AIM: To explore the anti-hepatitis B virus effect of RNA interference (RNAi) using small hairpin RNA (shRNA)expression vector.METHODS: Hepatitis B virus surface antigen green fluorescent protein (HBs-GFP) fusion vecto...AIM: To explore the anti-hepatitis B virus effect of RNA interference (RNAi) using small hairpin RNA (shRNA)expression vector.METHODS: Hepatitis B virus surface antigen green fluorescent protein (HBs-GFP) fusion vector and shRNA expression vectors were constructed and cotransfected transiently into HepG2 cells. mRNAs extracted from HepG2 cells were detected by real-time PCR. Fluorescence of HBs-GFP protein was detected by fluorescence-activated cell sorting (FACS). The effective shRNA expression vector was transfected into HepG2.2.15 cells. HBsAg and HBeAg in HepG2.2.15 cells were analyzed by radioimmunoassay (RIA) method.RESULTS: FACS revealed that shRNA targeting at HBsAg reduced the GFP signal by 56% compared to the control.Real-time PCR showed that HBs-GFP mRNA extracted from HepG2 cells cotransfected with pAVU6+27 and HBs-GFP expression plasmids decreased by 90% compared to the empty vector control. The expressions of HBsAg and HBeAg were also inhibited by 43% and 64%, respectively.CONCLUSION: RNAi using shRNA expression vector can inhibit the expression of HBsAg, providing a fresh approach to screening the efficient small interfering RNAs (siRNAs).展开更多
Objective:The aim of this study was to study the effect of Bcl-2 small hairpin RNA(shRNA) enhancing methotrexate(MTX)-induced apoptosis of Raji cells.Methods:Expression plasmid with Bcl-2 shRNA was transfected into Ra...Objective:The aim of this study was to study the effect of Bcl-2 small hairpin RNA(shRNA) enhancing methotrexate(MTX)-induced apoptosis of Raji cells.Methods:Expression plasmid with Bcl-2 shRNA was transfected into Raji cells by Lipofectmine 2000 and then treated with MTX.At 48 h of transfection,the expression level of Bcl-2 mRNA and protein was evaluated by RT-PCR and immunofluorescence.MTT assay was used to analyze cell proliferation at 24,48 and 72 h.Apoptosis was detected by Giemsa staining and flow cytometric cell cycle analysis.Results:After transfection with Bcl-2 shRNA,the expression levels of Bcl-2 mRNA and protein in Raji cells decreased(P < 0.05).Using Giemsa staining,cells transfected with Bcl-2 shRNA combined with MTX at 48 h displayed changes of apoptosis.MTX significantly inhibited the growth of cells after transfected with Bcl-2 shRNA(P < 0.05).Apoptotic rates of the Raji cells treated with Bcl-2 shRNA combined with MTX significantly increased(P < 0.05),compared with either control shRNA/MTX combination or MTX-treatment cells alone.Conclusion:Our results suggest the shRNA against Bcl-2 mRNA could increase MTX-induced apoptosis of Raji cells.展开更多
OBJECTIVE To investigate whether small hairpin RNA (shRNA)targeting Bcl-2 mRNA could inhibit the growth of lymphomatransplanted subcutaneously in nude mice.METHODS Recombinant Bcl-2 shRNA expression vector withgreen f...OBJECTIVE To investigate whether small hairpin RNA (shRNA)targeting Bcl-2 mRNA could inhibit the growth of lymphomatransplanted subcutaneously in nude mice.METHODS Recombinant Bcl-2 shRNA expression vector withgreen fluorescence protein (GFP) gene was constructed andpreserved in our lab. We evaluated the antitumor effect of the Bcl-2shRNA in vivo which was the model of nude mice bearing Rajicells xenografts. Human Raji cells were injected subcutaneouslyinto nude mice to establish lymphoma models. When thediameters of tumor were above 0.5 cm after Raji cells injection,the mice bearing tumor were randomly divided into four groups:saline control group, negative shRNA group, plasmid vectorgroup, Bcl-2 shRNA group. The polyethylenimine (PEI) was usedto transfect shRNA into tumor. The mixed PEI and shRNA wasinjected into tumors. The growth and size of tumor were observed.Tissue was stained by H&E for its pathological morphology. Theexpression of Bcl-2 mRNA in the tumor mass was detected byreverse transcription polymerase chain reaction (RT-PCR).RESULTS A significant difference in median tumor weightwas observed in mice treated with Bcl-2 shRNA, compared withthose in the groups of negative shRNA or plasmid vector or salinesolution (P< 0.05). Pathological evaluation was completed in allexcised tumors from nude mice bearing Raji cells xenografts.The tumor tissue of the mice treated with Bcl-2 shRNA showedapoptosis, serious necrosis of the cells and inflammatory cellsinfiltration. There was no change in the morphology of cellsamong negative shRNA, plasmid vector and saline solution group.In the group of the Bcl-2 shRNA, the expression levels of Bcl-2mRNA of the tumor tissue were effectively inhibited (P < 0.05).CONCLUSION The shRNA targeting at the Bcl-2 mRNAcould inhibit the growth of human lymphoma transplantedsubcutaneously in nude mice.展开更多
基金Supported by the National Natural Science Foundation of China, No. 30371270 the Major Program of Department of Science and Technology of Zhejiang Province, No. 2003C13015
文摘AIM: To explore the anti-hepatitis B virus effect of RNA interference (RNAi) using small hairpin RNA (shRNA)expression vector.METHODS: Hepatitis B virus surface antigen green fluorescent protein (HBs-GFP) fusion vector and shRNA expression vectors were constructed and cotransfected transiently into HepG2 cells. mRNAs extracted from HepG2 cells were detected by real-time PCR. Fluorescence of HBs-GFP protein was detected by fluorescence-activated cell sorting (FACS). The effective shRNA expression vector was transfected into HepG2.2.15 cells. HBsAg and HBeAg in HepG2.2.15 cells were analyzed by radioimmunoassay (RIA) method.RESULTS: FACS revealed that shRNA targeting at HBsAg reduced the GFP signal by 56% compared to the control.Real-time PCR showed that HBs-GFP mRNA extracted from HepG2 cells cotransfected with pAVU6+27 and HBs-GFP expression plasmids decreased by 90% compared to the empty vector control. The expressions of HBsAg and HBeAg were also inhibited by 43% and 64%, respectively.CONCLUSION: RNAi using shRNA expression vector can inhibit the expression of HBsAg, providing a fresh approach to screening the efficient small interfering RNAs (siRNAs).
基金Supported by the grants from the Natural Science Program Foundation of Guangdong Province(No.04010446)the Overseas Chinese Affairs Office of the State Council Key Discipline Construction Fund (No.51205002)
文摘Objective:The aim of this study was to study the effect of Bcl-2 small hairpin RNA(shRNA) enhancing methotrexate(MTX)-induced apoptosis of Raji cells.Methods:Expression plasmid with Bcl-2 shRNA was transfected into Raji cells by Lipofectmine 2000 and then treated with MTX.At 48 h of transfection,the expression level of Bcl-2 mRNA and protein was evaluated by RT-PCR and immunofluorescence.MTT assay was used to analyze cell proliferation at 24,48 and 72 h.Apoptosis was detected by Giemsa staining and flow cytometric cell cycle analysis.Results:After transfection with Bcl-2 shRNA,the expression levels of Bcl-2 mRNA and protein in Raji cells decreased(P < 0.05).Using Giemsa staining,cells transfected with Bcl-2 shRNA combined with MTX at 48 h displayed changes of apoptosis.MTX significantly inhibited the growth of cells after transfected with Bcl-2 shRNA(P < 0.05).Apoptotic rates of the Raji cells treated with Bcl-2 shRNA combined with MTX significantly increased(P < 0.05),compared with either control shRNA/MTX combination or MTX-treatment cells alone.Conclusion:Our results suggest the shRNA against Bcl-2 mRNA could increase MTX-induced apoptosis of Raji cells.
基金supported by a grant from Natural Science Program Foundation of Guangdong Province,China(No.04010446).
文摘OBJECTIVE To investigate whether small hairpin RNA (shRNA)targeting Bcl-2 mRNA could inhibit the growth of lymphomatransplanted subcutaneously in nude mice.METHODS Recombinant Bcl-2 shRNA expression vector withgreen fluorescence protein (GFP) gene was constructed andpreserved in our lab. We evaluated the antitumor effect of the Bcl-2shRNA in vivo which was the model of nude mice bearing Rajicells xenografts. Human Raji cells were injected subcutaneouslyinto nude mice to establish lymphoma models. When thediameters of tumor were above 0.5 cm after Raji cells injection,the mice bearing tumor were randomly divided into four groups:saline control group, negative shRNA group, plasmid vectorgroup, Bcl-2 shRNA group. The polyethylenimine (PEI) was usedto transfect shRNA into tumor. The mixed PEI and shRNA wasinjected into tumors. The growth and size of tumor were observed.Tissue was stained by H&E for its pathological morphology. Theexpression of Bcl-2 mRNA in the tumor mass was detected byreverse transcription polymerase chain reaction (RT-PCR).RESULTS A significant difference in median tumor weightwas observed in mice treated with Bcl-2 shRNA, compared withthose in the groups of negative shRNA or plasmid vector or salinesolution (P< 0.05). Pathological evaluation was completed in allexcised tumors from nude mice bearing Raji cells xenografts.The tumor tissue of the mice treated with Bcl-2 shRNA showedapoptosis, serious necrosis of the cells and inflammatory cellsinfiltration. There was no change in the morphology of cellsamong negative shRNA, plasmid vector and saline solution group.In the group of the Bcl-2 shRNA, the expression levels of Bcl-2mRNA of the tumor tissue were effectively inhibited (P < 0.05).CONCLUSION The shRNA targeting at the Bcl-2 mRNAcould inhibit the growth of human lymphoma transplantedsubcutaneously in nude mice.
