Objective To construct luciferase reporter plasmid containing synthetic CArG elements and to investigate their radiation-inducible property in tumor cells. Methods Insert chimeric regulation elements consisting of nine tandem-repeat copies of CArG sequence (CCATATAAGG) and CMV IE basal gene promoters into pGL3-Basic vectors to construct luciferase reporter plasmids. Tumor cells(HeLa, A549 and HepG2) were transiently transfected by reporter plasmids using lipofectamine, and transfected cells were irradiated by ?-ray with different doses. After 36 h, we assayed the level of reporter gene expression. Results The CArG elements could successfully induce the expression of a downstream reporter gene following irradiation, with maximal expression seen after 3 Gy irradiation. Conclusion The synthetic CArG elements are responsive to low dose of radiation and are able to enhance down-stream gene expression. It is expected to be the essential potential for future application of radiogenetic cancer therapy.

Humanized monoclonal antibodies(mAbs) are increasingly widely used in targeted therapy for cancer and some other major diseases.Complementarity-determining region(CDR) grafting makes quantities of humanized mAbs available.Herein,we provide an overview on the strategy and progress of CDR grafting.

Objective To upregulate Notch signaling in cancer cells by overexpression of active part of Notch1 and to examine the proliferation of the cells. Methods Four cancer cell lines were infected with retrovirus recombined with sequence encoding active part of Notch1.CBF-1 reporter plasmid was used to detect Notch signaling and proliferation assay was carried out by MTS method.Cell cycle analysis was synchronously conducted. Results The overexpression of the active part of Notch1 induced upregulation of Notch signaling,led to growth inhibition in Hela and HepG2 cell lines and growth boost in BGC-823 cell lines,while had no effect on Chang cell lines. Conclusion The upregulation of Notch signaling can exert various effects on different cancer cell lines which is critical to the gene therapy for cancers.

Objective To study the activity of the survivin gene promoter in several tumor cell lines and evaluate the possible application of this promoter in tumor gene therapy. Methods ①The expressions of survivin gene in A549,MDA-MB231 and HepG2 cell lines were detected by RT-PCR and Western blotting.②Tumor cells(A549,MDA-MB231,HepG2) were transiently transfected by reporter plasmids containing different length of survivin promoter using lipofectamine.And 48 h later,the level of reporter gene expression was analyzed.Results There were different levels of survivin expression in A549,MDA-MB231 and HepG2 cell lines.Transient transfection assay approved that pLuc-surP-987,pLuc-surP-596,pLuc-surP-269 and pLuc-surP-158 showed high activity and 269 bp survivin promoter demonstrated the highest activity. Conclusion In transcriptional level,survivin promoter can activate the reporter gene in several tumor cell lines.It is a potential candidate promoter in tumor gene therapy.

The goal of this study is to investigate the population pharmacokinetics of oral tacrolimus in Chinese renal transplant patients and to identify possible relationship between covariates and population parameters. Details of drug dosage history, sampling time and concentration of 802 data points in 58 patients were collected retrospectively. Before analysis, the 58 patients were randomly allocated to either the model building group (n=41) or the validation group (n=17). Population pharmacokinetic data analysis was performed using the nonlinear mixed-effects model (NONMEM) program on the model building group. The pharmacokinetics of tacrolimus was best described by a one compartment model with first-order absorption and elimination. Typical values of apparent clearance (CL/F), apparent volume of distribution (V/F) were estimated. A number of covariates including demographic index, clinical index and coadministration of other drugs were evaluated statistically for their influence on these parameters. The final population model related clearance with POD (post operative days), HCT (haematocrit), AST (aspartate aminotransferase) and coadministration of nicardipine and diltiazem. Predictive performance of the final model evaluated with the validation group showed insignificant bias between observed and model predicted concentrations. Typical value of CL/F and V/F was 21.7 L x h(-1) and 241 L, inter-patient variability (RSD) in CL/F and V/F was 41.6% and 49.7%, respectively. The residual variability (SD) between observed and model-predicted concentrations was 2.19 microg x L(-1). The population pharmacokinetic model of tacrolimus in Chinese renal transplant patients was established and significant covariates on the tacrolimus model were identified.
Administration, Oral ; Adolescent ; Adult ; Aged ; Asian Continental Ancestry Group ; Female ; Humans ; Immunosuppressive Agents ; administration & dosage ; blood ; pharmacokinetics ; Kidney Transplantation ; Male ; Metabolic Clearance Rate ; Middle Aged ; Models, Statistical ; Nonlinear Dynamics ; Retrospective Studies ; Tacrolimus ; administration & dosage ; blood ; pharmacokinetics ; Young Adult

OBJECTIVETo evaluate the effect of local application of vascular endothelial growth factor (VEGF) via adenovirus-mediated gene transfer on survival of full thickness flaps selected randomly in rats.

METHODSThirty Sprague-Dawley rats weighing 480-520 g were used in this study. A dorsal flap (8 cm x 2 cm) in full thickness with the pedicle located at the level of the iliac crest was designed. Then the rats received 1,012 pfu replication-deficient recombinant adenovirus carrying VEGF (AdCMV-VEGF group, n=10), 1,012 pfu recombinant beta-galactosidase adenovirus (AdCMV-Gal group, n=10) and 1 ml saline (saline group, n=10), respectively, in the distal two thirds of the proposed flap by means of subdermal injection at 8 different locations. Three days after treatment, the flaps were elevated as originally designed and sutured back in situ. The survival rate of the flaps was evaluated on day 7 after operation.

RESULTSThe survival rate of the flaps in the AdCMV-VEGF group increased significantly as compared with those of the AdCMV-Gal group (P<0.01) and the saline group (P<0.01). Immunohistochemical staining showed that VEGF was expressed in the survival flaps injected with AdCMV-VEGF. Histological analysis showed that more granulation tissues and angiogenesis were observed in the AdCMV-VEGF group than those in the AdCMV-Gal and the saline groups.

CONCLUSIONSLocal application of adenovirus-mediated VEGF165 cDNA may efficiently improve the survival of ischemic skin flaps.

Adenoviridae ; genetics ; Animals ; Endothelial Growth Factors ; genetics ; Genetic Therapy ; Intercellular Signaling Peptides and Proteins ; genetics ; Lymphokines ; genetics ; Male ; Neovascularization, Physiologic ; Rats ; Rats, Sprague-Dawley ; Surgical Flaps ; Transfection ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

Objective To construct a modified and enhanced thymidine kinase (TK) vector regulated by human telemerase catalytic subunit promoter (hTERT) promoter and cytomegaiovirus (CMV) enhancer and its killing effect on nasopharyngeal carcinoma in vitro and in vivo and its safety in vivo. Methods The pGL3-basic,as basic vector template,was linked and constructed into TK vector regulated by hTKRT promoter and CMV enhancer with mono-promoter vector as control. Enhanced TK expression was confirmed by fluorescent microscopy and real time fluorescent quantitative PCR. Telomerase activity was measured by stretch PCR. Tumour killing effects were examined by MTT and Boyden areole. The effects of enhanced TK on the invasiveness of tumor cell NPC 5-8F and the growth of xenograft implanted in nude mice were investigated. Results Compared with non-enhanced vector, TK expressed by the enhanced vector significantly increased in NPC 5-8F and MCF-7 cells,telomerase activity was positive in human in NPC 5-8F cells and breast cancer MCF-7 cells and negative in control human blood vessel endothelium ECV-304 cells. After ganciclovir (GCV) treatment, NPC 5-8F cell survival rate and invasiveness decreased and tumor progress of NPC xenograft implanted in nude mice was inhibited, without obvious toxicity effects on mouse liver and kidney. Conclusions The enhanced TK vector regulated by hTERT promoter and CMV enhancer can obviously and specifically inhibit and kill nasopharyngeal carcinoma cells in culture and nasopharyngeal carcinoma xenograft in nude mice in vivo, without obviously toxic side effects on nude mice. The targeted and enhanced TK gene vector with high performance may be a new tumour targeted gene therapy strategy clinically to aim directly at most malignant tumours including nasopharyngeal carcinoma, with more extensive anti-cancer spectrum.

Objective To investigate the protective role of brain-derived neurotrophic factor (BDNF) gene transfected bone-marrow mesenchymal stem cells (BMSC) on cochlear spiral ganglion cells (SGC) impaired by aminoglycoside antibiotics(AmAn). Methods The differentiation of BMSC transfected by BDNF gene (BDNF-BMSC) were detected with immunohistochemical examination of Nestin, neuronspecific enolase (NSE), and glial fibrillary acid protein (GFAP)antibody in vitro. BDNF gene transfected BMSC were transplanted into the cochleae of guinea pigs deafened by amikacin, while the control groups were designed in which artificial perilymphatic fluid(APF), BMSC or BDNF gene was injected into cochleae alone. The cochleae were obtained on the week 1,2 and 4 after injection, respectively, paraffin-embedded,and cut in a paramodiolar plane subsequently. The histopathological changes of cochleae were observed, the density of SGC was calculated by staining with HE, and the corresponding optical density (COD) was calculated with immunohistochemical staining using NSE antibody. And the protective role of various groups on the cochlear SGC were compared. Results The positive staining rate of BDNF gene transfected BMSC with Nestin, NSE and GFAP antibody were all higher than that of BMSC in vitro (P< 0.01). After transplantation into cochleae, the differences of SGC density and COD among various groups were all significant on the same time points(P < 0.05). The SGC density and COD of the BDNF gene transfected BMSC group were the highest. The SGC density and COD of various groups on week 4 were all obviously decreased than those on week 1 and 2 (P < 0.05). Conclusion AmAn-induced SGC damage could be depressed by BMSC, BDNF gene or BDNF gene transfected BMSC transplantion into cochleae, while BDNF gene transfected BMSC showed the best protective role.

Objective： This study was designed to evaluate TNF-α gene therapy for conquering multidrug resistance(MDR). Methods： By using recombinant retrovirus vector, TNF-α gene was transfected into multidrug-resistant human breast cancer cell line MCF-7/Adr. The TNF-α secreting cell clone MCF-7/Adr-TNF1 and MCF-7/Adr-TNF2 were obtained by G418 selection. The integrating and secreting of TNF-α were analyzed by PCR and ELISA method. Cell growth inhibiting and reversal effect of MDR on MCF-7/Adr cells by TNF-α gene were examined by cell account and MTT assay. The changing of intracellular ADR accumulation was analyzed by flow cytometric assay. Results： The level of TNF-α secreted by MCF-7/Adr-TNF1 and MCF-7/Adr-TNF2 were 1737 pg/ml (106 cells/48 h) and 2875 pg/ml respectively. Cancer cells showed lower growth rate after transfection, and the inhibition of growth rate were 32.4％ and 54.8％ in MCF-7/Adr-TNF1 and MCF-7/Adr-TNF2 in comparison with the control, respectively. At the same time, the resistance to ADR were reversed by 5.2 times and 19.3 times, and the intracellular ADR accumulation increased significantly. Conclusion： Drug resistance could be conquered by TNF-α gene therapy. Increasing intracellular drug accumulation may be the mechanism of reversing drug resistance.

BACKGROUNDCardiovascular complications of Kawasaki disease (KD) are a common cause of heart disease in pediatric populations. Previous studies have suggested a role for endothelial progenitor cells (EPCs) in coronary artery lesions associated with KD. However, long-term observations of EPCs during the natural progression of this disorder are lacking. Using an experimental model of KD, we aimed to determine whether the coronary artery lesions are associated with down-regulation of EPCs.

METHODSTo induce KD, C57BL/6 mice were administered an intraperitoneal injection of Lactobacillus casei cell wall extract (LCWE; phosphate buffered saline used as control vehicle). Study groups included: group A (14 days following LCWE injection), group B (56 days following LCWE injection) and group C (controls). Numbers of circulating EPCs (positively staining for both CD34 and Flk-1 while staining negative for CD45) were evaluated using flow cytometry. Bone marrow mononuclear cells were cultured in vitro to expand EPCs for functional analysis. In vitro EPC proliferation, adhesion and migration were assessed.

RESULTSThe model was shown to exhibit similar coronary artery lesions to KD patients with coronary aneurysms. Numbers of circulating EPCs decreased significantly in the KD models (groups A and B) compared to controls ((0.017 ± 0.008)% vs. (0.028 ± 0.007)%, P < 0.05 and (0.016 ± 0.007)% vs. (0.028 ± 0.007)%, P < 0.05). Proliferative, adhesive and migratory properties of EPCs were markedly impaired in groups A and B.

CONCLUSIONCoronary artery lesions in KD occur as a consequence of impaired vascular injury repair, resulting from excess consumption of EPCs together with a functional impairment of bone marrow EPCs and their precursors.

Animals ; Cell Adhesion ; physiology ; Cell Movement ; physiology ; Cell Proliferation ; Cells, Cultured ; Endothelial Cells ; cytology ; Flow Cytometry ; Male ; Mice ; Mice, Inbred C57BL ; Mucocutaneous Lymph Node Syndrome ; pathology ; Stem Cells ; cytology