Culture method of mesenchymal stem cells and application of mesenchymal stem cells in treatment of diabetes mellitus

Abstract

The invention relates to the technical field of biological medicines, in particular to a culture method of mesenchymal stem cells and application of the mesenchymal stem cells in treatment of diabetes mellitus. The umbilical cord mesenchymal stem cells are cultured by adopting a gel matrix, and the gel matrix is prepared from the following raw materials in parts by weight: 1 to 2 parts of cellulose nanofiber, 1 to 2 parts of p-toluenesulfonyl-arginine, 0.5 to 1 part of phenylalanine, 1 to 1.5 parts of chitosan, 0.9 to 1.2 parts of caffeic acid, 0.5 to 0.8 part of cysteine, 0.8 to 1.2 parts of polyvinyl alcohol and 1 to 2 parts of rosemary essential oil. The cultured mesenchymal stem cells are high in proliferation activity and patch rate, can effectively reduce blood sugar, and can be applied to preparation of drugs for treating diabetes mellitus.

Description

Technical Field

[0001] The present invention relates to the field of biological medicine technology, and particularly relates to a method for culturing mesenchymal stem cells and its application in the treatment of diabetes. Background Art

[0002] Diabetes is a common chronic metabolic disease, the main feature of which is long-term elevated blood glucose levels, which can lead to various complications, such as cardiovascular diseases, kidney diseases, retinopathy, neuropathy, etc., seriously affecting the quality of life and life expectancy of patients. At present, the treatment of diabetes mainly includes diet control, exercise therapy, drug therapy (such as insulin, oral hypoglycemic drugs, etc.) and islet transplantation. However, these treatment methods all have certain limitations. For example, drug therapy can only control blood glucose levels, cannot cure diabetes fundamentally, and may produce side effects after long-term use; although islet transplantation can effectively improve blood glucose control, it is difficult to be widely applied due to problems such as donor shortage and immune rejection. Mesenchymal stem cells are a type of adult stem cells with self-renewal ability and multi-directional differentiation potential, which are widely present in tissues such as umbilical cord, bone marrow, adipose tissue, dental pulp, placenta, etc., and have important application values in the treatment of many diseases. In recent years, with the gradual use of mesenchymal stem cells in the clinical treatment of many major diseases, the research on the treatment of diabetes with mesenchymal stem cells has also achieved great results. Clinical experimental results show that the reduction effect of mesenchymal stem cells on type 2 diabetic patients (HbAc1 reduction of 3%-5%) is better than that of traditional hypoglycemic drugs (HbAc1 reduction of 0.5%-1.5%). Among them, umbilical cord mesenchymal stem cells (UC-MSCs) have the characteristics of wide source, low immunogenicity, non-invasive, no ethical issues and low tumorigenicity, and are considered to be a better choice for MSCs in clinical applications. Summary of the Invention

[0003] Aiming at the deficiencies of the prior art, the present invention proposes a method for culturing mesenchymal stem cells and its application in the treatment of diabetes.

[0004] The present invention is achieved through the following technical solutions: A method for culturing mesenchymal stem cells, comprising the following steps: S1: Take a fresh umbilical cord, soak and wash it with physiological saline containing 1% penicillin-streptomycin, cut it into lengths of 1-3 cm, separate and remove the umbilical cord outer membrane, umbilical artery and vein, cut it into tissue blocks of 1 mm 3 in size, add complete medium to submerge the tissue blocks, and culture them under the conditions of 5% CO 2 , 37 °C and saturated humidity; S2: After culturing in step S1 for 72 h, the culture medium was completely replaced, and then half of the complete culture medium was replaced every 3 - 4 days. When the cell growth reached a confluence of 70 - 80%, it was digested and passaged with 0.20% trypsin to obtain passage 3 umbilical cord mesenchymal stem cells; S3: A gel matrix with a thickness of 2 mm was placed at the bottom of a six - well culture plate, and passage 3 umbilical cord mesenchymal stem cells in the logarithmic growth phase of proliferation were inoculated at an inoculation density of 1×10 5 cells / well, and 2.5 mL of complete culture medium was added to each well. The fresh complete culture medium was replaced every 2 - 3 days.

[0005] Further, the complete culture medium comprises the following components: 1% penicillin - streptomycin, 10% fetal bovine serum, and the balance is DMEM / F12 medium.

[0006] Further, the raw materials for preparing the gel matrix include the following components in parts by weight: 1 - 2 parts of cellulose nanofibers, 1 - 2 parts of tosyl - arginine, 0.5 - 1 part of phenylalanine, 1 - 1.5 parts of chitosan, 0.9 - 1.2 parts of caffeic acid, 0.5 - 0.8 part of cysteine, 0.8 - 1.2 parts of polyvinyl alcohol, and 1 - 2 parts of rosemary essential oil.

[0007] Further, the preparation method of the gel matrix comprises the following steps: (1) Mix cellulose nanofibers, tosyl - arginine, phenylalanine, Fmoc - Cl, EDC·HCl, and DMAP in DMF, stir at 400 - 500 rpm at room temperature for 14 - 16 h, centrifuge at 8000 rpm for 10 - 15 min, and wash the precipitate with deionized water and ethanol to obtain Fmoc - amino acid - nanofibers; (2) Ultrasonically disperse the Fmoc - amino acid - nanofibers obtained in step (1) in a 20% (v / v) piperidine / DMF solution at 300 - 400 W for 20 min, centrifuge at 8000 rpm for 10 - 15 min, wash the precipitate with DMF, and freeze - dry at - 40℃ to obtain amino acid - nanofibers; (3) At 50℃, dissolve chitosan in a 2% (v / v) acetic acid aqueous solution, cool to room temperature to obtain a chitosan solution. At 4℃, dissolve caffeic acid and EDC in a 70% (v / v) ethanol solution, add NHS, stir and react in an ice - water bath for 1 h, gradually add the chitosan solution dropwise, stir and react in an ice - water bath for 30 min, stir in the dark at room temperature for 24 h, adjust the pH value to 8 - 9, heat in a water bath at 50℃ for 20 - 30 min, dialyze for 24 h, change the water every 4 h, and freeze - dry at - 40℃ to obtain modified chitosan; (4) Mix polyvinyl alcohol and deionized water evenly, and place them in a water bath at 80 °C for 2 h. Then add cysteine, and use H 2 SO 4 to adjust the pH value to 2 - 3. Under nitrogen protection, place them in a water bath at 90 - 100 °C and stir at 300 - 400 rpm for 6 - 8 h. Dialyze with deionized water for 24 h, changing the water every 6 h, to obtain modified polyvinyl alcohol; (5) Take the modified chitosan obtained in step (3) and the modified polyvinyl alcohol obtained in step (4), add them to PBS buffer solution with pH 7.4, and heat in a water bath at 37 °C for 15 - 20 min to obtain a mixed solution. Take the amino acid - nanofibers obtained in step (2), mix them evenly in deionized water, add rosemary essential oil, and while stirring, add the mixed solution. Stir at 11000 - 13000 rpm for 3 - 5 min, let it stand at 37 °C for 20 - 30 min, stand at room temperature for 12 - 14 h, freeze at -20 °C for 10 - 12 h, and thaw at room temperature to obtain a gel matrix.

[0008] Furthermore, in step (1), the dosage ratio of the cellulose nanofibers, Fmoc - Cl, EDC·HCl, DMAP, and DMF is 1 g:0.75 g:1.25 g:0.08 g:50 mL.

[0009] Furthermore, in step (2), the dosage ratio of the 20% (v / v) piperidine / DMF solution to the cellulose nanofibers is 1 g:20 mL.

[0010] Furthermore, in step (3), the dosage ratio of the chitosan to the 2% (v / v) acetic acid aqueous solution is 1 g:50 mL.

[0011] Furthermore, in step (3), the mass concentration of caffeic acid in a 70% (v / v) ethanol solution is 20 mg / mL.

[0012] Furthermore, in step (3), the mass ratio of caffeic acid, EDC, and NHS is 3:3:2.

[0013] Furthermore, in step (4), the mass concentration of polyvinyl alcohol in deionized water is 10 - 15 mg / mL.

[0014] Furthermore, in step (5), the mass concentration of the amino acid - nanofibers in deionized water is 5 mg / mL.

[0015] Furthermore, in step (5), the mass concentration of the modified chitosan in PBS buffer solution is 10 - 20 mg / mL.

[0016] Furthermore, the present invention also provides the use of the mesenchymal stem cells in the preparation of a medicament for treating diabetes.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a method for culturing mesenchymal stem cells and its application in the treatment of diabetes. Through the design of the culture system and the development of a functionalized gel matrix, multi-dimensional improvements in cell activity, functional stability, and therapeutic efficacy have been achieved. The gel matrix prepared by the present invention forms a three-dimensional network structure, simulating the characteristics of the extracellular matrix and enhancing the cell adhesion rate. The present invention realizes the directional grafting of amino acids on the surface of nanofibers through the Fmoc-Cl protection strategy. By covalently binding cellulose nanofibers with hydrophobic amino acids, the hydrophobicity is enhanced. Using amino acid-nanofibers as stabilizers can enhance the emulsion stability, encapsulate rosemary essential oil, achieve a sustained release effect, continuously inhibit bacteria and antioxidants, help maintain the healthy growth state of cells, facilitate cell growth and proliferation, and enhance cell activity. The present invention grafts and modifies chitosan with caffeic acid, connecting phenolic hydroxyl groups on the surface of chitosan. The phenolic hydroxyl groups on caffeic acid are easily oxidized into quinone compounds, which can form Schiff base bonds with the amino groups of amino acid-nanofibers and crosslink with each other. At the same time, the double bond of caffeic acid in the modified chitosan can undergo an addition reaction with the mercapto group of cysteine-modified polyvinyl alcohol and crosslink with each other to form a three-dimensional network structure. The formed network structure can enhance the mechanical properties of the gel, provide attachment sites for cells, provide a biomimetic three-dimensional growth environment for mesenchymal stem cells, facilitate cell adhesion and proliferation, improve cell culture efficiency and quality, provide good support for cells, facilitate the formation of the cell skeleton and the maintenance of tension, thereby promoting cell adhesion, providing a microenvironment similar to that in vivo for cells, facilitating the interaction between cells and the gel matrix, making it easier for cells to adhere, and promoting cell adhesion. The mesenchymal stem cells obtained by this culture method have good biological activity, can effectively restore glucose homeostasis and relieve insulin resistance, improve islet function, and UC-MSCs can regulate the proportion of Treg / Th17 cells in the spleen and peripheral blood, weaken the activation of NLRP3 inflammasome to relieve type 2 diabetes mellitus (T2DM), supplement the function of damaged islet β cells, and regulate blood glucose levels. Description of the Drawings

[0018] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only those of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0019] Figure 1The blood glucose - lowering effects of the mesenchymal stem cells cultured in Example 1 of the present invention and Comparative Examples 1 - 4 Figure 2 The effects of the mesenchymal stem cells of the present invention on mouse islets. Among them, A is the eosin (H&E) staining of mouse pancreas, and B is the proportion of islet area in mouse pancreatic tissue Figure 3 The mesenchymal stem cells of the present invention regulate CD4 + CD25 + Foxp3 + The percentage of Treg and the mean fluorescence intensity of Foxp3 in the spleen tissue and peripheral blood of T2DM mice Figure 4 The mesenchymal stem cells of the present invention regulate CD4 + CD3 + The percentage of Th17 of IL - 17A and the mean fluorescence intensity of IL - 17A Figure 5 The HE staining and PAS staining of the liver of T2DM mice in the present invention Figure 6 The effects of the mesenchymal stem cells of the present invention on the levels of serum - related inflammatory factors in T2DM mice Figure 7 The effects of the mesenchymal stem cells of the present invention on the levels of NLRP3 - related transcription factors in the liver of T2DM mice Figure 8 The effects of the mesenchymal stem cells of the present invention on the expression levels of NLRP3 - related proteins in the liver of T2DM mice Figure 9 The effects of the mesenchymal stem cells of the present invention on the glucose consumption, glycogen synthesis content, and levels of NLRP - related transcription factors in HepG2 cells Figure 10 The effects of the mesenchymal stem cells of the present invention on the levels of inflammatory factors in the supernatant of HepG2 cells Figure 11 The effects of the mesenchymal stem cells of the present invention on the expression levels of NLRP - related proteins in HepG2 cells Detailed implementation manners

[0020] To make the objectives, technical solutions, and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in combination with specific embodiments. However, the present invention is not limited to the following embodiments. It should be noted that unless otherwise specified, the chemical reagents involved in the present invention are purchased through commercial channels

[0021] Example 1: This example provides a method for culturing mesenchymal stem cells, including the following steps S1: Take a fresh umbilical cord, soak and wash it with physiological saline containing 1% penicillin-streptomycin, cut it into 3 cm lengths, separate and remove the umbilical cord outer membrane, umbilical arteries and veins, and cut it into tissue blocks with a size of 1 mm 3 in size. Add complete medium (1% penicillin-streptomycin, 10% fetal bovine serum, and the balance is DMEM / F12 medium) to submerge the tissue blocks, and place them in an incubator at 5% CO 2 ₂, 37 °C with saturated humidity for culture; S2: After culturing in step S1 for 72 h, change the medium completely. Then, change half of the complete medium every 4 days. When the cell confluence reaches 80%, digest and passage them with 0.20% trypsin to obtain passage 3 umbilical cord mesenchymal stem cells; S3: Place a gel matrix with a thickness of 2 mm at the bottom of a six-well culture plate, inoculate passage 3 umbilical cord mesenchymal stem cells in the logarithmic growth phase of proliferation, and the inoculation density is 1×10 5 cells / well. Add 2.5 mL of complete medium to each well, and change the fresh complete medium every 3 days.

[0022] The preparation raw materials of the gel matrix include the following components in parts by weight: 2 parts of cellulose nanofibers, 2 parts of tosyl-arginine, 1 part of phenylalanine, 1.5 parts of chitosan, 1.2 parts of caffeic acid, 0.8 part of cysteine, 1.2 parts of polyvinyl alcohol, and 2 parts of rosemary essential oil.

[0023] The preparation method of the gel matrix includes the following steps: (1) Add 2 g of cellulose nanofibers, 2 g of tosyl-arginine, 1 g of phenylalanine, 1.5 g of Fmoc-Cl, 2.5 g of EDC·HCl, and 0.16 g of DMAP to 100 mL of DMF, mix well, stir at 500 rpm at room temperature for 14 h, centrifuge at 8000 rpm for 15 min, and wash the precipitate with deionized water and ethanol to obtain Fmoc-amino acid-nanofibers; (2) Ultrasonically disperse the Fmoc-amino acid-nanofibers obtained in step (1) in 40 mL of 20% (v / v) piperidine / DMF solution at 400 W for 20 min, centrifuge at 8000 rpm for 15 min, wash the precipitate with DMF, and freeze-dry at -40 °C to obtain amino acid-nanofibers; (3) At 50 °C, 1.5 g of chitosan was dissolved in 75 mL of 2% (v / v) acetic acid aqueous solution, cooled to room temperature to obtain a chitosan solution. At 4 °C, 1.2 g of caffeic acid and 1.2 g of EDC were dissolved in 60 mL of 70% (v / v) ethanol solution, 0.8 g of NHS was added, and the reaction was stirred in an ice-water bath for 1 h. Then it was added dropwise to the chitosan solution, and the reaction was stirred in an ice-water bath for 30 min, and then stirred in the dark at room temperature for 24 h. The pH value was adjusted to 9, heated in a water bath at 50 °C for 30 min, dialyzed for 24 h, and the water was changed every 4 h, and freeze-dried at -40 °C to obtain modified chitosan; (4) 1.2 g of polyvinyl alcohol was mixed with 80 mL of deionized water, heated in a water bath at 80 °C for 2 h, 0.8 g of cysteine was added, and the pH value was adjusted to 3 with H 2 SO 4 Under nitrogen protection, it was heated in a water bath at 100 °C and stirred at 400 rpm for 8 h, and dialyzed with deionized water for 24 h, and the water was changed every 6 h to obtain modified polyvinyl alcohol; (5) The modified chitosan obtained in step (3) and the modified polyvinyl alcohol obtained in step (4) were added to PBS buffer solution with pH 7.4. The mass concentration of the modified chitosan in the PBS buffer solution was 20 mg / mL, and it was heated in a water bath at 37 °C for 20 min to obtain a mixed solution. The amino acid-nanofibers obtained in step (2) were added to deionized water and mixed evenly. The mass concentration of the amino acid-nanofibers in deionized water was 5 mg / mL, 2 g of rosemary essential oil was added, and the mixed solution was added while stirring, stirred at 13000 rpm for 3 min, left standing at 37 °C for 30 min, left standing at room temperature for 14 h, frozen at -20 °C for 12 h, and thawed at room temperature to obtain a gel matrix.

[0024] Example 2: This example provides a method for culturing mesenchymal stem cells, which includes the following steps: S1: Take fresh umbilical cord, soak and wash it with physiological saline containing 1% penicillin-streptomycin, cut it into 1 cm length, separate and remove the umbilical cord membrane, umbilical artery and vein, cut it into tissue blocks with a size of 1 mm 3 Add complete medium (1% penicillin-streptomycin, 10% fetal bovine serum, and the balance is DMEM / F12 medium) to submerge the tissue blocks, and culture them under the conditions of 5% CO 2 2, 37 °C and saturated humidity; S2: After culturing in step S1 for 72 h, change the liquid in full volume, and then change half of the complete medium every 3 days. When the cell confluence reaches 70%, digest and passage with 0.20% trypsin to obtain passage 3 umbilical cord mesenchymal stem cells; S3: Place a gel matrix with a thickness of 2 mm at the bottom of a six-well plate culture plate, inoculate passage 3 umbilical cord mesenchymal stem cells in the logarithmic growth phase of proliferation, with an inoculation density of 1×10 5 cells / well, add 2.5 mL of complete medium to each well, and replace the fresh complete medium every 2 days.

[0025] The preparation raw materials of the gel matrix include the following components in parts by weight: 1 part of cellulose nanofibers, 1 part of tosyl-arginine, 0.5 part of phenylalanine, 1 part of chitosan, 0.9 part of caffeic acid, 0.5 part of cysteine, 0.8 part of polyvinyl alcohol, and 1 part of rosemary essential oil.

[0026] The preparation method of the gel matrix includes the following steps: (1) Add 1 g of cellulose nanofibers, 1 g of tosyl-arginine, 0.5 g of phenylalanine, 0.75 g of Fmoc-Cl, 1.25 g of EDC·HCl, and 0.08 g of DMAP to 50 mL of DMF, mix well, stir at 400 rpm at room temperature for 16 h, centrifuge at 8000 rpm for 10 min, and wash the precipitate with deionized water and ethanol to obtain Fmoc-amino acid-nanofibers; (2) Ultrasonically disperse the Fmoc-amino acid-nanofibers obtained in step (1) in 20 mL of 20% (v / v) piperidine / DMF solution at 300 W for 20 min, centrifuge at 8000 rpm for 10 min, wash the precipitate with DMF, and freeze-dry at -40°C to obtain amino acid-nanofibers; (3) At 50°C, dissolve 1 g of chitosan in 50 mL of 2% (v / v) acetic acid aqueous solution, cool to room temperature to obtain a chitosan solution. At 4°C, dissolve 0.9 g of caffeic acid and 0.9 g of EDC in 40 mL of 70% (v / v) ethanol solution, add 0.6 g of NHS, stir and react in an ice-water bath for 1 h, gradually add the chitosan solution dropwise, stir and react in an ice-water bath for 30 min, stir in the dark at room temperature for 24 h, adjust the pH value to 8, heat in a water bath at 50°C for 20 min, dialyze for 24 h, change the water every 4 h, and freeze-dry at -40°C to obtain modified chitosan; (4) Take 0.8 g of polyvinyl alcohol and mix it with 80 mL of deionized water, heat in a water bath at 80°C for 2 h, add 0.5 g of cysteine, and use H 2 SO 4 to adjust the pH value to 2. Under nitrogen protection, heat in a water bath at 90°C and stir at 300 rpm for 8 h, dialyze with deionized water for 24 h, change the water every 6 h, to obtain modified polyvinyl alcohol; (5) Take the modified chitosan obtained in step (3) and the modified polyvinyl alcohol obtained in step (4), add them to PBS buffer with a pH of 7.4. The mass concentration of the modified chitosan in the PBS buffer is 10 mg / mL. Heat in a water bath at 37 °C for 15 min to obtain a mixed solution. Take the amino acid-nanofibers obtained in step (2), add them to deionized water and mix well. The mass concentration of the amino acid-nanofibers in the deionized water is 5 mg / mL. Add 1 g of rosemary essential oil, and while stirring, add the mixed solution. Stir at 11000 rpm for 5 min, let stand at 37 °C for 20 min, let stand at room temperature for 12 h, freeze at -20 °C for 10 h, and thaw at room temperature to obtain a gel matrix.

[0027] Example 3: This example provides a method for culturing mesenchymal stem cells, which includes the following steps: S1: Take fresh umbilical cords, soak and wash them with physiological saline containing 1% penicillin-streptomycin, cut them into 2 cm lengths, separate and remove the umbilical cord outer membrane, umbilical arteries and veins, and cut them into tissue blocks with a size of 1 mm 3 in size. Add complete medium (1% penicillin-streptomycin, 10% fetal bovine serum, and the balance is DMEM / F12 medium) to submerge the tissue blocks, and place them in an incubator with 5% CO 2 2, at 37 °C and saturated humidity for culture; S2: After culturing in step S1 for 72 h, change the medium in full volume, and then change half of the complete medium every 3 days. When the cell confluence reaches 75%, digest and passage with 0.20% trypsin to obtain passage 3 umbilical cord mesenchymal stem cells; S3: Place a gel matrix with a thickness of 2 mm at the bottom of a six-well plate culture plate, inoculate passage 3 umbilical cord mesenchymal stem cells in the logarithmic growth phase, with an inoculation density of 1×10 5 cells / well, add 2.5 mL of complete medium to each well, and change the fresh complete medium every 2 days.

[0028] The raw materials for preparing the gel matrix include the following components in parts by weight: 1.5 parts of cellulose nanofibers, 1.5 parts of tosyl-arginine, 0.8 part of phenylalanine, 1.2 parts of chitosan, 1.02 parts of caffeic acid, 0.6 part of cysteine, 1 part of polyvinyl alcohol, and 1.5 parts of rosemary essential oil.

[0029] The method for preparing the gel matrix includes the following steps: (1) Add 1.5 g of cellulose nanofibers, 1.5 g of tosyl-arginine, 0.8 g of phenylalanine, 1.125 g of Fmoc-Cl, 1.875 g of EDC·HCl and 0.12 g of DMAP to 75 mL of DMF, mix well, stir at 450 rpm for 15 h at room temperature, centrifuge at 8000 rpm for 12 min, and wash the precipitate with deionized water and ethanol to obtain Fmoc-amino acid-nanofibers; (2) Ultrasonically disperse the Fmoc-amino acid-nanofibers obtained in step (1) in 30 mL of 20% (v / v) piperidine / DMF solution at 350 W for 20 min, centrifuge at 8000 rpm for 12 min, wash the precipitate with DMF, and freeze-dry at -40 °C to obtain amino acid-nanofibers; (3) At 50 °C, dissolve 1.2 g of chitosan in 60 mL of 2% (v / v) acetic acid aqueous solution, cool to room temperature to obtain a chitosan solution. At 4 °C, dissolve 1.02 g of caffeic acid and 1.02 g of EDC in 51 mL of 70% (v / v) ethanol solution, add 0.68 g of NHS, stir and react in an ice-water bath for 1 h, add the chitosan solution dropwise, stir and react in an ice-water bath for 30 min, stir in the dark at room temperature for 24 h, adjust the pH value to 8.5, heat in a water bath at 50 °C for 25 min, dialyze for 24 h, change the water every 4 h, and freeze-dry at -40 °C to obtain modified chitosan; (4) Take 1 g of polyvinyl alcohol and mix it with 80 mL of deionized water, heat in a water bath at 80 °C for 2 h, add 0.6 g of cysteine, use H 2 SO 4 Adjust the pH value to 2.5, stir at 350 rpm in a water bath at 95 °C for 7 h under nitrogen protection, dialyze with deionized water for 24 h, change the water every 6 h, to obtain modified polyvinyl alcohol; (5) Take the modified chitosan obtained in step (3) and the modified polyvinyl alcohol obtained in step (4), add them to PBS buffer solution with pH 7.4. The mass concentration of the modified chitosan in the PBS buffer solution is 15 mg / mL, heat in a water bath at 37 °C for 18 min to obtain a mixed solution. Take the amino acid-nanofibers obtained in step (2), mix them with deionized water. The mass concentration of the amino acid-nanofibers in the deionized water is 5 mg / mL, add 1.5 g of rosemary essential oil, add it to the mixed solution while stirring, stir at 12000 rpm for 4 min, stand at 37 °C for 25 min, stand at room temperature for 13 h, freeze at -20 °C for 11 h, and thaw at room temperature to obtain a gel matrix.

[0030] The difference between Comparative Example 1 and Example 1 is only that cellulose nanofibers are used to replace amino acid-nanofibers; The difference between Comparative Example 2 and Example 1 is only that chitosan is used to replace the modified chitosan; The difference between Comparative Example 3 and Example 1 is only that polyvinyl alcohol is used to replace the modified polyvinyl alcohol; The difference between Comparative Example 4 and Example 1 is only that rosemary essential oil is not added.

[0031] Experimental Example 1: Umbilical cord mesenchymal stem cells after culturing for 7 d in Example 1 and Comparative Examples 1-4 were collected for counting, and the proliferation multiple was calculated. The proliferation multiple = the total number of cells after culturing for 7 d / the total number of initially inoculated cells. At 24 h after inoculating the cells, cells in each group were randomly selected, and the cell supernatant (the supernatant contained non-adherent cells) was collected, washed twice with PBS, digested with 0.25% trypsin, and centrifuged to prepare a single-cell suspension for counting. The cell adhesion rate was calculated. The adhesion rate = (the number of adherent cells / the total number of harvested cells) × 100%. The results are shown in Table 1.

[0032] Table 1: The results in Table 1 showed that the cell proliferation multiple and adhesion rate of the Example 1 group were higher than those of Comparative Examples 1-4. The three-dimensional network structure formed by the gel matrix of the present invention was beneficial to cell adhesion and proliferation, improved cell biological activity, and could enhance the therapeutic effect of umbilical cord mesenchymal stem cells.

[0033] Experimental Example 2: Normal male C57BL6J mice (18 ± 2 g) at 6 weeks of age were raised in an SPF-level environment, with free access to water and food during the period, room temperature of 22.5 ± 2.5 °C, humidity of 50 - 70%, and a 12 h / 12 h light-dark cycle. The mice were divided into a model group and a normal group, and were given HFD (High-fat diet, 60 kcal% fat) (Ready Dietech) and normal mouse diet for 8 weeks, respectively. The mice in the model group were intraperitoneally injected with STZ 40 mg / kg for 3 consecutive days. One week after STZ injection, an oral glucose tolerance test (OGTTS) and an intraperitoneal insulin tolerance test (IPITTS) were performed to ensure the establishment of a type 2 diabetes mellitus (T2DM) mouse model. All mice were divided into 7 groups, with 8 mice in each group: Control group, blank group, Example 1 group, Comparative Example 1 group, Comparative Example 2 group, Comparative Example 3 group, Comparative Example 4 group. In the Example 1 group and Comparative Examples 1-4 groups, the established diabetic model mice were intraperitoneally injected with 1 × 10 6 umbilical cord mesenchymal stem cells suspended in 0.2 mL PBS (cultured for 7 d after inoculation) once a week for 5 consecutive weeks. The mice in the blank group were injected with an equal amount of PBS, and the mice in the Control group were injected with an equal amount of PBS as the normal group mice. Blood glucose was detected at the designated time points, and the results are as Figure 1 shown.

[0034] Figure 1 The results showed that after injection of umbilical cord mesenchymal stem cells, the blood glucose level of mice decreased significantly, indicating that umbilical cord mesenchymal stem cells can be applied to the treatment of diabetes. Among them, the hypoglycemic effect of Example 1 was the best. In Comparative Example 1, cellulose nanofibers were not modified, resulting in a decrease in emulsion stability, a reduction in the sustained-release effect, and a decrease in the biological activity of the cultured mesenchymal stem cells. In Comparative Example 2, chitosan was not modified, resulting in a decrease in crosslinking degree, a reduction in the stability of the gel matrix, and a decrease in cell activity. In Comparative Example 3, polyvinyl alcohol was not modified, resulting in a decrease in crosslinking degree, a reduction in stability, and a decrease in cell activity. In Comparative Example 4, rosemary essential oil was not added, resulting in a decrease in antioxidant activity and a decrease in cell biological activity.

[0035] Experimental Example 3: According to the method of Experimental Example 2, a mouse diabetes model was constructed. Mice in the normal group were injected with PBS as the Control group. Diabetic mice were injected with PBS and umbilical cord mesenchymal stem cells cultured for 7 days after inoculation with Example 1, which were the T2DM group and the UC-MSCs group, respectively. After 5 injections, the pancreatic tissues of each group of mice were fixed overnight in 4% paraformaldehyde solution before paraffin embedding. The tissues were cut into 5-μm thick sections, stained with hematoxylin-eosin (H&E), and the proportion of islet area in the pancreatic tissue was statistically analyzed. The results are as Figure 2 shown.

[0036] Figure 2 The results showed that the pancreatic islet volume of mice in the UC-MSC group was higher than that in the T2DM group. HE staining showed that T2DM could cause a decrease in the number of islet cells and a reduction in islet volume. Multiple injections of UC-MSCs could improve the decrease in the number of islet cells and the reduction in islet volume caused by T2DM. The umbilical cord mesenchymal stem cells of the present invention can effectively improve islet function.

[0037] Experimental Example 4: Mouse blood samples were collected in heparin sodium anticoagulant tubes, lymphocyte separation medium was added, and centrifuged at 20 °C and 700 g for 20 min. The buffy coat cells at the middle interface were aspirated and lysed with red blood cell lysate. A part of the cells was cultured in RPMI1640 medium containing 1% penicillin-streptomycin, 10% serum, and cell stimulation mixture (plus protein transport inhibitor) in a 37 °C, 5% CO 2 incubator for 6-8 hours, and then incubated with Th17 cell antibody. Another part of the cells was incubated with Treg cell antibody. The spleen was removed from T2DM mice and gently ground in RPMI1640 medium to extract spleen cells. The samples were filtered through a 70-μm filter and lysed with red blood cell lysate. A part of the spleen cells was cultured in RPMI1640 medium containing 1% penicillin-streptomycin, 10% serum, and cell stimulation mixture (plus protein transport inhibitor) in a 37 °C, 5% CO 2Cultivate for 6 - 8 hours in an incubator, then incubate with Th17 cell antibodies, and incubate another part of the cells with Treg cell antibodies. Use a flow cytometer to detect the proportions of Treg and Th17 cells in the mouse spleen and peripheral blood. For Th17 detection, after culturing with a cell stimulation mixture (plus a protein transport inhibitor) for 6 - 8 hours, the collected cells are blocked with 1% BSA in PBS at 4°C for 10 min, and incubated with cell surface antibodies CD3 - APC and CD4 - FITC at 4°C for 30 minutes. For the intracellular antibody IL - 17A - PE, permeabilize and fix the cells with an intracellular fixation and permeabilization buffer at 4°C in the dark for 30 min before staining. Finally, incubate the intracellular antibody IL - 17A - PE at 4°C in the dark for 30 min, wash the cells with PBS containing 1% FBS, and detect using a flow cytometer. For Treg cell detection, the collected cells are blocked with 1% BSA in PBS at 4°C for 10 min, and incubated with cell surface antibodies CD25 - APC and CD4 - FITC at 4°C for 30 min. For the intracellular antibody Foxp3 - PE, permeabilize and fix the cells with an intracellular fixation and permeabilization working solution at 4°C in the dark for 30 min before staining. Incubate the intracellular antibody Foxp3 - PE at 4°C in the dark for 30 min, wash the cells with PBS containing 1% FBS, and detect using a flow cytometer. The results are as Figure 3 and Figure 4 shown.

[0038] Figure 3 and Figure 4 The results showed that after UC - MSCs infusion, it could effectively regulate the balance of CD4 + CD25 + Foxp3 + Treg cells in the peripheral blood and spleen of T2DM mice. Compared with the T2DM group, the percentages of CD4 + CD25 + Foxp3 + Treg cells and the mean fluorescence intensity (MFI) of Foxp3 in the spleen and peripheral blood of mice in the UC - MSCs group increased significantly, and the percentages of CD4 + CD3 + IL - 17A + Th17 and the mean fluorescence intensity of IL - 17A decreased. Multiple injections of UC - MSCs could regulate the balance of Treg and Th17 cells in the peripheral blood and spleen of T2DM mice.

[0039] Experimental Example 5: The liver tissues of each group of mice were fixed overnight in a 4% paraformaldehyde solution before paraffin embedding. The tissues were cut into 5 - μm - thick sections and stained with hematoxylin - eosin (H&E) and periodic acid - Schiff (PAS). The results are as Figure 5As shown. After the injection experiment, the mice were fasted overnight for 12 h, blood samples of the mice were collected, centrifuged at 3000 rpm for 10 min at room temperature, the serum was collected, and the contents of IL-1β, IL-18, and TNF-α were measured using a specific ELISA kit. The results are as Figure 6 shown; Total RNA was extracted from mouse liver tissue cells using Trizol reagent (Transgen), reverse transcription was performed using a reverse transcription kit, q-PCR was performed using SYBR Green PCR master mix (EZBioscience) with specific gene primers, and the Roche Light Cycler 480 II system was used to amplify genes to measure the levels of NLRP3-related transcription factors (NLRP3, IL-1β, IL-18, IL-6, TNF-α, MCP-1). The results are as Figure 7 shown. Total protein was extracted from liver tissue cells. The primary antibodies were ASC, NLRP3, Cleaved Caspase-1, IkBα, p-IKBα, NFkB, p-NFkB, and β-actin. After blocking, the membrane was immunoblotted with the primary and secondary antibodies to measure the expression levels of NLRP3-related proteins. The results are as Figure 8 shown.

[0040] Figure 5 The results of HE staining showed that compared with the Control group, the liver structure in the T2DM group was disordered, the area of fatty degeneration increased, and the number of vacuolar changes increased. In the UC-MSCs group, the liver structure was significantly improved, the area of fatty degeneration decreased, and the number of vacuolar changes decreased; PAS staining showed that glycogen deposition decreased in the T2DM group and recovered in the UC-MSCs group. Figure 6 The results of serum ELISA showed that the levels of serum TNF-α, IL-1β, and IL-18 were significantly increased in the T2DM group, while their release was significantly decreased in the UC-MSCs group. Figure 7 The results of liver q-PCR analysis also showed that the gene expression levels of NLRP3, IL-1β, IL-18, IL-6, TNF-α, and MCP-1 were significantly decreased after UC-MSCs treatment. The expression of NLRP3-related proteins in the inflammatory signaling pathway was detected by western blot. Figure 8 The results showed that compared with the mice in the T2DM group, the expressions of ASC, NLRP3, and Cleaved Caspase-1 were significantly decreased in the mice in the UC-MSCs group. In addition, the expressions of p-IkBα and IkBα, p-NFkB and NFkB were significantly lower in the UC-MSCs group than in the T2DM group. The above results indicate that UC-MSCs can reduce liver injury and effectively improve the inflammatory state of target tissues in T2DM mice.

[0041] Experimental Example 6: HepG2 cells were cultured in L-DMEM in an incubator at 37 °C with 5% CO 2 2. After starvation for 16 h with serum-free DMEM containing 0.5% bovine serum albumin, 2×10 5 HepG2 cells were pretreated with 10 μg / mL LPS (lipopolysaccharide), and 0.25 mM PA (palmitic acid) was added for 24 h of culture. Then, they were co-cultured with 1×10 5 UC-MSCs through transwell for 24 h. Meanwhile, a group treated only with LPS and PA was set as a control (PA-LPS group), and HFF-1 was used as a negative control for UC-MSCs (HFF-1 group). Finally, the cells were washed twice with PBS and stimulated with 100 nM insulin for 3 hours. The supernatant was taken by centrifugation at 1000 rpm for 10 min at room temperature. The glucose consumption in the cell supernatant and the amount of cellular glycogen synthesis were detected using a glucose and glycogen assay kit. Total RNA was extracted from the cells using Trizol reagent (Transgen), and reverse transcription was performed using a reverse transcription kit to measure the levels of NLRP3-related transcription factors. The results are as Figure 9 shown. The contents of IL-1β and TNF-α were measured using a specific ELISA kit. The results are as Figure 10 shown. Total protein was extracted from HepG2 cells. The primary antibodies were ASC, NLRP3, Cleaved Caspase-1, IkBα, p-IKBα, NFkB, p-NFkB, and β-actin. After blocking, the membrane was immunoblotted with the primary and secondary antibodies to measure the expression levels of NLRP3-related proteins. The results are as Figure 11 shown.

[0042] Figure 9 Compared with Figure 10The results showed that compared with the Control group, PA-LPS treatment significantly reduced the glycogen synthesis content, and the secretion levels of IL-1β and TNF-α also increased significantly, indicating that PA-LPS could induce the expression of inflammatory mediators in insulin resistance of HepG2 cells. UC-MSCs were co-cultured with PA-LPS-treated HepG2 cells, and human fibroblasts HFF-1 were used as a negative control. The results showed that the UC-MSCs group could significantly increase glycogen synthesis and glucose consumption, and compared with the PA-LPS group, the release of inflammatory factors IL-1β and TNF-α in the UC-MSCs group was significantly reduced. The mRNA levels related to NLRP3 inflammation (NLRP3, IL-1β, IL-18, and TNF-α) showed lower expression in the UC-MSCs treatment group. Similarly, western blot detection of the expression of NLRP3-related proteins in the inflammatory signaling pathway showed that the expressions of ASC, NLRP3, and Cleaved Caspase-1 were significantly reduced after UC-MSCs treatment, and the phosphorylation levels of IkBα and NFkB were also significantly reduced. These results indicate that UC-MSCs can effectively reduce the inflammatory response, promote glucose utilization, and improve insulin sensitivity.

[0043] Those of ordinary skill in the art should understand that the discussion of any above embodiments is only exemplary, and is not intended to imply that the scope of the present invention is limited to these examples; under the concept of the present invention, the technical features in the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations in different aspects of the present invention as described above, which are not provided in detail for the sake of brevity.

Claims

1. A method for culturing mesenchymal stem cells, characterized in that: The following steps are involved: S1: Take fresh umbilical cord, wash it, separate and remove the umbilical cord outer membrane, umbilical artery and vein, cut it into tissue blocks, add complete culture medium to immerse it, and culture it at 5% CO2 and 37℃; S2: After the culture in step S1, the medium is changed, and when the cells grow to a confluence of 70-80%, they are digested and passaged to obtain P3 umbilical cord mesenchymal stem cells; S3: Place the gel matrix at the bottom of the culture plate, inoculate P3 umbilical cord mesenchymal stem cells in the logarithmic proliferation phase, add complete culture medium, and replace with new complete culture medium every 2-3 days; The complete culture medium includes the following components: 1% penicillin-streptomycin, 10% fetal bovine serum, and the remainder is DMEM / F12 culture medium; The raw materials for preparing the gel matrix include the following components in parts by weight: 1-2 parts of cellulose nanofibers, 1-2 parts of p-toluenesulfonyl-arginine, 0.5-1 parts of phenylalanine, 1-1.5 parts of chitosan, 0.9-1.2 parts of caffeic acid, 0.5-0.8 parts of cysteine, 0.8-1.2 parts of polyvinyl alcohol, and 1-2 parts of rosemary essential oil; The preparation method of the gel matrix comprises the following steps: (1) adding cellulose nanofibers, p-toluenesulfonyl-arginine, phenylalanine, Fmoc-Cl, EDC·HCl and DMAP into DMF, mixing, stirring, centrifuging, precipitating and washing to obtain Fmoc-amino acid-nanofibers; (2) ultrasonically dispersing the Fmoc-amino acid-nanofibers obtained in step (1) in a piperidine / DMF solution, centrifuging, washing the precipitate, and freeze-drying to obtain amino acid-nanofibers; (3) dissolving chitosan in an acetic acid aqueous solution, cooling to obtain a chitosan solution, dissolving caffeic acid and EDC in an ethanol solution at 4°C, adding NHS, stirring in an ice-water bath for reaction, adding dropwise to the chitosan solution, stirring in an ice-water bath for reaction, stirring in the dark at room temperature, adjusting the pH value to 8-9, heating in a water bath, dialyzing, and freeze-drying to obtain modified chitosan; (4) Mix polyvinyl alcohol and deionized water, place in a water bath, add cysteine, adjust the pH value to 2-3, stir in a water bath, and dialyze to obtain modified polyvinyl alcohol; (5) Take the modified chitosan obtained in step (3) and the modified polyvinyl alcohol obtained in step (4), add them to PBS buffer, heat them in a water bath to obtain a mixed solution, take the amino acid-nanofibers obtained in step (2), add them to deionized water and mix well, add rosemary essential oil, add the mixed solution while stirring, stir, let stand, freeze, and thaw to obtain a gel matrix.

2. The method for culturing mesenchymal stem cells according to claim 1, characterized in that: In step (1), the usage ratio of the cellulose nanofibers, Fmoc-Cl, EDC·HCl, DMAP and DMF is 1 g:0.75 g:1.25 g:0.08 g:50 mL.

3. The method for culturing mesenchymal stem cells according to claim 2, characterized in that: In step (2), the ratio of the 20% (v / v) piperidine / DMF solution to the cellulose nanofibers is 1 g:20 mL.

4. The method for culturing mesenchymal stem cells according to claim 3, characterized in that: In step (3), the ratio of chitosan to 2% (v / v) acetic acid aqueous solution is 1 g:50 mL.

5. The method for culturing mesenchymal stem cells according to claim 4, characterized in that: In step (3), the mass concentration of caffeic acid in 70% (v / v) ethanol solution is 20 mg / mL.

6. The method for culturing mesenchymal stem cells according to claim 5, characterized in that: In step (3), the mass ratio of caffeic acid, EDC and NHS is 3:3:

2.

7. The method for culturing mesenchymal stem cells according to claim 6, characterized in that: In step (4), the mass concentration of the polyvinyl alcohol in deionized water is 10-15 mg / mL.

8. The method for culturing mesenchymal stem cells according to claim 7, characterized in that: In step (5), the mass concentration of the amino acid-nanofibers in deionized water is 5 mg / mL.

9. The method for culturing mesenchymal stem cells according to claim 8, characterized in that: In step (5), the mass concentration of the modified chitosan in PBS buffer is 10-20 mg / mL.

Images