Summary： Alzheimer＇s disease （AD） is an age-related, progressive neurodegenerative disorder that occurs gradually and results in memory, behavior, and personality changes. Abnormal sphingolipid metabolism was repo...Summary： Alzheimer＇s disease （AD） is an age-related, progressive neurodegenerative disorder that occurs gradually and results in memory, behavior, and personality changes. Abnormal sphingolipid metabolism was reported in AD previously. This study aimed to investigate whether sphK1 could exacerbate the accumulation of amyloid protein （Aβ） and sharpen the learning and memory ability of the animal model of AD using siRNA interference. An adenovirus vector expressing small interfering RNA （siRNA） against the sphK1 gene （sphKl-siRNA） was designed, and the effects of sphKl-siRNA on the APP/PS1 mouse four weeks after treatment with sphKl-siRNA hippocampal injection were examined. SphK1 protein expression was confirmed by using Western blotting and ceramide content coupled with SIP secretion was evaluated by enzyme-linked immunosorbent assay （ELISA）. Aβ load was detected by immunohistochemical staining and ELISA. Morris water maze was adopted to test the learning and memory ability of the APP/PS 1 mice. A significant difference in the expression of sphK1 protein and mRNA was observed between the siRNA group and the control group. Aβ load in transfected mice was accelerated in vivo, with significant aggravation of the learn- ing and memory ability. The sphKl gene modulation in the All load and the learning and memory ability in the animal model of AD may be important for the treatment of AD.展开更多
The RNA interference(RNAi)phenomenon is a recently observed process in which the introduction of a double-stranded,small interfering RNA(siRNA)into a cell causes the specific degradation of a homologous single-strande...The RNA interference(RNAi)phenomenon is a recently observed process in which the introduction of a double-stranded,small interfering RNA(siRNA)into a cell causes the specific degradation of a homologous single-stranded RNA.It represents an exciting new technology that could have therapeutic applications for the treatment of viral infections.Since hepatitis G virus(HGV)genome is a positive-sense single-stranded RNA,the replication of HGV does not lead to an integrated DNA genome,suggesting a particularly attractive target for RNAi study that could eliminate viral RNA from infected cells.The eukaryotic expression vector pVAX.EH containing the cDNA sequences of the entire HGV structural genes and hygromycin resistance gene downstream from the encephalomyocarditis virus(ECMV)internal ribosome entry site(IRES)was constructed and transfected into human hepatoma cell Huh-7.The modified cleavage products of the structural proteins of HGV expressed in hygromycin-resistant cell line Huh-7-EH were confirmed by RT-PCR and Western blot methods.Two specific HGV E2 siRNAs(1-E2siRNA,2-E2 siRNA)synthesized with T7 RNA polymerase by transcription in vitro were transfected into the Huh-7-EH cells.With the analyses of Western blot and the formation of hygromycin-resistant colonies,the inhibitions of expression of HGV structural protein by two HGV E2siRNAs were detected and found lasting at least one week.The inhibition of 2-E2 siRNA was stronger and only 1%of the cells treated with 2-E2 siRNA formed hygromycin-resistant colonies.These results support that specific HGV 2-E2 siRNAs mediate the degradation of mRNA spanning from HGV structural gene cDNA to hygromycin resistance gene in a majority of cells.In conclusion,the Huh-7-EH cells expressing HGV structural proteins stably can be used as a cell model for studying the replication of HGV and RNAi and the enlargement of RNAi may exist,in mammalian cells.展开更多
Chronic liver diseases with different aetiologies rely on the chronic activation of liver injuries which result in a fibrogenesis progression to the end stage of cirrhosis and liver failure.Based on the underlying cel...Chronic liver diseases with different aetiologies rely on the chronic activation of liver injuries which result in a fibrogenesis progression to the end stage of cirrhosis and liver failure.Based on the underlying cellular and molecular mechanisms of a liver fibrosis,there has been proposed several kinds of approaches for the treatment of liver fibrosis.Recently,liver gene therapy has been developed as an alternative way to liver transplantation,which is the only effective therapy for chronic liver diseases.The activation of hepatic stellate cells,a subsequent release of inflammatory cytokines and an accumulation of extracellular matrix during the liver fibrogenesis are the major obstacles to the treatment of liver fibrosis.Several targeted strategies have been developed,such as antisense oligodeoxynucleotides,RNA interference and decoy oligodeoxynucleotides to overcome this barriers.With this report an overview will be provided of targeted strategies for the treatment of liver cirrhosis,and particularly,of the targeted gene therapy using short RNA and DNA segments.展开更多
基金supported by the National Natural Science Foundation of China (No. 81070879)
文摘Summary： Alzheimer＇s disease （AD） is an age-related, progressive neurodegenerative disorder that occurs gradually and results in memory, behavior, and personality changes. Abnormal sphingolipid metabolism was reported in AD previously. This study aimed to investigate whether sphK1 could exacerbate the accumulation of amyloid protein （Aβ） and sharpen the learning and memory ability of the animal model of AD using siRNA interference. An adenovirus vector expressing small interfering RNA （siRNA） against the sphK1 gene （sphKl-siRNA） was designed, and the effects of sphKl-siRNA on the APP/PS1 mouse four weeks after treatment with sphKl-siRNA hippocampal injection were examined. SphK1 protein expression was confirmed by using Western blotting and ceramide content coupled with SIP secretion was evaluated by enzyme-linked immunosorbent assay （ELISA）. Aβ load was detected by immunohistochemical staining and ELISA. Morris water maze was adopted to test the learning and memory ability of the APP/PS 1 mice. A significant difference in the expression of sphK1 protein and mRNA was observed between the siRNA group and the control group. Aβ load in transfected mice was accelerated in vivo, with significant aggravation of the learn- ing and memory ability. The sphKl gene modulation in the All load and the learning and memory ability in the animal model of AD may be important for the treatment of AD.
基金This work was supported by the National Basic Research(973)Progrum of China(No.2003CB514129)the National INatural Science Foundation of China(Grant No.30170514).
文摘The RNA interference(RNAi)phenomenon is a recently observed process in which the introduction of a double-stranded,small interfering RNA(siRNA)into a cell causes the specific degradation of a homologous single-stranded RNA.It represents an exciting new technology that could have therapeutic applications for the treatment of viral infections.Since hepatitis G virus(HGV)genome is a positive-sense single-stranded RNA,the replication of HGV does not lead to an integrated DNA genome,suggesting a particularly attractive target for RNAi study that could eliminate viral RNA from infected cells.The eukaryotic expression vector pVAX.EH containing the cDNA sequences of the entire HGV structural genes and hygromycin resistance gene downstream from the encephalomyocarditis virus(ECMV)internal ribosome entry site(IRES)was constructed and transfected into human hepatoma cell Huh-7.The modified cleavage products of the structural proteins of HGV expressed in hygromycin-resistant cell line Huh-7-EH were confirmed by RT-PCR and Western blot methods.Two specific HGV E2 siRNAs(1-E2siRNA,2-E2 siRNA)synthesized with T7 RNA polymerase by transcription in vitro were transfected into the Huh-7-EH cells.With the analyses of Western blot and the formation of hygromycin-resistant colonies,the inhibitions of expression of HGV structural protein by two HGV E2siRNAs were detected and found lasting at least one week.The inhibition of 2-E2 siRNA was stronger and only 1%of the cells treated with 2-E2 siRNA formed hygromycin-resistant colonies.These results support that specific HGV 2-E2 siRNAs mediate the degradation of mRNA spanning from HGV structural gene cDNA to hygromycin resistance gene in a majority of cells.In conclusion,the Huh-7-EH cells expressing HGV structural proteins stably can be used as a cell model for studying the replication of HGV and RNAi and the enlargement of RNAi may exist,in mammalian cells.
基金Supported by National Research Foundation of Korea Grant funded by the Korean Government,No.2012R1A1A401015639
文摘Chronic liver diseases with different aetiologies rely on the chronic activation of liver injuries which result in a fibrogenesis progression to the end stage of cirrhosis and liver failure.Based on the underlying cellular and molecular mechanisms of a liver fibrosis,there has been proposed several kinds of approaches for the treatment of liver fibrosis.Recently,liver gene therapy has been developed as an alternative way to liver transplantation,which is the only effective therapy for chronic liver diseases.The activation of hepatic stellate cells,a subsequent release of inflammatory cytokines and an accumulation of extracellular matrix during the liver fibrogenesis are the major obstacles to the treatment of liver fibrosis.Several targeted strategies have been developed,such as antisense oligodeoxynucleotides,RNA interference and decoy oligodeoxynucleotides to overcome this barriers.With this report an overview will be provided of targeted strategies for the treatment of liver cirrhosis,and particularly,of the targeted gene therapy using short RNA and DNA segments.
