| Objective establishing a more efficient and stable animal model of orthotopic liver transplantation in rat.Application of Breviscapine in the perioperative,and Clarifying whether it has a protective effect on ischemia/reperfusion injury after orthotopic liver transplantation in rats.Methods Three steps:1.Basing on the original two-cuff technique and integrating varieties of modified techniques of rat orthotopic liver transplantation model,perfusion by abdominal aortic, evertedly suturing anastomosis suprahepatic inferior vena cava by single thread,anastomosis portal vein and infrahepatic inferior vena cava by cuff methold, end-to-end anastomosis common bile duct by single-tube intra-stent.Improving the details in surgery and reducing blooding,reasonably rewarming and rehydration, shortening operative time and anhepatic phase.Observing postoperative survival rate and survival period.2.Randomly divided 40 experimental rats into treatment transplantation group and control transplantation group,each further divided into two sub-groups,repeatedly perform rat liver transplantation model.Observe the postoperative survival rate,6-hour and 24-hour survival rate,and 6-hour and 24-hour,obtained blood from the inferior vena cava for determination of serum ALT vitality,cut a part of liver tissues for observation by ordinary optical microscope.With SPSS13.0 statistical software used two independent samples t-test for statistical analysis.3.(1)Grouping 420 male SD rats randomly were divided into normal group,sham operatipon group,normal saline control group,low-dose treatment group and high-dose treatment group,except the normal group,each group divided into five point-in-time sub-groups,consist of 1 hour,3 hours,6 hours,12 hours and 24 hours, the normal group and sham operation group n=5,normal saline control group, low-dose treatmen group and high-dose treatment group n=10.(2)Administration normal group and sham operation group did not to any intervention;normal saline control group only normal saline injection;low-dose treatment group were given 5mg/kg Breviscapine saline;high-dose treatment group were given 10mg/kg Breviscapine saline.(3)Surgical methods normal group without any operationt;sham operation group cutting off the ligaments around the liver and ligate left subphrenic vein and hepatic artery;normal saline control group,low-dose treatment group and high-dose treatment group to liver transplantation.(4)Sampling and storage puncture the inferior vena cava and phlebotomize blood 3-4ml,centrifugalize,leave supernatant and store in -80℃;took a small piece of liver tissue storing in cold normal saline and immediately inspected apoptosis;took a piece of liver tissue storing in -80℃;take a piece of liver tissue storing in 10% neutral formalin.(5)Detection methods Recorded normal saline control group,low-dose treatment group and high-dose treatment group the time of graft cold preserved ischemia, anhepatic time,receptor operative time,respectively,to compare;by automatic biochemical Analysis,inspecedt serum ALTand AST vitality and TBil concentration; respectively,xanthine oxidase method,dithio nitrobenzoic acid method,thiobarbituric acid(TBA)inspected serum SOD and GSH-Px vitality and MDA concentration;grinded into the liver tissue,which storing in cold saline water,then filtered and centrifugalized,flow cytometry inspected liver cell apoptosis percentage by Annexin V-PE staining;thawed the frozen liver tissue,made into 10%homogenate solution,then filtered and centrifugalized,,by brodford method inspected protein concentration of every samples,respectively,by xanthine oxidase method,dithio nitrobenzoic acid method,thiobarbituric acid(TBA) inspected liver tissue SOD and GSH-Px vitality and MDA concentration;the liver tissues,storing in 10%neutral formalin,paraffin-embedded,sliced 4μm thick,shopped tablet,stained,and observed by ordinary optical microscope.(6)Statistical methods Showed data to mean±standard deviation(x±s).Using SPSS13.0 statistical sofware,compared between different groups by analysis of variance,within group by t test,a=0.05 as test standard.Results1.Established 120 cases(not including pretest)SD rat orthotopic liver transplantation model with improved two-cuff technique,the time of donor surgery need 17±1.5minutes,37±2.5 minutes about receptor,the anhepatic phase only 19±1.5 minutes,the success rate of surgery of liver transplantation reach 94.2%and the one week survival rate is 90.0%.2.Treatment transplantation group and control transplantation group,respective 20 cases were operrated,compared postoperative survival rate to the two groups,no statistical difference(P>0.05);in 6-hour the survival rate of treatment transplantation group were betterr than the control transplantation group,with statistical difference(P<0.05);in 24-hour the survival rate of treatment transplantation group were betterr than the control transplantation group,with statistical difference(P<0.05);in 6-hour treatment transplantation group serum ALT vitality were lower than the control transplantation group,with statistical difference(P<0.05);in 24-hour treatment transplantation group serun ALT vitality were lower than the control transplantation group,with statistical difference(P<0.05); pathological changes,such as,cell degeneration and necrosis,liver sinusoidal occlusion,inflammatory cell infiltration;The seriousness of treatment transplantation group was worse than the control transplantation group,with statistical difference(P<0.05). 3.(1)Operation time Graft cold ischemia storage time,among the C,L,H three groups no statistical significance(P> 0.05);anhepatic phase,among the C,L,H three groups no statistical significance(P> 0.05);receptor operation time,among the C,L, H three groups no statistical significance(P> 0.05).(2)Liver function ~ Serum ALT vitality,at different time-point,the N,L and H three groups significantly increased,N compared with S group has statistical significance(P <0.01);C group higher than the L group,there is statistical significance(P <0.05);L group higher than H group,there is difference(P <0.05);H group was significantly higher than the H group,a statistical significance(P <0.01); the 6-hour of each group are the peak(P <0.05,P <0.01).(~)Serum AST vitality,at different time-point,the N,L and H three groups significantly increased,N compared with S group was significant difference(P <0.01);C group higher than the L group, there was statistical significance(P <0.05);L group higher than H group,a dstatistical significance(P <0.05);H group was significantly higher than the H group,a statistical significance(P <0.01);the 6-hour of each group are the peak,there was statistical significance(P <0.05,P < 0.01).(~)Serum TBil concentration,the N,L and H three groups increased significantly,compared with N and S group has statistical significance(P <0.01);C group higher than the L group,there is difference(P <0.05); L group higher than H group,a statistical significance(P <0.05);H group was significantly higher than the H group,a statistical significance(P <0.01);the 6-hour of each group are the peak(P <0.05,P <0.01).(3)Serum SOD,GSH-Px vitality and MDA concentration(~ Serum SOD vitality, S group increased slightly,compared with the N group was statistical significance((P <0.05);and C,L and H group decreasing,compared with the N and S group was statistical significance(P <0.01);C group,reducing most significant,compared with the L group was statistical significance(P<0.05),C group compared with the H group was difference(P<0.01,P<0.05);H group lower than L group,compared with between the two groups was statistical significance(P<0.01,P<0.05);the 6-hour of each group,reducing mostly,compared with the 1-hour was statistical significance(P <0.01,P<0.05);from the 12-hour to the 24-hour gradually increased,the 24-hour compared with the 6-hour was statistical significance(P<0.01,P<0.05),24-hour compared with 1 hour,in L and H group,no statistical significance(P>0.05).②Serum GSH-Px vitality,C group,L group were significantly lower,respectively, compared with N,S group was statistical significance(P<0.01,P<0.05);and the H group improve better than the L group,H group compared with L group were statistical significance(P <0.05);the 6-hours of each groups reduced mostly, compared with 1 hour was statistical significance(P<0.01,P<0.05);12-hour to 24-hour gradually increased,24-hour compaed with 6-hour was statistical significance (P<0.01,P<0.05).③Serum MDA concentration,C group increased more than the N group and S group,and there were statistical significance(P<0.01,P<0.05);L group and H group decreased,compared with between the two was statistical significance(P <0.05);the 6-hour of each group was the peak,the 1-hour compared with the 6-hour was statistical significance(P<0.01);the 24-hour compared with the 6-hours was statistical significance(P<0.01,P<0.05).(4)Liver tissue SOD,GSH-Px vitality and MDA concentration①Liver tissue SOD vitality,S group increased slightly,compared with the N group,a statistical significance((P<0.05);and C,L and H group decreasing,compared with the N and S group was statistical significance(P<0.01);C group to reduce the most significant, compared with the L group was statistical significance(P<0.05),C group compared with the H group was difference(P<0.01,P<0.05);H group lower than L group, compared with between the two groups was statistical significance(P<0.01,P<0.05); the 6-hour of each group to reduce mostly,compared with the 1-hour was statistical significance(P<0.01,P<0.05);from 12-hour to 24-hour gradually increased,24-hour compared with 6 hours was statistical significance(P<0.01,P<0.05),24-hour compared with 1 hour,in L and H group,no statistical significance(P>0.05).②Liver tissue GSH-Px vitality,C group,L group were significantly lower,respectively, compared with N,S group was statistical significance(P<0.01,P<0.05);and the H group improve better than the L group,H group compared with L group were statistical significance(P<0.05);the 6-hours of each groups reduced mostly, compared with 1 hour was statistical significance(P<0.01,P<0.05);12-hour to 24-hour gradually increased,24-hour compared with 6-hour was statistical significance(P<0.01,P<0.05).③Liver tissue MDA concentration,C group increased more than the N group and S group,and there were statistical significance(P <0.01,P<0.05);L group and H group decreased,compared with between the two was statistical significance(P<0.05);the 6-hour of each group was the peak,the 1-hour compared with the 6-hour was statistical significance(P<0.01);the 24-hour compared with the 6-hours was statistical significance(P<0.01,P<0.05).(5)Hepatocyte apoptosis C group was the highest proportion of apoptosis,and C group compared with L group,was statistical significance(P<0.01);L group compared with the C group was statistical significance(P<0.05);while the effect of inhibiting apoptosis,the H group was stronger than L group,compared the two groups was statistical significance(P<0.05);the 6-hour was peak,then the proportion of apoptosis gradually decreased,the 6-hour compared with the 1-hour was statistical significance(P<0.01,P<0.05);the 24-hour compared with the 6-hour statistical significance(P<0.01,P<0.05).(6)Pathological changes in C group liver cells degeneration and necrosis, sinusoidal congestion and inflammatory cell infiltration,and so on,was most serious, L group in second,and the H group in third;the pathological injury grade,C group the highest,C group compared with L group was statistical significance(P<0.01,P <0.05);while the H group was lower than the L group,compared the two groups was statistical significance(P<0.01,P<0.05);the 6-hour more was most serious,,6 hours compared with other time-point were statistical significance(P<0.01,P<0.05).Conclusions1.By improved "two-cuff" method of orthotopic liver transplantation in rats,basing on skilled microsurgical skills,two mutually collaborate with streamline operations, improve detail and reduce errors and complications,then shorten the operation time, especially anhepatic phase,careful perioperative management,can improve the survival rate and time,and establish a more efficient and stable model of rat orthotopic liver transplantation for study.2.Application of an improved"two-cuff"method in rat model of orthotopic liver transplantation,the surgical success rate was high,and the operation process was more stable,the measures controlled similar,decreased the differences and impact of individuals caused by the operation.Application Breviscapine can improve the receptor's survival rate and time,can reduce the serum ALT vitality level of receptor, improve the liver histopathological changes.We believed that application breviscapine has a protective effect on ischemia/reperfusion injury of postoperative transplantation rat donor liver,the specific protective mechanism and the relationship with drug dose which need further study.3.By inhibiting the generation of oxygen free radicals,decreasing liver tissue and serum SOD and GSH-Px consumption,and increasing the SOD,GSH-Px vitality, relieving lipid peroxidation and protecting cell membrane,inhibiting liver tissue and serum MDA producing and concentration,inhibiting cell apoptosis,reduction of liver cell damage and improve liver function,alleviating the pathological changes in liver breviscapine which apply take the role of protective effect to liver and body ischemia/reperfusion injury after liver transplantation in rat,and with a certain degree of dose-effect relationship. |
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