Objective:Our aim is to investigate the relationship of VEGF-C,VEGF-D and lymph node metastasis in pancreatic cancer,and to elucidate the role and significance of the cancerous peripheral lymphatic tissue.Methods: The expression of VEGF-C and VEGF-D was assayed by means of immunohistochemistry in 30 pancreatic carcinomas.Results:The positive rates of VEGF-C、VEGF-D were 73% (22/30)、57% (17/30) respectively in pancreatic cancer.The expression of VEGF-C、 VEGF-D in cancerous invasive edge was significantly higher than that in the center of cancerous tissues.There was no correlation between the expression of VEGF-C、VEGF-Dand the site,differentiation,histology types.Ⅲ~Ⅳ stages of pancreatic cancer showed strong expressions of VEGF-C、VEGF-D than that Ⅰ~Ⅱ stages of pancreatic cancer.The positive lymph node in positive VEGF-C and VEGF-D group were higher than that in the negative group.Conclusion:VEGF-C and VEGF-D induced lymphangiogenisis in pancreatic cancer,promoted the tumor cell lymph metastasis.

AIM:Allogeneic hematopoietic stem cell transplantation for treatment of acute lymphoblastic leukemia characterized with high rate of relapse,especially in Ph+ patients.Presently,researchers focus on how to resolve relapse.Transplantation time,transplantation schedule and adoptive immunotherapy after transplantation are important.The study was performed to preliminarily observe treatment effectiveness after myeloablative stem cell transplantation,non-myeloablative transplantation after donor lymphocyte infusion and non-myeloablative transplantation followed by low-doses of Cyclosporin A.METHODS:Five patients were admitted at Department of Haematology of First People's Hospital between December 1998 and May 2007.Acute lymphoblastic leukemia patients were informed consent for allogeneic hematopoietic stem cell transplantation.The experiment was approved by hospital ethics committee.Among them,one patient was used the traditional preconditioning of busulfan and cyclophosphamide.Four cases were performed with non-myeloablative hematopoietic stem cell transplantation,and one of them was treated with reduced intensity regimen based on anti-thymocyte globulin donor lymphocyte infusion after transplantation;Three cases with fludarabine-based non-myeloablative transplantation were used low-dose Cyclosporin A after engraftment.Graft-versus-host disease prevention regimen was consisted of short-range methotrexate combined with Cyclosporin A.Haematopoiesis,chimerism,graft-versus-host disease and infection were observed after transplantation.RESULTS:All patients achieved successful engraftment.①One patient with mixed chimerism received eight donor lymphocyte infusion based on anti-thymocyte globulin-non-myeloablative transplantation and gradually converted into full donor chimerism with disease-free survival,and complicated acute graft-versus-host disease of skin and liver.②Three patients achieved full donor chimerism based on fuladarabine-non-myeloablative transplantation,one patient relapsed without graft-versus-host disease,and other two cases eliminated BCR/ABL fusion gene-positive cells with acute and chronic graft-versus-host disease.③One case after myeloablative transplantation relapsed and complicated with acute and chronic graft-versus-host disease.CONCLUSION:①The traditional,anti-thymocyte globulin or fludarabine-based non-myeloablative conditioning for transplantation in the treatment of adults with acute leukemia will be eligible for the successful implantation,and adoptive immunotherapy have graft-versus-leukemia effect.②The efficacy and complications of three transplantation strategies should be further studied.

Objective To investigate the effects of adefovir resistance related mutations in hepatitis B virus (HBV) reverse transcription (RT) region on the viral replication and hepatitis B surface antigen (HBsAg) secretion. Methods Twelve adefovir treated chronic hepatitis B (CHB) patients who experienced a viral breakthrough were enrolled in this study. The RT region was amplified by polymerase chain reaction (PCR) using HBV DNA extracted from sera as the template. PCR products were then sequenced and analyzed to find out mutation patterns. Full-length HBV genome was amplified from 4 representative serum samples followed by direct sequencing. The dominant strain was cloned into vector PHY106 to construct a recombinant plasmid containing the 1.1 unit of HBV genome Which was transfected into Huh7 cells. HBsAg and hepatitis B e antigen (HBeAg) expression were determined by enzyme-linked immunosorbent assay (ELISA), meanwhile intracellular HBV DNA level was determined by quantitative real-time PCR. Furthermore strain harboring rtA181T/sW172 · mutation and strain without rtA181 mutation were cotransfected into Huh7 cells. HBsAg and intracellular HBV DNA were also determined after transfection. Results Ten out the 12 patients enrolled in this study exhibited mutations conferring resistance to adefovir. The rtA181T mutation was detected in 5 cases, and the rtA181T/S+rtN236T mutation was observed in 4 cases. Different mutants showed variable HBsAg secretion competency in vitro. Despite the defect of HBsAg secretion of the rtA181T/sW172 · mutant, the replication efficiency was almost the same in different mutants. When the strains with and without rtA181 mutation were cotranfected into cells, the HBsAg level increased in accordance with the amount of stains without rtA181 mutation. However, the intracellular HBV DNA level was not changed significantly. Conclusions The rtA181 mutation is common in patients with adefovir resistance, of which the rtA181T mutation is the major pattern. In vitro analysis reveals that the rtA181T/sW172 · mutant is defective in HBsAg secretion which could be rescued by coexistence of wild-type strains. The replication efficiency in various mutants shows no obvious differences.

Objective To establish a novel and convenient method to study the phenotype of drug resistant hepatitis B virus (HBV) isolates,and to analyze the drug susceptibility by replacing the reverse transcriptase (RT) domain of wild-type HBV with that of the drug resistant HBV isolates.Methods Full length of HBV isolates was amplified and cloned from the sera of patients prior to nucleoside/nucleotide analogues (NA) treatment.Wild-type full-length HBV genomes was used to construct the recombinant expression plasmids PHY536207 (genotype B) and PHY97 (genotype C).The restriction enzyme sites were introduced in the upstream and downstream region of reverse transeription (RT) domain to construct plasmid,which were named as mPHY536207 and mPHY97,respectively.Lamivudine (LAM) resistant mutant and adefovir (ADV) resistant mutant were isolated and cloned to construct recombinant expression plasmids PHY634 and PHY6923,respectively.Subsequently,the RT domain of mPHY536207 was replaced by that of drug resistant mutant to construct the plasmids RT634 (LAM-resistant) and RT6923 (ADVresistant).The HBV constructs were transfected into Huh7 cells.The HBsAg levels in supernatant were determined by enzyme-linked immunosobent assay (ELISA),and the amount of intracellular HBV DNA was assayed by real-time polymerase chain reaction and Southern blot analysis.Results The plasmids PHY536207 and PHY97 containing genotype B and genotype C wild-type fulllength HBV genomes were constructed successfully,both of which could replicate in Huh7 cells.Intracellular HBV DNA extracted from cells in each of six-well culture plates was more than 1 × 107 copy/ mL,and the introduction of Pst Ⅰ restriction enzyme site did not affect the viral replication and HBsAg secretion.PHY634 and RT634,in which mutant RT domain was replaced into a wild type HBV expressing vector,exhibited the same HBV DNA replication under the treatment with different doses of LAM,the value of 50％ inhibitory concentration (IC50) was ＞100 μmol/L,while the IC50 of mPHY536207 was 0.18μmol/L.Moreover,wild-type isolate was sensitive to ADV (IC50 =1.2 μmol/L),while PHY6923 and RT6923 were resistant to ADV treatment (IC50 ＞100 μmol/L).Conclusion The phenotypic assay is successfully developed in this study based on replacing RT domain of wild-type HBV strains with that of clinical isolated drug resistant strain.

significantly save the time of diagnosis and facilitate the clinical application of large samples.