A mesenchymal stem cell preparation for treating diabetes and its preparation method

Abstract

The present invention relates to the field of biomedicine, specifically to a mesenchymal stem cell preparation for treating diabetes and its preparation method. The stem cell preparation comprises the following components: umbilical cord mesenchymal stem cells and naringin@SiO2. The raw materials for preparing the naringin@SiO2 include the following components by weight: 10-15 parts tetraethyl orthosilicate (TEOS), 4-6 parts 3-mercaptopropyloxysilane (MPTMS), 2-3 parts naringin, 0.8-1.2 parts succinic anhydride, and 2-3 parts cysteamine hydrochloride. The resulting stem cell preparation has enhanced synergy among its components, can repair pancreatic islet damage, and inhibit inflammatory responses.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a mesenchymal stem cell preparation for treating diabetes and a preparation method thereof. Background Art

[0002] Diabetes is a common chronic metabolic disease characterized by chronically elevated blood glucose levels, which can lead to numerous complications, such as cardiovascular disease, kidney disease, retinopathy, and neuropathy, severely impacting patients' quality of life and life expectancy. Currently, a variety of diabetes treatments are available, but all have limitations. Regarding medications, oral hypoglycemic agents such as sulfonylureas and biguanides can achieve a certain degree of blood glucose control. However, long-term use can lead to drug tolerance, reduced efficacy, and side effects such as hypoglycemia and gastrointestinal discomfort. While insulin injections effectively replenish insulin, they require strict control of dosage and timing. Frequent injections are extremely inconvenient for patients, and long-term use cannot prevent the further decline of pancreatic beta cell function. Pancreatic islet transplantation is a relatively effective treatment option, but due to the scarcity of donor organs, immune rejection is inevitable, requiring long-term use of immunosuppressants, which increases the risk of infection and other complications, limiting its widespread application. Mesenchymal stem cells (MSCs) have become a hot topic in diabetes treatment research due to their unique biological properties. MSCs possess multipotential differentiation potential and, under specific induction conditions, can differentiate into insulin-secreting cells, replenishing damaged pancreatic beta cells and restoring insulin secretion. At the same time, MSCs can also secrete a variety of cytokines and growth factors through paracrine effects, promoting the repair and regeneration of endogenous pancreatic beta cells, providing a new approach for the treatment of diabetes. Although mesenchymal stem cells have shown great potential in the treatment of diabetes, their current clinical application still faces many challenges. Among them, how to prepare safe, effective, stable and standardized mesenchymal stem cell preparations is a key issue. Mesenchymal stem cells obtained from different sources and different preparation methods have differences in cell activity, purity, biological function, etc., which affects the consistency and reliability of the treatment effect. Summary of the Invention

[0003] In view of the deficiencies of the prior art, the present invention provides a mesenchymal stem cell preparation for treating diabetes and a preparation method thereof.

[0004] The present invention is achieved through the following technical solutions:

[0005] A mesenchymal stem cell preparation for treating diabetes, comprising the following components: umbilical cord mesenchymal stem cells and naringin@SiO2.

[0006] Furthermore, the raw materials for preparing naringin@SiO2 include the following components in parts by weight: 10-15 parts of tetraethyl orthosilicate (TEOS), 4-6 parts of 3-mercaptopropyloxysilane (MPTMS), 2-3 parts of naringin, 0.8-1.2 parts of succinic anhydride, and 2-3 parts of cysteamine hydrochloride.

[0007] Furthermore, the preparation method of naringin@SiO2 comprises the following steps:

[0008] A1: CTAB, 1 mol / L sodium hydroxide solution, and 20-25 vol% ethanol aqueous solution were mixed, ultrasonically dispersed at 300W for 20-30 min, heated to 60-70°C, and TEOS and MPTMS were added dropwise at a rate of 0.1 mL / min. After the addition was complete, the reaction was continued for 2-3 hours. The mixture was cooled to room temperature, centrifuged at 8000-9000 rpm for 10-15 minutes, and the precipitate was washed with deionized water and vacuum dried. The mixture was dispersed in a mixed solution of anhydrous ethanol and 37% hydrochloric acid, heated under reflux for 24 hours, and centrifuged at 8000-9000 rpm for 10-15 minutes. The precipitate was washed with deionized water and vacuum dried to obtain modified porous silica.

[0009] A2: Dissolve naringin and succinic anhydride separately in pyridine. Slowly add the succinic anhydride solution dropwise to the naringin solution with stirring. React in a 35°C water bath for 14-16 hours. Dialyze the solution against deionized water for 2 days, replacing the dialysate every 4 hours. Freeze-dry to obtain naringin succinate.

[0010] A3: Dissolve the naringin succinate obtained in step A3 in DMF, add NHS / EDCI aqueous solution dropwise, stir for 2 h, adjust the pH to 4.8-4.9, add tris(2-carboxyethyl)phosphine (TCEP) and cysteamine hydrochloride, stir at 35°C for 8 h, adjust the pH to 4.8-4.9 during the process, dialyze against 0.008 M HCl in the dark for 24 h, wash the precipitate with 0.008 M HCl, and freeze-dry at -40°C to obtain modified naringin;

[0011] A4: The modified naringin obtained in step A3 and the modified porous silica obtained in step A1 were added to deionized water and mixed evenly. The mixture was stirred at 150-200 rpm at room temperature for 1-2 h. DMSO was added and the mixture was stirred for 4-5 h. The mixture was centrifuged at 10,000 rpm for 8-12 min. The precipitate was washed with anhydrous ethanol and deionized water, and dried under vacuum to obtain naringin@SiO2.

[0012] Furthermore, in step A1, the usage ratio of CTAB, 1 mol / L sodium hydroxide solution, 30 vol% ethanol aqueous solution and TEOS is 1 g:7 mL:150 mL:5 g.

[0013] Furthermore, in step A1, the volume ratio of ethanol to hydrochloric acid in the mixed solution of anhydrous ethanol and 37% hydrochloric acid is 10-12:1.

[0014] Furthermore, in step A2, the mass concentration of naringin in pyridine is 200 mg / mL.

[0015] Furthermore, in step A2, the mass concentration of succinic anhydride in pyridine is 80 mg / mL.

[0016] Furthermore, in step A3, the mass concentration of naringin succinate in DMF is 20 mg / mL.

[0017] Furthermore, in step A3, the mass concentrations of NHS and EDCI in the NHS / EDCI aqueous solution are 10 mg / mL and 25 mg / mL, respectively.

[0018] Furthermore, in step A3, the mass ratio of NHS to cysteamine hydrochloride is 2:1.

[0019] Furthermore, in step A3, the mass ratio of TCEP to cysteamine hydrochloride is 0.8-1.2:1.

[0020] Furthermore, in step A4, the mass concentration of the modified porous silica in deionized water is 5-8 mg / mL.

[0021] Furthermore, in step A4, the volume ratio of DMSO to deionized water is 1:5.

[0022] Furthermore, the present invention also provides a method for preparing the mesenchymal stem cell preparation for treating diabetes, comprising the following steps:

[0023] S1: Soak honeysuckle in PBS solution for 20-30 min, homogenize with a high-speed homogenizer, filter with medical sterile gauze, store the filtrate at 4°C overnight, perform gradient centrifugation at 1000g for 10 min, 2000g for 20 min, 5000g for 60 min, and 10000g for 60 min at 4°C, filter through a 0.22 μm filter membrane, and centrifuge the filtrate at 100,000g for 60 min. Discard the supernatant and resuspend the solution in complete culture medium to 20 μg / mL to obtain a honeysuckle exosome resuspension.

[0024] S2: Umbilical cord mesenchymal stem cells (hUCMSCs) were isolated, cultured and passaged according to conventional experimental methods. P4 hUCMSCs were seeded into six-well plates at 5×10 4cells, 2.5 mL complete medium, culture at 37°C, 5% CO2 for 24 h, remove the old medium, add 2 mL honeysuckle exosome resuspension, and continue to culture for 24 h;

[0025] S3: After culturing for 24 h in step S2, hUCMSCs were collected and resuspended in PBS to a density of 10 6 The mixture was stirred at 200-300 rpm for 30 min to obtain a mesenchymal stem cell preparation for treating diabetes.

[0026] Furthermore, the complete culture medium is DMED / F12 culture medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention provides a mesenchymal stem cell preparation for the treatment of diabetes. This formulation combines a thiol-functionalized mesoporous silica carrier with thiol-modified naringin, combined with umbilical cord mesenchymal stem cells pretreated with honeysuckle exosomes, to achieve a multi-component synergistic effect, enhancing anti-inflammatory and pancreatic islet protection. The present invention utilizes a CTAB template method combined with a mercaptosilane coupling agent to surface functionalize the silica, forming a modified porous silica with an ordered mesoporous structure. This carrier introduces thiol-active sites, forming covalent bonds with the subsequently modified naringin, thereby improving encapsulation efficiency. The present invention utilizes the advantages of hydroxyl compounds and acid anhydrides, such as good reactivity, high grafting rate, and easy control of the reaction process. The carboxyl group of naringin is modified through a succinic anhydride esterification reaction. Without changing the pharmacological activity of naringin, a reactive group easily reactive is introduced through the reaction of the hydroxyl group at the glycoside site of the naringin with the acid anhydride. The modified naringin then forms a covalent bond with cysteamine hydrochloride. The amino group in the cysteamine molecule undergoes an amidation reaction with the carboxyl group in the naringin succinate ester molecule to prepare a modified naringin containing a thiol reactive group. The modified naringin forms a disulfide bond with modified porous silica. The drug is loaded on a carrier through physical adsorption, hydrogen bond interaction, and covalent bond. A composite carrier system with multiple bonding effects is constructed through chemical modification, thereby controlling the release rate of the drug, significantly improving the drug loading efficiency and sustained-release performance, and effectively prolonging the drug action time. The present invention forms a multi-target treatment system through dual regulation of honeysuckle exosome pretreatment and drug carriers. By co-culturing honeysuckle exosomes with umbilical cord mesenchymal stem cells (hUCMSCs), the cell proliferation rate of hUCMSCs is significantly improved, the proliferation of hUCMSCs is effectively promoted, and the activity of hUCMSCs is increased, which is beneficial to improving the efficacy of the preparation. Combined with naringin@SiO2, it shows excellent anti-inflammatory effects in diabetic model mice, and by promoting β-cell regeneration, the proportion of pancreatic β cells is restored to normal levels. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only for the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0030] Figure 1 The effect of the honeysuckle exosomes of the present invention on the activity of hUCMSC;

[0031] Figure 2 The effects of the mesenchymal stem cell preparations described in Example 1 and Comparative Examples 1-4 of the present invention on IL-β levels;

[0032] Figure 3The effects of the mesenchymal stem cell preparations described in Example 1 and Comparative Examples 1-4 of the present invention on the proportion of β cells;

[0033] Figure 4 The encapsulation efficiency of the glycosides@SiO2 described in Examples 1-3 of the present invention and Comparative Example 2 is shown. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific examples, but the present invention is not limited to the following examples. It should be noted that, unless otherwise specified, the chemical reagents involved in the present invention were purchased through commercial channels.

[0035] Example 1: This example provides a mesenchymal stem cell preparation for treating diabetes, comprising the following components: umbilical cord mesenchymal stem cells and naringin@SiO2.

[0036] The raw materials for preparing naringin@SiO2 include the following components in parts by weight: 15 parts of tetraethyl orthosilicate (TEOS), 6 parts of 3-mercaptopropyloxysilane (MPTMS), 3 parts of naringin, 1.2 parts of succinic anhydride, and 3 parts of cysteamine hydrochloride.

[0037] The preparation method of naringin@SiO2 comprises the following steps:

[0038] A1: 3 g of CTAB, 21 mL of 1 mol / L sodium hydroxide solution, and 450 mL of 25 vol% ethanol aqueous solution were mixed, and ultrasonic dispersion was performed at 300 W for 30 min. The temperature was raised to 70°C, and 15 g of TEOS and 6 g of MPTMS were added dropwise at a rate of 0.1 mL / min. After the addition was complete, the reaction was continued for 3 h. The mixture was cooled to room temperature and centrifuged at 9000 rpm for 15 min. The precipitate was washed with deionized water and dried in vacuum. The mixture was dispersed in a mixed solution of ethanol and 37% hydrochloric acid (v:v = 12:1), heated under reflux for 24 h, and centrifuged at 9000 rpm for 15 min. The precipitate was washed with deionized water and dried in vacuum to obtain modified porous silica.

[0039] A2: Dissolve 3 g of naringin and 1.2 g of succinic anhydride in 15 mL of pyridine, respectively. Slowly add the succinic anhydride solution dropwise to the naringin solution while stirring. React in a 35°C water bath for 16 h. Dialyze the solution against deionized water for 2 days, replacing the dialysate every 4 h. Freeze-dry to obtain naringin succinate.

[0040] A3: Dissolve the naringin succinate obtained in step A3 in DMF at a ratio of 20 mg / mL, add dropwise an NHS / EDCI aqueous solution, wherein the mass concentrations of NHS and EDCI in the NHS / EDCI aqueous solution are 10 mg / mL and 25 mg / mL, respectively, and the mass ratio of NHS to cysteamine hydrochloride is 2:1, stir for 2 h, adjust the pH to 4.9, add 3.6 g of tris(2-carboxyethyl)phosphine (TCEP) and 3 g of cysteamine hydrochloride, stir at 35°C for 8 h, adjust the pH to 4.9 during the process, dialyze against 0.008 M HCl in the dark for 24 h, wash the precipitate with 0.008 M HCl, and freeze-dry at -40°C to obtain modified naringin;

[0041] A4: The modified naringin obtained in step A3 and the modified porous silica obtained in step A1 were added to deionized water and mixed evenly. The mass concentration of the modified porous silica in deionized water was 8 mg / mL. The mixture was stirred at 200 rpm at room temperature for 2 h. DMSO was added with a volume ratio of DMSO to deionized water of 1:5. The mixture was stirred for another 5 h. The mixture was centrifuged at 10,000 rpm for 12 min. The precipitate was washed with anhydrous ethanol and deionized water, and dried in vacuo to obtain naringin@SiO2.

[0042] This embodiment also provides a method for preparing the mesenchymal stem cell preparation for treating diabetes, comprising the following steps:

[0043] S1: Soak honeysuckle in PBS solution for 30 min, homogenize with a high-speed homogenizer, filter with medical sterile gauze, store the filtrate at 4°C overnight, perform gradient centrifugation at 1000g for 10 min, 2000g for 20 min, 5000g for 60 min, and 10,000g for 60 min at 4°C, filter through a 0.22 μm filter membrane, and centrifuge the filtrate at 100,000g for 60 min. Discard the supernatant and resuspend the filtrate to 20 μg / mL in complete culture medium (DMED / F12 medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin) to obtain a honeysuckle exosome resuspension.

[0044] S2: Umbilical cord mesenchymal stem cells (hUCMSCs) were isolated, cultured and passaged according to conventional experimental methods. P4 hUCMSCs were seeded into six-well plates at 5×10 4 cells, 2.5 mL complete medium, culture at 37°C, 5% CO2 for 24 h, remove the old medium, add 2 mL honeysuckle exosome resuspension, and continue to culture for 24 h;

[0045] S3: After culturing for 24 h in step S2, hUCMSCs were collected and resuspended in PBS to a density of 10 6 / mL, naringin@SiO2 was added to a concentration of 10 mg / mL, and stirred at 300 rpm for 30 min to obtain a mesenchymal stem cell preparation for treating diabetes.

[0046] Example 2: This example provides a mesenchymal stem cell preparation for treating diabetes, comprising the following components: umbilical cord mesenchymal stem cells and naringin@SiO2.

[0047] The raw materials for preparing naringin@SiO2 include the following components in parts by weight: 10 parts of tetraethyl orthosilicate (TEOS), 4 parts of 3-mercaptopropyloxysilane (MPTMS), 2 parts of naringin, 0.8 parts of succinic anhydride, and 2 parts of cysteamine hydrochloride.

[0048] The preparation method of naringin@SiO2 comprises the following steps:

[0049] A1: 2 g of CTAB, 14 mL of 1 mol / L sodium hydroxide solution, and 300 mL of 20 vol% ethanol aqueous solution were mixed, and ultrasonic dispersion was performed at 300 W for 20 min. The temperature was raised to 60°C, and 10 g of TEOS and 4 g of MPTMS were added dropwise at a rate of 0.1 mL / min. After the addition was complete, the reaction was continued for 2 h. The mixture was cooled to room temperature and centrifuged at 8000 rpm for 10 min. The precipitate was washed with deionized water and dried in vacuum. The mixture was dispersed in a mixed solution of anhydrous ethanol and 37% hydrochloric acid (v:v = 10:1), heated under reflux for 24 h, and centrifuged at 8000 rpm for 10 min. The precipitate was washed with deionized water and dried in vacuum to obtain modified porous silica.

[0050] A2: Dissolve 2 g of naringin and 0.8 g of succinic anhydride in 10 mL of pyridine, respectively. Slowly add the succinic anhydride solution dropwise to the naringin solution while stirring. React in a 35°C water bath for 14 h. Dialyze the solution against deionized water for 2 days, replacing the dialysate every 4 h. Freeze-dry to obtain naringin succinate.

[0051] A3: Dissolve the naringin succinate obtained in step A3 in DMF at a ratio of 20 mg / mL, add dropwise an NHS / EDCI aqueous solution, wherein the mass concentrations of NHS and EDCI in the NHS / EDCI aqueous solution are 10 mg / mL and 25 mg / mL, respectively, and the mass ratio of NHS to cysteamine hydrochloride is 2:1, stir for 2 h, adjust the pH to 4.8, add 1.6 g of tris(2-carboxyethyl)phosphine (TCEP) and 2 g of cysteamine hydrochloride, stir at 35°C for 8 h, adjust the pH to 4.8 during the process, dialyze with 0.008 M HCl in the dark for 24 h, wash the precipitate with 0.008 M HCl, and freeze-dry at -40°C to obtain modified naringin;

[0052] A4: The modified naringin obtained in step A3 and the modified porous silica obtained in step A1 were added to deionized water and mixed evenly. The mass concentration of the modified porous silica in deionized water was 5 mg / mL. The mixture was stirred at 150 rpm at room temperature for 1 h. DMSO was added at a volume ratio of DMSO to deionized water of 1:5. The mixture was stirred for another 4 h. The mixture was centrifuged at 10,000 rpm for 8 min. The precipitate was washed with anhydrous ethanol and deionized water, and dried in vacuo to obtain naringin@SiO2.

[0053] This embodiment also provides a method for preparing the mesenchymal stem cell preparation for treating diabetes, comprising the following steps:

[0054] S1: Soak honeysuckle in PBS solution for 20 min, homogenize with a high-speed homogenizer, filter with medical sterile gauze, store the filtrate at 4°C overnight, perform gradient centrifugation at 1000g × 10 min, 2000g × 20 min, 5000g × 60 min, and 10000g × 60 min at 4°C, filter through a 0.22 μm filter membrane, and centrifuge the filtrate at 100000g × 60 min. Discard the supernatant and resuspend the filtrate to 20 μg / mL in complete culture medium (DMED / F12 medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin) to obtain a honeysuckle exosome resuspension.

[0055] S2: Umbilical cord mesenchymal stem cells (hUCMSCs) were isolated, cultured and passaged according to conventional experimental methods. P4 hUCMSCs were seeded into six-well plates at 5×10 4 cells, 2.5 mL complete medium, culture at 37°C, 5% CO2 for 24 h, remove the old medium, add 2 mL honeysuckle exosome resuspension, and continue to culture for 24 h;

[0056] S3: After culturing for 24 h in step S2, hUCMSCs were collected and resuspended in PBS to a density of 10 6 / mL, naringin@SiO2 was added to a concentration of 10 mg / mL, and stirred at 200 rpm for 30 min to obtain a mesenchymal stem cell preparation for treating diabetes.

[0057] Example 3: A mesenchymal stem cell preparation for treating diabetes, comprising the following components: umbilical cord mesenchymal stem cells and naringin@SiO2.

[0058] The raw materials for preparing naringin@SiO2 include the following components in parts by weight: 12 parts of tetraethyl orthosilicate (TEOS), 5 parts of 3-mercaptopropyloxysilane (MPTMS), 2.5 parts of naringin, 1 part of succinic anhydride, and 2.5 parts of cysteamine hydrochloride.

[0059] The preparation method of naringin@SiO2 comprises the following steps:

[0060] A1: 2.4 g of CTAB, 16.8 mL of 1 mol / L sodium hydroxide solution, and 360 mL of 22 vol% ethanol aqueous solution were mixed, and ultrasonic dispersion was performed at 300 W for 25 min. The temperature was raised to 65°C, and 12 g of TEOS and 5 g of MPTMS were added dropwise at a rate of 0.1 mL / min. After the addition was complete, the reaction was continued for 2.5 h. The mixture was cooled to room temperature and centrifuged at 8500 rpm for 12 min. The precipitate was washed with deionized water and dried in vacuum. The mixture was dispersed in a mixed solution of anhydrous ethanol and 37% hydrochloric acid (v:v = 11:1), heated under reflux for 24 h, and centrifuged at 8500 rpm for 12 min. The precipitate was washed with deionized water and dried in vacuum to obtain modified porous silica.

[0061] A2: Dissolve 2.5 g of naringin and 1 g of succinic anhydride in 12.5 mL of pyridine, respectively. Slowly add the succinic anhydride solution dropwise to the naringin solution while stirring. React in a 35°C water bath for 15 h. Dialyze the solution against deionized water for 2 days, replacing the dialysate every 4 h. Freeze-dry to obtain naringin succinate.

[0062] A3: Dissolve the naringin succinate obtained in step A3 in DMF at a ratio of 20 mg / mL, add dropwise an NHS / EDCI aqueous solution, wherein the mass concentrations of NHS and EDCI in the NHS / EDCI aqueous solution are 10 mg / mL and 25 mg / mL, respectively, and the mass ratio of NHS to cysteamine hydrochloride is 2:1, stir for 2 h, adjust the pH to 4.9, add 2.5 g of tris(2-carboxyethyl)phosphine (TCEP) and 2.5 g of cysteamine hydrochloride, stir at 35°C for 8 h, adjust the pH to 4.8 during the process, dialyze against 0.008 M HCl in the dark for 24 h, wash the precipitate with 0.008 M HCl, and freeze-dry at -40°C to obtain modified naringin;

[0063] A4: The modified naringin obtained in step A3 and the modified porous silica obtained in step A1 were added to deionized water and mixed evenly. The mass concentration of the modified porous silica in deionized water was 6 mg / mL. The mixture was stirred at 180 rpm at room temperature for 1.5 h. DMSO was added at a volume ratio of DMSO to deionized water of 1:5. The mixture was stirred for another 4.5 h. The mixture was centrifuged at 10,000 rpm for 10 min. The precipitate was washed with anhydrous ethanol and deionized water, and dried in vacuo to obtain naringin@SiO2.

[0064] This embodiment also provides a method for preparing the mesenchymal stem cell preparation for treating diabetes, comprising the following steps:

[0065] S1: Soak honeysuckle in PBS solution for 25 min, homogenize with a high-speed homogenizer, filter with medical sterile gauze, store the filtrate at 4°C overnight, perform gradient centrifugation at 1000g for 10 min, 2000g for 20 min, 5000g for 60 min, and 10,000g for 60 min at 4°C, filter through a 0.22 μm filter membrane, and centrifuge the filtrate at 100,000g for 60 min. Discard the supernatant and resuspend the filtrate to 20 μg / mL in complete culture medium (DMED / F12 medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin) to obtain a honeysuckle exosome resuspension.

[0066] S2: Umbilical cord mesenchymal stem cells (hUCMSCs) were isolated, cultured and passaged according to conventional experimental methods. P4 hUCMSCs were seeded into six-well plates at 5×10 4 cells, 2.5 mL complete medium, culture at 37°C, 5% CO2 for 24 h, remove the old medium, add 2 mL honeysuckle exosome resuspension, and continue to culture for 24 h;

[0067] S3: After culturing for 24 h in step S2, hUCMSCs were collected and resuspended in PBS to a density of 10 6 / mL, naringin@SiO2 was added to a concentration of 10 mg / mL, and stirred at 250 rpm for 30 min to obtain a mesenchymal stem cell preparation for treating diabetes.

[0068] The only difference between Comparative Example 1 and Example 1 is that no modified naringin is added.

[0069] The only difference between Comparative Example 2 and Example 1 is that no MPTMS is added to prepare porous silica, and naringenin is used instead of modified naringenin.

[0070] The only difference between Comparative Example 3 and Example 1 is that naringin@SiO2 is replaced by naringin.

[0071] The only difference between Comparative Example 4 and Example 1 is that the honeysuckle exosomes are not co-cultured with hUCMSCs, that is, in step S2, the honeysuckle exosomes heavy suspension is replaced by complete culture medium.

[0072] Experimental Example 1: hUCMSC cells at generation P4 were seeded into 96-well plates at 3×10 cells per well. 3 The cells were cultured at 37°C and 5% CO2 for 24 h, the old culture medium was removed, and 20 μg / mL of honeysuckle exosome resuspension was added. The control group was added with complete culture medium. After culturing for 24 h and 48 h, the old culture medium was removed, 100 μL of CCK-8 solution was added to each well, and the cells were incubated for 2 h. The absorbance at 450 nm was detected using a microplate reader. The results are shown in Figure 2. Figure 1 shown.

[0073] Figure 1 The results showed that honeysuckle exosomes can significantly increase the cell proliferation rate of hUCMSC, effectively promote hUCMSC proliferation, and improve hUCMSC activity, indicating that the hUCMSC in the mesenchymal stem cell preparation for treating diabetes of the present invention has better biological activity, which is beneficial to improving the therapeutic effect.

[0074] Experimental Example 2: Six-week-old normal male C57BL / 6J mice (18 ± 2 g) were selected and housed in an SPF environment with free access to water and food. The room temperature was 22.5 ± 2.5 ° C, the humidity was 50-70%, and the light-dark cycle was 12 h / 12 ​​h. They were given a high-fat diet (60 kcal% fat) (ReadyDietech) for 8 weeks. STZ 40 mg / kg was injected intraperitoneally for 3 consecutive days. One week after STZ injection, an oral glucose tolerance test (OGTT) and an intraperitoneal insulin tolerance test (IPITTS) were performed to ensure the establishment of a type 2 diabetes mouse model. All mice were divided into 7 groups, with 8 mice in each group: a control group, an Example 1 group, and a comparative example 1-4 group. The diabetic model mice in the Example 1 and comparative example 1-4 groups were intraperitoneally injected with the stem cell preparations of Example 1 and comparative examples 1-4, with a single injection of 200 μL once a week for 5 consecutive weeks. At the same time, diabetic mice were injected with an equal amount of PBS as a control group. One week after the last administration, peripheral blood of rats was collected and centrifuged at 3400 g for 10 min, and the supernatant was collected to obtain serum. The IL-1β concentration in the serum was determined by ELISA kit. The results were as follows: Figure 2 As shown. Rats were sacrificed, and pancreatic tissue was taken and placed in 30% sucrose for dehydration overnight. It was then embedded in embedding agent and stored at -80°C for later use. Pancreatic tissue was sliced ​​into 5 μm sections. Tissue sections were incubated with 0.5% Triton-X100 at room temperature for 15 min, washed three times with PBS, and blocked with serum for 30 min at room temperature. After blocking, insulin and glucagon were added as primary antibodies, and incubated at 4°C overnight. Cells not bound to the primary antibody were washed with PBS, and fluorescent secondary antibodies were added. The sections were incubated at room temperature for 2 h, and the percentage of β cells was counted by fluorescence microscopy. The results are shown as follows: Figure 3 shown.

[0075] Figure 2 The results showed that the IL-1β levels in Example 1 and Comparative Examples 1-4 were significantly lower than those in the control group. The levels of the pro-inflammatory factor IL-1β in the serum of mice treated with the mesenchymal stem cell preparation were significantly reduced. Figure 3The results showed that the β cell percentages of Example 1 and Comparative Examples 1-4 were significantly higher than those of the control group, with Example 1 having the best effect. Comparative Example 1 did not add modified naringin, and therefore could not exert a synergistic therapeutic effect, resulting in a decreased therapeutic effect. Comparative Example 2 did not modify the porous silica and naringenin, but only used physical adsorption and weak hydrogen bond interactions to load the drug, resulting in a reduced loading of the active ingredient, a decreased long-term sustained release, and a decreased therapeutic effect. Comparative Example 3 did not load naringin, resulting in a decreased long-term sustained release. Comparative Example 4 did not co-culture honeysuckle exosomes with mesenchymal stem cells, resulting in decreased cell activity and a decreased therapeutic effect. The above results indicate that the mesenchymal stem cell preparation for treating diabetes of the present invention can effectively inhibit the decrease in pancreatic β cells, increase the decrease in β cell percentage caused by diabetes, thereby improving the body's hyperglycemic state, promoting insulin secretion by pancreatic cells to exert a hypoglycemic effect, and simultaneously inhibiting inflammatory responses in the body.

[0076] Experimental Example 3: According to the methods of Examples 1-3 and Comparative Example 2, naringin@SiO2 was prepared and the encapsulation efficiency (%) of the active ingredient was determined. The results are as follows: Figure 4 shown.

[0077] Figure 4 The results showed that the encapsulation efficiency of Examples 1-3 was significantly higher than that of Comparative Example 2. The naringin@SiO2 of the present invention can load more active ingredients and improve the therapeutic effect. Comparative Example 2 did not modify naringin and porous silica, but only loaded the active ingredients through physical adsorption and weak hydrogen bond interactions, resulting in a lower loading amount and decreased long-term effectiveness.

[0078] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present invention is limited to these examples. Within the scope of the present invention, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the present invention as described above, which are not provided in detail for the sake of simplicity.

Claims

1. A method for preparing a mesenchymal stem cell preparation for treating diabetes, characterized in that: The following steps are involved: S1: Soak honeysuckle in PBS solution, homogenize, filter through gauze, store the filtrate at 4°C overnight, perform gradient centrifugation at 4°C, filter through a 0.22 μm filter membrane, centrifuge the filtrate at 100,000 g, discard the supernatant, and resuspend in complete culture medium to obtain a honeysuckle exosome resuspension; S2: hUCMSCs were isolated, cultured, and passaged according to conventional experimental methods. P4 hUCMSCs were inoculated into six-well plates and cultured. The old culture medium was removed and the heavy suspension of honeysuckle exosomes was added and cultured again. S3: After culturing in step S2, hUCMSCs are collected, resuspended in PBS, and naringin@SiO2 is added and stirred to obtain a mesenchymal stem cell preparation for treating diabetes; The raw materials for preparing the naringin@SiO2 include the following components in parts by weight: 10-15 parts of TEOS, 4-6 parts of MPTMS, 2-3 parts of naringin, 0.8-1.2 parts of succinic anhydride, and 2-3 parts of cysteamine hydrochloride; The preparation method of naringin@SiO2 comprises the following steps: A1: CTAB, 1 mol / L sodium hydroxide solution, and 20-25 vol% ethanol aqueous solution were mixed, sonicated, heated, and TEOS and MPTMS were added dropwise. After the addition was complete, the reaction was continued, cooled, centrifuged, the precipitate was washed, dried, and dispersed in a mixed solution of anhydrous ethanol and 37% hydrochloric acid, heated to reflux, centrifuged, the precipitate was washed, and vacuum dried to obtain modified porous silica. A2: Dissolve naringin and succinic anhydride in pyridine separately. Slowly add the succinic anhydride solution dropwise to the naringin solution while stirring. React in a water bath, dialyze, and freeze-dry to obtain naringin succinate. A3: Dissolve the naringin succinate obtained in step A3 in DMF, add NHS / EDCI aqueous solution dropwise, stir, adjust the pH to 4.8-4.9, add TCEP and cysteamine hydrochloride, stir, adjust the pH to 4.8-4.9, dialyze against 0.008 M HCl in the dark, wash the precipitate with 0.008 M HCl, and freeze-dry to obtain modified naringin; A4: The modified naringin obtained in step A3 and the modified porous silica obtained in step A1 are added to deionized water, mixed evenly, and stirred. DMSO is added, stirring is continued, and the mixture is centrifuged. The precipitate is washed with anhydrous ethanol and deionized water, and vacuum dried to obtain naringin@SiO2.

2. The method for preparing a mesenchymal stem cell preparation for treating diabetes according to claim 1, wherein: In step A1, the ratio of CTAB, 1 mol / L sodium hydroxide solution, 30 vol% ethanol aqueous solution, and TEOS is 1 g:7 mL:150 mL:5 g.

3. The method for preparing a mesenchymal stem cell preparation for treating diabetes according to claim 2, wherein: In step A1, the volume ratio of ethanol to hydrochloric acid in the mixed solution of anhydrous ethanol and 37% hydrochloric acid is 10-12:

1.

4. The method for preparing a mesenchymal stem cell preparation for treating diabetes according to claim 3, wherein: In step A2, the mass concentration of naringin in pyridine is 200 mg / mL; the mass concentration of succinic anhydride in pyridine is 80 mg / mL.

5. The method for preparing a mesenchymal stem cell preparation for treating diabetes according to claim 4, characterized in that: In step A3, the mass concentration of naringin succinate in DMF is 20 mg / mL.

6. The method for preparing a mesenchymal stem cell preparation for treating diabetes according to claim 5, characterized in that: In step A3, the mass concentrations of NHS and EDCI in the NHS / EDCI aqueous solution are 10 mg / mL and 25 mg / mL, respectively.

7. The method for preparing a mesenchymal stem cell preparation for treating diabetes according to claim 6, characterized in that: In step A3, the mass ratio of NHS to cysteamine hydrochloride is 2:

1.

8. The method for preparing a mesenchymal stem cell preparation for treating diabetes according to claim 7, characterized in that: In step A3, the mass ratio of TCEP to cysteamine hydrochloride is 0.8-1.2:

1.

9. The method for preparing a mesenchymal stem cell preparation for treating diabetes according to claim 8, characterized in that: In step A4, the mass concentration of the modified porous silica in deionized water is 5-8 mg / mL; and the volume ratio of DMSO to deionized water is 1:

5.

10. A mesenchymal stem cell preparation for treating diabetes prepared by the method according to any one of claims 1 to 9.

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