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Objective To investigate the effect of CaMK Ⅱ expression on apoptosis of rat hepatocytes BRL-3A.Methods Rat BRL-3A cells were stable passage were cultured.The CaMK Ⅱ γ protein (LV-CaMK Ⅱ γ group) and CaMK Ⅱ γshRNA (shRNA group) lentiviral expression systems were constructed.The corresponding blank vectors (LV-NC group and shRNA-NC group) and normal saline (CON group) were perfused into the control groups.The expression levels of CaMK Ⅱ,Cyt C and MF proteins were detected by Western blotting,and the apoptosis rate of BRL-3A cells was measured by Tunel method.Results The protein expression of CaMK Ⅱ,Cyt C and AIF in LV-CaMK Ⅱ γ group was significantly higher than that in CON group (P＜0.05).The protein expression of CaMK Ⅱ,Cyt C and AIF in shRNA group was significantly lower than that in CON group (P＜ 0.05).There was no significant difference among CON group,LV-NC group and shRNA-NC group (P＞0.05).At the same time point,the apoptosis rate of hepatocytes in LV-CaMK Ⅱ γ group was significantly higher than that in CON group (P＜0.05).At the same time point,the apoptosis rate of hepatocytes in shRNA group was significantly higher than in CON group (P＜0.05).There was no significant difference in the apoptosis of hepatocytes among CON group,LV-NC group and shRNA-NC group (P＞0.05).Conclusion The specific CaMK Ⅱ signaling pathway can inhibit the apoptosis of BRL-3A cells,while the enhanced CaMK Ⅱ signaling pathway promotes the apoptosis of BRL-3A cells.
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Objective To discuss the differential expression of hepatic stress proteins after reduced-size liver transplantation in rats.Methods The specimens of liver tissues were procured on 1 d,3 d and 7 d after the improved model of reduced-size liver transplantation in rats.Then,the two-dimensional electrophoresis of these specimens was compared with that of the original liver tissues of normal donors and recipients.The differentially expressed protein spots were selected with the standard of change times greater than 10 or less than 1 /10 and then were analyzed and identified by mass-spectrometric technique and data bases.Results Seventy-two differentially expressed protein spots were found in total.And the 32 kinds of proteins were identified with definite function through mass spectrometry and a series of identifications.The expression difference of heat shock protein-8 and hypertrophy agonist reactive protein was larger,amounting 7% (5 /72)of all differential proteins.Conclusions This study provides fundamental research data for studying the relation between liver ischemia-reperfusion injury after liver transplant and the above differential proteins of stress reaction in transplant liver which are found after reduced-size liver transplantation in rats.
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BACKGROUND:At present, the proteome is a mature technology that has been applied in basic research fields related to liver transplantation. But, it has been not reported in research related to reduced-size liver transplantation. OBJECTIVE:To explore the expression of differential proteins related to hepatic energy metabolism fol owing reduce-size liver transplantation in rats by using by proteomic technology. METHODS:The improved model of reduced-size liver transplantation was used in this experiment. The donor was health female Lewis rats and the recipient was male Wistar rats for liver transplantation. The difference between the donor and the recipient was about 20 g. The weight of donor liver/the weight of recipient donor was approximately equal to 50%. The donor liver tissue was harvested and trimmed to the required size. The portal vein and infrahepatic vena cava were cannulated, and the biliary tract was implanted into the donor bile duct for transplantation. Then the donor was transplanted into the recipient after the removal of original liver tissue. Hepatic specimens were harvested by 1, 3 and 7 days after reduced-size liver transplantation. Then, the harvested specimens were compared with the normal donor and recipient liver tissue that were previously harvested and frozen, to generate two-dimensional gel electrophoresis profile using proteome technology. Then tandem mass spectrometry and databases analysis were performed after two-dimensional electrophoresis for identifying differential protein stains. RESULTS AND CONCLUSION:In this experiment, 72 differential protein stains with over lo-fold changes were selected. After identification, 32 proteins showed clear functions, and among them three differential proteins (ATP synthase beta subunit, electron-transferring flavoprotein beta peptide and proton-transferring ATP synthase) were involved in the process of cel energy metabolism. The proteins were distributed on 1 and 7 days after reduce-size liver transplantation, accounting for 6%.
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Abstract BACKGROUND: Looking for the early diagnosis of acute rejection indicators after liver transplantation can assess the risk after liver transplantation quickly and effectively, and T lymphocytes play the significant role in acute rejection. OBJECTIVE:To observe the relationship between acute rejection and variation of expression of T cel subset in blood after liver transplantation in rhesus monkey. METHODS: The sixteen liver transplant models in rhesus monkey which were constructed successfuly by the method of “double-cuff and one support tube” were divided into two groups randomly: experiment group (no treated by immunosuppressant in perioperative period) and control group (treated by immunosuppressant in perioperative period). Then the blood specimen and liver tissue respectively were colected at 6, 12, 24 and 72 hours after operation. The levels of alanine transferase, aspartate aminotransferase, and total bilirubin were detected with the fuly automatic biochemical analyser. The levels of CD4+/CD8+were tested by flow cytometry. The liver tissue in rhesus monkey after liver transplantation was detected by hematoxylin-eosin staining. The degree of acute rejection was evaluated by Banff Score System. RESULTS AND CONCLUSION: Acute rejection appeared in the experiment group at 12, 24, and 72 hours after liver transplantation. Levels of alanine transferase, aspartate aminotransferase, and total bilirubin were significantly higher in the experimental group than in the control group at 24 and 72 hours after transplantation (P < 0.05). The expression of CD4+/CD8+of the experiment group and control group began to rise at 6 hours after surgery, but the experiment group increased the most obvious. CD4+/CD8+ expression was significantly greater in the experimental group than in the control group at 24 and 72 hours after transplantation (P < 0.05). Morphological pathology was severer, and Banff score was higher in the experiment group than in the control group at 72 hours (P < 0.05). These data suggested that the variation of expression of CD4+/CD8+was earlier than the change of liver tissue pathology and the change of liver function in the early acute rejection after liver transplantation. The rise of level of CD4+/CD8+ after liver transplantation indicated the increase of celular immunity in body, which had an important role in the early diagnosis of acute rejection after liver transplantation.
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BACKGROUND:The proteome is a highlight technology in medical research fields lately, and has been reported to be applied in basic research fields related to liver transplantation. However, it has not been heard that the proteome has been used in research related to reduced-size liver transplantation. OBJECTIVE: To study expression of hepatic differential proteins related to signal transduction using proteomics after reduced-size liver transplantation in rats. METHODS:On the basis of successful establishment of rat models of reduced-size liver transplantation, transplanted liver tissues were obtained at 1, 3 and 7 days after transplantation. Postoperative liver tissue and normal donor, receptor liver tissues were subjected to solid pH gradient two-dimensional gel electrophoresis. Two-dimensional gel electrophoresis patterns were set up. Differentialy expressed protein spots were identified using tandem mass spectrometry analysis and database. RESULTS AND CONCLUSION:Seventy-two differential protein stains were found taking 10 times measure. Finaly, 32 proteins with clear functions were identified. Of them, four proteins participated in signal transduction, and they distributed at 3 and 7 days after liver transplantation, accounting for 6%. Results verified that on the basis of successful and stable establishment of rat models of reduced-size liver transplantation, proteomics technology was utilized to study differential proteins involving in signal transduction after reduced-size liver transplantation, and this study provides data for further deep investigation of regulating MicroRNA of these proteins.
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BACKGROUND:Tumor necrosis factor-αis an inflammatory cytokine involved in the immune response and increasing graft antigen expression. OBJECTIVE:To investigate the relationship between tumor necrosis factor-αin the liver tissue and acute rejection after liver transplantation in a rhesus monkey. METHODS:Liver transplant models in rhesus monkey were constructed by the improved vascular dual cuff, supporting tube of biliary tract and artery anastomosis method. The successful models were randomly divided into experimental group (no immunosuppressant treatment in perioperative period) and control group (treated by immunosuppressant in perioperative period). Then the blood samples and liver tissue were col ected at 6, 12, 24, and 72 hours after surgery. Al ograft rejections of liver transplantation were monitored by liver function tests, hematoxylin-eosin staining and Banff score. Final y, the expression level of tumor necrosis factor-αwas detected by western blot analysis and immunohistochemistry technique. RESULTS AND CONCLUSION:The expression of tumor necrosis factor-αin the experimental group and control group began to increase at 6 hours, reached the peak at 12 hours, and then decreased at 24-72 hours. The changes of expression level were the most obvious in the experimental group. At 6, 12, 24 and 72 hours, the expression of tumor necrosis factor-αin the experimental group was significantly higher than that in the control group (P<0.05). This change appeared earlier than pathological changes in the liver and liver function. Variations in the expression of tumor necrosis factor-αafter liver transplantation have important implications for early diagnosis of acute rejection after liver transplantation.
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BACKGROUND:Interleukin-6 is an important cytokine in the immune inflammatory response, strongly links with graft rejection reaction, and plays an important role in diagnosis of graft rejection and evaluation of anti-rejection. OBJECTIVE:To measure the expression of interleukin-6 in acute rejection of the liver transplantation in the rhesus monkey, and to evaluate the value as an early diagnosis of acute rejection after liver transplantation. METHODS:A total of 16 rhesus monkeys were used as the object and randomly divided into experimental group (no treated by immunosuppressant in perioperative period), and control group (treated by immunosuppressant in perioperative period). The al ograft orthotopic liver transplantation models were established in those monkeys. Then serum and liver tissue were col ected at 6, 12, 24, and 72 hours after surgery. Al ograft rejection was monitored by liver function tests, and hematoxylin-eosin staining of liver and Banff score. Final y, the expression levels of interleukin-6 were detected by enzyme linked immunosorbent assay and immunohistochemistry.RESULTS AND CONCLUSION:Acute graft rejection reaction appeared at 12, 24 and 72 hours after liver transplantation in the experimental group. The expressions of alanine aminotransferase, aspartate aminotransferase and total bilirubin were significantly higher in the experimental group than in the control group at 24 and 72 hours (P<0.05). Histological manifestations were severer and Banff score was higher in the experimental group at 72 hours than in the control group (P<0.05). Interleukin-6 levels were significantly higher in the serum and liver tissue of experimental group than in the control group at 12, 24 and 72 hours after liver transplantation (P<0.05), especial y at 72 hours. Results suggested that interleukin-6 possibly participated in rejection after liver transplantation. The expression of interleukin-6 was probably of significance in the early diagnosis of acute rejection after orthotopic liver transplantation in rhesus monkeys.
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Objective To explore the effect of nano-hydroxyapatite/collagen scaffolds incorporating ADM-PLGA microspheres in repairing large bone defects of rabbit femoral condyle. Methods Animal models of bone defects were induced in 21 New Zealand white rabbits by drilling holes in bilateral femoral lateral condyles , and the rabbits were equally divided into 3 groups:group A as the control group with the defects untreated , group B treated by filling with nano-hydroxyapatite/collagen scaffolds (NHAC), and group C treated by filling with the nano-hydroxyapatite/collagen scaffolds incorporating ADM-PLGA microspheres (ADM-PLGA-NHAC). At week 12 after implanting , the rabbits were all sacrificed for the implanted scaffolds , which were then examined by X-ray , and Micro-CT 3D reconstruction and in histology for evaluation of the new bone formation. Results X-ray, Micro-CT and the measurement and analysis of BMD indicated thatthere was no significant differencein the new bone formation between group B and group C (P > 0.05). The histological examination revealed that. 12 weeks after operation an evident number of new born bones were seen on the implanted scaffolds in groups B and C , while very few were seen scattering in group A. Conclusion The nano-hydroxyapatite/collagen scaffolds incorporating ADM-PLGA microspheres is effective in repairing bone defect without influencing the prosthetic process.
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BACKGROUND:Nuclear factor-κB as an important nuclear transcription factor, is a converge point for various signal transduction pathways, and participate in the regulation of reactive substances gene expression such as the immune cel proliferation, differentiation and apoptosis. The nuclear factor-κB plays an important role in humoral and cel ular immune. OBJECTIVE:To investigate the relationship between the nuclear factor-κB p65 protein expression and acute rejection in transplanted liver tissue of rhesus monkey. METHODS:The rhesus monkey recipients were randomly divided into two groups:acute rejection group and control group. The acute rejection group did not received anti-rejection treatment after liver transplantation, and the control group was given anti-rejection treatment during and after liver transplantation. The blood samples were col ected at 6, 12, 24 and 72 hours after transplantation, and the automatic biochemical analyzer was used to detect the levels of alanine aminotransferase and total bilirubin. Hematoxylin-eosin staining of transplanted liver tissue was performed to observe the morphological structure and rejection. The degree of rejection was evaluated according to the Banff scoring system, and the expression of nuclear factor-κB p65 in the liver tissue was detected with Western blot. RESULTS AND CONCLUSION:When the acute rejection occurred after liver transplantation, the liver function change was observed after liver histopathological examination, the expression of nuclear factor-κB p65 in the liver tissue was up-regulated, and the degree of acute rejection was increased. In the early stage of acute rejection, the liver function and pathology were changed slightly, while the expression of nuclear factor-κB p65 was significantly increased. The results indicate that the detection of nuclear factor-κB p65 in the transplanted liver tissue has great significance for the early diagnosis of acute rejection after liver transplantation, and the nuclear factor-κB may be the new target for control ing the acute rejection.
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BACKGROUND: Various factors contribute to the establishment of liver transplantation models in rhesus monkey, the rate of successful operation and long-term survival are very low. OBJECTIVE: To analyze the cause of early death following liver transplantation in rhesus monkey. METHODS: Liver transplantation models were fabricated with the classical and modified methods in rhesus monkeys. Operation of donor was performed quickly by a big crucial incision of abdomen. The improved double-cuff of the portal vein and inferior vena cava were finished, in addition to stay pipe of biliary tract in the process of repairing donor liver. Operation of the receptor was performed by classical orthotopic liver transplantation. RESULTS AND CONCLUSION: A total of 25 pairs of rhesus monkeys were successfully for establishing liver transplantation models. Seven rhesus monkeys died within early stage of post-operation, including six out of nine monkeys died by using the classical approach and one out of sixteen monkeys died by using the improved approach. There were five of seven monkeys died of intra-abdominal hemorrhage, one died of primary graft nonfunction and one died of respiratory failure. Results indicated that, the major death cause after classical orthotopic liver transplantation in rhesus monkey is abdominal hemorrhage. The improved methods of liver transplantation apparently reduce the hemorrhage and raise early survival rate following liver transplantation.