Objective:We investigate whether replication-competent adenovirus Ad5(E1+E3+) could enhance the expression of hCNTF secreted by celllines after replication-defective adenovirus Ad5-hCNTF infection in vitro.Methods:Plasmid encoding human CNTF was digested and subcloned into shuttle plasmid pshuttle to obtain a recombinant plasmid pshuttle-hCNTF.Using Ad-EASY system,the lineared and dephosphorylated shuttle vector and pAdEasy-1 plasmid which contain most of the human adenovirus serotype 5 genome were cotransfomed into BJ5183 bacteria,a recombination event then take place and result in the production of recombinant AdEasy plasmid,then the plasmid carrying adenovirus major genome and target gene was transfected into package cells AD-293 celllines to obtain a replication-defective adenovirus Ad5-hCNTF.Results:The recombinant adenovirus Ad5-hCNTF were analyzed by PCR and the expression of hCNTF in retinal pigment epithelium(RPE) cells after Ad infection were examined by Western blotting,and the ability of the cell to secrete CNTF was measured with a sandwich enzyme-linked immunosorbent assay(ELISA).the results indicated that Ad5-hCNTF contained the target gene,and were capable of infecting RPE celllines and produce the protein into the supernatant.Conclusion:We also have demonstrated that combined with different amount of replication-competent adenovirus Ad5(E1+E3+) in vitro,the expression of hCNTF secreted by celllines after Ad infection was enhanced significantly,and the enhancement of gene expression was observed in a concentration-dependent manner,and it offers basis for further study of gene therapy by Ad.

Purpose: This study aims to generate a more potent oncolytic adenovirus, Ad.hTERT-E1A/CMV-CD, which combines therapeutic gene and oncolytic effect. Methods: A human telomerase reverse transcriptase (hTERT) gene promoter was used to regulate the expression of adenoviral immediate-early gene 1A (E1A) to induce selective replication of recombinant adenovirus in tumor cells. To further enhance antitumor effect, a cytomegalovirus (CMV) promoter-driven Escherichia coli cytosine deaminase (CD) gene expression cassette was further incorporated into E1 region and the antitumor effect of this novel adenovirus was evaluated in vitro and in vivo. Results: Ad.hTERT-E1A/CMV-CD was capable to selectively replicate and lyse in various human tumor cell lines, including NCIH460, SW1990, and HeLa, while causing no damage to primary fibroblasts. The combined therapy of Ad.hTERT-E1A/CMV-CD with prodrug 5-fluorocytosine (5-FC) elicited a greater killing effect on tumor cells than Ad.hTERT-E1A/CMV-CD alone, and it synergistically suppressed tumor growth in BALB/c nude mice bearing human lung tumor. Conclusions: As telomerase is reactivated in a broad spectrum of tumors and prodrug 5-FC is much safe than its metabolized 5-fluorouracil, a chemotherapeutic agent in the treatment of many malignancies, Ad.hTERT-E1A/CMV-CD in combination with 5-FC may be a potential strategy for the treatment of a wide range of solid tumors.

Background Non-small cell lung cancer (NSCLC) is the leading cause of cancer related mortality, any improvements in therapeutic strategies are urgently required. In this study we generated a novel 'suicide gene' armed oncolytic adenoviral vector and investigated its antitumor effect both in vitro and in vivo. Methods Since the up-regulated expression of human telomerase reverse transcriptase (hTERT) is a hallmark of alltypes of NSCLC, we chose hTERT promoter to transcriptionally control E1A gene expression to obtain adenoviral replication in NSCLC. In order to further enhance anti-tumor effect of this oncolytic adenoviral vector, we inserted a 'suicide gene' i.e. Herpes Simplex Virus Thymidine Kinase (HSV-TK) into oncolytic adenoviral vector to engineer a novel armed oncolytic adenoviral vector 'Ad.hTERT-E1A-TK'. Results Ad.hTERT-E1A-TK efficiently killed different types of tumor cells including two types of NSCLC cells in vitro , causing no damage to normal primary fibroblasts. Furthermore, Ad.hTERT-E1A-TK infection combined with administration of prodrug gancyclovir (GCV) resulted in more potent cytotoxicity on NSCLC cells, and synergistically suppressed human NSCLC tumor growth in nude mice. Conclusion The results from this study showed that Ad.hTERT-E1A-TK/GCV could be a potent but safe anti-tumor strategy for NSCLC biotherapy.

A fusion gene called Ig-BDNF, in which brain-derived neurotrophic factor cDNA fused to the 3' end of signal peptide of Ig coding sequence, was constructed by PCR, digested and subcloned into shuttle plasmid pSNAV to obtain a recombinant plasmid pSNAV-Ig-BDNF. Then the plasmid encoding fusion protein was transfected into 293 cell lines and the stably transfected clones were selected with neomycin. AAV1 containing Ig-BDNF fusion gene vectors were obtained by super-infection by Herpes virus. The resultant adeno-associated virus vectors AAV-Ig-BDNF were confirmed by PCR, Western blotting and a sandwich enzyme-linked immunosorbent assay (ELISA) after infection of 293 cell lines. The results indicated that AAV-Ig-BDNF contained the target gene, and infected cells and produced the fusion protein into the supernatant. The content of BDNF in medium per 5x104 cells over a 24 h incubation period reached 1000 pg/mL. With the help of non-replicative adenovirus during AAV-Ig-BDNF infection, the expression of BDNF increased 7-8 fold, and the enhancement of BDNF gene expression was observed in a concentration-dependent manner. These results suggested that a functional AAV-Ig-BDNF was successfully constructed and it offers basis for further study for gene therapy of neural degeneration diseases.

Objective To construct signal transduction and activators of transcription 3 (STAT3) short hairpin RNA (shRNA) expression vector and to investigate its inhibitory effects on STAT3 expression, cell proliferation and apoptosis of human pancreatic cancer. Methods Three pairs of hairpin-like oligonucleotide sequences specific for human STAT3 gene were designed and synthesized. The annealed oligonucleotide fragments were subcloned into pRNAT-U6.1/Neo plasmid. The STAT3 shRNA expressing vectors were confirmed by PCR and DNA sequencing. STAT3 mRNA and protein expression were examined by using reverse transcription polymerase chain reaction (RT-PCR) and Western blot, respectively. MTT assay and flow cytometry were performed to detect the state of cell proliferation and cell apoptosis, respectively. Results PCR and DNA sequencing showed that the oligonucleotide fragments were correctly inserted into pRNAT-U6.1/Neo plasmid. STAT3 expression and cell proliferation in the transfected cells was inhibited significantly by three STAT3 shRNA expressing vectors (P<0.05). ConclusionSTAT3 shRNA expression vector can effectively inhibit the expression of STAT3. Silencing of STAT3 with RNAi can significantly inhibit the proliferation and promotes the apoptosis of pancreatic cancer cells and may provide a novel therapeutic target for treating pancreatic cancer.

Objective To construct signal transduction and activators of transcription 3 (STAT3) short hairpin RNA (shRNA) expression vector and to investigate its inhibitory effects on STAT3 expression, cell proliferation and apoptosis of human pancreatic cancer. Methods Three pairs of hairpin-like oligonucleotide sequences specific for human STAT3 gene were designed and synthesized. The annealed oligonucleotide fragments were subcloned into pRNAT-U6.1/Neo plasmid. The STAT3 shRNA expressing vectors were confirmed by PCR and DNA sequencing. STAT3 mRNA and protein expression were examined by using reverse transcription polymerase chain reaction (RT-PCR) and Western blot, respectively. MTT assay and flow cytometry were performed to detect the state of cell proliferation and cell apoptosis, respectively. Results PCR and DNA sequencing showed that the oligonucleotide fragments were correctly inserted into pRNAT-U6.1/Neo plasmid. STAT3 expression and cell proliferation in the transfected cells was inhibited significantly by three STAT3 shRNA expressing vectors (P0.05). ConclusionSTAT3 shRNA expression vector can effectively inhibit the expression of STAT3. Silencing of STAT3 with RNAi can significantly inhibit the proliferation and promotes the apoptosis of pancreatic cancer cells and may provide a novel therapeutic target for treating pancreatic cancer.

OBJECTIVE:To investigate whether adeno-associated virus (AAV) could enhance its infection efficiency on cancer cells when combined with non-replicable adenovirus (Ad-null) in vitro as well as in vivo and to study its underlying mechanisms.METHODS:AAV2 particle was added into NCI-H460 tumor cell lines alone or in combination with different amount of adenovirus. 1 to 7 days after transduction, cells were observed and recorded with fluorescence microscope, the expression levels of report gene EGFP in tumor cells were examined by using flow cytometry and Western blotting, the expression of report genes luciferase was analyzed with luminometer to obtain the relative light units. After the establishment of tumor model, the nude mouse were administrated with AAV2 or AAV2 + Ad-null in tumors, and then tested their infection efficiency and expression levels with roperscientific bioluminescence tumor imaging system.RESULTS:The results obtained with the help of flow cytometry and luciferase assay suggested that the infection efficiency of AAV2 was enhanced significantly when combined with low dose Ad-null in vitro, the infection efficiency of AAV2 alone was 6.4% and it reached 55.2% when combined with 10 MOI Ad-null, Western blotting assay demonstrated that the protein expression level of reporter gene in tumor cells enhanced when combined with 10 MOI Ad-null compared with AAV2 infection alone, and the enhancement of reporter gene expression was observed in a concentration-dependent manner; real-time PCR analysis confirmed that Ad-null enhanced the mRNA level of AAV2-EGFP but not the copies of genomic DNA of AAV2-EGFP. Ad-null significantly augmented the infection efficiency when tested on NCI-H460 tumor model. With the help of Ad-null, the signal of luciferin in nude mouse was 4.5 times more than that of control.CONCLUSION:The infection efficiency of AAV was enhanced significantly when combined with low dose Ad-null in vitro and in vivo, and it offers basis for further study of gene therapy by AAV.

Herpes simplex virus type-1 thymidine kinase (HSV-1TK) and Escherichia coli cytosine deaminase (CD) fusion protein was designed using InsightII software. The structural rationality of the fusion proteins incorporating a series of flexible linker peptide was analyzed, and a suitable linker peptide was chosen for further investigated. The recombinant plasmid containing the coding regions of HSV-1TK and CD cDNA connected by this linker peptide coding sequence was generated and subsequently transfected into the human embryonic kidney 293 cells (HEK293). The Western blotting indicated that the recombinant fusion protein existed as a dimer with a molecular weight of approximately 90 kDa. The toxicity of the prodrug on the recombinant plasmid-transfected human lung cancer cell line NCIH460 was evaluated, which showed that TKglyCD-expressing cells conferred upon cells prodrug sensitivities equivalent to that observed for each enzyme independently. Most noteworthy, cytotoxicity could be enhanced by concurrently treating TKglyCD-expressing cells with prodrugs GCV and 5-FC. The results indicate that we have successfully constructed a HSV-1TKglyCD fusion gene which might have a potential application for cancer gene therapy.

Objective:To establish a novel neuroglioma transplantation model in rats to demonstrate the dynamic growth of tumor.Methods:pEGFP-N1,a eukaryotic plasmid containing enhanced green fluorescent protein(EGFP) gene,was transfected into the rats glioma cell line C6 with the help of liposome lipofectamine.Then the cell clones(C6-gfp) stably expressing EGFP was obtained through neomycin G418 screening and subcloning.The growth of C6 and C6-gfp cells were observed using Cell Counting Kit-8(CCK-8).Then the C6-gfp cells were transplanted into Wistar rat's cranium.The tumor formation was observed using B ultrasound and H-E staining.The EGFP expression in the tumor was detected using fluorescence stereomicroscope and anti-GFP immunohistochemistry.Results:Flow cytometry showed that 97.7% of the screened C6-gfp cells were positive of EGFP.There was no obvious difference in the growth curves of C6 and C6-gfp cells(P0.05).The tumor formation rate in the rat's cranium was 70% after C6-gfp transplantation.The dynamic growth of tumor could be observed using B ultrasound and the expression of green fluorescence could be observed under the fluorescence stereomicroscope.H-E staining showed large nuclei in glioma cells,deep dying with more nuclear divisions.New vessels could be seen in the tumor.Anti-GFP immunohistochemistry showed GFP-positive cells with distinct intensity.Conclusion:A C6-gfp cell line stably expressing GFP has been successfully established in the present study and it can readily form tumors in the cranium,which lays a foundation for further study of prevention and treatment of glioma.

Objective:To construct a recombinant plasmid containing the suicide gene CD and study the expression of CD gene in HEK293 cert.Methods:A fragment containing full-lengh coding region of CD was subcloned into HAtag-tagged vector pcDNA3.1/HA-myc -His(-)Z to construct recombinant plasmid pcDNA3,1/HA-myc-His(-)Z-CD.CD was identified by enzyme digestion of EcoRI/ BamHI and sequence,then rite positive recombinant plasmid was transfected into HEK293 cells using a routine lipofectamine method.After 48h, total protein was extracted and the expression of the CD gene in transfected HEK293 cells was identified by western blot.Results:A fragment of 5.5kb and inserted fragment of 1.3kb were got by cutting positive recombinant plasmid of pcDNA3.1/HA-myc-His(-)Z-CD with EcoRI/ BamHI.Automatic DNA sequence analysis demonstrated that sequence of the recombinant plasmid pcDNA3.1/HA-myc-His(-)Z-CD was totally the same with that published in GenBank.The expression of CD gene was detected by western blot.Conclusion:pcDNA3.1/HA-myc- His(-)Z-CD was successfully constructed.

腺相关病毒(AAV)能感染非分裂期的细胞,对宿主几无致病性及能使宿主长期稳定的表达外源基因,是神经系统疾病基因治疗的常用载体。不同血清型的AAV,由于分子结构与细胞结合机制的差异,在组织细胞中常表现出不同的转导效率。为了寻求更有效的中枢神经系统疾病基因治疗的方法,有必要对各型AAV在神经系统组织细胞内的转导特性进行深入的研究。