| Background: Oxygen on the body’s metabolism,growth and development,maintaining the homeostasis is an irreplaceable role.Lack of oxygen suppliment or oxygen metabolism disorders will cause cells,tissues,organs appearing dysfunction,followed by a lot of pathophysiological phenomena and a series of clinical symptoms.Researchers found that hypoxia and hypoxia have a serious impact on the function of islet grafts.There are numerous reports about this phenomenon,but the mechanism of how ischemia and hypoxia caused the loss of graft function has rarely been reported.Diabetes is one of the chronic diseases that threaten the human health.The rapid increase in the population and the trend towards younger age have drawn wide attention from the medical community and social the public.It has become one of the most intractable chronic diseases in our country and even in the world.The traditional treatment is extremely limited for controlling blood glucose by subcutaneous insulin and oral hypoglycemic drugs.Even complete insulin replacement therapy cann,t completely avoid the occurrence of diabetic complications,but also cann,t completely replace the pancreatic function,resulting in a sharp decline in the quality of life of patients.However,the survival rate of islet graft is completely low and the loss of function after transplantation is still the most prominent issue that hinder the development of the islet transplantation.In recent years,the researchers found that human islet amyloid polypeptide(h IAPP)was deposited in the cell membrane of pancreatic β-cells in an autopsy of islet transplant patients.h IAPP is a 37 amino acid peptide that co-exists with insulin in pancreatic β cells and secrete an endocrine hormone at the same time by a certain percentage.The misfolded spatial conformation of h IAPP will result the deposition of h IAPP in islet beta cells.Therefore,we suspect that ischemia and hypoxia during islet transplantation will affect the deposition of h IAPP.However,there is none of report on this aspect.In this study,h IAPP-INS1 cell model was established to further verify the relationship between hypoxia and h IAPP in pancreatic β-cell.From a new perspective,we can clarify how hypoxia affects the deposition of h IAPP and provide a new idea for the prevention and treatment of diabetes and islet transplanation.Methods: 1.Insulin-producing rat tumor cell line(INS1)was stably transfected with lentivirus to establish a new insulinoma cell line with puromycin resistance(h IAPP-INS1).The h IAPP-INS1 cells were screened with puromycin2μg/ml for 14 days to establish the h IAPP-INS1 cell and the control group CON-INS1 cell.The expression of h IAPP gene in h IAPP-INS1 cells and CON-INS1 cells was detected by q RT-PCR.The expression of h IAPP gene in h IAPP-INS1 and INS-INS1 cells was detected by fluorescence microscopy.The fluorescence intensity expressed by h IAPP in h IAPP-INS1 cells and CON-INS1 cells after thioflavin S staining was detected by chemiluminescence analyzer.2.The h IAPP-INS1 cells model and CON-INS1 cells model were cultured under hypoxia for 6h,12 h,24 and 48 h respectively.The proliferation activity of h IAPP-INS1 cells and CON-INS1 cells were detected by MTS in different time periods of hypoxia.Fluorescence microscopy was used to observe the expression of h IAPP-INS1 cells and CON-INS1 cells.The change of h IAPP deposition was examined by chemiluminescence analyzer.The fluorescence intensity of h IAPP-INS1 cells and CON-INS1 cells were detected by ELISA to evaluate the changes of insulin secretion ability of h IAPP-INS1 cells and CON-INS1 cells stimulated by low glucose(2m M)and high glucose(20m M)at different hypoxia time.Different autophagy-related protein expression(LC3-II and P62)were detected in h IAPP-INS1 cells and CON-INS1 cells during different hypoxia environment by Western blot.Results: 1.Establishment and Validation of h IAPP-INS1 Cell Line and CON-INS1 Cell Line Model:(1)The h IAPP expression was significantly up-regulated in h IAPP-INS1 cells compared with CON-INS1 cells,p <0.0001.(2)Compared with CON-INS1 cells,the fluorescence intensity of h IAPP-INS1 cells increase significantly,p <0.0001.2.The study of hypoxia on h IAPP deposition and cell function in h IAPP-INS1 cell line and CON-INS1 cell :(1)The activity of h IAPP-INS1 cell and CON-INS1 cell decreased gradually with hypoxia time.The activity of h IAPP-INS1 cells decreased significantly from 12 h and was significantly different from that in hypoxia 6h group and hypoxia 24 h group(P <0.05).(2)Fluorescence intensity of the CON-INS1 group has no change with hypoxia time;the fluorescence intensity of h IAPP-INS1 group increased after 12 h hypoxia,there was a significant difference between hypoxia 12 h group and hypoxia 6h group(P <0.05),there was also significant difference with hypoxia 24 h group(p <0.05).(3)h IAPP-INS1 cells and CON-INS1 cells had no significant changes that insulin secretion with hypoxia12 h for hypoxia24 h in 2m M glucose stimulated(p> 0.05),respectively insulin secretion was significantly decreased when stimulated by 20 m M glucose(p <0.05).(4)The results of Western blot showed that the expression of LC3-Ⅱ in h IAPP-INS1 cells and CON-INS1 cells was significantly increased from 0-12 h under hypoxia environment(P <0.05),P62 levels were significantly decreased from 0-12 h under hypoxia environment(P<0.05),indicating that autophagy was enhanced;The expression of LC3-Ⅱ was decreased from 12-24 h under hypoxia environment(P <0.05),however,P62 level was increased(p <0.05)indicating that autophagy was reduced,consistenting with the deposition of h IAPP.Conclusion: 1.h IAPP-INS1 cell model and CON-INS1 cell model were established successfully.2.Insulin secretion was limited stimulated under high glucose(20m M)with hypoxia 12 h and hypoxia 24 h in h IAPP-INS1 cells and CON-INS1 cells.3.The autophagy of h IAPP-INS1 cells and CON-INS1 cells were enhanced by mitochondrial pathway before hypoxia 12 hours and reduce the deposition of h IAPP in cells.The cell injury increased and autophagic function decreased after hypoxia 12 hours. |
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