The Influence of PEG Modification on EGF-PLL Binding Capacities to EGFR / 生物化学与生物物理进展

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.

Structural and functional analysis of 2.2 kb spliced variant of hepatitis B virus genomes isolated from liver tissues from hepatocellular carcinoma patients / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To study the structure and function of 2.2 kb spliced variant of HBV genome from liver tissues of hepatocellular carcinoma patients.</p><p><b>METHODS</b>HBV 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.</p><p><b>RESULTS</b>Semi-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.</p><p><b>CONCLUSIONS</b>The 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.</p>

Expression and function of hepatocellular carcinoma-related gene pp1158 / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study in vitro and in vivo protein expression and biological function of gene pp1158, a hepatocellular carcinoma (HCC)-related gene.</p><p><b>METHODS</b>pp1158 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.</p><p><b>RESULTS</b>In 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.</p><p><b>CONCLUSION</b>pp1158 is a new candidate tumor suppressor gene of HCC.</p>

Isolation of cDNA Clones Using PAC and BAC Probes / 复旦学报（医学版）

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.

Controlled live dendritic cell vaccine mediates potent antitumor immune responses / 中华妇产科杂志

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

In vitro experimental study of gene therapy for ovarian cancer with thymidine kinase gene of herpes simplex virus mediated by a non-viral GE7 delivery system / 中华妇产科杂志

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.

Effect on growth of human hepatoma cells by antisense gene of type Ⅱ insulin-like growth factor receptor transfection / 中华消化杂志

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.

Detection of Replication-Competent Retrovirus / 中国肿瘤生物治疗杂志

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.

In vitro Inhibitory Effect on Growth of Human Glioma Cells with p21 Gene Mediated by a Novel Gene Delivery System / 中国肿瘤生物治疗杂志

Objective: To investigate the effects of novel targeted non-viral vector in gene therapy of human glioma. Methods: The EGF-R targeting gene delivery system GE7 was constructed. Human Glioma cell line U251 was transfected in vitro with ?－gal as reportor gene and p21 as therapeutic gene using this gene delivery system. By means of the assay of ?-galactosidase staining, Western blotting, in situ end labeling apoptosis cells and DNA ladder, the transferring of exogenous genes and the apoptosis of the tumor cells were examined.Results: It was showed that gene transfer efficiency is over 80%. When transfected with p21 gene, the growth of cells was inhibited significantly, and the apoptosis was detected in the transfected cell by the methods of in situ end labeling and DNA ladder. Conclusion: The GE7 gene delivery system has the ability to transfer exogenous gene to tumor cells and the expression of the therapeutic gene can inhibit the growth of the cells.

Conceptual consideration of cancer,challenges and opportunities for cancerbiotherapy / 中国肿瘤生物治疗杂志

The outcome of current cancer therapy is far from satisfactory despite the considerable advancements have been achievedin life science and medicine.When looking for more potent drugs or technologies for cancer therapeutics,we shouht re-evaluate ourtraditional perception of caneer and have introspection whether we have some misunderstandings of cancer biology and the relationshipbetween cancer and host.In this commentary,the authors outlined their conceptual consideration of cancer,and proposed theirviewpoints about challenges and opportunities for cancer biotherapy.

The Influence of Polycationic Polypetride on the Transfective Efficacy of NonViral Vector / 中国肿瘤生物治疗杂志

Objective: Purpose to investigate the different in vitro function of targetable non-viral vector containing poly-L-lysine or protamine. Methods: Using GV1 and GV2 targetable non-viral vectors, the influences of the poly-L-lysine and protamine on in vitro gene transfer efficiency and the course of gene expression were observed. Results: ?-galactosidase was expressed at intermediate level (50% ) in A375 cells using a complex containing either protamine or poly-L-lysine. Howerver, in case of ABAE cells, ?－galactosidase expression level was low (20% ) transferred with a comPlex containing protamine. On the contrary, ?－ galactosidase expression was at high level (70% ) provided that protamine was replaced with poly-L-lysine. In addition, ?-galac- tosidase activity reached the peak at the 6th day after transfection with the complex containing protamine. The expression was not altered with subsequent subcultures, at least for 3 passages. Using poly-L-lysine, the expression peak in A375 reached the peak at the 7th day after transfection, but the level declined along with subsequent passages of cells. Conclusion: The apllication of protamine in VEGF receptor mediated gene delivery system was limited.

Expression of cancer gene products of IGF-Ⅱ, IGF-Ⅱ receptors, CSF-1 receptors in primary hepatic cancer and non-cancerous liver tissue / 第三军医大学学报

The expression of cancer gene products of insuline-like growth factor Ⅱ (IGF-Ⅱ),IGF-Ⅱ receptors (IGF-Ⅱ-R),and colony-stimulating factor 1 receptors (CSF-1-R) /c-fms in 17 cases of human primary hepatic cancer,the non-cancerous liver tissue adjacent to the cancer,and normal liver tissue was studied with immunocytochemistry (ABC),Western blot and Northern blot techniques.It was found that the expression of IGF-Ⅱ,IGF-Ⅱ-R and CSF-1-R was significantly higher in the cancers than in normal liver tissues,and the expression of IGF-Ⅱ and IGF-Ⅱ-R was higher in the non-cancerous liver tissues than in the cancers.It was noteworthy that the expression of IGF-Ⅱ in both the cancerous and non-cancerous hepatic tissues was characterized by its fetal type.However the expression of CSF-1-R was distinctly higher in the cancers than in the non-cancerous hepatic tissue.These findings,the authors believe,imply that the aberrant overexpression of IGF-Ⅱ,IGF-Ⅱ-R and CSF-l-R might be related to the mechanism of auto-crine-stimulated growtth of the cells of human primary hepatic cancer.

Expression of Multidrug Resistance Gene, in Human Hepatocellular Carcinoma Tissues / 第二军医大学学报

We have cloned full-length MDR1 cDNA from human normal liver tissue in previous study. Using this MDR1 cDNA as probe, we observed the MDR1 gene expression in human hepatocellular carcinoma treated with and without chemotherapy by Northern blot. The results showed that expression of MDR1 gene in hepatocellular carcinoma tissues was higher than that in their adjacent normal liver tissues; and enhanced MDR1 gene expression was also observed in hepatocellular carcinoma treated with chemotherapic agents. We also explored a method for quantitative analysis of MDR1 gene expression in hepatocellular carcinoma by polymerase chain reaction. This study suggests that overexpression of MDR1 gene may be responsible for the intrinsic and acquired drug resistance of human hepatocellular carcinoma, and PCR is a preferable method for quantitative analysis of MDR1 gene expression in hepatocellular carcinoma.

Expression of hepatocellular carcinoma-associated protein 1 in peripheral blood of patients with acute leukemia / 第二军医大学学报

0. 05). Conclusion: The HCAPl can be expressed in both normal peripheral white blood cells and the leukemic cells, and there is no difference in the protein levels between them.

Cloning of Full-length Multidrug Resistance Gene, in Human Normal Liver / 第二军医大学学报

Overexpression of multidrug resistance gene, (MDR1) is responsible for the resistance of anticancer agents in cancer cells. In this study, we designed three pairs of DNA primers to clone MDR, cDNA from human normal liver by polymerase chain reaction. The sites of these primers in MDR cDNA are (I)-64-46 (5') and 1680-1698 (3'); (II) 1471-1489 (5') and 2905-2923 (3'); (III)2729-2747 (5') and 3845-3863 (3). The three reactions were underwent after the human normal liver mRNA was reverse transcripted into single strand DNA. The lengths of PCR products are 1762bp, 1452 bp and 1134bp, respectively. The former two products were subcloned in pBluscript SK, respectively and the latter product was subcloned in pGEM7Z (named as pMDR1.7, pMDR1.4 and pMDRl.l, respectively). Cloned genes were certificated as MDR1 cDNA by sequence analysis. Full-length MDR, cDNA was obtained after further subcloning. Full-length MDR1 cDNA we obtained will be a very important tool in molecular diagnosis and gene therapy of anticancer drug resistance.