Replication-incompetent Adenovirus Vector-mediated MDA-7/IL-24 Selectively Induces Growth Suppression and Apoptosis of Hepatoma Cell Line SMMC-7721

In order to investigate the effect of replication-incompetent adenovirus vector expressing MDA-7/IL-24 on tumor growth and apoptosis of human hepatocellular carcinoma （HCC） cell line SMMC-7721 and normal liver cell line L02, the recombinant replication-incompetent Ad.mda-7 virus vector was constructed and infected into the HCC cell line SMMC-7721 and normal liver cell line L02. RT-PCR was performed to examine the expression of MDA-7 mRNA. The concentrations of MDA-7/IL-4 in culture superuatants were determined by using ELISA. MTT and Hoechst staining assay were applied to observe the inhibitory and killing effects of MDA-7 on the HCC cells. By using flow cytometry, the apoptosis, cell cycle and proliferation of SMMC-7721 and L02 cells were measured. The results showed recombinant replication-incompetent virus expressing MDA-7/IL-24 was constructed successfully, and RT-PCR revealed that it could mediate the high expression of the exogenous gene MDA-7/IL-24 in SMMC-7721 and L02 cells. The expression of MDA-7/IL-24 proteins in the culture superuatant was detectable by ELISA. Ad.mda-7 infection induced apoptosis and growth suppression in SMMC-7721 cells and an increased percentage of HCC cells in the GyM phase of the cell cycle, but not in L02 cells. It was concluded that mda-7/IL-24 gene, mediated with replication-incompetent adenovirus vector, could selectively induce growth suppression and apoptosis in HCC cell line SMMC-7721 but without any toxic side-effect on normal liver line L02.     

Inhibitory Effects of Anti-sense PTTG on Malignant Phenotype of Human Ovarian Carcinoma Cell Line SK-OV-3

To construct eukaryotic expression vector expressing full length anti-sense pituitary tumor transforming gene (PTTG) mRNA and observe its blocking effect on the potential invasion of human ovarian carcinoma cell line SK-OV-3. PCR primers containing designed enzyme cut sites were used for cloning full-length PTTG gene fragment, and the resulting PCR product was inserted into the eukaryotic vector pcDNA3. 1 in the antisense direction. The recombinant vector was then transfected into SK-OV-3 by Lipofectamine. The positive cell clone was screened by G418, PTTG and bFGF at protein level expression were detected by Western blot. The biological behavior change of transfection positive cells was observed by colony formation in soft agar assay. Our results showed that SK-OV-3 clones stably expressing full-length recombinant pcDNA3. 1-PTTGas were obtained. The expressions of PTTG and bFGF protein in transfected cells were decreased by 61.5% and 52.3%, respectively as compared with non-transfected ones. The number of colony formation was reduced significantly in transfected cells as compared with empty vector transfected and non-transfected cells. It is concluded that the recombinant vector pcDNA3. 1-PTTGas is a novel tool and provides an alternative anti-sense gene therapy targeted at PTTG in human carcinoma.     

Construction and identification of a vector expressing TRAM siRNA in mammalian cells

Objective：To construct and identify a vector expressing TRAM siRNA in mammalian cells.Methods :It was constructed that the vector named R-pSUPER-EGFP used to transcribe functional TRAM siRNA. Two of pair 64 nt TRAM gene specific target sequences were inserted into the downstream of the H1 promoter. The recombinants were transformed into E. coli JM109, and finally their veracity was confirmed by double cutting with the enzymes and sequencing. R-pSUPER-EGFP was transfected into RAW264. 7 cell by using LipofectamineTM2000, and the expression of TRAM was detected by Western blotting. Results .. Two different recombinant plasmids containing corresponding TRAM gene specific target sequences were constructed, transfected into RAW264.7 cell line successively, which can specifically restrain expression of TRAM protein. Conclusion :The optimizing method in constructing the recombinant vector serves other plasmid-based RNA interference research. Therefore, the recombinant vectors establish the basis for research on the function of TRAM gene.     

Immune Killing Activity of Lymphocytes on Hela Cells Expressing Interleukin-12 In Vitro

The killing effects of lymphocytes on Hela cells expressing interleukin-12 （IL-12） in vitro were explored. By using gene transfection technique, full length IL-12 gene was transfected into Hela cells. The expression of IL-12 in Hela cells was detected quantitatively by ELISA; Changes in killing effects of lymphocytes on Hela cells expressing IL-12 were observed by MTT. It was found that Hela cells could express IL- 12 between 24 h and 72 h after transfection. Killing activity of lymphocytes on Hela cells expressing IL-12 was significantly enhanced. It was concluded by cell transfection technique, Hela cells could express 1L-12 and were more easily killed by lymphocytes.     

Cloning,Eukaryotic Expression of Human ISG20 and Preliminary Study on the Effect of Its Anti-HBV

Human ISG20 gene was cloned and the effect of its anti-HBV was primarily studied. The ISG20 gene was amplified from HeLa cells by RT-PCR and recombinant vector expressing ISG20 was constructed by genetic engineering. The overexpression of ISG20 in HepG2 cells was detected by Western blot and the levels of secretion of HBs antigen and HBe antigen tested by ELISA. The results showed that： （1） Sequence of ISG20 cloned was consistent to that published in Genebank; （2） Recombinant vector expressing ISG20 could be expressed in HepG2 cells by transfection; （3） The overexpression of ISG20 protein could reduce the levels of the secretion of HBs antigen and HBe antigen in transfected HepG2 cells. It was suggested that the overexpression of recombinant ISG20 in culture cells could reduce the synthesis of HBV proteins.     

Tumor cell- specific gene transfer with retroviral vectors displaying single- chain antibody

Objective To specifically deliver the therapeutic gene to cancer cells and construct target retroviral vectors by inserting the single- chain variable antibody fragment into the retroviral envelope. Methods Single- chain antibody expression vector pET - 20bScfv was constructed. Binding activity of the genetically engineered single- chain variable antibody fragment was verified by enzyme- linked immunosorbent assay (ELISA) and Western blot. At the same time, by means of polymerase chain reaction (PCR)- directed mutagenesis, the appropriate cloning site EcoT22 Ⅰ/Sal Ⅰ was generated at the N- terminus of receptor- binding SU domain in the MoMLV env polypetide. Then the single- chain antibody gene, encoding a functional antibody, was inserted into the cloning site. The Scfv- env fusion gene fragment was subcloned into CMV expression vector. The Lac- Z retrovirus that co- displayed the Scfv- env chimeric protein and wild- type env protein was produced by transfection of] Ψ2 cells with retroviral plasmid and the fusion gene expressing plasmid. To confirm the specificity of the recombinant retrovirus, infection assays and competitive inhibition assays were performed. Results The results of ELISA and Western blot showed that the genetically engineered single- chain variable antibody fragment could bind to the SHG44 cells surface membrane antigen. Virus- binding assay, viral infection and competitive inhibition assays confirmed that the harvested virus efficiently bound to and infected SHG44 cancer cells expressing the relative membrane antigen specially via the recognition of the target antigen. Conclusion These results imply that insertion of Scfv into the retroviral envelope can specifically deliver the interested gene into specific antigen- producing cancer cells.     

Labeling embryonic stem cells with enhanced green fluorescent protein on the hypoxanthineguanine phosphoribosyl transferase locus

Objective To label embryonic stem(ES)cells with enhanced green fluorescent protein(EGFP)on the hypoxanthineguanine phosphoribosyl transferase(HPRT) gene locus for the first time to provide a convenient and efficient way for cell tracking and manipulation in the studies of transplantation and stem cell therapy.Methods Homologous fragments were obtained by polymerase chain reaction(PCR),from which the gene targeting vector pHPRT-EGFP was constructed.The linearized vector was introduced into ES cells by electoporation.The g4186tG cell clones were obtained after selection with G418 and 6TG media.The integration patterns of these esistant cell clones were identified with Southern blotting. Results EGFP expressing ES cells on the locus of HPRT were successfully generated.They have normal properties,such as karyotype,viability and differentiation ability.The green fluorescence of EGFP expressing cells was maintained in propagation of the ES cells for more than 30 passages and in differentiated cells.Cultured in suspension,the “green”ES cells aggregated and formed embryoid bodies, retaining the green fluorescence at varying developmental stages.The“green”embryoid bodies could expand and differentiate into various types of cells,exhibiting ubiquitous greem fluorescence.Conclusions This generation of “green”targeted ES cells is described in an efficient protocol for obtaining the homologous fragments by PCR.Introducing the marker gene in the genome of ES cells,we should be able to manipulate them in vitro and use them as vehicles in cell-replacement therapy as well as for other biomedical and research purposes.     

In vitro effect of p21^WAF-1／CIPI gene on growth of human glioma cells mediated by EGFR targeted non-viral vector GE7 system

To construct the EGFR targeted non-viral vector GE7 system and explore the in vitro effect of p21^WAF-1/CIP1 gene on growth of human glioma cells mediated by the GE7 system. Methods: The EGFR targeted non-vi-ral vector GET gene delivery system was constructed. The malignant human glioma cell llne U251MG was transfected in vitro with β-galactosidase gene( reporter gene) and p21^WAF-1/CIP1 gene (therapeutic gene) using the GET system. By means of X-gal staining, MTS and FACS, the transfection efficiency of exogenous gene and apoptosis rate of tumor cells were examined. The expression of p21^WAF-1/CIP1 gene in transfected U251MG cell was examined by immunohistochemitry staining. Results: The highest transfer rate of exogenous gene was 70%. After transfection with p21^WAF-l/CIP1 gene,the expression of WAF-1 increased remarkably and steadily; the growth of U251MG cells were inhibited evidently.FACS examination showed G1 arrest. The average apoptosis rate was 25.2%. Conclusion: GE7 system has the ability to transfer exogenous gene to targeted cells efficiently, and expression of p21^WAF-l/CIP1 gene can induce apoptosis of glio-ma cell and inhibit its growth.     

Inhibition of NF-κB by mutant IκBα enhances TNF-α-induced apoptosis in HL-60 cells by controlling bcl-xL expression

Backgound The aim of this study was to explore whether the inhibition of nuclear factor-κB (NF-κB)activation by mutant IκBα (S32,36→A) can enhance TNF-α-induced apoptosis of leukemia cells and to investigate the possible mechanism. Methods The mutant IκBα gene was transfected into HL-60 cells by liposome-mediated techniques. G418 resistant clones stably expressing mutant IκBα were obtained by the limiting dilution method. TNF-α-induced NF-κB activation was measured by electrophoretic mobility shift assay (EMSA). The expression of bcl-xL was detected by RT-PCR and Western blot after 4 hours exposure of parental HL-60 and transfected HL-60 cells to a variety of concentrations of TNF-α. The percentage of apoptotic leukemia cells was evaluated by flow cytometry (FCM). Results Mutant IκBα protein was confirmed to exist by Western blot. The results of EMSA showed that NF-κB activation by TNF-α in HL-60 cells was induced in a dose-dependent manner, but was almost completely inhibited by mutant IκBα repressor in transfected cells. The levels of bcl-xL mRNA and protein in HL-60 cells increased after exposure to TNF-α, but changed very little in transfected HL-60 cells. The inhibition of NF-κB activation by mutant IκBα enhanced TNF-α-induced apoptosis. Thecytotoxic effects of TNF-α were amplified in a time- and dose-dependent manner. Conclusions NF-κB activation plays an important role in the resistance to TNF-α-induced apoptosis. The inhibition of NF-κB by mutant IκBα could provide a new approach that may enhance the antileukemia effects of TNF-α or even of other cytotoxic agents.     

Construction and identification of recombinant lentivirus-mediated gene transfer system for rat transducer of regulated CREB activity 1

Objective:To construct a recombinant lentivirus vector which carries SD rat transducer of regulated CREB activity-1(TORC1) gene and examine its ability to express the TORC1 gene in vitro.Methods:The coding sequence of SD rat TORC1 gene was amplified using PCR and cloned into pGC-FU vector.293T cells were transfected using Lipofectamine 2000 and packaged for the recombinant lentivirus particles.When the cloned sequence was identified to be right,the recombinant lentivirus particles were amplified in a large quantity.The titer of virus was determined by real-time PCR and the level of TORC1 expression was examined by Western blot. Results:The recombinant lentivirus vector carrying TORC1 was constructed successfully and could express TORC1 at a high level in 293T cells in vitro,and the titer determined by real-time PCR was 2×108 TU/ml.Conclusion:The recombinant lentivirus vector could express TORC1 gene at a high level,and was very helpful in the study of exploring the effect of TORC1 on spinal cord injury.     

Targeted silencing of heparanase gene by small interfering RNA inhibits invasiveness and metastasis of osteosarcoma cells

The effects of targeted silencing of heparanase gene by small interfering RNA(siRNA) on invasiveness and metastasis of osteosarcoma cells(MG63 cells) were investigated in the present study.Two complementary oligonucleotide strands were synthesized and inserted into pGenesil-1 vector based on the mRNA sequence of heparanase gene.The expression vector containing short hairpin RNA(pGenesil-shRNA) was constructed successfully.MG63 cells were randomly allocated into 3 groups:blank group,empty vector(pGenesil) transfected group and expression vector(pGenesil-shRNA) transfected group.Under the induction of Lipofectamine 2000,the recombinants were transfected into MG63 cells.Heparanase gene expression level was detected by RT-PCR and Western blotting.Cell prolifera-tion was measured by MTT assay.Cell invasiveness and metastasis were examined by cell adhesion and Transwell-ECM assays.HUVECs migration assay was applied for the detection of angiogenesis.As compared with negative controls,the mRNA and protein expression levels of heparanase were down-regulated by 76.1%(P<0.01) and 75.3%(P<0.01) respectively in the pGenesil-shRNA transfected group.Meanwhile,the proliferation,adhesiveness,invasiveness and angiogenesis properties of MG63 cells were all significantly inhibited.It was suggested that targeted silencing of heparanase gene by siRNA could dramatically inhibit the invasiveness and metastasis of osteosarcoma cells.     

Enhanced expression of HLA class I molecules in human hepatocellular carcinoma cells transduced with gamma-interferon gene.

OBJECTIVE To investigate the expression of exogenous gamma-interferon gene in human hepatocellular carcinoma cells following retroviral transduction and the effect on the expression of surface HLA class I molecules.

METHODS Retroviral vector pLXSN was used to introduce human gamma-interferon (IFN-gamma) gene into four different human hepatocellular carcinoma cell lines (HCC). The G418-resistant colonies were isolated and cloned. The integration and expression of IFN-gamma gene were determined by PCR and RT-PCR analysis. A bioassay method was used to test the amount of IFN-gamma secreted by gene modified HCC cells. The expression of HLA class I molecules in HCC cells were analyzed by flow cytometry using indirect fluorescence staining.

RESULTS Four different HCC cell lines were successfully transduced with human IFN-gamma gene using retroviral vector. The integration and expression of IFN-gamma gene were shown only in the transduced cells. All four genetically modified HCC cells can secrete varied amount of IFN-gamma and demonstrate a significant up-regulation of surface HLA class I antigens. One specific HLA class I antigen, HLA-A2, has almost the same degree of increase as that of the total HLA class I molecules after transduction with IFN-gamma gene.

CONCLUSIONS Gene modification with IFN-gamma gene can significantly enhance the expression of HLA class I molecules in HCC cells and may increase its immunogenicity. These gene modified tumor vaccines can be helpful in tumor biotherapy.

     

In vitro effect of p2l~(WAF-1/CIP1) gene on growth of human glioma cells mediated by EGFR targeted non-viral vector GE7 system

Objective: To construct the EGFR targeted non-viral vector GE7 system and explore the in vitro effect of p21WAF-1/CIPI gene on growth of human glioma cells mediated by the GE7 system. Methods: The EGFR targeted non-viral vector GE7 gene delivery system was constructed. The malignant human glioma cell line U251MG was transfected in vitro with β-galactosidase gene ( reporter gene) and p21WAF-1/CIPI gee (therapeutic gene) using the GE7 system. By means of X-gal staining, MTS and FACS, the transfection efficiency of exogenous gene and apoptosis rate of tumor cells were examined. The expression of p21WAF-1/ CIPI gene in transfected U251MG cell was examined by immunohistochemis-try staining. Results: The highest transfer rate of exogenous gene was 70% . After transfection with p21WAF-1/CIPI gene, the expression of WAF-1 increased remarkably and steadily; the growth of U251MG cells were inhibited evidently. FACS examination showed G1 arrest. The average apoptosis rate was 25.2%. Conclusion: GE7 system has the     

Construction of genetically engineered macrophages expressing Smad6 andSmad7 genes with adeno-associated virus

To construct the genetically engineered macrophages expressing Smad6 and Smad7 genes with adeno-associated virus (AAV). Methods: The plasmids containing peDNA3-Smad6/Flag and peDNA3-Smad7/Flag were digested with BamH I and Xho I , respectively. Then the Smad6/Flag and Smad7/Flag gene segments obtained were cloned into plasmid pAAV-MCS respectively to construct the recombinant pAAV-Smad6/Flag and pAAV-Smad7/Flag plasmids. The resulting recombinant plasmids (pAAV-Smad6/Flag or pAAV-Smad7/Flag) or pAAV-LacZ plasmid were co-transfected into the HEK 293cells with pHelper and pAAV-RC by calcium-phosphate precipitation method. Recombinant AAV-2 viral particles were prepared from infected HEK293 cells and then were used to infect mouse macrophages. The expressions of Smad6and Smad7 in macrophages were detected by immunocytochemical staining and expression of b-galactosidase was evaluated by X-gal staining. Results: The recombinant AAV vector containing Smad6 or Smad7 genes was successfully constructed. More than 95% macrophage cells expressed X-gal and Smad6 and Smad7 genes at 72 h after infection. Conclusion: These results indicate that the genetically engineered macrophages can express Smad6 and Smad7 proteins effectively, laying the foundation for the studies of TGF-β-induced diseases in vivo and highlighting the feasibility of macrophage-based gene therapy.     

The effects of antisense PTEN gene transfection on the growth and invasion of glioma cells

Objective:To study the effects of antisense PTEN gene on the growth and invasion of glioma cells. Methods: A pcDNA3. 1/Hygro(-) recombinant plasmid containing antisense PTEN gene fragment was constructed. Glioma cells of primary culture were transfected with antisense PTEN gene vector and stably transfected clones were selected. Then, the different growth and invasion abilities and the different MMP9 mRNA expressions of three kinds of cells were observed, including the transfected cells, untrans-fected cells and the cells transfected with empty vector. Results:The abilities of growth and invasion of the transfected cells and the expressions of MMP9 mRNA were obviously enhanced. Conclusion: Antisense PTEN gene could have a negative impact on the growth and invasion of primary culture glioma cells.     

Construction of ER-β siRNA Expression Vector and Its Expression in Human Periodontal Ligament Fibroblast Cells

Objective To construct vectors expressing siRNA against human ER-β gene and study the inhibition effect of ER-β-siRNA on expression of ER-β in human periodontal ligament （HPDL） cells. Methods ER-β-specific siRNA gene was synthesized and cloned into pSilencer3.1-Hl-neo vector. The plasmid was identified by DNA sequencing. The constructed ER-β-siRNA was transfected into HPDL cells. The expression of ER-β in the levels of mRNA was detected by RT-PCR. Results It was confirmed by sequencing that the plasmid had been constructed successfully. The result of RT-PCR showed that Sequence-specific siRNAs targeting ER-β significantly down regulated the expression of ER-β gene in HPDL cells. Conclusion The ER-β siRNA plasmid was constructed successfully. ER-β gene expression of HPDL cells was inhibited by RNAi.