ABSTRACT
Objective To study the effects of cleavage of Bcl-2 by two DNAzymes on apoptosis of human hepatoma cell line (HepZ1).Methods Two “10-23” DNAzymes(DzT and DzTi) targeting Bcl-2 mRNA and their analogues(DzT' and DzTi') were synthesized and used to cleave Bcl-2 mRNA in vitro and in BEL-7402 cells.The RT-PCR was performed to assess the cleaving efficiency.Expression of Bcl-2 protein was determined by immunofluorescent method.Cell apoptosis was detected by flow cytometry.Results The unmodified Enzymes DzT,and its modified form DzTi,which had an added 3'-inverted thymidine,could effectively cleave Bcl-2 mRNA in vitro.After transfected into BEL-7402 cells,DzTi exhibited more powerful cleaving ability than DzT,significantly down-regulated the level of Bcl-2 protein(P ＜0.01) and inhibited the cell growth(P ＜0.05).The results of flow cytometry suggested that the apoptosis rate of DzT and DzTi significantly increased,appeared apoptotic peak.Cell cycle was delayed in DzT and DzTi group,proportion of cells in G0/G1 increased,S phase cells decreased.Conclusion The synthesized DNAzymes could effectively cleave Bcl-2 mRNA,decrease the level of Bcl-2 protein and induce hepatoma cells apoptosis.
ABSTRACT
Objective To construct a recombinant bacterial vaccine which can express specific 10-23 deoxyribozyme(DZ) in macrophage, identify the intracellular production of specific 10-23DZ and detect the activity of this recombinant bacterial vaccine on inhibiting the expression of TACO gene in macrophage.Methods The pSDE02 was obtained by inserting the replicon of Mycobacterium into pSDE01, a plasmid which can express 10-23DZ in eukaryotic cells. The expression sequence of DZ1, a 10-23DZ targeting the TACO mRNA of macrophage designed in our previous study was synthesized and inserted into pSDE02. The resulted plasmid was named pDZM01. pDZM01 was then transferred into Mycobacterium smegmatis by electroperation. The recombinant M. smegmatis, named rMs-DZ1 was screened on low-salt LB medium containing Zeocin and identified by Colony PCR. The targeted delivery property of recombinant M. smegmatis was observed by Ziehl-Heelson stain and GFP expression observation via fluorescence microscope. rMs-DZ1 was used to infect RAW264.7 cells and the expression of DZ1 in macrophage was identified by dot-blot assay. At 24 h and 48 h after infection, total RNA and proteins were extracted and the TACO mRNA and protein expression level was assayed by RT-PCR and western-blot respectively. Results Restrictive analysis and sequencing data showed that the Mycobacterium-eukaryotic cell shuttle plasmid pSDE02 and pDZM01 was successfully constructed. rMs-DZ1 was confirmed by colony PCR. When engulfed by macrophage, rMs-DZ1 would express DZ1 in RAW264.7 cells and inhibit the expression of taco gene. When compared to uninfected macrophage, rMs-DZ1 significantly reduced the taco mRNA by 67.90% and 57.14% and down-regulated the expression of TACO protein by 53.85% and 68.92% at 24 h and 48 h respectively. Conclusion A recombinant M. smegmatis vaccine was successfully constructed which could generate specific 10-23DZ in macrophage and inhibit the expression of target gene of interest. To our knowledge, this is the first bacterial vector which can express intracellularly 10-23DRz in targeted manner. This study may further prompt the feasibility of using 10-23 DNAzyme to achieve effective and targeted gene silence.
ABSTRACT
Objective To investigate the effects of deoxyribozyme(DZ) against respiratory syncytial virus(RSV) infection in BALB/c mice and nude mice. Methods RSV infected BALB/c mice and nude mice were nasally dripped with DZ. Pulmonary viral titers were detected by plaque forming experiment,and viral mRNA expression was assayed by RT-PCR. Leukocytes and the subgroup cells in bronchoalveolar lavage fluid (BALF) were counted, cytokines of TNF-α, IL-12, IFN-γand IL-10 in BALF were assayed by ELISA. Pulmonary histopathology was examined to realize the inflammation of airway. Results Pulmonary titers of 0.2 mg, 0.4 mg and 0.8 mg DZ treated BALB/c mice were lg(3.65 ±0.12) PFU/g lung,lg( 3.25 ± 0.10) PFU/g lung and lg( 3.03 ±0.08 ) PFU/g lung, decreased as compared with that of infected control BALB/c mice lg(4.35 ± 0.11 ) PFU/g lung ( P<0.05 ). Meanwhile viral titers of 0.2 mg,0.4 mg and 0.8 mg DZ treated nude mice were lg(4.82 ±0.15) PFU/g lung, lg(4.47 ±0.12) PFU/g lung and lg(4.21 ±0.11 ) PFU/g lung, declined dramatically as compared with that of infected control nude mice lg(6.23 ± 0.15) PFU/g lung( P<0.01 ). 0.2 mg, 0.4 mg and 0.8 mg DZ reduced BALB/c mice pulmonary viral mRNA expression by 30.51% ,47.38% ( P<0.05 ) and 53.97% ( P<0.01 ) and nude mice by 36.59% (P ＜0.05 ), 48.72%, 59.78% ( P<0.01 ) respectively as compared with their infected control groups. In 0.4 mg DZ treated BALB/c mice and nude mice, total numbers of leukocytes in BALF were decreased dramatically and pulmonary histology was significantly improved compared with their infected controls( P<0.05 ). And the treatment of 0.4 mg DZ reduced productions of TNF-α, IL-12 and IFN-γin BALF of RSV infected nude mice ( P<0.05 ). Conclusion DZ effectively inhibits viral replication and reduces airway inflammation in RSV infected BALB/c mice and nude mice, and the effects in nude mice are more significant. DZ is a potential therapeutic agent against RSV infection in vivo.
ABSTRACT
Isocitrate lyase (ICL) which plays a pivotal role in the glyoxylate cycle of mycobacterium tuberculosis(MTB) could be a potential target against mycobacterium tuberculosis.Deoxyribozyme(DRz) is a small single-strand,enzymatic DNA molecule.Assistant by sketch of secondary structure,appropriate and not appropriate target sequences of MTB ICL mRNA were predicted.Two DRzs were designed targeting above sequences respectively.To affirm feasibility of this kind of prediction,in vitro cleavage of MTB ICL mRNA segment by DRz-ICLcf and DRz-ICLcj were performed.The results showed that sketch of secondary structure could indeed predict potential target of DRz.This research will provide basis for developing DRz as a novel antimicrobial agent against MTB.