Mesenchymal stem cell preparation for treating diabetes and preparation method thereof

Abstract

The invention relates to the technical field of biological medicines, in particular to a mesenchymal stem cell preparation for treating diabetes and a preparation method thereof. The stem cell preparation is prepared from the following components: umbilical cord mesenchymal stem cells and naringin coated SiO2, and preparation raw materials of the naringin coated SiO2 are prepared from the following components in parts by weight: 10 to 15 parts of tetraethoxysilane (TEOS), 4 to 6 parts of 3-mercaptopropyl oxysilane (MPTMS), 2 to 3 parts of naringin, 0.8 to 1.2 parts of succinic anhydride and 2 to 3 parts of cysteamine hydrochloride. The prepared stem cell preparation has synergistic blood lineage of each component, and can repair pancreas islet injury and inhibit inflammatory response.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, 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, the main feature of which is the long-term increase in blood sugar levels, which can lead to a variety of complications, such as cardiovascular disease, kidney disease, retinopathy, neuropathy, etc., which seriously affect the patient's quality of life and life expectancy. There are various treatment methods for diabetes at present, but they all have limitations. In terms of drug treatment, although oral hypoglycemic drugs such as sulfonylureas and biguanides can control blood sugar to a certain extent, long-term use is prone to drug tolerance, reduced efficacy, and may also cause side effects such as hypoglycemia and gastrointestinal discomfort. Although insulin injection can effectively supplement insulin, the dosage and injection time must be strictly controlled. Frequent injections bring great inconvenience to patients, and long-term use cannot prevent the further decline of pancreatic β cell function. Islet transplantation is a relatively effective treatment method, but due to the scarcity of donor organs, immune rejection reactions are difficult to avoid, and long-term use of immunosuppressants is required, which increases the risk of infection and other complications, limiting its wide application. Mesenchymal stem cells (MSCs) have become a hot topic in diabetes treatment research due to their unique biological characteristics. MSCs have multidirectional differentiation potential. Under specific induction conditions, they can differentiate into insulin-secreting cells, supplement damaged pancreatic β cells, and rebuild insulin secretion function. At the same time, MSCs can also secrete a variety of cytokines and growth factors through paracrine effects, promote the repair and regeneration of endogenous pancreatic β cells, and provide a new approach for the treatment of diabetes. Although mesenchymal stem cells have shown great potential in the treatment of diabetes, their current clinical applications still face 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 differ in terms of 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: A mesenchymal stem cell preparation for treating diabetes, comprising the following components: umbilical cord mesenchymal stem cells, naringin@SiO 2 .

[0005] Furthermore, the naringin@SiO 2The raw materials for preparation 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.

[0006] Furthermore, the naringin@SiO 2 The preparation method comprises the following steps: 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, TEOS and MPTMS were added dropwise at a rate of 0.1 mL / min, and the reaction was continued for 2-3 h after the addition was completed. The mixture was cooled to room temperature, centrifuged at 8000-9000 rpm for 10-15 min, the precipitate was washed with deionized water, vacuum dried, dispersed in a mixed solution of anhydrous ethanol and 37% hydrochloric acid, heated to reflux for 24 h, centrifuged at 8000-9000 pm for 10-15 min, the precipitate was washed with deionized water, and vacuum dried to obtain modified porous silica; A2: Dissolve naringin and succinic anhydride in pyridine respectively, slowly add the succinic anhydride solution to the naringin solution under stirring, react in a water bath at 35°C for 14-16 h, dialyze with deionized water for 2 days, replace the dialysate every 4 h, 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 for 2 h, adjust the pH to 4.8-4.9, add tri(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 with 0.008 M HCl for 24 h in the dark, wash the precipitate with 0.008 M HCl, and freeze-dry at -40°C to obtain modified naringin; A4: Take the modified naringin obtained in step A3 and the modified porous silica obtained in step A1, add them into deionized water and mix them evenly, stir them at 150-200 rpm for 1-2 h at room temperature, add DMSO, continue stirring for 4-5 h, centrifuge at 10000 rpm for 8-12 min, wash the precipitate with anhydrous ethanol and deionized water, and vacuum dry to obtain naringin@SiO 2 .

[0007] 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.

[0008] 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.

[0009] Furthermore, in step A2, the mass concentration of naringin in pyridine is 200 mg / mL.

[0010] Furthermore, in step A2, the mass concentration of succinic anhydride in pyridine is 80 mg / mL.

[0011] Furthermore, in step A3, the mass concentration of naringin succinate in DMF is 20 mg / mL.

[0012] 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.

[0013] Furthermore, in step A3, the mass ratio of NHS to cysteamine hydrochloride is 2:1.

[0014] Furthermore, in step A3, the mass ratio of TCEP to cysteamine hydrochloride is 0.8-1.2:1.

[0015] Furthermore, in step A4, the mass concentration of the modified porous silica in deionized water is 5-8 mg / mL.

[0016] Furthermore, in step A4, the volume ratio of DMSO to deionized water is 1:5.

[0017] Furthermore, the present invention also provides a method for preparing the mesenchymal stem cell preparation for treating diabetes, comprising the following steps: 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×10min, 2000g×20min, 5000g×60min, and 10000g×60min at 4°C, filter through a 0.22μm filter membrane, centrifuge the filtrate at 100000g×60 min, discard the supernatant, and resuspend with complete culture medium to 20μg / mL to obtain a heavy suspension of honeysuckle exosomes; S2: Umbilical cord mesenchymal stem cells (hUCMSC) were isolated, cultured and subcultured according to conventional experimental methods. P4 hUCMSC were inoculated into six-well plates, with 5×10 4 cells, 2.5 mL complete medium, 37°C, 5% CO 2 After culturing for 24 h, the old culture medium was removed and 2 mL of honeysuckle exosome resuspension was added, and the culture was continued for 24 h. S3: After culturing for 24 h in step S2, hUCMSCs were collected and resuspended in PBS to a density of 10 6 / mL, add naringin@SiO 2 The mixture was stirred at 200-300 rpm for 30 min to a concentration of 10 mg / mL to obtain a mesenchymal stem cell preparation for treating diabetes.

[0018] Furthermore, the complete culture medium is a DMED / F12 culture medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin.

[0019] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a mesenchymal stem cell preparation for treating diabetes. By constructing a composite system of thiol-functionalized mesoporous silica carrier and thiol-modified naringin, combined with umbilical cord mesenchymal stem cells pretreated with honeysuckle exosomes, multi-component synergistic enhancement, anti-inflammatory and pancreatic islet protection effects are enhanced. The present invention uses a CTAB template method combined with a thiol silane coupling agent to perform surface functionalization on silica to form a modified porous silica with an ordered mesoporous structure. The carrier forms a covalent bond with the subsequently modified naringin by introducing a thiol active site, thereby improving the encapsulation rate. The invention utilizes the advantages of good reactivity, high grafting rate, easy control of reaction process and the like of hydroxyl compounds and acid anhydrides, and performs carboxyl modification on naringin through esterification reaction of succinic anhydride, and introduces reactive groups that are easy to react by reaction of hydroxyl groups at glycoside sites with acid anhydride without changing the pharmacological activity of naringin, and then forms a covalent bond with cysteamine hydrochloride, and prepares modified naringin containing thiol reactive groups through amidation reaction of amino groups in cysteamine molecules and carboxyl groups in naringin succinate molecules, and forms a disulfide bond with modified porous silica, and loads the drug on a carrier through physical adsorption, hydrogen bond interaction and covalent bond, and constructs a composite carrier system with multiple bond interactions through chemical modification means, controls the release rate of the drug, significantly improves the drug loading efficiency and sustained-release performance, and effectively prolongs the drug action time. The present invention forms a multi-target treatment system through dual regulation of honeysuckle exosome pretreatment and drug carrier. 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. 2 , showed excellent anti-inflammatory effects in diabetic model mice, and restored the proportion of pancreatic β cells to normal levels by promoting β cell regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are only for the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 The effect of the honeysuckle exosomes of the present invention on the activity of hUCMSC; 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; Figure 3 The 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; Figure 4 The pizoglycoside @SiO 2 The encapsulation rate. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific embodiments, but 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.

[0023] Example 1: This example provides a mesenchymal stem cell preparation for treating diabetes, comprising the following components: umbilical cord mesenchymal stem cells, naringin@SiO 2 .

[0024] Naringin@SiO 2 The raw materials for preparation 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.

[0025] Naringin@SiO 2 The preparation method comprises the following steps: 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 completed, the reaction was continued for 3 h, and the mixture was cooled to room temperature, centrifuged at 9000 rpm for 15 min, and 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 to reflux for 24 h, and centrifuged at 9000 pm for 15 min. The precipitate was washed with deionized water and dried in vacuum to obtain modified porous silica. A2: Dissolve 3 g of naringin and 1.2 g of succinic anhydride in 15 mL of pyridine, and slowly add the succinic anhydride solution to the naringin solution under stirring. React in a water bath at 35°C for 16 h, dialyze with deionized water for 2 days, replace the dialysate every 4 h, and freeze-dry to obtain naringin succinate. A3: Dissolve the naringin succinate obtained in step A3 in DMF at a ratio of 20 mg / mL, add NHS / EDCI aqueous solution dropwise, 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 tri(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 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; A4: Take the modified naringin obtained in step A3 and the modified porous silica obtained in step A1, add them into deionized water and mix them evenly. The mass concentration of the modified porous silica in deionized water is 8 mg / mL. Stir at 200 rpm for 2 h at room temperature. Add DMSO. The volume ratio of DMSO to deionized water is 1:5. Continue stirring for 5 h. Centrifuge at 10000 rpm for 12 min. Wash the precipitate with anhydrous ethanol and deionized water, and vacuum dry to obtain naringin@SiO 2 .

[0026] This embodiment also provides a method for preparing the mesenchymal stem cell preparation for treating diabetes, comprising the following steps: 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×10min, 2000g×20min, 5000g×60min, and 10000g×60min at 4°C, filter through a 0.22μm filter membrane, centrifuge the filtrate at 100000g×60 min, discard the supernatant, and resuspend to 20μg / mL with complete culture medium (DMED / F12 medium containing 10% fetal bovine serum, 100U / mL penicillin and 100μg / mL streptomycin) to obtain a heavy suspension of honeysuckle exosomes; S2: Umbilical cord mesenchymal stem cells (hUCMSC) were isolated, cultured and subcultured according to conventional experimental methods. P4 hUCMSC were inoculated into six-well plates, with 5×10 4 cells, 2.5 mL complete medium, 37°C, 5% CO 2 After culturing for 24 h, the old culture medium was removed and 2 mL of honeysuckle exosome resuspension was added, and the culture was continued for 24 h. S3: After culturing for 24 h in step S2, hUCMSCs were collected and resuspended in PBS to a density of 10 6 / mL, add naringin@SiO 2 The mixture was stirred at 300 rpm for 30 min until the concentration reached 10 mg / mL, thereby obtaining a mesenchymal stem cell preparation for treating diabetes.

[0027] Example 2: This example provides a mesenchymal stem cell preparation for treating diabetes, comprising the following components: umbilical cord mesenchymal stem cells, naringin@SiO 2 .

[0028] Naringin@SiO 2 The raw materials for preparation 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.

[0029] Naringin@SiO 2 The preparation method comprises the following steps: 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 completed, the reaction was continued for 2 h, and the mixture was cooled to room temperature, centrifuged at 8000 rpm for 10 min, 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 (v:v=10:1), heated to reflux for 24 h, and centrifuged at 8000 pm for 10 min. The precipitate was washed with deionized water and vacuum dried to obtain modified porous silica. 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 to the naringin solution under stirring. React in a water bath at 35°C for 14 h. Dialyze with deionized water for 2 days, replace the dialysate every 4 h, and freeze-dry to obtain naringin succinate. A3: Dissolve the naringin succinate obtained in step A3 in DMF at a ratio of 20 mg / mL, add NHS / EDCI aqueous solution dropwise, 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 tri(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; A4: Take the modified naringin obtained in step A3 and the modified porous silica obtained in step A1, add them into deionized water and mix them evenly. The mass concentration of modified porous silica in deionized water is 5 mg / mL. Stir at 150 rpm for 1 h at room temperature. Add DMSO. The volume ratio of DMSO to deionized water is 1:5. Continue stirring for 4 h. Centrifuge at 10000 rpm for 8 min. Wash the precipitate with anhydrous ethanol and deionized water, and vacuum dry to obtain naringin@SiO 2 .

[0030] This embodiment also provides a method for preparing the mesenchymal stem cell preparation for treating diabetes, comprising the following steps: 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×10min, 2000g×20min, 5000g×60min, and 10000g×60min at 4°C, filter through a 0.22μm filter membrane, centrifuge the filtrate at 100000g×60 min, discard the supernatant, and resuspend to 20μg / mL with complete culture medium (DMED / F12 medium containing 10% fetal bovine serum, 100U / mL penicillin and 100μg / mL streptomycin) to obtain a honeysuckle exosome resuspension; S2: Umbilical cord mesenchymal stem cells (hUCMSC) were isolated, cultured and subcultured according to conventional experimental methods. P4 hUCMSC were inoculated into six-well plates, with 5×10 4 cells, 2.5 mL complete medium, 37°C, 5% CO 2 After culturing for 24 h, the old culture medium was removed and 2 mL of honeysuckle exosome resuspension was added, and the culture was continued for 24 h. S3: After culturing for 24 h in step S2, hUCMSCs were collected and resuspended in PBS to a density of 10 6 / mL, add naringin@SiO 2 The mixture was stirred at 200 rpm for 30 min until the concentration reached 10 mg / mL, thereby obtaining a mesenchymal stem cell preparation for treating diabetes.

[0031] Example 3: A mesenchymal stem cell preparation for treating diabetes, comprising the following components: umbilical cord mesenchymal stem cells, naringin@SiO 2 .

[0032] Naringin@SiO 2 The raw materials for preparation 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.

[0033] Naringin@SiO 2 The preparation method comprises the following steps: 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 completed, the reaction was continued for 2.5 h. The mixture was cooled to room temperature, centrifuged at 8500 rpm for 12 min, the precipitate was washed with deionized water, vacuum dried, dispersed in a mixed solution of anhydrous ethanol and 37% hydrochloric acid (v:v=11:1), heated to reflux for 24 h, centrifuged at 8500 pm for 12 min, the precipitate was washed with deionized water, and vacuum dried to obtain modified porous silica; 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 to the naringin solution under stirring. React in a water bath at 35°C for 15 h. Dialyze with deionized water for 2 days, replace the dialysate every 4 h, and freeze-dry to obtain naringin succinate. A3: Dissolve the naringin succinate obtained in step A3 in DMF at a ratio of 20 mg / mL, add NHS / EDCI aqueous solution dropwise, 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 tri(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 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; A4: Take the modified naringin obtained in step A3 and the modified porous silica obtained in step A1, add them into deionized water and mix them evenly. The mass concentration of modified porous silica in deionized water is 6 mg / mL. Stir at 180 rpm for 1.5 h at room temperature. Add DMSO. The volume ratio of DMSO to deionized water is 1:5. Continue stirring for 4.5 h. Centrifuge at 10000 rpm for 10 min. Wash the precipitate with anhydrous ethanol and deionized water, and dry it in vacuum to obtain naringin@SiO 2 .

[0034] This embodiment also provides a method for preparing the mesenchymal stem cell preparation for treating diabetes, comprising the following steps: 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×10min, 2000g×20min, 5000g×60min, and 10000g×60min at 4°C, filter through a 0.22μm filter membrane, centrifuge the filtrate at 100000g×60 min, discard the supernatant, and resuspend to 20μg / mL with complete culture medium (DMED / F12 medium containing 10% fetal bovine serum, 100U / mL penicillin and 100μg / mL streptomycin) to obtain a heavy suspension of honeysuckle exosomes; S2: Umbilical cord mesenchymal stem cells (hUCMSC) were isolated, cultured and subcultured according to conventional experimental methods. P4 hUCMSC were inoculated into six-well plates, with 5×10 4 cells, 2.5 mL complete medium, 37°C, 5% CO 2 After culturing for 24 h, the old culture medium was removed and 2 mL of honeysuckle exosome resuspension was added, and the culture was continued for 24 h. S3: After culturing for 24 h in step S2, hUCMSCs were collected and resuspended in PBS to a density of 10 6 / mL, add naringin@SiO 2 The mixture was stirred at 250 rpm for 30 min to a concentration of 10 mg / mL to obtain a mesenchymal stem cell preparation for treating diabetes.

[0035] The only difference between Comparative Example 1 and Example 1 is that no modified naringin is added.

[0036] The difference between Comparative Example 2 and Example 1 is that the porous silica is prepared without adding MPTMS, and naringenin is used instead of modified naringenin.

[0037] The difference between Comparative Example 3 and Example 1 is that naringin is used instead of naringin@SiO 2 .

[0038] The only difference between Comparative Example 4 and Example 1 is that the co-culture of honeysuckle exosomes and hUCMSCs is not used, that is, in step S2, the honeysuckle exosome heavy suspension is replaced by complete culture medium.

[0039] Experimental Example 1: P4 hUCMSC cells were inoculated into 96-well plates, with 3×10 cells per well. 3 cells, 37°C, 5% CO 2After culturing for 24 h, the old culture medium was removed and 20 μg / mL of honeysuckle exosome resuspension was added. Complete culture medium was added to the control group. After culturing for 24 h and 48 h, the old culture medium was removed and 100 μL of CCK-8 solution was added to each well. The cells were incubated for 2 h and the absorbance at 450 nm was detected using an enzyme reader. The results are shown in Table 1. Figure 1 shown.

[0040] Figure 1 The results showed that honeysuckle exosomes can significantly increase the cell proliferation rate of hUCMSC, effectively promote the proliferation of hUCMSC, and improve the activity of hUCMSC, indicating that the hUCMSC in the mesenchymal stem cell preparation for treating diabetes of the present invention has better biological activity and is conducive to improving the therapeutic effect.

[0041] Experimental Example 2: Select 6-week-old normal male C57BL / 6J mice (18±2g) and raise them in an SPF environment. During this period, they were free to drink water and eat, with a room temperature of 22.5±2.5°C, humidity (50-70%), and a 12h / 12h light-dark cycle. Give HFD (High-fatdiet, 60 kcal% fat) (ReadyDietech) for 8 weeks. Intraperitoneal injection of STZ 40 mg / kg for 3 consecutive days. One week after STZ injection, oral glucose tolerance test (OGTT) and intraperitoneal insulin tolerance test (IPITTS) were performed to ensure the establishment of type 2 diabetic mouse model. All mice were divided into 7 groups, each with 8 mice: control group, Example 1 group, comparative example 1-4 group, Example 1 and comparative example 1-4 group The stem cell preparations of Example 1 and comparative example 1-4 were intraperitoneally injected into diabetic model mice, with a single injection of 200 μL, once a week, for 5 consecutive weeks, and the diabetic mice were injected with an equal amount of PBS as the control group. One week after the last administration, peripheral blood was collected from rats and centrifuged at 3400 g for 10 min, and the supernatant was aspirated to obtain serum. The IL-1β concentration in the serum was determined by ELISA kit. The results were as follows: Figure 2 The rats were killed, and the pancreatic tissue was taken and placed in 30% sucrose for dehydration overnight, then embedded with embedding agent and stored at -80°C for later use. The pancreatic tissue was sliced ​​into 5 μm sections. The tissue sections were incubated with 0.5% Triton-X100 at room temperature for 15 min, washed three times with PBS, and blocked with serum at room temperature for 30 min. After blocking, Insulin and Glucagon primary antibodies were added, incubated at 4°C overnight, and cells that were not bound to the primary antibody were washed with PBS. Then, fluorescent secondary antibodies were added and incubated at room temperature for 2 h. The percentage of β cells was observed and counted under a fluorescence microscope. The results are shown in the figure. Figure 3 shown.

[0042] Figure 2The results showed that the IL-1β levels in Example 1 and Comparative Examples 1-4 were significantly lower than those in the control group, and the levels of pro-inflammatory factors in the serum of mice treated with the mesenchymal stem cell preparation were significantly reduced. Figure 3 The 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, could not exert a synergistic therapeutic effect, and the therapeutic effect decreased; Comparative Example 2 did not modify porous silica and naringin, only used physical adsorption and weak hydrogen bond interactions to load drugs, the loading amount of effective ingredients was reduced, the long-term sustained release was reduced, and the therapeutic effect was reduced; Comparative Example 3 did not load naringin, and the long-term sustained release was reduced; Comparative Example 4 did not use honeysuckle exosomes and mesenchymal stem cells for co-culture, cell activity decreased, and the therapeutic effect decreased; the above results show that the mesenchymal stem cell preparation for the treatment of diabetes of the present invention can effectively inhibit the reduction of pancreatic β cells, improve the reduction of β cell percentage caused by diabetes, thereby improving the body's hyperglycemia, promoting pancreatic cells to secrete insulin and thus play a hypoglycemic effect, while inhibiting the inflammatory response in the body.

[0043] Experimental Example 3: According to the methods of Examples 1-3 and Comparative Example 2, naringin@SiO 2 , determine the effective ingredient encapsulation rate (%), the results are as follows Figure 4 shown.

[0044] Figure 4 The results showed that the encapsulation efficiency of Examples 1-3 was significantly higher than that of Comparative Example 2. 2 More effective ingredients can be loaded to improve the therapeutic effect. In Comparative Example 2, naringin and porous silica were not modified, and the effective ingredients were loaded only through physical adsorption and weak hydrogen bond interactions, resulting in a reduced loading amount and decreased long-term effectiveness.

[0045] 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. Under the concept 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 with 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 with complete culture medium to obtain a heavy suspension of honeysuckle exosomes; S2: hUCMSCs were isolated, cultured and subcultured according to conventional experimental methods. P4 hUCMSCs were inoculated into six-well plates, cultured, the old culture medium was removed, and the heavy suspension of honeysuckle exosomes was added and cultured continuously; S3: After culturing in step S2, hUCMSCs are collected, resuspended with PBS, naringin@SiO2 is added, and stirred to obtain a mesenchymal stem cell preparation for treating diabetes; The raw materials for preparing 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, ultrasonicated, heated, TEOS and MPTMS were added dropwise, and the reaction was continued after the addition was completed, cooled, centrifuged, precipitated and washed, dried, dispersed in a mixed solution of anhydrous ethanol and 37% hydrochloric acid, heated to reflux, centrifuged, precipitated and washed, and vacuum dried to obtain modified porous silica; A2: Dissolve naringin and succinic anhydride in pyridine respectively, slowly add the succinic anhydride solution to the naringin solution under 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 during the process, dialyze with 0.008 M HCl in the dark, wash the precipitate with 0.008 M HCl, and freeze-dry to obtain modified naringin; A4: Take the modified naringin obtained in step A3 and the modified porous silica obtained in step A1, add them into deionized water, mix them evenly, stir, add DMSO, continue stirring, centrifuge, wash the precipitate with anhydrous ethanol and deionized water, and vacuum dry to obtain naringin@SiO2.

2. The method for preparing a mesenchymal stem cell preparation for treating diabetes according to claim 1, characterized in that: 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.

3. The method for preparing a mesenchymal stem cell preparation for treating diabetes according to claim 2, characterized in that: 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, characterized in that: 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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