl ester derivatives: Add tyrosine to a mixture of hydrochloric acid and methanol, react at 60°C for 8 hours, and after the reaction is completed, separate the precipitate under reduced pressure. Add 80% ethanol to the precipitate and recrystallize it twice 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 dissolving solution, add lauroyl chloride-benzene solution to the dissolving solution, react at 70°C for 4 hours, cool to 25°C, continue to react for 1 hour, and after the reaction is completed, let it stand for 48 hours, wash with water and dry to obtain a crude product, and add methanol to the crude product and recrystallize it twice 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 to 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.

[0045] Preparation of cytokine composition: tyrosine methyl ester derivative, caffeic acid methyl ester and surfactin are added to PBS buffer to obtain an auxiliary material solution, and then mesenchymal stem cell solution is added to the auxiliary material solution to obtain a cytokine composition. The amount of tyrosine methyl ester derivative added to PBS buffer is 0.1 mg / ml, the amount of caffeic acid methyl ester added to PBS buffer is 0.08 mg / ml, the amount of surfactin added to PBS buffer is 0.015 mg / ml, and the volume ratio of the amount of mesenchymal stem cell solution used to the amount of auxiliary material solution used is 1:1.

[0046] Embodiment 2: The preparation of mesenchymal stem cell solution is the same as that in Example 1.

[0047] The preparation of tyrosine methyl ester derivatives is the same as in Example 1.

[0048] 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 the other conditions and parameters are the same as in Example 1.

[0049] Embodiment 3: The preparation of mesenchymal stem cell solution is the same as that in Example 1.

[0050] The preparation of tyrosine methyl ester derivatives is the same as in Example 1.

[0051] 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.

[0052] Embodiment 4: The preparation of mesenchymal stem cell solution is the same as that in Example 1.

[0053] The preparation of tyrosine methyl ester derivatives is the same as in Example 1.

[0054] Preparation of cytokine composition: tyrosine methyl ester derivative, caffeic acid methyl ester, surfactin and mannitol hexaacetate are added to PBS buffer to obtain an auxiliary material solution, and then mesenchymal stem cell liquid is added to the auxiliary material solution to mix and obtain a cytokine composition. The amount of tyrosine methyl ester derivative added to PBS buffer is 0.1 mg / ml, the amount of caffeic acid methyl ester added to PBS buffer is 0.08 mg / ml, the amount of surfactin added to PBS buffer is 0.015 mg / ml, the amount of mannitol hexaacetate added is 0.05 mg / ml, and the volume ratio of the amount of mesenchymal stem cell liquid used to the amount of auxiliary material solution used is 1:1.

[0055] Embodiment 5: The preparation of mesenchymal stem cell solution is the same as that in Example 1.

[0056] The preparation of tyrosine methyl ester derivatives is the same as in Example 1.

[0057] 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.

[0058] Embodiment 6: The preparation of mesenchymal stem cell solution is the same as that in Example 1.

[0059] The preparation of tyrosine methyl ester derivatives is the same as in Example 1.

[0060] 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.

[0061] Comparative Example 1: The preparation of mesenchymal stem cell solution is the same as that in Example 1.

[0062] The preparation of tyrosine methyl ester derivatives is the same as in Example 1.

[0063] 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, and other conditions and parameters are the same as in Example 1.

[0064] Comparative Example 2: The preparation of mesenchymal stem cell solution is the same as that in Example 1.

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

[0066] 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, and other conditions and parameters are the same as in Example 1.

[0067] Comparative Example 3: The preparation of mesenchymal stem cell solution is the same as that in Example 1.

[0068] The preparation of tyrosine methyl ester derivatives is the same as in Example 1.

[0069] 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, and other conditions and parameters are the same as in Example 1.

[0070] Comparative Example 4: The preparation of mesenchymal stem cell solution is the same as that in Example 1.

[0071] The preparation of tyrosine methyl ester derivatives is the same as in Example 1.

[0072] 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 the PBS buffer is 0.005 mg / ml, and the other conditions and parameters are the same as in Example 1.

[0073] Experimental Example 1: The tyrosine methyl ester derivative prepared in Example 1 was characterized by infrared spectroscopy analysis in the scanning range of 500-4000cm -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 -1 It is the characteristic absorption peak of C=O, 1500-1600cm -1 is the characteristic absorption peak of CN, 1300-1500cm -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.

[0074] Experimental Example 2: Observation of bone marrow mesenchymal stem cell aging by β-galactosidase method: Bone marrow mesenchymal stem cells were inoculated in DMEM / F12 culture medium. When the cells were 50% fused, they were replaced with serum-free DMEM / F12 culture medium. The control group did not add it. The remaining groups were added with the cytokine composition prepared by Examples 1-6 and Comparative Examples 1-3, respectively, and the rat bone marrow mesenchymal stem cells were treated and intervened. After 72 hours of intervention, they were stained with an aging-related 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 in the total number of observed cells, i.e., the cell senescence rate, was calculated. The volume ratio of the amount of the cytokine composition used to the amount of serum-free DMEM / F12 culture medium used was 1:4.5.

[0075] The results are as follows Figure 2 As shown, after the cytokine composition prepared in Example 1 of the present invention intervenes in bone marrow mesenchymal stem cells, the aging rate of bone marrow mesenchymal stem cells is 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 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 any one of tyrosine methyl ester derivatives and 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 surfactant used is too low, and has no obvious effect on reducing the aging rate of bone marrow mesenchymal stem cells.

[0076] Experimental Example 3: In vitro immunosuppression of cytokine combination: Human peripheral blood mononuclear cells were labeled with CFSE and activated with CD3 and CD28 monoclonal antibodies, and then co-cultured with bone marrow mesenchymal stem cells and cytokine combination for 72 hours. The control group was co-cultured only with bone marrow mesenchymal stem cells, and the proliferation of PBMCs was detected by flow cytometry, and the inhibition rate of CD3+ total T cells was calculated.

[0077] The results are as follows Figure 3As shown, the proliferation of CD3+ total T cells co-cultured with the cytokine composition prepared in Example 1 of the present invention is significantly reduced, and the inhibition rate is 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 on the basis of tyrosine methyl ester derivatives and caffeic acid methyl ester, mannitol hexaacetate can be used , and further improve the inhibition rate of CD3+ total T cells, that is, improve immunosuppression; Example 4 is compared with Examples 5 and 6, indicating that the usage of mannitol hexaacetate is proportional to the inhibition rate of CD3+ total T cells within a certain range; Example 1 is compared with Comparative Examples 2 and 3, and the inhibition rate of CD3+ total T cells is higher than that of 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 CD3+ total T cells; Example 1 is compared with Comparative Example 4, indicating 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 CD3+ total T cells.

[0078] 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 are not intended to limit the implementation methods of the technology of the present invention in any form. Any technical personnel in this field 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. 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 its 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 and the objective existence of infinite specific structures, ordinary technicians in this technical field can make several improvements, modifications or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the concept and technical solution of the invention 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 obtained by adding a tyrosine methyl ester derivative, caffeic acid methyl ester and surfactin into a PBS buffer; the tyrosine ester derivative is obtained by reacting tyrosine, methanol and lauroyl chloride.

2. A cytokine composition according to claim 1, Features: The volume ratio of the mesenchymal stem cell solution to the auxiliary material solution is 1:0.8-1.

5.

3. A method for preparing a cytokine composition, include: S1: First, bone marrow mesenchymal stem cells are inoculated into DMEM culture medium and subcultured to P4-P6 generations. Then, P4-P6 bone marrow mesenchymal stem cells are inoculated into serum-free DMEM / F12 culture medium for culture. After the culture is completed, the supernatant is collected and subjected to microfiltration and ultrafiltration centrifugation to obtain mesenchymal stem cell fluid; S2: adding mesenchymal stem cell solution to the auxiliary material solution and mixing to obtain a cytokine composition.

4. A method for preparing a cytokine composition according to claim 3, Features: The subculture temperature in S1 is 35-37°C and the culture conditions are 4%-6% CO 2 The subculture time is when the cells grow to 70-85% confluence.

5. A method for preparing a cytokine composition according to claim 3, Features: The culture time of the serum-free DMEM / F12 culture medium in the S1 is 36-60 hours.

6. A method for preparing a cytokine composition according to claim 3, Features: The temperature of the ultrafiltration centrifugation in S1 is 0-12°C, the speed is 1500-3500rpm, and the ultrafiltration membrane used for ultrafiltration is 5-10kD.

7. A method for preparing a cytokine composition according to claim 3, Features: The auxiliary material solution in S2 is obtained by adding tyrosine methyl ester derivative, caffeic acid methyl ester and surfactin into PBS buffer.

8. A method for preparing a cytokine composition according to claim 3 or 7, Features: The amount of the tyrosine methyl ester derivative added to the PBS buffer is 0.08-0.3 mg / ml, the amount of caffeic acid methyl ester added to the PBS buffer is 0.05-0.25 mg / ml, and the amount of surfactin added to the PBS buffer is 0.009-0.017 mg / ml.

9. The method for preparing a cytokine composition according to claim 3, Features: The volume ratio of the amount of the mesenchymal stem cell solution used in the S2 to the amount of the auxiliary material solution used is 1:0.8-1.

5.

10. Use of a cytokine composition in the preparation of an anti-aging and frailty preparation, comprising the cytokine composition according to any one of claims 1 to 2 or the cytokine composition prepared by the method according to any one of claims 3 to 9.

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