OBJECTIVETo establish and evaluate a rabbit model of fecal incontinence.

METHODSTwelve normal adult male New Zealand rabbits were randomly divided into experimental group and control group. The nerve innervating the external anal sphincter, namely the fourth sacral nerve, was functionally located and selectively damaged with local injection of 50 g/L ropivacaine in the experimental group, and normal saline injection was administered in the control group. The changes in the resting anal pressure was examined before and after the surgery, and the electromyogram (EMG) of the external anal sphincter was recorded for comparison with the pathological changes of the fourth sacral nerve.

RESULTSCompared with the control group, the experimental group exhibited significantly decreased resting anal pressure after the surgery. The EMG of the experimental group showed abnormal nerve conduction velocity of the fourth sacral nerve, suggesting successful nerve block. Transmission electron microscope revealed irreversible pathological changes in the ultrastructure of the axons of the fourth sacral nerve.

CONCLUSIONThis method allows successful establishment of fecal incontinence in rabbits, which facilitates further in vivo study of artificial sphincters for treatment of anal incontinence.

Amides ; administration & dosage ; Anal Canal ; physiopathology ; Animals ; Disease Models, Animal ; Electromyography ; Fecal Incontinence ; Lumbosacral Plexus ; Male ; Nerve Block ; Rabbits ; Random Allocation

OBJECTIVETo study the selective cytotoxic effect of lentivirus-mediated double suicide gene (CD/TK) against human gastric carcinoma cells SGC-7901 in vitro.

METHODSSGC-7901 cells were infected with FGW-KDRP-CD/TK vector and the infection efficiency was observed under a fluorescence microscope. The morphological changes of the infected cells were observed by Giemsa staining. Flow cytometry (FCM) was employed for cell cycle analysis, and the expression of CD/TK was detected by RT-PCR. The infected cells were then treated with the prodrugs ganciclovir (GCV) and/or 5-fluorocytosine (5-FC) at different concentrations, and the cytotoxic effects were evaluated using MTT method.

RESULTSThe infection efficiency of the lentiviral vector in SGC-7901 cells increased with the titer of the virus, which produced no significant effect on the cancer cell morphology in vitro or on the percentages of G0-G1, G2-M and S phase cells (P>0.05). RT-PCR demonstrated the expression of CD/TK gene in SGC-7901 cells infected by FGW-KDRP-CD/TK. The infected cells were highly sensitive to the prodrugs with a dose-dependent cytotoxic effect within a specific concentration range of the drugs, whereas the non-infected cells were not sensitive to the prodrugs. Combined use of the two prodrugs produced an obviously stronger inhibitory effect than either of the them (P<0.05). When combined, GCV and 5-FC at the concentration of 0.1+40, 1+80, 10+160, and 100+320 mg/L demonstrated a synergetic effect with a CDI<1.

CONCLUSIONLentivirus-mediated CD/TK fusion gene system can selectively kill gastric cancer cells, and the two prodrugs show a synergistic cytotoxic effect.

Adenocarcinoma ; genetics ; pathology ; Cell Line, Tumor ; Cytosine Deaminase ; biosynthesis ; genetics ; Cytotoxins ; pharmacology ; Genes, Transgenic, Suicide ; genetics ; Genetic Therapy ; Genetic Vectors ; genetics ; Humans ; Lentivirus ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Stomach Neoplasms ; genetics ; pathology ; Thymidine Kinase ; biosynthesis ; genetics ; Vascular Endothelial Growth Factor Receptor-2 ; genetics ; metabolism

OBJECTIVETo investigate the selective killing of colorectal tumor cells by lentivirus-mediated double suicide gene under the regulation of KDR promoter.

METHODS293T packaging cells were transfected with the plasmid FGW-KDRP-CD/TK to obtain the infectious viruses. KDR-expressing LoVo cells and LS174T cells that did not produce KDR were transfected with the recombinant virus, and the transfection efficiency was evaluated by the fluorecence microscope. RT-PCR was employed to examine the expression of CDglyTK. After treatment of the cells with 5-FC and GCV, the killing effects on the two cell lines were evaluated.

RESULTSThe recombinant construct showed similar infection rate of the two cell lines. RT-PCR demonstrated that CDglyTK gene was expressed only in LoVo cells infected with FGW-KDRP-CD/TK but not in LS147T cells, and the sensitivity of the two cell lines to the prodrugs was significantly different (P<0.001). The killing effect of the double suicide gene was much stronger than that of single suicide gene administered (P<0.001).

CONCLUSIONThe double suicide gene driven by KDR promoter has specific killing effect on the KDR-expressing colorectal tumor cells.

Antimetabolites ; pharmacology ; Apoptosis ; drug effects ; Cell Line ; Cell Line, Tumor ; Colorectal Neoplasms ; genetics ; metabolism ; pathology ; Cytosine Deaminase ; genetics ; metabolism ; Flow Cytometry ; Flucytosine ; pharmacology ; Ganciclovir ; pharmacology ; Genes, Transgenic, Suicide ; genetics ; Genetic Vectors ; genetics ; Humans ; Lentivirus ; genetics ; Promoter Regions, Genetic ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; Thymidine Kinase ; genetics ; metabolism ; Transfection ; Vascular Endothelial Growth Factor Receptor-2 ; genetics

Aged ; External Fixators ; Female ; Follow-Up Studies ; Fractures, Bone ; complications ; physiopathology ; surgery ; therapy ; Humans ; Male ; Middle Aged ; Osteoporosis ; complications ; Tomography, X-Ray Computed ; Treatment Outcome

OBJECTIVETo investigate the clinical value of 64-slice computed tomographic angiography (CTA)-based virtual colonoscopy in the diagnosis of colonic tumors.

METHODSPhilips/Brilliance 64 CT volumetric scanning was performed in 8 patients with colonic cancer and 2 with colonic polypi identified by postoperative pathological examination. Mimics software was used for surface rendering of the intestine with the Marching Cubes algorithm for 3-dimensional (3D) virtual endoscope (VE) reconstruction and CTA-based 3D reconstruction of the large intestine and the surrounding structures. The location, volume and appearance of the lesions displayed by the virtual techniques were compared with the pathological results.

RESULTSThe 3D reconstruction was successfully completed in all the 10 cases, and the imaging diagnoses showed a total match with the pathological diagnoses. No significant differences were found between virtual endoscopy and CT virtual endoscopy. Virtual colonoscopy combined with digital model reconstruction provided valuable information for accurate identification of the position of the lesions and the complex adjacent anatomical structures.

CONCLUSIONVirtual colonoscopy based on 64-slice CTA, when combined with 3D reconstruction technique, allows accurate display of the colonic lesions and potential metastasis, which can be crucial for clinical staging and surgical planning of colonic cancer.

Adult ; Angiography ; methods ; Colorectal Neoplasms ; diagnostic imaging ; therapy ; Female ; Humans ; Image Processing, Computer-Assisted ; methods ; Imaging, Three-Dimensional ; Male ; Middle Aged ; Tomography, Spiral Computed

OBJECTIVETo evaluate the effect of adenovirus-mediated double suicide gene (CD/TK) for selective killing of breast cancer cells.

METHODSVascular endothelial growth factor (VEGF)-expressing MCF-7 cells and normal human mammary epithelial cells that did not express VEGF were infected with the adenovirus containing VEGFP-CD/TK-GFP genes. CD/TK gene expression in the infected cells was detected by RT-PCR. After treatment of the infected cells with GCV and/or 5-FC, the cell growth status was evaluated using MTT assay, and the cell cycle changes were detected with flow cytometry. In nude mice bearing human breast cancer, the recombinant adenovirus vector was injected directly into the tumor followed by intraperitoneal injection of the prodrugs GCV and/or 5-FC, and the subsequent tumor growth was observed.

RESULTSThe recombinant adenovirus achieved similar infection rates in MCF-7 and human mammary epithelial cells, and the rates increased gradually with the multiplicity of infection (MOI) of the virus. RT-PCR demonstrated the presence of CD/TK gene product in infected MCF-7 cells, but not in the infected mammary epithelial cells. The infected MCF-7 cells, but not the mammary epithelial cells, were highly sensitive to the pro-drugs. The CD/TK fusion gene system showed significantly greater efficiency than either of the single suicide gene in killing the target cells (P<0.01). At the MOI of 100, treatment of the infected cells with the pro-drugs resulted in increased cell percentage in G(0)-G(1) phase and decreased percentage in S phase. In nude mice bearing MCF-7 cell-derived subcutaneous tumor, treatment with the double suicide gene system significantly inhibited the tumor growth, showing much stronger effect than either of the single suicide gene (P<0.01).

CONCLUSIONThe adenovirus-mediated CD/TK double suicide gene driven by VEGF promoter combined with GCV and 5-FC treatment can be an effective therapy against experimental breast cancer, and produces much greater efficacy than the single suicide gene CD/TK combined with GCV or 5-FC.

Adenoviridae ; genetics ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Cytosine Deaminase ; genetics ; metabolism ; Female ; Flow Cytometry ; Flucytosine ; pharmacology ; Ganciclovir ; pharmacology ; Genes, Transgenic, Suicide ; genetics ; Genetic Therapy ; methods ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Recombinant Fusion Proteins ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Thymidine Kinase ; genetics ; metabolism ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

To improve the targeting of adenovirus vector for gene therapy, a fusion gene sCAR-EGF, in which epidermal growth factor gene was fused to the 3' end of extracellular Coxsackie virus-adenovirus receptor gene, was constructed and cloned into shuttle plasmid pDC315 to obtain a recombinant plasmid pDC315-sCAR-EGF. With the AdMax system, AD-293 cells were co-transfected with pDC315-sCAR-EGF and adenovirus genomic plasmid pBHGloxdeltaE13cre. Through high efficiency site specific recombination, a replication-defective adenovirus Ad5-CMV-sCAR-EGF was constructed. The recombinant adenovirus was analyzed by PCR and Western blotting, the results indicated that Ad5-CMV-sCAR-EGF contained the fusion gene sCAR-EGF, and the adenovirus infected cells was induced to produce and secrete the fusion protein into the supernatant. We have demonstrated that the fusion protein sCAR-EGF is helpful for elevating the infection efficiency of Ad5-CMV-luc with the reporter gene in vitro, which providing a new approach to the gene therapy for tumors overexpressing EGFR.
Adenoviridae ; genetics ; Cell Line ; Coxsackie and Adenovirus Receptor-Like Membrane Protein ; Enzyme-Linked Immunosorbent Assay ; Epidermal Growth Factor ; analysis ; genetics ; Genetic Therapy ; Humans ; Neoplasms ; therapy ; Polymerase Chain Reaction ; Receptors, Virus ; analysis ; genetics ; Recombinant Fusion Proteins ; biosynthesis

OBJECTIVETo evaluate the specific killing effects of combination of recombinant adenovirus mediated double suicide gene driven by KDR promoter and survivin antisense oligonucleotide(ASODN) on colorectal cancer cells and vascular endothelial cells.

METHODSThe 293 packaging cells were transfected with the plasmids of pAdEasy-CDglyTK and the recombinant adenovirus were generated. The KDR expressive cells of SW620, ECV304 were infected with adenovirus, meanwhile survivinASODN was transferred into the same cells. The infection rate of adenovirus and transfection efficiency of survivinASODN were observed and the expression of CDglyTK was detected by RT-PCR. The expression of survivin was measured by Western blot. The killing effects and bystander effects on SW620, ECV304 were examined through MTT method.

RESULTSThe cells which were infected with the adenovirus mediated double suicide gene could be transfected with the survivin ASODN and the infection rate was not affected as well as the transfection efficiency. The high expression of CDglyTK gene was found in SW620, ECV304 cells infected with recombinant adenovirus and survivin ASODN decreased the survivin protein level. The survival rate of gene therapy group was significantly lower than that of negative group. The combination of survivin ASODN and AdKDR-CDglyTK gene therapy showed significantly lower survival rate of SW620 and ECV304 cells as compared with the AdKDR-CDglyTK or survivin ASODN used alone (P<0.05). The survival rate was slightly lower in GCV 100 microg/ml, 5-FC 2000 microg/ml than that AdKDR-CDglyTK used alone (P>0.05). The combined therapy of AdKDR-CDglyTK and survivin ASODN showed synergistic killing efficacy and more significant bystander effects.

CONCLUSIONThe combined gene therapy of AdKDR-CDglyTK system and survivin ASODN has stronger specific killing effects on colorectal cancer cells and vein endothelial cells.

Adenoviridae ; genetics ; Cell Line, Tumor ; Colorectal Neoplasms ; genetics ; metabolism ; Endothelial Cells ; metabolism ; Genes, Transgenic, Suicide ; genetics ; Humans ; Inhibitor of Apoptosis Proteins ; Microtubule-Associated Proteins ; genetics ; Oligonucleotides, Antisense ; genetics ; Receptors, Vascular Endothelial Growth Factor ; genetics ; Transcription Initiation Site

OBJECTIVETo study the effect of adenovirus (Ad)-mediated fusion gene system driven by KDR promoter on the proliferation of human stomach adneocarcinoma SCG7901.

METHODSThe KDR-expressing SCG7901 cells and HepG2 cells that did not express KDR were both transfected with AdEasy-KDR-CDglyTK followed by treatment with the prodrugs 5-FC and/or GCV at different concentrations. The killing effects of the transfection on the cells were evaluated.

RESULTSThe expression of green fluorescent protein (GFP) was observed in 95% of the infected SCG7901 and HepG2 cells with the multiple of infection (MOI) of the Ads of 100. Transfection of SCG7901 and HepG2 cells did not produce significant changes in the cell growth, and the infected cells exhibited different sensitivities to the two prodrug: SCG7901 cells infected with rAd were highly sensitive to the prodrugs, but the infected HepG2 cells were not (P<0.01). The killing effect of CDglyTK fusion gene on the target cells was much stronger than that of either the single suicide gene (P<0.01).

CONCLUSIONCDglyTK fusion gene system driven by KDR promoter selectively kills the KDR-CDglyTK SCG7901 cells and inhibits their proliferation.

Adenocarcinoma ; genetics ; metabolism ; pathology ; Adenoviridae ; genetics ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Dose-Response Relationship, Drug ; Flucytosine ; pharmacology ; Ganciclovir ; pharmacology ; Genes, Transgenic, Suicide ; genetics ; Genetic Vectors ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Humans ; Neovascularization, Pathologic ; genetics ; metabolism ; pathology ; Prodrugs ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Stomach Neoplasms ; genetics ; metabolism ; pathology ; Transfection ; Vascular Endothelial Growth Factor Receptor-2 ; biosynthesis ; genetics

OBJECTIVETo study the effect of adenovirus (Ad)-mediated fusion gene system driven by the KDR promoter on the proliferation of human colon adenocarcinoma SW620 cells.

METHODSThe KDR-expressing SW620 cells and LS174T cells not expressing KDR were both infected with AdEasy-KDR-CDglyTK followed by treatment with the prodrugs 5-FC and/or ganciclovir at different concentrations. The effect of the transfection on the cell proliferation was evaluated.

RESULTSThe expression of green fluorescent protein (GFP) was observed in 95% of the infected SW620 and LS174T cells with a multiplicity of infection (MOI) of 100. Significant difference was not founded in the growth of SW620 and LS174T cells with or without the transfection. The infected SW620 cells exhibit high sensitivity to the prodrugs, but the infected LS174T cells did not (P<0.01). The CDglyTK fusion gene produced much stronger killing effect of on the target cells than either of the single suicide genes (P<0.01).

CONCLUSIONCDglyTK fusion gene system driven by the KDR promoter selectively kills the KDR-CDglyTK SW620 cells and inhibits the cell proliferation.

Adenocarcinoma ; pathology ; Adenoviridae ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Colonic Neoplasms ; pathology ; Cytosine Deaminase ; genetics ; Genes, Transgenic, Suicide ; genetics ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Promoter Regions, Genetic ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; Thymidine Kinase ; genetics ; Transfection ; Vascular Endothelial Growth Factor Receptor-2 ; genetics ; metabolism