Adolescent ; Catheterization ; adverse effects ; Endocarditis, Bacterial ; etiology ; Female ; Heart Septal Defects, Ventricular ; therapy ; Humans ; Postoperative Complications

Adult ; Female ; Humans ; Myocardial Infarction ; Puerperal Disorders

Adolescent ; Arteriovenous Fistula ; therapy ; Catheter Ablation ; methods ; Coronary Artery Disease ; therapy ; Female ; Heart Defects, Congenital ; therapy ; Humans ; Male ; Middle Aged

Aged ; Coronary Angiography ; methods ; Coronary Artery Disease ; diagnostic imaging ; Female ; Femoral Artery ; Humans ; Male ; Middle Aged ; Prospective Studies ; Radial Artery

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

BACKGROUNDRevTet-On gene expression system was used to deliver the suicide gene tk to human breast cancer cell line MCF-7 and control the tk gene expression level. The animal model of human breast cancer on severe combined immune deficiency (SCID) mice was set up to explore the suicide gene therapy by the regulation of Tet-On.

METHODSHerpes simplex virus-thymidine kinase (HSVtk) gene was inserted into the plasmid pRevTRE and the recombinant retroviral vector pRevTRE/HSVtk was constructed. Using modified calcium phosphate co-precipitation method, two transfections, pRevTRE/HSVtk and pRevTet-On were performed for MCF-7 cell line and selected by hygromycin B and G418. MCF-7 cell line that stably expressed Tet-regulated tk gene was established. HSVtk gene expression in the MCF/TRE/tk/Tet-On cell line was under the control of Doxycycline (Dox). Cell viability was also determined by MTT assay, whereas HSVtk gene expression was analyzed by reverse transcription-PCR (RT-PCR).

RESULTSMCF/TRE/tk/Tet-On cell survival rate was decreased from 100% to less than 20% when ganciclovir (GCV) concentration was increased from 0 to 1000 microg/ml at 1 microg/ml of Dox after 72 hours of GCV administration. At 1 microg/ml of GCV concentration, the cell numbers decreased from 7 x 10(4) cells/ml to 2 x 10(4) cells/ml when Dox concentration was increased from 0 to 1500 ng/ml after 72 hours culture. In addition, bystander effects were generated in vitro when 10% - 25% of transduced MCF-7 cells were mixed in untransduced MCF-7 cells. On the other hand, the human breast cancer models in SCID mice were set up. The tk gene was expressed with the regulated character after MCF/TRE/tk/Tet-On cells were implanted into the female SCID mice 7 days after Dox induction followed by intraperitoneally administration of GCV for 23 days. Subcutaneous tumors in SCID mice that were implanted with MCF/TRE/tk/Tet-On cells shrank remarkably after Dox and GCV administration as compared with the control.

CONCLUSIONThe human breast tumor cells (MCF-7) expressing HSVtk gene can be eradicated by administration of GCV and induced with tetracycline or its derivative Dox in vitro and in vivo.

Animals ; Breast Neoplasms ; therapy ; Bystander Effect ; Cell Line, Tumor ; Cell Survival ; Doxycycline ; pharmacology ; Ganciclovir ; pharmacology ; Genes, Transgenic, Suicide ; Genetic Therapy ; methods ; Genetic Vectors ; Herpesviridae ; genetics ; Humans ; Mice ; Mice, SCID ; Retroviridae ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Thymidine Kinase ; genetics ; Transfection

OBJECTIVETo study the changes of morphology and erectile function of the cavernous tissues in spontaneously hypertensive rats.

METHODSSpontaneously hypertensive male rats (SHR) (n = 15) and normotensive Wistar-Kyoto rats (WKY) (n = 15) were studied for 20 weeks. Systolic blood pressure (SBP) was measured weekly by the tail/cuff method. Erectile function was tested by injecting apomorphine (APO). The expression alpha-smooth muscle actin (alpha-SMA) and collagen III was examined by immunohistochemistry.

RESULTSSHR showed a higher systolic blood pressure (205.7 +/- 11.9 vs 114.3 +/- 10.2 mm Hg) and a lower erection frequency (0.6 +/- 0.5 vs 2.4 +/- 0.6). The expression of alpha-smooth muscle actin and collagen III in the cavernous tissues in the SHR was significantly higher than in the WKY.

CONCLUSIONThe erectile function of the penis is markedly affected by hypertension, and the pathological changes may be one of the most important mechanisms of decreased erectile function in SHR.

Actins ; biosynthesis ; Animals ; Apomorphine ; administration & dosage ; Blood Pressure ; physiology ; Collagen Type III ; biosynthesis ; Hypertension ; pathology ; physiopathology ; Immunohistochemistry ; Male ; Penile Erection ; physiology ; Penis ; metabolism ; pathology ; Rats ; Rats, Inbred SHR ; Rats, Inbred WKY

OBJECTIVETo investigate the effects of hydroxycamptothecin (HCPT) on proliferation and apoptosis of rat hepatic stellate cells (HSC).

METHODSRat HSC line (HSC-T6) and rat hepatocyte line (BRL-3A) were treated with different concentrations of HCPT (0, 0.008, 0.016, 0.031, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32 mg/L respectively) for 24 h. Cell proliferation was assessed by MTT colorimetric assay, apoptosis was detected with PI staging followed by flow cytometry, and by DNA ladder assay. The morphological change of apoptosis was observed under transmission electron microscopy (TEM).

RESULTSMTT assay indicated that HCPT significantly inhibited the proliferation of HSC-T6 and BRL-3A in a dose-dependent manner. 24 h after the treatment with different concentrations of HCPT (0.25, 0.5, 1 mg/L), the apoptosis rate (13.46%+/-2.42%, 26.25%+/-5.65%, 47.05%+/-8.76%, respectively) in HSC-T6 was significantly higher than that in control cells (4.89%+/-1.80%, F = 34.24, P less than 0.01). 24 h after 0.5 mg/L HCPT treatment, cell shrinkage, nucleoli disappearance, chromatin condensation were found under TEM, and DNA ladder was demonstrated by agarose gel electrophoresis.

CONCLUSIONHCPT could significantly inhibit proliferation and induce apoptosis of HSC-T6 in a dose-dependent manner.

Animals ; Apoptosis ; drug effects ; Camptothecin ; analogs & derivatives ; pharmacology ; Cell Line ; Cell Proliferation ; drug effects ; Hepatic Stellate Cells ; drug effects ; Rats

OBJECTIVETo explore the HSVtk gene expression mediated by the retroviral vector and to obtain high titer recombinant retroviral virus.

METHODSThe recombinant vector pRevTRE/HSVtk was constructed by inserting HSVtk gene into pRevTRE. The recombinant retrovirus, which was produced from cloned PA317 cells screened by hygromycin B after "micro-pingpong" technique transferring with pRevTRE/HSVtk plasmids DNA by using modified calcium phosphate precipitation method. HSVtk gene expression was performed on target cells and virus titers were detected in different cultured temper, time and sodium butyrate concentration.

RESULTSThe recombinant retroviral vector pRevTRE/HSVtk was constructed and HSVtk gene expression was detected on target cells after they were infected with the recombinant retrovirus.

CONCLUSIONHigh titer of retroviruses could be obtained in the culture medium of PA317 cell line through "micro-pingpong" technique at 30 hours and 10 mmol/L sodium butyrate concentration followed by frozen ultrafiltration.

Animals ; Breast Neoplasms ; enzymology ; pathology ; virology ; Cell Line ; Cell Line, Tumor ; Female ; Gene Expression Regulation, Enzymologic ; Genetic Vectors ; Humans ; Mice ; NIH 3T3 Cells ; Recombination, Genetic ; Retroviridae ; genetics ; Simplexvirus ; enzymology ; genetics ; Thymidine Kinase ; biosynthesis ; genetics ; Titrimetry ; Transfection

BACKGROUNDPercutaneous vertebroplasty (PVP) has become a popular procedure for painful vertebral osteoporotic fracture (VOF), with immediate pain relief and improved mobility; however, polymethylmethacrylate (PMMA) injected into the vertebral body is not absorbable and little information is available concerning the long-term results. In this retrospective study, we evaluated the long-term clinical results and radiological changes after PVPs for VOFs.

METHODSFifty-one patients with VOFs were treated by PVPs with PMMA between 2000 and 2004. After > 7 years of follow-up, eight patients had died from causes unrelated to the intervention and 12 patients were lost to follow-up, thus leaving 31 patients available for evaluation with an average length of follow-up of 9.2 years (follow-up rate, 72.1%). Among these 31 patients, the PMMA was injected at 43 levels with a mean volume of 4.3 ml per level (range, 2 - 6 ml). The pain was assessed with a visual analog scale (VAS), and the mobility was graded as walking without difficulty (grade 1), walking with assistance (grade 2), and bedridden (grade 3). Plain radiographs and computed tomography (CT) were obtained and assessed pre-operatively, immediately post-operatively, and after 7 years of follow-up. The PMMA, vertebral height, and Cobb angle were assessed and compared.

RESULTSAll of the patients experienced pain relief and improved mobility after intervention and during the follow-up period. Cement leakage was detected in post-operative CT scans in 9 of 51 patients, but without neurological compromise. For the 31 patients followed up over 7 years, the VAS decreased from 8.3 ± 2.6 pre-operatively, to 2.1 ± 1.6 immediately post-operatively, and 1.0 ± 0.9 at the final follow-up evaluation, with significantly improved mobility. Additional compression fractures occurred at adjacent levels in three patients, and there were no new fractures at the augmented vertebrae. Based on a review of the radiographs, neither loose nor displaced cement was detected. The changes in vertebral height and Cobb angle were not significant. On CT scans, the cement closely contacted or infiltrated the trabecular bone. The boundary between the cement and trabecular bone was indistinct and there was no evident radiolucent gap between the cement and trabecular bone.

CONCLUSIONSAt an average follow-up of 9.2 years, PVPs provided sustained pain relief and improved mobility in patients with VOFs. The PMMA injected into the vertebral body combined closely with the host trabecular bone without adverse reactions.

Aged ; Aged, 80 and over ; Female ; Humans ; Male ; Middle Aged ; Osteoporotic Fractures ; surgery ; Polymethyl Methacrylate ; therapeutic use ; Retrospective Studies ; Spinal Fractures ; surgery ; Vertebroplasty ; methods