Adipose tissue-derived stem cell factor composition as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to an adipose tissue-derived stem cell factor composition as well as a preparation method and application thereof. The adipose tissue-derived stem cell factor composition is prepared from the following components in parts by weight: 120 to 150 parts of adipose tissue-derived stem cell culture supernate, 1 to 7 parts of BPC157, 5 to 10 parts of cyanidin-3-O-glucoside and 0.5 to 1 part of isovaleraldehyde. Water content and cytokine content detection experiments prove that the composition disclosed by the invention can effectively maintain the stability and activity of cytokines and is beneficial to long-term storage of cytokine products. Furthermore, anti-aging detection experiment data also fully shows that the composition disclosed by the invention can be used for preparing the medicine for resisting skin aging.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to an adipose-derived mesenchymal stem cell factor composition, a preparation method thereof and application thereof. BACKGROUND

[0002] Mesenchymal stem cells (MSCs) are a kind of progenitor cells derived from mesoderm, which have high in vitro proliferation ability and high self-renewal ability, and are considered to be one of the most promising means for treating various diseases of the human body. With the in-depth study of MSCs, scholars have found that MSCs not only exist in bone marrow, but also exist in dental pulp, periosteum, adipose tissue, umbilical cord blood and other tissues. Adipose-derived mesenchymal stem cells (ADMSCs) are a kind of stem cells derived from adipose tissue, which are favored by scholars in the field of biotechnology due to the advantages of abundant source, easy in vitro expansion and culture, and no ethical controversy.

[0003] ADMSCs can secrete a variety of autocrine and paracrine factors, including cytokines, growth factors and extracellular matrix proteases. Cytokines are a class of substances that can specifically bind to cell receptors, have multiple functions such as regulating innate immunity and adaptive immunity, hematopoiesis, cell growth and repair of damaged tissues. Cytokines play a role in the body through paracrine, autocrine or endocrine, and have multiple physiological properties such as pleiotropism, overlapping, antagonism and synergy, and participate in various important physiological functions of the human body. In recent years, cytokines have been applied in many fields, but many studies have found that cytokine freeze-dried powder has the problems of poor stability and reduced activity, so it cannot better play a role. Therefore, it is necessary to improve the composition of the cytokine protective agent to improve the stability and activity of the cytokine. SUMMARY

[0004] The first object of the present application is to provide an adipose-derived mesenchymal stem cell factor composition, which can maintain the stability and activity of the cytokine, and is beneficial to the long-term preservation of the cytokine product.

[0005] The second object of the present application is to provide a preparation method of an adipose-derived mesenchymal stem cell factor composition, which is simple and easy to operate.

[0006] The third object of the present application is to provide an application of an adipose-derived mesenchymal stem cell factor composition.

[0007] In order to achieve the above objects, the technical scheme adopted by the present application is: A kind of fat mesenchymal stem cell factor composition, including the following ingredients: fat mesenchymal stem cell culture supernatant 120-150 parts, BPC157 1-7 parts, cyanidin-3-O-glucoside 5-10 parts, isovaleraldehyde 0.5-1 part;Wherein the amino acid sequence of BPC157 is as follows: Gly-Glu-Pro-Pro-Pro-Gly-Lys-Pro-Ala-Asp-Asp-Ala-Gly-Leu-Val.

[0008] Further, the fat mesenchymal stem cell factor composition includes the following ingredients: fat mesenchymal stem cell culture supernatant 131 parts, BPC157 4 parts, cyanidin-3-O-glucoside 7 parts, isovaleraldehyde 0.5 parts.

[0009] Further, the process of obtaining the fat mesenchymal stem cell culture supernatant is as follows: the fat mesenchymal stem cells are subjected to hypoxia treatment under the condition that the oxygen content is 1-5% for 6-10h, then the fat mesenchymal stem cells after hypoxia treatment are inoculated into DMEM medium added with 10% FBS, 100~250U / mL penicillin and 100~250U / mL streptomycin for culture, and the fat mesenchymal stem cell culture supernatant is collected.

[0010] Further, the fat mesenchymal stem cells are P2~P5 generation fat mesenchymal stem cells.

[0011] Further, the inoculation density of the fat mesenchymal stem cells in the DMEM medium is 1.5×10 4 ~6.5×10 4 per mL.

[0012] Further, the culture time is 48~72h, and the culture condition is 37℃, 5% CO2.

[0013] Further, the fat mesenchymal stem cell culture supernatant is further subjected to filtration treatment; the specific process of the filtration treatment is as follows: the fat mesenchymal stem cell culture supernatant is pressurized and sequentially passes through microporous filters of 100 μm, 70 μm, 40 μm and 25 μm, and is concentrated by a filter membrane of 2~5KD.

[0014] The preparation method of the above-mentioned fat mesenchymal stem cell factor composition includes the following steps: each component is weighed according to the weight ratio, mixed uniformly and then freeze-dried for preservation.

[0015] The application of the above-mentioned fat mesenchymal stem cell factor composition is to apply the fat mesenchymal stem cell factor composition to skin anti-aging.

[0016] Compared with the prior art, the beneficial effects of the present application mainly lie in that: The present application improves the composition of the adipose mesenchymal stem cell factor protective agent, and innovatively uses BPC157, cyanidin-3-O-glucoside and isovaleraldehyde as the protective agent of the adipose mesenchymal stem cell factor. Further, the water content and cytokine content detection experiment proves that the composition of the present application can effectively maintain the stability and activity of the cytokine, and is beneficial to the long-term preservation of the cytokine product. The present application also detects the anti-aging ability of the adipose mesenchymal stem cell factor-containing composition, and the experimental data shows that the composition of the present application can be used for preparing an anti-skin aging drug. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 are P2, P4 and P5 adipose mesenchymal stem cell morphology charts, wherein Figure A is a P2 adipose mesenchymal stem cell morphology chart, Figure B is a P4 adipose mesenchymal stem cell morphology chart, and Figure C is a P5 adipose mesenchymal stem cell morphology chart; Figure 2 is a detection chart of the content of hydroxyproline in the skin after using the adipose mesenchymal stem cell factor-containing composition of Examples 1-3 and Comparative Examples 1-4. DETAILED DESCRIPTION

[0018] The technical solutions of the present application are further described below in combination with the specific embodiments. However, the protection scope of the present application is not limited to this: the materials, reagents and the like used in the following examples can be obtained from commercial channels unless otherwise specified.

[0019] I. Examples Example 1 The example provides an adipose mesenchymal stem cell factor composition, which is composed of 131 parts of adipose mesenchymal stem cell culture supernatant, 4 parts of BPC157, 7 parts of cyanidin-3-O-glucoside and 0.5 parts of isovaleraldehyde. The amino acid sequence of BPC157 is as follows: Gly-Glu-Pro-Pro-Pro-Gly-Lys-Pro-Ala-Asp-Asp-Ala-Gly-Leu-Val.

[0020] The acquisition process of the adipose mesenchymal stem cell culture supernatant is as follows: P2 adipose mesenchymal stem cells are subjected to hypoxic treatment for 6h under the condition of 1% oxygen content, and then the P2 adipose mesenchymal stem cells after hypoxic treatment are inoculated into a 24-well plate, and the 24-well plate is added with DMEM medium containing 10% FBS, 200U / mL penicillin and 180U / mL streptomycin, and the inoculation density of the P2 adipose mesenchymal stem cells in the DMEM medium is 1.5x104 Cells / mL. The 24-well plate was placed in a cell culture incubator at 37°C and 5% CO2 saturated humidity for 48 hours. The culture supernatant was pressurized and passed sequentially through microporous filters of 100μm, 70μm, 40μm and 25μm. The supernatant was then concentrated through a 3KD filter membrane to obtain the final product.

[0021] The method for obtaining P2 generation adipose-derived mesenchymal stem cells is as follows: (1) Wash human adipose tissue samples with D-Hanks solution, centrifuge, add 1% type II collagenase and shake to digest to obtain digestion solution; (2) Add an equal volume of D-Hanks salt solution to the digestion solution from step (1) to dilute, centrifuge, and obtain cell pellet; (3) Resuspend the cell pellet from step (2) in complete culture medium (DMEM medium with 10% FBS and 1% penicillin-streptomycin antibiotics) to obtain a cell suspension; (4) Mix the cell suspension from step (3) at a ratio of 2×10 4 / cm 2 The cells were seeded at a density of 100% into T75 flasks and cultured in a cell culture incubator at 37°C and 5% CO2 saturated humidity. The culture medium was changed every 3 days. When the cell confluence reached more than 80%, the old culture medium was discarded, and the cells were digested with recombinant trypsin solution and passaged at a ratio of 1:3 to obtain P2 generation adipose-derived mesenchymal stem cells for later use.

[0022] This embodiment also provides a method for preparing the above-mentioned adipose-derived mesenchymal stem cell factor composition, the specific preparation process of which is as follows: Weigh each component according to the above weight ratio, mix them evenly, pre-freeze at -80℃ for 8 hours, and then freeze-dry in a freeze dryer (pressure 35Pa, temperature -10℃) for 24 hours to obtain freeze-dried powder, which is stored at 4℃.

[0023] Example 2 This embodiment provides an adipose-derived mesenchymal stem cell factor composition, which comprises 120 parts of adipose-derived mesenchymal stem cell culture supernatant, 1 part of BPC157, 5 parts of cyanidin-3-O-glucoside, and 0.8 parts of isovaleraldehyde. The amino acid sequence of BPC157 is as follows: Gly-Glu-Pro-Pro-Pro-Gly-Lys-Pro-Ala-Asp-Asp-Ala-Gly-Leu-Val.

[0024] The process for obtaining the culture supernatant of adipose-derived mesenchymal stem cells (ADMSCs) was as follows: P4 ADMSCs were subjected to hypoxia treatment at an oxygen content of 5% for 10 hours. Then, the hypoxia-treated P4 ADMSCs were seeded into 24-well plates. DMEM medium containing 10% FBS, 100 U / mL penicillin, and 250 U / mL streptomycin was added to the 24-well plates. The seeding density of P4 generation ADMSCs in DMEM medium was 3 × 10⁻⁶. 4 Cells / mL, 24-well plates were placed in a cell culture incubator at 37℃ and 5% CO2 saturated humidity for 56h, and the culture supernatant was pressurized and passed sequentially through microporous filters of 100μm, 70μm, 40μm and 25μm, and concentrated through a 5KD filter membrane to obtain the final product.

[0025] The method for obtaining P4 generation adipose-derived mesenchymal stem cells is the same as the method for obtaining P2 generation adipose-derived mesenchymal stem cells in Example 1.

[0026] This embodiment also provides a method for preparing the above-mentioned adipose-derived mesenchymal stem cell factor composition, the specific preparation process of which is as follows: Weigh each component according to the above weight ratio, mix them evenly, pre-freeze at -80℃ for 8 hours, and then freeze-dry in a freeze dryer (pressure 35Pa, temperature -10℃) for 24 hours to obtain freeze-dried powder, which is stored at 4℃.

[0027] Example 3 This embodiment provides an adipose-derived mesenchymal stem cell factor composition, which comprises 150 parts of adipose-derived mesenchymal stem cell culture supernatant, 7 parts of BPC157, 10 parts of cyanidin-3-O-glucoside, and 1 part of isovaleraldehyde. The amino acid sequence of BPC157 is as follows: Gly-Glu-Pro-Pro-Pro-Gly-Lys-Pro-Ala-Asp-Asp-Ala-Gly-Leu-Val.

[0028] The process for obtaining the culture supernatant of adipose-derived mesenchymal stem cells (ADMSCs) was as follows: P5 generation ADMSCs were subjected to hypoxia treatment at an oxygen content of 3% for 8 hours. Then, the hypoxia-treated P5 generation ADMSCs were seeded into 24-well plates. DMEM medium containing 10% FBS, 250 U / mL penicillin, and 100 U / mL streptomycin was added to the 24-well plates. The seeding density of P5 generation ADMSCs in the DMEM medium was 6.5 × 10⁻⁶. 4 Cells / mL, 24-well plates were placed in a cell culture incubator at 37℃ and 5% CO2 saturated humidity for 72h. The culture supernatant was pressurized and passed sequentially through microporous filters of 100μm, 70μm, 40μm and 25μm, and concentrated through a 2KD filter membrane to obtain the final product.

[0029] The method for obtaining P5 generation adipose-derived mesenchymal stem cells is the same as the method for obtaining P2 generation adipose-derived mesenchymal stem cells in Example 1.

[0030] This embodiment also provides a method for preparing the above-mentioned adipose-derived mesenchymal stem cell factor composition, the specific preparation process of which is as follows: Weigh each component according to the above weight ratio, mix them evenly, pre-freeze at -80℃ for 8 hours, and then freeze-dry in a freeze dryer (pressure 35Pa, temperature -10℃) for 24 hours to obtain freeze-dried powder, which is stored at 4℃.

[0031] II. Comparative Example Comparative Example 1 This comparative example provides an adipose-derived mesenchymal stem cell factor composition, which differs from Example 1 in that: this adipose-derived mesenchymal stem cell factor composition consists of 131 parts of adipose-derived mesenchymal stem cell culture supernatant, 7 parts of cyanidin-3-O-glucoside, and 0.5 parts of isovaleraldehyde, while the rest are consistent with Example 1.

[0032] Comparative Example 2 This comparative example provides an adipose-derived mesenchymal stem cell factor composition, which differs from Example 1 in that: this adipose-derived mesenchymal stem cell factor composition consists of 131 parts of adipose-derived mesenchymal stem cell culture supernatant, 4 parts of BPC157, and 0.5 parts of isovaleraldehyde, while the rest are consistent with Example 1.

[0033] Comparative Example 3 This comparative example provides an adipose-derived mesenchymal stem cell factor composition, which differs from Example 1 in that: this adipose-derived mesenchymal stem cell factor composition consists of 131 parts of adipose-derived mesenchymal stem cell culture supernatant, 4 parts of BPC157, and 7 parts of cyanidin-3-O-glucoside, while the rest are consistent with Example 1.

[0034] Comparative Example 4 This comparative example provides an adipose-derived mesenchymal stem cell factor composition, which differs from Example 1 in that: this adipose-derived mesenchymal stem cell factor composition consists only of 131 parts of adipose-derived mesenchymal stem cell culture supernatant, while the rest is consistent with Example 1.

[0035] III. Experimental Examples Experimental Example 1 This experimental example observes the morphology of P2, P4, and P5 generation adipose-derived mesenchymal stem cells obtained in Examples 1-3. The observation method is as follows: The P2, P4, and P5 generation adipose-derived mesenchymal stem cells obtained in Examples 1-3 were observed under an inverted microscope to examine their cell morphology. The results are shown below. Figure 1 As shown.

[0036] Figure 1 These are morphological images of P2, P4, and P5 generation adipose-derived mesenchymal stem cells. Figure 1 A in the diagram is a morphological representation of P2 generation adipose-derived mesenchymal stem cells; Figure 1 Image B is a morphological diagram of P4 generation adipose-derived mesenchymal stem cells; Figure 1 Image C is a morphological diagram of P5 generation adipose-derived mesenchymal stem cells. (Source: [Insert Image Here]) Figure 1 It can be seen that the cell morphology in all three sets of images is elongated spindle-shaped and uniform, which is consistent with the morphological characteristics of adipose-derived mesenchymal stem cells.

[0037] Experimental Example 2 This experiment tested the stability of the lyophilized powders obtained in Examples 1-3 and Comparative Examples 1-4. The testing method is as follows: The moisture content of the freeze-dried powders obtained in Examples 1-3 and Comparative Examples 1-4 after 0 months and 6 months of storage was determined using the Karl Fischer method. The results are shown in Table 1. Note: Storage for 0 months means the experiment was conducted immediately after freeze-drying.

[0038] Table 1 As shown in Table 1, the freeze-dried powders of Examples 1-3 have low moisture content; the moisture content of the freeze-dried powders does not increase significantly with the gradual increase of storage time.

[0039] Furthermore, Table 1 shows that the freeze-dried powders of Comparative Examples 1-4 have a high water content; the water content of the freeze-dried powders increases significantly with increasing storage time. In summary, the composition of the present invention can effectively maintain the stability of adipose-derived mesenchymal stem cell factors, which is beneficial for the long-term preservation of cytokine products.

[0040] Experimental Example 3 This experiment detected the cytokine content in the lyophilized powders obtained in Examples 1-3 and Comparative Examples 1-4. The detection method is as follows: The lyophilized powders of Examples 1-3 and Comparative Examples 1-4, stored for 0 months and 6 months respectively, were mixed with solvent (4g VC and 6g hyaluronic acid dissolved in 100mL sterile water) at a ratio of 1:3. Fibroblast growth factor (FGF) levels were detected using an ELISA kit, and the results are shown in Table 2. Note: The initial concentration represents the cytokine content in the lyophilized powder stored for 0 months in each experimental group.

[0041] Table 2 As shown in Table 2, the initial concentrations of the freeze-dried powders in Comparative Examples 1-4 were lower than those in Examples 1-3 after freeze-drying, and the cytokine content decreased significantly after 6 months of storage. Based on the above experimental results, the composition of the present invention can maintain the activity of cytokines, which is beneficial for the long-term preservation of cytokine products.

[0042] Experiment Example 4 This experimental example uses the lyophilized powders obtained in Examples 1-3 and Comparative Examples 1-4 to prepare anti-skin aging drugs, and tests their anti-aging ability. The testing methods are as follows: Preparation of anti-skin aging drugs Experimental group: The lyophilized powder of adipose-derived mesenchymal stem cell factor composition of Examples 1-3 and Comparative Examples 1-4, which had been stored for 6 months, was mixed with solvent (4g VC and 6g hyaluronic acid dissolved in 100mL sterile water) at a ratio of 1:3 to obtain the composition solution of each experimental group for later use. The anti-skin aging drug consists of the following ingredients: 5% sodium hyaluronate, 2% butylene glycol, 0.05% disodium hydrogen phosphate, 0.05% sodium dihydrogen phosphate, 0.8% carbomer, 2% glyceryl stearate citrate, 0.5% vitamin C, 1.5% phenoxyethanol, 12% solution of the above composition, and the balance being deionized water, for later use.

[0043] Control group: physiological saline, ready for use.

[0044] Constructing a subacute aging model Eighty mice with similar physiological conditions were selected and divided into groups of 10. The mice were subcutaneously injected with 2% D-galactose 200 mg / kg in the neck and back for 8 consecutive weeks.

[0045] Experimental Procedure Hair was removed from the backs of mice in each group by 10 cm. 2 The anti-skin aging medications for each experimental group were applied to the hair removal area, while the control group received an equal volume of saline solution. Forty days later, after death at the cervical dislocation site, skin samples were collected from the treated area. The hydroxyproline content of an equivalent area of ​​skin was measured using a skin hydroxyproline kit. The results are shown below. Figure 2 As shown.

[0046] Figure 2 This is a graph showing the detection of hydroxyproline content in the skin after using the drug containing the adipose-derived mesenchymal stem cell factor compositions of Examples 1-3 and Comparative Examples 1-4. Figure 2 It is evident that the hydroxyproline content in the skin significantly increased after using the drug containing the adipose-derived mesenchymal stem cell factor composition of Examples 1-3. Based on the above experimental results, it is concluded that the adipose-derived mesenchymal stem cell factor composition of the present invention can be used in the preparation of drugs for anti-skin aging.

[0047] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. Based on the present invention, some modifications or substitutions can be made, but these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of protection claimed by the present invention.

Claims

1. A composition of adipose-derived mesenchymal stem cell factors, characterized in that, It includes the following components: 120-150 parts of adipose-derived mesenchymal stem cell culture supernatant, 1-7 parts of BPC157, 5-10 parts of cyanidin-3-O-glucoside, and 0.5-1 part of isovaleraldehyde.

2. The adipose-derived mesenchymal stem cell factor composition as described in claim 1, characterized in that, The process for obtaining the culture supernatant of adipose-derived mesenchymal stem cells is as follows: adipose-derived mesenchymal stem cells are treated with hypoxia for 6-10 hours under conditions of 1-5% oxygen content, and then the hypoxia-treated adipose-derived mesenchymal stem cells are seeded into DMEM medium supplemented with 10% FBS, 100-250 U / mL penicillin, and 100-250 U / mL streptomycin and cultured, and the culture supernatant of adipose-derived mesenchymal stem cells is collected.

3. The adipose-derived mesenchymal stem cell factor composition as described in claim 2, characterized in that, The adipose-derived mesenchymal stem cells are P2-P5 generation adipose-derived mesenchymal stem cells.

4. The adipose-derived mesenchymal stem cell factor composition as described in claim 2, characterized in that, The adipose-derived mesenchymal stem cells were seeded at a density of 1.5 × 10⁻⁶ in DMEM medium. 4 ~6.5×10 4 per mL.

5. The adipose-derived mesenchymal stem cell factor composition as described in claim 2, characterized in that, The culture time is 48~72h, and the culture conditions are 37℃ and 5%CO2.

6. The adipose-derived mesenchymal stem cell factor composition as described in claim 2, characterized in that, The adipose-derived mesenchymal stem cell culture supernatant is further subjected to filtration treatment; the specific process of filtration treatment is as follows: the adipose-derived mesenchymal stem cell culture supernatant is pressurized and passed sequentially through microporous filters of 100μm, 70μm, 40μm, and 25μm, and concentrated through a 2~5KD filtration membrane.

7. The method for preparing the adipose-derived mesenchymal stem cell factor composition according to any one of claims 1 to 6, characterized in that, Includes the following steps: Weigh each ingredient according to the specified weight ratio, mix them thoroughly, and then freeze-dry for storage.

8. The application of the adipose-derived mesenchymal stem cell factor composition according to any one of claims 1 to 6, characterized in that, The adipose-derived mesenchymal stem cell factor composition is used in the preparation of drugs for anti-skin aging.

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