Objective:To investigate the specific interference of OX40 gene expression induced by RNAi technique in 293T cell lines transfected with rat OX40 gene.Methods:293T cells were transfected with recombined plasmid pEGFP-N1-GFP/OX40,and the positive cell clones were selected by fluorescence protein observation and RT-PCR.One specific dicer siRNA targeted to OX40 mRNA was designed and synthesized,which shared no homology with exons of known human gene.Quantitative real-time PCR was performed to measure the inhibitory rate of target gene expression by comparing OX40 mRNA concentrations before and after siRNA transfection.Results:10 nmol/L siRNA-OX40 elicited the highest level of gene silence in 293T cells which was transfected with siRNA after 48 h (68.3?8.7)%);The time of maximal inhibitory effect was at 48-72 h [(61.7?8.4)%,(39.6?5.6)%].Conclusion:The exogenous OX40 expression can be significantly inhibited by treatment with specific siRNA in a dose and time -dependent manner in 293T cells,which may provide a useful profile for further investigation of inhibition of OX40 protein,and a promising control approach for preventing immune reaction.

Objective To summarize the clinical experience of liver transplantation in the treatment of hepatocellular carcinoma(HCC).Methods From June 2004 to March 2007,70 consecutive HCC patients underwent liver transplantation in our hospital,including classic orthotopic liver transplantation in 41 cases,and piggyback liver transplantation in 29 cases.All data of patients were retrospectively analyzed.Results All liver transplantation were successfully conducted.The average warm ischemia duration was 4.5 minutes,and cold ischemia duration was 8 hours.There were 3 cases of postoperative deaths.Surgical complications were intra-abdominal hemorrhage in 2 cases,and biliary anastomotic stricture in 4 cases.Sixty-seven cases were followed up for 12-33(average 21) months,10 cases had recurrence of liver cancer after transplatation and 1 dead.Conclusions Liver transplatation can used on selected patients with HCC.Successful liver transplantation relies on good quality of liver graft,and idealized technique of vascular and bile duct reconstruction are key factors of liver transplantation.Proper postoperative management can effectively reduce the complications of operation.

Objective To investigate the effect of blockading OX40-OX40L co-stimulatory signaling on the survival time of liver allograft in rat.Methods siRNA-expression vectors were constructed to targeting OX40.3～5 minutes before DA to Lewis orthotopic liver transplantation was performed,5×109 pfu of targeting OX40 siRNA plasmid DNA were diluted in 5 ml of phosphate buffered saline(PBS)and inlected intravenously into recipient Lewis rat over a period of 10 seconds.Serum IL-2 and IFN-γ levels were assayed by ELISA,and mix lymphocyte response(MLR)were tested by 3H-thymidine.Results The survival time of recipients in siRNA treatment group(74.0±9.3)was significantly longer than that in control group[(7.3±0.5)days].In experiment group,the inflammatory cell infihration and liver tissue structure destruction were very slight.The concentration of serum IL-2 was much lower in siRNA treatment group[(46±8.4)pg/ml]than that in control group[(286.5±14.6)pg/ml].Meanwhile,the concentration of serum IFN-γ was much lower in siRNA treatment group [(202.7±14.6)pg/ml]than that in control group[(1682.7±87.9)pg/ml].Conclusion Administration of OX40-siRNA can blockade OX40-OX40L co-stimulatory signaling pathway.hence inhibit the rejection of liver allograft.

Hilar cholangiocarcinoma (HCC) is a rare tumor with a poor prognosis.With the development of high definition imaging technology,improvement of surgical instruments,optimization of perioperative surgical strategies and accumulation of surgical experiences,the radical resection rate of HCC is significantly improved.Operation is the main method of treatment for HCC,and radical resection is important for a long-term survival of HCC patients.The clinical data of 66 patients with HCC who were admitted to the Beijing Youan Hospital from April 2004 to April 2012 were retrospectively analyzed.The key points in surgical procedure and prognosis of patients were investigated.

Objective To investigate technical skills on outflow reconstruction in right lobe graft adult-adult living donor liver transplantation for avoiding of venous congestion. Methods The clinical data of 21 donors and recipients who underwent right lobe living donor liver transplantation were analyzed retrospectively. Donor's standard liver volume was between 1150. 1 and 1629. 8 cm3,graft weight was between 585 and 920 g, the ratio of graft volume to recipient's estimated standard liver volume (GV/ESLV) was between 43 % and 67 %, graft-recipient weight ratio (GRWR) was between 0. 82 % and 1.59 %, the ratio of remnant liver volume to donor's standard liver volume(RLV/SLV) was between 32 % and 55 %, all graft macrosteatosis was less than 10 %. For graftwith middle hepatic vein (MHV), a triangle large orifice was made by joining MHV to right hepatic vein (RHV), then anastomosed to recipient' s enlarged orifice of RHV. For graft without MHV, if tributary of MHV＞5 mm, autologous or allogenic blood vessel was used as interposition graft to connect to IVC, and if no large MHV tributary, graft RHV was anastomosed to IVC directly. Graft's right portal vein was anastomosed to main trunk of recipient's portal vein, graft's right hepatic artery to recipient's hepatic artery, and graft's right hepatic duct to recipient's right hepatic duct. Results Among the 21 right lobe grafts, 4 right lobe grafts had MHV, 17 right lobe grafts had no MHV.Autologous greater saphenous veins were adopted in 2 cases, cryopreserved iliac arteries were adopted in 5 cases, and RHV was anastomosed directly to IVC in 10 cases. Outflow was all patent in 7 cases having reconstruction of MHV tributaries one month after operation. One-year survival rate was 75 %, 85. 7 % and 70 % respectively in MHV group, MHV tributaries reconstructed group and RHV directly anastomosed to IVC group with the difference being not significance among these three groups (P＞0. 05). Biliary complications occurred in 7 cases during the follow-up period. One case developed small-for-size syndrome, which was cured by splenic artery embolization. No severe complication occurred in donors. All donors returned to normal life during a follow-up period of 6 to 31 months. Conclusion If outflow tract was reconstructed properly, right lobe graft without MHV has equivalent clinical outcomes to right lobe graft with MHV. Using of autologous or allogenic blood vessel as interposition vessel graft for right lobe graft without MHV is an effective modality to prevent hepatic congestion and secure functional graft volume to meet recipients metabolic demand.

Objective To compare the curative results of three different therapies for earlyintermediate stage primary liver cancer. Methods The data of 428 patients with early-intermediate stage primary liver cancer treated with one of three curative therapies from April 2004 to July 2010 in our center were analyzed retrospectively. The patients were divided non-randomly into three groups: group A liver-cancer resection (n = 231), group B radio-frequency ablation (RFA) (n = 63), and group C liver transplantation (n=134). The 1-, 3-, 5-year accululative survival and recurrence rate in each group were compared. Results The accumulative 1-, 3-, 5-year survival rates were 93.3%, 71.9%, 57.2% for group A; 86.7%, 46.5%, 38.8% for group B; 95.7%, 78.3%, 72.1% for group C,respectively. The 1-, 3-, 5-year recurrence rates were 30. 3% , 49. 7%, 68. 6% for group A; 39. 3% , 58. 7% , 79. 3% for group B; 7. 0% , 12. 1% , 12. 1% for group C,respectively. There was a highly significant difference between groups A, B and C in the survival rates and the recurrence rates. The 5-year survival rate was significantly higher for group C than group A and group B (P<0. 01, P<0. 001), and the recurrence rate of 1, 3, 5-years were significantly lower for group C than for group A and B (P<0. 001). Conclusion Liver transplantation was the most effective therapy for the early-intermediate stage primary liver cancer.

Objective To evaluate the safety of programmed cell death protein 1 (PD-1) inhibitor in the treatment of primary liver cancer (liver cancer) before liver transplantation. Methods Clinical data of 7 recipients given with PD-1 inhibitor before liver transplantation for liver cancer were retrospectively analyzed. The incidence of immune-related adverse event (irAE) and clinical prognosis of the recipients were summarized. The safety of PD-1 inhibitor in recipients prior to liver transplantation for liver cancer was evaluated. Results Seven recipients were treated with PD-1 inhibitor with 1-20 courses before liver transplantation for liver cancer. The time interval from drug withdrawal to liver transplantation was 6-120 d. Five recipients suffered from irAE of different degrees, including fatigue in 3 cases, fever in 2 cases, alopecia in 2 cases, rash in 1 case, nausea in 1 case and myocarditis in 1 case, respectively. A majority of these irAE were classified as grade Ⅰ-Ⅱ. One recipient died from grade Ⅴ irAE (fatal myocarditis). One recipient developed rejection at postoperative 7 d, which were mitigated after glucocorticoid pulse therapy combined with increased dosage of tacrolimus. Conclusions PD-1 inhibitor can be applied in preoperative treatment before liver transplantation for liver cancer. Nevertheless, the incidence of irAE and postoperative rejection should be intimately monitored.

Objective To evaluate the influence of hepatitis B surface antigen positive or antihepatitis B core positive donors on HBV allograft re-infection or de novo hepatitis B and recipients and grafts survival after liver transplantation.Methods Between June 2004 and December 2011,510 liver transplants were performed at our department while 387 patients were followed up.Among them,9 patients received hepatitis B surface antigen positive grafts,50 patients received anti-hepatitis B core positive grafts,and 328 patients received HBV marks negative grafts.The rate of HBV allograft reinfection or de novo hepatitis B and accumulative recipients as well as grafts survival were compared.Results All recipients with hepatitis B surface antigen positive donors remained hepatitis B surface antigen carriers after operation.HBV allograft re-infection occurred in one recipient of anti-hepatitis B core positive donor group. Five recipients with HBV marks negative donors appeared hepatitis B surface antigen positive,including two cases of Lamivudine resistance leading to HBV allograft reinfection and three cases of de novo hepatitis B from non-related diseases. The 1-,3-,5-year accumulative survival rate in anti-hepatitis B core positive grafts group,hepatitis B surface antigen positive grafts group and HBV marks negative grafts group was 100％,86％,43％; 87％,79％,57％; and 87％,80％,79％,respectively (Log-rank =1.287,P =0.525).And the 1-,3-,5-year accumulative grafts survival rate in these three groups was 100％,86％,43％; 85％,77％,56％;and 86％,79％,77％,respectively (Log rank=1.288,P =0.525).During the follow-up period,no graft loss or death was found to be related to the HBV allograft re-infection or de novo hepatitis B.Conclusion Liver grafts from anti-hepatitis B core positive donors do not increase the risk of graft loss or recipient death due to HBV allograft re-infection or de novo hepatitis B under effective antiviral therapy.Hepatitis B surface antigen positive donors are feasible to save lives or prolong life in emergency situation.

Objective To investigate the correlations between expression of CASC2 and hepatocellular carcinoma(HCC) prognosis.Methods A total of 129 patients including 80 males and 49 females with HCC were includedin this study,ranging from 21 to 73 years in Xuanwu Hospital of Capital Medical University and Beijing You'an Hospital were retrospectively analyzed from September 2007 to January 2014.Expression of CASC2 was assessed using reverse transcription quantitative-polymerase chain reaction in HCC tissue and the adjacent normal tissue.The correlations between CASC2 mRNA level and clinicopathological parameters was investigated.The relationship between the expression of CASC2 and the prognosis of patients with HCC was analyzed by Kaplan-Meier method.A log-rank analysis was performed to identify group differences.Univariate and multivariate Cox analysis were used to analyze the variables affecting the patient's prognosis.Results In 129 HCC samples,the level of CASC2 expression (0.84 ± 0.05) was lower than (3.35 ± 0.11) adjacent normal tissue (P ＜ 0.05).There were significant differences between CASC2 expression and tumor size,histological differentiation,and tumor stage in 129 HCC speciments.The median expression level of CACS2 in HCC tissues,0.84-fold,was used as the cut-off value to divide the 129 patients into two groups:low-expression group (n =72) and high-expression group (n =57).Overall survival rate of HCC patients with high CACS2 expression was significantly higher than those of patients with low CACS2 expression(P ＜0.05).Multivariate analysis indicated that histological differentiation (HR =0.20,95％ CI:0.05 ～ 0.59),tumor stage (HR =1.71,95％ CI:1.02 ～ 2.99) and CACS2 expression (HR =O.51,95％ CI:O.08 ～0.92) were an independent predictor of overall survival.Conclusion Low expression of CACS2 might be associated with the occurrence and development of HCC.

Objective:The study aimed to study the efficacy and safety of combined dual therapy using anti-programmed death (PD)-1 and tyrosine kinase inhibitor (TKI) with combined triple therapy using anti-PD-1, TKI and locoregional intervention triple therapy in patients with postoperative refractory recurrent liver cancer.Methods:Patients with postoperative refractory recurrent liver cancer who had undergone either anti-PD-1 and TKI dual therapy or anti-PD-1, TKI and locoregional intervention triple therapy between July 2016 and March 2019 at the First Medical Center, Chinese PLA General Hospital were retrospectively studied. Tumor responses were assessed by the modified response evaluation criteria in solid tumors and overall survival and progression free survival were compared. Adverse events were evaluated according to the National Cancer Institute Common Terminology Criteria for Adverse Events.Results:Of 63 patients who were included in this study, there were 25 patients in the dual therapy group (16 males and 9 females, aged 54.3±8.8 years) and 38 patients in the triple therapy group (31 males and 7 females, aged 55.5±8.4 years). The 1-year survival rate of the triple therapy group was significantly higher than the dual therapy group (94.5%vs 54.9%) ( P<0.01). The disease control rate was 64.0% (16/25) in the dual therapy group and 84.2% (32/38) in the triple therapy group, and the difference was not significant ( P>0.05). The incidence of treatment-related adverse events in the triple therapy group and the dual therapy group were 78.9% (30/38) and 80% (20/25), respectively. There was no treatment-related death in the 2 groups. Conclusions:Anti-PD-1 and TKI dual therapy and anti-PD-1, TKI and locoregional intervention triple therapy were effective and tolerable treatments for postoperative refractory recurrent liver cancer. The latter treatment had a significantly better clinical benefit on survival outcomes.