Stem cell preparation for improving diabetes disease course and preparation method and application thereof

Abstract

The invention discloses a stem cell preparation for improving the course of diabetes and a preparation method and application thereof, and belongs to the technical field of biological medicines.The stem cell preparation is prepared from, by weight, 0.5%-1.2% of dental pulp mesenchymal stem cells, 1.5%-4.5% of manystem extract, 1.1%-2.6% of saikoside C and 3%-6% of glycerin, and the stem cell preparation is obtained by evenly mixing the components with a proper amount of normal saline. The dental pulp mesenchymal stem cells with immunoregulation ability are adopted, and the synergistic effect of the manystem extract, the saikoside C and the glycerol is combined, so that the blood glucose level can be effectively regulated and controlled, and a potential strategy can be provided for intervention of the pathological process of diabetes mellitus.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a stem cell preparation for improving the course of diabetes, its preparation method, and its application. Background Technology

[0002] Diabetes mellitus is a chronic hyperglycemic metabolic disease caused by insufficient insulin secretion or insulin resistance. According to data from the International Diabetes Federation (IDF), the number of patients worldwide has exceeded 500 million, and the incidence rate is rapidly increasing. The clinical harm of this disease mainly stems from its complications (such as cardiovascular disease, retinopathy, nephropathy, neuropathy, and diabetic foot), which are not only leading causes of disability and death but also impose a heavy burden on patients and society.

[0003] Human pluripotent stem cells (including embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs)) possess the potential for unlimited proliferation and multi-directional differentiation into functional pancreatic β-cells, providing a sustainable cell source for cell replacement therapy in diabetes. Through in vitro directed differentiation techniques, these stem cells can be induced to form functional pancreatic organoids or heterogeneous cell clusters (such as those containing pancreatic β-cells and α-cells). Theoretically, they can mimic the function of natural pancreatic islets, sensing blood glucose changes and secreting insulin, thus achieving precise regulation of blood glucose homeostasis. This scientific concept not only effectively alleviates the clinical dilemma of donor islet shortage but also provides new ideas for revolutionizing diabetes treatment strategies.

[0004] Currently, clinical treatment of diabetes mainly relies on exogenous insulin injections and oral hypoglycemic drugs. However, while these existing technologies can control blood sugar levels to some extent, they cannot cure the disease and pose problems such as the need for lifelong medication, the risk of hypoglycemia, and complications. In addition, the application of stem cells in the clinical treatment of diabetes still faces three major challenges: (1) transplanted cells are susceptible to attack by the host's immune system; (2) slow vascularization process leads to insufficient nutrition and oxygen supply; and (3) the microenvironment of hypoxia, inflammation, and malnutrition leads to low cell survival rate.

[0005] Therefore, developing a stem cell preparation that effectively protects transplanted cells, improves the transplanted microenvironment, promotes rapid cell vascularization, and effectively avoids immune rejection is of decisive significance for advancing the clinical translation of stem cell therapy for diabetes. Summary of the Invention

[0006] Therefore, the present invention provides a stem cell preparation for improving the course of diabetes, its preparation method and application, in order to solve the related technical problems existing in the prior art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: According to a first aspect of the present invention, a stem cell preparation for improving the course of diabetes is provided, comprising the following components: dental pulp mesenchymal stem cells, bergamot nail extract, saikosaponin C and glycerol.

[0008] Furthermore, each component, by weight percentage, contains 0.5-1.2% dental pulp mesenchymal stem cells, 1.5-4.5% Buddha's fingernail extract, 1.1-2.6% saikosaponin C, and 3-6% glycerol. These components are mixed evenly and brought to a final volume using physiological saline to obtain the stem cell preparation.

[0009] Furthermore, the concentration of dental pulp mesenchymal stem cells was 1 x 10⁻⁶. 6 ~10 7 per mL.

[0010] Furthermore, the dental pulp mesenchymal stem cells were passaged to P3-5 generations.

[0011] According to a second aspect of the present invention, a method for preparing a stem cell preparation for improving the course of diabetes is provided, comprising the following steps: (1) Passage dental pulp mesenchymal stem cells to P3-5 generation cells for later use; (2) Weigh and prepare the Buddha's fingernail extract; (3) Weigh out dental pulp mesenchymal stem cells, bergamot extract, saikosaponin C and glycerol according to the proportion. First, dissolve saikosaponin C and glycerol in physiological saline at 5°C. Add bergamot extract and mix well. Finally, slowly add cell suspension and mix gently. Then, adjust the volume with physiological saline to obtain stem cell preparation.

[0012] Furthermore, the preparation method of dental pulp mesenchymal stem cells includes the following steps: Cell resuscitation: Thaw 1 mL of cell suspension in a cryovial by rapid shaking in a 37°C water bath. Add 5 mL of mouse bone marrow mesenchymal stem cell complete culture medium containing 10% fetal bovine serum and penicillin-streptomycin, mix well, centrifuge at 800 rpm for 5 min, and discard the supernatant. Add 4-6 mL of the above complete culture medium, mix well, and seed into culture flasks. Incubate overnight at 37°C in a 5% CO2 incubator. Change the medium the next day. When the cell density reaches 80%-90%, passage the cells. Cell passage: Discard the culture supernatant, rinse the cells 1-2 times with PBS free of calcium and magnesium ions; add 1-2 mL of 0.25% trypsin, digest at 37°C for 1-2 min, and observe the cell digestion under a microscope; after complete digestion, add 5 mL of culture medium containing 10% fetal bovine serum to stop digestion; gently pipette to collect the cell suspension, centrifuge at 800 rpm for 10 min, discard the supernatant, add 1-2 mL of culture medium and mix well; seed into new culture flasks at a ratio of 1:3, and passage to P3-5 for later use.

[0013] Furthermore, the dental pulp mesenchymal stem cells were P4 generation cells.

[0014] The preparation method of Buddha's fingernail extract is as follows: Raw material pretreatment: Dry and pulverize the Buddha's fingernail; Solution impregnation: Place the Buddha's fingernail powder in an Erlenmeyer flask and add an appropriate amount of water or ethanol solvent; Ultrasonic-assisted extraction: Place the conical flask in an ultrasonic cleaner or a professional ultrasonic extractor, set the power to 300W, and perform ultrasonic extraction at 50~60℃ for 20~40 minutes. Post-processing and concentration: After extraction, the solution is cooled and filtered, then the filtrate is concentrated under reduced pressure and dried to obtain the Buddha's fingernail extract.

[0015] According to a third aspect of the present invention, the use of stem cell preparations in the preparation of drugs for improving the course of diabetes is provided.

[0016] The present invention has the following advantages: This application develops a stem cell preparation based on dental pulp mesenchymal stem cells (DPSCs), comprising 0.5-1.2% DPSCs, 1.5-4.5% *Cinnamomum camphora* extract, 1.1-2.6% *Saikosaponin C*, and 3-6% glycerol, obtained by mixing and adjusting the volume with physiological saline. This preparation utilizes DPSCs with immunomodulatory capabilities, combined with the synergistic effects of *Cinnamomum camphora* extract, *Saikosaponin C*, and glycerol, to effectively regulate blood glucose levels, providing a potential strategy for intervention in the pathological progression of diabetes. Detailed Implementation

[0017] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] To address the technical problems existing in the prior art, according to a first aspect of the present invention, a stem cell preparation for improving the course of diabetes is provided, comprising the following components: dental pulp mesenchymal stem cells, bergamot nail extract, saikosaponin C, and glycerol.

[0019] Furthermore, each component, by weight percentage, contains 0.5-1.2% dental pulp mesenchymal stem cells, 1.5-4.5% Buddha's fingernail extract, 1.1-2.6% saikosaponin C, and 3-6% glycerol. These components are mixed evenly and brought to a final volume using physiological saline to obtain the stem cell preparation.

[0020] Furthermore, the concentration of dental pulp mesenchymal stem cells was 1 x 10⁶ to 10⁷ cells / mL.

[0021] Furthermore, the dental pulp mesenchymal stem cells were passaged to P3-5 generations.

[0022] According to a second aspect of the present invention, a method for preparing a stem cell preparation for improving the course of diabetes is provided, comprising the following steps: (1) Passage dental pulp mesenchymal stem cells to P3-5 generation cells for later use; (2) Weigh and prepare the Buddha's fingernail extract; (3) Weigh out dental pulp mesenchymal stem cells, bergamot extract, saikosaponin C and glycerol according to the proportion. First, dissolve saikosaponin C and glycerol in physiological saline at 5°C. Add bergamot extract and mix well. Finally, slowly add cell suspension and mix gently. Then, adjust the volume with physiological saline to obtain stem cell preparation.

[0023] Furthermore, the preparation method of dental pulp mesenchymal stem cells includes the following steps: Cell resuscitation: Thaw 1 mL of cell suspension in a cryovial by rapid shaking in a 37°C water bath. Add 5 mL of mouse bone marrow mesenchymal stem cell complete culture medium containing 10% fetal bovine serum and penicillin-streptomycin, mix well, centrifuge at 800 rpm for 5 min, and discard the supernatant. Add 4-6 mL of the above complete culture medium, mix well, and seed into culture flasks. Incubate overnight at 37°C in a 5% CO2 incubator. Change the medium the next day. When the cell density reaches 80%-90%, passage the cells. Cell passage: Discard the culture supernatant, rinse the cells 1-2 times with PBS free of calcium and magnesium ions; add 1-2 mL of 0.25% trypsin, digest at 37°C for 1-2 min, and observe the cell digestion under a microscope; after complete digestion, add 5 mL of culture medium containing 10% fetal bovine serum to stop digestion; gently pipette to collect the cell suspension, centrifuge at 800 rpm for 10 min, discard the supernatant, add 1-2 mL of culture medium and mix well; seed into new culture flasks at a ratio of 1:3, and passage to P3-5 for later use.

[0024] Furthermore, the dental pulp mesenchymal stem cells were P4 generation cells.

[0025] The preparation method of Buddha's fingernail extract is as follows: Raw material pretreatment: Dry and pulverize the Buddha's fingernail; Solution impregnation: Place the Buddha's fingernail powder in an Erlenmeyer flask and add an appropriate amount of water or ethanol solvent; Ultrasonic-assisted extraction: Place the conical flask in an ultrasonic cleaner or a professional ultrasonic extractor, set the power to 300W, and perform ultrasonic extraction at 50~60℃ for 20~40 minutes. Post-processing and concentration: After extraction, the solution is cooled and filtered, then the filtrate is concentrated under reduced pressure and dried to obtain the Buddha's fingernail extract.

[0026] According to a third aspect of the present invention, the use of stem cell preparations in the preparation of drugs for improving the course of diabetes is provided.

[0027] To better illustrate the technical effects of this application, the following embodiments are also provided.

[0028] Preparation Example 1 The mouse bone marrow mesenchymal stem cells used in this application were purchased from Shanghai Xinyu Biotechnology Co., Ltd., and cultured as mouse bone marrow mesenchymal stem cells.

[0029] Cell resuscitation: Thaw 1 mL of cell suspension in a cryovial by rapid shaking in a 37°C water bath. Add 5 mL of 10% fetal bovine serum and penicillin-streptomycin-containing mouse bone marrow mesenchymal stem cell complete culture medium, mix well, centrifuge at 800 rpm for 5 min, and discard the supernatant. Add 4-6 mL of the above complete culture medium, mix well, and seed into culture flasks. Incubate overnight at 37°C in a 5% CO2 incubator. Change the medium the next day. When the cell density reaches 80%-90%, passage the cells. Cell passage: Discard the culture supernatant, rinse the cells 1-2 times with PBS free of calcium and magnesium ions; add 1-2 mL of 0.25% trypsin and digest at 37°C for 1-2 min, observing the cell digestion under a microscope; after complete digestion, add 5 mL of culture medium containing 10% fetal bovine serum to stop digestion; gently pipette to collect the cell suspension, centrifuge at 800 rpm for 10 min, discard the supernatant, add 1-2 mL of culture medium and mix well; seed into a new culture flask at a ratio of 1:3 and passage to P4 for later use.

[0030] Preparation Example 2 The preparation method of Buddha's fingernail extract is as follows: Raw material pretreatment: Dry and pulverize the Buddha's fingernail; Solution impregnation: Place the Buddha's fingernail powder in an Erlenmeyer flask and add an appropriate amount of water or ethanol solvent; Ultrasonic-assisted extraction: Place the conical flask in an ultrasonic cleaner or a professional ultrasonic extractor, set the power to 300W, and perform ultrasonic extraction at 50~60℃ for 20~40 minutes. Post-processing and concentration: After extraction, the solution is cooled and filtered, then the filtrate is concentrated under reduced pressure and dried to obtain the Buddha's fingernail extract.

[0031] Example 1 A stem cell preparation for improving the course of diabetes, comprising, by weight percentage, 0.5% dental pulp mesenchymal stem cells, 1.5% bergamot nail extract, 1.1% saikosaponin C, and 3% glycerol, mixed and brought to a final volume with physiological saline to obtain the stem cell preparation.

[0032] The preparation method of stem cell preparations includes the following steps: (1) Passage dental pulp mesenchymal stem cells to P4 generation cells for later use; (2) Weigh and prepare the Buddha's fingernail extract; (3) Weigh out dental pulp mesenchymal stem cells, bergamot extract, saikosaponin C and glycerol according to the proportion. First, dissolve saikosaponin C and glycerol in physiological saline at 5°C. Add bergamot extract and mix well. Finally, slowly add cell suspension and mix gently. Then, adjust the volume with physiological saline to obtain stem cell preparation.

[0033] Example 2 The difference between Example 2 and Example 1 is that the stem cell preparation formula contains, by weight percentage, 0.85% bone marrow mesenchymal stem cells, 3% bergamot extract, 1.85% saikosaponin C and 4.5% glycerol, and the specific experimental steps are the same as in Example 1.

[0034] Example 3 The difference between Example 3 and Example 1 is that the stem cell preparation formula, by weight percentage, includes 1.2% bone marrow mesenchymal stem cells, 4.5% bergamot nail extract, 2.6% saikosaponin C and 6% glycerol, and the specific experimental steps are the same as in Example 1.

[0035] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that no bone marrow mesenchymal stem cells were added.

[0036] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that no Buddha's fingernail extract was added.

[0037] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that Saikosaponin C was not added.

[0038] Comparative Example 4 The difference between Comparative Example 4 and Example 1 is that no bergamot nail extract and saikosaponin C were added.

[0039] The following experiments were conducted using the stem cell preparations from Examples 1-3 and Comparative Examples 1-4: Establishment of a diabetic model in SD rats Rats with an initial weight of 220-250g were selected and fed a high-fat, high-sugar diet for 5-9 weeks, followed by streptozotocin injection (50mg / kg). Blood glucose concentration in the tail vein of the rats was measured using a glucometer. Individuals with two consecutive blood glucose values ​​≥16.7mmol / L were selected to confirm successful modeling and used as the diabetic model group for subsequent experiments.

[0040] Seventy diabetic rats were randomly divided into seven groups: experimental groups 1-3, comparative groups 1-4, and a blank control group. Each group received a 1 mL injection via tail vein daily. After four weeks of experimentation, the rats were fasted for six hours and then administered 20% glucose by gavage. The average blood glucose level (mmol·L⁻¹) was measured. -1 The results are shown in the table below:

[0041] The results in the table above show that in diabetic rats, blood glucose levels remained high for 30-60 minutes after glucose administration, with a slight decrease at 120 minutes. Compared to the diabetic group, the blood glucose levels in experimental groups 1-3 and control groups 1-4 administered the preparation were significantly lower within 60 minutes compared to the model group. This result indicates that the glycemic regulation function of the experimental and control groups was improved compared to the diabetic group. Further analysis showed that experimental groups 1-3 exhibited good glycemic regulation effects at different concentrations, while the improvement in glycemic regulation function in control groups 1-3 was significantly reduced when any key component was missing. Notably, control group 4, which simultaneously lacked both bergamot extract and saikosaponin C, showed a further reduction in glycemic regulation function, with an effect lower than that of control groups 1-3 lacking only a single component. These data fully demonstrate that the stem cell preparation developed in this application has an ameliorative effect on impaired glycemic regulation function, and that the improvement effect is maximized when all four components are present simultaneously.

[0042] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A stem cell preparation for improving the course of diabetes, characterized in that, It contains the following components: dental pulp mesenchymal stem cells, bergamot nail extract, saikosaponin C, and glycerin.

2. The stem cell preparation for improving the course of diabetes as described in claim 1, characterized in that, The components, by weight percentage, include 0.5-1.2% dental pulp mesenchymal stem cells, 1.5-4.5% Buddha's fingernail extract, 1.1-2.6% saikosaponin C, and 3-6% glycerol. They are mixed and brought to a final volume with physiological saline to obtain a stem cell preparation.

3. The stem cell preparation for improving the course of diabetes as described in claim 1, characterized in that, The concentration of the dental pulp mesenchymal stem cells was 1 x 10⁻⁶. 6 ~10 7 per mL.

4. The stem cell preparation for improving the course of diabetes as described in claim 1, characterized in that, The dental pulp mesenchymal stem cells were passaged to generation P3-5.

5. A method for preparing a stem cell preparation for improving the course of diabetes, characterized in that, It includes the following steps: (1) Passage dental pulp mesenchymal stem cells to P3-5 generation cells for later use; (2) Weigh and prepare the Buddha's fingernail extract; (3) Weigh out dental pulp mesenchymal stem cells, bergamot extract, saikosaponin C and glycerol according to the proportion. First, dissolve saikosaponin C and glycerol in physiological saline at 5°C. Add bergamot extract and mix well. Finally, slowly add cell suspension and mix gently. Then, adjust the volume with physiological saline to obtain stem cell preparation.

6. The method for preparing the stem cell preparation for improving the course of diabetes as described in claim 5, characterized in that, The method for preparing the dental pulp mesenchymal stem cells comprises the following steps: Cell resuscitation: Thaw 1 mL of cell suspension cryovial by rapid shaking in a 37°C water bath, add 5 mL of 10% fetal bovine serum and penicillin-streptomycin-containing mouse bone marrow mesenchymal stem cell complete culture medium, mix well, centrifuge at 800 rpm for 5 min, and discard the supernatant; add 4-6 mL of the above complete culture medium, mix well, and seed into culture flasks, and incubate overnight at 37°C in a 5% CO2 incubator; change the medium the next day, and passage when the cell density reaches 80%-90%; Cell passage: Discard the culture supernatant, rinse the cells 1-2 times with PBS free of calcium and magnesium ions; add 1-2 mL of 0.25% trypsin, digest at 37°C for 1-2 min, and observe the cell digestion under a microscope; after complete digestion, add 5 mL of culture medium containing 10% fetal bovine serum to stop digestion; gently pipette to collect the cell suspension, centrifuge at 800 rpm for 10 min, discard the supernatant, add 1-2 mL of culture medium and mix well; seed into new culture flasks at a ratio of 1:3, and passage to P3-5 for later use.

7. The method for preparing the stem cell preparation for improving the course of diabetes as described in claim 5, characterized in that, The dental pulp mesenchymal stem cells are P4 generation cells.

8. The method for preparing the stem cell preparation for improving the course of diabetes as described in claim 5, characterized in that, The preparation method of the Buddha's fingernail extract includes the following steps: Raw material pretreatment: Dry and pulverize the Buddha's fingernail; Solution impregnation: Place the Buddha's fingernail powder in an Erlenmeyer flask and add an appropriate amount of water or ethanol solvent; Ultrasonic-assisted extraction: Place the conical flask in an ultrasonic cleaner or a professional ultrasonic extractor, set the power to 300W, and perform ultrasonic extraction at 50~60℃ for 20~40 minutes. Post-processing and concentration: After extraction, the solution is cooled and filtered, then the filtrate is concentrated under reduced pressure and dried to obtain the Buddha's fingernail extract.

9. The use of the stem cell preparation according to any one of claims 1 to 4 in the preparation of a drug for improving the course of diabetes.

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