Application Of β-cells Induced By Adipose Mesenchymal Stem Cells Loaded With PDA-PLGA Hydrogel Biological Scaffold On The Treatment Of Diabetes

Diabetic(DM)is a chronic non-communicable disease with high morbidity and mortality.Diabetes causes a series of vascular events that affect most tissues and is the leading cause of renal failure,vision loss,ischemic heart disease,stroke and peripheral arterial occlusive disease.Islet β-cell failure is the key link leading to the occurrence and development of diabetes.The current antidiabetic drugs can only relieve or relieve symptoms and delay the progress of the disease.There are no drugs for islet β-cell regeneration and can not fundamentally cure the disease.Islet transplantation can effectively transport insulin and reduce the level of blood glucose in the body,so islet transplantation may be the best way to treat diabetes.However,due to insufficient supply and quality of islets,problems such as post-transplantation immune rejection limit the wide application of islet transplantation.In recent years,some achievements in clinical research on stem cell beds are expected to solve this problem.Stem cells can self-renew and secrete a variety of important cytokines,and are an important source of cells for pancreatic islet transplantation.Among them,adipose-derived mesenchymal stem cells,as an important family member of stem cells,can be induced to differentiate into a variety of cells such as fat,bone,pancreatic islet β-cells,and myocardium under specific conditions,and therefore have broad prospects in the treatment of diabetes by cell transplantation.In clinical practice,most pancreatic islet transplantation involves transplanting cells to the liver,but the cells are easily lost during the transplantation process and only have a short-term effect.Therefore,in the process of islet cell transplantation,the choice of transplantation site and transplantation carrier also restricts the success of islets.Studies have found that the microenvironment of skeletal muscles can be transplanted,and it is convenient for blood glucose monitoring after transplantation.This suggests that our muscle tissue,as a very important target organ for glucose utilization,has great potential as a site for islet cell transplantation.However,agglomerated pancreatic islet cells may affect cell survival due to internal hypoxia during transplantation.Therefore,it is very important to prepare suitable degradable bio-scaffolds.The cell scaffold is an important carrier for the growth and material exchange of transplanted cells.As a highly hydrophilic polymer scaffold,hydrogel has been widely used in various fields of biomedical engineering.Recently,some studies have shown that stem cells loaded with hydrogel as a cell scaffold can promote wound healing.In addition,people are also committed to developing in-situ injectable hydrogels that can spontaneously and rapidly gel under physiological conditions,and to modify the hydrogel niche to meet the needs of different cell culture and tissue engineering applications.Sodium alginate(SA)is a natural polysaccharide extracted from brown algae.It has high hydrophilicity,biodegradability,and good biocompatibility.It is a traditional hydrogel material and can be used as a cell scaffold.Provide a suitable microenvironment for cell transplantation.As an important target organ for glucose utilization,skeletal muscle can provide suitable conditions for cell proliferation and is a potential transplantation site.However,the grafts in the skeletal muscle part are very susceptible to being squeezed by the internal muscles.Therefore,our grafts are required to have a certain degree of mechanical force to allow the cells to survive for a long time at the transplantation site.Polylactic acid-glycolic acid copolymer(PLGA)is a degradable,non-toxic and harmless polymer material.It is currently used in many clinical studies.The research team found that PLGA stents were coated with polydopamine(PDA)in the early stage.In addition to the original advantages of the obtained PDA-PLGA stent,the hydrophilicity and mechanical properties have also been greatly improved.At present,there is no report that PDA-PLGA-coated sodium alginate hydrogel is loaded with adipose-derived mesenchymal stem cells to induce β-cells for skeletal muscle transplantation to treat diabetes.In this paper,sodium alginate hydrogel scaffolds were prepared by ion cross-linking method and characterized,and the scaffolds with high water content and good mechanical properties were obtained.By preparing polydopamine(PDA)coated PLGA scaffold and wrapping it with sodium alginate hydrogel,a biological scaffold system with strong mechanical properties and high biocompatibility was obtained.The PDA-PLGA-Hydrogel scaffold was transplanted with Rinm5 f into the skeletal muscle of diabetic rats to evaluate the effectiveness of the scaffold.Adipose mesenchymal stem cells,which are rich in resources and can be used for autologous transfusion,were used as stem cells to differentiate into functional islet β cells by adenovirus overexpression of PDX-1 and induction of multiple factors,that is,they can continuously secrete basic insulin and have the characteristics of glucose-dependent insulin release.Functional islet cells were transplanted into skeletal muscle with biodegradable scaffold coated with PDA-PLGA alginate hydrogel to make it easy to monitor and adapt to the microenvironment,and a cell therapy system with sustainable treatment of diabetes was obtained.The following is the research content of this thesis.1.PDA-PLGA-Hydrogel biological scaffolds were prepared.In this experiment,the biological scaffold of islet transplantation was prepared by using the synthetic polymer PDA-PLGA with good mechanical properties to encapsulate the natural polymer sodium alginate hydrogel with high biocompatibility.Sodium alginate hydrogels were prepared by crosslinking different concentrations of sodium alginate and Ca2+.The mechanical properties and water content of different concentrations of sodium alginate hydrogels were tested,and the sodium alginate hydrogels suitable for transplantation in vivo were selected.the results showed that there was a positive correlation between the concentration of sodium alginate and the mechanical properties of the hydrogels,and a negative correlation between the water content and the mechanical properties of the hydrogels.Therefore,the hydrogel with2% sodium alginate concentration has better mechanical properties and water content is more suitable to be used as an internal filler.Further detection of the internal and external structure,swelling and cell biosafety of sodium alginate hydrogel showed that 2% sodium alginate hydrogel could provide the living environment of islet cells without affecting the material exchange between islet cells and the outside world.The PDA-PLGA biological scaffold system was prepared by rolling the PDA-PLGA-Hydrogel scaffold into a cylinder and filling it with sodium alginate hydrogel.At the animal level,PDA-PLGA scaffolds and PDA-PLGA-Hydrogel scaffolds were transplanted with ADSCs cells into rats for 3 days,7 days and 14 days,respectively.The results showed that compared with simple PDA-PLGA scaffolds,the survival rate of biological scaffolds coated with hydrogel was higher,and it was more suitable to be used as a carrier for cell transplantation.The islet cells of PDA-PLGA-Hydrogel loaded with Rinm5 f were transplanted into the skeletal muscle of diabetic rats.The results showed that the graft could reduce the blood glucose of rats and have a certain therapeutic effect on diabetes.PDA-PLGA-Hydrogel biological scaffold system can be used for the treatment of cell transplantation.2.Establishment of islet cell system induced by rat mesenchymal stem cells.In this part of the experiment,adipose mesenchymal stem cells were over-expressed PDX-1 and combined with multiple factors to differentiate into insulin-secreting cells,to establish an efficient,simple and reproducible induction method.Firstly,rat mesenchymal stem cells were isolated and cultured,their surface markers were identified by flow,and their differentiation potential was identified by adipogenic differentiation.the results showed that rat mesenchymal stem cells with high purity and differentiation potential were isolated and isolated.Furthermore,the PDX-1 gene was transferred into adipose mesenchymal stem cells by adenovirus transfection,and the expression of PDX-1 was detected by western-blot and immunofluorescence.It was found that PDX-1 was significantly expressed in the transfection group compared with the non-transfection group.Finally,the cells were induced to differentiate into insulin-secreting cells by multiple factors((2% B27 activin-A,10 n M exendin-4,0.1m M taurine 20ng/μL b FGF,20ng/ μL EGF,1% BSAMague 1%ITSMagi 10ng/μL HGF,10 m M nicotinamide,5m M taurine,10ng/μL HGF),and their morphology and function were detected.The results showed that the induced cells were stained scarlet by dithizone staining,and the positive expression of insulin was detected by immunofluorescence.And insulin secretion increased under the stimulation of high concentration of glucose,and had the function of glucose-dependent insulin secretion.The above studies showed that adipose mesenchymal stem cells were successfully induced into islet cell system by overexpression of PDX-1 and combined with multi-factor induction,which provided a source of seed cells for islet transplantation in the treatment of diabetes.3.Study on the application of PDA-PLGA biological scaffold in islet cell transplantation induced by stem cells.A rat type I diabetes model was established by injecting streptozotocin(STZ).The islet cells induced by adipose-derived mesenchymal stem cells were inoculated into sodium alginate hydrogel,wrapped with PDA-PLGA,and transplanted into diabetic rats In skeletal muscle,monitor fasting blood glucose changes in rats,tissue sections to detect pathological changes(HE staining and Masson staining),immunofluorescence to detect insulin and vascular CD31 levels,to explore the combination of islet cells and scaffolds induced by adipose-derived mesenchymal stem cells The therapeutic effect of the body on diabetes.The results show that the PDA-PLGA bio-scaffold carrying islet cells can reduce fasting blood glucose in the skeletal muscle of diabetic rats 4 weeks before transplantation,but after 5 weeks of treatment,it is found that the insulin fluorescence in the stent is significantly reduced,the hypoglycemic effect disappears,HE and The results of Masson staining indicated that the hydrogel inside the stent was degraded during the initial stage of transplantation,and the CD31 fluorescence intensity was not high,indicating insufficient neovascularization,which may be the reason why the effect cannot be maintained.To sum up,this study illustrates that 1.PDA-PLGA-Hydrogel biological scaffolds have strong anti-skeletal muscle mechanical pressure properties,and better biosafety can be used in islet cell skeletal muscle transplantation to play a therapeutic role;2.Overexpression of PDX-1 combined with multiple factors can induce adipose mesenchymal stem cells to differentiate into islet β cells,continuously secrete insulin and have the characteristics of glucose dependent insulin secretion,which provides a rich source of islet cells for diabetic cell therapy.3.The transplantation of islet cell-biological scaffold complex in rat skeletal muscle has the characteristics of continuous insulin secretion and biodegradation,and has a certain therapeutic effect on diabetes,but in view of the lack of treatment duration,the transplantation needs to be further improved.In this study,we optimized the preparation method of adipose mesenchymal stem cells to differentiate into islet cells,and provided a new strategy for stem cells to differentiate into islet cells.The islet cell-biological scaffold skeletal muscle transplantation system was established to provide a new idea for the treatment of diabetes.