Ischemic preconditioning decreases C-X-C chemokine expression and neutrophil accumulation early after liver transplantation in rats

AIM: Polymorphonuclear neutrophil (PMN) plays a major role in liver ischemia/reperfusion injury. Protective effect of ischemic preconditioning (IP) has been confirmed in liver ischemia/reperfusion injury. The purpose of this study was to investigate the effect of IP on C-X-C chemokine expression and PMNs recruitment eady after liver transplantation.METHODS: Male Sprague-Dawley rats were used as donors and recipients of orthotopic liver transplantation (OLT). The donor liver was stored 24 hours in University of Wisconsin(UW) solution at 4 ℃ pre-implantation, IP was done by damp of the portal vein and hepatic artery of the donor liver for 10 minutes followed by reperfusion for 10 minutes before harvesting. The neutrophilic infiltration in liver was quantified using a myeloperoxidase (MPO) assay. Intragrafl: expression of macrophage inflammatory protein-2 (MIP-2) mRNA was investigated with in situ hybridization, The serum levels of MIP-2 and tumor necrosis factor (TNF)-α were also monitored,RESULTS: After liver transplantation without IP, the hepatic MPO increased significantly compared with sham operated group. In IP group, PMN in liver indicated by MPO was reduced significantly. In situ hybridization showed no MIP-2 mRNA in sham group but dramatic expression in hepatocytes in non-IP group. In IP group, MIP-2 mRNA was significantly down-regulated. Similarly, serum MIP-2 and TNF-α levels were significantly elevated in non-IP group and both were reduced in IP group.CONCLUSION: IP might protect graft liver from preservation-reperfusion injury after OLT through down-regulating C-X-C chemokine expression of hepatocytes, and alleviating PMNs recruitment after reperfusion.

Inhibitory effect of antisense vascular endothelial growth factor 165 eukaryotic expression vector on proliferation of hepatocellular carcinoma cells

AIM:To construct antisense VEGF165 eukaryotic expression vector PCDNA3-as-VEGF165 and to study its expression and effect on the proliferation of hepatocarcinoma SMMC-7721 cells.METHODS: VEGF165 cDNA was inserted into polylinker sites of eukaryotic expression vector PCDNA3 to construct PCDNA3-as-VEGF165.Then the vector was transferred into human hepatocarcinoma cell strain SMMC-7721 with cation lipofectamine 2000 mediated methods to evaluate the expression of VEGF protein and the inhibitory effect on the proliferation of hepatocarcinoma SMMC-7721 cells.RESULTS:The detection indicated the presence of VEGF cDNA in normally cultured SMMC-7721 cells by PCR.VEGF mRNA expression was notably decreased in SMMC-7721 cells by RT-PCR after PCDNA3-as-VEGF165 transfection. The expression of VEGF protein was dramatically inhibited (142.01±7.95 vs 1625.52±64.46 pg·ml^-1, P<0.01) 2 days after transfection,which correlated with the dose of PCDNA3-as-VEGF165 gene.VEGF protein was most expressed in PCDNA3 transferred SMMC-7721 cells but few in PCDNA3-as-VEGF165 transferred cells by immunohistochemical staining. The apoptotic rate of hepatocarcinoma SMMC-7721 cells was significantly promoted (17.98±0.86% vs 4.86±0.27%, P<0.01) and the survival rate was notably decreased (80.99±3.20% vs 93.52±3.93%, P<0.05) due to antisense VEGF165 by flow cytometry (FCM). The transfection of antisense VEGF165 gene resulted in the inhibitory effect on the proliferation of hepatocarcinoma cells by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and the death of all hepatocarcinoma cells on day 6 after transfection.CONCLUSION:It is confirmed that antisense VEGF165 can inhibit the expression of VEGF protein, interfere with the proliferation and induce the apoptosis of hepatocarcinoma cells in our study. Antisense VEGF165 gene therapy may play an important role in the treatment of human hepatocarcinoma.

Abdominal aorta transplantation after programmed cryopreservation

AIM:To study the morphologic and cellular immunologic changes after homologous transplantation of the abdominal aorta in rats after programmed cryopreservation (-196℃).METHODS:Abdominal aorta was harvested from anesthetized Spraque Dawley (SD) rats for cryopreservation (group B) or immediate implantation (group A). The survival rates and apoptotic rates of aortic endothelial cells (ECs) were examined. The patency rates, histology and cellular immunologic changes of the abdominal aorta were examined on days 1, 3, 7, 14, 30, 60 after transplantation respectively.RESULTS:The survival rate of ECs after programmed cryopreservation was 90.1±1.79%, about 3.4% lower than that of uncryopreservation (93.5±1.96%). The apoptotic rates of ECs was increased after cryopreservation (7.15% vs 4.86%, P<0.05). The patency rate of group B was significantly higher than that of group A (91.6±12.9% vs62.5±26.2%, P<0.01). CD4/CD8 ratio, TCRαβ and CD11b/CD18 ratio of group B were significantly lower than those of group A (P<0.05). Revivification of the cryopreserved abdominal aorta showed normal adventitia and intact smooth muscle cells.CONCLUSION:Cryopreservation can reduce homologous abdominal aortic antigenecity. Even if without administration of immunosuppressive agents, it is still feasible to implement homologous artery grafting in rats.