| [Background] The existing critical shortage of grafts is still a major problem in clinical liver transplantation. A feasible way to expand the existing donor organ pool could be aggressive use of marginal grafts. As the rate of primary nonfunction after liver transplantation can reach 15%, selective pretreatment of grafts appears to be a promising concept in order to optimize organ function after transplanta -tion. The function of nonparenchymal liver cells appears to be crucial to the success of liver transplantation. Kupffer cells play a key role in the pathophysiology of hepatic ischemia/reperfusion injury. Kupffer cells in the transplanted liver has a dual role : They protect the transplanted liver transplant also can cause liver damage. One of their main functions is the elimination of toxins such as endotoxin and bacteria from splanchnic circulation. Activation of Kupffer cells during graft reperfusion by endotoxin leads to the production of reactive oxygen species, stimulates lipid peroxygenation, and augments the release of mediators such as thromboxane,prostaglandins, leukotrienes, tumor necrosis factor TNFa, interleukins, and proteases. Kupffer cells are also of relevance with regard to antigen presentation and specific immune response. Then modulating the donor liver Kupffer cells, The transplanted liver function and immune response to any kind of impact is worth exploring.Animal experiments have shown that the gadolinium chloride can effectively inhibit the activation of Kupffer cells, but has no direct impact on liver cells, endothelial cells, the body circulating mononuclear cells and the other macrophage function; Glycine is a non-essential amino acids the human body. In recent years, it was found to protect, a variety of entities organ ischemia-reperfusion injuryt, in addition, it has immunosuppressive effects on the body. So we choose gadolinium chloride and glycine modulate Kupffer cells of the donor liver to explore the its effecting and possible mechanism on allograft function in immune tolerance and rejection of liver transplantation. To further selection regutalors for Kupffer cell function, it provide the basis for the experimental and theoretical basis for Specific protection of liver function and reduce acute rejection after liver transplantation and clinical studies.[Objective] (1) To investigate the technique for performing orthotopic liver transplantation (OLT) stably in Wistard→Wistard, Wistard→SD rats and choose suitable, reliable orthotopic liver transplantation in the rat model of the same strain and acute rejection. (2) To study the effects on function of donor kupffer cells by modulation of GdCI3 and glycine,research on the liver function of liver transplantation and its possible mechanism. (3) To investigate the hepatic allograft rejection afected by the functions of Kupffer cells in rats, explore its possible mechanisms and find out the exact roles of Kuppffer cells in inducing the liver allograft tolerance. By this three-part experiment. We hope to further clarify the Kupffer cells in acute rejection of rat liver transplantation and the transplanted liver function, provid regutalors for Kupffer cell function and the basis for research and clinical application to protecting the liver transplant and reducing immune rejection.[Methods] (1) Orthotopic liver transplantation in Wistard→Wistard and Wistard→SD rats were performed with the modified two-cuf method that described by Kamada. Liver tissues and blood samples were collected. Recipient survival, histopathological characteristics of hepatic allogfrats were investigated. Plasm levels of alanine aminotransferase (ALT), aspartase aminotransferase(AST), and total bilirubin (TBIL) were measured with an automatic biochemical analyser. Pathological changes in liver were observed,at the same time, immune rejection in rat liver transplantation model were assessed. (2)In one treatment group donor animals were pretreated with a single intravenous injection of GdCI3 (10 mg/kg, 1 ml;) 1 h before graft harvesting. In a second treatment group organ donors were treated in the same way with glycine (300 mm, 1 ml;), The third group, pretreated with an equal volume of physiologic saline,served as the control group. After 3 days, 5 days and 7 days of OLT ,the blood serum was obtained by liver supra hepatic inferior vena cave, the supernatant after centrifugation was preserved in -70℃refrigerator for inspection. 1cm2 liver right anterior leaf tissue were cut and made of wax blocks and electron microscopy specimens, part of the liver after three days of preparation for hepacyties. The TBIL,ALT and AST of serum, TNFαIL→1,Expression of Fas ligand protein in Kupfer cells ,lymphocytes apoptosis rate by Flow cytometry. The corresponding pathological specimens of optical and electron microscopy observation of Kupffer cells were performed. (3) According to the above method, Wistard→SD rat model of liver transplantation was constructed. In one treatment group donor animals were pretreated with a single intravenous injection of GdCl3 1 h before graft harvesting..The a second treatment group organ donors were treated in the same way with glycine.The third group, pretreated with an equal volume of physiologic saline.served as the control group. The liver function and histopathological characteristics of hepatic allogfrats were investigated 7day after operation. IL-10 and TGFβcytokine levels in plasma were also measured with enzyme linked immunosorbent assay (ELISA), respectively. Expression of FasL on Kupfer cells and apoptosis rate of lympocytes in liver grafts were detected using immunohistochemical staining and Flow cytometry. This study was conducted to investigate the survival of recipients in which the function of Kupfer cells were altered. ALL data were expressed as means±SEM. All calculations were performed by use of the SPSS procedures P value of <0.05 was considered statisticaly significant.[RESULTS] (1)The technique orthotopic liver transplantation surgery were same in Wistard→SD and Wistard→Wistard rats, the technical training group completed a total of 46 cases of liver transplantation in rats, only seven patients survived more than 24 hours and six cases for the late-operative survival. Strains with orthotopic liver transplantation group completed 38 cases, 36 cases were successfully completed 37 cases of liver transplantation rejection Group. 33 cases surgery was successful. Postoperative liver transplantation with variant strains ALT, AST, and TB decreased from three days to five days, five to seven days will remain stable and exclusive group after 3, 5, 7 days ALT, AST and the gradual increase in TB. These two groups showed significant differences (P <0.05). Liver transplantation with variant strains all of the transplanted liver histopathological examination revealed no acute rejection performance. However, Wistard→SD group of immune rejection can be seen clearly. After three days, RAI mean 3.67±1.33, rose to 5.22 ± 1.62 after five days, 7.06 ± 0.78 after seven days. The longest survival time was 12 days, versus 33 days in the same strain rats of liver transplantation. (2)Changes in Kupffer cells, within 24 hours after transplantation, all three groups of rats were survived (100%). Gadolinium and glycine survival rate was 87.5%, the control group was 75.0%. Compared with the control group, P <0.01, there is significantly different. After gadolinium chloride and glycine treatment after liver transplantation. The serum AST,ALT and TBIL level significantly were higher than that of preoperation. But changes in the experimental group was significantly lower than the control group, the difference was statistically significant (P<0.05), the serum TNFα and IL-1 were significantly increased in two liver transplantation groups. The changes in glycine group was significantly lower in the control and gadolinium chloride group, the IL-10 levels in gadolinium chloride group was lower than that of the control group,but TNF-α significantly was higher than that of the control group, the difference was statistically significant (P<0.05).After three days immunohistochemical staining of liver tissue kupffer cells scattered in the protein FasL expression in gadolinium chloride group, other cells was not expressing FasL. kupffer liver cells FasL part expression in liver cells and lymphocytes positive staining was observed in Glycine group. FasL expression in the control group was higher after three days, more kupfer cells were positive. Experimental group with the control group, there was a significant difference. FasL in kupffer after seven days, immunohistochemical staining positive cells were 3.21 ±0.07% area, 7.03 ±0. 86% and 7.24 ±0.31 %. Experimental group and control group, there was a significant difference (P<0.05). glycine group and gadolinium chloride group also are significant differences (P<0.05). FCM was observed low rate of hepatic cells apoptosis of gadolinium chloride group versus the control group are significant differences. However glycine group and the control group showed no significant difference. Pathological changes in the three groups after three days, to minimize the inflammatory infiltration glycine group, the second group of gadolinium chloride, with the control group showed significant differences. The electron microscope compared with the control group. Kupffer cell function in Group gadolinium chloride and glycine group have been inhibited by suppressing, important gadolinium chloride group was the most sever. (3)Acute rejection in the present study, survival rates of gadolinium chloride was significantly lower in rats, followed by the control group, the glycine group was the highest survival rate, liver function index in gadolinium chloride group was significantly higher than that in glycine group, in the glycine treatment of liver function were significantly lower than the control group (P<0.05). Group A after liver transplantation in rats with the lowest levels of TGFβand IL-10 was significantly lower than the control group. TGFβlevel of glycine group was higher than the control group.The difference was statistically significant (P<0.05). Kupffer cells FasL with Immunohistochemistry (SP) staining in experiment group was observed decreased versus the control group, there was a significant difference (P<0.05).there is a significant difference (P<0.05) between glycine group and gadolinium chloride group. After 7 days lymphocyte apoptosis rate of apoptosis,gadolinium chloride group, was low rate, there is high proportion of lymphocyte apoptosis in glycine and control group (lymphocyte apoptosis index: Gadolinium group 2.83±0.42%, glycine group 7.81±1.06%, control group 7.64±1.28%); Kupffer cells amount of the liver tissue specimens was accumulated by electron microscope. Glycine group and control group was significantly higher than that of gadolinium chloride group and; Banff and pathological specimens to exclude activity index for each group is given an average score of RAI. Gadolinium Group was found the most sever immunological rejection, that in the glycine group was mild .there is a significant difference versus the control group.[Conclusions] (1) Liver transplantation from donor Wistar rats to recipient Wistar and SD rats can result in tolerance and acute rejection model. Wistard →SD rats of acute rejection in orthotopic liver transplantation is an ideal model of high rejection. It can be used to research immune rejection of Liver transplantation. (2) Regulating KCs function can affect liver function and allograft rejection in liver transplantation. This function is express through Fas, T lymphocytes apoptosis ,IL-1, IL-10 cytokines , TNFα and TGFβ to achieve. (3) Regulation of Gadolinium for Kupffer cells has a dual effection on transplanted liver function GdCl3 could block KCs function, down regulated FAS expression, the release of inflammatory factor and cytokines, thereby protecting transplanted liver function. But GdCl3 can inhibit the function of reducing KC Fas expression and the release of TGFβ ,IL-10 cytokines, aggravate the hepatic immunologic rejection. (4) Glycine may result of the glycine receptor inhibition of intracellular calcium release of Kupffer cells, reduceing the release of cytokines and Fas expression and apoptosis in the liver,so it play a protective role in the injury of liver transplantation.It can increase the release of cytokines TGFβ and Inhibite T-lymphocyte cells and reduce their toxicity ,so reduce the intensity of the hepatic immuneologic rejection. Glycine is a good regulator Kupffer cells, but there is a need to further explore the clinical application of safe and effective dosage. The release mechanism of TGF-β and IL-10 of glycine effecting on Kupffer cells functional regulation also need to do indepth research.
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