ABSTRACT
Intra-arterial chemotherapy (IAC) refers to the injection of chemotherapeutics into patients' eyes through arteria ophthalmica in order to local chemotherapy for retinoblastoma (RB).The common drugs in the treatment of RB include melphalan,topotecan and carboplatin.IAC can form the high-concentration drug effects on local areas.It can effectively kill tumors and significantly reduce the occurrence rate of complications after intravenous chemotherapy.Application of IAC can cure patients with RB in B,C,D and E stage in international criteria,which can effectively improve eye protection rate.Complications of IAC include eyelids edema,chemosis,blepharoptosis,ocular motility disorders,vitreous hemorrhage,ischemia of choroid or retina,transient myelosuppression,etc.It should be further conduction of long-term and large-sample cases studies on how to improve curative effects and reduce serious eye complications.
ABSTRACT
Background Adeno-associated virus-based vector is one of most efficient vehicles.It presents with a long-term and efficient transfer and expression of therapeutic genes with minimal toxicity.But its delayed-and low-efficient transgene expression limits the application of AAV vector.To explore an improving method of AAV infecting RPE cells is the hot spot. Objeetive Present study was to investigate whether adeno-associated virus (AAV)combined with low dose non-replieable adenovirus(Ad-null)can enhance its infection efficieney on RPE ceils in vitro. Methods Human RPE cells were isolated from the donate eyeballs under the approval of the Ethic Committee of this hospital.The cells were cultured in DMEM containing 10%fetal bovine serum.AAV particles with enhanced green fluorescence protein(EGFP)were added into the medium alone or in combination with different amount of adenovirus for 30 days.The cells were detected under the fluorescence microscope,and the protein expression levels of report gene EGFP in RPE cells were analyzed with Western blotting assay. Results Melanin granules could be found in cultured RPE cells.EGFP was expressed in RPE cells at 2 days after AAV-EGFP infection and peaked at 12 days and remained for about 3-week duration,showing the green influorescence under the influorescence mwroscope.After the cells were infected by AAV2-EGFP with 0.01 to 1000 MOI Ad-null respectively,the number of cells with green influorescence was obviously increased with the enhanced infiuorescence intensity.The enhance of the infection efficiency began in the 0.1 MOI Ad-null group and peaked in 10 MOI Ad-null group.Dead cells were exhibited in the 100 or more MOI Ad-nulor group.Western blotting assay demonstrated that the protein expression level of EGFP in RPE cells enhanced significantly in 1 and 10 MOI Ad-null groups compared with only AAV infection group. Conclusion These finding suggested that the infection efficiency of AAV can be improved significantly when it is used with low dose Ad-null in vitro.This offers a basis for further study of gene therapy.
ABSTRACT
Objective To construct a new conditionally replicative adenovirus (CRAds) targeting carcinoembryonic antigen (CEA) positive colorectal cancer cells. Methods The DNA fragment of the CEA gene promoter was amplified through PCR and cloned into the vector carrying fusion reporter gene EGFP-Luc to construct expression plasmid pCEA-EGFPLuc. The constructed plasmid pCEA-EGFPLuc was transfected into CEA positive and negative cells by liposome. The activity of CEA gene promoter was evaluated by detecting the expression of EGFP and luciferase activity. The conditionally replicative adenovirus Ad.CEA-E1A/CMV-TK carrying suicide gene HSVtk was constructed, in which the E1A gene was controlled under CEA promoter. CEA positive(Lovo and SW620)and negative tumor cells(HeLa) were infected with Ad.CEA-E1A/ CMV-TK. The selective cytotoxicity of Ad.CEA-E1A/CMV-TK and the synergistic effect of the virus with GCV in CEA positive tumor cells were evaluated by the expression of E1A, cytopathic effect and cell survival rate. Results CEA promoter possesses a good specificity as well as high activity. The expression of E1A only presented in CEA positive tumor cells. After infection with Ad. CEA-E1A/CMV-TK, the cell survival rates of Lovo and SW620 were (36.72±2.49)% and (39.82±4.76)%, respectively, significantly lower than that of Hela[(87.44±2.76)%1 (P<0.01). When combined with GCV, Ad.CEA-E1A/CMV-TK had better oncolytic effect on Lovo and SW620 cells, with cell survival rates of (17.26±3.65) % and (23.93±5.40) %, respectively, significantly lower than those without GCV[(36.72±2.49) % and (39.82±4.76) %, respectively] (P<0.01). Conclusion Ad. CEA-E1A/CMV-TK under the control of CEA promoter has selective cytotoxic effect on CEA positive colorectal cancer cells, and the effect can be enhanced when combined with GCV.