| IntroductionType 1 diabetes is the result of autoimmune destruction of insulin-producing p-cells within the islets of Langerhans. The precise mechanisms for cytokine-in-duced (3-cell of type 1 diabetes have yet to defined . The accumulating evidence suggests that programmed cell death ( apoptosis) is the main form of -cell death in these disorders. Zumsteg et al reported nitric oxide ( NO) production and Fas/Fas ligand (FasL) interaction are two independent pathways of cyto-kine-induced murine -cell damage, Moreover NO of high concentration is a terminal effective factor in cell injury stimulated by CK. IL-1 , alone or in combination with TNF- and IFN-, induces in rodent and human islets the transcription of the inducible nitric oxide synthase (iNOS) gene. This enzyme catalyzes the generation of NO.Cyclic 3' ,5'-adenosine monophosphate ( cyclic AMP) is an important physiological amplifier of insulin secretion. Agents that increase cyclic AMP levels by activating adenylyl cyclase , or by inhibiting cyclic AMP phosphosdiesterase markedly augment glucose-induced insulin release. There are 11 PDE families ( PDE1~ PDE11 ) , several subtypes, and numerous isofonn splice variants. The foreign test has showed that islet cells express both PDE3 and PDE4. However , the effects of the two PDE inhibitors on suppressing NO production and elevating cAMP level of islet cells have not been extensively studied.In this test, which with isolated pancreatic islet cells from Wistar rats were cultured in vitro, further studied the effects of NO production, insulin secretion , cAMP levels and cell activity ( MTT assay) in IL-1 -induced islet cells and the protective effect of specific PDE3 inhibitor( Cilostamide, CIL) and PDE4 in-hiLitor(Rolipram, ROL) on islet cells and their mechanisms.MaterialsDrugs and Reagents; ( 1 )IL-l,CIL,ROL were purchased from Shanghai Kangchen Biotechnology co. , LTD; (2) Type V collagenase test kit, NO test kit and insulin radio-immunity test kit were obtained from Beijing Biotinge Biotechnology CO. ,LTD; (3)1 I-cAMP radio-immunity test kit was purchased from the Laboratory of isotope of Shanghai University of Traditional Chinese Medicine; (4) RPMI-1640 medium was obtained from Zhengzhou Sino-American Biotechnology CO. , LTD; ( 5 ) Bovine serum albumin ( BSA ) , colorimetric [ 3-(4^5-dimethylthythiazol-2-yl)-2, 5-diphenyltetrazolium bromide]( MTT) and dimethyl sulfoxide ( DMSO) were gifts from the Department of Cell Biology and were products of Sigma.Animals ; Male Wistar rats were purchased from Laboratory animal center of China Medical University.Instruments; Phase contrast Microscope, Cleaning Cabinet, C02 Incubator, Filter, cell Counting plate, Spectrophotometer( Daojing,Japan,UV160).Methods1. Islet Isolation and Culture; Male Wistar rats weighing 100-150g were anaesthetized with sodium pentobarbital (3% , 1. 2-1. 3ml/kg). The pancreas was removed under aseptic condition, washed , chopped and digested in 38T1 water with collagenase type V for 23min. the islets were washed two times with Hanks and filtered through a 80um metal grid to remove undigested tissue and debris. The islets were collected and cultured in RPMI-1640 medium containing 10% fetal calf serum, 2g/L glucose, 100IU/ml penicillin, 100mg/ml streptomycin at 37 C in an atmosphere of 5% C02 and 95% air using an incubator. The RPMI-1640 medium was changed each 2 days. After 96h, the medium was centrifuged at 100 x g and islet cells were collected. Subseoquently, the islet cells were seeded at a density of 6.5 105 islet cells/ml/well in 24-well plates.2. Groups and Measurement; The islet cells were divided into 9 groups(n(1) control(2)added30U/mlIL-lp( 3 ) added 30U/ml IL-1 p ,200mmol/L CIL(4)added 30U/ml IL-lp,400mmol/L CIL(5)added 30U/ml IL-1600mmol/L CIL(6)added 30U/ml IL-1 ,50mmol/L ROL(7)added 30U/ml IL-lpJOOmmol/L ROL(8)added 30U/ml IL-1 ,200mmol/L ROL(9)added 30U/ml IL-1 ,200mmol/L CIL,50mmol/L ROLThe 100ul culture supernatant of above -mentioned islet cells were collec...
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