Objective:To investigate the effects of chemotherapeutic agent Etoposide on transgene expression mediated by recombinant replication-defective adenovirus in tumor cell lines.Methods:Cultured tumor cells,including NCI-H446,NCI-H460,A549,SMMC-7721,SGC7901,SKBR-3,and BTT were infected by Ad-CMV-EGFP alone(MOI being 1 and 10)or in combination with Etoposide at different final concentrations(0.2,2,20,40,80,100 and 200 μg/ml).GFP positive cell rates and the mean intensities of GFP fluorescence in tumor cells were detected by Fluorescence Activated Cell Sorting(FACS)after cultured with different strategies.EGFP protein expression in tumor cells was analyzed by Western blotting.Quantitative analysis of mRNA and DNA copies of EGFP in tumor cells were performed by RT-PCR and real-time PCR.Results:FACS results indicated that Etoposide efficiently enhanced the mean intensities of EGFP fluorescence to different degrees in all 7 cell lines but had no evident effects on the EGFP positive rate.Twenty-four hours after cultured at the presence of 10 MOI Ad5-CMV-EGFP and 40 μg/ml Etoposide,the mean intensities of EGFP fluorescence in NCI-H446,NCI-H460,A549,SMMC-7721,SGC7901,SKBR-3,and BTT cells were respectively 3.3,3.5,3.1,6.2,7.0,5.4,and 3.4 folds that cultured with 10 MOI Ad5-CMV-EGFP alone.Western blotting showed that EGFP protein expression in cells co-cultured with Ad5-CMV-EGFP and Etoposide was 2-5 times that cultured with 10 MOI Ad5-CMV-EGFP alone.EGFP mRNA expression had a similar tendency as EGFP protein,but the copies of EGFP DNA had no evident changes.Conclusion:Etoposide can enhance transgene expression mediated by recombination replication-defective adenovirus in several tumor cell lines,which may play a role at the transcriptional level.

Diabetic retinopathy (DR) is one of the most common diabetic complications. In lots of researches,there were indications that retinoneurocyte degeneration appeared in the early stage of diabetic retinopathy. And angiogenesis and proliferative membrane , which were induced by vascular endothelial growth factor (VEGF) or pigment epithelium-derived factor (PEDF), appeared in the late stage of diabetic retinopathy, which may cause poor vision or even blindness. At present, there is no effective treatment for diabetic retinopathy. The aim of this review is to present the progress and current researches of gene therapy for DR.

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.