ABSTRACT
Objective To investigate the effect of alpha 1-antitrypsin (A1AT) concerning in reducing the injury of transplanted islets by pancreas exocrine cells and promoting proliferation of the pancreas B cells.Method The pancreases of mice were digested with collagenase,islets were isolated artificially,and pancreatic exocrine cells were collected.In purified islet group (n =6),100 islets were seeded into a 6 well culture plate.In experimental group(n =6),100 islets were co-cultured with equal volume of pancreas exocrine cells,and 0.5 mg/mL A1AT was added into a 6-well culture plate.In control group(n =6),100 islets were co-cultured with equal volume of pancreas exocrine cells.After 48 h,insulin content of islets in each well and trypsin concentration in the supernatant of each well were measured.The islets were cultured in low sugar and high sugar 1640 medium,then glucose stimulated insulin secretion (GSIS) test was carried out.In vivo,8-9-week old male BALB/C mice were induced with STZ (190 mg/kg body weight,i.p) to establish the diabetic model and randomly divided into two groups.In experimental(n =10) and control(n =10) groups,250 islets and the equal volume of pancreatic exocrine cells were transplanted into different regions of left kidney subcapsule,resepctively.The experimental group was injected with A1AT (83 mg/kg,qd,i.p) for 28 days after operation,and the control group was injected with the same amount of normal saline (qd,i.p) for 28 days.Both two groups were given EDU (5 μg/g,qd,i.p) for 28 days.The blood glucose level was monitored continually.Nephrectomies were performed after 28 days.The expression of anti-amylase antibodies in the renal subcapsule was detected by immunohistochemical staining,and the proliferation of islet beta cells was examined using immunofluorescence staining.Result Insulin levels and insulin stimulation index in the control group were decreased as compared with those in the purified islet group; those in the experimental group were higher than in the control group,but lower than in the purified islet group.Trypsin concentration in the control group was increased as compared with the purified islet group,that in the experimental group was lower than the control group,but higher than in the purified islet group (all P＜0.01).After islets transplantation,the blood glucose levels in control and experimental groups were normal,but those in the control group recovered later than in the experimental group (P＜0.01).At 3rd day after nephrectomy,the blood glucose levels were ＞21 mmol/L in both two groups.A large number of anti-amylase antibody-positive cells were found in the renal subcapsule in the control group while little seen in the experimental group after 28 days.The immunofluorescence showed that the insulin +/EDU + B cells in the experimental group were more than those in the control group.Conclusion Conclusion Co-culture of islets and pancreatic exocrine cells with A1AT can prevent islet cells from damage caused by trypsin.A1AT could inhibit the secretion of pancreatic amylase from pancreatic acinar cells and promote proliferation of islet beta cells.
ABSTRACT
Objective To investigate the protective effects of α-1 antitrypsin on human islets injured by protease released from pancreas exocrine cells. Methods ( 1 ) in vivo experiment. Parts of the cadaveric pancreas was digested with collagenase, islets were selected artificially, and pancreatic exocrine cells were collected. 8-9 weeks olds male BALB/c-Nu nude mice were induced into diabetic mice with STZ (240 mg/kg body weight, i. p) and randomly divided into two groups: the control group (n = 6), 250 islets were transplanted into left kidney subcapsule of diabetic nude mice; cotransplant group (n = 7), 250 islets and the equal volume of pancreatic exocrine cells were transplanted into different regions of left kidney subcapsule. Blood glucose level was monitored. Nephrectomies were performed after 28 days. The expression of anti-amylase antibodies in subcapsule was detected by using immunohistochemical staining. (2) Islets culture: Three groups were randomly set up. Group 1: purified islet group, 250 islets were incubated into a 6-well culture plate; Group 2: non-purified islet group, 250 purified islets and equal volume of exocrine cells were incubated; Group 3: nonpurified islet + Al AT group, 250 purified islets and equal volume of exocrine cells were incubated with α-1 antitrypsin added (0. 5 mg/ml). After 48 h, insulin content of islets in each well and trypsin concentration in the supernatant of each well were measured. Results 10000 islets were collected.After islets transplantation, the blood glucose levels in control and co-transplant groups were normal,but a delayed islet function in reversing diabetes was in the co-transplant group, and ehe mice in both groups became hyperglycemic after nephrectomy. A large number of anti-amylase antibody-positive cells were found in renal subcapsule in the co-transplant group while little seen in the control group.Insulin levels in the non-purified islet group were decreased as compared with purified islet group,those in the non-purified islet group + A1AT group were higher than in the non-purified islet group,but lower than in the purified islet groups. Trypsin concentration in the non-purified islet group was increased as compared with purified group, that in the non-purified islet group + A1AT group was lower than the non-purified islet group, but higher than in the purified islets group (all P＜0. 01).Conclusion Protease released from acinar cells during pancreatic digestion has detrimental effect on islet function after transplantation. Co-cultivation of islets and pancreatic exocrine cells with A1AT added can prevent islet cell damage caused by trypsin.