An oral stem cell factor preparation and its preparation method and application

Abstract

The present invention relates to the field of biotechnology, and in particular to an oral stem cell factor preparation and a preparation method thereof. The preparation method of the oral stem cell factor preparation comprises the following steps: (1) adding mussel mucin to a stem cell factor concentrate and mixing evenly; (2) adding synephrine to the mixture of step (1), and freeze-drying to obtain the product. The present invention innovatively adds mussel mucin and synephrine components to the stem cell factor concentrate. Among them, the synergistic effect of mussel mucin and synephrine can effectively maintain the activity of stem cell factor. The preparation method provided by the present invention has simple steps, and the obtained oral stem cell factor preparation can not only improve insulin secretion, but also has anti-inflammatory effect.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an oral stem cell factor preparation and a preparation method and application thereof. Background Art

[0002] Diabetes mellitus (DM) is a common chronic metabolic disease caused by insufficient insulin and characterized by high blood sugar. With the accelerated pace of life, the incidence of diabetes has shown an increasing trend year by year, so the research and development of hypoglycemic drugs has become a research hotspot. However, traditional oral hypoglycemic drugs have limited therapeutic effects on diabetes and even have many adverse reactions.

[0003] In recent years, mesenchymal stem cells (MSCs) have attracted much attention in the field of biotechnology due to their multiple differentiation potentials, ability to participate in immune responses, ability to self-renew and replicate, and ability to regulate the local microenvironment and thus affect the functions of other cells. Mesenchymal stem cells also have an important function, which is to secrete a variety of cytokines and growth factors in a paracrine manner. Cytokines can affect changes in the physiological or pathological state of the body, thereby playing an important role in the stability of the body's internal environment. With the deepening of research, scholars have found that cytokines can reduce fasting blood sugar in diabetic patients, improve the function of pancreatic cells and their sensitivity to insulin, thereby reducing the intake of exogenous insulin in diabetic patients. However, the current method of preserving cytokines often results in the problem of poor activity of cytokines. Based on this problem, it is necessary to improve the protective agent of cytokines to effectively maintain the activity of cytokines. Summary of the invention

[0004] In order to overcome the deficiencies of the prior art, one of the objectives of the present invention is to provide a method for preparing an oral stem cell factor preparation, which has simple steps and is easy to operate.

[0005] The second object of the present invention is to provide an oral stem cell factor preparation that can significantly improve insulin secretion and has anti-inflammatory effects.

[0006] The third purpose of the present invention is to provide an application of an oral stem cell factor preparation, which has broad application prospects.

[0007] One of the purposes of the present invention is achieved by the following technical solution:

[0008] A method for preparing an oral stem cell factor preparation comprises the following steps:

[0009] (1) adding mussel mucin to the stem cell factor concentrate and mixing evenly;

[0010] (2) Add synephrine to the mixture of step (1), and freeze-dry to obtain the product.

[0011] Furthermore, the protein content of the stem cell factor concentrate is 1 to 1.5 mg / mL.

[0012] Furthermore, the masses of mussel mucin and synephrine added to every 100 mL of the stem cell factor concentrate are: 0.5-6 g mussel mucin and 0.1-1 g synephrine.

[0013] Furthermore, the preparation process of the stem cell factor concentrated solution is: collecting culture supernatant during stem cell culture and concentrating the supernatant to obtain the concentrated solution.

[0014] Furthermore, the stem cell is one of adipose mesenchymal stem cells, umbilical cord mesenchymal stem cells, and bone marrow mesenchymal stem cells.

[0015] Furthermore, the freeze-drying process is: lowering the temperature to -55 to -40°C at a rate of 5 to 9°C / min, maintaining freezing for 2 to 4 hours, and then placing in a vacuum freeze dryer at a pressure of 15 to 40Pa and a temperature of -30 to -10°C for freeze drying for 24 to 36 hours.

[0016] The second object of the present invention is achieved by the following technical solution:

[0017] An oral stem cell factor preparation is prepared by the above-mentioned preparation method of the oral stem cell factor preparation.

[0018] The third object of the present invention is achieved by adopting the following technical solution:

[0019] The application of the above oral stem cell factor preparation in the preparation of blood sugar regulating drugs.

[0020] Compared with the prior art, the present invention has the following beneficial effects: the present invention innovatively adds mussel mucin and synephrine components to the stem cell factor concentrate, wherein the synergistic effect of mussel mucin and synephrine can effectively maintain the activity of stem cell factor.

[0021] The preparation method provided by the invention has simple steps, and the prepared oral stem cell factor preparation can not only improve the secretion of insulin but also has anti-inflammatory effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a graph showing the insulin content in rats after treatment with the oral stem cell factor preparation of the present invention;

[0023] Figure 2 This is a graph showing the results of IL-1β and IL-6 levels in rats after treatment with the oral stem cell factor preparation of the present invention. DETAILED DESCRIPTION

[0024] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments. It should be noted that, under the premise of no conflict, the embodiments described below or the technical features can be arbitrarily combined to form new embodiments. The specific conditions not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, unless otherwise specified, are conventional products obtained through commercial channels.

[0025] The following examples take adipose-derived mesenchymal stem cells as an example to illustrate the preparation method of the oral stem cell factor preparation of the present invention and the technical effects that can be achieved.

[0026] Example 1

[0027] A method for preparing an oral stem cell factor preparation, the specific preparation process is as follows:

[0028] (1) Human adipose tissue was digested and separated by 1% type II collagenase, and D-Hanks buffer was added for dilution. Cell pellets were collected after centrifugation, and the cell pellets were resuspended in DMEM / F12 medium containing 10% FBS, 100 U / mL penicillin, and 100 U / mL streptomycin. The cells were cultured in a cell culture incubator at 37°C and 5% CO2 until the cell confluence reached more than 80%, and the cells were subcultured at a ratio of 1:3. The medium was changed every 2 days during the culture process to obtain subcultured P3 adipose-derived mesenchymal stem cells; the P3 adipose-derived mesenchymal stem cells were subjected to hypoxia treatment in an environment with an oxygen content of 7% for 8 hours, and the P3 adipose-derived mesenchymal stem cells after hypoxia treatment were then inoculated into a cell culture incubator containing 10% FBS, 100U / mL penicillin, 100U / mL streptomycin DMEM / F12 medium, restore the oxygen content to normal, culture the cells until the cell confluence reaches more than 75%, change to serum-free DMEM / F12 medium, starve culture for 24 hours, collect the culture supernatant, put the supernatant in a centrifuge, adjust the speed of the centrifuge to 2500rpm, centrifuge for 3 minutes, and then ultrafiltration and concentration through an ultrafiltration membrane to obtain a stem cell factor concentrate with a protein content of 1.5mg / mL, which is used for standby use; add mussel mucin to the stem cell factor concentrate prepared above, the amount of mussel mucin added is 4g of mussel mucin per 100mL of stem cell factor concentrate, and mix well;

[0029] (2) Add synephrine to the mixture of step (1), with the amount of synephrine added being 0.5 g synephrine per 100 mL of stem cell factor concentrate, then cool to -50°C at a rate of 7°C / min, maintain freezing for 3 hours, and then freeze-dry in a vacuum freeze dryer at a pressure of 25 Pa and a temperature of -20°C for 28 hours to obtain an oral stem cell factor preparation.

[0030] An oral stem cell factor preparation is prepared by adopting the preparation method of the oral stem cell factor preparation.

[0031] Example 2

[0032] A method for preparing an oral stem cell factor preparation, the specific preparation process is as follows:

[0033] (1) Human adipose tissue was digested and separated by 1% type II collagenase, and D-Hanks buffer was added for dilution. Cell pellets were collected after centrifugation, and the cell pellets were resuspended in DMEM / F12 medium containing 10% FBS, 100 U / mL penicillin, and 100 U / mL streptomycin. The cells were cultured in a cell culture incubator at 37°C and 5% CO2 until the cell confluence reached more than 80%, and the cells were subcultured at a ratio of 1:3. The medium was changed every 2 days during the culture process to obtain subcultured P3 adipose-derived mesenchymal stem cells; the P3 adipose-derived mesenchymal stem cells were subjected to hypoxia treatment in an environment with an oxygen content of 7% for 8 hours, and the P3 adipose-derived mesenchymal stem cells after hypoxia treatment were then inoculated into an environment with an oxygen content of 8% FBS, 100U / mL penicillin, 100U / mL streptomycin DMEM / F12 medium, restore the oxygen content to normal, culture the cells until the confluence reaches more than 75%, change to serum-free DMEM / F12 medium, starve culture for 18 hours, collect the culture supernatant, put the supernatant in a centrifuge, adjust the speed of the centrifuge to 2500rpm, centrifuge for 3min, and then ultrafiltration and concentration through an ultrafiltration membrane to obtain a stem cell factor concentrate with a protein content of 1.2mg / mL, which is set aside; add mussel mucin to the stem cell factor concentrate prepared above, the amount of mussel mucin added is 0.5g mussel mucin per 100mL stem cell factor concentrate, and mix well;

[0034] (2) Add synephrine to the mixture of step (1), wherein the amount of synephrine added is 0.1 g of synephrine per 100 mL of stem cell factor concentrate, then cool to -55°C at a rate of 5°C / min, maintain freezing for 4 hours, and then place in a vacuum freeze dryer at a pressure of 15 Pa and a temperature of -30°C for lyophilization for 24 hours to obtain an oral stem cell factor preparation.

[0035] An oral stem cell factor preparation is prepared by adopting the preparation method of the oral stem cell factor preparation.

[0036] Example 3

[0037] A method for preparing an oral stem cell factor preparation, the specific preparation process is as follows:

[0038] (1) Human adipose tissue was digested and separated by 1% type II collagenase, and D-Hanks buffer was added for dilution. Cell pellets were collected after centrifugation, and the cell pellets were resuspended in DMEM / F12 medium containing 10% FBS, 100 U / mL penicillin, and 100 U / mL streptomycin. The cells were cultured in a cell culture incubator at 37°C and 5% CO2 until the cell confluence reached more than 80%, and the cells were subcultured at a ratio of 1:3. The medium was changed every 2 days during the culture process to obtain subcultured P3 adipose-derived mesenchymal stem cells; the P3 adipose-derived mesenchymal stem cells were subjected to hypoxia treatment in an environment with an oxygen content of 5% for 6 hours, and the P3 adipose-derived mesenchymal stem cells after hypoxia treatment were then inoculated into a cell culture incubator containing 10% FBS, 100U / mL penicillin, 100U / mL streptomycin DMEM / F12 medium, restore the oxygen content to normal, culture the cells until the confluence reaches more than 75%, change to serum-free DMEM / F12 medium, starve culture for 10 hours, collect the culture supernatant, put the supernatant in a centrifuge, adjust the speed of the centrifuge to 2500rpm, centrifuge for 3 minutes, and then ultrafiltration and concentration through an ultrafiltration membrane to obtain a stem cell factor concentrate with a protein content of 1mg / mL, which is used for standby; add mussel mucin to the stem cell factor concentrate prepared above, the amount of mussel mucin added is 6g of mussel mucin per 100mL of stem cell factor concentrate, and mix well;

[0039] (2) Add synephrine to the mixture of step (1), with the amount of synephrine added being 1 g of synephrine per 100 mL of stem cell factor concentrate, then cool to -40°C at a rate of 9°C / min, maintain freezing for 2 hours, and then freeze-dry in a vacuum freeze dryer at a pressure of 40 Pa and a temperature of -10°C for 36 hours to obtain an oral stem cell factor preparation.

[0040] An oral stem cell factor preparation is prepared by adopting the preparation method of the oral stem cell factor preparation.

[0041] Comparative Example 1

[0042] An oral stem cell factor preparation, which differs from Example 1 in that mussel mucin is not added in step (1) of the preparation process, and the rest is the same as Example 1.

[0043] Comparative Example 2

[0044] An oral stem cell factor preparation, which differs from Example 1 in that synephrine is not added in step (2) of the preparation process, and the rest is the same as Example 1.

[0045] Comparative Example 3

[0046] An oral stem cell factor preparation, which is different from Example 1 in that: mussel mucin is not added in step (1) of the preparation process, and synephrine is not added in step (2) of the preparation process, and the rest is the same as Example 1.

[0047] The cytokine content in the oral stem cell factor preparation prepared by the present invention is detected below, and the specific detection process is as follows.

[0048] The oral stem cell factor preparations of Example 1-3 and Comparative Example 1-3 stored at 4°C for 6 months were dissolved in water, and the content of epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) in each oral stem cell factor preparation was detected using an ELISA kit. The relevant experimental data of the oral stem cell factor preparation stored for 0 months were the experimental data obtained by testing immediately after freeze-drying. The detailed test results are shown in Table 1.

[0049] Table 1

[0050]

[0051] It can be seen from Table 1 that compared with the oral stem cell factor preparation stored for 0 month, the stem cell factor in the oral stem cell factor preparation of Examples 1-3 after 6 months of storage was not significantly reduced; compared with the oral stem cell factor preparation stored for 0 month, the stem cell factor in the oral stem cell factor preparation of Comparative Examples 1-3 after 6 months of storage was significantly reduced.

[0052] An analysis of the reasons for the above experimental results showed that the synergistic effect of mussel mucin and synephrine can effectively maintain the activity of stem cell factor.

[0053] The following is the detection of insulin content, IL-1β, and IL-6 content in rats after treatment with the oral stem cell factor preparation of the present invention. The specific detection process is as follows.

[0054] Dissolve 0.1 g of the oral stem cell factor preparation of Example 1-3 or Comparative Example 1-3 stored at 4° C. for 6 months in 10 mL of water for later use;

[0055] Dissolve 4 g of mussel mucin in 100 mL of water to obtain a 0.04 g / mL mussel mucin solution for later use;

[0056] Dissolve 0.5 g of synephrine in 100 mL of water to obtain a 0.005 g / mL synephrine solution for later use.

[0057] A total of 100 rats with similar growth status were selected, 10 rats were used as the normal group and fed with basic feed, and the rest were not treated in any way. The other 90 rats were used as the experimental group to construct a hyperglycemia animal model. The method for constructing the hyperglycemia animal model was as follows: 90 rats were fed with high sugar, and the feed used in the feeding was: 55% basic feed, 18% lard, 23% sucrose, and 3% egg yolk. After feeding for 8 weeks, the rats were fasted for 12 hours, and 32 mg / Kg of streptozotocin was intraperitoneally injected. The above feed was continued for one week to complete the construction of the hyperglycemia animal model.

[0058] The animals in the above-mentioned hyperglycemia group were treated, wherein the experimental group was gavaged with 2 mL of 0.1 g / mL oral stem cell factor preparations of Examples 1-3 and Comparative Examples 1-3, the control group was not given drug treatment, the mussel mucin group was gavaged with 2 mL of 0.04 g / mL mussel mucin solution, and the synephrine group was gavaged with 2 mL of 0.005 g / mL synephrine solution.

[0059] After the treatment, the tail vein blood of rats in each group was collected for the detection of insulin, IL-1β, and IL-6 levels. The detailed test results are shown in Figure 1-2 .

[0060] Figure 1 This is a graph showing the results of insulin content in rats after treatment with the oral stem cell factor preparation of the present invention. Figure 2 This is a graph showing the results of the IL-1β and IL-6 levels in rats after treatment with the oral stem cell factor preparation of the present invention. Figure 1 The insulin content of the control group was compared with that of the normal group. The present invention successfully established a rat diabetes model.

[0061] observe Figure 1 By comparing the insulin content of the control group with that of Example 1-3 and Comparative Example 1-3, it is known that the oral stem cell factor preparation of Example 1-3 can significantly improve the secretion of insulin, and the effect is better than that of Comparative Example 1-3.

[0062] observe Figure 2 It is known that the IL-1β and IL-6 levels in rats were reduced after treatment with the oral stem cell factor preparation of the present invention, which indicates that the oral stem cell factor preparation of the present invention has an anti-inflammatory effect.

[0063] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A method for preparing an oral stem cell factor preparation, characterized in that: The following steps are involved: (1) Add mussel mucin to the stem cell factor concentrate and mix well; (2) adding synephrine to the mixture of step (1), and freeze-drying to obtain the product; the protein content of the stem cell factor concentrate is 1-1.5 mg / mL; the mass of mussel mucin and synephrine added to every 100 mL of the stem cell factor concentrate is: 0.5-6 g mussel mucin and 0.1-1 g synephrine; The preparation process of the stem cell factor concentrate is as follows: collecting culture supernatant during stem cell culture and concentrating the supernatant to obtain the concentrate; the stem cells are adipose mesenchymal stem cells; The freeze-drying process is: lowering the temperature to -55~-40°C at a rate of 5~9°C / min, maintaining freezing for 2~4 hours, and then placing in a vacuum freeze dryer at a pressure of 15~40Pa and a temperature of -30~-10°C for freeze-drying for 24~36 hours.

2. An oral stem cell factor preparation, characterized in that: The preparation method according to claim 1 is used for preparation.

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