Gdolinium Chloride's Effect On Ischemia Reperfusion Injury To Rat Receptor's Liver

Partâ… :The Influence of Gadolinium Chloride on Kupffer Cell ActivityObjectives:To explore the effects of gadolinium chloride on the activation of the kupffer cells and release of cell factors,and to elucidate its possible mechanism.Methods:Kupffer Cells were isolated and purified by collagenase perfusion.The isolated and purified cells were then divided into 3 groups: Group A,control group in which kupffer cells were cultured normally; Group B,LPS group in which LPS was added into the growth medium to a final concentration of 10mg/l;Group C,GdCl₃+LPS growth medium to a final concentration of 2mmol/l and 24 hours later added LPS into the growth medium to a final concentration of 10mg/l.After 6h culturing to each group,the morphology of kupffer cells were watched under an inverted microscope,the activities of kupffer cells were tested by carbon phagocytic test,the purity was measured by anti-ED-2 immunohistochemical staining,and the concentration of IL-1 and TNF-αin the supernatant were measured by ELISA.Tests in each group were repeated for 8 times to obtain the mean value of each index as the final result.Results:The shape of newly isolated kupffer cells were globose,and suspended in the growth medium;After 6 hours culturing,most of them turned flat and adhering to the wall,pseudopodium formation occurred in few cells;Pseudopodium formation were found in most of the cells after 24h;After about 48h,all cells were extended totally with bigger size in star-shape or polygonal shape;Under a microscope after Hematoxyhn-Eosin staining,typical star-shape cells with renal-shape nuclus were observed.The purity ratio of each group was 90%,and the activity ratio was more than 96%.The phagocytic ability of group C had higher than phagocytic ability and less cell shape change than group B;Concentration of TNF-αand IL-1 in growth medium of group B was much higher than that of group A and C(P＜0.05).Conclusions:(1) Situ collagenase perfusion is a good method for kupffer cell isolation and purifying,by which the purity ratio and the activity ratio reached 90%and 96%respectively;(2) Gdolinium chloride can inhibit the phagocytic ability of kupffer cells and as a result reduce its ability of releasing cytokines such as TNF-αand IL-1. Partâ…¡Establishing Rats Model of Orthotopic Liver TransplantationObjective:To establish orthotopic liver transplantation model to provide ideal animal model for liver transplantation research.Methods:We chose the modified Kamada's two-cuff technique to establish orthotopic liver transplantation models,and in this test the animals were closed colony Sprague-Dawley male rats with recipient rats a little larger in size than the donors.10%glucose was given to the recipient rats after transplantation,and Penicillin was injected for the following 3 days with 100ku per day,and immunosuppressive drugs were not used.Results:100 cases of stable stage models were made in this test, with donor harvest time 45-60min,warm ischemic duration 0-0.5min, donor preparation time 12-15min,cold storage of donor duration 2h, anhepatic phrase 18-22min,24h survival rate 92.0%(92/100).8 cases were failed because of failure of operation or death within 24 hours:3 cases of anesthetic accident,4 cases of hemorrhoea in the operation process,1 case of diaphragmatic muscle's disruption inducing pneumothorax,9 cases died in 1 days to 7 days,which included 4 cases of infection,3 cases of biliary leakage and 2 cases of unknown reason. Conclusion:(1) Reducing operation time and anhepatic phrase as well as microsurgery skills are key factors for operations.(2) The choice of modified Kamada's two-cuff technique is reliable and stable which would be a powerful support for successive laboratory research. Partâ…¢The Effect of Gdolinium Chloride on Ischemia Reperfusion Injury of Rat Receptor's LiverObjective:To study the effect of Gdolinium Chloride on rat-receptor's transplanted livers.Methods:Donors and receptors were all male SD rats which were randomly divided into 3 groups,n=8:Group A,sham group;Group B: with Saline pretreatment;Group C:with gdolinium chloride pretreatment. The modified Kamada's two-cuff technique was used to build rat liver transplantation model,and the rats will killed after 24 hours.The ALT, AST,AKP andγ-GT were measured in serum,and the pathomorphology changes of liver tissue were observed after HE staining and analyzed by semi-quantitative method using Suzuki's score standard;The cell apoptosis was measured by TUNEL and the apoptosis index was calculated;In liver tissue,the TNF-α,IL-1 level were measured by ELISA.Result:Compared with Group A,the serum AST,ALT,AKP andγ-GT level obviously were increased in Group C and Group B(P＜0.05); In pathologic histology tests,the extent of IRI were all more obviously increased Group B and Group C.Compared with Group B,the serum AST,ALT,AKP andγ-GT levels were decreased in Group C(P＜0.05); the concentration of IL-1 and TNF-α,the apoptosis index and the Suzuki's score were also significantly in group C(P＜0.05).Conclusion:(1) Gdolinium chloride could inhabit the release of Cytokines of Kupffer cells;(2) Gdolinium chloride could protect the transplanted liver from ischemia reperfusion injury;(3) The protective effect of gdolinium chloride on ischemia reperfusion injury of transplanted liver might result from reducing cytokine release in Kupffer cells.