| ObjectiveThe pancreas transplant is one of surgery therapies used chiefly for patients suffering from juvenile - onset insulin - dependent diabetes mellitus( IDDM). In 1996, Kelly et al first performed simultaneous pancreas and kidney transplants on a patient with IDDM complicated by renal failure. While the number of pancreatic transplants has risen with the recent stabilization of the transplant procedure ,the results of pancreas transplants are poor compared to those for kidney , heart,or liver transplants, meanwile,ischemia 鈥?reperfusion injury in the pancreas is one of the main causes of the loss of the graft, continuance ischemia of pancreas may lead tissue trauma and cell perish. Though it is important for the tissue that reperfusion on pancreas in the early stage, but white blood cell of circulation that attach to vascular wall, accumulate, transude vascular and infiltrate organs may lead to cell putrescence of organs, and activated complement as well as produce of activated oxygen result in organs trauma of ischemia more severity, namely reperfusion injury, with participate of various cell factors, they lead to organ cell apoptosis and necrosis, it is ideal method of attenuate reperfusion that start - up protect mechanism of endogenesis of economy by the way of pretreat-ment, and improve organ endure for the ischemia reperfusion. in the present stud-y, we investigated whether prior heat load stress as well as prior adrenergic stimulation reduce ischemia - reperfusion injury of rat model of pancreas transplant, then the mechanism of such favorable effect of hsp is assessed and it provide experiment base for clinic to alleviate ischemia - reperfusion injury by precondition.Material and MethodMale wistar rats, weighting 250g - 270g, are obtained from animal department of china medical university . antibody used for western blotting inducible hsp70(rabbit anti -rat )is obtained from BEI JING ZHONG SHANG biological technology ltd. TNF - a and HSP70 immunohistochemical package are obtained from WU HAN BOSTER biological technology ltd. pancreas transplant model described by lee et al. the pancreas was transplanted into the abdomen arteries and veins, rats were divided three parts( HPC , IPC ,C ). there were five rats in every part. HPC , IPC parts were used to induce hsp70, and C was control part. Collection of specimens and determination of hsp70 in the pancreas for 0, 6,12,24,36 and 48 hours after heat loading and administration of isoprotereol. We performed pancreas transplant 12 hours after heat loading(H) and 24 hours administration of isoprotereol (I) when a maximum level of hsp had been induced. The control group consisting of normal rats was performed pancreas transplant as well as. grafts were determined for immunohistochemica hsp70 and TNF - a after reperfusion for 6 hours, blood of rats was determined changes of serum amylase. method: 1. measurement of hsp70 level by western blotting; pancreas samples obtained immediately after sacrifice were homogenized and ahquots( 10ug protein) subjected to SDS electrophoresis. And separated proteins were transferred to nitrocellulose membranes, induction of hsp70 was monitored using antibody to inducible hsp70 (1 : 400). Labeled proteins were visualized u-sing horseradish peroxidase coupled secondary antibody (1:600) and quantitated using a gelatin image analysis system. 2. immunolocalisation of hsp70 and TNF- : pancreas samples were fixed (24 hours) in neutral buffered formalin, embedded in paraffin, sectioned (5um) ,and mounted on superfrost/plus slides, endogenous peroxidase activity was quenched with 3% hydrogen peroxide and non- specific binding was blocked by 1 % BSA. the sections were then incubated with primary antibody ( anti - mouse hsp70 1:50, anti - mouse TNF - 1: 80 ) o-vernight at 4C. after rinsing, the sectionswere treated with secondary antibody for 30 minutes, the antigen - antibody complex was visualized using the DABmethod,and slides were then counterstained and rinsing and coverslipped with permount. the microscope i...
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