| BACKGROUNDSince the Starzl performed the first clinical liver transplantation in 1963,the liver transplantation is the only effective treatment for the end-stage liver diseases and has been widely accepted.but,the ischemia-reperfusion injury (IRI) are often lead to primary graft non-function (PNF), and it is considered the largest reason of re-transplantation except rejection.so,how can we reduce the ischemia-reperfusion injury is regarded as one of the hot issues in the field of liver surgery.Murry has first put forward to the concept of ischemic preconditioning(IPC)in 1986,it is defined as rapid intermittent periods of reperfusion and ischemia before ischemia,which can made it a signifieant tolerance in a longer Period of time after ischemia and reduce reperfusion injury. In recent years, researchers had found that can protect the tissues and organs by rapid intermittent periods of reperfusion and ischemia in the early phase of repefusion after long ischemia of the tissues and organs, and it has similar protective effects with IPC.thus,they put forward to the concept of ischemic postconditioning (IPO).However,the IPC has lost the best time for treatment when the ischemia reperfusion injury is formed,and the IPO is the measure after ischemia which it can make up for the lack of IPC treatment IRI, then it have more clinical value.Currently the protection mechanisms of IPO were not yet entirely clear.The ischemic postconditioning is play its role through many possible mechanisms such as oxygen free radical, calcium overload, polymorphonuclear neutrophil, cytokine, cell apoptosis and mitochondrial and so on.The death way of sinusoidal endothelial cells and liver cells is the apoptosis in the hepatic ischemia-reperfusion injury.Recently, the studies of mitochondria have shown that mitochondria plays a pivot role in apoptosis, and the early of ischemia reperfusion injury is mainly in the changes of mitochondrial structure.mitochondrial dysfunction has become an important mechanism of the hepatic ischemia-reperfusion injury, mitochondrial is play a key role in during the IPO fight IRI.The critical moment of reperfusion injury is mainly occurred in a few minutes when the beginning of ischemia-reperfusion,then it is the tiome of IPO,so IPO is a early intervention mechanism which has an important protective effect on hepatic ischemia reperfusion injury, it may be through intervention mitochondrial structure and function and reduce liver cells apoptosis to achieve. So it is important to study for IPO protect liver cell apoptosis from the angles of mitochondria, and the mitochondrial permeability transition pore is closely related with the IPO.we are further study of whether the pathway of mitochondrial is can inhibiting the ischemia reperfusion injury by observing the apoptosis of liver cells and the morphology and function of mitochondrial.It can provide theory based on continuous study on protective mechanisms of ischemic postconditioning on hepatic graft and targets of anti-ischemia-reperfusion injury.OBJECTIVETo establish a reliable animal model of rat orthotopic liver transplantation (OLT) with modified "doubl-cuff"technique,and investigate the protective effects and the possible mechanisms of the mechanical IPO on reduce the ischemia-reperfusion injury. it will provides a new method for the prevention and treatment hepatic ischemia-reperfusion injury in clinical.MATERIALS AND METHODS1.Animal Model:Healthy male Sprague Dawley (S-D) Rats weighing 200-250g were used to establish an improved model of rat liver transplantation based on Kamada's "doubl-cuff"technique.2.Animal groups:Fourty healthy male Sprague Dawley (S-D) Rats were randomly divided into four groups:â‘ sham-operation group:only free liver ligament; â‘¡schemia and reperfusion group (I/Rgroup):The portal vein were persistent reperfusion after implantation the graft;â‘¢ischemic postconditioning group(IPO group):animals were given six 60-second episodes of ischemia at 60-seeond intervals for reperfusion before persistent reperfusion of portal vein,then fully liberalized.3.Detection:Separate groups of rats were killed at 6h after their portal vein were opened, and the samples were collected for further analysis. The hepatocellular function (ALT, AST) was analyzed; The levels of Bcl-2 mRNA and Bax mRNA were detected by RT-PCR. The levels of cyt-c protein were detected by Western Blotting. The apoptosis and necrosis of liver tissue and the mitochondrial transmembrane potential were detected by flow cytomertry (FCM).The changes of liver tissues and mitochondrial structure Were observed by HE staining and electron microscope.RESULTS1. Compared with Sham group,the levels of ALT and AST in I/R group and IPO group were significantly higher(P<0.05),and the levels of ALT and AST in I/R were more higher(P<0.05).the difference was statistically significant.2.RT-PCR show that:the levels of Bax mRNA in I/R group and IPO group were significantly higher,the levels of Bax mRNA in I/R was higher than that in IPO group,(P<0.05). Compared with Sham group,the levels of Bcl-2 mRNA in IPO group and I/R group were significantly lower,the levels of Bcl-2 mRNA in I/R was significantly lower than the IPO group (P<0.05).the difference was statistically significant.3.Western Blotting showed that:Compared with Sham group,the levels of Cyt-c protein in I/R group and IPO group were significantly higher(P<0.05),and the levels of Cyt-c protein in I/R were more higher(P<0.05). the difference was statistically significant.4. Flow cytometry showed that:Compared with Sham group,the levels of apoptosis and necrosis in I/R group and IPO group were significantly higher, (P<0.05);the I/R group was significantly higher than the IPO group,(P<0.05).the difference was statistically significant. Mitochondrial transmembrane potential (MTP) of Liver Cell in the I/R group and IPO group was significantly lower than the Sham group (P <0.05);the I/R group was significantly lower than the IPO, group(P<0.05).the difference was statistically significant.5. HE staining showed that:the structural of hepatic lobule is complete in Sham group, and the structure of portal area and liver cells is normal;the hepatic lobule central vein and hepatic sinusoid is obvious extravasated blood in I/R group,and the liver cells are widely edema;the structural of hepatic lobule is the basic integrity in IPO group,then the liver cells and portal area is almost normal, and only a few liver cells is mild edema.6.Electron microscopy showed that:The changes of ultrastructure is severe in I/R group,the integrity of liver cells structure is destroyed,mitochondria is swelling and in part by the vacuolization; the IPO can still the integrity of liver cells structure,and the mitochondria is lighter swelling than the I/R group.CONCLUTION1. The modified mold of hepatic ischemia reperfusion injury is a stable, reliable, convenient and practicable mold.2.Ischemic postconditioning have a protective effect on cold ischemia-reperfusion injury of rat liver transplantation,it can effectively reduce the ischemia reperfusion injury,reduce liver cell apoptosis and improving liver function.3. The mitochondrial Pathway is one of the ways in which the IPO reduce HIRI.The mechanism may be increase the expression of antiapoptosis gene Bcl-2,inhibit the expression of proapoptosis gene Bax and improve the ratio to inhibiting of MPTP opening,further to maintain the stability of mitochondrial transmembrane potential (or higher) and protective the structure and function of mitochondria by mechanical ischemic postconditioning in the mitochondrial pathway.lt can reduced the release of Cyt-c and other apoptosis proteins, to prevent the liver cells apoptosis, and eventually reduce reperfusion injury.
|
PDF Full Text Request