Objective An adeno associated virus (AAV)vector targeted to hepatoma cells was constructed in order to be used in the apoptosis inducing gene therapy of liver cancer. Methods Firstly, primers containing specific enzyme cutting sites was designed and used to amplify the alpha fetoprotein promoter(AFP promoter) from human genome; Secondly, the promoter was cloned into the eukyrocyte expressing plasmid pTR UF5,resulting in the recombinant AAV vector plasmid containing the reporter gene( rAAV AFP GFP ); thirdly, blunted ligation was applied to construct the recombinant AAV vector plasmid containing the p53 gene( rAAV AFP p53 ). Results Recombinant adeno associated virus plasmid was constructed successfully that carried human wild type p53 gene under the control of human AFP promoter. Conclusion Theoretically the recombinant adeno associated virus plasmid rAAV AFP p53 we constructed could be used in gene therapy for hepatocellular carcinoma.

ObjectiveTo use MAGE-1 antigen as tumor vaccine for the treatment of hepatocellular carcinoma (HCC).MethodsIrradiated peripheral blood mononuclear cells (PBMCs) pulsed with a MAGE-1 peptide (NYKCRFPEI) were used as antigen presenting cells (APC). The PBMCs from the same patient were stimulated with APCs on every 7th day for 4 times to elicit cytotoxic T lymphocytes (CTLs). The cytotoxicity of CTLs to various kinds of target cells was detected with the method of lactate dehydrogenase (LDH) releasing assay. Results The number of PBMCs increased by 32 folds during 28 days of culture. When effective cells: target cells (E∶T) was 10∶1, CTLs exhibited 62.5% cytotoxicity against autogenous lymphoblasts pulsed with the peptide of MAGE-1 antigen (NYKCRFPEI), 40.25% cytotoxicity against cells of BEL7405, a HCC cell line expressing both MAGE-1 and HLA-A24, compared with 17.88% cytolysis observed against autogenous lymphoblasts, 19.55% against cells of HLE, a HCC cell line expressing MAGE-1, negatively expressing HLA-A24. The cytotoxicity against cells of QGY7701, a HCC cell line expressing neither MAGE-1 nor HLA-A24 was much lower (1.6%). When E∶T was 3.3∶1, the cytotoxicity of CTL against peptide pulsed lymphoblasts was 53.6%, while against autogenous lymphoblasts, cells of HLE and cells of QGY7701 was much lower, 15.6%, 13% and 1% respectively. After 4h culture, most cells of BEL7405 were adhered with several CTL, few cells of QGY7701 were adhere with CTL. ConclusionsMAGE-1 peptide NYKCRFPEI in vitro successfully induced CTL with the ability of specifically killing target cells expressing both MAGE-1 and HLA-A24.

ObjectiveTo evaluate the effect of combined gene transfection with p53,p16 and p21 on the growth of human liver cancer cell lines.MethodsAfter transducing adeno associated virus(AAV) mediated p53, p16 or/and p21 genes to human hepatic carcinoma cell lines HLE,HepG2,QGY 7701,QGY 7703,BEL 7402,SMMC 7721, gene expression and tumor inhibition were studied in vitro and in BALB/c mouse model.ResultsAdeno associated virus mediated p53, p16 or p21 encoding gene could express in BEL 7402 cell line. Each individual type of recombinant AAV effected a significant induction of tumor cell apoptosis both in vitro and in vivo, the rate of apoptotic cells in vitro is about 30% and that of tumor growth inhibition is about 30～44%. And the apoptosis inducing efficiency was the highest after the tumor cell line was transfected by three recombinant AAV simultaneously, with a rate of 56% (in vitro) and 65% (in vivo).ConclusionNot only could all of the exogenous wild type p53, p16 and p21 genes mediated by AAV inhibit the growth of liver cancer cell lines, but also can the efficiency be significantly elevated by combined gene transfection.

0.05). Nine HLA-A2-restricted CTL epitopes of CT10 were found. Conclusion CT10 mRNA is expressed with a high percentage and specificity in hepatocellular carcinomas. The CT10 gene product is potential target for antigen-specific immunotherapy of hepatocellular carcinoma.

Objective To explore the possibility of immune therapy for hepatocellular carcinoma (HCC) with MAGE-3 antigen peptide (FLWGPRALV). Methods The expression of HLA-A2 in HCC patients were examined with microcytotoxicity assay .The mRNA of MAGE-3 genes in the tumor tissues were detected by RT-PCR, the CD8 positive T lympocytes were separated as effector cells with immunobeads from peripheral blood mononuclear cells (PBMCs) of those patients. The irradiated autologous CD8 negative PBMC were pulsed with MAGE-3 peptide as APC. Some effector cells were co-cultured with APC, and some not as the control. After 14 days′ culture with IL-2, the frequency of effector cells secreting IFN-? in response to MAGE-3 peptide were monitored by IFN-? secretion assay. The hepatocellular carcinoma cell line HLE was used as the target cells.ResultsNine HCC patients were HLA-A2 positive in 25 HCC patients, and 3 of them were MAGE-3 mRNA positive. After two weeks′ culture, the effector cells increased by 4-6 folds. The frequency of CD8 positive cells secreting IFN-? in response to MAGE-3 peptide was 22.0% in 3 MAGE-3 mRNA positive patients(n=3),and 0.5% in 6 negative patients( n=6) (?

Objective To investigate the precautionary effect of fibrin glue on postoperative bleeding and bile leakage when used in partial hepatic resection.Methods House-hold rabbit partial hepatectomy model was prepared and 40 rabbits were divided into sham operation(SO) group(n=10),control group(n=10) and protocol group(n=20) randomly.The SO rabbits received laparotomy only,while both the control and protocol group received partial hepatectomy.Fibrin glue was used in the protocol group following electric coagulation therapy while the control group received electric coagulation therapy only.The animals' diet,weight and adverse effects were observed then,and the celiac drainage volume,levels of hemoglobin(Hg),WBC and TBil in celiac outflow and ALT,AST,LDH and TBil in rabbit serum were checked on schedule after operation.Results The average blood loss volume and operation time in the protocol group were(16.0?2.7) ml and(23.7?2.9) min respectively,which were lower than those in the control group((20.8?3.5) ml and(27.3?2.9) min(P

Objective To evaluate the diagnostic procedures and treatment choice of primary malignant tumor of the duodenum.Methods The clinical data of 54 cases with primary malignant tumor of the duodenum at Peking University People's Hospital from 1995 to 2005 were analyzed retrospectively.Resuits Tumors located in the first,second,third and fourth parts in 6 cases(10%),44 cases(82%),2 cases(4%),and 2 cases(4%)respectively,and among them,tumors within papillary area accounted for 86%(38 cases)of all cases.Fifty cases(92%)were of adenocarcinoma,2 cases(4%)of mucinous adenoearcinoma carcinoid and undifferentiated carcinoma for 1 case each(2%)respectively.The main clinical presentations included jaundice,upper abdominal pain,weight loss,abdominal distention,nausea and vomiting.gastrointestinal obstruction and abdominal mass.The accuracy rate of duodenoscopy and ERCP in preoperative diagnosis was 94%,and 78%respectively.Preoperative associated cholecystopathy accounted for 37%.Panceaticoduodenectomy was performed in 38 cases,duodenectonmy in 1 ease,palliative resection of tumor in 9 cases,and tumor was inoperable in 6 cases.Resection rate was 89%,and radical resection rate was 72%.The postoperative 3-and 5-year survival rate was 41%and 22%respectively.Patients after palliative resection died from 3 months to 24 months and all patients who did not undergo a surgery died within 6 months.Conclusions Tumors located in papillary region account for the majority of primary malignant tumors of the duodenum and are mainly of adenocarcinoma.Specific signs on abdominal examination are few.The symptoms of advanced stage are complicated,associated cholecystopathy is relatively frequent.Endoscopy and ERCP examination are the main diagnostic tools.the pancreatoduodenectomy is the first choice of therapy for patients with primary duodenal carcinoma.

Objective To evaluate the experience in the diagnosis and surgical treatment of primary retroperitoneal tumor (PRT). Methods Clinical data of PRT 63 cases from January 1990 to March 2007 confirmed by pathology were retrospectively analyzed including clinical manifestation, surgical procedures, pathological examination and follow-up. Results Tumors were benign in 25 patients and malignant in 38. The main symptoms and signs were abdominal mass. The Youdon's index of CT examination for distinguishing benign from malignant tumors was 85%. The complete surgical resection rate was 88% for benign tumors and 68% for malignances. Removal of the involved organs or vessels was needed in 40% patients in complete surgical resection group. Postoperative recurrence was high in those the tumor was not completely removed and those the tumor was malignant. The 5-year survival rate for complete surgical resection of benign tumors was 83.6% ,and that of malignant tumors was 27.3%. After a median follow-up of 36 months (range 5 ～ 168)for the resection of malignant tumors, the local recurrence rate was 53%. Median time between initial surgery and recurrence was 25 months (range 3 ～ 108). For local recurrence of malignant tumor, the complete surgical resection rate was 62.5%. One patient died of intraabdominal hemorrhage in the first day after operation. Conclusion For PRT patients, early diagnosis is often difficult. Preoperative imaging results are essential to predict the surgical resectability. The optimal treatment of patients with PRT is radical resection, an bloc organ resection if necessary. Regularly postoperative follow-up is mandatory for early finding recurrence.

Objective To analyze the risk factors for delayed gastric emptying (DGE) after pancreaticoduodenectomy.Methods Clinical data of 213 patients who underwent pancreaticoduodenectomy at our hospital from January 1996 to December 2011 was retrospectively analyzed.Results The overall incidence of DGE was 40.8％ (87/213).The incidence of grade A,grade B and grade C DGE was 14.1％ (30/213),14.5 ％ (31/213) and 12.2％ (31/213) respectively.Median postoperative hospital stay was significantly prolonged in patients with DGE:30.5,32 and 61 days for grade A,B and C respectively versus 21 days in patients without DGE (x2 =66.171,P =0.000).Univariate analysis showed that operation time (≥420 min),intraoperative blood loss (≥ 1000 ml),Child alimentary reconstruction and pancreatic fistula were risk factors for postoperative DGE.Multivariate analysis using Logistic regression identified three variables as independent risk factors associated with postoperative DGE,namely,Child alimentary reconstruction (OR =2.098),intraoperative blood loss (≥ 1000 ml) (OR =2.525) and pancreatic fistula (OR =4.821).Grade C DGE was more frequently seen in patients suffering from postoperative pancreatic fistula.Conclusions The incidence of DGE after pancreaticoduodenectomy is still high.DGE prolongs the postoperative hospital stay significantly.The incidence of DGE could be reduced by Roux-en-Y reconstruction and reducing intraoperative blood loss.Postoperative pancreatic fistula is significantly associated with DGE,especially grade C DGE.

Objective To study the value of flow cytometry in identifying metastatic CK positive and negative nonhematopoietic neoplasms in bone marrow. Methods Twenty-six cell lines representing ten epithelial neoplasms, one lymphoma cell line and one human T cell lymphoblast-like cell line were purchased from American Tissue Culture Collection. From July 2009 to June 2010, five nonhematopoietic neoplasms,fifteen hematopoietic neoplasms and fifteen control patients with complete remession after hematopoietic stem cell transplantation were collected in Beijing Daopei Hospital. Cryopreserved cell lines were thawed and cultured until they entered log phase. After permeabilization, cell lines were analyzed by staining with cytoplasmic CK-FITC antibody using four-color flow cytometer. The percent CK positivity was measured by comparing with negative control. Bone marrow samples were stained with membrane and cytoplasmic antibodies according to our routine methods. Based on lineage markers and blast markers as well as CK expression, the relevant hematopoietic diseases were diagnosed or excluded according to 2008 World Health Organization diagnosis standards. Results All epithelial neoplasm cell lines expressed CK, with average positive percentage 81.1%. All the lymphoid tumor cell lines didn't express CK. Two epithelial neoplasms were CK positive, 100. 0% in thyroid carcinoma and 98. 2% in lung carcinoma, respectively. Hematopoietic tumor and control samples didn't express CK. They expressed relevant hematopoietic markers, such as CD45 as well as lineage markers, or CD138 and cytoplasmic immunoglobulin light chain. Three nonepithelial nonhematopoietic neoplasms didn't express CK. CK positive or negative nonhematopoietic neoplasms didn't express hematopoietic markers such as CD45, HLA-ABC and HLA-DR DP DQ, as well as lineage specific markers. Besides, CK positive might be helpful to suggest epithelial origin. Conclusion Flow cytometry with hematopoietic markers and CK can effectively exclude hematopoietic tumor and identify metastatic CK positive and negative nonhematopoietic neoplasms in bone marrow.