| Objective: Clinical islet transplantation can be expected to become one of the most effective therapy for the patients of type 1 diabetes with the establishment and improvement of Edmonton protocol.Islet transplantation has the advantages of surgical safety,minimal invasion and can be performed easily.However,shortage of pancreas donors,the side effect of immunosuppression,and poor graft survival restrain the success and worldwide application of islet transplantation.In clinical islet transplantation,after isolation and purification,islets are cultured in vitro for 24–72 h before transplantation to enable the quality control of the isolated islets,the immunosuppressive pretreatment of recipients,the transportation of islets to remote transplant centers,and minimizing post-transplantation leukocyte-mediated reaction,and a 48-h cultivation period tends to be the standard practice accepted by most islet transplantation centers.Human serum albumin is used as a supplementation of islet culture media,which provides the necessary osmotic pressure for islet survival.However,it lacks the abundant growth factors and extracellular matrix molecules existing in serum,which causes a serum-deprived condition that may impair islet viability and function.It is reported that up to 20% of islets were lost during pretransplant culture period,which exacerbates the problem of islet shortage and restrains the success of islet transplantation.Therefore,the efficiency of islet transplantation can be improved by preventing and reducing islet cell apoptosis induced by serumdeprivation and protecting islet viability and function.Human amniotic membrane extract is a safe biomaterial with abundant resources,which has been utilized in the treatment of clinical corneal diseases.Studies show that amniotic membrane extract can promote wound healing and tissue regeneration,reduce inflammation and prevent oxidative stress-induced injury,and promote cell proliferation.Consequently,this study intended to establish a model of serum deprivation stress of mouse primary islets cultured in vitro before islet transplantation,verifying the protective effect of amniotic membrane extract on primary islets of mice under serum deprivation stress through a series of experiments,and to explore the possible molecular mechanism of the protective effect of human amniotic membrane extract on islets,so as to provide a new idea for clinical islet transplantation and for pre-transplantation islet preservation.Methods: 1.Collect human amniotic membrane from healthy pregnant women after caesarean section and prepare human amniotic membrane extract.The total protein of human amniotic membrane was assessed using a standard Bradford protein assay.2.AO/EB kits were used to detect the viability of serum-deprived islets after exposure to different concentrations of human amniotic membrane extract(0-1.5mg/m L)for 48 hours.3.TUNEL assay was used to detect the apoptosis rate of serum-deprived islet cells after exposure to different concentrations of human amniotic membrane extract(0-1.5mg/m L)for 48 hours.4.Glucose stimulated insulin secretion function of serum-deprived islet cells after exposure to different concentrations of human amniotic membrane extract(0-1.5mg/m L)for 48 hours was assessed using Insulin ELISA kits.5.The expression levels of phosphorylated Akt and ERK1/2 in islet treated by human amniotic membrane extract were determined by western blot.6.The expression levels of apoptosis related proteins such as Bcl2,BAX and cleaved caspase 3 in islet treated by human amniotic membrane extract were determined by western blot.7.Diabetic recipient for syngeneic islet transplantation was induced by injecting streptozotocin(STZ)into male C57BL/6 mice.8.Islet transplantation was performed underneath the kidney capsule.9.The metabolic function of islet graft was evaluated by monitoring non-fasting blood glucose,body weight and intra-peritoneal glucose tolerance test(IPGTT).10.The histopathological characteristics of islet graft were observed by HE staining,immunohistochemical staining and immunofluorescence staining.11.Graft insulin content was detected by Insulin ELISA kit.12.The proteomics of human amniotic membrane extract was assessed by LCMS/MS analysis.13.The concentrations of growth factors TIMP-1,EGF,HGF and IL-1RA were determined by respective ELISA kits.Results: 1.Amniotic membrane extract was prepared successfully,total protein content was 2.65±0.41 mg/m L,and amniotic membrane extract could be preserved in-80?.2.After cultured for 48 hours in vitro,the viability of serum deprivation stressed islets was significantly reduced compared to normal serum cultured islets(P<0.0001),while human amniotic membrane extract supplementation significantly increased the viability of serum deprivation stressed islets,and reaching a peak at the concentration of 0.5 mg/m L(P<0.001).3.After cultured for 48 hours in vitro,the apoptosis rate of serum deprivation stressed islets was significantly higher than normal serum cultured islets(P<0.0001).And supplementation of human amniotic membrane extract in culture medium significantly reduced islet apoptosis induced by serum deprivation,and supplementation of human amniotic membrane extract at the concentrations of 0.5mg/m L and 1.0 mg/m L mostly benefited islets(P<0.0001).4.After cultured for 48 hours in vitro,the insulin secretion levels were significantly reduced in serum deprivation stressed islets compared to normal serum cultured islets under high glucose stimulation(16.8 m M)(P<0.0001),but the insulin secretion levels between these two groups did not differ under low glucose condition(2.8 m M).Supplementation of human amniotic membrane extract did not affect their insulin secretion under low glucose condition(2.8 m M),but supplementation of human amniotic membrane extract at the concentrations of 0.5 mg/m L and 1.0 mg/m L significantly increased the insulin secretion levels of serum-deprived islets under high glucose stimulation(16.8 m M),and reaching a peak at the concentration of 0.5 mg/m L(P<0.0001).5.After cultured for 48 hours in vitro,human amniotic membrane extract significantly increased the expression levels of phosphorylated Akt(P<0.01)and ERK1/2(P<0.001)in serum deprivation stressed islets.6.After cultured for 48 hours in vitro,human amniotic membrane extract significantly increased the Bcl2/BAX ratio(P<0.01),while significantly reduced the expression level of cleaved caspase 3 in serum deprivation stressed islets.7.Syngeneic islet transplantation underneath the kidney capsule was established successfully.Recipients receiving human amniotic membrane extract cultured islet revealed better blood glucose control compared to recipients receiving serum-deprived islet(P<0.0001).The body weights of recipient mice were significantly higher in human amniotic membrane extract group compared to serum deprivation group(P<0.0001).The percent of diabetic recipients achieving euglycemia was significantly higher in human amniotic membrane extract group compared to serum deprivation group(P<0.01).8.Recipients receiving human amniotic membrane extract cultured islet revealed better blood glucose clearance ability compared to recipients receiving serum-deprived islet(P<0.0001).9.Histopathological results showed that pancreatic islets cultured with human amniotic membrane extract could be locally colonized and survived after transplantation,and insulin and glucagon-positive cells were clearly visible in islet grafts.10.The graft insulin content of islet grafts that were treated with human amniotic membrane extract was significantly higher than serum deprivation stressed islet grafts(P<0.0001).11.LC-MS/MS analysis revealed that the component of human amniotic membrane extract was stable,including key proteins modulating endothelial cell proliferation,angiogenesis,apoptosis and immune regulation.12.Human amniotic membrane extract contained key growth factors that were pivotal for islet survival,and these growth factors were stable in-80? cryopreservation.Conclusion: Human amniotic membrane extract supplementation improved islet viability and function,and decreased islet apoptosis during serum-deprived culture,and the optimal concentration of human amniotic membrane extract is 0.5 mg/m L.Human amniotic membrane extract significantly increased phosphorylation of Akt and ERK1/2,increased the Bcl2/BAX ratio,while decreased the expression of cleaved caspase 3.Moreover,transplantation of serum-deprivation stressed islets that were cultured in AME into diabetic mice revealed better blood glucose control and improved islet graft survival.The component of human amniotic membrane extract was stable,including key proteins modulating apoptosis and pivotal growth factors for islet survival. |
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