1.ImmunologicalProtectiononLiverXenotransplantationbyBlockingtheDonorLiverwithSerumofRecipient2.Effects OfAntisenseOliggnucleotidesofPKC-αand10-HCPTontheGrowthandProliferationandApoptosisofHuman Hepatoma Cell Lines HepG₂ And QGY In Vitro And I

【Objective】 In order to search the new way to prevent the the hyperacute rejection (HAR) of liver xenotransplantation. We established an experimental model of orthotopic liver xenotransplantation from guinea-pig (GP)-to- Sprague-Dawley (S.D.) rat, and observeed the effect on HAR through blocking the xenograft with recipient blood serum before transplantation, and explored the possible mechanism of immunological protection on liver xenotransplantation. The results would provide evidence for clinical liver transplantation about the preventive strategy of rejection. 【Methods】 â‘  Twenty SD rats were pair-matched between donor and recipient randomly. The models of rat orthotopic liver transplantation were reproduced using the modified two-cuff technique, and hepatectomys were performed on another ten rats as a control. â‘¡ Ten GPs and thirty SD rats were matched randomly. The models of orthotopic liver xenotransplantation from GP-to-Rat was established in the same way above, and orthotopic liver transplantations from rat to rat were operated as a control. â‘¢ Before transplantation, the blocking solution was dispensed by the way as follows. The rat blood serum was collected by the tube intubated into abdominal aorta and inactivated at 45℃ in water bath for 30 minutes, and mixed with Ringer solution, the GP blood serum was prepared in the same way as a control. Their concentrations were controlled at 0.1% and were kept in icebox at 4℃. Twenty GPs and rats were choiced respectively and pair-matched between GP donor and rat recipient randomly. The donor livers were preperfused with Ringer solution contained 12.5u/ml of heparin at first, and perfused again for 5 minutes with blocking serum prepared above. Then orthotopic liver xenotransplantations were performed immediately. The effect of blocking the liver xenograft with recipient blood serum on HAR was evaluated by observing the time and intension of rejction, and mortality and survival of recipients. â‘£ Donor livers of thirty GPs were perfused with Ringer solution, then blocked by 0.5% SD rat serum inactivated and not inacvited respectively, Ringer solution as a control. The livers pretreated above were collected and their histopathological patterns were detected in order to observe the effect on donor liver tissue in vitro. Thirty livers of GPs were preconditioned by the same way above, then transplanted into thirty rat recipients randomly. HAR was observed immediately and the livers were collected at once for histopathological examination. The differences of hepatic histopathology among different experiments respectively in vitro and in vivo were valued in order to deduce their possible mechanism of immunological protection on liver xenotransplantation. 【Results】① The models of rat orthotopic liver transplantation were reproduced successfully, the time without liver could be controlled within 23～25min,the survival rates at different time amoung 0 hour, 24 hour, and 1 week after transplantation could be 90%, 80% and 60%. However, the rate of control group only survived for about 1¹.5 hours. â‘¡ The model of orthotopic liver xenotransplantation from GP-to-Ratwas established successfully. The survival rate of recipient after liver xenotransplantation from GP to rat was 80%. The liver became dark purple, inadequate reperfusion and significant edema immediatly with heart beating faster and spasms after revascularization, and the recipients died about 0.5 to 1hours after xenotransplantation. â‘¢The survival rate and time of recipients from experimental group, which donor liver were blocked with rat serum, were improved significantly, and its histological damages including thrombosis, interstitial bleeding and edema were alleviated markebly compared with that from control group(p<0.01). â‘£ In vitro, no evidence of histological damage the GP donor liver blocked by rat serum inactivated were found, however, there were still more evident histological damage of donor liver blocked by rat serum not inactivated than that of control group (p<0.01). In vivo, the histological damage of liver pretreated by rat serum inactivated and not inactivated above after transplantation was lighter significantly than that of control group (p<0.01). 【Conclution】The model of orthotopic liver xenotransplantation from GP-to-Rat is a useful rodent model for either exploration of mechanisms of immunological responses or examination of the immunosuppressive effects of pharmacological agents. Blocking the donor liver with recipient blood serum could significantly prolong the time and alleviate the intension of hyperacute rejection happened in liver xenotransplantation from GP to SD in some extent. It suggested that there was immune protective effect on liver xenotransplantation in some degree by blocking the donor with recipient blood serum. No significant damage and inadequate reperfusion of liver xenograftwas found after that the GP donor liver was blocked by rat serum in vitro. It indicated that blocking the donor with recipient blood serum was safe to the donor liver. Although blocking the donor with recipient blood serum could not block the HAR completely, the intension of HAR and the degree of immunological damage were alleviated significantly. The reason may be that the classic pathway of complement was blocked because the nature antibody cannot find the spot on the xenograft to bind, however, the alternative pathway of complement could be still activated in another way. The potential mechanism should be further investigated. Our results suggested blocking the donor with recipient blood serum could be a potential strategy to prevent HAR of liver xenotransplantation, and could provide a new idea for the prevention of rejection on clinical liver transplantation.【Objective】In order to search more sensitive pharmacological agents to hepatocellular cancer (HCC), two human hepatoma cell lines QGY and HepG2 were employed to investigate the inhibition of antisense oligonucleotides of PKC-α (PKC-α asODN) and 10-HCPT on their growth and proliferation, and explore the induction of PKC-α asODN on the apoptosis of HepG2 in vitro, then observe the effect of 10-HCPT on the volumes of tumors transplanted by HepG2 in the subrenal capsule of normal immunocompetent mouse. The data would provide evidence for clinical interventional therapy of HCC. 【Methods】The human hepatoma cell lines HepG2 and QGY were cultured and subcultured in RPMI1640 cell culture medium containing 15% fetal bovine serum (FBS) in vitro. â‘  HepG2 cells in exponential growth term were prepared, and different concentrations of PKC-α asODN and a random unspecific sequence of PKC-α (PKC-α rODN) as a control were transfected into HepG2 cells by lipofectin (LP). The cell growth index was detected by MTT colorimetric assay and the clone formation rates of HepG2 and QGY were observed by soft agar assay, respectively. The apoptpsis rate of HepG2 treated with PKC-α asODN was assayed by flow cytometry (FCM). â‘¡ HepG2 and QGY cells in exponential growth term were treated bydifferent concentrations of 10-HCPT for 48 hours, and treated by 100μg/mL of 10-HCPT for different times, respectively. The cell growth index (GI) was detected by MTT colorimetric assay and the clone formation rates of HepG2 and QGY were observed by soft agar assay, respectively. â‘¢ In order to study the effect on the growth of HepG2 in vivo, the tumor model was established by implanting HepG2 cells into the subrenal capsule of KM mice, which were treated by 10-HCPT, saline as a control, through abdominal cavity injection everyday, and the growth of the xenografts was measured on the sixth day after transplantation. 【Results】① The GI of HepG2 transfected by PKC-α asODN with concentrations ranging from 0.10μM to 1.00μM were lower significantly than that of control groups (p<0.05). The clone formation rates of HepG2 transfected by PKC-α asODN from 0.05μM to 1.00μM were lower very significantly than that of control groups (p<0.01), and there were dose-dependent relationship among them. The apoptpsis rates of HepG2 treated with PKC-α asODN from 0.50μM to 1.00μM were significantly higher than that of control group. â‘¡ The GI of HepG2 and QGY cells treated by 10-HCPT with concentration 10³60μg/mL and 30³60μg/mL were significantly lower than that of control groups respectively (p<0.05), and the inhibitive effect increased with concentrations ranging from 30 to 360μg/mL. There was a dose-dependent relationship among them. The effects of 100μg/mL of 10-HCPT on the growth of QGY and HepG2 cells for 24 hours were lower significantly than that of control groups (p<0.05). The inhibition treated for 36⁷2 hours were very significant (p<0.01),and not significant among them (p>0.05).The clone formation rates of QGY and HepG2 of experimental groups were lower significantly than that of control groupsrespectively (p<0.01). â‘¢ The volumes of xenografts treated in vivo by 10-HCPT from 3×10^-3 mg/30g to 9×10^-3 mg/30g weight were smaller than that of control group very significantly (p<0.01). 【 Conclution 】 PKC-α asODN could inhibit the growth and proliferation of HepG2 and induce its apoptosis in vitro, the effect could correlate with blocking PKC-α as one of key cell signal transduction. It indicated that PKC-α plays an impotant role in the progress of HCC. 10-HCPT, as a specific inhibitive agent of Topo-1, could inhibit the growth and proliferation of human hepatoma cell lines QGY and HepG2 effectively in vitro, and decrease the volumes of xenografts of HepG2 significantly in vivo. The data above could provide evidence for the use of 10-HCPT in the clinical interventional therapy of HCC. The finding that blocking the PKC-α and Topo-1 of human hepatoma cell lines could inhibit their growth and proliferation, and induce apoptosis of HepG2, indicated that PKC-α and Topo-1 could be targets for HCC targeted therapy.