Cationic polymers are being developed quickly as gene delivery vectors. For in vivo gene delivery, the cationic polymers are usually further modified by hydrophilic polymer grafting or ligand conjugation, which have been shown to increase the vector stability, gene delivery efficiency and specificity greatly. Some previous research had shown that modified hydrophilic polymer may partly shield the targeting ligand and result in poor delivery specificity. Developing a method to evaluate the influence of PEG modification on targeting delivery is particularly critical to cationic polymer design and gene therapy development. One of most commonly used cationic polymer polylysine (PLL) was chosen as a model. Targeting ligand epidermal growth factor(EGF)was conjugated with PLL to form PLL-EGF. Then hydrophilic polymer polyethylene glycol (PEG) with molecular mass 7 000 and 20 000 were used to modify PLL-EGF respectively to generate PEG7000-g-PLL-EGF and PEG20000-g-PLL-EGF. In BIAcore experiments, epidermal growth factor receptor (EGFR) was conjugated onto BIAcore chip and various PEG modified PLL-EGF solutions were flowed over the chip. By observing the change of RU value, the specific interaction of EGF to EGFR was compared. Compared with PLL-EGF, PEG modified PLL-EGF showed lower association rate and higher disassociation rate to EGFR. Furthermore, compared to PEG7000 modified PLL-EGF, PEG20000 modified PLL-EGF got lower association rate and higher disassociation rate to EGFR. The Scatchard analysis results showed that the interactions between EGFR and PLL-EGF or PEG-PLL-EGF are non-linear. It can be concluded that PEG modification indeed reduced the association rate and enhanced the dissociation rate of EGF to EGFR. The length of PEG chain was also a key factor to influence interaction between ligand and receptor. The results showed that it was critical important to evaluate the influence of PEG modification on delivery specificities. The BIAcore method developed in this paper can successfully evaluate the influence, which would be important for cationic polymer design and its application as potential non-viral gene delivery vectors.

BACKGROUNDTo study the structure and function of 2.2 kb spliced variant of HBV genome from liver tissues of hepatocellular carcinoma patients.

METHODSHBV genomes were amplified by using PCR from paired hepatocellular carcinoma tissues and peritumor tissues. The 3.2 kb full-length HBV genome and 2.2 kb spliced variant were separately cloned and sequenced. Hep G2 cells were co-transfected with full-length HBV DNA and 2.2 kb spliced variant, and after transfection, HBV DNAs from intracellular core particles were harvested and specific primers were used in PCR to evaluate the interactions between spliced variant and full-length counterpart in replication.

RESULTSSemi-quantification by scanning density showed that 2.2 kb spliced variant was present in all tumor and peri-tumor samples studied. Sequence analyes revealed that the 5 terminus packaging signal for pregenomic and X and PreC/C genes were retained. When full-length HBV DNA was co-transfected with 2.2 kb, the replication signal of 3.2 kb HBV genome was increased 3-7 times.

CONCLUSIONSThe 2.2 kb HBV spliced variant was present in liver tissues, and relative content was higher in tumor tissues than that in the peri-tumor tissues. This spliced variant could enhance the replication of full-length HBV genome, which suggested the possible role of the variant in the pathogenesis of development of hepatocellular carcinoma.

Adult ; Aged ; Carcinoma, Hepatocellular ; virology ; DNA, Viral ; analysis ; Female ; Genetic Variation ; Genome, Viral ; Hepatitis B virus ; genetics ; Humans ; Liver ; virology ; Liver Neoplasms ; virology ; Male ; Middle Aged ; RNA, Viral ; analysis ; Sequence Analysis, DNA

OBJECTIVETo study in vitro and in vivo protein expression and biological function of gene pp1158, a hepatocellular carcinoma (HCC)-related gene.

METHODSpp1158 was expressed with fusion expression vector pET-32a in E. Coli-BL21 (DE3), and rabbit anti-pp1158 fusion protein polyclonal antibody was prepared. The biological function and differential expressions of pp1158 were studied by in vitro colony formation assay nude mouse in vivo tumor formation assay of transfected BEL7402 cell line and immunohistochemistry and Western blot. Differential expression of pp1158 in human fetal tissues were examined by Northern blot.

RESULTSIn vitro experiment showed that pp1158 inhibited colony formation rate of transfected BEL 7402 cells, with an inhibition rate of 58.3% (P < 0.01). Tumor formation assay indicated that tumor formation of pCMV-Script-1158 transfected BEL 7402 cell line was significantly inhibited (P < 0.05) as compared with that of the control group. Immunohistochemical assay showed that pp1158 was expressed in human tissue in the following sequence: normal liver >/= noncancerous liver tissue > HCC. Western blot indicated that a 60kD protein (pp1158 protein) was expressed in BEL 7402 cells transfected with pCMV-Script-pp1158 DNA, while it was detected in BEL 7402 cells transfected with pCMV-Script vector DNA. Northern blot showed pp1158 was expressed in the placenta at a very high level, heart, liver, muscle, pancreas and lung but expressed poorly in the brain and kidney.

CONCLUSIONpp1158 is a new candidate tumor suppressor gene of HCC.

Angiopoietin-like 4 Protein ; Angiopoietins ; Animals ; Blotting, Northern ; Blotting, Western ; Carcinoma, Hepatocellular ; genetics ; metabolism ; pathology ; Female ; Gene Expression Regulation, Neoplastic ; Genes, Tumor Suppressor ; Humans ; Intercellular Signaling Peptides and Proteins ; Liver Neoplasms ; genetics ; metabolism ; pathology ; Male ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data ; Neoplasm Proteins ; genetics ; metabolism ; Neoplasm Transplantation ; Neoplasms, Experimental ; genetics ; metabolism ; pathology ; Proteins ; genetics ; RNA, Messenger ; genetics ; metabolism ; Transplantation, Heterologous ; Tumor Cells, Cultured

Animals ; Carcinoma, Hepatocellular ; genetics ; therapy ; Genetic Therapy ; methods ; Humans ; Liver Neoplasms, Experimental ; genetics ; therapy ; Treatment Outcome

Purpose　To introduce a method to isolate cDNA clones using bacteriophage P1-derived artificial chromosome (PAC) or bacterial artificial chromosome (BAC) as probe for hybridization and try to find some novel genes related to hepatocellular carcinoma.　Methods　PAC 579 (D17S926 locus) and BAC 1529 (D17S1529 locus) in the deletion region of chromosome 17p13.3 in human hepatocellular carcinoma were chosen to screening the human liver cDNA library as probe for hybridization. The isolated positive cDNA clones were partially sequenced, then analyzed by computer comparison and Southern blot.　Results　After three cycles of screening, 78 and 8 candidate positive cDNA clones were isolated using PAC 579 and BAC 1529 probes respectively. Further analysis indicated 18 cDNA clones isolated by PAC 579 probe and 5 cDNA isolated by BAC 1529 probe were potential novel genes related to hepatocellular carcinoma.　Conclusions　The isolation of cDNA clones using PAC and BAC probes is effective and practical.

Objective To investigate the efficiency of antitumor immune responses induced by a controlled live dendritic cell(DC)vaccine Methods DC precursors were isolated from Fischer 344 rat bone marrow and cultured with granulocyte macrophage colony stimulating factor and interleukin 4 The rat ovarian tumor cell line NuTu 19 was genetically modified by retroviral mediated suicide gene(HSV 1 TK), and the positive clones were selected using G418 Live DC vaccine was then fused with DC and NuTu 19/TK cell by polyethylene glycol The characteristics of live DC vaccine were assayed with flow cytometry and confocal laser scanning microscopy The specific expression of HSV 1 TK gene in live DC vaccine was evaluated by RT PCR and western blot The sensitivity of live DC vaccine to ganciclovir (GCV) was evaluated by methylthiazoletetrazolium assay In vivo, rats vaccinated twice with live DC vaccine were compared to those vaccinated with killed DC vaccine, unfused DC and NuTu 19/TK cell or phosphate buffered saline Seven days following the last immunization, the rats were sacrificed to test the specific cytotoxic T lymphocyte (CTL) activity by lactate dehydrogenase release assay, or challenged with NuTu 19 and tumor incidence was observed Results The fusion efficiency was approximately (23?14) Live DC vaccine displayed an up regulated expression of major histocompatibility complex (MHC) IIOX6 (87 6?3 4)%, costimulatory molecule B 1 2 (71 1?9 3)%, integrin OX 62 (68 0?7 4)%, and adhesion ICAM 1 (77 1?2 0)%, and specifically expressed HSV 1 TK gene. Our data showed that spleen T lymphocytes from rats vaccinated with live vaccine displayed enhanced CTL aetivity (61 8?8 3)% contrast to that of rats vaccinated with killed vaccines (26 0?3 8)% ( P

Objective To investigate gene transfer efficiency of a novel target non-viral vector GE7 and effects of herpes simplex virus thymidine kinase (HSV 1-tk)/ganciclovir(GCV) mediated by it in vitro. Methods The epidermal growth factor receptor (EGF-R) target gene delivery system GE7 was constructed.Human ovarian cancer cell line CAOV3 was transfected in vitro with ?-galactosidase(?-gal) as reporter gene and HSV 1-tk gene as therapeutic gene using this gene delivery system.By means of the assay of X-gal staining, Northern blotting, cell growth-inhibiting curve and so on,the transferring efficiency of exogenous genes and killing effects are observed. Results It showed that gene transfer efficiency is over 80%.When 10 mg/L GCV was put into ovarian cells transfected with HSV 1-tk gene, 95% of cells were killed, and the apoptosis ratio reached up to 30. Conclusions The GE7 gene delivery system is an effective and safe delivery system.GE7/ HSV 1-tk /GCV therapeutic gene system is appraising for ovarian cancer.

Objective To look for mutations of the p53 gene in leukimic patients and study the relationship between abnormalities in p53 gene and leukemogenesis.Methods The peripheral blood and Bone Marrow Samples were collected from 36 patients with various leukemia types including 14 cases of lymphocytic leukemia [8 cases of acute lymphocytic leukemia (ALL), 4 cases of chronic lymphocytic leukemia (CLL), 2 cases of hairy cell leukemia (HCL)] and 22 cases of myelocytic leukemia [11 cases of acute non-lymphocytic leukemia (ANLL), 11 cases of chronic myelocytic leukemia (CML)]. DNA structures of exon 5-8 of the p53 gene were scanned by PCR-SSCP (single strand conformation polymorphism analysis of polymerase chain reaction products). The appropriate DNA fragments were amplified, Purified and sequenced directly.Results By PCR-SSCP analysis, shifts in electrophoretic mobility of the p53 gene were detected in 3 of 14 patients with lymphocytic leukemia (2 ALL and 1 CLL), but none in 22 patients with myelocytic leukemia including one in blastic crisis. Direct nucleotide sequencing in one patient with ALL showed transition of CTG to CAG at codon 257 of exon 7, resulting in a change of its encoded amino acids from aspartic acid to valine. To our knowledge, the mutation at this codon has not been previously reported hitherto.Conclusions The p53 gene mutations are specifically associated with lymphocytic leukemia. Alternations of the p53 gene may play a certain role in leukemogenesis in some cases of lymphocytic leukemia.

Objective To study the growth inhibition of human hepatoma cells tranfected with type Ⅱ insulin like growth factor receptor (IGF ⅡR) antisense gene. Methods We constructed the recombinant eucaryotic expression plasmids of IGF ⅡR sense and antisense genes, which were transfected into SMMC 7721 human hepatoma cell line using calcium phosphate coprecipitation, and selected in DMEM supplemented with 500 ?g/ml G418(Geneticin) for 2～3 weeks. Then the total number of colonies formed was counted. The effect of IGF ⅡR antisense gene transfection on endogenous IGF ⅡR mRNA levels was examined by Northern blot, and the growth rate and the ability of the transfected cells to form colonies in soft agar medium were also examined. Results The hepatoma cells transfected with IGF ⅡR antisense gene exhibited significant reduction in endogenous IGF ⅡR messenger RNA(mRNA) levels and loss of their ability for anchorage independent growth in soft agar. The IGF ⅡR antisense gene supressed the formation of colonies of SMMC 7721 cells resistant to G418 without alteration on cell growth curve. Conclusion The IGF ⅡR antisense gene expression effectively blocks IGF Ⅱ to IGF ⅡR autocrine and/or paracrine signal transduction pathway and reverses certain aspects of the cell malignant phenotype.

Retroviral vectors are the most used gene delivery vehicle in human gene therapy.When the therapeutic gene is packagedfrom producer cell line, one has to determined whether thereplication-competent retrovirus(RCR) is being generated. In this study, two methods, namely the marker rescue assay and RT/PCR, were employed to detect RCR in the cells. While the marker rescue assay can detect RCR with a limit at 6?102CFU/ml, RT/PCR can be used to detect RCR at as low as lCFU/103ml. Combining the specificity of the marker rescue assay and the sensitivity of RT/PCR, both assays together should serve as an adequate test for detecting the generation of RCR in retroviral producer cell lines.