Culture medium, stem cell secreta and application of stem cell secreta in treatment of myocardial fibrosis and anti-inflammation

Abstract

The invention discloses a culture medium, stem cell secretions and application thereof, the culture medium comprises a traditional Chinese medicine fermentation product and a basic culture medium, the traditional Chinese medicine fermentation product is a fermentation product obtained by fermenting a traditional Chinese medicine composition through a probiotic agent, and the traditional Chinese medicine composition comprises the following components in parts by weight: 10-15 parts of astragalus membranaceus, 6-10 parts of citron and 6-10 parts of fingered citron. The traditional Chinese medicine leavening contained in the culture medium provided by the invention is obtained by fermenting astragalus membranaceus, citron and fingered citron as matrixes and probiotics as fermentation strains, and the culture medium can promote generation or secretion of some active cell factors (stem cell secretions) in stem cells; the active cytokines can significantly inhibit Ang II induced myocardial fibroblast excessive proliferation, and stem cell secretions containing the active cytokines have a significant anti-inflammatory effect.

Description

Technical Field

[0001] The present invention relates to the fields of stem cells and cardiovascular diseases, and in particular to a culture medium, stem cell secretions and their applications in the treatment of myocardial fibrosis and anti-inflammation. Background Art

[0002] Myocardial fibrosis is characterized by abnormal proliferation of myocardial fibroblasts, excessive aggregation of collagen fibers, excessive elevation of collagen content or alteration of collagen components. It is a common pathological manifestation of various cardiovascular diseases such as hypertension, coronary heart disease, and myocardial infarction at a certain stage, and can lead to congestive heart failure, malignant arrhythmia, and even sudden cardiac death. Mesenchymal stem cells not only have the ability of self-renewal but also have the potential of multi-directional secretion. Currently, research results show that mesenchymal stem cells can reduce impaired cardiac function through paracrine and autocrine effects.

[0003] Based on the potential of stem cells, the present application has developed a culture medium. The traditional Chinese medicine ferment in this culture medium is obtained by fermenting a traditional Chinese medicine composition with astragalus membranaceus, citron, and fingered citron as substrates and probiotic bacteria as fermentation strains. Then we continued to study the effect of this culture medium on stem cells and found that it can promote stem cells to secrete a certain active cytokine, and this active cytokine has a significant inhibitory effect on the excessive proliferation of angiotensin II-induced myocardial fibroblasts. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a culture medium, stem cell secretions and their applications in the treatment of myocardial fibrosis and anti-inflammation to solve the problems raised in the above technical background.

[0005] To achieve the above purpose, the present invention is realized through the following technical solutions:

[0006] In a first aspect, the present invention provides a culture medium, comprising a traditional Chinese medicine ferment and a basal medium. The traditional Chinese medicine ferment is a ferment obtained by fermenting a traditional Chinese medicine composition with a probiotic agent. The traditional Chinese medicine composition comprises 10-15 parts by weight of astragalus membranaceus, 6-10 parts by weight of citron, and 6-10 parts by weight of fingered citron.

[0007] Further, the traditional Chinese medicine composition comprises 12 parts by weight of astragalus membranaceus, 8 parts by weight of citron, and 8 parts by weight of fingered citron.

[0008] Further, the probiotic agent is an acidophilus lactobacillus fermentation broth, wherein the concentration of acidophilus lactobacillus in the acidophilus lactobacillus fermentation broth is 10 7 ~10 8 CFU / mL.

[0009] Further, the preparation method of the acidophilus lactobacillus fermentation broth is as follows:

[0010] (1) Under aseptic conditions, the well-preserved Lactobacillus acidophilus was inoculated into MRS medium for activation culture and used as the seed liquid for fermentation culture;

[0011] (2) The Lactobacillus acidophilus seed liquid obtained in step (1) was inoculated into MRS medium at an inoculation amount of 2 - 3 v / v%, and cultured at 36 - 38 °C for 20 - 24 h to obtain a Lactobacillus acidophilus fermentation broth with a concentration of 10 7 ~10 8 CFU / mL.

[0012] Furthermore, the preparation method of the traditional Chinese medicine fermented product is as follows:

[0013] (1) After mixing the components contained in the traditional Chinese medicine composition in proportion, 8 - 10 times the amount of distilled water was added for extraction. After extracting 3 times, the filtrates were combined and concentrated to a concentrated solution with a crude drug concentration of 0.5 - 1.0 g / ml;

[0014] (2) A probiotic agent was added to the concentrated solution at an addition amount of 0.5 - 2 v / v%. The fermentation temperature was 35 °C - 40 °C, and the fermentation time was 2.5 h - 4 h to obtain the traditional Chinese medicine fermented product.

[0015] Furthermore, the addition amount of the traditional Chinese medicine fermented product is 0.1 - 2.0 v / v%.

[0016] Furthermore, the basic medium is DMEM / F12, DMEM or α - MEM.

[0017] In the second aspect, the present invention provides a stem cell secretion, which is the supernatant after culturing stem cells in the above-mentioned medium.

[0018] Furthermore, the stem cells are adipose mesenchymal stem cells.

[0019] In the third aspect, the present invention provides the application of the above-mentioned stem cell secretion in the preparation of a drug for preventing or treating myocardial fibrosis.

[0020] In the fourth aspect, the present invention provides the application of the above-mentioned stem cell secretion in the preparation of an anti - inflammatory product.

[0021] The innovation of the present invention lies in that the fermented product obtained by fermenting a traditional Chinese medicine composition (Astragalus membranaceus, Citrus medica var. sarcodactylis, and Citrus medica) with a probiotic agent can promote the production or secretion of certain active cytokines in stem cells, and these active cytokines can significantly inhibit the excessive proliferation of cardiac fibroblasts induced by Ang II, indicating the potential to develop an innovative drug for the prevention and treatment of myocardial fibrosis. Moreover, Astragalus membranaceus, Citrus medica var. sarcodactylis, and Citrus medica act synergistically with Lactobacillus acidophilus, and none of them can be absent. Only the fermented product obtained by fermenting Astragalus membranaceus, Citrus medica var. sarcodactylis, and Citrus medica with Lactobacillus acidophilus can promote the production of active cytokines, thereby inhibiting the excessive proliferation of cardiac fibroblasts induced by Ang II; at the same time, the stem cell secretions also have a significant anti-inflammatory effect.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] 1. The present application provides a culture medium, and the traditional Chinese medicine fermented product contained in this culture medium is obtained by fermenting Astragalus membranaceus, Citrus medica var. sarcodactylis, and Citrus medica as substrates with probiotics as fermentation strains. This culture medium can promote the production or secretion of certain active cytokines in stem cells (stem cell secretions), and these active cytokines can significantly inhibit the excessive proliferation of cardiac fibroblasts induced by Ang II, indicating the potential to develop an innovative drug for the prevention and treatment of myocardial fibrosis.

[0024] 2. The present application provides a culture medium, and the traditional Chinese medicine fermented product contained in this culture medium is obtained by fermenting Astragalus membranaceus, Citrus medica var. sarcodactylis, and Citrus medica as substrates with probiotics as fermentation strains. This culture medium can promote the production or secretion of certain active cytokines in stem cells, and the stem cell secretions containing these active cytokines have a significant anti-inflammatory effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It shows the influence of the stem cell secretions of the present invention on the secretion of Col I and Col III by HCFB;

[0026] Figure 2 It shows the influence of the stem cell secretions of the present invention on the secretion of TGF-β1 and CTGF by HCFB. DETAILED DESCRIPTION OF THE INVENTION

[0027] The following specific examples illustrate the implementation manners of the present invention, and those skilled in the art can easily understand the other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation manners, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, without conflict, the following examples and the features in the examples can be combined with each other.

[0028] Culture medium

[0029] Example 1

[0030] A culture medium comprising a traditional Chinese medicine ferment and a basal medium, wherein the addition amount of the traditional Chinese medicine ferment is 2.0 v / v% of the basal medium. The basal medium is DMEM / F12 (Gibco). The traditional Chinese medicine ferment is a ferment obtained by fermenting a traditional Chinese medicine composition with Lactobacillus acidophilus fermentation broth. The traditional Chinese medicine composition comprises 10 parts of Astragalus membranaceus, 10 parts of Citrus medica var. sarcodactylis, and 6 parts of Citrus medica var. sarcodactylis by weight; the concentration of Lactobacillus acidophilus in the Lactobacillus acidophilus fermentation broth is 10 8 CFU / mL.

[0031] In this example, the preparation method of the Lactobacillus acidophilus fermentation broth is as follows:

[0032] (1) Under aseptic conditions, the preserved Lactobacillus acidophilus (product number GIM1.731, purchased from Shanghai Huzheng Biotechnology Co., Ltd.) was inoculated into MRS medium for activation culture and used as the seed liquid for fermentation culture;

[0033] (2) The Lactobacillus acidophilus seed liquid obtained in step (1) was inoculated into MRS medium at an inoculation amount of 3 v / v%, and cultured at 37 °C for 24 h to obtain a Lactobacillus acidophilus fermentation broth with a concentration of 10 8 CFU / mL.

[0034] In this example, the preparation method of the traditional Chinese medicine ferment is as follows:

[0035] (1) After mixing the components contained in the traditional Chinese medicine composition in proportion, 10 times the amount of distilled water was added for extraction. After extraction three times, the filtrates were combined and concentrated to a concentrated solution with a crude drug concentration of 1.0 g / ml;

[0036] (2) The Lactobacillus acidophilus fermentation broth was added to the concentrated solution at an addition amount of 0.5 v / v%, the fermentation temperature was 35 °C, and the fermentation time was 3.5 h to obtain the traditional Chinese medicine ferment.

[0037] Example 2

[0038] A culture medium comprising a traditional Chinese medicine ferment and a basal medium, wherein the addition amount of the traditional Chinese medicine ferment is 1.0 v / v% of the basal medium. The basal medium is DMEM (Gibco). The traditional Chinese medicine ferment is a ferment obtained by fermenting a traditional Chinese medicine composition with Lactobacillus acidophilus fermentation broth. The traditional Chinese medicine composition comprises 12 parts of Astragalus membranaceus, 8 parts of Citrus medica var. sarcodactylis, and 8 parts of Citrus medica var. sarcodactylis by weight; the concentration of Lactobacillus acidophilus in the Lactobacillus acidophilus fermentation broth is 10 8 CFU / mL.

[0039] In this example, the preparation method of the Lactobacillus acidophilus fermentation broth is as follows:

[0040] (1) Under aseptic conditions, the well-preserved Lactobacillus acidophilus was inoculated into MRS medium for activation culture and used as the seed liquid for fermentation culture;

[0041] (2) The Lactobacillus acidophilus seed liquid obtained in step (1) was inoculated into MRS medium at an inoculation amount of 3 v / v%, and cultured at 37 °C for 24 h to obtain a Lactobacillus acidophilus fermentation broth with a concentration of 10 8 CFU / mL.

[0042] In this example, the preparation method of the traditional Chinese medicine fermented product is as follows:

[0043] (1) After mixing the components contained in the traditional Chinese medicine composition in proportion, 8 times the amount of distilled water was added for extraction. After 3 extractions, the filtrates were combined and concentrated to a concentrated solution with a crude drug concentration of 0.8 g / ml;

[0044] (2) A probiotic agent was added to the concentrated solution at an addition amount of 1.0 v / v%. The fermentation temperature was 38 °C and the fermentation time was 4 h to obtain the traditional Chinese medicine fermented product.

[0045] Example 3

[0046] A medium contains a traditional Chinese medicine fermented product and a basal medium. The addition amount of the traditional Chinese medicine fermented product is 0.2 v / v% of the basal medium. The basal medium is α-MEM (Gibco). The traditional Chinese medicine fermented product is a fermented product obtained by fermenting a traditional Chinese medicine composition with Lactobacillus acidophilus fermentation broth. The traditional Chinese medicine composition contains 15 parts of Astragalus membranaceus, 6 parts of Citrus medica var. sarcodactylis, and 10 parts of Citrus medica var. sarcodactylis by weight; the concentration of Lactobacillus acidophilus in the Lactobacillus acidophilus fermentation broth is 10 7 CFU / mL.

[0047] In this example, the preparation method of the Lactobacillus acidophilus fermentation broth is as follows:

[0048] (1) Under aseptic conditions, the well-preserved Lactobacillus acidophilus was inoculated into MRS medium for activation culture and used as the seed liquid for fermentation culture;

[0049] (2) The Lactobacillus acidophilus seed liquid obtained in step (1) was inoculated into MRS medium at an inoculation amount of 2 v / v%, and cultured at 37 °C for 20 h to obtain a Lactobacillus acidophilus fermentation broth with a concentration of 10 7 CFU / mL.

[0050] In this example, the preparation method of the traditional Chinese medicine fermented product is as follows:

[0051] (1) After mixing the components contained in the traditional Chinese medicine composition in proportion, 8 times the amount of distilled water was added for extraction. After 3 extractions, the filtrates were combined and concentrated to a concentrated solution with a crude drug concentration of 0.5 g / ml;

[0052] (2) Add probiotic agents to the concentrated solution at an addition amount of 2.0 v / v%, with a fermentation temperature of 40 °C and a fermentation time of 2.5 h to obtain a traditional Chinese medicine fermented product.

[0053] Comparative Example 1

[0054] This example is similar to Example 2, except that: the traditional Chinese medicine composition does not contain astragalus membranaceus. Specifically: a culture medium comprising a traditional Chinese medicine fermented product and a basal medium, wherein the addition amount of the traditional Chinese medicine fermented product is 1.0 v / v% of the basal medium. The basal medium is DMEM. The traditional Chinese medicine fermented product is a fermented product obtained by fermenting a traditional Chinese medicine composition with Lactobacillus acidophilus fermentation broth. The traditional Chinese medicine composition contains 8 parts of citron and 8 parts of fingered citron by weight; the concentration of Lactobacillus acidophilus in the Lactobacillus acidophilus fermentation broth is 10 8 CFU / mL.

[0055] Comparative Example 2

[0056] This example is similar to Example 2, except that: the traditional Chinese medicine composition does not contain citron. A culture medium comprising a traditional Chinese medicine fermented product and a basal medium, wherein the addition amount of the traditional Chinese medicine fermented product is 1.0 v / v% of the basal medium. The basal medium is DMEM. The traditional Chinese medicine fermented product is a fermented product obtained by fermenting a traditional Chinese medicine composition with Lactobacillus acidophilus fermentation broth. The traditional Chinese medicine composition contains 12 parts of astragalus membranaceus and 8 parts of fingered citron by weight; the concentration of Lactobacillus acidophilus in the Lactobacillus acidophilus fermentation broth is 10 8 CFU / mL.

[0057] Comparative Example 3

[0058] This example is similar to Example 2, except that: the traditional Chinese medicine composition does not contain fingered citron. A culture medium comprising a traditional Chinese medicine fermented product and a basal medium, wherein the addition amount of the traditional Chinese medicine fermented product is 1.0 v / v% of the basal medium. The basal medium is DMEM. The traditional Chinese medicine fermented product is a fermented product obtained by fermenting a traditional Chinese medicine composition with Lactobacillus acidophilus fermentation broth. The traditional Chinese medicine composition contains 12 parts of astragalus membranaceus and 8 parts of citron by weight; the concentration of Lactobacillus acidophilus in the Lactobacillus acidophilus fermentation broth is 10 8 CFU / mL.

[0059] Comparative Example 4

[0060] This example is similar to Example 2, with the difference being that the traditional Chinese medicine fermentate is not fermented by the fermentation broth of Lactobacillus acidophilus. A culture medium contains a traditional Chinese medicine fermentate and a basal medium, and the addition amount of the traditional Chinese medicine fermentate is 1.0 v / v% of the basal medium. The basal medium is DMEM. The traditional Chinese medicine fermentate is a fermentate directly obtained by fermenting a traditional Chinese medicine composition, and the traditional Chinese medicine composition contains 12 parts of Astragalus membranaceus, 8 parts of Citrus medica var. sarcodactylis, and 8 parts of Citrus medica var. bergamia by weight;

[0061] In this example, the preparation method of the traditional Chinese medicine fermentate is as follows:

[0062] (1) After mixing the components contained in the traditional Chinese medicine composition in proportion, add 8 times the amount of distilled water for extraction. After extracting 3 times, combine the filtrates and concentrate to a concentrated solution with a crude drug concentration of 0.8 g / ml;

[0063] (2) Ferment the concentrated solution at a fermentation temperature of 38°C for 4 h to obtain the traditional Chinese medicine fermentate.

[0064] Stem cell secretions

[0065] Example 4 of this application provides a stem cell secretion, which is the supernatant of adipose mesenchymal stem cells cultured in the culture medium provided in Example 1.

[0066] The method for obtaining the stem cell secretion is as follows: Take adipose mesenchymal stem cells (product number LM-H202, purchased from Shanghai Lianmai Biological Engineering Co., Ltd.), and inoculate them in a culture dish at a density of 1×10 6 cells / mL. When the cell confluence reaches about 80%, replace it with the culture medium provided in Example 1, continue to culture for 48 h, then collect the supernatant of the cell culture medium, centrifuge and filter the supernatant to obtain the stem cell secretion, and store it at 4°C for later use, the same below.

[0067] Example 5 of this application provides a stem cell secretion, which is the supernatant of adipose mesenchymal stem cells cultured in the culture medium provided in Example 2.

[0068] Example 6 of this application provides a stem cell secretion, which is the supernatant of adipose mesenchymal stem cells cultured in the culture medium provided in Example 3.

[0069] Comparative Example 5 of this application provides a stem cell secretion, which is the supernatant of adipose mesenchymal stem cells cultured in the culture medium provided in Comparative Example 1.

[0070] Comparative Example 6 of this application provides a stem cell secretion, which is the supernatant of adipose mesenchymal stem cells cultured in the culture medium provided in Comparative Example 2.

[0071] Comparative Example 7 of this application provides a stem cell secretion, which is the supernatant of adipose mesenchymal stem cells cultured in the culture medium provided in Comparative Example 3.

[0072] Comparative Example 8 of this application provides a stem cell secretion, which is the supernatant of adipose mesenchymal stem cells cultured in the culture medium provided in Comparative Example 4.

[0073] Comparative Example 9 of this application provides a stem cell secretion, which is the supernatant of adipose mesenchymal stem cells cultured in DMEM culture medium.

[0074] Efficacy study of stem cell secretions

[0075] I. Inhibitory effect of stem cell secretion on the excessive proliferation of cardiac fibroblasts induced by angiotensin II (Ang II)

[0076] 1. Cell culture: Human cardiac fibroblasts HCFB (product number AC339420, purchased from Shanghai Zeye Biotechnology Co., Ltd.); culture HCFB cells in a culture flask containing H-DMEM culture medium, and the culture medium contains 10% fetal bovine serum, 100 μg / mL streptomycin, and 100 IU / mL penicillin by volume fraction, and place them in a cell culture incubator for culture.

[0077] 2. Determination of the survival rate (cytotoxicity) of cardiac fibroblasts by MTT method

[0078] 2.1 Administration grouping: Set 5 groups, including 1 control group and 4 experimental groups. Specifically:

[0079] Control group: DMEM culture medium;

[0080] Experimental group 1: DMEM culture medium + 0.5 v / v% of the stem cell secretion of Example 5;

[0081] Experimental group 2: DMEM culture medium + 1.0 v / v% of the stem cell secretion of Example 5;

[0082] Experimental group 3: DMEM culture medium + 3.0 v / v% of the stem cell secretion of Example 5;

[0083] Experimental group 4: DMEM culture medium + 5.0 v / v% of the stem cell secretion of Example 5;

[0084] 2.2 Test method

[0085] The human cardiac fibroblasts HCFB cells in the logarithmic growth phase were resuspended in H-DMEM medium containing 10% fetal bovine serum by volume. The cells were seeded at 5000 cells per well in a 96-well plate, 200 μL per well. After culturing for 24 h, the original medium was discarded, and the cells were washed twice with PBS and then replaced with serum-free DMEM medium and cultured overnight. The serum-free DMEM medium was aspirated, and then 200 μL of the culture medium for the drug administration groups (the culture media for the control group and experimental groups 1-4) was added for drug administration culture, with 5 replicates in each group; After continued culturing for 48 h, 20 μL of 5 mg / mL MTT solution was added to each well and cultured for 4 h. The culture media in each well were discarded, 150 μL of DMSO solution was added to each well, and after shaking for 10 min, the absorbance A value at 570 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader. The survival rate of cardiac fibroblasts was calculated according to the following formula, as shown in Table 1:

[0086] Survival rate = (A value of cells in the experimental group / A value of cells in the control group) × 100%

[0087] Table 1 Effects of stem cell secretions at different concentrations on the survival rate of HCFB

[0088] Group Survival rate (%) 0.5 v / v% 106.6±1.5 1 v / v% 104.4±1.4 3 v / v% 103.1±1.1 5 v / v% 97.9±1.1

[0089] As can be seen from Table 1, the stem cell secretions of the present invention did not show an obvious inhibitory effect on the proliferation of human cardiac fibroblasts HCFB cells at a concentration of 0.5-5 v / v%.

[0090] 3. Detection of the inhibitory effect of stem cell secretions on the proliferation ability of Ang II-stimulated cardiac fibroblasts HCFB by the MTT method

[0091] 3.1 Drug administration grouping: 10 groups were set up, including 1 blank control group, 1 model group (Ang II group), and 8 experimental groups, specifically as follows:

[0092] Blank control group: DMEM culture medium;

[0093] Ang II group: DMEM culture medium containing 0.1 μM Ang II;

[0094] Experimental groups: DMEM culture medium containing 0.1 μM Ang II + 1 v / v% stem cell secretions corresponding to Examples 4-6 and Comparative Examples 5-9;

[0095] 3.2 Test method

[0096] The human cardiac fibroblasts HCFB cells in the logarithmic growth phase were resuspended in H-DMEM medium containing 10% fetal bovine serum by volume. The cells were seeded at 5000 cells / well in a 96-well plate, 200 μL per well. After culturing for 24 h, the original medium was discarded, and the cells were washed twice with PBS and then replaced with serum-free DMEM medium for overnight culture. The serum-free DMEM medium was aspirated, and then 200 μL of the culture medium for drug administration groups (the culture media of the blank control group, Ang II group, and experimental groups) was added for drug administration culture, with 5 replicates in each group; After continued culturing for 48 h, 20 μL of 5 mg / mL MTT solution was added to each well, and after culturing for 4 h, the culture medium in each well was discarded, 150 μL of DMSO solution was added to all wells, and after shaking for 10 min, the absorbance A value at 570 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader, as shown in Table 2:

[0097] Table 2 Effects of each group on the proliferation of HCFB

[0098]

[0099] As can be seen from Table 2, compared with the Ang II group, the OD values of the proliferation of cardiac fibroblasts in the Example 4-6 groups were significantly decreased, indicating that the stem cell secretions of the present invention can significantly inhibit the proliferation effect of Ang II on HCFB cells.

[0100] 4. ELISA detection of the secretion of type I collagen (Col I), type III collagen (Col III), TGF-β1, and CTGF in cardiac fibroblasts

[0101] 4.1 Drug administration grouping: 10 groups were set up, including 1 blank control group, 1 model group (Ang II group), and 8 experimental groups. Specifically:

[0102] Blank control group: DMEM culture medium;

[0103] Ang II group: DMEM culture medium containing 0.1 μM Ang II;

[0104] Experimental groups: DMEM culture medium containing 0.1 μM Ang II + 1 v / v% corresponding stem cell secretions of Examples 4-6 and Comparative Examples 5-9;

[0105] 3.2 Test method

[0106] The human cardiac fibroblasts HCFB cells in the logarithmic growth phase were resuspended in H-DMEM medium containing 10% fetal bovine serum by volume. The cells were seeded at 1×10 5 cells / well in a 12-well plate, 2 mL per well. Incubated at 37 °C, 5% CO 2Cultured under the given conditions, after the cells adhered to the wall, discard the culture medium, wash twice with PBS, replace with serum-free DMEM culture medium and culture overnight. Aspirate and discard the serum-free DMEM culture medium, then add 2 ml of the culture medium for drug administration groups (the culture media of the blank control group, AngⅡ group and experimental group) for drug administration culture, with 5 replicates in each group; continue to culture for 48 h and then collect the cell supernatants of each group. Use the double antibody sandwich Elisa method to detect the secretion of ColⅠ, ColⅢ, TGF-β1 and CTGF in the cell supernatants of each group.

[0107] The specific operation of the Elisa method is as follows:

[0108] (1) Coating: Dilute the primary antibodies ColⅠ, ColⅢ, TGF-β1 and CTGF appropriately with 0.05 M carbonate coating buffer at pH 9.6 (for example, dilute to a protein content of 1 - 10 μg / ml, preferably 2 μg / ml). Add 100 μl of the corresponding primary antibody dilution to each reaction well of a 96-well enzyme-linked immunosorbent assay (ELISA) plate. Set 5 replicate wells for each primary antibody dilution in each group and incubate overnight at 4°C. The next day, discard the solution in the wells and wash 3 times with PBST, 3 min each time.

[0109] (2) Blocking: Block with 1% BSA at 37°C for 1 hour. Discard the solution in the wells and wash 3 times with PBST, 3 min each time.

[0110] (3) Sample addition: Add 100 μl of the cell culture supernatant of the corresponding group to the above-coated reaction wells, incubate at 37°C for 1 hour, discard the solution in the wells, and wash 3 times with PBST, 3 min each time.

[0111] (4) Secondary antibody incubation: Add 100 μl of the secondary antibody diluted 4000-fold to each well, incubate at 37°C for 1 h, and wash 5 times with PBST, 3 min each time.

[0112] (5) Color development: Add 100 μl of TMB color development solution to each well and develop color in the dark for 3 - 5 min.

[0113] (6) Termination: When the liquid in the well turns yellow, add 50 μl of 2 M H 2 SO 4 Termination solution to stop color development.

[0114] (7) Measure the absorbance value at a wavelength of 450 nm. The absorbance values are shown in Table 3, Figure 1 and Figure 2 as shown.

[0115] Table 3 Effects of each group on the secretion of ColⅠ, ColⅢ, TGF-β1 and CTGF by HCFB

[0116]

[0117]

[0118] As can be seen from Table 3, Figure 1 and Figure 2 compared with the blank control group, the contents of ColⅠ, ColⅢ, TGF-β1 and CTGF in the supernatant of myocardial fibroblasts in the AngⅡ group were significantly increased, and the difference was statistically significant; compared with the AngⅡ group and Comparative Examples 5-9, the contents of ColⅠ, ColⅢ, TGF-β1 and CTGF in the supernatant of myocardial fibroblasts in Examples 4-6 groups were significantly decreased, and the difference was statistically significant. It shows that the fermented product obtained by fermenting the traditional Chinese medicine composition (Astragalus membranaceus, Citrus medica, and Citrus medica var. sarcodactylis) with a probiotic agent can promote the production or secretion of certain active cytokines in stem cells, and these active cytokines can significantly inhibit the excessive proliferation of myocardial fibroblasts induced by AngⅡ, indicating the potential to develop an innovative drug for the prevention and treatment of myocardial fibrosis. Moreover, Astragalus membranaceus, Citrus medica, and Citrus medica var. sarcodactylis act synergistically with Lactobacillus acidophilus, and none of them can be absent. Only the fermented product obtained by fermenting Astragalus membranaceus, Citrus medica, and Citrus medica var. sarcodactylis with Lactobacillus acidophilus can promote the production of active cytokines, thereby inhibiting the excessive proliferation of myocardial fibroblasts induced by AngⅡ.

[0119] In summary, the stem cell secretions in this application reduce the contents of type I collagen and type III collagen in the supernatant of myocardial fibroblasts, and reduce the contents of CTGF and TGF-β1 in the supernatant of myocardial fibroblasts, indicating that the stem cell secretions can inhibit the excessive proliferation of myocardial fibroblasts induced by AngⅡ, suggesting the potential to develop an innovative drug for the prevention and treatment of myocardial fibrosis.

[0120] II. Anti-inflammatory properties of stem cell secretions

[0121] Inflammatory mouse model: 48 healthy Kunming KM mice (purchased from Nanjing Junke Biological Engineering Co., Ltd.), SPF grade, male, were fed with cobalt-60 irradiated sterilized basal diet. After 2 days of adaptive feeding under conventional conditions in the animal house, the body weight of the mice was maintained at 20±2 g. 100 μl / mouse of xylene was evenly applied to both sides of the right auricle of the mice to cause inflammation, and the left ear was not treated.

[0122] Grouping: The modeled inflammatory mice were randomly divided into 8 groups, namely 1 model group, 1 positive control group, and 6 test groups, with 6 mice in each group; among them, the model group was smeared with normal saline, 0.15 ml / mouse; the positive control group was smeared with dexamethasone acetate ointment, and the dosage was 0.15 ml / mouse; the test groups were smeared with the stem cell secretions (diluted with normal saline) obtained from Example 5 and Comparative Examples 5-9 at 1 v / v%, and the dosage was 0.15 ml / mouse.

[0123] Test method: 100 μl of xylene per mouse was evenly applied to both the inner and outer surfaces of the right auricle of the mouse to cause inflammation. 30 minutes after inflammation induction, 0.15 ml of the test substance sample of each group was evenly applied to both the inner and outer surfaces of the right auricle of each mouse in each group. One hour after the administration of the test substance sample, the mice were sacrificed by cervical dislocation. Both ears were cut along the auricle baseline. Ear pieces were taken from the same part of both ears with a puncher with a diameter of 8 mm, weighed, and the swelling degree and inhibition rate of each group were calculated. The results are shown in Table 4;

[0124] Among them, the swelling degree (mg) = weight of the right ear - weight of the left ear; inhibition rate = [(swelling degree of the model group - swelling degree of the test group) / swelling degree of the model group] × 100%.

[0125] Table 4

[0126]

[0127]

[0128] As can be seen from Table 4, compared with the model group, the positive control group, Example 5, and Comparative Examples 5 - 9 all had significant inhibitory effects on xylene-induced ear swelling in mice. Compared with Comparative Examples 5 - 9, Example 5 had a significant inhibitory effect on xylene-induced ear swelling in mice, indicating that the fermented product obtained by fermenting the traditional Chinese medicine composition (Astragalus membranaceus, Citrus medica, and Citrus medica var. sarcodactylis) with a probiotic agent can promote the production or secretion of certain active cytokines in stem cells, and the stem cell secretions containing these active cytokines have a significant inhibitory effect on xylene-induced ear swelling in mice, which shows that the stem cell secretions of the present invention have a significant anti-inflammatory effect.

[0129] The above-described embodiments only represent the specific embodiments of the present invention, and the description thereof is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A culture medium, characterized in that The invention comprises a Chinese medicine fermentation product and a basic culture medium, wherein the Chinese medicine fermentation product is a fermentation product obtained by fermenting a Chinese medicine composition with a probiotic agent, and the Chinese medicine composition comprises 10 to 15 parts of astragalus, 6 to 10 parts of citron, and 6 to 10 parts of bergamot by weight.

2. A culture medium according to claim 1, characterized in that It is the supernatant of adipose mesenchymal stem cells after being cultured in a culture medium; the culture medium comprises a Chinese medicine fermentation product and a basic culture medium, the Chinese medicine fermentation product is a fermentation product obtained by fermenting a Chinese medicine composition with a probiotic agent, and the Chinese medicine composition comprises 12 parts of astragalus, 8 parts of citron, and 8 parts of bergamot by weight; the probiotic agent is a Lactobacillus acidophilus fermentation liquid, wherein the concentration of Lactobacillus acidophilus in the Lactobacillus acidophilus fermentation liquid is 10 7 ~10 8 CFU / mL, the addition amount of the traditional Chinese medicine fermentation product is 0.1~2.0v / v%, and the basic culture medium is DMEM.

3. A culture medium according to claim 2, characterized in that The preparation method of the Lactobacillus acidophilus fermentation liquid comprises the following steps: (1) Under sterile conditions, the preserved Lactobacillus acidophilus was inoculated into MRS medium for activation and culture, and then used as the seed liquid for fermentation culture; (2) The Lactobacillus acidophilus seed solution obtained in step (1) was inoculated into MRS medium at an inoculum amount of 2-3 v / v%, and cultured at 36-38°C for 20-24 hours to obtain a concentration of 10 7 ~10 8 CFU / mL of Lactobacillus acidophilus fermentation broth.

4. A culture medium according to claim 2, characterized in that The preparation method of the Chinese medicine fermentation product is: (1) After mixing the ingredients of the Chinese medicine composition according to a certain proportion, 8 to 10 times the amount of distilled water is added for extraction. After three extractions, the filtrates are combined and concentrated to a concentrated solution having a crude drug concentration of 0.5 to 1.0 g / ml; (2) Adding probiotics in an amount of 0.5-2 v / v% to the concentrated solution, the fermentation temperature is 35°C-40°C, the fermentation time is 2.5 h-4 h, and a Chinese medicine fermentation product is obtained.

5. A culture medium according to claim 4, characterized in that The addition amount of the Chinese medicinal fermentation product is 0.1-2.0 v / v%.

6. A culture medium according to claim 1, characterized in that The basic culture medium is DMEM / F12, DMEM or α-MEM.

7. Stem cell secretion, characterized in that It is the supernatant obtained after stem cells are cultured in the culture medium according to any one of claims 1 to 7.

8. Use of the stem cell secretion according to claim 7 in the preparation of a drug for preventing or treating myocardial fibrosis.

9. Use of the stem cell secretion according to claim 8 in the preparation of anti-inflammatory products.

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