| 1.Background and ObjectiveIslet transplantation is one of the effective methods to treat end-stage type 1diabetes mellitus,but there are still many problems to be solved in order to improve the islet transplantation outcome.Among them,the loss of graft function caused by immune damage,hypoxia/reoxygenation damage effect,nonspecific inflammatory response and apoptosis is the biggest obstacle that islet transplantation is facing at present.The survival and function maintenance of transplanted islets depend on adequate nutrition supply and blood oxygen,and the delay and insufficiency of revascularization process will lead to islet death and early functional loss.Therefore,early rapid and sufficient vascular reconstruction plays an important role in islet transplantation.The decrease of oxygen supply leads to the colonization and activation of hypoxia inducible factor HIF,which in turn leads to the transcription of vascular endothelial growth factor?VEGF?.Although the molecular mechanism of islet revascularization is unclear,islet cells may be presumed to be a source of VEGF,which is an important factor in initiating revascularization and maintaining vascular permeability of islet grafts.To explore the ways to improve the islet cell hypoxia/reoxygenation injury after islet transplantation and how to prevent the adverse effects of revascularization on transplanted islets is the key to improve the transplantation outcome.The physiological function of SST is mediated by somatostatin receptor?SSTR?on cell membrane.SSTR is a glycoprotein with seven transmembrane segments.It has been confirmed that SSTR includes five different molecular subtypes SSTR 15.It has been reported that SST can directly inhibit pancreatic secretion and amylase secretion by acting with SSTR on PACS.when SSTR2 is activated,it can inhibit pancreaticenzymesynthesisandsecretionbyregulatingcell electrophysiological activities and ion channels.Our preliminary study showed that pancreatic exocrine cells and islet cells were transplanted into the left renal capsule of diabetic mice simultaneously,the secretion and release of trypsin and amylase from pancreatic exocrine cells delayed the functional recovery of transplanted pancreatic islet cells,but the mechanism is unclear.In this study,the effects of octreotide?OCT?on the growth activity and apoptosis of pancreatic acinar cells?AR42J?and pancreatic?cells?min 6?in vitro were investigated under normal oxygen/hypoxia conditions,and the effects of octreotide on the expression of VEGF protein and HIF-?protein were analyzed.Furthermore,by establishing a diabetic mouse model,islet cells and pancreatic exocrine cells were transplanted into the left renal capsule of diabetic mice,and then octreotide was injected subcutaneously after transplantation to explore the possible mechanism of octreotide on the function recovery of transplanted islet cells in mice,in order to provide theoretical basis and new basis for elucidating the effect of octreotide on the function recovery of transplanted islet cells.2.Method2.1 Pancreatic exocrine cells?AR42J?were cultured and divided into experimental group and control group.The experimental group was treated with octreotide,and then cultured under normal oxygen/hypoxia conditions.The growth activity of pancreatic exocrine cells in octreotide and control group were detected by CC-K8 method respectively.The expression of VEGF protein in the supernatant of the two groups was detected by ELISA.The expression difference of HIF-?protein in the experimental group and control group was detected by western blot method.The effect of octreotide intervention group and control group on apoptosis of pancreatic exocrine cells was detected by flow cytometry.2.2 The expression of HIF-1?in min6 cells treated with different concentrations of CoCl2 was detected by qPCR.The effects of different concentrations of CoCl2on the expression of HIF-1?in min 6 cells were investigated.The hypoxia positive control group of min 6 cells was prepared with CoCl2 concentration which could induce up-regulation of HIF-1?expression.Islet?cells?min 6?were cultured and divided into experimental group and control group.The experimental group was treated with octreotide,and then cultured under normal oxygen/hypoxia conditions.The growth activity of islet?cells in octreotide and control group was detected by CCK8 method,and the expression of VEGF protein in the supernatant of the two groups was detected by ELISA.The expression difference of HIF-?protein was detected by western blot method.The effect of octreotide intervention group and control group on islet?cell apoptosis was detected by flow cytometry.2.3 C57BL/6 mice were anesthetized and then the pancreas was expanded by retrograde perfusion of low temperature v-collagenase in situ through pancreaticobiliary duct.Then the islets were isolated by low temperature centrifugation with Histopaque-10771.The islets were picked manually with200?l micropipette under inverted microscope.The quantity and purity of isolated and purified islets were detected by specific staining with dithizone.The islet cells were digested with 4%trypsin and then stained with trypan blue to calculate the activity of islet cells.The insulin secretion of islets stimulated by low glucose and high glucose was detected by ELISA,and the function of islets was determined by insulin stimulation index.Pancreatic exocrine cells were cultured for 1 h and 4 h respectively,the relative expression of trypsin was measured by ELISA.2.4 C57BL/6 mice were induced into diabetic mice by intraperitoneal injection of streptozotocin one week before islet transplantation or operation.Then they were randomly divided into islet and pancreatic exocrine cells co-transplantation group?n=10?,islet transplantation group?n=10?and sham operation group?n=10?.Blood glucose levels were measured by Roche blood glucose meter at different time intervals after operation.On the 14th day after operation,5diabetic mice were randomly selected from the sham operation group,and five diabetic mice which were considered diabetes mellitus cured in the simple islet transplantation group and the co-transplantation group were randomly selected,respectively.IPGTT was performed to compare the differences of islet glucose tolerance among the three groups.The left kidney of C57BL/6 mice in the simple transplantation group and the co-transplantation group was excised 28days after operation.The expression of insulin and trypsin in the left kidney of each group was detected by immunochemical staining.2.5 Totally 48 C57BL/6 male diabetic mice were randomized into experimental group?co-transplantation group+octreotide intervention n=24?and control group?co-transplantation group n=24?.C57BL/6 mice islets and pancreatic exocrine cells were isolated by collagenase v digestion.The mice were anesthetized by intraperitoneal injection of 1%pentobarbital sodium at 0.01 ml/g.200 islets and equal volume of pancreatic exocrine cells were transplanted to the upper and lower poles of the left renal capsule in diabetic mice,respectively.After operation,the blood glucose concentrations in the experimental group and the control group were measured.On the 21st day after operation,IPGTT test was used to compare the islet glucose tolerance between the experimental group and the control group.Three mice in the experimental group and the control group were randomly selected on the 1st,3rd,7th and 14th day after islet transplantation to remove the left kidney containing graft for pathological examination.Seven days after surgery,the grafts under the left renal capsule in the experimental group and the control group were detected by immunofluorescence with double staining of insulin and?-amylase to investigate the difference in the expression of insulin and amylase.Early postoperative period?day 1,3,7,14?in the two groups,the expression of p53and bax protein was detected by immunohistochemical method,the apoptosis of transplanted islets was detected by TUNEL method,and the proliferation of islet cells was detected by EDU and insulin double staining immunofluorescence method.3.Result3.1 In vitro experimental study showed that octreotide concentration of 10-8M could promote the proliferation of AR42J cells.Octreotide can inhibit the expression of vascular endothelial growth factor VEGF and hypoxia inducible factor HIF-1a in pancreatic exocrine cells under hypoxia.The apoptosis of pancreatic exocrine cells in octreotide group and control group was detected by flow cytometry.The results showed that octreotide could significantly inhibit apoptosis of pancreatic exocrine cells under normal oxygen condition?p<0.05?,and octreotide could also promote apoptosis of pancreatic exocrine cells under hypoxia condition?p<0.05?.Octreotide can reduce the necrosis of pancreatic exocrine cells?AR42J?under normal oxygen conditions?p<0.05?,but has no effect on cell necrosis under hypoxia conditions.3.2 Hypoxia positive control cells were prepared by CoCl2 at 400?mol/l for12h.CCK-8 detection results showed that octreotide?OCT?could inhibit the proliferation of islet?cells?min 6?under normal oxygen/hypoxia conditions.After 9 hours of culture,the supernatant of min 6 cells in hypoxia group and normal oxygen group was collected and detected by ELISA kit.There was no difference in the relative expression of VEGF between the two groups?p>0.05?.Under normal oxygen condition,the relative expression of VEGF in negative control group?no OCT group?was not different from that in OCT co-culture group?p>0.05?,that is,OCT had no significant effect on the expression of VEGF in min 6 cells under normal oxygen condition.However,the relative expression of VEGF in the group without OCT was higher than that in the group with OCT co-culture?p<0.05?,which indicated that the expression of VEGF could be down-regulated after oct acted on min 6 cells under hypoxia.The apoptosis rate of control group and octreotide group was3.52±0.31%vs 5.20±0.38%under normal oxygen condition.However,the apoptosis rates of the control group and octreotide group were 11.40±0.62%and6.12±1.07%respectively under hypoxic condition.There was no statistical difference in apoptosis rate between control group and octreotide group under normal oxygen condition?t=4.203,p>0.05?,indicating that octreotide had no effect on apoptosis of min 6 cells under normal oxygen condition.The apoptosis rate of octreotide group was significantly lower than that of control group?t=5.803,p<0.05?,which indicated that octreotide could inhibit the apoptosis of min 6 cells under hypoxic condition,which was a protective mechanism for the functional recovery of transplanted islets.3.3 Pancreatic islet were isolated from mice by retrograde collagenase perfusion in common bile duct.On average?130±20?high quality islets were obtained from each C57BL/6 mouse.The activity and purity of the islets were over 90%.The insulin secretion was?0.458±0.086?ng/islets in low glucose group?2.8mmol/glucose?,?0.955±0.122?ng/islets in high glucose group?16.7 mmol/l glucose?,and the insulin stimulation index was 2.08.This indicates that the function of isolated and purified islet is good.Pancreatic exocrine cells were cultured for 1 h and 4 h respectively.The trypsin content of the supernatant was determined by ELISA,and the results were?4.283±0.289?ng/ml and?5.435±0.203?ng/ml,respectively.3.4 After islet transplantation,the blood glucose of mice in both groups decreased gradually.The time required for diabetic mice in islet transplantation group to restore normal blood glucose concentration was shorter than that in co-transplantation group?p<0.05?.The ratio of diabetic mice with blood glucose concentration>11.1 mmol/l after islet transplantation was lower in islet transplantation group than in co-transplantation group?p<0.05?.At 3 days after left nephrectomy,the blood glucose of recipient rats in both transplantation group and co-transplantation group was>21 m mol/l,and hyperglycemia was recovered.However,the blood glucose concentration of diabetic mice in sham operation group had no significant fluctuation before and after operation.A large number of positive anti-insulin antibodies were found under renal capsule in both simple and co-transplantation groups,which proved to be islet?cells transplanted.A large number of anti-amylase antibody positive particles were found around the islet cell mass in the co-transplantation group,while no significant positive particles were found in the islet transplantation group alone.3.5 In C57BL/6 diabetic mice,the blood glucose concentrations in octreotide group and control group decreased gradually after transplantation of islet and pancreatic exocrine cells at the upper and lower poles of the left renal capsule,respectively.In octreotide group,the average time of recovering normoglycemia was 10 days after islet transplantation,while in control group,the time of recovering normoglycemia was 14 days after islet transplantation?p<0.05?.21days after operation,IPGTT showed that the blood glucose of octreotide group was lower than that of control group at different time points?p<0.05?,combined with the comparison results of the blood glucose area under the curve?p<0.05?,which showed that the mice in experimental group were more tolerant to the glucose tolerance test than that of the control group.Seven days after operation,mice renal subcapsular islet graft were examined by pathology.The results of immunofluorescence detection with anti-insulin antibody and anti-alpha-amylase showed that the graft was transplanted islet cells and could secrete insulin normally.The islet survival rate in octreotide group was higher than that in control group.The expression of p53 and bax in octreotide group was significantly higher than that in control group on the first day after operation?p<0.05?.On the 3rd and 7th day after operation,there was no difference in p53 expression between octreotide group and control group?p>0.05?,but the relative expression of bax in octreotide group was lower than that in control group?p<0.05?,which indicated that octreotide could inhibit the apoptosis of transplanted islets.The results of EDU and insulin immunofluorescence showed that islet?cell proliferation was not found in the experimental group and the control group on the 1st,3rd and 7th day after islet transplantation,but it was found that islet?cell proliferation occurred in the experimental group and the control group on the 14th day after islet transplantation.The increment rate of octreotide group was 13.9%±1.2%on average and 5.8%±0.6%in the control group?p<0.05?.4.Conclusion4.1 In vitro experiments showed that octreotide could promote the proliferation of AR42J cells.The proliferation of islet?cells?min 6?was inhibited to some extent.4.2 VEGF protein expression was up-regulated in pancreatic exocrine cells?AR42J?under hypoxia.Octreotide can inhibit the expression of vascular endothelial growth factor?VEGF?protein and down-regulate the expression of hypoxia inducible factor?HIF-1??protein in AR42J cells.Octreotide may promote AR42J apoptosis under hypoxia,but has no significant effect on cell necrosis.4.3 Octreotide can down-regulate the expression of VEGF protein in min6 cells under hypoxia.The relative expression of HIF-1?protein in min6 cells was higher than that in normal oxygen condition,and octreotide could up-regulate the expression of HIF-1?protein in islet?cells?min6?under normal oxygen/hypoxia condition.HIF-1?overexpression may play an important role in islet revascularization.In this study,we first found that octreotide could inhibit the apoptosis of islet?cells?min6?under hypoxia by flow cytometry.4.4 Highpurity and highquality islets could be obtained by retrograde perfusion of low-temperature v-collagenase into pancreaticobiliary duct to digest the pancreas and separating the islets of mice by low-temperature centrifugal chromatographycombinedwithasingleconcentrationgradient Histopaque-10771.4.5 C57BL/6 diabetic mice were successfully induced by intraperitoneal injection of STZ at 160 mg/kg.Islet cells and pancreatic exocrine cells were transplanted to the upper and lower poles of the left renal capsule,respectively,to establish a diabetic mouse model of islet cells and pancreatic exocrine cells co-transplantation.4.6 In vivo study of C57BL/6 diabetic mice model of islet and pancreatic exocrine cells co-transplantation showed that octreotide could reduce the damage of pancreatic exocrine cells to transplanted islet and reduce the time required for normoglycemia in diabetic mice.Meanwhile,IPGTT test suggested that octreotide could improve the tolerance of islet to glucose tolerance test in mice with normal blood glucose after islet transplantation,and inhibit apoptosis and promote proliferation of islet cells after islet transplantation.We conclude that the mechanism may be related to p53 upregulation and bax downregulation after octreotide treatment,which may promote the repair of early islet injury and inhibit the apoptosis of transplanted islet?cells.On the other hand,octreotide can down-regulate the expression of VEGF in islet?cells in the early stage of islet transplantation,so as to reduce the energy consumption of ATP in the early stage of islet transplantation and improve the survival of islets.Octreotide can promote the up-regulation of HIF-1?expression and may play an important role in the revascularization and cell proliferation of transplanted islets.Furthermore,octreotide can reduce the damage of pancreatic exocrine cells to transplanted islets by promoting pancreatic exocrine cells apoptosis under hypoxia. |
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