CN115569194A - Temperature-sensitive hydrogel with diabetes treatment effect, kit, use method and application of temperature-sensitive hydrogel - Google Patents

Temperature-sensitive hydrogel with diabetes treatment effect, kit, use method and application of temperature-sensitive hydrogel Download PDF

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CN115569194A
CN115569194A CN202211337782.9A CN202211337782A CN115569194A CN 115569194 A CN115569194 A CN 115569194A CN 202211337782 A CN202211337782 A CN 202211337782A CN 115569194 A CN115569194 A CN 115569194A
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islet
final concentration
gel
cells
cell
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王哲
屈艳玲
明磊国
董玲娟
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Shaanxi Zhonghong Kerui Institute Of Regenerative Medicine Co ltd
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Shaanxi Zhonghong Kerui Institute Of Regenerative Medicine Co ltd
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    • A61K31/167—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the nitrogen of a carboxamide group directly attached to the aromatic ring, e.g. lidocaine, paracetamol
    • A—HUMAN NECESSITIES
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    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1858—Platelet-derived growth factor [PDGF]
    • A61K38/1866—Vascular endothelial growth factor [VEGF]
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    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
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    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
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    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics

Abstract

The invention 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 medicines. The temperature-sensitive hydrogel is formed by mixing human umbilical vein endothelial cell suspension, mixed liquid of P407 with the final concentration of 15-20% and sodium hyaluronate with the final concentration of 0.6-0.8%, vascular endothelial growth factor with the final concentration of 50ng/mL, alkaline fibroblast growth factor with the final concentration of 10ng/mL, lidocaine hydrochloride with the final concentration of 0.5-2%, tumor necrosis factor alpha monoclonal antibody with the final concentration of 100-200 mg/mL and islet cell-alginic acid gel fiber sections with the final concentration of 2-5% and the length of less than or equal to 1mm; the temperature-sensitive hydrogel is safe and non-toxic, low in immunogenicity, low in cost and good in treatment effect; the reagent is combined with the sterile consumable to prepare the kit, so that the kit is convenient to use and can be better applied to the treatment of diabetes.

Description

Temperature-sensitive hydrogel with diabetes treatment effect, kit, use method and application of temperature-sensitive hydrogel
Technical Field
The invention belongs to the technical field of diabetes treatment medicines, and particularly relates to a temperature-sensitive hydrogel with a diabetes treatment effect, a kit, a use method and application thereof.
Background
Diabetes is a chronic disease of glucose metabolism disorder of a body caused by hypofunction or failure of pancreatic islets, and as the disease course is prolonged, patients can suffer from systemic vasculopathy, so that multiple organs are damaged, and the life and health are seriously threatened. Diabetes is largely divided into two major categories, type 1 diabetes and type 2 diabetes. Type 1 diabetes accounts for about 10% of the total number of diabetic patients, more than 70% of islet cells are destroyed, insulin cannot be secreted in sufficient quantity to maintain normal blood sugar level, and patients need to inject insulin for stabilizing blood sugar for life, so that serious hypoglycemia reaction can occur, and various diabetic complications can occur. Type 2 diabetes is common, accounting for more than 80% of all diabetic patients, and one third of the middle and late stage patients also develop type 1 diabetes.
At present, common treatment methods such as insulin injection and the like can only regulate blood sugar to a normal level in a staged manner, and cannot simulate the accurate regulation of normal insulin secretion time and dosage by normal insulin, so that the risk of hypoglycemia and diabetic complications cannot be avoided, and most of hypoglycemic drugs have strong side effects and further damage the physical health of patients. In order to improve the quality of life of patients, researchers began to treat diabetes using islet transplantation techniques. Because the diabetes is caused by the damaged function of the pancreatic islets, the pancreatic islet transplantation can radically cure the diabetes theoretically, but actually, the treatment method still has many problems which need to be solved urgently at present, for example, the pancreatic islet separation and extraction process is time-consuming and labor-consuming, the cost is high, and the purification is required by using equipment such as a COBE.2991 cell separator; like other organ transplantation techniques, donor shortages severely limit the widespread use of islet transplantation techniques; strong immunological rejection exists after allogeneic or xenogeneic islet transplantation; in addition, the current islet transplantation needs to be performed by B-ultrasonic guided percutaneous hepatic portal vein puncture and tube placement and then instillation, or kidney capsule is transplanted downwards, the operation is complex, a specialized doctor is needed, and a highly specialized supporting facility is needed in the implementation process, which are obstacles for the islet transplantation in clinical application.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a temperature-sensitive hydrogel with a diabetes treatment effect, a kit, a use method and application thereof, so that the diabetes treatment is simple, the immunogenicity is lower, the cost is lower and the treatment effect is better.
In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:
the invention discloses a temperature-sensitive hydrogel with a diabetes treatment effect, which is formed 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%, a vascular endothelial growth factor with a final concentration of 50ng/mL, an alkaline fibroblast growth factor with a final concentration of 10ng/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 islet cells-alginic acid gel fiber segments with a final concentration of 2-5%;
wherein the final concentration of human umbilical vein endothelial cells is 5-8 multiplied by 10 6 The islet cell-alginic acid gel fiber section is a gel fiber section which is prepared by mixing islet cells, physiological saline and alginate, has the length of less than or equal to 1mm and the diameter of 100-300 mu m.
Preferably, the final concentration of islet cells in the islet cell-alginic acid gel fiber segment is 3-6 × 10 6 Per mL, the final concentration of alginate is 1.5-3%.
Preferably, the human umbilical vein endothelial cells and islet cells are both low immunogenic cells.
Preferably, the islet cells are derived from umbilical cord stem cells by induction with an islet cell induction solution.
The invention also discloses application of the temperature-sensitive hydrogel with the diabetes treatment effect in preparing a medicament for treating diabetes.
The invention also discloses a kit with the function of treating diabetes, which comprises a sterile reagent and a sterile consumable, wherein the sterile reagent comprises normal saline, sodium alginate, a calcium chloride solution, a mixed solution of P407 and sodium hyaluronate, a vascular endothelial growth factor, an alkaline fibroblast growth factor, lidocaine hydrochloride and a tumor necrosis factor-alpha monoclonal antibody; the sterile consumables include a 1mL syringe, a 2mL syringe, a 30G needle, a 14G needle, a single-sided blade, a plate, and a 100 μm cell screen.
Preferably, the kit further comprises an islet cell induction solution and a dithizone staining solution.
Further preferably, the islet cell inducing solution comprises an islet cell A1 inducing solution and an islet cell A2 inducing solution, the islet cell A1 inducing solution is composed of a H-DMEM high-sugar medium, 1.2mg/mL nicotinamide, and β -mercaptoethanol having a substance concentration of 0.07 ‰, and the islet cell A2 inducing solution is composed of a DMEM/F12 high-sugar medium, 2% fbs, 2% b27, and 1.2mg/mL nicotinamide.
The invention also discloses a using method of the kit with the diabetes treatment effect, firstly, mixing the islet cells, normal saline and sodium alginate to obtain islet cell-sodium alginate gel; secondly, placing a calcium chloride solution in a plate, sucking islet cells-sodium alginate gel by using a 1mL syringe with a 30G needle, extruding the islet cells-sodium alginate gel in the calcium chloride solution at a constant speed while drawing lines in parallel to obtain gel fibers with uniform thickness, washing the gel fibers with normal saline, cutting the gel fibers into sections, adding the calcium chloride solution to calcify the gel fibers, washing the gel fibers with normal saline, and filtering the gel fibers by using a 100-micron cell screen to obtain islet cells-alginic acid gel fiber sections; mixing the islet cell-alginic acid gel fiber segment with the mixed solution of human umbilical vein endothelial cells, P407 and sodium hyaluronate, vascular endothelial growth factor, basic fibroblast growth factor, lidocaine hydrochloride and tumor necrosis factor-alpha monoclonal antibody uniformly to obtain temperature-sensitive hydrogel, and finally injecting the temperature-sensitive hydrogel into the subcutaneous space of a diabetic patient by using a 2mL injector with a 14G needle.
Preferably, the islet cell-alginic acid gel fiber section is gel fiber with the length less than or equal to 1mm cut by a single-sided blade.
Compared with the prior art, the invention has the following beneficial effects:
according to the temperature-sensitive hydrogel with the diabetes treatment effect, the islet cells-alginic acid gel fiber section is used as an islet analogue, a large number of islet cells are wrapped inside the islet cell, the diameter of the islet cells is 100-300 microns and is close to the size of natural islets, the islet cells can survive for a long time after external capillary vessels are formed, and the islet cells can replace natural islets of an organism to secrete insulin and control blood sugar; the alginate material in the fiber segment has low autoimmunity, biological inertia, long retention time in vivo, and outer layer of alginate capable of reacting with Ca 2+ The microcapsule combined with eggshell-shaped structure wraps alginate inside to form gel with various shapes, has higher mechanical strength, can provide a proper living environment for islet cells wrapped inside, and the calcified membrane surface holes allow small molecular substances such as nutrients and metabolites secreted by cells to pass through the membrane for exchange, while immune cells are isolated outside to reduce immunological rejection. The lidocaine hydrochloride and the tumor necrosis factor alpha monoclonal antibody can greatly inhibit inflammatory reaction generated by injection administration and delay gel degradation time to 15 days at the same time of fully utilizing the advantage of the injection administration favorable for survival of transplanted microcapsules, and have sufficient time for ensuring angiogenesis of transplanted substances. At the same time, in order to further raise angiogenesis speed, said invention mixes a large quantity of human umbilical vein vascular endothelial cells in the temp. -sensitive gel, and under the action of vascular endothelial growth factor and basic fibroblast growth factor, it can quickly raise angiogenesis speedThe capillary vessels are quickly formed, so that cell death caused by ischemia and hypoxia problems is solved for the islet substitutes, the survival rate of the transplant is greatly improved, and the disease symptoms of the diabetic patients are better reversed. P407 in the temperature-sensitive hydrogel is a temperature-sensitive gel material, the low temperature is in a liquid state, the experimental operation is convenient, and the temperature is increased to be converted into a gel state after the hydrogel enters a human body, so that a certain form can be maintained; the low-crosslinking sodium hyaluronate can prevent P407 from degrading too fast, and has insufficient space to form capillary vessels, so that the blood and oxygen can not be supplied to islet cells in the gel fiber section; the P407 is mixed with the low-crosslinking sodium hyaluronate, can be used as a substrate to protect human umbilical vein vascular endothelial cells in the umbilical vein vascular endothelial cells, provides enough space and time to help capillary vessel generation, supplies and supplies blood and oxygen to islet cells in a fiber section, and improves the survival rate of the islet cells. The temperature-sensitive hydrogel can be degraded by tissues, is safe and non-toxic, has low immunogenicity and low cost, has a simple method for treating diabetes and a good effect, and can be applied to the treatment of diabetes.
In the kit with the diabetes treatment effect, normal saline can clean and dilute islet cells, islet cells-alginic acid gel fiber segments, sodium alginate and the like in an aseptic reagent; sodium alginate as a natural polymer material has excellent biocompatibility, and can ensure long-time survival of cells in the process of preparing islet cells-alginic acid gel fiber sections; the calcium chloride solution can be combined with the sodium alginate on the outer layer to form an eggshell-shaped structure, and the sodium alginate in the inner layer is wrapped to form gel with various shapes, such as gel balls, gel fibers and the like; the P407 and the low-crosslinking sodium hyaluronate are used for preparing gel with temperature-sensitive property, and can provide time and space for capillary angiogenesis; the vascular endothelial growth factor and the basic fibroblast growth factor can accelerate angiogenesis, provide nutrition for islet cells in the gel and greatly improve the survival rate; on one hand, the lidocaine hydrochloride can perform local anesthesia to reduce discomfort after initial injection, and on the other hand, the lidocaine hydrochloride can reduce inflammatory reaction after initial injection; the tumor necrosis factor alpha monoclonal antibody can regulate immunity and reduce inflammatory reaction within a long period of time (about 2 weeks), so that the survival time of cells in gel is prolonged (a large amount of immune cells are gathered in the inflammatory reaction, and foreign matters such as foreign cells and the like are killed); operate with the help of aseptic consumptive material, convenient to use can obtain a large amount of islet analogs that can supply the injection in aseptic laboratory through this kit, and whole easy operation need not to use equipment such as cell separation machine to separate the islet, requires greatly reduced to hardware facilities and technical staff, and the application promotion of being convenient for, and the gained islet cell of single experiment and endothelial cell can adorn the cryopreservation to supply follow-up repetitious usage.
Furthermore, the islet cell inducing solution can induce the umbilical cord stem cells into islet cells with low immunogenicity, secrete insulin, and is not easy to induce immunological rejection; the dithizone staining solution can stain the islet cells into reddish brown, and the islet cells induced by the stem cells are stained and identified, so that the induction success is ensured.
The use method of the kit with the diabetes treatment effect, provided by the invention, has the advantages of simple subcutaneous injection, convenience in monitoring, time saving, high flexibility, repeated operation, no need of B-ultrasonic-guided percutaneous hepatic portal vein puncture tube placement and then instillation or renal subcontracting transplantation, operation time saving for hours, greatly reduced infection risk, and avoidance of complications of hepatic hemorrhage, portal vein thrombosis, portal vein high pressure and the like.
Drawings
FIG. 1 is a diagram of human umbilical cord mesenchymal stem cells of the present invention;
FIG. 2 is a diagram of human umbilical vein endothelial cells of the present invention;
FIG. 3 is a diagram of islet cells-alginic acid gel fiber segments according to the present invention;
FIG. 4 is a diagram of islet cells-alginate gel beads of the present invention;
FIG. 5 is a graph comparing cell proliferation in gel fiber segments and gel spheres of the present invention;
FIG. 6 is a comparison of the blood inflammatory factors of diabetic mice of the present invention.
Detailed Description
In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that the terms "first," "second," and the like in the description and claims of the present invention and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in other sequences than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
The invention is described in further detail below with reference to the accompanying drawings:
the invention provides a temperature-sensitive hydrogel preparation kit with a diabetes treatment effect, which comprises a sterile reagent needing to be frozen and a sterile consumable material needing to be stored at normal temperature, wherein the sterile reagent comprises: islet cell inducing solution, dithizone staining solution, physiological saline, 5% alginate, 1.5% calcium chloride solution, a mixed solution of 25% P407 (poloxamer 407) and 1% low cross-linked sodium Hyaluronate (HA), vascular Endothelial Growth Factor (VEGF), basic fibroblast growth factor (bFGF), lidocaine hydrochloride, and tumor necrosis factor-alpha monoclonal antibody; the sterile consumable comprises: 1mL syringe x 2, 2mL syringe x 2, 30G needle x 2, 14G needle x 2, single sided blade x 2, plastic plate x 2 and 100 μm cell screen x 2;
wherein, the aseptic reagent needs to be stored in a frozen way, the aseptic consumables need to be stored at normal temperature, and the quality guarantee period is 1 year; the islet cell inducing liquid comprises islet cell A1 inducing liquid and islet cell A2 inducing liquid, wherein the islet cell A1 inducing liquid is composed of an H-DMEM high-glucose medium, 1.2mg/mL nicotinamide and 0.07 ‰ (V/V) beta-mercaptoethanol, and the islet cell A2 inducing liquid is composed of a DMEM/F12 high-glucose medium, 2%; the dithizone staining solution is prepared by dissolving dithizone powder in dimethyl sulfoxide; the alginate is sodium alginate, potassium alginate or ammonium alginate.
The invention provides a use method of a temperature-sensitive hydrogel preparation kit with a diabetes treatment effect, which comprises the following steps:
1. preparing cell materials:
1) Islet cell induction
Inducing umbilical cord stem cells (UC-MSCs) into islet cells by using an islet cell inducing solution;
wherein, the UC-MSCs can be replaced by diabetic autologous isolated adipose-derived stem cells (ADSCs) or diabetic autologous isolated Bone Marrow Stem Cells (BMSC) for the diabetic; the islet cells induced by the islet cell inducing solution can be replaced by human fetal islet cells or neonatal porcine islet cells.
2) Islet cell identification
Performing staining identification on the islet cells obtained in the step 1) of the step 1 by using a dithizone staining solution: a reddish brown cell cluster can be seen, which indicates that the islet cells are successfully induced;
3) Culture and morphological identification of Human Umbilical Vein Endothelial Cells (HUVECs)
Separating HUVECs from human umbilical vein tissues by an enzyme digestion method, and carrying out subculture; observing under an inverted microscope, wherein the HUVECs are in a typical cobblestone shape, and the cell nucleus is obvious; the source of the cell can be commercial cells, the generation of P3-P5 needs to be identified by CD31/vWF immunofluorescence, and the purity is more than 90%.
2. Preparation of islet analogue (islet cells-sodium alginate gel fiber segment):
suspending the islet cells obtained in step 1) with normal saline, adding 5% sodium alginate, and mixing uniformly to obtain the islet cell-sodium alginate gel with the final cell concentration of 3-6 × 10 6 Per mL, the final concentration of sodium alginate is 1.5-3%;placing 1.5% calcium chloride solution in a plate, sucking islet cells-sodium alginate gel by using a 1mL injector, extruding the islet cells-sodium alginate gel in 1.5% calcium chloride solution at a constant speed through a 30G needle, simultaneously drawing lines in parallel to obtain gel fibers with uniform thickness, pouring out the 1.5% calcium chloride solution, cleaning the gel fibers for 3 times by using normal saline, removing liquid, cutting the gel fibers in the plate into small sections along the long axis by using a single-sided blade, wherein the length of the small sections is less than or equal to 1mm, adding the 1.5% calcium chloride solution into the plate, slightly shaking to uniformly distribute the gel fiber sections, soaking for 2min, removing the liquid, cleaning for 3 times by using the normal saline, and filtering by using a 100 mu m cell screen to obtain the islet cells-sodium alginate gel fiber sections with calcified surfaces, wherein the islet cells-sodium alginate gel fiber sections serve as islet analogs for standby.
3. Preparing composite temperature-sensitive gel:
suspending the human umbilical vein endothelial cells obtained in the step 3) in normal saline, uniformly mixing with 25% of P407+1% low-crosslinking sodium hyaluronate, vascular endothelial growth factor, alkaline fibroblast growth factor, lidocaine hydrochloride and the tumor necrosis factor-alpha monoclonal antibody, adding the islet cells-sodium alginate gel fiber segments prepared in the step 2, and uniformly mixing to obtain the composite temperature-sensitive gel;
wherein the final concentration of human umbilical vein endothelial cells is 5-8 × 10 6 The medicine composition is prepared from the following raw materials, by weight, per mL, 50ng/mL of vascular endothelial growth factor, 10ng/mL of bFGF, 15-20% of P407, 0.6-0.8% of HA, 2-5% of islet cell-alginic acid gel fiber section, 0.5-2% of lidocaine hydrochloride and 100-200 mg/mL of tumor necrosis factor alpha monoclonal antibody.
4. Treatment of diabetes:
the type 1 diabetes model was induced by disruption of mouse pancreatic beta cells with streptozotocin. And (2) injecting the composite temperature-sensitive gel obtained in the step (3) into the groin of a model mouse subcutaneously, wherein 1mL of the gel is injected on one side, the gel is formed after entering the body, the gel is uniformly pressed after injection, the gel distribution area is enlarged, the gel thickness is reduced, endothelial cells in the gel can quickly form blood vessels under the action of vascular endothelial growth factor and basic fibroblast growth factor double factors, the blood vessels are communicated with peripheral tissue blood vessels, nutrient substances are conveyed, lidocaine hydrochloride and a tumor necrosis factor alpha monoclonal antibody are combined to reduce the inflammatory reaction caused by transplantation, the gel volume loss is delayed, the survival of implanted islet cells is ensured through multiple functions, and insulin is secreted, so that the long-term blood sugar reducing effect is achieved.
Example 1
1. Preparing cell materials:
1) Islet cell induction
Separating and culturing human umbilical cord mesenchymal stem cells (UC-MSC) from umbilical cord of healthy newborn, culturing to P5 generation, observing under microscope, and arranging cells in form of long spindle, uniform size and vortex; detecting the expression of cell surface markers CD29, CD44 and CD90 as positive and the expression of CD34 and CD45 as negative by a flow cytometer, and identifying the cell as the UC-MSC cell.
Culturing UC-MSC of P5 generation at 5 × 10 5 Inoculating each cell/well in a 6-well plate, culturing the cells with 70-80% of cell fusion degree the next day, replacing the culture medium with islet cell A1 induction liquid, removing the islet cell A1 induction liquid after continuously culturing for 24h, adding the islet cell A2 induction liquid for continuous induction, replacing the islet cell A2 induction liquid every 2-3 days, and observing the induction condition under a microscope: when the induction lasts for about 10 days, islet-like cell masses appear in a scattered distribution, and the induction lasts for about 30 days to obtain islet cells;
wherein the islet cell A1 inducing liquid comprises an H-DMEM high-sugar culture medium, 1.2mg/mL nicotinamide and 0.07 thousandth (V/V) beta-mercaptoethanol; the islet cell A2-inducing solution comprises DMEM/F12 high-glucose medium, 2% FBS, 2% B27 and 1.2mg/mL nicotinamide.
2) Islet cell identification
Mixing 10 mu L of dithizone staining solution with 1mL of islet cell A2 inducing solution, performing staining identification on the islet cells induced in the step 1), adding a 6-well plate, incubating for 30min in an incubator at 37 ℃, washing for 3 times by PBS, observing under a microscope, and staining cell clusters to be reddish brown, which indicates successful induction.
Wherein the dithizone staining solution is prepared by dissolving dithizone powder in dimethyl sulfoxide, and the concentration is 10mg/mL.
3) Culture and morphological identification of Human Umbilical Vein Endothelial Cells (HUVECs)
HUVECs primary cultureThe cultivation method comprises the following steps: in a super clean bench, the umbilical cord is washed clean with PBS solution, the umbilical vein is found, and the umbilical vein is repeatedly washed clean by sucking PBS with a 20mL syringe and injecting the PBS into the umbilical vein. One end of the umbilical vein was closed with hemostatic forceps, and the other end was perfused with 0.2% collagenase i (PBS solution) until the umbilical vein was full, and the umbilical vein was closed with hemostatic forceps. The umbilical cord was placed in sterile PBS solution and incubated at 37 ℃ for about 10 min. In the incubation process, the enzyme solution in the umbilical vein is pumped/injected for 1 time every 3min, so that the collagenase is promoted to be fully and uniformly contacted with the vein wall. The umbilical cord was removed, the liquid on the surface of the umbilical cord was blotted with sterile gauze, the enzyme solution was aspirated into a 50mL centrifuge tube, and the tube was neutralized by adding an equal volume of 10% FBS-containing medium. Then, a 2ml syringe is used for sucking sterile PBS solution to flush the umbilical vein, the flushing solution is also collected, after the flushing solution is fully mixed, the cells are collected by centrifugation, and the culture medium is resuspended. Inoculating the cell suspension into a cell culture flask, and placing at 5% CO 2 Culturing at 37 deg.C, changing culture solution after 24 hr, and removing non-adherent cells. And changing the solution every 2-3 days, and carrying out normal subculture to obtain the human umbilical vein endothelial cells.
And (3) morphological identification of HUVECs: when observed by an inverted microscope, HUVECs cells are typically cobblestone-like and the nuclei are evident as shown in FIG. 2.
2. Preparation of islet analogue (islet cells-sodium alginate gel fiber segment):
digesting and centrifuging the islet cells obtained in the step 1), suspending the islet cells with normal saline, adding 5% sodium alginate, and uniformly mixing to obtain the islet cell-sodium alginate gel with the final cell concentration of 3 multiplied by 10 6 Per mL, final concentration of sodium alginate is 1.5%; placing 1.5% calcium chloride solution in a plate, sucking islet cells-sodium alginate gel with a 1mL syringe, extruding at constant speed in 1.5% calcium chloride solution through a 30G needle while drawing a line in parallel to obtain gel fiber with a diameter of 100-300 μm, discarding 1.5% calcium chloride solution, cleaning gel fiber with normal saline for 3 times, removing liquid, cutting gel fiber into small segments with a single-sided blade along a long axis in the plate, wherein the length is less than or equal to 1mm, adding 1.5% calcium chloride solution in the plate, gently shaking to uniformly distribute gel fiber segments, soaking for 2min, removing liquid, and soaking with a single-sided bladeWashing with normal saline for 3 times, filtering with 100 μm cell screen to obtain pancreatic islet cells-sodium alginate gel fiber segment with calcified surface, and using as pancreatic islet analogue for standby, wherein the shape chart is shown in FIG. 3, the diameter of the pancreatic islet cells-sodium alginate gel fiber segment is 100-300 μm, and the length is less than or equal to 1mm;
meanwhile, a 1mL syringe is used for sucking the islet cells-sodium alginate gel, a 30G needle is used for extruding at a constant speed, 1.5% calcium chloride solution is dripped into the islet cells-sodium alginate gel, after soaking for 2min, liquid is removed, and the islet cells-sodium alginate gel ball is prepared by washing for 3 times with normal saline, wherein the shape chart is shown in figure 4, the diameter is 1000-1500 mu m, and the proliferation and survival conditions of the cells in the gel ball and the gel fiber section are compared, and the result is shown in figure 5, so that the islet cells-sodium alginate gel ball is better in proliferation and survival conditions in the gel fiber section, and is more suitable for being transplanted as an islet analogue.
3. Preparing composite temperature-sensitive gel:
suspending the human umbilical vein endothelial cells obtained in the step 3) in normal saline, uniformly mixing the human umbilical vein endothelial cells with a mixed solution of 25 percent of P407 and 1 percent of low-crosslinking sodium hyaluronate, vascular endothelial growth factor, alkaline fibroblast growth factor, lidocaine hydrochloride and tumor necrosis factor-alpha monoclonal antibody, adding the islet cells-sodium alginate gel fiber segments prepared in the step 2, and uniformly mixing to obtain the composite temperature-sensitive gel.
Wherein the final concentration of human umbilical vein endothelial cells is 5 × 10 6 The traditional Chinese medicine composition comprises the following components of a/mL solution, a final concentration of Vascular Endothelial Growth Factor (VEGF) is 50ng/mL, a final concentration of bFGF is 10ng/mL, a final concentration of P407 is 15%, a final concentration of HA is 0.6%, an islet cell-alginic acid gel fiber section is 2%, lidocaine hydrochloride is 0.5%, and tumor necrosis factor alpha monoclonal antibody is 100mg/mL.
4. Treatment of diabetes:
1) Preparation of streptozotocin liquid (STZ)
Preparing 2.1% citric acid aqueous solution and 2.94% sodium citrate aqueous solution, mixing the two solutions, adjusting the pH value to 4.2-4.5, adding streptozotocin for dissolution to obtain the product with the final concentration of 10mg/mL, and performing sterile filtration to obtain the product.
2) Establishment of type I diabetes mouse model
Taking 30 SPF-grade ICR male mice of 6 weeks old, adaptively feeding for one week, and performing intraperitoneal injectionSTZ was modeled after fasting and water deprivation for 12 hours prior to administration, and body weight (g) and fasting blood glucose (mmol/L) were measured. Note that STZ is used as it is, and is protected from light in the whole course, and the dosage is 50mg kg -1 Injections were given 1 time per day for 6 consecutive days. After 1 week of the last injection, the mice are fixed by a mouse fixer, the blood sugar content of tail veins of the mice is detected by a rapid blood sugar detector, and the continuous 3d random blood sugar is more than or equal to 16.7mmol/L, which indicates that the animal model is successfully constructed. A total of 17 type 1 diabetic mice were obtained.
3) Diabetic mouse treatment
16 type 1 diabetic mice were divided into 2 groups, 8 of which were used for each of the control and treatment groups.
Placing the composite temperature-sensitive gel obtained in the step 3 at 4 ℃ for half an hour to be in a liquid state, uniformly mixing, sucking into a 2mL injector, performing subcutaneous injection at the groin position of a diabetic mouse in a treatment group by using a 14G needle, wherein the volume of one side is 1mL, the composite temperature-sensitive gel forms a semisolid state after entering the body, uniformly pressing after the injection is completed, expanding the gel distribution area and reducing the gel thickness; injecting equal amount of blank gel into the control group;
wherein the blank gel is prepared from 5% sodium alginate, 1.5% calcium chloride solution, 25% P407 and 1% low cross-linked sodium hyaluronate and physiological saline by the same procedure.
4) Treatment outcome monitoring
The monitoring of the blood sugar level and the body weight of the mice is continuously carried out for 3 months, and the specific conditions are as follows:
after the islet analogue is injected and transplanted, the blood sugar level of the mice in a non-fasting state is detected for 2 times per week, the mice in the control group and the treatment group are weighed for 1 time per week, the blood sugar monitoring results of the mice in the control group and the treatment group are shown in a table 1, and the weight monitoring results of the mice in the control group and the treatment group are shown in a table 2.
Table 1: blood glucose monitoring results of control and treated mice
Figure BDA0003915800390000121
Table 2: body weight monitoring results of mice in control group and treatment group
Figure BDA0003915800390000122
The results in table 1 show that the blood sugar of the treated mice is continuously lower than 11.1mmol/L at the beginning of 5 weeks, which indicates that the islet graft normally plays a blood sugar reducing function, and the survival rate of the treated mice is 83% in an observation period of 3 months; the blood sugar of the control group mice is more than or equal to 16.7mmol/L, and the control group mice die in sequence in an observation period of 3 months. The results in Table 2 show that the body weight of the control mice decreased continuously from week 2, while the body weight of the treated mice showed a tendency to increase.
And (3) collecting blood from the tail vein 24h after injection, wherein the blood collecting result is as follows:
the serum cytokines IL-6 and IFN-gamma levels are detected by an ELISA method, the result is shown in figure 6, and the result shows that compared with a control group, the inflammatory factors in the blood of mice in a treatment group after injection are obviously reduced, which indicates that the combination of lidocaine hydrochloride and the tumor necrosis factor alpha monoclonal antibody can reduce the inflammatory reaction caused by transplantation, ensure the survival of implanted islet cells and secrete insulin, thereby playing the role of reducing blood sugar for a long time.
Example 2
1. Preparing cell materials:
1) Islet cell induction
Autologous Bone Marrow Stem Cells (BMSC) isolated from diabetic patients, cultured to P4 generation at 5X 10 5 Inoculating each cell/well cell into a 6-well plate, replacing a culture medium with islet cell A1 induction liquid when the cell fusion degree reaches 70-80%, continuously culturing for 24h, removing the islet cell A1 induction liquid, adding the islet cell A2 induction liquid for continuous induction, and replacing the islet cell A2 induction liquid every 2-3 days to obtain the islet cells;
wherein the islet cell A1 inducing liquid comprises an H-DMEM high-sugar culture medium, 1.2mg/mL nicotinamide and 0.07 thousandth (V/V) beta-mercaptoethanol; the islet cell A2-inducing solution comprises DMEM/F12 high-glucose medium, 2% FBS, 2% B27 and 1.2mg/mL nicotinamide.
2) Islet cell identification
Mixing 10 mu L of dithizone staining solution with 1mL of islet cell A2 inducing solution, performing staining identification on the islet cells induced in the step 1), adding a 6-well plate, incubating for 30min in an incubator at 37 ℃, washing for 3 times by PBS, and observing the induction condition under a microscope.
Wherein the dithizone staining solution is prepared by dissolving dithizone powder in dimethyl sulfoxide, and the concentration is 10mg/mL.
3) Culture and morphological identification of Human Umbilical Vein Endothelial Cells (HUVECs)
The procedure is as in example 1.
2. Preparation of islet analogue (islet cells-sodium alginate gel fiber segment):
digesting and centrifuging the islet cells obtained in the step 1), suspending the islet cells with normal saline, adding 5% sodium alginate, and uniformly mixing to obtain the islet cell-sodium alginate gel with the final cell concentration of 3.8 multiplied by 10 6 Per mL, final concentration of sodium alginate is 2%; placing 1.5% calcium chloride solution in a plate, sucking islet cells-sodium alginate gel by using a 1mL injector, extruding the islet cells-sodium alginate gel in 1.5% calcium chloride solution at a constant speed through a 30G needle, simultaneously drawing a line in parallel to obtain gel fibers with the diameter of 100-300 mu m, discarding 1.5% calcium chloride solution, cleaning the gel fibers for 3 times by using normal saline, removing liquid, cutting the gel fibers into small sections with a single-sided blade in the plate along the long axis, wherein the length is less than or equal to 1mm, adding 1.5% calcium chloride solution into the plate, slightly shaking to uniformly distribute the gel fiber sections, soaking for 2min, removing the liquid, cleaning for 3 times by using normal saline, and filtering by using a 100 mu m cell screen to obtain the islet cells-sodium alginate gel fiber sections with calcified surfaces, wherein the islet cells-sodium alginate gel fiber sections serve as islet analogs for standby.
3. Preparing composite temperature-sensitive gel:
suspending the human umbilical vein endothelial cells obtained in the step 3) in normal saline, uniformly mixing the human umbilical vein endothelial cells with a mixed solution of 25 percent of P407 and 1 percent of low-crosslinking sodium hyaluronate, vascular endothelial growth factor, alkaline fibroblast growth factor, lidocaine hydrochloride and tumor necrosis factor-alpha monoclonal antibody, adding the islet cells-sodium alginate gel fiber segments prepared in the step 2, and uniformly mixing to obtain the composite temperature-sensitive gel.
Wherein the final concentration of human umbilical vein endothelial cells is 6.6 × 10 6 The total concentration of the VEGF is 50ng/mL, the final concentration of the bFGF is 10ng/mL, the final concentration of the P407 is 17%, the final concentration of the HA is 0.68%, the islet cell-alginic acid gel fiber section is 3%, the lidocaine hydrochloride is 1.1%, and the tumor necrosis factor alpha monoclonal antibody is 160mg/mL.
Example 3
1. Preparing cell materials:
1) Islet cell induction
Separating adipose-derived stem cells (ADSCs) from diabetic patients, culturing to P3 generation, and culturing at 5 × 10 5 Inoculating each cell/well cell into a 6-well plate, replacing a culture medium with islet cell A1 induction liquid when the cell fusion degree reaches 70-80%, continuously culturing for 24h, removing the islet cell A1 induction liquid, adding the islet cell A2 induction liquid for continuous induction, and replacing the islet cell A2 induction liquid every 2-3 days to obtain the islet cells;
wherein the islet cell A1 inducing liquid comprises an H-DMEM high-sugar culture medium, 1.2mg/mL nicotinamide and 0.07 thousandth (V/V) beta-mercaptoethanol; the islet cell A2-inducing solution comprises DMEM/F12 high-glucose medium, 2% FBS, 2% B27 and 1.2mg/mL nicotinamide.
2) Islet cell identification
Mixing 10 mu L of dithizone staining solution with 1mL of islet cell A2 inducing solution, performing staining identification on the islet cells induced in the step 1), adding a 6-well plate, incubating for 30min in an incubator at 37 ℃, washing for 3 times by PBS, and observing the induction condition under a microscope.
Wherein the dithizone staining solution is prepared by dissolving dithizone powder in dimethyl sulfoxide, and the concentration is 10mg/mL.
3) Culture and morphological identification of Human Umbilical Vein Endothelial Cells (HUVECs)
The procedure is as in example 1.
2. Preparation of islet analogue (islet cells-sodium alginate gel fiber segment):
digesting and centrifuging the islet cells obtained in the step 1), suspending the islet cells with normal saline, adding 5% sodium alginate, and uniformly mixing to obtain the islet cell-sodium alginate gel with the final cell concentration of 6 multiplied by 10 6 Per mL, final concentration of sodium alginate is 3%; the 1.5% calcium chloride solution was placed in a dish and injected with 1mLThe method comprises the steps of sucking islet cells-sodium alginate gel by an injector, extruding the islet cells-sodium alginate gel in a 1.5% calcium chloride solution at a constant speed through a 30G needle, simultaneously drawing a line in parallel to obtain gel fibers with the diameter of 100-300 mu m and the uniform thickness, discarding the 1.5% calcium chloride solution, cleaning the gel fibers for 3 times by using normal saline, removing liquid, cutting the gel fibers into small sections with the length of less than or equal to 1mm along a long axis by using a single-sided blade in a plate, adding the 1.5% calcium chloride solution into the plate, slightly shaking to uniformly distribute the gel fiber sections, soaking for 2min, removing the liquid, cleaning for 3 times by using the normal saline, and filtering by using a 100 mu m cell screen to obtain the islet cells-sodium alginate gel fiber sections with the calcified whole surface for later use as islet analogs.
3. Preparing composite temperature-sensitive gel:
suspending the human umbilical vein endothelial cells obtained in the step 1 and 3) by using physiological saline, uniformly mixing the human umbilical vein endothelial cells with a mixed solution containing 25 percent of P407 and 1 percent of low cross-linked sodium hyaluronate, vascular endothelial growth factor, alkaline fibroblast growth factor, lidocaine hydrochloride and tumor necrosis factor-alpha monoclonal antibody, adding the islet cells-sodium alginate gel fiber segments prepared in the step 2, and uniformly mixing to obtain the composite temperature-sensitive gel.
Wherein the final concentration of human umbilical vein endothelial cells is 8 × 10 6 The total concentration of the VEGF is 50ng/mL, the final concentration of the bFGF is 10ng/mL, the final concentration of the P407 is 20%, the final concentration of the HA is 0.8%, the islet cell-alginic acid gel fiber segment is 5%, the lidocaine hydrochloride is 2%, and the tumor necrosis factor alpha monoclonal antibody is 200mg/mL.
Example 4
1. Preparing cell materials:
1) Culture and morphological identification of Human Umbilical Vein Endothelial Cells (HUVECs)
The procedure is as in example 1.
2. Preparation of islet analogue (islet cells-sodium alginate gel fiber segment):
digesting and centrifuging human fetal islet cells, suspending with normal saline, adding 5% sodium alginate, and mixing to obtain islet cell-sodium alginate gel with final cell concentration of 4.5 × 10 6 Per mL, final concentration of sodium alginate is 1.6%; the 1.5% calcium chloride solution was placed in a petri dish and aspirated with a 1mL syringeTaking islet cells-sodium alginate gel, extruding the islet cells-sodium alginate gel in a 1.5% calcium chloride solution at a constant speed through a 30G needle, simultaneously drawing a line in parallel to obtain gel fibers with the diameter of 100-300 mu m and the uniform thickness, discarding the 1.5% calcium chloride solution, cleaning the gel fibers for 3 times by using normal saline, removing liquid, cutting the gel fibers into small sections with the length of less than or equal to 1mm along a long axis by using a single-sided blade in a plate, adding the 1.5% calcium chloride solution into the plate, slightly shaking to uniformly distribute the gel fiber sections, soaking for 2min, removing the liquid, cleaning for 3 times by using the normal saline, and filtering by using a 100 mu m cell screen to obtain the islet cells-sodium alginate gel fiber sections with calcified surfaces, wherein the islet cells-sodium alginate gel fiber sections are used as islet analogs for standby.
3. Preparing composite temperature-sensitive gel:
suspending the human umbilical vein endothelial cells obtained in the step 1) with physiological saline, uniformly mixing the human umbilical vein endothelial cells with a mixed solution of 25 percent of P407 and 1 percent of low-crosslinking sodium hyaluronate, vascular endothelial growth factor, alkaline fibroblast growth factor, lidocaine hydrochloride and tumor necrosis factor-alpha monoclonal antibody, adding the islet cells-sodium alginate gel fiber segments prepared in the step 2, and uniformly mixing to obtain the composite temperature-sensitive gel.
Wherein the final concentration of human umbilical vein endothelial cells is 7.2 × 10 6 The total concentration of the VEGF is 50ng/mL, the final concentration of the bFGF is 10ng/mL, the final concentration of the P407 is 16%, the final concentration of the HA is 0.7%, the islet cell-alginic acid gel fiber segment is 4%, the lidocaine hydrochloride is 1.5%, and the tumor necrosis factor alpha monoclonal antibody is 140mg/mL.
Example 5
1. Preparing cell materials:
1) Culture and morphological identification of Human Umbilical Vein Endothelial Cells (HUVECs)
The procedure is as in example 1.
2. Preparation of islet analogue (islet cells-sodium alginate gel fiber segment):
digesting and centrifuging the newborn pig islet cells, suspending the newborn pig islet cells with normal saline, adding 5% sodium alginate, and uniformly mixing to obtain the islet cell-sodium alginate gel with the final cell concentration of 5 multiplied by 10 6 Per mL, final concentration of sodium alginate is 3%; the 1.5% calcium chloride solution was placed in a dish and the islets were aspirated with a 1mL syringeThe cell-sodium alginate gel is extruded in 1.5 percent calcium chloride solution at a constant speed by a 30G needle and simultaneously drawn in parallel to obtain gel fibers with the diameter of 100-300 mu m and uniform thickness, 1.5 percent calcium chloride solution is discarded, the gel fibers are washed by normal saline for 3 times to remove liquid, the gel fibers are cut into small sections with the length of less than or equal to 1mm along the long axis by a single-sided blade in a flat dish, 1.5 percent calcium chloride solution is added into the flat dish, the gel fiber sections are evenly distributed by slight shaking, the liquid is removed after the gel fibers are soaked for 2min, the gel fibers are washed by normal saline for 3 times and filtered by a 100 mu m cell screen to obtain the islet cells-sodium alginate gel fiber sections with calcified surfaces, and the islet cells-sodium alginate gel fiber sections are used as islet analogues for standby.
3. Preparing composite temperature-sensitive gel:
suspending the human umbilical vein endothelial cells obtained in the step 1) with physiological saline, uniformly mixing the human umbilical vein endothelial cells with a mixed solution of 25 percent of P407 and 1 percent of low-crosslinking sodium hyaluronate, vascular endothelial growth factor, alkaline fibroblast growth factor, lidocaine hydrochloride and tumor necrosis factor-alpha monoclonal antibody, adding the islet cells-sodium alginate gel fiber segments prepared in the step 2, and uniformly mixing to obtain the composite temperature-sensitive gel.
Wherein the final concentration of human umbilical vein endothelial cells is 6.3 × 10 6 The total concentration of the VEGF is 50ng/mL, the final concentration of the bFGF is 10ng/mL, the final concentration of the P407 is 18%, the final concentration of the HA is 0.72%, the islet cell-alginic acid gel fiber segment is 2%, the lidocaine hydrochloride is 1.9%, and the tumor necrosis factor alpha monoclonal antibody is 100mg/mL.
The above-mentioned contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and any modification made on the basis of the technical idea of the present invention falls within the protection scope of the claims of the present invention.

Claims (10)

1. The temperature-sensitive hydrogel with the diabetes treatment effect is characterized by being formed by mixing human umbilical vein endothelial cell suspension, mixed liquid of P407 with the final concentration of 15-20% and sodium hyaluronate with the final concentration of 0.6-0.8%, vascular endothelial growth factor with the final concentration of 50ng/mL, alkaline fibroblast growth factor with the final concentration of 10ng/mL, lidocaine hydrochloride with the final concentration of 0.5-2%, tumor necrosis factor alpha monoclonal antibody with the final concentration of 100-200 mg/mL and islet cell-alginic acid gel fiber segments with the final concentration of 2-5%;
wherein the final concentration of human umbilical vein endothelial cells is 5-8 × 10 6 The islet cell-alginic acid gel fiber section is a gel fiber section which is prepared by mixing islet cells, physiological saline and alginate, has the length of less than or equal to 1mm and the diameter of 100-300 mu m.
2. The temperature-sensitive hydrogel according to claim 1, wherein the final concentration of islet cells in the islet cell-alginic acid gel fiber segment is 3-6 x 10 6 Per mL, the final concentration of alginate is 1.5-3%.
3. The temperature-sensitive hydrogel having the effect of treating diabetes mellitus according to claim 1, wherein human umbilical vein endothelial cells and islet cells are low-immunogenicity cells.
4. The temperature-sensitive hydrogel according to claim 1, wherein the islet cells are induced from umbilical cord stem cells by an islet cell-inducing solution.
5. Use of the temperature-sensitive hydrogel having a diabetes therapeutic effect according to any one of claims 1 to 4 in the preparation of a medicament for treating diabetes.
6. A kit with a diabetes treatment effect is characterized by comprising a sterile reagent and a sterile consumable, wherein the sterile reagent comprises normal saline, sodium alginate, a calcium chloride solution, a mixed solution of P407 and sodium hyaluronate, a vascular endothelial growth factor, an alkaline fibroblast growth factor, lidocaine hydrochloride and a tumor necrosis factor-alpha monoclonal antibody; the sterile consumables include a 1mL syringe, a 2mL syringe, a 30G needle, a 14G needle, a single-sided blade, a plate, and a 100 μm cell screen.
7. The kit for treating diabetes according to claim 6, further comprising an islet cell-inducing solution and a dithizone staining solution.
8. The kit for diabetes therapy according to claim 7, wherein said islet cell inducing solution comprises an islet cell A1 inducing solution and an islet cell A2 inducing solution, said islet cell A1 inducing solution is composed of a high-glucose H-DMEM medium, 1.2mg/mL nicotinamide, and β -mercaptoethanol having a substance concentration of 0.07 ‰, and said islet cell A2 inducing solution is composed of a high-glucose DMEM/F12 medium, 2% FBS, 2% B27, and 1.2mg/mL nicotinamide.
9. The use of the kit for diabetes treatment according to any one of claims 6 to 8, wherein firstly islet cells, physiological saline and sodium alginate are mixed to obtain an islet cell-sodium alginate gel; secondly, placing a calcium chloride solution in a plate, sucking islet cells-sodium alginate gel by using a 1mL syringe with a 30G needle, extruding the islet cells-sodium alginate gel in the calcium chloride solution at a constant speed while drawing lines in parallel to obtain gel fibers with uniform thickness, washing the gel fibers with normal saline, cutting the gel fibers into sections, adding the calcium chloride solution to calcify the gel fibers, washing the gel fibers with normal saline, and filtering the gel fibers by using a 100-micron cell screen to obtain islet cells-alginic acid gel fiber sections; mixing the islet cell-alginic acid gel fiber segment with the mixed solution of human umbilical vein endothelial cells, P407 and sodium hyaluronate, vascular endothelial growth factor, basic fibroblast growth factor, lidocaine hydrochloride and tumor necrosis factor-alpha monoclonal antibody uniformly to obtain temperature-sensitive hydrogel, and finally injecting the temperature-sensitive hydrogel into the subcutaneous space of a diabetic patient by using a 2mL injector with a 14G needle.
10. The method for using the kit with the effect of treating diabetes mellitus as claimed in claim 9, wherein the islet cell-alginic acid gel fiber section is gel fiber with the length less than or equal to 1mm cut by a single-sided blade.
CN202211337782.9A 2022-10-28 2022-10-28 Temperature-sensitive hydrogel with diabetes treatment effect, kit, use method and application of temperature-sensitive hydrogel Pending CN115569194A (en)

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