ABSTRACT
<p><b>OBJECTIVE</b>To explore the feasibility of IGF2 imprinting system in target gene therapy for tumors.</p><p><b>METHODS</b>The mouse H19 enhancer, DMD and promoter H19 were amplified by PCR from mouse genomic DNA and then cloned into the plasmid pDC312. The EGFP and DT-A fragments were amplified by PCR and cloned into the recombinant plasmid, and then the shuttle plasmid were transfected into HEK293 cells together with the adenoviral vector Ad5, namely, Ad-EGFP and Ad-DT-A. Adenovirus hexon gene expression was applied to confirm the presence of adenovirus infections. The effect of the IGF2 imprinting system was tested by fluorescence microscopy. RT-PCR and Western blotting after transfection of the recombinant adenoviral vectors into cancer cells were used to show loss of IGF2 imprinting (LOI) and maintenance of IGF2 imprinting (MOI), respectively. The anti-tumor effect was assessed by MTT and flow cytometry after the HCT-8 (LOI). Human breast cancer cell line MCF-7 (MOI) and human normal gastric epithelial GES-1 (MOI) cell line were transfected with Ad-DT-A in vitro. The anti-tumor effect was detected by injecting the Ad-DT-A in nude mice carrying HCT-8 tumors.</p><p><b>RESULTS</b>The expression of EGFP protein, DT-A mRNA and DT-A protein were seen to be positive only in the HCT-8 tumor cell line. Infection with Ad-DT-A resulted in obviously growth inhibition in HCT-8 cells (75.4 ± 6.4)% compared with that in the control group, and increased the percentage of apoptosis in the HCT-8 cells (20.8 ± 5.9)%. The anti-tumor effect was further confirmed by injecting the recombinant adenoviruses in HCT-8 tumor-bearing nude mice, and the results showed that the Ad-DT-A inhibited the tumor growth, with on inhibition rate of 36.4%.</p><p><b>CONCLUSIONS</b>The recombinant adenoviruses carrying IGF2 imprinting system and DT-A gene have been successfully constructed, while Ad-DT-A can effectively kill the tumor cells showing loss of IGF2 imprinting. It might play an important role in future target gene therapy against malignant tumors based on loss of IGF2 imprinting.</p>
Subject(s)
Animals , Female , Humans , Mice , Adenoviridae , Genetics , Apoptosis , Breast Neoplasms , Genetics , Pathology , Colonic Neoplasms , Genetics , Pathology , Therapeutics , Diphtheria Toxin , Genetics , Genetic Therapy , Methods , Genetic Vectors , Genomic Imprinting , Green Fluorescent Proteins , Genetics , Insulin-Like Growth Factor II , Genetics , Metabolism , MCF-7 Cells , Mice, Nude , Neoplasm Transplantation , Peptide Fragments , Genetics , Plasmids , RNA, Messenger , Metabolism , Random Allocation , Recombinant Fusion Proteins , Genetics , TransfectionABSTRACT
<p><b>AIM</b>To investigate the preventive effects of Panax notoginseng saponins (PNS) and Ginkgo biloba extracts (GbE) on acute oxygen toxicity and the possible mechanisms.</p><p><b>METHODS</b>Mice were injected intraperitoneally with PNS and GbE for 5 days, then were exposed to 500 kPa hyperbaric oxygen (HBO) for 60 min, the convulsion latency, times and interval were observed. Moreover, reactive oxygen (RO) unit, MDA, NO, GSH levels and GSH-Px, CAT, MAO activities of mice brain were determined after they were exposed to HBO for 15 min.</p><p><b>RESULTS</b>PNS and GbE could markedly prolong the convulsion latency and interval, reduce convulsion times, decrease contents of MDA and NO in mice brain, keep RO unit, GSH and GSH-Px at higher levels, but had no effects on CAT and MAO activities.</p><p><b>CONCLUSION</b>PNS and GbE could effectively prevent acute oxygen toxicity, which were related to their antioxidant activities.</p>