Objective:To investigate the application value of hepatic vein outflow tract reconstruction with ringed polytetrafluoroethylene vascular in right lobe living donor liver trans-plantation.Methods:The retrospective and descriptive study was conducted. The clinicopatho-logical data of 4 donors and 4 recipients undergoing right lobe living donor liver transplantation in Nanjing Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School and 17 donors and 17 recipients undergoing right lobe living donor liver transplantation in the First Affiliated Hospital with Nanjing Medical University from June 2015 to August 2018 were collected. Of 21 donors, there were 10 males and 11 females, aged from 35 to 57 years, with a median age of 46 years. The median body mass of 21 donors were 64 kg, with a range from 56 to 72 kg. Of 21 recipients, there were 16 males and 5 females, aged from 21 to 68 years, with a median age of 42 years. The median body mass of 21 recipients were 63 kg, with a range from 47 to 77 kg. Observation indicators: (1) surgical and postoperative situations; (2) follow-up. Follow-up was conducted by outpatient examination or telephone interview to detect graft function, tumor recurrence, vascular graft complications, patency of vascular graft and survival of recipients up to August 2020. All recipients will be followed up for all their lives. Measurement data with normal distribution were represented as Mean±SD and measurement data with skewed distribution were represented as M (range). Count data were represented as absolute numbers or percentages. The Kaplan-Meier method was used to calculate patency rates of hepatic vein outflow tract and survival rates to draw patency curve and survival curve. Results:(1) Surgical and postoperative situations: the operation time, the weight of donor graft, graft to recipient weight ratio and duration of hospital stay of 21 donors were (367±72)minutes, (557±68)g, 0.89%±0.16% and (10+2)days, respectively. No major complication requiring reoperation or intervention occurred in any of the 21 donors. One donor undergoing mild bile leakage preserved peritoneal drainage for one week. All 21 recipients underwent classic orthotopic liver transplantation successfully. The time of hepatic vein outflow tract reconstruction in donor graft, operation time and time of anhepatic phase of 21 recipients were (24±4)minutes, (326±66)minutes and (42±6)minutes, respectively. The number of reconstructed middle hepatic vein in hepatic segment 5 and 8 were 18 and 15, with the diameter of (6.1±1.3)mm and (7.2±1.2)mm, respectively. The number of reconstructed inferior right hepatic vein were 10, with the diameter of (6.3±1.3)mm. The postoperative treatment time at intensive care unit and duration of hospital stay of 21 recipients were (1.5±0.9)days and (22.6±6.7)days, respectively. Ten of 21 recipients underwent postoperative complications. Five recipients underwent graft dysfunction including the level of alanine aminotransferase and aspartate aminotransferase >1 000 IU/L and the level of bilirubin slightly increasing, combined with increased ascites. Enhanced computed tomography scan showed congestion in the right anterior of graft and thrombosis in the middle hepatic vein of hepatic segment 5 and segment 8. All 5 recipients undergoing graft dysfunction recovered with normal liver function and ascites decreasing after symptomatic treatment including liver protection therapy, anticoagulation and albumin infusion. Two recipients underwent inferior vena cava thrombosis and intractable pleural effusion one month after operation. Vena cava venography examination showed thrombosis in the graft vascular. Of the 2 recipients, one case with collateral circulation formation recovered undergoing balloon dilatation and stent placement combined with anticoagulation therapy of warfarin. The other one case recovered after anticoagulation therapy of warfarin. One recipient undergoing bile leakage and abdominal infection with klebsiella pneumoniae recovered after symptomatic treatment. Two recipients undergoing abdominal infection or pulmonary infection recovered after symptomatic treatment. There was no serious complication or death during perioperative period. (2) Follow-up: all 21 recipients were followed up for 10 to 57 months, with a median follow-up time of 38 months. During the follow-up, no recipient underwent graft dysfunction and 2 recipients had tumor recurrence at postoperative 6 months. Six of the 21 recipients died within 2 years after operation including 3 cases dying of tumor recurrence, 2 cases dying of acute hemorrhage and 1 case dying of liver failure. There was no death caused by vascular graft complica-tions. The postoperative 1, 3, 6-month, and 1-year and 2-year potency rates of hepatic vein outflow tract in 21 recipients were 88.4%, 88.4%, 82.4%, 68.0% and 42.1%, respectively. The 6-month, 1-year and 2-year overall survival rates in 21 recipients were 100%, 94.4%, 71.4%, respectively.Conclusion:Application of hepatic vein outflow tract reconstruction with ringed polytetrafluoroethylene vascular in right lobe living donor liver transplantation is safe and feasible.

objective To investigate preoperative donor and recipient assessment,choice of surgical options in living donor liver transplantation(LDLT).Methods The clinical data of 95 patients who underwent LDLT from January 1995 to October 2007 in our center were retrospectively analyzed.Of all,92 recipients were benign end-stage liver disease patients (including 45 patients with Wilson disease),and 3 were malignant hepatic carcinbma patients.Results Thirty-one fight lobes without middle hepatic vein(MHV),3 right lobes with MHV,51 left lobes with MHV.and 10 left lobes or left lateral lobes without MHV were obtained.All the donors recovered after operation. Recipients with benign end-stage liver disease were followed up for 1 to 86 months,and the 1-,3-,5-year accumulative survival rates were 89%(82 cases),78%(71 cases)and 73%(67 cases),respectively. The 1-,3-,5-year survival rates of patients with Wilson disease were 92%(42 cases),89%(40 cases)and 76%(34 cases),respectively. For the 3 patients with malignant hepatic carcinoma,2 died and 1 was alive and well. The copper metabolism was back to normal in both donors and recipients. Conclusions Establishment of a system for the safety of donors is basic for LDLT. The key to raise the recipients' survival rates is to choose the optimal surgical approach. LDLT is effective in treating Wilson disease.

AIM: To clone EBV-LMP2A gene, construct and identify the recombinant retroviral vector and stable cell strains expressing EBV LMP2A. METHODS: The full-length EBV LMP2A gene was generated by RT-PCR amplification from B95.8 cells which contain complement nucleotide sequence of EBV LMP2A gene. The gene was ligated to T-vector and sequenced to construct retroviral vector consisting with LMP2A. To produce retroviral virus, packing cells, 293T cells were co-transfected with recombinant retroviral expression vector pGEZ-LMP2A and two auxiliary viral vectors pHIT456 and pHIT60 by lipofectAMINE2000. Viral titration was performed according to the instructions of the manufacturer. To establish L929 cell line stable expressing LMP2A, L929 cells were infected with recombinant retrovirus three times and selected by Zeocine. The Zeocine-resistant clones (L929/LMP2A) were screened for LMP2A expression by RT-PCR and Western blot. RESULTS: The recombinant retrovirus vector carrying LMP2A gene was constructed successfully. Transfection yield a titer of 5×10~8 infectious particles/L. The infected L929 cells were selected by Zeocine. Results of RT-PCR and Western blot indicated that L929 transgenetic cells could stably express EBV-LMP2A. CONCLUSION: The L929 cell line stably expressing LMP2A provides suitability for extraction of the LMP2A protein and preparations of the vaccine for the therapy of EBV-associated diseases.

Primary liver cancer, especially hepatocellular carcinoma, poses a serious threat to the life and health of the Chinese people. Given the insidious onset of liver cancer, less than 30% of hepatocellular carcinoma patients are considered for radical treatment at the initial diagnosis. Systemic anti-tumor therapy plays an important role in the treatment of advanced hepatocellular carcinoma. Immunotherapy of hepatocellular carcinoma has developed rapidly, and an increasing number of immunotherapy drugs, which can better control the progress of hepatocellular carcinoma and prolong the survival of patients, have become first- and second-line treatment options. This article reviews briefly the progress of immunotherapy for hepatocellular carcinoma in recent years.

OBJECTIVETo sum up the clinical experience of liver transplantation.

METHODA retrospective study was made in 11 patients receiving living donor liver transplantation (LDLT)/and 14 patients having orthotopic liver transplantation (OLT), including one time operation of reduced size liver retransplantation and one time operation of cadaveric liver retransplantation.

RESULTSThe voluntary donors were a sister and 10 mothers of recipients. The location of graft included 3 patients of segment II, III, part of IV (not including intermediate hepatic veins), 6 patients of segment II, III, IV (including intermediate hepatic veins), and 2 patients of V, VI, VII, VIII (not including intermediate hepatic veins). The weight range of graft was 270 - 620 g. Twenty-four recipients achieved a long-term survival and retained normal liver function during the follow-up. Only 1 patient died from serious rejection on the 72nd day postoperatively. Ten patients with hepatitis B cirrhosis were treated with lamivudine and anti-HBVIg, and HBV-DNA in serum was negative during the follow-up for 4 approximately 21 months. Copperoxidase, ceruloplasmin and main indexes of liver function became normal in all patients with Wilson's Disease. Postoperative complications included abdominal hemorrhage (2 patients), acute respiratory distress syndrome (5), acute rejection (4), and acute renal function failure (2).

CONCLUSIONSThe wise solution to improve the result of liver transplantation and optimize liver resources is the "multimodal approach", by which all kinds of techniques for liver transplantation including CLT, LDLT and RSLT should well developed.

Adolescent ; Adult ; Child ; Female ; Humans ; Liver Transplantation ; adverse effects ; methods ; Living Donors ; Male ; Middle Aged ; Reoperation

Objective To investigate the role of Glutathione peroxidase 2(GPX2)in the occurrence and progres-sion of intrahepatic cholangiocarcinoma(ICC).Methods The Omicshare website was used to analyze GPX2 ex-pression levels in ICC.The expression levels in ICC were validated using quantitative real-time reverse transcription PCR(RT-qPCR),Western blot,and immunohistochemistry.Stable GPX2 knockdown and overexpression HuC-CT1 cell lines were constructed.The effects of GPX2 on ICC cell proliferation,migration,apoptosis,and epithelial-mesenchymal transition(EMT)were investigated using colony formation assays,cell counting kit-8(CCK-8)as-says,wound healing assays,Transwell assays,and flow cytometry.A mouse model of cholangiocarcinoma was con-structed to assess GPX2 expression in mouse cholangiocarcinoma tissues.Results Based on the analysis results from the Omicshare website,GPX2 was generally upregulated in intrahepatic cholangiocarcinoma(ICC)(P＜0.001).Western blot(P＜0.000 1),RT-qPCR(P＜0.001),and immunohistochemistry experiments showed that,compared to adjacent non-cancerous tissues,the expression of GPX2 was significantly elevated in ICC.When GPX2 was knocked down,the colony formation rate of cells decreased significantly(P＜0.01),and the prolifera-tion capacity was reduced(P＜0.001).Conversely,overexpression of GPX2 led to a significant increase in colony formation rate(P＜0.01)and enhanced proliferation capacity(P＜0.01).Results from wound healing and Tran-swell assays demonstrated that GPX2 knockdown slowed down cell wound healing(P＜0.01)and reduced migra-tion ability(P＜0.01).Additionally,GPX2 knockdown resulted in an increase in E-cadherin(P＜0.01)and a decrease in N-cadherin(P＜0.01)and Vimentin(P＜0.05).On the other hand,overexpression of GPX2 accel-erated wound healing(P＜0.05)and enhanced migration ability(P＜0.05),while E-cadherin expression de-creased(P＜0.05)and N-cadherin(P＜0.01)and Vimentin(P＜0.001)expression increased.Flow cytometry for apoptosis and Western blot experiments indicated that GPX2 knockdown increased the apoptosis rate(P＜0.001),decreased the expression of Bcl-2(P＜0.001),and increased the expression of BAX(P＜0.01).But overexpression of GPX2 reduced the apoptosis rate(P＜0.01),increased Bcl-2 expression(P＜0.000 1),and decreased BAX expression(P＜0.001).Finally,elevated levels of GPX2 were observed in a mouse model of cholangiocarcinoma.Conclusion GPX2 is highly expressed in human and mouse cholangiocarcinoma tissues,and it may enhance cholangiocarcinoma cell proliferation and migration,promote tumor cell EMT,and inhibit tumor cell apoptosis.

Objective To investigate the expression of urocanase domain containing 1(UROC1)in Hepatocellular carcinoma(HCC)and its effect on the development of HCC.Methods UROC1 expression and prognostic data in tumor and para-tumor tissues from protein mass spectrometry and TCGA database were analyzed.Immunohistochem-ical staining,real time quantitative polymerase chain reaction(qPCR)and Western blot experiments were utilized to verify the expression of UROC1 in HCC.The effect of UROC1 expression level on tumor differentiation was ana-lyzed by HE staining to determine the tumor differentiation level.Hep3B and LM3 cell lines stably overexpressing UROC1 and its mutants were constructed for in vitro phenotyping experiments.Effects of UROC1 on HCC cell pro-liferation and migration were explored by cell counting kit-8(CCK-8)assay,colony formation,EdU assay,scratch assay and Transwell assay.Results The results of database analysis showed that UROC1 was generally downregu-lated in HCC tissues,and patients in the UROC1 low-expression group had a worse prognosis.Immunohistochemi-cal staining and scoring,qPCR and Western blot experiments verified the low expression of UROC1 in HCC.Im-munohistochemical staining of tumor tissues with different differentiation levels demonstrated that the poorer the dif-ferentiation of HCC tissues,the lower the expression level of UROC1.CCK-8,colony formation and EdU assays suggested that overexpression of UROC1 inhibited the proliferation of HCC cells.The results of scratch and Tran-swell assays showed that overexpression of UROC1 inhibited the migration of HCC cells.However,the results of the above experimental phenotypes after active site mutation converged with those of the control group.Conclusion UROC1 is lowly expressed in HCC tissues,and overexpression of UROC1 in HCC cells may inhibit the ability of cell proliferation and migration.

Objective To investigate the effect of N-myc downstream regulatory gene 1(NDRG1)on Hepatocellu-lar carcinoma(HCC)and whether NDRG1 affects the sensitivity of HCC to Sorafenib.Methods The expression level of NDRG1 in HCC was predicted by TCGA database,and verified by Western blot(WB)and Immunohisto-chemistry(IHC).NDRG1 knockout cell lines were constructed,followed by cell counting kit-8(CCK-8),EdU,cell scratches and Transwell experiment to investigate the effects of NDRG1 and its combination with Sorafenib on the proliferation,migration and invasion of HCC cells.In addition,flow cytometry was used to detect the apoptosis of HCC cells.The effect of NDRG1 and Sorafenib on HCC tumor formation was studied in vivo by subcutaneous tumor bearing in nude mice.WB and IHC were used to determine the pathway regulating the sensitivity of HCC to Sorafenib.Results WB and IHC confirmed that NDRG1 is highly expressed in HCC,consistent with the results of TCGA data.Tumor functional experiments showed that NDRG1 knockout or Sorafenib stimulation weakened the proliferation,migration,and invasion ability of HCC cells,and increased tumor cell apoptosis.However,NDRG1 knockout combined with Sorafenib further weakened the proliferation,migration and invasion ability of HCC cells,and further increased tumor cell apoptosis(P＜0.000 1).Mouse subcutaneous tumor model showed that NDRG1 knockout or Sorafenib stimulation reduced the tumor volume and quality,while NDRG1 knockout combined with Sorafenib further reduced the tumor volume and quality(P＜0.000 1).The WB and IHC results indicated that NDRG1 knockout combined with Sorafenib could reduce the phosphorylation level of Erk1/2.Conclusion NDRG1 is highly expressed in HCC,which promotes the proliferation,migration and invasion of HCC cells,and restricts apoptosis.NDRG1 knockout enhances HCC sensitivity to Sorafenib by reducing ERK signaling pathway phosphorylation.

Objective To investigate the mechanism by which human hypoxia up-regulation 1(HYOU1)regulates pancreatic cancer development.Methods Bioinformatic and immunohistochemical staining analyses of HYOU1 ex-pression level in pancreatic tumor tissues,normal and paracancerous tissues and its correlation with patients'surviv-al.Quantitative real-time PCR(qPCR)and Western blot were used to clarify the expression level of HYOU1 in a number of human pancreatic ductal adenocarcinoma cell lines and a normal human pancreatic ductal cell line.Two cell lines with the highest expression levels of HYOU1,BXPC-3 and Panc-1,were selected to knock out HYOU1 by CRISPR-Cas9,and then cell survival,proliferation and migration of these cells were examined by cell counting kit-8(CCK-8),colony formation assay and wound healing experiment separately,as well as apoptosis was detected by flow cytometry.Subsequently,the protein levels of the PI3K/Akt signaling including PI3K,p-PI3K,Akt,and p-Akt were detected by Western blot in parental and HYOU1-ablated BXPC-3 and Panc-1 cells.Cell proliferation was also examined in HYOU1-ablated cells after treatment of recilisib,an activator of the PI3K/Akt pathway.Results The expression of HYOU1 in pancreatic tumor tissues was significantly higher than that in normal tissues,and the patients with high expression of HYOU1 had a much shorter survival compared to the patients with low HYOU1(P＜0.01).Immunohistochemical staining of pancreatic cancer specimens showed that the expression of HYOU1 was higher in tumor tissues than in paracancerous tissues(P＜0.01).The mRNA and protein levels of HYOU1 were higher in all pancreatic cancer cell lines compared to the human normal pancreatic ductal cell(P＜0.001,P＜0.01).HYOU1 ablation inhibited BXPC-3 and Panc-1 cells survival,proliferation and migration,and promoted early cell apoptosis.In addition,loss of HYOU1 decreased PI3K/Akt signaling activity,whereas the PI3K/Akt ac-tivator Recilisib reversed the effects of HYOU1 ablation on cell survival and proliferation.Conclusion HYOU1 promotes pancreatic cancer progression by activating the PI3K/Akt signaling pathway.

Liver cancer is the sixth most common type of cancer worldwide and the third leading cause of cancer-related deaths.Approximately 30％of patients are diagnosed at an early stage of liver cancer,at which point cura-tive treatments such as partial liver resection and liver transplantation can be performed,leading to a median overall survival exceeding 60 months.However,most patients are diagnosed at an advanced stage of liver cancer,where treatment options often include targeted therapies and immune checkpoint inhibitors.Systemic treatments including immune checkpoint inhibitors play a crucial role in significantly improving the prognosis of patients with advanced liver cancer.Therefore,in-depth exploration of the immune microenvironment characteristics of liver cancer and ac-tive identification of biomarkers for immunotherapy are crucial to advancing and improving immunotherapy for liver cancer.