Experiment Research Of Differential Proteins Related To Liver Regeneration After Rat Reduced-size Liver Transplantation And Immune Hyporesponsiveness Induced By Liver Regeneration

Objective:It was the foundation and prerequisite for the next research of liver regeneration and it's immune hyporesponsiveness that The improved model of reduced-size liver transplantation in the rat was investigated.Methods:1.The donors were female and the receptors were male,which were healthy SD rats and weight range was 260-280 g,weight of the receptor was more than that of the donor,about 10g.2.Operation of donor was performed by only one person with the naked eye,during which reduced-size donor liver was performed.The handle of self-made cuff was placed in the good front of portal vein and inferior vena cava respectively,which the tied ligature of pyloric veins was turned inside out of the self-made cuff, furthermore,the tied ligature was placed in the left of the self-made cuff;the same to inferior vena cava except that the tied ligature of right renal vein was placed in the right of the self-made cuff.Then the portal vein and inferior vena cava were received washing with self-made perfusate respectively.3.Operation of the receptor was performed by two person with the naked eye,with improved dual-cuff technique of Kamada and stay pipe of biliary tract.The fixed points of left and right were connected by anastomosis of "8" type turning inside out while inosculating inferior vena cava.4.The pre-improved model of reduced-size liver transplantation in rat was performed by reported in the literature,in which lobes of rat liver were removed in liver repair basin.5.The symptoms and signs> all complications,survival conditions,liver function change of rats were observed after liver transplantation in the improved and pre-improved model groups.Results:1.The average operation time of gaining the donor and preparating the donor liver were 32±2 minutes and 6±2 minutes respectively.The average operation time of gaining the receptor and the anhepatic were 40±3 minutes and 14±3 minutes respectively.And cold preservation time of liver donor was 51±3 minutes.The general successful rate was 92%,three-day survival rate was 85%and two-week survival rate was 83%.The postoperative complications was reduced clearly.the contrast was significant difference(P<0.05).2.ALT values of ldpo(days post operation),3dpo and 7dpo in improved group were reduced progressively in pre-improved group in comparation with that of pre-improved group,which was significantly difference(P<0.05) in two groups at the same time.But ALT values of 14dpo and 21dpo in improved group were non-significant difference(P>0.05) in comparation with that of pre-improved group.3.Compared with pre-improved group,TBIL values in improved group were reduced progressively advanced at 1dpo,3dpo and 7dpo,which were significantly difference (P<0.05) in two groups at the same time.But the level of 14dpo and 21dpo in two groups were non-significant difference(P>0.05).4.Compared with pre-improved group,XCHE values in improved group was progressive advanced at 1dpo,3dpo and 7dpo,which were significantly difference (P<0.05) in two groups at the same time.But the level of 14dpo and 21dpo in two groups were non-significant difference(P>0.05).5.Compared with pre-improved group,AMON values in improved group was reduced progressively at ldpo and 3dpo,which were significant difference(P<0.05) in two groups at the same time.But the level of it in two groups at 7dpo,14dpo and 21dpo were non-significant difference(P>0.05) at the same time.Conclusions:1.It was better method of obtaining high quality and satisfied reduced-size liver in the rat that lobes of rat liver in operating on donor liver were removed after liver donor perfusion.The operation of donor and receptor was intercoordination,which may shorten cold preservation time of liver donor;The improved vascular anastomosis may shorten anhepatic time and reduce bleeding complications;It was worthy of being accepted model of reduced-size liver transplantation in the rat that the improved model may reduce in-operation and post-operation complications after liver transplantation,and raise succeed rate of rat liver transplantation and survival rate of receptor.2.It was maybe the foundation and prerequisite for hepatic rebirth and immune experimental research after liver transplantation that improved model of reduced-size liver transplantation in the rat was established successfully. Objective:To search for the differential proteins related to liver regeneration after transplantation,it was studied on the base of the success in the reduce-size liver transplantation in rat applying of technology of proteomics.Methods:1.Experimental donors were healthy and female Lewis rats,weight range was 200-230 g.The receptor were healthy and male Wistar rats,weight range was 220-250 g.2.Models of reduced-size liver transplantation in rats were established successfully, which weight ratio of the donor and the receptor liver was about 50%.The postoperative rats accepted immunosuppressive treatment of KF506,with 10mg/kg·d.The postoperative liver specimens were harvested by 1 d,3 d and 7 d after reduce-size liver transplantation.And the normal liver specimens of the donors and the receptors were harvested during reduce-size liver transplantation,which were kept in a -70℃refrigerator for the proteomics test.3.The harvested liver tissue specimens were accepted proteomic test and analysis.The first step,comparative proteome analysis with postoperative liver tissue and normal donor,receptor liver tissue.Namely,they were accepted MS-MS spectrometry after two-dimensional electrophoresis(2-DE) for identifying proteins stains with differentiation exceeding 10 times.At last,the function of differential proteins related to liver regeneration was analyzed after reduce-size liver transplantation in rats.Results:1.The results showed the high-resolution and reproduciblity 2-DE patterns about postoperative liver tissue and normal donors and receptors,and found 72 differential protein stains by taking 10 times measure.2.Seventy-two differentially expressed protein spots were all identified by MS-MS spectrometry and PMF.At last,Forty proteins were identified successfully.3.The identified proteins mostly participated in physiology pathway:signal transduct -ion,stress defence,redox homeostasis,carbohydrate metabolism,energy, aminoacid metabolism and cell cytoskeleton,and so on.Some of which participated in liver regeneration after reduce-size liver transplantation by means of direct and indirect physiology pathway.Conclusion:1.Reduce-size liver transplantation models in rats were established successfully,with Lewis to Wistar.2.Sevety-two differential protein spots and forty proteins were identified successfully. 3.The differential proteins related to liver regeneration were found successfully after reduce-size liver transplantation,which maybe participate in liver regeneration after reduce-size liver transplantation through two aspects as follows:on one hand,to promote liver regeneration by direct physiology pathway;on other hand,to promote liver regeneration by indirect physiology pathway.4.provided fundamental research results for next research related to liver regeneration mechanism after reduce-size liver transplantation.Objective:Liver regeneration and how many the recipients' bone marrow stem cells differentiated to hepatocytes,and it's possible mechanism of immune hyporesponsiveness induced by liver regeneration after removing immunosuppressive drugs were studied on the basis of successful murine model of reduced-size liver transplantation.Methods:1.Experimental donors were healthy and female Lewis rats,whose weights varied from 200 to 230 g.Recipients were healthy and male Wistar rats,whose weights varied from 240 to 380 g.2.Models were operated via method of improved Lewis-Wistar reduced-size liver transplantation;details can be seen in the first part.3.Experimental groups:(1) Group A:whole liver transplantation group.The donors' weight was less than that of receptors.(2) Group B:50%liver transplantation group.The ratio of donors' to recipients' liver weights was about 50%.(3) Group C:30%liver transplantation group.The ratio of donors' to recipients' liver weights was about 30%.4.The entire experiment can be divided into two parts.(1) To observe situations of liver regeneration among groups that accepted different volume liver transplantations.After operations,used FK506 as immunosuppressive drug,whith 0.1 mg/kg·d.On the first day and 3rd day and 7th day after transplantation,sampled serum and liver tissue for relevant tests.(2) To observe low immunological reject reaction induced by liver regeneration. When liver regeneration followed partial liver transplantation finished, removed usage of FK506,then observed rejection among 3 groups.On the 0 dpo,3 dpo,7 dpo and 11 dpo after removal FK506,sampled serum and liver tissue for relevant tests.5.Survival situation and rejection reaction of rats were observed and judged:If rat died within 3 days after operation,the rat would be excluded,another would be compensated.The degree of rejection was judged by Banff score system.6.The level of ALT,AST,TBIL in serum and level of serum cytokines IL-2,IFN-γ, IL-4,IL-10 and TGF-β₁ were tested.7.Levels of IL-6 and TNF-αin liver tissues were detested by ELISA,which related to regeneration after liver transplantation,and the level of TGF-β₁ related to rejection was detested.8.To detect variation of IL-6,TNF-α,PCNA and Ki-67 related regeneration after liver transplantation,liver tissues immunochemical methods were performed.And then, expression variation of ICAM-1,NFκB/p65 and TGF-β₁ related to rejection after operation was detested in same methods.9.Y chromosome in liver tissues was detested by hybridization in situ,In ordor to prove bone marrow stem cells of recipients differentiated into hepatocytes.10.To observe morphous and rejection reaction of liver tissues.Liver tissues were treated by hematoxylin and eosin stain,and seen under electron microscopy and detested by Tunnel methods to showe celluar apoptosis,Results:1.In the early stage of regeneration followed reduced-size liver transplantation, regeneration was accelerated(on the second day of operation,it got to climax),then decelerated.On the 7th day of operation,liver' weight achieved to as big as that of pre-transplantation.2.In the process of regeneration,Y chromosome positive rate was the highest in the group C,lower in group B,lowest in group A.3.In the process of regeneration,levels of IL-6 and TNF-a in liver tissues were ascend -ing in early stage(the summit appeared in the second day),then they were descending.4.When observing rejection,levels of IL-2 and IFN-γin serum were the highest in group A,lower in group B,the lowest in group C.On the opposite,the levels of IL-4 and IL-10 in serum were the highest in group C,lower in group B,the lowest in group A.5.The serum level of TGF-Pi were ascending at the beginning,then carried on descending.When it was ascending,group A appeared fastest and highest,then the group B,but group C was last one.On the other hand,in the process of TGF-β₁ descending,it can be seen to decrease fastest and most in group A,then group B, group C followed.Meanwhile,TGF-β₁ level in liver tissues appeared increasing persistently,same mode as IL-2 and IFN-γin serum,the most apparent is group A, group B is the second,at last,group C.6.By immunochemical examination of ICAM-1,NFκB/p65,TGF-β₁,hepatocyte apoptosis and hematoxylin and eosin stain analysis,the most severely imflammation reaction was observed in group A,better in group B,then group C.Conclusions:1.Lewis-Wistar improved reduced-size liver transplantation models was builted successfully.2.In the process of liver regeneration after improved reduced-size liver transplantation, bone marrow stem cells differentiated into hepatocytes.3.On the basis of complete liver regeneration after improved reduced-size liver transplantation,most severel rejection was observed in group A,better in group B, and best in group C after removing FK506.4.The mechanism of low immunological reaction induced by improved reduced-size liver transplantation maybe relate to the process of bone marrow stem cells differentiated into hepatocytes,and multiple factors maybe contribute to this process.