Capsid Proteins ; genetics ; metabolism ; Simplexvirus ; genetics ; metabolism

Animals ; Apoptosis ; Herpes Simplex ; physiopathology ; virology ; Humans ; MicroRNAs ; genetics ; metabolism ; Simplexvirus ; genetics ; physiology ; Virus Latency

Aged ; Aged, 80 and over ; Esophagus ; pathology ; virology ; Herpes Simplex ; diagnosis ; genetics ; pathology ; Humans ; In Situ Hybridization ; Male ; Middle Aged ; Simplexvirus ; genetics

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.
Brain-Derived Neurotrophic Factor ; biosynthesis ; genetics ; Cell Line ; Dependovirus ; genetics ; metabolism ; Genetic Vectors ; genetics ; Humans ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Recombination, Genetic ; Simplexvirus ; genetics ; Transfection

In order to investigate the application of recombinant adeno-associated virus (rAAV) vector containing Tet regulation system and HSVtk gene in cancer gene therapy, pAAV/TRE/HSVtk/Tet-On was constructed and identified with PCR and restriction enzyme digestion. Packaging cells HEK293 were cotransfected with plasmids pAAV/TRE/HSVtk/Tet-On, pAAV-RC and pAAV-helper to produce infectious rAAV, and CsCl2 densitygradient centrifugation method was performed for purification and concentration of rAAV. The viruses were then transduced into MCF-7 cells. The results of dot blot hybridization indicate that the rAAV can transfer the target gene into MCF-7 cells. MTT assay showed that GCV could kill AAV-infected MCF-7 cells under the induction of Dox. The data demonstrated that rAAV containing Tet regulation system and HSVtk gene was successfully obtained, and could be used for further investigation of in vivo and in vitro experiments.
Cell Line, Tumor ; Dependovirus ; genetics ; metabolism ; Doxycycline ; pharmacology ; Ganciclovir ; pharmacology ; Genes, Transgenic, Suicide ; genetics ; Genetic Therapy ; Genetic Vectors ; genetics ; Humans ; Simplexvirus ; enzymology ; genetics ; Thymidine Kinase ; genetics ; Transfection

OBJECTIVETo investigate the effect of herpes simplex virus-thymidine kinase (HSV-TK) gene transduced into T cell clone on the treatment of ulcerative colitis (UC) in rat.

METHODSThe T cell clone transduced with HSV-tk was infused into 27 rats with UC. Changes of stimulation index (SI), CD4(+), CD8(+), IL-13, IL-4 were detected, and pathological changes before and after infusion was compared.

RESULTSIn the second and third day after tk+ T clone infusion, the inflammation of colon was assimilated. Two weeks later, the colon began to renovate and mend. The average SI was 7.39+/-1.24 before infusion, and 2.67+/-0.87 after infusion (P<0.05). Peripheral blood levels of CD4(+), CD8(+) or IL-13 and IL-4 in therapeutic group were significantly decreased as compared to control group (P<0.05).

CONCLUSIONHSV-tk gene transduced into T lymphocyte clone and infused back is effective for UC gene therapy and target design in rat.

Animals ; Colitis, Ulcerative ; therapy ; Genes, Transgenic, Suicide ; Genetic Therapy ; Male ; Rats ; Rats, Wistar ; Simplexvirus ; genetics ; T-Lymphocytes ; Thymidine Kinase ; genetics ; Viral Proteins ; genetics

OBJECTIVETo study the effect of cotransfection of genes of insulin-like growth factor I (IGF-I) and herpes simplex virus thymidine kinase (HSV-tk) on wound healing.

METHODSThirty male Wistar rats were inflicted with 30% TBSA full-thickness scald. They were then divided into A group (4.6 microg pcDNA3.1/IGF-I+Lipofectamine 2000+saline), B group (3.6 microg pcDNA3.1/HSV-tk+Lipofectamine 2000+saline), C1 group and C2 group (2.3 microg pcDNA3.1/IGF-I+1.8 microg pcDNA3.1/HSV-tk+Lipofectamine 2000+saline), and D group (3.0 microg pcDNA3.1+Lipofectamine 2000+saline) according to the random number table, with 6 rats in each group. The above-mentioned mixtures were subcutaneously injected into left back of each rat the moment after injury and on post scald day (PSD) 7, 14, 21, and 28. Gancyclovir (2.5 mg/100 g) was hypodermically injected into rats in C2 group on PSD 29, 30, 31, 32. Changes in body weight of rats were measured. Wound healing rates were calculated. On PSD 35, the expressions of IGF-I gene in local wound and liver tissue were determined with immunohistochemical staining. The serum expression of IGF-I was determined with radioimmunoassay. Expression of HSV-tk gene in local wound was determined with RT-PCR. Apoptosis of fibroblast in C1 and C2 groups was observed under transmission electron microscope. Data were processed with one-way analysis of variance and Turkey method.

RESULTSBody weight of rats in A, C1, and C2 groups increased from PSD 7 through 35, and the difference between former three groups and B, D groups was statistically significant (with F value respectively 2.764, 4.519, 5.009, 13.449, 5.877, P values all below 0.05). Wound healing rates of rats in A, C1, and C2 groups were higher than those in B, D groups (with F value respectively 5.286, 100.880, 152.380, 127.850, 147.750, P values all below 0.05). IGF-I gene was positively expressed in wound fibroblast in A, C1 and C2 groups, but negatively in liver tissues of all the rats. There was no significant statistical difference among groups in serum content of IGF-I [from (1185+/-170) to (1270+/-130) ng/mL, F=0.355, P=0.838]. HSV-tk gene was positively expressed in rat skin tissue in B, C1 and C2 groups. Fibroblast apoptosis was observed under transmission electron microscope in C2 group, but it was not observed in C1 group.

CONCLUSIONSCotransfection of pcDNA3.1/IGF-I and pcDNA3.1/HSV-tk mediated by liposome can promote wound healing, and inhibit the scar proliferation to some extent.

Animals ; Burns ; genetics ; metabolism ; therapy ; Insulin-Like Growth Factor I ; genetics ; metabolism ; Male ; Rats ; Rats, Wistar ; Simplexvirus ; enzymology ; Thymidine Kinase ; genetics ; metabolism ; Transfection ; Wound Healing

OBJECTIVETo investigate the in vitro effect of HSV-tk/GCV using a hTERT promoter-driven vector system on Skov3 ovarian cancer cells.

METHODSAn expression vector (pBTdel-279-tk) containing tk gene under the hTERT promoter was constructed by molecular biological methods, and then was transfected into Skov3 ovarian cancer cells, normal ovarian epithelial cells (NOEC) and human embryonic lung fibroblast by cationic liposome. Following the transfection with tk, GCV was added, and MTT and flow cytometry methods were applied to investigate its antitumor effect. RT-PCR was used to detect the tk gene in ovarian cancer cells and normal cells after the transfection of pcDNA3-tk or pBTdel-279-tk.

RESULTSpBTdel-279-tk/GCV system induced apoptosis in hTERT-positive ovarian cancer cells, but not in hTERT-negative normal ovarian epithelial cells and fibroblasts. The hTERT promoter system was almost as efficient in inducing cancer cell death as the CMV promoter. tk gene was expressed in Skov3 cells and NOEC after pcDNA3-tk transfection, while positive was only in ovarian cancer cells after pBTdel-279-tk transfection.

CONCLUSIONThe telomerasespecific transfer of the tk gene under the hTERT promoter is a novel targeting approach for the treatment of ovarian cancer and may lead to an effective and specific gene therapy.

Apoptosis ; genetics ; Cystadenocarcinoma, Serous ; genetics ; pathology ; DNA-Binding Proteins ; Female ; Ganciclovir ; pharmacology ; Genetic Therapy ; Humans ; Ovarian Neoplasms ; genetics ; pathology ; Promoter Regions, Genetic ; genetics ; Simplexvirus ; genetics ; Telomerase ; genetics ; Thymidine Kinase ; genetics ; Transfection ; Tumor Cells, Cultured

OBJECTIVETo investigate the therapeutic effects of adenovirus-mediated transfer of the herpes simplex virus thymidine rinase gene (HSV-tk) used by several methods and the dose-effect relationship.

METHODSDiverse doses (1 x 10(9) PFU, 1 x 10(10) PFU, 1 x 10(11) PFU) of adenovirus-mediated transfer of HSV-tk were given by intraparenchymatous, intravenous and intraperitoneal injection, and ganciclovir (GCV) (100 mg.kg(-1).d(-1)) was injected into the cavity of the peritoneum to treat human hepatocarcinoma. The change of tumors size was observed and the fragments of HSV-tk gene were tested.

RESULTSIn nude mice after intraparenchymatous injection and high-dose (1 x 10(11) PFU) intravenous injection, the tumors were suppressed significantly (t = 13.1, 12.4, P < 0.01) and lots of fragments of HSV-tk gene were observed. In mice after intraperitoneal injection and low-dose (1 x 10(9) PFU, 1 x 10(10) PFU) intravenous injection, no suppressive effect was observed (t = 1.8, 1.0, 2.1, 1.1, 0.8, P > 0.05) with few or without fragments in the tumors.

CONCLUSIONSAdenovirus-mediated transfer of the HSV-tk by intraparenchymatous or intravenous injection is effective in treatment of hepatocarcinoma in nude mice, but intraperitoneal injection has no therapeutic effect.

Adenoviridae ; genetics ; Animals ; Gene Transfer Techniques ; Genetic Therapy ; methods ; Humans ; Liver Neoplasms, Experimental ; therapy ; Mice ; Mice, Inbred BALB C ; Simplexvirus ; enzymology ; Thymidine Kinase ; genetics ; Tumor Cells, Cultured

Cytokines ; secretion ; Cytotoxicity, Immunologic ; Graft vs Leukemia Effect ; Humans ; Immunophenotyping ; Lymphocyte Activation ; Retroviridae ; genetics ; Simplexvirus ; enzymology ; T-Lymphocytes ; immunology ; Thymidine Kinase ; genetics ; Transfection