Deoxyribozymes Targeting Period1 Gene Block Its Expression In Vivo And In Vitro

Drug addcition results from the long-term abuse of certain drugs, which has been a medical and social problem paid close attention to. But, treatment of psychic dependence of drug abuse is still troublesome presently. Based on the correlation between the circdian gene period 1 (per1) and drug addiction, the deoxyribozyme (DRz) targeting per1was used a tool of blocking the per1 expression in order to make a further resarch and explore a novel therapy anout drug addition.10-23 deoxyribozymes (DRz 164 and DRz256) directed against per1 was designed and synthesized to serve as a gene inhibitor. An in vitro transcription plasmid pcDNA3.1(+)-per1_164:256 was constructed. Following the in vitro transcription, cleavage reactions in vitro were carried out by mixing DRz 164 /DRz256 with DIG-labeled per1 mRNA component in certain reaction conditions. The cleavage band was detected by DIG nuclei acid detection kit. The eukaryotic expression plasmid pcDNA3-per1 and DRz164/DRz256 were introduced into NIH3T3 cells by lipofectamine. The effects of deoxyribozymes on per1 were studied by reverse transcript-polymerase chain reaction (RT-PCR) and flow cytometry (FCM). The cleavage reactions showed that both DRzl64 and DRz256 had specific cleavage activities against the per1 mRNA component. The in vitro cleavage ratio reached about 63% byDRzl64, and 50.5% by DRz256. Analysis of PCR and FCM indicated that DRzl64/DRz256 can highly block the expression of perl gene in cellular milieu.Mice were housed under a 12-h Iightyl2-h dark cycle and divided into 3 groups randomly. The morphine (10 mg/kg, s.c.) -induced dependence in mice was observed in a conditioned place preference test after Lev. pretreatment with the deoxyribozyme (In g/mouse). Cellular nuclear extracts and cytoplasmic fractions of mouse brain were prepared every 4 h after the last injection of morphine/saline. Perl protein was detected by western blot technology. In the following, c-Fos protein and MAPKs were analysized by western blot technology. Lev. pretreatment with deoxyribozyme (In g/mouse) attenuated this morphine (10 mg/kg, s.c.) -induced place preference in morphine-dependent mice by ANOVA between the groups. The content of Perl protein in nuclear and cytoplasm fractions in the mice pretreatment with DRzl64 decreased but in the morphine-dependent mice increased. The expression of c-Fos protein in the mice pretreatment with dDRzl64 decreased and in the morphine-dependent mice increased. Analysis of MAPKs indicated that the expression of MAPKs in the mice pretreatment with dDRzl64 increased and in the morphine- dependent mice decreased.The human neuroblastoma cells SH-SY5Y were cultured and differentiated by retinoic acid. First, SH-SY5Y cells were pretreated with DRzl64. After 24h cells were incubated with 1640 containg 50% horse serum for 2 h. and then incubated with the 1640 without serum in the presence of chronic morphine (10 u M) exposure for 48 h. perl expression was detected by RT-PCR at different time and basic cAMP concentration was determined by radioimmunoasssay. In addtion, MAPK pathway was detected by western blot. The results displayed that perl expression was attenuated in SH-SY5Ycells pretretment with DRzl64. The change of cAMP concentrationwas divided into two stages, which differed from the morphine control cells. MAPK signaling pathways were induced by acute morphine treatment. The effect of DRzl64 on MAPK pathway was more marked than that of morphine. Cells with chronic morphine exposure show no siginificant increase of MAPK but cells pretreatment with DRzl64 persisted.