A temperature-sensitive hydrogel with diabetes treatment effect, kit, method of use and applications thereof

Abstract

The application discloses a temperature-sensitive hydrogel with a diabetes treatment effect, a kit, a use method and application thereof, and belongs to the technical field of diabetes treatment drugs. The temperature-sensitive hydrogel is formed by mixing a human umbilical vein endothelial cell suspension, a mixed solution with a final concentration of 15-20% of P407 and a final concentration of 0.6-0.8% of sodium hyaluronate, a final concentration of 50 ng / mL of vascular endothelial growth factor, a final concentration of 10 ng / mL of basic fibroblast growth factor, a final concentration of 0.5-2% of lidocaine hydrochloride, a final concentration of 100-200 mg / mL of tumor necrosis factor alpha monoclonal antibody and a final concentration of 2-5% of islet cell-alginic acid gel fiber segments with a length of less than 1 mm. The temperature-sensitive hydrogel is safe, non-toxic, low in immunogenicity, low in cost and good in treatment effect. The reagent is combined with sterile consumables to form a kit, and the kit is convenient to use and can be better applied to the treatment of diabetes.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of diabetes treatment drugs, and particularly relates to a temperature-sensitive hydrogel with a diabetes treatment effect, a kit, a use method and application thereof. BACKGROUND

[0002] Diabetes is a chronic disease caused by decreased or failed pancreatic islet function, leading to systemic vascular disease and multiple organ damage, which seriously threatens life and health. Diabetes is mainly divided into two categories: type 1 diabetes and type 2 diabetes. Type 1 diabetes accounts for about 10% of the total number of diabetes patients, and more than 70% of its islet cells are destroyed, which cannot timely and adequately secrete insulin to maintain normal blood glucose levels. Patients need to inject insulin to stabilize blood glucose, which may cause severe hypoglycemia and various complications of diabetes. Type 2 diabetes is more common, accounting for more than 80% of the total number of diabetes patients, and one-third of patients in the middle and late stages also develop type 1 diabetes.

[0003] Current common treatment methods such as insulin injection can only regulate blood glucose to normal levels in stages, and cannot simulate the precise adjustment of normal islets to insulin secretion time and dose, thus cannot avoid the risk of hypoglycemia and the occurrence of diabetes complications. Moreover, most hypoglycemic drugs have strong side effects, which can further damage the health of patients. In order to improve the quality of life of patients, researchers have begun to use islet transplantation technology to treat diabetes. Because the occurrence of diabetes is caused by impaired islet function, islet transplantation can theoretically cure diabetes, but in practice, this treatment method still has many problems to be solved, such as the islet separation and extraction process which is not only time-consuming and labor-intensive, but also expensive, requiring the use of COBE.2991 cell separation machine and other equipment for purification; similar to other organ transplantation technologies, the shortage of donors seriously limits the wide application of islet transplantation technology; there is strong immune rejection after allogeneic or xenogeneic islet transplantation; and current islet transplantation needs to be guided by B-ultrasound to perform percutaneous liver portal vein puncture and infusion, or subrenal capsule transplantation, which is a complex operation requiring professional physicians and highly specialized supporting facilities. All of the above are obstacles to the application of islet transplantation in clinical practice. SUMMARY

[0004] In order to overcome the shortcomings of the prior art, the purpose of the present application is to provide a temperature-sensitive hydrogel with a diabetes treatment effect, a kit, a use method and application thereof, which makes diabetes treatment simple, has lower immunogenicity, lower cost and better treatment effect.

[0005] In order to achieve the above purpose, the present application adopts the following technical solutions:

[0006] The application discloses a temperature-sensitive hydrogel with a diabetes treatment effect, which is prepared by mixing a human umbilical vein endothelial cell suspension, a mixed solution of P407 with a final concentration of 15-20% and sodium hyaluronate with a final concentration of 0.6-0.8%, vascular endothelial growth factor with a final concentration of 50 ng / mL, basic fibroblast growth factor with a final concentration of 10 ng / mL, lidocaine hydrochloride with a final concentration of 0.5%-2%, tumor necrosis factor alpha monoclonal antibody with a final concentration of 100-200 mg / mL and pancreatic islet cell-alginic acid gel fiber segments with a final concentration of 2-5%.

[0007] The final concentration of the human umbilical vein endothelial cells is 5-8*10 6 / mL, and the pancreatic islet cell-alginic acid gel fiber segments are gel fiber segments with a length of less than 1 mm and a diameter of 100-300 mu m, which are prepared by mixing pancreatic islet cells, physiological saline and alginic acid salt.

[0008] Preferably, in the pancreatic islet cell-alginic acid gel fiber segments, the final concentration of the pancreatic islet cells is 3-6*10 6 / mL, and the final concentration of the alginic acid salt is 1.5-3%.

[0009] Preferably, the human umbilical vein endothelial cells and the pancreatic islet cells are both low immunogenic cells.

[0010] Preferably, the pancreatic islet cells are obtained by inducing umbilical cord stem cells by an islet cell induction solution.

[0011] The application further discloses application of the temperature-sensitive hydrogel with the diabetes treatment effect in preparation of a diabetes treatment drug.

[0012] The application further discloses a kit with a diabetes treatment effect, which comprises sterile reagents and sterile consumables, the sterile reagents comprise physiological saline, sodium hyaluronate, a calcium chloride solution, a mixed solution of P407 and sodium hyaluronate, vascular endothelial growth factor, basic fibroblast growth factor, lidocaine hydrochloride and tumor necrosis factor alpha monoclonal antibody, and the sterile consumables comprise a 1 mL syringe, a 2 mL syringe, a 30G needle, a 14G needle, a single-sided blade, a petri dish and a 100 mu m cell screen.

[0013] Preferably, the kit further comprises an islet cell induction solution and a dithizone staining solution.

[0014] More preferably, the islet cell induction solution includes islet cell A1 induction solution and islet cell A2 induction solution. The islet cell A1 induction solution consists of H-DMEM high glucose medium, 1.2 mg / mL nicotinamide, and β-mercaptoethanol at a concentration of 0.07‰. The islet cell A2 induction solution consists of DMEM / F12 high glucose medium, 2% FBS, 2% B27, and 1.2 mg / mL nicotinamide.

[0015] This invention also discloses a method for using the aforementioned reagent kit for treating diabetes. First, islet cells, physiological saline, and sodium alginate are mixed to obtain islet cell-sodium alginate gel. Next, calcium chloride solution is placed in a petri dish, and the islet cell-sodium alginate gel is drawn using a 1mL syringe with a 30G needle. While being extruded uniformly into the calcium chloride solution, parallel lines are drawn to obtain uniformly sized gel fibers. These fibers are washed with physiological saline, cut into segments, and calcified with calcium chloride solution. They are then washed again with physiological saline and filtered through a 100μm cell sieve to obtain islet cell-algin gel fiber segments. The islet cell-algin gel fiber segments are then mixed with a mixture of human umbilical vein endothelial cells, P407, and sodium hyaluronate, vascular endothelial growth factor, basic fibroblast growth factor, lidocaine hydrochloride, and tumor necrosis factor-α monoclonal antibody to obtain a thermosensitive hydrogel. Finally, the thermosensitive hydrogel is injected subcutaneously into a diabetic patient using a 2mL syringe with a 14G needle.

[0016] Preferably, the pancreatic islet cell-alginic acid gel fiber segment is a gel fiber with a length ≤1mm cut by a single-edged blade.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] This invention provides a thermosensitive hydrogel with therapeutic effects on diabetes. The islet cell-alginic acid gel fiber segment acts as an islet analog, encapsulating a large number of islet cells with a diameter of 100-300 μm, close to the size of natural islets. After external capillary formation, these cells can survive for a long time and can replace the body's natural islets in secreting insulin to control blood sugar. The alginate material in the fiber segment has low immunogenicity and is biologically inert, allowing it to remain in the body for a long time. The outer alginate layer can interact with Ca... 2+Microcapsules with an eggshell-like structure encapsulate internal alginate, forming gels of various shapes with high mechanical strength. This provides a suitable environment for the encapsulated pancreatic islet cells. The pores on the calcified membrane surface allow small molecules such as nutrients and cellular metabolic products to pass through and exchange, while immune cells are isolated, reducing immune rejection. Lidocaine hydrochloride and tumor necrosis factor-alpha monoclonal antibody fully utilize the advantages of injectable administration for the survival of transplanted microcapsules, while significantly inhibiting the inflammatory response induced by injectable administration and delaying gel degradation for up to 15 days, providing ample time for angiogenesis in the transplanted tissue. Furthermore, to further accelerate angiogenesis, this invention mixes a large number of human umbilical vein endothelial cells into the thermosensitive gel. Under the influence of vascular endothelial growth factor and basic fibroblast growth factor, these cells rapidly form capillaries, addressing cell death caused by ischemia and hypoxia in the islet replacement, greatly improving transplant survival and better reversing the disease symptoms of diabetic patients. The P407 in this thermosensitive hydrogel is a temperature-sensitive gel material. It is liquid at low temperatures, facilitating experimental handling, and transforms into a gel state upon entering the body due to increased temperature, maintaining a certain shape. Low-crosslinked sodium hyaluronate prevents P407 from degrading too quickly, thus preventing insufficient space for capillary formation and ensuring adequate blood and oxygen supply to the pancreatic islet cells within the gel fiber segments. The mixture of P407 and low-crosslinked sodium hyaluronate acts as a matrix, protecting the human umbilical vein endothelial cells and providing sufficient space and time to promote capillary formation, thereby improving the blood and oxygen supply to the pancreatic islet cells within the fiber segments and increasing their survival rate. This thermosensitive hydrogel is biodegradable, safe, non-toxic, has low immunogenicity, and is inexpensive. It offers a simple and effective treatment method for diabetes and can be applied to the treatment of diabetes.

[0019] This invention provides a reagent kit for the treatment of diabetes. In the sterile reagent, physiological saline washes and dilutes islet cells, islet cell-alginic acid gel fiber segments, and sodium alginate. Sodium alginate, as a natural polymer material, has excellent biocompatibility, ensuring long-term cell survival during the preparation of islet cell-alginic acid gel fiber segments. Calcium chloride solution combines with the outer layer of sodium alginate to form an eggshell-like structure, encapsulating the inner sodium alginate to form gels of various shapes, such as gel spheres and gel fibers. P407 and low-crosslinked sodium hyaluronate are used to prepare a thermosensitive gel, providing time and space for capillary angiogenesis. Vascular endothelial growth factor and basic fibroblast growth factor accelerate angiogenesis, providing nutrition to the islet cells in the gel. It significantly improves survival rates; lidocaine hydrochloride provides local anesthesia, reducing initial discomfort after injection, and also reduces the inflammatory response after initial injection; tumor necrosis factor-alpha monoclonal antibody modulates immunity and reduces the inflammatory response for a relatively long period (about 2 weeks), thereby prolonging the survival time of cells in the gel (the inflammatory response causes a large number of immune cells to aggregate and kill foreign cells and other foreign substances); it is easy to use with sterile consumables, and a large number of injectable islet analogs can be obtained in a sterile laboratory using this kit. The overall operation is simple, eliminating the need for islet separation equipment such as cell separators, greatly reducing the requirements for hardware facilities and technical personnel, facilitating application and promotion, and the islet cells and endothelial cells obtained from a single experiment can be aliquoted and cryopreserved for subsequent multiple uses.

[0020] Furthermore, the islet cell induction solution can induce umbilical cord stem cells into islet cells with low immunogenicity, which secrete insulin and are less likely to induce immune rejection; the dithizone staining solution can stain islet cells reddish-brown, and the staining identification of stem cell-induced islet cells can ensure successful induction.

[0021] The present invention provides a method for using a reagent kit with diabetes treatment effects. The subcutaneous injection is simple, easy to monitor, time-saving, highly flexible, and can be repeated. It does not require ultrasound-guided percutaneous hepatic portal vein puncture and catheterization for infusion, or subcapsular renal transplantation, saving several hours of surgical time, greatly reducing the risk of infection, and avoiding complications such as liver hemorrhage, portal vein thrombosis, and portal hypertension. Attached Figure Description

[0022] Figure 1 This is a diagram of human umbilical cord mesenchymal stem cells used in this invention.

[0023] Figure 2 This is a diagram of human umbilical vein endothelial cells according to the present invention;

[0024] Figure 3 This is a diagram of pancreatic islet cell-alginic acid gel fiber segments of the present invention;

[0025] Figure 4 This is an image of pancreatic islet cells-alginic acid gel spheres from the present invention;

[0026] Figure 5 This is a comparison diagram of cell proliferation in the gel fiber segments and gel spheres of the present invention;

[0027] Figure 6 This is a comparison chart of blood inflammatory factors in diabetic mice according to the present invention. Detailed Implementation

[0028] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0029] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0030] The present invention will now be described in further detail with reference to the accompanying drawings:

[0031] This invention provides a thermosensitive hydrogel preparation kit with therapeutic effects on diabetes, comprising sterile reagents requiring cryopreservation and sterile consumables for room temperature storage. The sterile reagents include: islet cell induction solution, dithizone staining solution, physiological saline, 5% alginate, 1.5% calcium chloride solution, a mixture of 25% P407 (poloxam 407) and 1% low-crosslinked sodium hyaluronate (HA), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), lidocaine hydrochloride, and tumor necrosis factor-α monoclonal antibody. The sterile consumables include: two 1mL syringes, two 2mL syringes, two 30G needles, two 14G needles, two single-edged blades, two plastic petri dishes, and two 100μm cell sieves.

[0032] The sterile reagents must be frozen, and the sterile consumables must be stored at room temperature, with a shelf life of 1 year. The islet cell induction solution includes islet cell A1 induction solution and islet cell A2 induction solution. The islet cell A1 induction solution consists of H-DMEM high glucose medium, 1.2 mg / mL nicotinamide, and 0.07‰ (V / V) β-mercaptoethanol. The islet cell A2 induction solution consists of DMEM / F12 high glucose medium, 2% FBS, 2% B27, and 1.2 mg / mL nicotinamide. The dithizone staining solution is prepared by dissolving dithizone powder in dimethyl sulfoxide. The alginate is sodium alginate, potassium alginate, or ammonium alginate.

[0033] The present invention provides a method for using a thermosensitive hydrogel preparation kit with therapeutic effects on diabetes, comprising the following steps:

[0034] 1. Preparation of cell materials:

[0035] 1) Pancreatic islet cell induction

[0036] Umbilical cord stem cells (UC-MSCs) were induced into pancreatic islet cells using an islet cell induction solution.

[0037] Among them, UC-MSCs can be replaced with autologous adipose-derived stem cells (ADSCs) or autologous bone marrow stem cells (BMSCs) isolated from diabetic patients for use by diabetic patients themselves; islet cells induced by islet cell induction solution can be replaced with human fetal islet cells or newborn porcine islet cells.

[0038] 2) Identification of pancreatic islet cells

[0039] The pancreatic islet cells obtained in step 1) were identified by staining with dithizone staining solution: reddish-brown cell clusters were visible, indicating that the pancreatic islet cells were successfully induced.

[0040] 3) Culture and morphological identification of human umbilical vein endothelial cells (HUVECs)

[0041] HUVECs were isolated from human umbilical vein tissue by enzymatic digestion and passaged. Under an inverted microscope, the HUVECs cells showed a typical cobblestone appearance with prominent nuclei. They can be obtained from commercially available cells, P3 to P5 generations, and need to be identified by CD31 / vWF immunofluorescence with a purity of over 90%.

[0042] 2. Preparation of islet analogues (islet cells-sodium alginate gel fiber segments):

[0043] The pancreatic islet cells obtained in step 1) are suspended in physiological saline, and 5% sodium alginate is added and mixed evenly to obtain pancreatic islet cell-sodium alginate gel with a final cell concentration of 3-6 × 10⁻⁶. 6 / mL, sodium alginate final concentration 1.5-3%; place 1.5% calcium chloride solution in a petri dish, use a 1mL syringe to draw pancreatic islet cell-sodium alginate gel, and squeeze it out at a uniform speed through a 30G needle into the 1.5% calcium chloride solution while drawing parallel lines to obtain uniformly thick gel fibers. Discard the 1.5% calcium chloride solution, wash the gel fibers three times with physiological saline, remove the liquid, and use a single-edged blade to cut the gel fibers in the petri dish into small segments along the long axis with a length ≤1mm. Add 1.5% calcium chloride solution to the petri dish, gently shake to distribute the gel fiber segments evenly, soak for 2min, remove the liquid, wash three times with physiological saline, filter through a 100μm cell sieve to obtain pancreatic islet cell-sodium alginate gel fiber segments with completely calcified surfaces, which can be used as islet analogs for later use.

[0044] 3. Preparation of composite temperature-sensitive gel:

[0045] The human umbilical vein endothelial cells obtained in step 1, 3) are suspended in physiological saline, mixed with 25% P407 + 1% low cross-linked sodium hyaluronate, vascular endothelial growth factor, basic fibroblast growth factor, lidocaine hydrochloride and tumor necrosis factor-α monoclonal antibody, and then the islet cell-sodium alginate gel fiber segment prepared in step 2 is added and mixed to obtain the composite thermosensitive gel.

[0046] The final concentration of human umbilical vein endothelial cells was 5–8 × 10⁻⁶. 6 / mL, final concentration of vascular endothelial growth factor 50ng / mL, final concentration of bFGF 10ng / mL, final concentration of P407 15-20%, final concentration of HA 0.6-0.8%, pancreatic islet cell-algin gel fiber segment 2-5%, lidocaine hydrochloride 0.5%-2%, tumor necrosis factor α monoclonal antibody 100-200mg / mL.

[0047] 4. Diabetes treatment:

[0048] A type 1 diabetes model was induced in mice by disrupting pancreatic β-cells with streptozotocin. Then, the composite thermosensitive gel obtained in step 3 was subcutaneously injected into the groin of the model mice, 1 mL per side. After the gel entered the body, it solidified. After injection, the gel was evenly pressed to expand its distribution area and reduce its thickness. The endothelial cells in the gel rapidly formed blood vessels under the dual action of vascular endothelial growth factor and basic fibroblast growth factor, connecting with the blood vessels of surrounding tissues and transporting nutrients. The combined use of lidocaine hydrochloride and tumor necrosis factor-α monoclonal antibody reduced the inflammatory response caused by transplantation and delayed gel volume loss. These multiple effects ensured the survival of the implanted pancreatic islet cells and their insulin secretion, thus achieving a long-term hypoglycemic effect.

[0049] Example 1

[0050] 1. Preparation of cell materials:

[0051] 1) Pancreatic islet cell induction

[0052] Human umbilical cord mesenchymal stem cells (UC-MSCs) were isolated and cultured from the umbilical cords of healthy newborns, cultured to passage P5, and observed under a microscope. Figure 1 The cells were elongated spindle-shaped, uniform in size, and arranged in a whorl pattern. Flow cytometry analysis showed that the expression of cell surface markers CD29, CD44, and CD90 was positive, while the expression of CD34 and CD45 was negative, thus identifying them as UC-MSC cells.

[0053] UC-MSCs cultured to passage P5 were divided into 5 × 10⁶ cells / year. 5 Cells were seeded per well in 6-well plates. On the second day, when the cell confluence was 70-80%, the culture medium was replaced with pancreatic islet cell A1 induction medium. After culturing for another 24 hours, the pancreatic islet cell A1 induction medium was removed, and pancreatic islet cell A2 induction medium was added for continuous induction. The pancreatic islet cell A2 induction medium was changed every 2-3 days, and the induction was observed under a microscope. When the induction was completed, scattered islet-like cell clusters appeared. The induction was continued until about 30 days, and pancreatic islet cells were obtained.

[0054] The induction solution for pancreatic islet cells A1 included H-DMEM high glucose medium, 1.2 mg / mL nicotinamide, and 0.07‰ (V / V) β-mercaptoethanol; the induction solution for pancreatic islet cells A2 included DMEM / F12 high glucose medium, 2% FBS, 2% B27, and 1.2 mg / mL nicotinamide.

[0055] 2) Identification of pancreatic islet cells

[0056] Mix 10 μL of dithizone staining solution with 1 mL of pancreatic islet cell A2 induction solution to stain and identify the pancreatic islet cells induced in step 1). Add the mixture to a 6-well plate and incubate at 37°C for 30 min. Wash the cells three times with PBS and observe them under a microscope. If the cell clusters are stained reddish-brown, the induction is successful.

[0057] The dithizone staining solution was prepared by dissolving dithizone powder in dimethyl sulfoxide at a concentration of 10 mg / mL.

[0058] 3) Culture and morphological identification of human umbilical vein endothelial cells (HUVECs)

[0059] Primary culture method for HUVECs: In a clean bench, rinse the umbilical cord with PBS solution to locate the umbilical vein. Use a 20mL syringe to inject PBS into the umbilical vein and rinse repeatedly. Clamp one end of the umbilical vein with hemostats, and infuse the other end with 0.2% collagenase I (prepared with PBS solution) until the umbilical vein is full. Clamp the umbilical vein again with hemostats. Place the umbilical cord in sterile PBS solution and incubate at 37°C for approximately 10 minutes. During incubation, aspirate / inject the enzyme solution from the umbilical vein every 3 minutes to promote thorough and uniform contact between the collagenase and the vein wall. Remove the umbilical cord, blot dry the surface with sterile gauze, and transfer the enzyme solution to a 50mL centrifuge tube. Add an equal volume of culture medium containing 10% FBS to neutralize. Then, use a 2mL syringe to flush the umbilical vein with sterile PBS solution, collecting the flushing fluid as well. Mix thoroughly, centrifuge to collect the cells, and resuspend in the culture medium. The cell suspension was seeded into cell culture flasks and incubated in a 5% CO2, 37°C incubator. After 24 hours, the culture medium was changed, and non-adherent cells were removed. The medium was then changed every 2–3 days, and the cells were passaged normally to obtain human umbilical vein endothelial cells.

[0060] Morphological identification of HUVECs: Observation was performed using an inverted microscope, such as... Figure 2 HUVECs cells have a typical cobblestone appearance and prominent nuclei.

[0061] 2. Preparation of islet analogues (islet cells-sodium alginate gel fiber segments):

[0062] The pancreatic islet cells obtained in step 1) are digested and centrifuged, suspended in physiological saline, and then mixed with 5% sodium alginate to obtain pancreatic islet cell-sodium alginate gel with a final cell concentration of 3 × 10⁻⁶. 6 / mL, sodium alginate final concentration 1.5%; place 1.5% calcium chloride solution in a petri dish, use a 1mL syringe to draw up islet cell-sodium alginate gel, and squeeze it out at a uniform speed through a 30G needle into the 1.5% calcium chloride solution while drawing parallel lines to obtain gel fibers of uniform thickness with a diameter of 100-300μm. Discard the 1.5% calcium chloride solution, wash the gel fibers three times with physiological saline, remove the liquid, and cut the gel fibers into small segments along the long axis with a single-edged blade in a petri dish, with a length ≤1mm. Add 1.5% calcium chloride solution to the petri dish, gently shake to distribute the gel fiber segments evenly, soak for 2min, remove the liquid, wash three times with physiological saline, filter through a 100μm cell sieve, and obtain islet cell-sodium alginate gel fiber segments with completely calcified surface, which are used as islet analogs for later use. Morphological image see Figure 3 The diameter of the islet cell-sodium alginate gel fiber segment is 100-300 μm and the length is ≤1 mm;

[0063] Simultaneously, using a 1mL syringe, islet cell-sodium alginate gel was drawn up and squeezed out at a uniform rate through a 30G needle. A 1.5% calcium chloride solution was then added, and the mixture was soaked for 2 minutes. The liquid was then removed, and the mixture was washed three times with physiological saline to obtain islet cell-sodium alginate gel spheres. Morphological images are shown below. Figure 4 The diameter is 1000-1500 μm. The proliferation and survival of cells in gel spheres and gel fiber segments were compared. Figure 5 The results showed that cells in the gel fiber segments proliferated and survived better, making them more suitable as islet analogs for transplantation.

[0064] 3. Preparation of composite temperature-sensitive gel:

[0065] The human umbilical vein endothelial cells obtained in step 1, 3) are suspended in physiological saline, mixed with a mixture of 25% P407 and 1% low cross-linked sodium hyaluronate, vascular endothelial growth factor, basic fibroblast growth factor, lidocaine hydrochloride and tumor necrosis factor-α monoclonal antibody, and then the pancreatic islet cell-sodium alginate gel fiber segment prepared in step 2 is added and mixed to obtain the composite thermosensitive gel.

[0066] The final concentration of human umbilical vein endothelial cells was 5 × 10⁻⁶. 6 / mL, final concentration of vascular endothelial growth factor 50ng / mL, final concentration of bFGF 10ng / mL, final concentration of P407 15%, final concentration of HA 0.6%, islet cell-algin gel fiber segment 2%, lidocaine hydrochloride 0.5%, tumor necrosis factor α monoclonal antibody 100mg / mL.

[0067] 4. Diabetes treatment:

[0068] 1) Prepare streptozotocin solution (STZ)

[0069] Prepare a 2.1% citric acid aqueous solution and a 2.94% sodium citrate aqueous solution. Mix the two solutions, adjust the pH to 4.2-4.5, add streptozotocin to dissolve, and the final concentration is 10 mg / mL. Filter aseptically to obtain the final solution.

[0070] 2) Establish a type 1 diabetic mouse model

[0071] Thirty 6-week-old SPF-grade male ICR mice were used to establish the mouse model via intraperitoneal injection of STZ after one week of acclimatization. Mice were fasted and deprived of water for 12 hours before administration. Body weight (g) and fasting blood glucose (mmol / L) were then measured. STZ should be prepared fresh and administered immediately, protected from light throughout the process, at a dose of 50 mg / kg. -1 The mice were injected once daily for six consecutive days. One week after the last injection, the mice were restrained with a mouse restraint device, and the blood glucose level in the tail vein was measured using a rapid blood glucose meter. A random blood glucose level ≥16.7 mmol / L for three consecutive days indicated successful establishment of the animal model. A total of 17 type 1 diabetic mice were obtained.

[0072] 3) Treatment of diabetic mice

[0073] Sixteen type 1 diabetic mice were divided into two groups, with eight mice in each group, namely the control group and the treatment group.

[0074] The composite thermosensitive gel obtained in step 3 was placed at 4°C for half an hour and became liquid. After mixing, it was drawn into a 2mL syringe and injected subcutaneously into the groin of diabetic mice in the treatment group using a 14G needle. The injection was 1mL per side. After the composite thermosensitive gel entered the body, it formed a semi-solid state. After the injection, it was pressed evenly to expand the gel distribution area and reduce the gel thickness. The control group was injected with an equal amount of blank gel.

[0075] The blank gel was prepared by the same steps using 5% sodium alginate, 1.5% calcium chloride solution, 25% P407 and 1% low crosslinked sodium hyaluronate, and physiological saline.

[0076] 4) Monitoring of treatment outcomes

[0077] For three consecutive months, the blood glucose levels and body weight of mice were continuously monitored. The specific results are as follows:

[0078] After islet analogue injection and transplantation, the blood glucose level of mice under non-fasting conditions was measured twice a week, and the mice were weighed once a week. The blood glucose monitoring results of mice in the control group and the treatment group are shown in Table 1, and the weight monitoring results of mice in the control group and the treatment group are shown in Table 2.

[0079] Table 1: Blood glucose monitoring results in mice of the control and treatment groups

[0080]

[0081] Table 2: Weight monitoring results of mice in the control and treatment groups

[0082]

[0083] Table 1 shows that the blood glucose levels of mice in the treatment group remained below 11.1 mmol / L starting from week 5, indicating that the pancreatic islet grafts were functioning normally in lowering blood glucose. During the 3-month observation period, the survival rate of mice in the treatment group was 83%. In the control group, blood glucose levels were all ≥16.7 mmol / L, and all mice in the control group died during the 3-month observation period. Table 2 shows that the body weight of mice in the control group decreased continuously starting from week 2, while the body weight of mice in the treatment group showed an increasing trend.

[0084] Blood was drawn from the tail vein 24 hours after injection. The results are as follows:

[0085] Serum cytokine IL-6 and IFN-γ levels were detected by ELISA, and the results are as follows: Figure 6The study found that, compared with the control group, the number of inflammatory factors in the blood of mice in the treatment group was significantly reduced after injection. This indicates that the combined use of lidocaine hydrochloride and tumor necrosis factor-α monoclonal antibody can reduce the inflammatory response caused by transplantation, ensure the survival of implanted pancreatic islet cells, secrete insulin, and thus play a long-term role in lowering blood sugar.

[0086] Example 2

[0087] 1. Preparation of cell materials:

[0088] 1) Pancreatic islet cell induction

[0089] Bone marrow stem cells (BMSCs) were isolated from diabetic patients, cultured to passage P4, and then subjected to a reaction at a rate of 5 × 10⁻⁶. 5 Cells were seeded per well in 6-well plates. When the cell confluence reached 70-80%, the culture medium was replaced with pancreatic islet cell A1 induction medium. After culturing for 24 hours, the pancreatic islet cell A1 induction medium was removed, and pancreatic islet cell A2 induction medium was added for continuous induction. The pancreatic islet cell A2 induction medium was replaced every 2-3 days to obtain pancreatic islet cells.

[0090] The induction solution for pancreatic islet cells A1 included H-DMEM high glucose medium, 1.2 mg / mL nicotinamide, and 0.07‰ (V / V) β-mercaptoethanol; the induction solution for pancreatic islet cells A2 included DMEM / F12 high glucose medium, 2% FBS, 2% B27, and 1.2 mg / mL nicotinamide.

[0091] 2) Identification of pancreatic islet cells

[0092] Mix 10 μL of dithizone staining solution with 1 mL of pancreatic islet cell A2 induction solution to stain and identify the pancreatic islet cells induced in step 1). Add the mixture to a 6-well plate and incubate at 37°C for 30 min. Wash the plate three times with PBS and observe the induction process under a microscope.

[0093] The dithizone staining solution was prepared by dissolving dithizone powder in dimethyl sulfoxide at a concentration of 10 mg / mL.

[0094] 3) Culture and morphological identification of human umbilical vein endothelial cells (HUVECs)

[0095] The steps are the same as in Example 1.

[0096] 2. Preparation of islet analogues (islet cells-sodium alginate gel fiber segments):

[0097] The pancreatic islet cells obtained in step 1) were digested and centrifuged, suspended in physiological saline, and then mixed thoroughly with 5% sodium alginate to obtain pancreatic islet cell-sodium alginate gel with a final cell concentration of 3.8 × 10⁻⁶. 6 / mL, sodium alginate final concentration 2%; place 1.5% calcium chloride solution in a petri dish, use a 1mL syringe to draw pancreatic islet cell-sodium alginate gel, and squeeze it out at a uniform speed through a 30G needle into the 1.5% calcium chloride solution while drawing parallel lines to obtain gel fibers with a diameter of 100-300μm and uniform thickness. Discard the 1.5% calcium chloride solution, wash the gel fibers three times with physiological saline, remove the liquid, and cut the gel fibers into small segments along the long axis with a single-edged blade in a petri dish, with a length ≤1mm. Add 1.5% calcium chloride solution to the petri dish, gently shake to distribute the gel fiber segments evenly, soak for 2min, remove the liquid, wash three times with physiological saline, filter through a 100μm cell sieve, and obtain pancreatic islet cell-sodium alginate gel fiber segments with complete surface calcification, which can be used as islet analogs for later use.

[0098] 3. Preparation of composite temperature-sensitive gel:

[0099] The human umbilical vein endothelial cells obtained in step 1, 3) are suspended in physiological saline, mixed with a mixture of 25% P407 and 1% low cross-linked sodium hyaluronate, vascular endothelial growth factor, basic fibroblast growth factor, lidocaine hydrochloride and tumor necrosis factor-α monoclonal antibody, and then the pancreatic islet cell-sodium alginate gel fiber segment prepared in step 2 is added and mixed to obtain the composite thermosensitive gel.

[0100] The final concentration of human umbilical vein endothelial cells was 6.6 × 10⁻⁶. 6 / mL, final concentration of vascular endothelial growth factor 50ng / mL, final concentration of bFGF 10ng / mL, final concentration of P407 17%, final concentration of HA 0.68%, islet cell-algin gel fiber segment 3%, lidocaine hydrochloride 1.1%, tumor necrosis factor α monoclonal antibody 160mg / mL.

[0101] Example 3

[0102] 1. Preparation of cell materials:

[0103] 1) Pancreatic islet cell induction

[0104] Autologous adipose-derived stem cells (ADSCs) were isolated from diabetic patients, cultured to passage P3, and then subjected to a reaction at a ratio of 5 × 10⁻⁶. 5 Cells were seeded per well in 6-well plates. When the cell confluence reached 70-80%, the culture medium was replaced with pancreatic islet cell A1 induction medium. After culturing for 24 hours, the pancreatic islet cell A1 induction medium was removed, and pancreatic islet cell A2 induction medium was added for continuous induction. The pancreatic islet cell A2 induction medium was replaced every 2-3 days to obtain pancreatic islet cells.

[0105] The induction solution for pancreatic islet cells A1 included H-DMEM high glucose medium, 1.2 mg / mL nicotinamide, and 0.07‰ (V / V) β-mercaptoethanol; the induction solution for pancreatic islet cells A2 included DMEM / F12 high glucose medium, 2% FBS, 2% B27, and 1.2 mg / mL nicotinamide.

[0106] 2) Identification of pancreatic islet cells

[0107] Mix 10 μL of dithizone staining solution with 1 mL of pancreatic islet cell A2 induction solution to stain and identify the pancreatic islet cells induced in step 1). Add the mixture to a 6-well plate and incubate at 37°C for 30 min. Wash the plate three times with PBS and observe the induction process under a microscope.

[0108] The dithizone staining solution was prepared by dissolving dithizone powder in dimethyl sulfoxide at a concentration of 10 mg / mL.

[0109] 3) Culture and morphological identification of human umbilical vein endothelial cells (HUVECs)

[0110] The steps are the same as in Example 1.

[0111] 2. Preparation of islet analogues (islet cells-sodium alginate gel fiber segments):

[0112] The pancreatic islet cells obtained in step 1) are digested and centrifuged, suspended in physiological saline, and then mixed thoroughly with 5% sodium alginate to obtain pancreatic islet cell-sodium alginate gel with a final cell concentration of 6 × 10⁻⁶. 6 / mL, sodium alginate final concentration 3%; place 1.5% calcium chloride solution in a petri dish, use a 1mL syringe to draw pancreatic islet cell-sodium alginate gel, and squeeze it out at a uniform speed through a 30G needle into the 1.5% calcium chloride solution while drawing parallel lines to obtain gel fibers with a diameter of 100-300μm and uniform thickness. Discard the 1.5% calcium chloride solution, wash the gel fibers three times with physiological saline, remove the liquid, and cut the gel fibers into small segments along the long axis with a single-edged blade in a petri dish, with a length ≤1mm. Add 1.5% calcium chloride solution to the petri dish, gently shake to distribute the gel fiber segments evenly, soak for 2min, remove the liquid, wash three times with physiological saline, filter through a 100μm cell sieve, and obtain pancreatic islet cell-sodium alginate gel fiber segments with complete surface calcification, which can be used as islet analogs for later use.

[0113] 3. Preparation of composite temperature-sensitive gel:

[0114] The human umbilical vein endothelial cells obtained in step 1, 3) are suspended in physiological saline, mixed with a mixture of 25% P407 and 1% low cross-linked sodium hyaluronate, vascular endothelial growth factor, basic fibroblast growth factor, lidocaine hydrochloride and tumor necrosis factor-α monoclonal antibody, and then the pancreatic islet cell-sodium alginate gel fiber segment prepared in step 2 is added and mixed to obtain the composite thermosensitive gel.

[0115] The final concentration of human umbilical vein endothelial cells was 8×10⁻⁶. 6 / mL, vascular endothelial growth factor final concentration 50ng / mL, bFGF final concentration 10ng / mL, P407 final concentration 20%, HA final concentration 0.8%, islet cell-algin gel fiber segment 5%, lidocaine hydrochloride 2%, tumor necrosis factor α monoclonal antibody 200mg / mL.

[0116] Example 4

[0117] 1. Preparation of cell materials:

[0118] 1) Culture and morphological identification of human umbilical vein endothelial cells (HUVECs)

[0119] The steps are the same as in Example 1.

[0120] 2. Preparation of islet analogues (islet cells-sodium alginate gel fiber segments):

[0121] Human fetal pancreatic islet cells were digested and centrifuged, suspended in physiological saline, and then mixed thoroughly with 5% sodium alginate to obtain islet cell-sodium alginate gel with a final cell concentration of 4.5 × 10⁻⁶. 6 / mL, sodium alginate final concentration 1.6%; place 1.5% calcium chloride solution in a petri dish, use a 1mL syringe to draw pancreatic islet cell-sodium alginate gel, and squeeze it out at a uniform speed through a 30G needle into the 1.5% calcium chloride solution while drawing parallel lines to obtain gel fibers with uniform thickness of 100-300μm in diameter. Discard the 1.5% calcium chloride solution, wash the gel fibers three times with physiological saline to remove the liquid, and cut the gel fibers into small segments along the long axis with a single-edged blade in a petri dish, with a length ≤1mm. Add 1.5% calcium chloride solution to the petri dish, gently shake to distribute the gel fiber segments evenly, soak for 2min, remove the liquid, wash three times with physiological saline, filter through a 100μm cell sieve to obtain pancreatic islet cell-sodium alginate gel fiber segments with completely calcified surface, which can be used as islet analogs for later use.

[0122] 3. Preparation of composite temperature-sensitive gel:

[0123] The human umbilical vein endothelial cells obtained in step 1) are suspended in physiological saline, mixed with a mixture of 25% P407 and 1% low cross-linked sodium hyaluronate, vascular endothelial growth factor, basic fibroblast growth factor, lidocaine hydrochloride and tumor necrosis factor-α monoclonal antibody, and then mixed with the pancreatic islet cell-sodium alginate gel fiber segment prepared in step 2 to obtain the composite thermosensitive gel.

[0124] The final concentration of human umbilical vein endothelial cells was 7.2 × 10⁻⁶. 6 / mL, final concentration of vascular endothelial growth factor 50ng / mL, final concentration of bFGF 10ng / mL, final concentration of P407 16%, final concentration of HA 0.7%, islet cell-algin gel fiber segment 4%, lidocaine hydrochloride 1.5%, tumor necrosis factor α monoclonal antibody 140mg / mL.

[0125] Example 5

[0126] 1. Preparation of cell materials:

[0127] 1) Culture and morphological identification of human umbilical vein endothelial cells (HUVECs)

[0128] The steps are the same as in Example 1.

[0129] 2. Preparation of islet analogues (islet cells-sodium alginate gel fiber segments):

[0130] Newborn porcine islet cells were digested and centrifuged, suspended in physiological saline, and then mixed thoroughly with 5% sodium alginate to obtain islet cell-sodium alginate gel with a final cell concentration of 5 × 10⁻⁶. 6 / mL, sodium alginate final concentration 3%; place 1.5% calcium chloride solution in a petri dish, use a 1mL syringe to draw pancreatic islet cell-sodium alginate gel, and squeeze it out at a uniform speed through a 30G needle into the 1.5% calcium chloride solution while drawing parallel lines to obtain gel fibers with a diameter of 100-300μm and uniform thickness. Discard the 1.5% calcium chloride solution, wash the gel fibers three times with physiological saline, remove the liquid, and cut the gel fibers into small segments along the long axis with a single-edged blade in a petri dish, with a length ≤1mm. Add 1.5% calcium chloride solution to the petri dish, gently shake to distribute the gel fiber segments evenly, soak for 2min, remove the liquid, wash three times with physiological saline, filter through a 100μm cell sieve, and obtain pancreatic islet cell-sodium alginate gel fiber segments with complete surface calcification, which can be used as islet analogs for later use.

[0131] 3. Preparation of composite temperature-sensitive gel:

[0132] The human umbilical vein endothelial cells obtained in step 1) are suspended in physiological saline, mixed with a mixture of 25% P407 and 1% low cross-linked sodium hyaluronate, vascular endothelial growth factor, basic fibroblast growth factor, lidocaine hydrochloride and tumor necrosis factor-α monoclonal antibody, and then mixed with the pancreatic islet cell-sodium alginate gel fiber segment prepared in step 2 to obtain the composite thermosensitive gel.

[0133] The final concentration of human umbilical vein endothelial cells was 6.3 × 10⁻⁶. 6 / mL, final concentration of vascular endothelial growth factor 50ng / mL, final concentration of bFGF 10ng / mL, final concentration of P407 18%, final concentration of HA 0.72%, islet cell-algin gel fiber segment 2%, lidocaine hydrochloride 1.9%, tumor necrosis factor α monoclonal antibody 100mg / mL.

[0134] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A temperature-sensitive hydrogel having a therapeutic effect on diabetes, characterized by, The temperature-sensitive hydrogel is mixed by a human umbilical vein endothelial cell suspension, a mixture of poloxamer 407 with a final concentration of 15-20% and low cross-linked sodium hyaluronate with a final concentration of 0.6-0.8%, vascular endothelial growth factor with a final concentration of 50 ng / mL, basic fibroblast growth factor with a final concentration of 10 ng / mL, lidocaine hydrochloride with a final concentration of 0.5-2%, tumor necrosis factor alpha monoclonal antibody with a final concentration of 100-200 mg / mL, and pancreatic islet-alginic acid gel fiber segments with a final concentration of 2-5%. The final concentration of the human umbilical vein endothelial cells is (5-8)×10 6 / mL, and the islet cell-alginate gel fiber segment is prepared as follows: mixing islet cells, normal saline and sodium alginate to obtain islet cell-sodium alginate gel; using a 1 mL syringe with a 30 G needle to suck the islet cell-sodium alginate gel, drawing lines in parallel at a constant speed while extruding in a calcium chloride solution to obtain gel fibers with uniform thickness, washing with normal saline, cutting, adding calcium chloride solution for calcification, washing with normal saline again, filtering with a 100 μm cell screen to obtain gel fiber segments with a length of ≤1 mm and a diameter of 100-300 μm.

2. The temperature-sensitive hydrogel having a therapeutic effect for diabetes according to claim 1, wherein the hydrogel is a hydrogel having a therapeutic effect for diabetes, which is prepared by mixing the compound of claim 1 with a pharmaceutically acceptable carrier. The final concentration of the islet cells in the islet cell-alginic acid gel fiber section is (3-6) x 10 6 / mL, and the final concentration of sodium alginate is 1.5%-3%.

3. The thermosensitive hydrogel with diabetic therapeutic effect according to claim 1, characterized in that, The human umbilical vein endothelial cells and the pancreatic islet cells are both low immunogenic cells.

4. The thermosensitive hydrogel with diabetic therapeutic effect according to claim 1, characterized in that, The pancreatic islet cells are induced by pancreatic islet cell induction solution from umbilical cord stem cells.

5. The temperature-sensitive hydrogel with a diabetes treatment effect according to any one of claims 1-4 is used for preparing a diabetes treatment drug.

6. A kit having a diabetes treating effect, characterized by, The kit comprises sterile reagents and sterile consumables, the sterile reagents comprising pancreatic islet cells, physiological saline, sodium alginate, calcium chloride solution, human umbilical vein endothelial cells, a mixture of poloxamer 407 and low cross-linked sodium hyaluronate, vascular endothelial growth factor, basic fibroblast growth factor, lidocaine hydrochloride, and tumor necrosis factor-alpha monoclonal antibody; and the sterile consumables comprising a 1 mL syringe, a 2 mL syringe, a 30 G needle, a 14 G needle, a single-blade knife, a petri dish, and a 100 μm cell screen. The kit is used as follows: firstly, the pancreatic islet cells, physiological saline, and sodium alginate are mixed to obtain pancreatic islet cell-sodium alginate gel; secondly, the calcium chloride solution is placed in a petri dish, the pancreatic islet cell-sodium alginate gel is sucked by a 1 mL syringe with a 30 G needle, and the gel fibers with uniform thickness are obtained by drawing lines in parallel while uniformly extruding the gel into the calcium chloride solution; the gel fibers are washed with physiological saline, cut, calcified by adding the calcium chloride solution, washed with physiological saline again, and filtered by a 100 μm cell screen to obtain pancreatic islet-alginic acid gel fiber segments with a length of ≤1 mm and a diameter of 100-300 μm; the pancreatic islet-alginic acid gel fiber segments are mixed with the human umbilical vein endothelial cells, the mixture of poloxamer 407 and low cross-linked sodium hyaluronate, the vascular endothelial growth factor, the basic fibroblast growth factor, the lidocaine hydrochloride, and the tumor necrosis factor-alpha monoclonal antibody to obtain the temperature-sensitive hydrogel of claim 1; and finally, the temperature-sensitive hydrogel is injected subcutaneously into a diabetes patient by a 2 mL syringe with a 14 G needle.

7. The kit having a diabetes treatment effect according to claim 6, wherein The kit further comprises pancreatic islet cell induction solution and dithizone staining solution.

8. The kit having a diabetes treatment effect according to claim 7, characterized by, The pancreatic islet cell induction solution comprises pancreatic islet cell A1 induction solution and pancreatic islet cell A2 induction solution, the pancreatic islet cell A1 induction solution is composed of H-DMEM high-sugar culture medium, 1.2 mg / mL nicotinamide, and 0.07‰ β-mercaptoethanol, and the pancreatic islet cell A2 induction solution is composed of DMEM / F12 high-sugar culture medium, 2% FBS, 2% B27, and 1.2 mg / mL nicotinamide.

9. A method for preparing a thermosensitive hydrogel with therapeutic effects on diabetes as described in claim 1, characterized in that, The pancreatic islet cells, physiological saline and sodium alginate are mixed to obtain pancreatic islet cell-sodium alginate gel; secondly, the calcium chloride solution is placed in a flat dish, the pancreatic islet cell-sodium alginate gel is taken by a 1 mL syringe with a 30 G needle, and the gel fibers with uniform thickness are obtained by drawing lines in parallel while uniformly extruding in the calcium chloride solution, the gel fibers are washed by physiological saline, cut into segments, added into the calcium chloride solution for calcification, washed by physiological saline again, filtered by a 100 μm cell screen to obtain pancreatic islet cell-sodium alginate gel fiber segments; the pancreatic islet cell-sodium alginate gel fiber segments are mixed with a mixed solution of human umbilical vein endothelial cells, poloxamer 407 and low cross-linked sodium hyaluronate, vascular endothelial growth factor, basic fibroblast growth factor, lidocaine hydrochloride and tumor necrosis factor-α monoclonal antibody to obtain a temperature-sensitive hydrogel.

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