Comparative Immunization in BALB/c Mice with Recombinant Replication-Defective Adenovirus Vector and DNA Plasmid Expressing a SARS-CoV Nucleocapsid Protein Gene

In order to investigate immunogenicity in the induction of humoral and cellular immune responses, severe acute respiratory syndrome associated coronavirus （SARS-CoV）-N gene recombinant replication-defective adenoviral vector, rAd-N, was generated and immunized BALB/c mice in a pcDNA3.1-N prime-rAd-N boost regimen. After humoral and cellular immune response detection, different levels of SARS-CoV N protein specific antibodies and interferon-γ （IFN-γ） secretion are shown compared to controls. The humoral immune response was induced more effectively by the DNA priming and recombinant adenovirus boosting regimen. There is a significant difference between heterogeneous and homologous vaccinations. The heterogeneous combinations were all higher than those of the homologous combinations in the induction of anti-N antibody response. Among the three heterogeneous combinations, pcDNA3.1-N/pcDNA3.1-N/pcDNA3.1-N/rAd-N induced the strongest antibody response. In the induction of IFN-γ production, the homologous combination of rAd-N/rAd-N/rAd-N/rAd-N was significantly stronger than that of pcDNA3.1-N/pcDNA3.1-N/pcDNA3.1-N/pcDNA3.1-N, but was relatively weaker than the heterogeneous combination of pcDAN3.1-N/pcDAN3.1-N/pcDAN3.1-N/rAd-N. This combination was a most efficient immunization regimen in induction of SARS-CoV-N-specific （IFN-γ） secretion just as the antibody response. These results suggest that DNA immunization followed by recombinant adenovirns boosting could be used as a potential SARS-CoV vaccine.

Recombinant adenovirus vector Ad-hIL-10 protects grafts from cold ischemia-reperfusion injury following orthotopic liver transplantation in rats

BACKGROUND: Interleukin 10 (IL-10), a Th2 type cytokine, modulates inflammatory responses by inhibiting the production of proinflammatory cytokines. This study was designed to investigate the protective effects of adenovirus-mediated human IL-10 (Ad-hIL-10) gene transfer on protecting grafts from cold ischemia-reperfusion injury following orthotopic liver transplantation in rats. METHODS: Adenoviruses encoding hIL-10 or beta-galactosidase (Ad-lacZ) were injected via the superior mesenteric vein into prospective donor animals. The donor liver was harvested 48 hours after transduction, and stored for 12 hours at 4 degrees C in lactated Ringer's solution prior to transplantation. The rats were divided into saline, Ad-lacZ, and Ad-hIL-10 groups. Liver function test, histopathological examination, reverse transcriptase-polymerase chain reaction (RT-PCR), and Western blotting were performed at 24 hours after transplantation in the three groups. RESULTS: Liver function (ALT and AST) was significantly improved, and the Suzuki score was significantly decreased in the Ad-hIL-10 group. The levels of hepatic TNF-alpha, MIP-2, ICAM-1 mRNA, and NF-kappa B protein in the Ad-hIL-10 group were significantly decreased. The expression of hIL-10 mRNA was detected by RT-PCR in Ad-hIL-10-treated grafts but not in controls treated with saline or Ad-lacZ. CONCLUSIONS: Donor pretreatment with Ad-hIL-10 down-regulates the expression of proinflammatory cytokines TNF-alpha, MIP-2, and ICAM-1 mRNA. hIL-10 protects against hepatic cold ischemia-reperfusion injury, at least in part, by suppressing NF-kappa B activation and subsequent expression of proinflammatory mediators. (Hepatobiliary Pancreat Dis Int 2010; 9: 144-148)

Adenovirus vector expressing mda-7 selectively kills hepatocellular carcinoma cell line Hep3B

BACKGROUND: Melanoma differentiation associated gene-7 (mda-7) is a novel tumor suppressor gene, which has suppressor activity in a broad spectrum of human cancer cells both in vitro and in vivo through activation of various intracellular signaling pathways. In this study, we investigated the potential effect of mda-7 on human hepatocellular carcinoma (HCC) in vitro. METHODS: Cells from the human HCC cell line Hep3B and the human liver cell line L-02 were assigned to three groups. One was cultured in Dulbecco's modified Eagle's medium without serum (control). The others were transfected with adenovirus expressing the mda-7 gene (Ad.mda-7) or adenovirus vector serving as negative control (Ad.vec). The expression of MDA-7 and Bcl-2 proteins in Hep3B and L-02 cells was confirmed by the reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay. The methyl thiazolyl tetrazolium colorimetric assay and flow cytometry were used to assess tumor cell proliferation and the cell cycle. Hoechst and Annexin-V/propidium iodide staining were used to study mda-7 gene expression in Hep3B and L-02 cells. The expression of MDA-7, Bcl-2 and Bax proteins were detected by Western blotting. RESULT S: The mda-7 gene was expressed in Hep3B and L-02 cells. The protein concentrations of MDA-7 in supernatants were 790 and 810 pg/ml, respectively. mda-7 induced Hep3B growth suppression and apoptosis, compared with Ad.mda-7 and control (P<0.01). In addition, cell block in G2/M was identified by exposure of HCC cells to secreted MDA-7 protein, but this was not found in L-02. The gene expression of Bcl-2 was markedly decreased in Hep3B but not in L-02. CONCLUSIONS: mda-7 selectively induces growth inhibition and apoptosis in the HCC cell line Hep3B but not in the normal liver cell line L-02 via downregulating the antiapoptosis protein Bcl-2. It could be an ideal gene for gene therapy in HCC.

Immunological tolerance of human hepatocyte xenograft induced by adenovirus vector-mediated CTLA4Ig gene transfer

BACKGROUND:Systemic administration of CTLA4Ig has been applied in inducing immunological tolerance of hepatocyte implants,but has potential for systemic immune inhibition.This study was designed to induce hepatocyte immunological tolerance by locally expressing CTLA4Ig in an attempt to improve the effectiveness of cell transplantation.METHODS:A normal human liver cell line(L02) was transfected with adenovirus vector containing the CTLA4Ig gene(Ad-CTLA4Ig-EGFP) in vitro,and the expression of CTLA4Ig by transfected cells was assessed by fluorescent imaging and immunocytochemical staining.Transfected cells then were injected into the spleen of Sprague-Dawley rats,the survival of cells was determined by immunohistochemistry,and the immune status was examined through CD4 + and CD69 + T cellcounts and ELISA detection of IL-2 in peripheral blood.RESULTS:L02 cells expressed CTLA4Ig in the cytoplasm for >4 weeks.Surviving L02 cells were observed in the experimental group at 3 and 4 weeks post-transplantation,while none was detected in the control group.Furthermore,the percentages of CD4 + and CD4 + CD69 + T cells in the CTLA4-transfected group were 24.5% and 45.1%,markedly lower than those in the control group at 4 weeks post-transplantation(P<0.01).Furthermore,the IL-2 level was also lower in the CTLA4transfected group than in the control group.CONCLUSION:Adenovirus-mediated CTLA4Ig gene transfer into human hepatocytes has the potential to become an effective method of inducing immunological tolerance in hepatocyte transplantation.

Construction of adenovirus vectors encoding the lumican gene by gateway recombinant cloning technology

AIM： To construct adenovirus vectors of lumican gene by gateway recombinant cloning technology to further understand the role of lumican gene in myopia. METHODS： Gateway recombinant cloning technology was used to construct adenovirus vectors. The wild-type （wt） and mutant （mut） forms of the lumican gene were synthesized and amplified by polymerase chain reaction （PCR）. The lumican cDNA fragments were purified and ligated into the adenovirus shuttle vector pDown- multiple cloning site （MCS）-/internal ribozyme entry site （IRES）/enhanced green fluorescent protein （EGFP）. Then the desired DNA fragments were integrated into the destination vector pAV.Desld yielding the final expression constructs pAV.Exld-CMV〉wt-lumican/IRES/ EGFP and pAV.Exld-cytomegalovirus （CMV） 〉mutlumican/IRES/EGFP, respectively.RESULTS： The adenovirus plasmids pAV.Exld-CMV〉 wt-lumican/IRES/EGFP and pAV.Exld-CMV 〉mutlumican/IRESlEGFP were successfully constructed by gateway recombinant cloning technology. Positive clones identified by PCR and sequencing were selected and packaged into recombinant adenovirus in HEK293 cells. CONCLUSION： We construct adenovirus vectors containing the lumican gene by gateway recombinant cloning technology, which provides a basis for investigating the role of lumicangene in the pathogenesis of high myopia.

Construction and characterization of calreticulin-HBsAg fusion gene recombinant adenovirus expression vector

AIM: To generate recombinant adenoviral vector con-taining calreticulin (CRT)-hepatitis B surface antigen (HBsAg) fusion gene for developing a safe, effective and HBsAg-specific therapeutic vaccine.METHODS: CRT and HBsAg gene were fused using polymerase chain reaction (PCR), endonuclease diges-tion and ligation methods. The fusion gene was cloned into pENTR/D-TOPO transfer vector after the base pairs of DNA (CACC) sequence was added to the 5′ end. Adenoviral expression vector containing CRT-HBsAg fusion gene was constructed by homologous recombinan-tion. The human embryo kidney (HEK) 293A cells were transfected with linearized DNA plasmid of the recombi-nant adenoviral vector to package and amplify recombi-nant adenovirus. The recombinant adenovirus titer was characterized using the end-dilution assay. The expres-sion of the CRT/HBsAg fusion protein in Ad-CRT/HBsAg infected 293A cells was detected by Western blotting.RESULTS: The CRT-HBsAg fusion gene was char-acterized by PCR and sequencing and its length and sequence were confirmed to be accurate. The CRT-HB-sAg fusion gene recombinant pENTR/D-TOPO transfer vector was constructed. The recombinant adenoviral vector, Ad-CRT/HBsAg, was generated successfully. The titer of Ad-CRT/HBsAg was characterized as 3.9 × 1011 pfu/mL. The CRT-HBsAg fusion protein was ex-pressed by HEK 293A cells correctly. CONCLUSION: CRT/HBsAg fusion gene recombinant replication-defective adenovirus expression vector is constructed successfully and this study has provided an experimental basis for further studies of Hepatitis B vi-rus gene therapy.

In situ tumor vaccination with adenovirus vectors encoding measles virus fusogenic membrane proteins and cytokines

AIM: To evaluate whether intratumoral expression of measles virus fusogenic membrane glycoproteins H and F (MV-FMG), encoded by an adenovirus vector Ad.MV-H/ F, alone or in combination with local coexpression of cytokines (IL-2, IL-12, IL-18, IL-21 or GM-CSF), can serve as a platform for inducing tumor-specifi c immune responses in colon cancer. METHODS: We used confocal laser scanning microscopy and flow cytometry to analyze cell-cell fusion after expression of MV-FMG by dye colocalization. In a syngeneic bilateral subcutaneous MC38 and Colon26 colon cancer model in C57BL/6 and BALB/c mice, we assessed the effect on both the directly vector-treated tumor as well as the contralateral, not directly vector- treated tumor. We assessed the induction of a tumor- specific cytotoxic T lymphocyte (CTL) response with a lactate dehydrogenase (LDH) release assay. RESULTS: We demonstrated in vitro that transduction of MC38 and Colon26 cells with Ad.MV-H/F resulted in dye colocalization, indicative of cell-cell fusion. In addition, in the syngeneic bilateral tumor model we demonstrated a signifi cant regression of the directly vector-inoculated tumor upon intratumoral expression of MV-FMG alone or in combination with the tested cytokines. We observed the highest anti-neoplastic efficacy with MV-FMG and IL-21 coexpression. The degree of tumor regression of the not directly vector-treated tumor correlated with the anti-neoplastic response of the directly vector-treated tumor. This regression was mediated by a tumor-specifi c CTL response. CONCLUSION: Our data indicate that intratumoral expression of measles virus fusogenic membraneglycoproteins is a promising tool both for direct tumor treatment as well as for tumor vaccination approaches that can be further enhanced by cytokine coexpression.

Gene therapy for allergic rhinitis with recombinant adenovirus vector containing CTLA4Ig in mice

Objective: To examine the role of recombinant adenovirus vector containing CTLA4Ig gene(Ad-CTLA4Ig) in the treatment of induced allergic rhinitis in mice.Methods: Allergic rhinitis was induced by sensitizing and challenging with ovalbumin(OVA).Ad-CTLA4Ig was intraperitoneally injected 30 min before OVA challenge.Adenovirus vector without inserted CTLA4Ig cDNA served as the control.The symptoms and morphological changes of nasal mucosa of each group were observed, and the serum levels of IgE against OVA were detected with ELISA.Results: There were no obvious symptoms and pathological changes in Ad-CTLA4Ig treated group, in which the serum OVA-specific IgE levels were significantly lower than that in control groups(P< 0.05).Conclusion: Ad-CTLA4Ig prevents and treats allergic rhinitis of mice,implying the possibility of the usage of Ad-CTLA4Ig against allergic rhinitis in clinic in future.

CONSTRUCTION OF THE DICISTRONIC ADENOVIRUS VECTOR EXPRESSING BIOACTIVE HUMAN INTERLEUKIN-12

The full-length cDNA encoding the subunits p40 and p35 of human interleukin12(hIL12) were cloned separately by RTPCR, linked together by internal ribosomal entry site (IRES) of encephalomyocarditis virus which initiates capindependent translation to form a dicistronic gene fragment. The dicistronic fragment was placed between the cytomegalovirus (CMV) promoter and SV40 polyA signal to form a dicistronic expression cassette. Subsequently, the dicistronic expression cassette was inserted into E1 region of Ad5 genome in cosmid vector pAx1cw of E1substitution type. By homologous recombination with EcoT22Idigested Ad5 DNATPC in 293 cells, the replicationdeficient recombinant adenoviruses of hIL12 were generated efficiently. After infected with hIL12 recombinant adenoviruses in vitro, 293 cells, human hepatocellular carcinoma cells HepG2, and primary human skin fibroblasts expressed and secreted hIL12 at comparable levels (30～60ng/ 106cells/24hr), which could stimulate the proliferation and IFNγ production of human lymphoblasts. These suggest that the dicistronic adenovirus vector of hIL12 could effectively mediate the expression of bioactive hIL12 and might be used in cancer gene therapy.

Construction and Expression of Human PTEN Tumor Suppressor Gene Recombinant Adenovirus Vector

The recombinant defective adenovirus vector carrying human PTEN tumor suppressor gene was constructed by using AdEasy-1 system and its expression was detected in human breast cancer cell line MDA-MB-468. Human PTEN cDNA was cloned into adenovirus shuttle plasmid pAdTrack-CMV to generate a recombinant plasmid pAdTrack-CMV-PTEN, then homologeous recombination was carried out in the E. coli BJ5183 by contransforming linearized shuttle vector with adenovirus backbone plasmid pAdEasy-1. The newly recombined defective adenovirus vector Ad- PTEN containing green fluorescent protein （GFP） was packaged and propagated in 293 cells. After being purified by cesium chloride gradient centrifugation, the adenovirus was transfected into human breast cancer cell line MDA-MB-468 in vitro. The expression of PTEN mRNA and protein in infected human breast cancer cell line MDA-MB-468 was detected by RT-PCR and Western blot respectively. The recombinant defective adenovirus vector carrying PTEN gene was constructed successfully. The viral titer of purified adenovirus was 2. 5 × 10^10 pfu/mL, and about 70 % breast cancer cells were infected with Ad-PTEN when multiplicity of infection （MOI） reached 50. The exogenous PTEN mRNA and protein were expressed in MDA-MB-468 cells infected with Ad-PTEN by RT-PCR and Western blot. The recombinant defective adenovirus vector of PTEN gene was constructed successfully using AdEasy-1 system rapidly, which paved a sound foundation for gene study of breast cancer.

THE ROLE OF RECOMBINANT Rb GENE ADENOVIRUS VECTOR IN THE GROWTH OF LUNG ADENOCARCINOMA CELLS

Objective: To study the role of the most extensively studied tumor suppressor gene, retinoblastoma (Rb) gene, on the growth of lung adenocarcinoma cell line GLC 82 and explore a gene therapy approach for lung adenocarcinoma Methods: The recombinant Rb gene adenovirus vector was constructed, the control virus which carries LacZ gene was producted by the same method Infection effects were detected by biochemical staining of β gal and immunohistochemical analysis of Rb protein The Rb cDNA of infected cells were determined by PCR The cell growth rate and cell cycle were observed by cell counting and flow cytometry Results: The constructed recombinant adenovirus vector could infect effectively the cells with high level expression of Rb cDNA and Rb protein The transfection of wild type Rb gene could suppress GLC 82 cell proliferation and decrease the cellular DNA synthesis Conclusions: These results showed the possibility of using recombinant Rb gene adenovirus vector in the gene therapy of cancer to inhibit the growth of cancer

CONSTRUCTION OF A RECOMBINANT ADENOVIRUS VECTOR OF HUMAN PAPILLOMAVIRUS TYPE 16 L1_E7C

Objective: Human papillomaviruses are closely associated with human cervical cancer, especially HPV types 16 and 18. At present, HPV can not be produced in large quantity; it also has tumorgenicity and these properties of HPV have seriously hampered the development of HPV vaccine. HPV type 16 LI proteins can assembled into virus-like particles (VLP), which are morphologically identical to the nature virion. In order to develop the recombinant adenovirus vectors of HPV, we constructed a recombinant adenovirus shuttle plasmid pCA14 L1-E7C. Methods: Human papillomavirus type 16 LI open reading frame without terminator codon (TAA) (5559–7152) and E7C (682–855) were amplified using PCR. The L1 and E7C fragments were inserted into pGEM-T easy vectors by T-A strategy, named pTAL1 and pTAE7C. pTAL1 was cut with Hind III and BgIII, the pTAE7C with BamHI and ClaI. The L1 DNA fragment, E7C and pBluesscript SK were ligated together using T4 DNA ligase. pBSL1-E7C and pBSL1-E7C was digested with Hind III and Xhol. The L1-E7C fragment was inserted into adenovirus shuttle plasmids pCA14, named pCA14L1-E7C. DNA sequence results indicated that The L1-E7C DNA fragment can encode the HPV16L1-E7 fusion protein correctly. Results: The L1 and E7C DNA fragments were amplified by PCR and recombinant plasmid pTAL1, pTAE7C, pBSL1-E7C and pCA14L1-E7C were constructed correctly. The pCA14L1-E7C can be used in the further research work, cotransfected the 293 cell with the parent adenovirus pBHG10. Conclusion: Our results indicated that we have constructed a HPV16L1-E7 fusion DNA fragments and the adenovirus shuttle plasmids pCAL1-E7C for the further research.

Preparation and identification of monoclonal antibodies against the adenovirus vector

Objective:To prepare and identify monoclonal antibodies(McAbs)against the capsid proteins of adenovirus vector.Methods:BALB/c mice were immunized with a mixture of the purified adenovirus vector(Adv)and Al(OH)3.McAbs were produced using cell fusion technique in a conventional way.The sensitivity and specificity of monoclonal antibodies was identified by indirect enzyme linked immunosorbent assay(ELISA),immunocytochemical staining and Western blotting. Results:Six strains of hybridoma cells(A4H11,A8C7,F1H5,G1D2,G4E3 and H2G8)that can stably secrete the IgG1 McAb against Adv were obtained.After 3 months subculture and low concentration of serum adapting culture,six strains retained their stability to secrete McAb.The ascites titers were between 1:106 and 1:108.Western blot analysis demonstrated that all the McAbs reacted with one protein(about 114 kDa)which is present in wild type 3 adenovirus(wtAd3),wild type 5 adenovirus (wtAd5),wild type 7 adenovirus(wtAd7)and adenovirus vector.Conclusion:Successfully prepared six strains of hybridoma cell secreted monoclonal antibodies against the hexon proteins of adenovirus vector,and provided the substantial foundation of preclinical research of adenovirus vectors.

Construction and expression of SET gene and siRNA recombinant adenovirus vectors

Objective:To construct SET gene recombinant adenovirus vector and SET gene small interfering RNA(SiRNA) recombinant adenovirus vector for over-expression or knock-down of SET levels. Methods:The cDNA sequence of SET was cloned by reverse transcriptive polymerase chain reaction(RT-PCR) and the SET gene fragment was subcloned into adenovirus shuttle plasmid pAdTrack-CMV to construct the shuttle plasmid pAdTrack-SET.The shuttle plasmid pAdtrack-SET was transformed into BJ5183 cells with the adenoviral backbone pAdEasy-1 to obtain the homologous recombinant Ad-CMV-SET and the recombinant Ad-CMV-SET was packaged and amplified in the AD293 cells.The expression of SET in AD293 cells was detected by Western blot.In addition,we constructed SET gene SiRNA recombinant adenovirus vector(Ad-H1-SiRNA/SET) and its efficacy of knockdown of SET protein was detected in infected GC-2spd(ts) cells by Western blot. Results:The recombinant adenovirus vectors,both SET gene recombinant adenovirus vector Ad-CMV-SET and SET gene SiRNA recombinant adenovirus vector Ad-H1-SiRNA/SET,were proven to be constructed successfully by the evidence of endonulease digestion and sequencing.AD293 cells infected with either recombinant adenovirus vector of Ad-CMV-SET or Ad-H1-SiRNA/SET were observed to express GFP.The expression of SET protein was up-regulated significantly in AD293 cells infected with SET gene recombinant adenovirus vector.On the contrast, SET protein was significantly down-regulated in the GC-2spd(ts) cells infected with Ad-H1-SiRNA/SET (P<0.05) and the knockdown efficiency was approximately 50%-70%. Conclusion:The recombinant adenovirus vector Ad-CMV-SET and Ad-H1-SiRNA/SET were successfully constructed and effectively expressed in germ cells and somatic cells.It provides an experimental tool for further study of SET gene in the physiological and pathophysiological mechanism of reproduction-related diseases.

Construction of the recombinant adenovirus vectors of CALB_2 gene and small interfering RNA,and application in testicular Leydig cells

Objective:To construct the recombinant adenovirus vectors of calretinin(CALB_2) gene and small interfering RNA(siRNA),for over-expression or knock-down of CALB_2,as the basis of functional investigation of CALB_2 in testicular Leydig cells. Methods:The cDNA sequence of CALB_2 was cloned by the reverse transcriptive polymerase chain reaction (RT-PCR).A CALB_2 gene fragment was sub-cloned into adenovirus shuttle plasmid pAdTrack-CMV to construct the shuttle plasmid pAdTrack-CALB_2.Then it was transformed into BJ5183 cells with the adenoviral backbone pAdEasy-1 to obtain the homologous recombinant AdCMV-CALB_2.The recombinant AdCMV-CALB_2 was further packaged and amplificated in AD293 cells.The expression of CALB_2 protein in AD293 cells was detected by Western blotting.CALB_2 protein was over-expressed in mouse Leydig cell line(MLTC-1 cells) by the constructed AdCMV-CALB_2. CALB_2 gene siRNA recombinant adenovirus vector(Ad-H1-siRNA/CALB_2 was also constructed simultaneously. Its efficacy was detected in AD293 cells by Western blotting. Results:The CALB_2 gene recombinant adenovirus vector AdCMV-CALB_2 and the CALB_2 gene siRNA recombinant adenovirus vector Ad-H1-siRNA/CALB_2 were constructed successfully by endonulease digestion and sequencing. AD293 cells infected with AdCMV-CALB_2 or Ad-H1-SiRNA/CALB_2 significantly expressed GFP protein. The expression of CALB_2 protein was significantly up-regulated in AD293 cells infected with AdCMV-CALB_2 plasmids, while the expression of CALB_2 protein was down-regulated by 60%in the CALB_2 cells infected with Ad-H1-SiRNA/CALB_2. MLTC-1 cells did not markedly express CALB_2 protein,while MLTC-1 cells infected with AdCMV-CALB_2 expressed CALB_2 protein at a high level. Conclusions:The recombinant adenovirus vectors of AdCMV-CALB_2 and Ad-H1-SiRNA/CALB_2 were successfully constructed.Both vectors effectively expressed in AD293.CALB_2 protein was over-expressed in the cultured MLTC-1 cells by AdCMV-CALB_2.These vectors of CALB_2 gene and Leydig cell line are useful tools for investigating the testicular function.

Inhibition of Metastatic Progression of SSTR2 Gene Transfection Mediated by Adenovirus in Human Pancreatic Carcinoma Cells

The inhibition of metastatic progression of Somatostatin receptor type 2 （SSTR2） gene transfection mediated by adenovirus in human pancreatic carcinoma cells and the mechanisms involved in this effect were studied. The full-length human SSTR2 cDNA was introduced into the pancreatic cancer cell line BXPC-3 by adenovirus-mediated transfection. Stable expression of mRNAs and protein of SSTR2 was detected by RT-PCR and Western-blot. The Matrigel-coated Transwell was used to detect the migratory and invasive ability of SSTR2-expressing cells, Adv-GFP control cells and mock control cells. Furthermore, the expression of matrix metalloproteinase-2 （MMP-2） and tissue inhibitor of metalloproteinase-2 （TIMP-2） was detected by RT-PCR in these cells. The stable expression of SSTR2 was detected in BXPC-3 transfected by Adv-GFP-SSTR2. A dramatic decrease of BXPC-3 expressing sst2 cells migrating through a Matrigel-coated filter was observed, as compared with Adv-GFP control and mock control cells （P〈0. 01）. Moreover, the expression of MMP-2 mRNA was significantly reduced in the SSTR2-expressing cells and converse- ly the expression of TIMP-2 mRNA was significantly increased in the SSTR2-expressing cells when compared with the Adv-GFP control and mock control （P〈0. 01）. The expression of reintroduced human SSTR2 gene in BXPC-3 cells by Adv-GFP-SSTR2 had the anti-migratory and anti-invasive effects, and the mechanisms involved in this effect may be due to the down-regulated expression of MMP-2 and up-regulated expression of TIMP-2.

Construction and identification of recombinant adenovirus-mediated gene transfer system for rat vascular endothelial growth factor

Objective： To construct the recombinant adenovirus vector carrying rat vascular endothelial growth factor（VEGF）, as preparation for genetic transfection that follows. Methods： Rat VEGF was obtained by using RT-PCR amplification and then cloned into the shutter plasmid pDC316. Subsequently, this newly constructed plasmid pDC316-VEGF, after identification by nuclease digestion analysis and sequencing analysis, was transfected into human embryonic kidney cells HEK293 by Lipofectamine 2000 mediation, together with adenovirus-packaging plasmid pBHGE3. Based on the homologous recombination of the two plasmids within HEK293 cells, the recombinant adenovirus vector carrying VEGF and VDC316-VEGF was created. VDC316-VEGF was subsequently identified using PCR, purified using repeated plaque passages, proliferated using freezing and melting within HEK293 cells, and titrated using 50% Tissue Culture Infective Dose（TCID50） assay. Results：The newly constructed recombinant adenovirus was confirmed to carry rat VEGF based on PCR results, and its titration value determined based on TCID50 assay was 3 × 10^9 pfu/ml. Conclusion：The recombinant adenovirus carrying rat VEGF was successfully constructed. The newly constructed adenovirus can produce a ufficiently high titration value within HEK293 cells, providing a reliable tool for genetic transfection in further gene therapy researches.

Adenovirus-Mediated Herpes Simplex Virus Thymidine Kinase Gene Transfer Driver by KDR Promoter in Treatment of Experimental Human HepatocelLular Carcinoma in Nude Mice

Objective： To investigate the therapeutic efficacy of adenovirus-mediated herpes simplex virus thymidine kinase （HSV-tk） gene transfer under the driving of KDR promoter （AdKDR-tk） in combination of ganciclovir （GCV） against human hepatocellular carcinoma in nude mice. Methods： HepG2 cell line was implanted subcutaneously into 32 nude mice, which were subsequently divided into 4 groups （n=8 each group）： Ganciclovir group （Ⅰ）, Ad group （Ⅱ）, AdCMV-tk/GCV group （under the driving of CMV promoter） （Ⅲ） and AdKDR-tk/GCV group （Ⅳ）. Then intratumoral injection of recombinant adenovirus or Ad was performed in all nude mice, and repeated 24 h later. For the following 10 d GCV was given at a dose of 100 mg/（kg· d）, ip. All the treated animals were killed to evaluate the tumor weight and the histopathological changes and the microvessel density of tumors after the treatment was determined. Results Compared with group Ⅰ, the tumor inhibitory rate was 12.3% in group Ⅲ and 24.5% in group Ⅳ; the inhibition rates were significantly different between group Ⅲand Ⅳ （P〈0.05）. The mean MVDs in group Ⅰ, Ⅱ, Ⅲ and Ⅳ were 37.4±8.6, 30.6±7.8, 27.6±7.1, and 10.7±4.1 （microvessels/mm^2）, respectively. Significant differences were found between group Ⅲ and Ⅱ （P〈0.05）, Ⅳ and Ⅱ （P〈0.01）, and Ⅳ and Ⅲ （P〈0.01）. Conclusion： Intratumoral injection of AdKDR-tk results in marked inhibition of HCC growth through inhibition angiogenesis in nude mice. It may be a new treatment approach for human HCC,

In Vitro Anti-tumor Immune Response Induced by Dendritic Cells Transfected with Recombinant Adenovirus Carrying Mutant K-ras Genes

Summary： The specific anti-tumor immune response induced by mouse bone marrow dendritic cells （DCs） lransfected with recombinant adenovirus carrying mutant k-ras genes was investighted. DCs were generated from mouse bone marrow in the presence of rmGM-CSF （3.3 ng/mL） and rmIL-4 （1.3 ng/mL） and detected by FACS, and then transfecled with the recombinant adenovirus encoding mutant k ras gene. The efficacy of transfection and T cell stimulating activity of DCs were detected. CTL activity of the mice vaccinated with DCs was observed. The resuhs showed thai DCs had dendritic veiled morphology. BmDCs highly expressed B7-1（80%）, B7-2（77%）, MHC Ⅱ （70%）, CDllc （65%）, CD40 （70%） and CD54 （96%） with FACS, and no significant difference in the expression was observed before and after the transfection （P〈0.05）. The DCs transfeeled by mutant k-ras gene could significantly stimulate lymphoeytes proliferation as compared with those transfeeted by Ad e or non-modified DCs （P〈0.05）. DC vaccine transfected by mutant k-ras gene could induce CTL activity against Lewis lung cancer, but not against B16. The specific eytotoxicity against Lewis lung cancer in Ad-k-ras/12-transdueed DC group was signifieantly higher than those in the control, vector and non transfeeted DCs groups （P〈0.05）. It was concluded that special antitumor response could be induced by DCs transfected with recombinant adenovirus carrying mutant k-ras genes.

In vitro suppression of vascular smooth muscle cell proliferation using adenovirus-mediated transfer of the cd gene

Objective : To develop a treatment strategy for restenosis with a replication-deficient recombinant adenovirus (AdCMVCD) containing the cd gent whose gene product phosphorylates the prodrug 5-flurocytosine to form a nucleoside analog that inhibits DNA synthesis. Methods: Cultured primary rabbit smooth muscle cell (SMC) infected with AdCMVCD was incubated in 5- flurocytosine-containing medium and assayed with a Coulter Counter on day 2, 6, 10, and 14 for the establish- ment of proliferation curves. In addition, to observe an inhibitory effect on neighboring SMC, only a portion of the SMC population received the cd transgene. Results : The antiproliferative effect of AdCMVCD was dependent on both concentation of virus and concentration of 5-flurocytosine . Transformed cells demonstrated a bystander effect wherein SMC expressing cd gene exerted an antiproliferative effect on neighboring cells that did not express cd gene . Conclusion:Theresult demonstrates the potential utility of adenovirus-mediated cd gene transfer for the treatment of restenosis after balloon injury.